Treatment of autoimmune diseases with engineered immune cells

Abstract

The present invention includes methods and compositions for treating autoimmune diseases with engineered immune cells including cytotoxic T cells and natural killer (NK) cells. The engineered immune cells include a chimeric antigen receptor (CAR). Also disclosed are methods of making the engineered cells, methods of administration, and treatment regimens.

Description

【Technical Field】 【0001】 The present invention relates to therapies using engineered T cells (CAR-T cells) that express chimeric antigen receptors, and more specifically, to methods of treating autoimmune diseases using CAR-T cells. 【Background Art】 【0002】 Lupus and rheumatoid arthritis are two of the most common autoimmune diseases, affecting an estimated 5 million and 14 million people worldwide, respectively. Lupus (systemic lupus erythematosus, SLE) affects women of childbearing age. Rheumatoid arthritis (RA) affects both sexes between the ages of 35 and 50 and often results in disability. SLE and RA are autoimmune diseases for which there is no cure, and symptoms are often inadequately managed with drug therapy. Autoimmune diseases result from abnormal activation of the immune system, which includes the inappropriate targeting of B and T cells against "self" or self-antigens. Current treatments include high-dose corticosteroids to effect systemic immunosuppression. 【0003】 Lupus is characterized by the presence of B cells with antibodies against nuclear proteins. Therapies developed for B cell lymphoma (B cell depletion therapies) have been successfully used to manage lupus and multiple sclerosis. These therapies include monoclonal antibodies (mAbs) that target CD19, CD20, B cell maturation antigen (BCMA), or BAFF-R. Unfortunately, mAb therapies typically require weekly intravenous administration, and beneficial effects are seen 6 weeks after the first infusion. For some patients, symptoms recur 9 months after the infusion. 【0004】 For example, rituximab (Rituxan®) is an anti-CD20 antibody that targets B cells. Rituximab has been shown to be effective against lupus. However, unlike the treatment of tumors, the management of autoimmune diseases requires repeated administration of therapeutic agents, and resistance develops over time. 【0005】 There is a need for a more reliable and potent therapy for lupus and RA that is well tolerated by patients. SUMMARY OF THE INVENTION 【0006】 The present invention includes methods and compositions for treating autoimmune diseases with engineered immune cells including T cells and natural killer (NK) cells. The engineered immune cells include chimeric antigen receptors (CARs). CAR-T cells or CAR-NK cells are administered at doses far lower than the doses of the same CAR-T cells or CAR-NK cells used for the treatment of B cell malignancies. In one embodiment, the present invention is a method of treating an autoimmune disease in a patient, the method comprising administering to the patient an amount of a composition comprising engineered immune cells that target CD19, thereby improving one or more symptoms of the autoimmune disease in the patient. In some embodiments, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), type 1 diabetes (T1D), Sjögren's syndrome, and multiple sclerosis (MS). In some embodiments, the patient is human. In some embodiments, one or more symptoms of the autoimmune disease are selected from the group consisting of proteinuria, alopecia, increased IgM and IgG antibody titers, the presence of anti-nuclear protein IgG or IgM in serum, increased B cell counts in plasma, and the presence of skin lesions or discoloration. In some embodiments, the antibody-producing cells are B cells. In some embodiments, the engineered immune cells that target CD19 are CAR-T cells that express an anti-CD19 chimeric antigen receptor (CAR). In some embodiments, the engineered immune cells that target CD19 are CAR-natural killer (NK) cells that express an anti-CD19 chimeric antigen receptor (CAR). In some embodiments, the engineered immune cells that target CD19 are allogeneic. In some embodiments of the method, the allogeneic immune cells include armed genomic modifications. In some embodiments, the armed genomic modification includes inactivation of the PDCD1 gene. 【0007】 In some embodiments, the anti-CD19 CAR comprises an anti-CD19 scFv, a transmembrane domain, and an intracellular stimulatory domain. In some embodiments, the anti-CD19 CAR further comprises a signal peptide and a hinge. In some embodiments, the anti-CD19 CAR comprises FMC63, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the anti-CD19 CAR is encoded by a nucleic acid comprising a coding sequence for the anti-CD19 CAR and a promoter. In some embodiments, the nucleic acid is integrated into the genome of the engineered immune cell. In some embodiments, the integration of the nucleic acid encoding the anti-CD19 CAR is performed using a CRISPR nuclease and a nucleic acid targeting nucleic acid (NATNA). In some embodiments, prior to integration, the nucleic acid encoding the anti-CD19 CAR is delivered into the immune cell via a viral vector. 【0008】 In some embodiments, the amount of the composition administered to the patient includes the dose of engineered immune cells targeting CD19, and the dose of engineered immune cells targeting CD19 is equivalent to 1 / 1000 of the dose used to treat B cell malignancies with engineered immune cells targeting CD19. In some embodiments, the amount of the composition administered to the patient includes 10,000 to 100,000 engineered immune cells targeting CD19. In some embodiments, the amount of the composition administered to the patient includes 100 to 1,000 engineered immune cells targeting CD19 per kilogram of the patient's body weight. In some embodiments, the amount of the composition administered to the patient includes approximately 40,000 engineered immune cells targeting CD19. In some embodiments, the amount of the composition administered to the patient includes approximately 600 engineered immune cells targeting CD19 per kilogram of the patient's body weight. In some embodiments, the amount of the composition administered to the patient includes 600,000 or fewer engineered immune cells targeting CD19. In some embodiments, the amount of the composition administered to the patient includes 10,000 or fewer engineered immune cells targeting CD19 per kilogram of the patient's body weight. 【0009】 In some embodiments, administration is performed intravenously. In some embodiments, administration is performed 2 to 4 times per year. In some embodiments, prior to administration, the patient undergoes lymphodepletion. In some embodiments, lymphodepletion includes administration of a compound selected from the group consisting of cyclophosphamide, fludarabine, azathioprine, methotrexate, mycophenolic acid, calcineurin inhibitors, and vorinostat. In some embodiments, lymphodepletion includes administering cyclophosphamide at 60 mg / kg per day for a maximum of 2 days. In some embodiments, lymphodepletion further includes administering fludarabine at 25 mg / m 2 for a maximum of 5 days. 【0010】 In some embodiments, the method further includes evaluating the patient for improvement in one or more symptoms selected from the group consisting of proteinuria, alopecia, increased IgM and IgG antibody titers, the presence of anti-nuclear protein IgG or IgM in serum, increased B cell count in plasma, and the presence of skin lesions or discoloration. In some embodiments, the method further includes increasing the dose of engineered immune cells targeting CD19 administered to the patient if no improvement is observed. 【0011】 In some embodiments, the composition further includes one or more pharmaceutically acceptable excipients. In some embodiments, the one or more excipients are selected from the group consisting of carbohydrates, inorganic salts, antibacterial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. In some embodiments, the composition further includes a cryoprotectant. In one embodiment, the present invention is a composition for treating an autoimmune disease, the composition comprising engineered immune cells targeting CD19 in an amount equivalent to 1 / 1000 of the s dose used to treat B cell malignancies with engineered immune cells targeting CD19. In some embodiments, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), type 1 diabetes (T1D), Sjögren's syndrome, and multiple sclerosis (MS). In some embodiments, the engineered immune cells targeting CD19 are CAR-T cells expressing an anti-CD19 chimeric antigen receptor (CAR). In some embodiments, the engineered immune cells targeting CD19 are CAR-natural killer (NK) cells expressing an anti-CD19 chimeric antigen receptor (CAR). In some embodiments, the engineered immune cells targeting CD19 are allogeneic. In some embodiments of the composition, the allogeneic immune cells include armed genomic modifications. In some embodiments, the armed genomic modification includes inactivation of the PDCD1 gene. 【0012】 In some embodiments, the anti-CD19 CAR comprises an anti-CD19 scFv, a transmembrane domain, and an intracellular stimulatory domain. In some embodiments, the anti-CD19 CAR further comprises a signal peptide and a hinge. In some embodiments, the anti-CD19 CAR comprises FMC63, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. 【0013】 In some embodiments, the composition comprises 10,000 to 10,000,000 engineered immune cells targeting CD19. In some embodiments, the amount of the composition administered to a patient comprises 100 to 100,000 engineered immune cells targeting CD19 per kilogram of the patient's body weight. In some embodiments, the amount of the composition administered to a patient comprises approximately 40,000 engineered immune cells targeting CD19. In some embodiments, the amount of the composition administered to a patient comprises approximately 600 engineered immune cells targeting CD19 per kilogram of the patient's body weight. In some embodiments, the amount of the composition administered to a patient comprises no more than 600,000 engineered immune cells targeting CD19. In some embodiments, the amount of the composition administered to a patient comprises no more than 10,000 engineered immune cells targeting CD19 per kilogram of the patient's body weight. In some embodiments, the amount of the composition administered to a patient comprises no more than 40,000,000 engineered immune cells targeting CD19. In some embodiments, the amount of the composition administered to a patient comprises no more than 60,000 engineered immune cells targeting CD19 per kilogram of the patient's body weight. 【0014】 In some embodiments, the composition further comprises one or more pharmaceutically acceptable excipients. In some embodiments, the one or more excipients are selected from the group consisting of carbohydrates, inorganic salts, antibacterial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. In some embodiments, the composition further comprises a cryoprotectant. 【0015】 In some embodiments, the present invention is a method for treating an autoimmune disease in a patient, comprising administering to the patient an amount of a composition comprising engineered immune cells that express an anti-CD19 CAR comprising FMC63, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain, wherein the immune cells have been evaluated for in vitro activity against B cells. In some embodiments, the activity against B cells is evaluated as cytotoxicity in co-culture with B cells comprising the composition. In some embodiments, the B cells comprising the composition are selected from plasma, a PBMC fraction, and a B cell fraction. In some embodiments, the co-culture has an effector cell:target cell ratio of 1:10 to 10:1, such as 1:8 to 8:1. 【0016】 In some embodiments, the activity against B cells is evaluated as a reduction in antibody secretion by the B cells. In some embodiments, the reduction in antibody secretion is evaluated by measuring the total IgG concentration in a culture comprising B cells. In some embodiments, the culture comprising B cells is selected from plasma, a PBMC fraction, and a B cell fraction. In some embodiments, the reduction in antibody secretion is evaluated by measuring the concentration of IgG characteristic of the autoimmune disease in a culture comprising B cells. In some embodiments, the culture comprising B cells is selected from plasma, a PBMC fraction, and a B cell fraction. BRIEF DESCRIPTION OF THE DRAWINGS 【0017】 【Figure 1】 Shows a nucleic acid expression construct encoding an anti-CD19 chimeric antigen receptor (CAR) designated CB-010. 【Figure 2】 Diagram of the gene editing steps used to generate CAR-T cells "CB-010" with the CAR construct shown in FIG. 1, and the phenotype of the resulting CB-010. 【Figure 3】 Shows the results of the in vitro cytotoxicity evaluation of CB-010. 【Figure 4】The results of the in vitro cytotoxicity evaluation of CB-010 are shown separately for the cell fractions derived from SLE and the cell fractions derived from RA. 【Figure 5】 Measurement of the concentration of autoantibodies in the co-cultures of CB-010 with the cell fractions derived from SLE and the cell fractions derived from RA is shown. 【Mode for Carrying Out the Invention】 【0018】 Definitions Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. See Sambrook et al., Molecular Cloning, A Laboratory Manual, 4th th Ed. Cold Spring Harbor Lab Press (2012). 【0019】 The following definitions are provided to assist in the understanding of the present disclosure. 【0020】 The term "therapeutic benefit" refers to the effect of improving a patient's condition with respect to the medical treatment of that condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, the treatment of cancer can include, for example, a reduction in tumor size, a reduction in tumor invasiveness, a reduction in tumor growth rate, or the prevention of metastasis, or an extension of the overall survival (OS) or progression-free survival (PFS) of a patient having cancer. 【0021】 The terms "pharmaceutically acceptable" and "pharmacologically acceptable" refer to the fact that a molecular entity and composition do not cause adverse, allergic, or other harmful reactions in a patient. For example, pharmaceutically and pharmacologically acceptable preparations should meet the standards defined by the FDA Office of Biological Standards. 【0022】 The terms "pharmaceutically acceptable carrier" and "excipient" refer to aqueous solvents (e.g., water, aqueous alcohol solutions, physiological saline, sodium chloride, Ringer's solution, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters), as well as dispersion media, coatings, surfactants, gels, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, stabilizers, binders, disintegrants, lubricants, sweetening agents, flavoring agents, and coloring agents. The concentrations and pH of the various components in the pharmaceutical composition are adjusted according to well-known parameters for each component. 【0023】 The term "domain" refers to a region in a polypeptide that is folded into a specific structure independently of other regions. 【0024】 The term "adoptive cell" refers to a cell that can be genetically modified for use in cell therapy treatment. Examples of adoptive cells include macrophages and lymphocytes including T cells and natural killer (NK) cells. 【0025】 The term "cell therapy" refers to the treatment of a disease or disorder using genetically modified cells. The term "adoptive cell therapy (ACT)" refers to a therapy using genetically modified adoptive cells. Examples of ACT include T cell therapy, CAR-T cell therapy, natural killer (NK) cell therapy, and CAR-NK cell therapy. 【0026】 The term "lymphocyte" refers to white blood cells that are part of the vertebrate immune system. Lymphocytes include T cells, e.g., CD4 + and / or CD8 + cytotoxic T cells, alpha / beta T cells, gamma / delta T cells, and regulatory T cells. Lymphocytes also include natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, and antigen-presenting cells (APCs), e.g., dendritic cells. Lymphocytes also include tumor-infiltrating lymphocytes (TILs). 【0027】 The terms "effective amount" and "therapeutically effective amount" of a composition, e.g., a cell therapy composition, refer to an amount of the composition that is sufficient to bring about a desired response in a patient to whom the composition is administered. In the context of administering a combination of therapeutic compounds, the effective amount of each therapeutic compound in the combination may differ from the effective amount of each therapeutic compound when administered alone. 【0028】 The terms "peptide", "polypeptide", and "protein" are interchangeable and refer to polymers of amino acids, including natural and synthetic (unnatural) amino acids, and amino acids not found in naturally occurring proteins, e.g., peptidomimetics, and D optical isomers. The polypeptide can be branched or linear and can be interrupted by non-amino acid residues. The terms also encompass amino acid polymers that have been modified via acetylation, disulfide bond formation, glycosylation, lipidation, phosphorylation, cross-linking, or conjugation (e.g., with a label). A polypeptide need not contain the full-length amino acid sequence of a reference molecule, but can contain only that portion of the reference molecule necessary for the polypeptide to retain its desired activity. For example, polypeptides that include the full-length protein, fragments thereof, polypeptides having amino acid deletions, additions, and substitutions are encompassed by the terms "protein" and "polypeptide" so long as the desired activity is retained. For example, polypeptides having 95%, 90%, 80%, 70%, or less sequence identity to a reference polypeptide are included so long as the desired activity is retained by the polypeptide. The determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm, e.g., BLAST, NBLAST, and XBLAST, which are described in Altschul, et al. (1990, J. Mol. Biol. 215:403-410) and are available from the National Center for Biotechnology Information (NCBI). 【0029】 The terms "CRISPR" (clustered regularly interspaced short palindromic repeats), "CRISPR-Cas" (CRISPR-associated proteins), and "CRISPR system" are of prokaryotic origin and refer to genome editing tools that include a nucleic acid guide molecule and an endonuclease guided by a sequence-specific nucleic acid capable of cleaving a target nucleic acid strand at a site complementary to the sequence in the nucleic acid guide. 【0030】 The term "NATNA" (nucleic acid targeting nucleic acid) refers to the nucleic acid guide molecule of the CRISPR system. NATNA can be composed of two nucleic acid-targeting polynucleotides ("dual guide") including CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). NATNA can be composed of a single nucleic acid-targeting polynucleotide ("single guide") including crRNA and tracrRNA connected by a fusion region (linker). CrRNA can include a targeting region and an activation region. TracrRNA can include a region capable of hybridizing to the activation region of crRNA. The term "targeting region" refers to a region capable of hybridizing to a sequence in a target nucleic acid. The term "activation region" refers to a region that interacts with a polypeptide, for example, a CRISPR nuclease. 【0031】 B cells that produce autoantibodies are a proven cause of at least one of autoimmune diseases such as lupus (SLE and other forms of lupus), rheumatoid arthritis (RA), type 1 diabetes (T1D), Sjögren's syndrome, and multiple sclerosis (MS). A common feature of activated B cells is surface expression of CD19. Anti-CD19 cytotoxic T cells, including autologous and allogeneic CAR-T cell therapies, have been shown to effectively reduce the number of CD19-expressing malignant B cells in patients. Attempts to attack autoimmune B cells with CAR-T cells in mouse models are described in US Patent Application Publication No. 2018 / 0264038 Chimeric antigen receptor (CAR) T cells as therapeutic interventions for auto-and alloimmunity, US Patent Application Publication No. 2020 / 078403 Use of chimeric antigen receptor modified cells to treat autoimmune disease, and US Patent Application Publication No. 2020 / 0085871 Methods of using cytotoxic T cells for treatment of autoimmune disesases. 【0032】 The present invention describes the use of low-dose, well-tolerated allogeneic anti-CD19 CAR-T cells for managing symptoms of autoimmune diseases in humans. 【0033】 In some embodiments, the present invention includes the use of adoptive cells for treating or alleviating autoimmune diseases including lupus, rheumatoid arthritis, type 1 diabetes (T1D), Sjögren's syndrome, and multiple sclerosis (MS), including adoptive cells. The adoptive cells of the present invention include lymphocytes such as T cells, CAR-T cells, NK cells, iPSC-derived NK (iNK) cells, and CAR-NK cells. 【0034】 In some embodiments, the present invention uses T cells isolated from healthy donors. In some embodiments, the T cells are obtained from a blood sample of a healthy donor via leukapheresis. Techniques for isolating lymphocytes are well known in the art; see, for example, Smith, J.W. (1997) Apheresis techniques and cellular immunomodulation, Ther. Apher. 1:203-206. In some embodiments, the present invention is directed to CD4 + T cells (T helper cells) depleted T cell compositions. In some embodiments, the present invention uses T cell compositions that are substantially free of CD4 + T cells. 【0035】 In some embodiments, the present invention uses natural killer (NK) cells isolated from healthy donors, for example, from peripheral blood mononuclear cells (PBMCs), leukapheresis products (PBSCs), bone marrow, or cord blood, by methods well known in the art; see, for example, Spanholtz, J. et al., (2011) Clinical-grade generation of active NK cells from cord blood hematopoietic progenitor cells for immunotherapy using a closed-system culture process, PloS one, 6(6), e20740, and Shah, N., et al., (2013) Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with anti-myeloma activity. PloS one, 8(10), e76781. 【0036】 In some embodiments, the present invention uses NK cells obtained by differentiating human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs). NK cells differentiated from iPSCs are referred to as iNK cells. 【0037】 In some embodiments, the NK cells are allogeneic and are haplotyped to match the patient at one or more HLA loci, one or more KIR loci, or both. 【0038】 In some embodiments, the isolated NK cell composition is depleted of CD3 + cells. In some embodiments, the isolated NK cell composition is enriched for CD56 + cells. In some embodiments, the isolated NK cell composition is enriched for CD45 + cells. In some embodiments, the isolated NK cell composition is enriched for CD56 + / CD45 + cells. In some embodiments, a quality control measurement or characterization step is applied to the isolated NK cell composition, and the quality control measurement or characterization step determines, for example, the percentage of CD56 + / CD3 - , CD45 + / CD3 - cells, CD56 + / CD45 + , or CD56 + / CD45 + / CD3 - in the composition. In some embodiments, the present invention uses an NK cell composition that is substantially free of CD3 + cells. 【0039】 In some embodiments, the isolated lymphocytes are characterized with respect to specificity, frequency of each subtype, and function. In some embodiments, the isolated lymphocyte population is a specific subset of T cells, such as CD8 + , CD25 + , or CD62L+ is enriched for. See, e.g., Wang et al., Mol. Therapy-Oncolytics (2016) 3:16015. In some embodiments, the isolated NK cell composition is CD56 + / CD45 + cells are enriched for. 【0040】 In some embodiments, quality control measurements or characterization steps are applied to the cell-containing composition. In some embodiments, the quality control measurement or characterization step is determining the percentage of CD56 + / CD45 + cells in the composition by flow cytometry. 【0041】 In some embodiments, after isolation, the lymphocytes are activated to promote proliferation and differentiation into specified lymphocytes. For example, T cells can be activated using soluble CD3 / 28 activator, or magnetic beads coated with anti-CD3 / anti-CD28 monoclonal antibodies. 【0042】 In some embodiments, the present invention is a method of treating an autoimmune disease in a patient, the method comprising administering to the patient a composition comprising immune cells that express a protein targeting CD19. In some embodiments, the immune cells are selected from T cells, natural killer (NK) cells, iNK cells. In some embodiments, the immune cells are selected from CAR-T cells, CAR-NK cells. 【0043】 In some embodiments, the protein targeting CD19 is an anti-CD19 T cell receptor. In some embodiments, the anti-CD19 T cell receptor in a chimeric antigen receptor (CAR). In some embodiments, the immune cells are CAR-T cells or CAR-NK cells. 【0044】 In some embodiments, the CAR comprises an extracellular domain comprising a CD19 binding region, a transmembrane domain, and one or more intracellular co-activation (co-stimulatory) and activation (stimulatory) domains. 【0045】 In some embodiments, the CD19 binding region of the CAR is derived from a monoclonal antibody. In some embodiments, the CD19 binding region comprises a single-chain variable fragment (scFv) or a variable portion of the heavy chain (V HH ) of a camelid single-domain antibody (V H ) fragment or a variable portion of the light chain (V L ) fragment. These fragments can be derived from a monoclonal antibody. The single-chain variable fragment (scFv) has the ability to bind to CD19. The scFv is composed of the Fv regions of the immunoglobulin heavy chain (H chain) and light chain (L chain) linked via a spacer sequence. In some embodiments, the CD19-binding scFv is FMC63; see Nicholson et al., (1997) Construction and characterization of a functional CD19 specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma, Mol. Immunol. 34:1157. 【0046】 In some embodiments, the transmembrane domain of the CAR is derived from a membrane-bound or transmembrane protein. For example, the transmembrane domain of the CAR can be the transmembrane domain of the T cell receptor alpha chain or beta chain, CD3 zeta chain, CD28, CD3 epsilon chain, CD2, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, DNAM1, NKp44, NKp46, NKG2D, 2B4, or GITR. In some embodiments, the transmembrane domain of the CAR is the CD8 transmembrane domain. In some embodiments, the transmembrane domain of the CAR is the CD8A transmembrane domain. 【0047】 The intracellular signaling domain of the CAR is involved in the activation of one or more effector functions of immune cells expressing the CAR. In some embodiments, the intracellular signaling domain of the CAR comprises a portion or the whole of the sequence of the CD3 zeta chain, CD3 epsilon chain, CD2, CD28, CD27, OX40 / CD134, 4-1BB / CD137, ICOS / CD278, IL-2R beta / CD122, IL-2R alpha / CD132, DAP10, DAP12, DNAM1, TLR1, TLR2, TLR4, TLR5, TLR6, MyD88, CD40, or a combination thereof. In some embodiments, the intracellular domain of the CAR consists of 4-1BB and the CD3 zeta chain. 【0048】 In some embodiments, the CAR comprises a hinge domain. In some embodiments, the hinge domain of the CAR is the CD8 hinge domain. In some embodiments, the hinge domain of the CAR is the CD8A hinge domain. 【0049】 An exemplary anti-CD19 CAR is shown in FIG. 1. The CAR comprises a signal sequence, an anti-CD19 scFv, a CD8 hinge domain, a transmembrane domain, 4-1BB, and a CD3 zeta intracellular domain. 【0050】 In some embodiments, the CAR is a fully human protein or is humanized to reduce immunogenicity in a human patient. In some embodiments, the nucleic acid sequence encoding the CAR is optimized for codon usage in human cells. 【0051】 The nucleic acid encoding the CAR can be introduced intracellularly as a genomic DNA sequence or a cDNA sequence. The cDNA sequence contains an open reading frame for translation of the CAR and, in some embodiments, further contains untranslated elements that improve, for example, the stability or the rate of translation of the CAR mRNA. 【0052】 In some embodiments, the cells (T cells, natural killer (NK) cells, iNK cells, CAR-T cells, or CAR-NK cells) used to treat autoimmune diseases further comprise genomic modifications that confer arming of the cells against attack by the recipient autoimmune disease patient's immune system. In some embodiments, the arming modification comprises protection from recognition by the host's cytotoxic T cells. Cytotoxic T cells recognize MHC class I antigens. MHC class I molecules are composed of a beta 2 microglobulin (B2M) associated with the heavy chain of an HLA-I protein (selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G) on the surface of the cell. The B2M / HLA-I complex on the surface of allogeneic cells is recognized by cytotoxic CD8 + T cells, and when the HLA-I is recognized as non-self, the allogeneic cells are killed by the T cells. In some embodiments, the cells of the invention comprise arming genomic modifications that include disruption of the B2M gene and thus disruption of MHC class I antigen recognition and cytotoxic T cell attack. 【0053】 In some embodiments, the arming genomic modifications include disruption of recognition by the host's NK cells. NK cells recognize cells lacking MHC-I proteins as "missing self" and kill such cells. NK cells are inhibited by HLA-I molecules that include HLA-E, the least polymorphic HLA-I protein. In some embodiments, the cells of the invention comprise a first arming genomic modification that includes disruption of the B2M gene and thus disruption of MHC class I antigen recognition and cytotoxic T cell attack, and further comprise a second arming genomic modification that includes insertion of an HLA-E gene fused to the beta 2 microglobulin (B2M) gene and thus expression of an HLA-E / B2M construct, hiding the cells from attack by NK cells. See, for example, Gornalusse et al., (2017) HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells, Nat. Biotechnol. (2017) 35:765-772. 【0054】 In some embodiments, the arming modification comprises transcriptionally silencing or disrupting one or more immune checkpoint genes. In some embodiments, the one or more immune checkpoint genes are selected from PD1 (encoded by the PDCD1 gene), CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, and 2B4, as disclosed in U.S. Patent Application Publication No. 2015 / 0017136, Methods for engineering allogeneic and highly active T cell for immunotherapy. 【0055】 In some embodiments, patients receiving treatment with immune cells expressing a protein that targets CD19 are monitored to assess clinical symptoms of autoimmune disease. The symptoms are expected to decrease with the treatment described herein. In some embodiments, patients are evaluated for clinical symptoms of autoimmune disease prior to administration of immune cells expressing a protein that targets CD19. In some embodiments, patients are evaluated hourly, daily, weekly, or monthly after the first administration of T cells expressing a protein that targets CD19. In some embodiments, patients are evaluated in relation to a daily, weekly, or monthly dosing regimen of immune cells expressing a protein that targets CD19. 【0056】 In some embodiments, the clinical symptoms of autoimmune disease include one or more of proteinuria, alopecia, organomegaly, presence of hypercellular glomeruli, IgG tissue deposition, IgM and IgG antibody titers, and presence of IgG or IgM antinuclear antibodies in serum, increase in the total number or concentration of B cells in plasma, and presence of skin lesions or discoloration. Accordingly, patients are evaluated for clinical symptoms of autoimmune disease by one or more of urinalysis, blood analysis (including complete blood count), and physical examination. 【0057】 In some embodiments, the total number or concentration of B cells in plasma is evaluated by flow cytometry. In some embodiments, the presence of IgG or IgM antinuclear antibodies in serum is evaluated by ELISA. 【0058】 In some embodiments, the patient is evaluated for the presence and relative numbers of immune cells expressing a protein targeting CD19, such as T cells, NK cells, CAR-T cells, or CAR-NK cells. In some embodiments, the presence and relative numbers of the cells are evaluated by one or more methods selected from flow cytometry, ELISA, fluorescence microscopy, fluorescence in situ hybridization (FISH), PCR, and RT-PCR, which aim to detect the presence of a protein targeting CD19, a gene encoding a protein targeting CD19, or an mRNA encoding a protein targeting CD19, respectively. 【0059】 In some embodiments, the anti-CD19 CAR is encoded by a nucleic acid construct introduced into cells (T cells, natural killer (NK) cells, or iNK cells) used to treat autoimmune diseases. In some embodiments, the anti-CD19 CAR expression construct includes a coding sequence for a CAR targeting CD19 and a promoter. 【0060】 In some embodiments, the anti-CD19 CAR expression construct is introduced via an expression vector or RNA encoding the anti-CD19 CAR protein. In some embodiments, the target cells are contacted in vitro, in vivo, or ex vivo with a nucleic acid encoding an anti-CD19 CAR. 【0061】 In some embodiments, the vector is a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector). A preferred vector is non-replicating in the target cell. In some embodiments, the vector is selected from or designed based on SV40, EBV, HSV, or BPV. The vector incorporates a protein expression sequence. In some embodiments, the expression sequence is codon-optimized for expression in mammalian cells. In some embodiments, the vector also incorporates regulatory sequences including, for example, transcription activator binding sequences, transcription repressor binding sequences, enhancers, and introns. In some embodiments, the viral vector provides a constitutive or inducible promoter. In some embodiments, the promoter is selected from EF1α, PGK1, MND, Ubc, CAG, CaMKIIa, and β-actin promoters. In some embodiments, the promoter is selected from the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, the mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, the β-interferon promoter, the hsp70 promoter, and the EF-1α promoter. In some embodiments, the promoter is the EF-1α promoter. In some embodiments, the promoter is the MND promoter. 【0062】 In some embodiments, the viral vector provides a transcription terminator or polyadenylation signal. In some embodiments, the transcription terminator or polyadenylation signal is the BGH transcription terminator and polyadenylation signal. 【0063】 In some embodiments, the vector is a plasmid selected from prokaryotic plasmids, eukaryotic plasmids, and shuttle plasmids. 【0064】 In some embodiments, the expression vector includes one or more selectable markers. In some embodiments, the selectable marker is an antibiotic resistance gene or other negative selectable marker. In some embodiments, the selectable marker includes a protein having an mRNA that is transcribed together with the CAR mRNA targeting CD19, and the polycistronic transcript is cleaved before translation. 【0065】 In some embodiments, the expression vector includes a polyadenylation site. In some embodiments, the polyadenylation site is the SV-40 polyadenylation site. 【0066】 In some embodiments, the coding sequence of the CAR targeting CD19 is introduced into the cell via a viral vector, such as an AAV vector (AAV6), or any other suitable viral vector capable of delivering an appropriate payload. In some embodiments, to facilitate homologous recombination, the coding sequence binds to homology arms located 5' (upstream or left) and 3' (downstream or right) of the insertion site at the desired insertion site in the genome. In some embodiments, the homology arms are about 500 bp in length. In some embodiments, the sequence encoding the CAR targeting CD19, together with the homology arms, is cloned into a viral vector plasmid. The plasmid is used to package the sequence into the virus. 【0067】 An exemplary nucleic acid construct is shown in FIG. 1. In addition to the CAR coding region, the construct includes an EF1α promoter, a left homology arm (LHA), and a right homology arm (RHA). 【0068】 In some embodiments, cells (T cells, natural killer (NK) cells, or iNK cells) are contacted with a viral vector, whereby the genetic material delivered by the vector is integrated into the genome of the target cells and then expressed in or on the cells. Transduced and transfected cells can be tested for transgene expression using methods well known in the art, such as fluorescence-activated cell sorting (FACS), microfluidics-based screening, ELISA, or Western blot. 【0069】 In some embodiments, the coding sequence for a CAR targeting CD19 is introduced into cells (T cells, natural killer (NK) cells, or iNK cells) as a "naked" nucleic acid by electroporation, for example, as described in U.S. Patent No. 6,410,319. 【0070】 In some embodiments, an engineered CRISPR system is introduced into cells (T cells, natural killer (NK) cells, or iNK cells). In some embodiments, the CRISPR system comprises a nucleic acid-guided endonuclease and a nucleic acid targeting nucleic acid (NATNA) guide (e.g., a CRISPR guide RNA selected from a single guide RNA incorporating elements of tracrRNA, crRNA, or tracrRNA and crRNA in a single molecule). 【0071】 In some embodiments, the NATNA is selected from the embodiments described in U.S. Patent No. 9,260,752. Briefly, the NATNA can comprise, in 5' to 3' order, a spacer extension, a spacer, a minimal CRISPR repeat, a single guide connector, a minimal tracrRNA, a 3'tracrRNA sequence, and a tracrRNA extension. In some instances, the nucleic acid targeting nucleic acid can comprise, in any order, a tracrRNA extension, a 3'tracrRNA sequence, a minimal tracrRNA, a single guide connector, a minimal CRISPR repeat, a spacer, and a spacer extension. 【0072】 In some embodiments, the guide nucleic acid targeting a nucleic acid can comprise a single guide NATNA. The NATNA comprises a spacer sequence that can be engineered to hybridize to a target nucleic acid sequence. The NATNA further comprises a CRISPR repeat comprising a sequence that can hybridize to a tracrRNA sequence. Optionally, the NATNA can have a spacer extension and a tracrRNA extension. These elements can comprise elements that can contribute to the stability of the NATNA. The CRISPR repeat and the tracrRNA sequence can interact to form a double-stranded structure in base pairs. The structure can facilitate the binding of an endonuclease to the NATNA. 【0073】 In some embodiments, the single guide NATNA comprises a spacer sequence located 5' of a first duplex comprising a hybridization region between a minimal CRISPR repeat and a minimal tracrRNA sequence. The first duplex can be interrupted by a bulge. The bulge can facilitate the recruitment of an endonuclease to the NATNA. Following the bulge, there can be a first stem comprising a linker connecting the minimal CRISPR repeat and the minimal tracrRNA sequence. The nucleotides in the last pair at the 3' end of the first duplex can be connected to a second linker that connects the first duplex to an intermediate tracrRNA. The intermediate tracrRNA can comprise one or more additional hairpins. 【0074】 In some embodiments, the NATNA can comprise a dual guide nucleic acid structure. The dual guide NATNA comprises a spacer extension, a spacer, a minimal CRISPR repeat, a minimal tracrRNA sequence, a 3’ tracrRNA sequence, and a tracrRNA extension. The dual guide NATNA does not include a single guide connector. Instead, the minimal CRISPR repeat sequence comprises a 3’ CRISPR repeat sequence, the minimal tracrRNA sequence comprises a 5’ tracrRNA sequence, and the dual guide NATNA can hybridize via the minimal CRISPR repeat and the minimal tracrRNA sequence. 【0075】 In some embodiments, the NATNA is an engineered guide RNA (CRISPR hybrid RDNA or chRDNA) comprising one or more DNA residues. In some embodiments, the NATNA is selected from the embodiments described in U.S. Patent No. 9,650,617. Briefly, some chRDNAs for use with type II CRISPR systems can be composed of two strands that form a secondary structure comprising an upper duplex region, a lower duplex region, a bulge, a targeting region, a nexus, and an activation region composed of one or more hairpins. The nucleotide sequence immediately downstream of the targeting region can contain varying proportions of DNA and RNA. Other chRDNAs can be single guide D(R)NAs for use with type II CRISPR systems, and the single guide D(R)NA comprises a targeting region, and a lower duplex region, an upper duplex region, a fusion region, a bulge, a nexus, and an activation region composed of one or more hairpins and an activation region. The nucleotide sequence immediately downstream of the targeting region can contain varying proportions of DNA and RNA. For example, the targeting region can contain DNA, or a mixture of DNA and RNA, and the activation region can contain RNA, or a mixture of DNA and RNA. 【0076】 In some embodiments, the components of the CRISPR system are introduced into the cell in the form of nucleic acids. In some embodiments, the components of the CRISPR system are introduced into the cell in the form of a nucleic acid-guided endonuclease and DNA encoding a NATNA guide. In some embodiments, the gene encoding the nucleic acid-guided endonuclease (e.g., a CRISPR nuclease selected from Cas9 and Cas12a) is inserted into a plasmid capable of replicating in the cell. In some embodiments, the gene encoding the NATNA guide is inserted into a plasmid capable of replicating in the cell. 【0077】 In some embodiments, the components of the CRISPR system, namely, the nucleic acid-guided endonuclease and the NATNA guide, are introduced into the cell in the form of an RNA, such as mRNA, encoding the nucleic acid-guided endonuclease together with the NATNA guide. 【0078】 In some embodiments, the components of the CRISPR system, namely, the nucleic acid-guided endonuclease and the NATNA guide, are introduced into the cell as a pre-assembled ribonucleoprotein complex. In some embodiments, the components of the CRISPR system, namely, the nucleic acid-guided endonuclease and the NATNA guide, are introduced into the cell via any combination of different means. For example, the endonuclease is introduced as DNA via a plasmid containing the gene encoding the endonuclease, and the guide is introduced in its final form as RNA (or RNA containing DNA nucleotides). 【0079】 In some embodiments, the nucleic acids encoding the components of the CRISPR system, namely, the nucleic acid-guided endonuclease and the NATNA guide, are introduced into the cell via electroporation. 【0080】 In some embodiments, the components of the CRISPR system, namely, the nucleic acid encoding a nucleic acid-guided endonuclease, are introduced into the cell in the form of mRNA, for example, as described in U.S. Patent No. 10,584,352, and are introduced via electroporation of viral pseudotransduction as described therein. 【0081】 In some embodiments, the coding sequence for a CAR targeting CD19 is inserted into a double-strand break in the genome of a cell (T cell, natural killer (NK) cell, or iNK cell). In some embodiments, the introduction of the coding sequence is concurrent with the inactivation of another gene by insertion of the CAR gene (gene knockout and concurrent gene knock-in). In some embodiments, the insertion site and the inactivated gene are TRAC, CBLB, PDCD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, and 2B4. In some embodiments, the CAR sequence targeting CD19 is inserted into the T cell receptor alpha (TRAC) gene. 【0082】 In some embodiments, CD19-targeting (anti-CD19) CAR-T cells are allogeneic. Allogeneic CAR-T cells may include arming modifications that protect the allogeneic cells from attack by the patient's (recipient's) immune system. In some embodiments, the arming modification includes transcriptionally silencing or disrupting one or more immune checkpoint genes. In some embodiments, the checkpoint gene is PDCD1 (encoding the PD-1 protein). 【0083】 Programmed cell death protein 1 (PD-1), also known as CD279 and encoded by the gene PDCD1, is a cell surface receptor that plays an important role in the downregulation of the immune system and the promotion of self-tolerance by suppressing T cell inflammatory activity. PD-1 binds to its cognate ligand, "programmed death ligand 1", also known as PD-L1, CD274, and B7 homolog 1 (B7-H1), or to another of its ligands, PD-L2, also known as CD273. PD-1 protects against autoimmunity through a dual mechanism that promotes programmed cell death (apoptosis) in antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis in anti-inflammatory regulatory T cells. Through these mechanisms, PD-1 binding of PD-L1 inhibits the immune system and thus prevents autoimmune disorders, but also prevents the immune system from killing cancer cells. Therefore, mutating or knocking out the expression of PD-1 (e.g., by disrupting the PDCD1 gene) can be beneficial in T cell therapy. 【0084】 In some embodiments, the immune checkpoint gene is disrupted using an endonuclease that specifically cleaves a nucleic acid strand within the target sequence of the gene to be disrupted. Strand cleavage by a sequence-specific endonuclease results in a nucleic acid strand break that can be repaired by non-homologous end joining (NHEJ). NHEJ is an imperfect repair process, and an imperfect repair process can result in direct religation, but more frequently results in the deletion, insertion, or substitution of one or more nucleotides in the target sequence. Such deletions, insertions, or substitutions of one or more nucleotides in the target sequence can result in missense or nonsense mutations in the protein coding sequence, either of which can eliminate the production of the protein or cause the production of a non-functional protein. 【0085】 In some embodiments, the arming modification includes targeted cleavage and repair of the PDCD1 gene that results in gene inactivation. In some embodiments, the PDCD1 gene is disrupted by cleavage of the PDCD1 locus in exon 2 of the PDCD1 gene on human chromosome 2 by a CRISPR-Cas endonuclease (e.g., Cas9) and a guide polynucleotide (NATNA). In some embodiments, the guide polynucleotide (NATNA) is a CRISPR hybrid RNA-DNA polynucleotide (chRDNA). 【0086】 In some embodiments, the anti-CD19 CAR-T cells are evaluated for their activity against B cells. In some embodiments, the anti-CD19 CAR-T cells are evaluated for their activity against B cells derived from patients diagnosed with an autoimmune disease. 【0087】 In some embodiments, the activity of the anti-CD19 CAR-T cells against B cells is evaluated in vitro as cytotoxicity against B cells. 【0088】 In some embodiments, the in vitro evaluation of the cytotoxic properties of the anti-CD19 CAR-T cells uses target cells or a target cell line. In some embodiments, the target cells are primary cells obtained from a human blood sample. In some embodiments, the human sample is from a patient diagnosed with an autoimmune disease. In some embodiments, the human sample is a control sample obtained from a subject without an autoimmune disease. In some embodiments, the sample is processed to extract a blood fraction, e.g., peripheral blood mononuclear cells (PBMCs), B cells, or non-B cells. 【0089】 In some embodiments, the target cells are an established lymphoid cell line. In some embodiments, the target cells are an established B cell line. In some embodiments, the target cells are an established lymphoid tumor cell line of a B cell tumor cell line. 【0090】 In some embodiments, the expression of CD19 in target cells is confirmed before evaluating the cytotoxicity of anti-CD19 CAR-T cells. In some embodiments, the expression of CD19 is confirmed by flow cytometry with an anti-CD19 antibody, staining with a labeled conjugate anti-CD19 antibody, fluorescence in situ hybridization, Western blot, or any other method known in the art for detecting the expression of proteins on the cell surface. 【0091】 In some embodiments, the cytotoxicity of anti-CD19 CAR-T cells is evaluated as the lysis of B cells in vitro. B cell lysis can be evaluated by co-culturing anti-CD19 CAR-T cells (effector cells or effectors) with a cell population containing or consisting of B cells. The co-culture can be established at different effector:target ratios (E:T ratios). In some embodiments, the E:T ratio ranges from about 0.1:1 (1:10) to about 10:1. In some embodiments, two or more E:T ratios within the selected range are evaluated. In some embodiments, two or more or all of the E:T ratios selected from 0.125:1 (1:8), 0.25:1 (1:4), 0.5:1 (1:2), 1:1, 2:1, 4:1, 8:1 are evaluated. 【0092】 In some embodiments, cell lysis is detected by labeling target cells with a combination of a cell-permeable, stable fluorescent dye (e.g., CellTrace™ Violet (CTV), ThermoFisher Scientific, Carlsbad, Calif.) and a viability dye, and measuring specific lysis by flow cytometry. Cytotoxicity can also be determined by using target cells expressing luciferase in co-culture with effector cells and measuring bioluminescence. Additionally, time-lapse imaging can be used to determine cell lysis, which is determined by either incorporating a viability dye and measuring an increase in fluorescence, or using cells containing a fluorescent reporter and measuring a decrease in fluorescence. Also, impedance-based systems, such as the xCELLigence system (Agilent, Santa Clara, Calif.), can provide dynamic real-time monitoring of cell lysis. 【0093】 In some embodiments, control experiments are performed to evaluate the lysis of cell populations consisting of non-B cells by anti-CD19 CAR-T cells. In some embodiments, control experiments are performed to evaluate the lysis of cell populations containing both B cells and non-B cells (e.g., PBMC) by anti-CD19 CAR-T cells. 【0094】 In some embodiments, B cell lysis by anti-CD19 CAR-T cells is compared in primary cell samples from autoimmune patients and primary cell samples from subjects without autoimmune disease. 【0095】 In some embodiments, the anti-CD19 CAR-T cell population that results in the highest percentage of B cell lysis is selected for administration to patients with autoimmune diseases. In some embodiments, an anti-CD19 CAR-T cell population that results in a high percentage of B cell lysis but has low non-B cell lysis is selected for administration to patients with autoimmune diseases. 【0096】 In some embodiments, the activity of anti-CD19 CAR-T cells against B cells is evaluated in vitro as a decrease in autoantibody secretion by B cells. In some embodiments, autoantibody secretion is evaluated by co-culturing anti-CD19 CAR-T cells (effector, E) with a cell population containing B cells (target, T). In some embodiments, the co-culture is at an E:T ratio in the range of about 1:10 to about 10:1. In some embodiments, the co-culture is at an E:T ratio of about 1:1. In some embodiments, the autoantibodies in the co-culture supernatant are evaluated qualitatively or quantitatively. The autoantibodies can be evaluated as total IgG in the supernatant. Specific species of autoantibodies (e.g., anti-dsDNA IgG characteristic of SLE) can be detected by antibody-based or antibody-conjugate-based assays, such as Western blot or ELISA, and similar secondary antibody-based methods with colorimetric, chemiluminescent, or fluorescent detection methods. Anti-dsDNA antibodies can also be detected using a Farr radioimmunoassay that measures radiolabeled dsDNA bound to the anti-dsDNA antibody, or using a Crithidia luciliae indirect immunofluorescence test (CLIFT). 【0097】 In some embodiments, the invention includes a composition comprising cells (T cells, natural killer (NK) cells, or iNK cells) that express a protein targeting CD19. In some embodiments, the composition comprises cytotoxic CAR-T cells or CAR-NK cells that express an anti-CD19 chimeric antigen receptor (CAR). In some embodiments, the composition comprises cells and one or more pharmaceutically acceptable excipients. Exemplary excipients include, but are not limited to, carbohydrates, inorganic salts, antibacterial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water, alcohol, polyols, glycerin, vegetable oils, phospholipids, and surfactants. Carbohydrates, such as sugars, derivatized sugars such as alditols, aldonic acids, esterified sugars, and / or sugar polymers, may be present as excipients. Specific carbohydrate excipients include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, and starch; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, and myo-inositol. The excipient can also include inorganic salts or buffers such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium monophosphate, sodium diphosphate, and combinations thereof. 【0098】 In some embodiments, the composition further comprises an antibacterial agent to prevent or inhibit microbial growth. In some embodiments, the antibacterial agent is selected from benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimerosal, and combinations thereof. 【0099】 In some embodiments, the composition further comprises an antioxidant added to prevent lymphocyte deterioration. In some embodiments, the antioxidant is selected from ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, disodium sulfite, and combinations thereof. 【0100】 In some embodiments, the composition further comprises a surfactant. In some embodiments, the surfactant is selected from polysorbates, sorbitan esters, lipids such as phospholipids (lecithin and other phosphatidylcholines), phosphatidylethanolamine, fatty acids and fatty acid esters, steroids such as cholesterol. 【0101】 In some embodiments, the composition further comprises a cryoprotectant, such as 3% - 12% dimethyl sulfoxide (DMSO) or 1% - 5% human albumin. 【0102】 The number of adoptive cells in the composition, such as T cells, NK cells, CAR-T cells, or CAR-NK cells, varies depending on several factors, but optimally is a therapeutically effective dose per vial. 【0103】 The minimum or optimal therapeutically effective dose can be determined experimentally by repeatedly administering incremental amounts of the composition and determining which amount results in a reduction in the symptoms of the autoimmune disease. 【0104】 The maximum or optimal therapeutically effective dose can be determined experimentally by repeatedly administering decremental amounts of the composition and determining which amount results in a reduction in the symptoms of the autoimmune disease without causing undesirable side effects or with an acceptable level of undesirable side effects. 【0105】 The present invention includes the step of administering to a patient a composition comprising immune cells (T cells, NK cells, or iNK cells) that express a protein targeting CD19. 【0106】 In some embodiments, prior to administration of the immune cells, the patient receives lymphodepleting pre-treatment to reduce any immune system attack against the administered immune cells. In some embodiments, the patient is pre-treated with an immunosuppressive agent known to be safe and effective against autoimmune diseases, see, for example, Fava A., and Petri, M. (2019) Systemic lupus erythematosus: diagnosis and clinical management, J. Autoimmun. 96: 1-13. 【0107】 In some embodiments, the immunosuppressive agent is cyclophosphamide, which has a history of use in lupus patients and is an alkylating agent known to deplete T and B cells. 【0108】 In some embodiments, the immunosuppressive agent is azathioprine, which is a purine analogue with a history of use in lupus patients. 【0109】 In some embodiments, the immunosuppressive agent is methotrexate, which has a history of use in lupus patients and is a metabolic antagonist known to suppress pro-inflammatory T cells. 【0110】 In some embodiments, the immunosuppressive agent is mycophenolic acid, which depletes guanosine nucleotides, has a history of use in lupus patients, and is a drug known to inhibit the proliferation of T and B cells. 【0111】 In some embodiments, the immunosuppressive agent is a calcineurin inhibitor (e.g., cyclosporine) that has a history of use in lupus patients and is known to reduce T cell activity. 【0112】 In some embodiments, lymphodepletion comprises a cyclophosphamide regimen. In some embodiments, lymphodepletion comprises administering cyclophosphamide at 60 mg / kg per day for 2 days. 【0113】 In some embodiments, lymphodepletion further comprises a fludarabine regimen. In some embodiments, lymphodepletion comprises administering fludarabine at 25 mg / m 2 per day for 5 days. 【0114】 At the end of lymphodepletion pre-treatment, the patient is administered a composition comprising immune cells expressing anti-CD19 CAR at 600,000 or fewer (equivalent to 10 4 cells / kg or fewer). In some embodiments, the patient is administered 40,000 anti-CD19 allogeneic CAR-T cells (equivalent to 600 cells / kg). 【0115】 The dosages of CD19-targeting cells (e.g., anti-CD19 CAR-T cells and CAR-NK cells) required to treat autoimmune diseases are substantially lower than the dosages of CAR-T or CAR-NK cells required to treat tumors. In addition, the dosages of allogeneic CAR-T or CAR-NK cells required to achieve a therapeutic effect on tumors are substantially lower than the dosages of autologous CAR-T or CAR-NK cells. Table 1 lists the relative dosages of autologous anti-CD19 CAR-T cells YESCARTA®, BREYANZI®, and KYMRIAH® compared to the experimental allogeneic anti-CD19 CAR-T cell composition CB-010. CB-010 has resulted in greater overall and complete responses in patients (Source: Abstract for European Hematology Association (EHA), 12 May 2022 CB-010 Clinical Program Update). 【Table 1】 【0116】 Mackensen et al. achieved remission in SLE patients treated with autologous anti-CD19 CAR-T cells administered at a dose of 10 6 cells / kg (4×10 7 to 9×10 7 cells per patient). Mackensen et al., (2022) Anti-CD19 CAR T cell therapy for refractory system lupus erythematosus, Nat. Med. 28:2124. This dose is in the range of 2 to 10 times lower than the doses used to treat B cell malignancies with autologous CAR-T cells (Table 1). 【0117】 In addition, it has been demonstrated that the administration of allogeneic CAR-T cells has led to better responses in patients than the administration of autologous CAR-T cells. Table 2 lists the efficacy (measured as overall response rate (ORR) and complete response (CR)) brought about by autologous anti-CD19 CAR-T cells YESCARTA®, BREYANZI®, and KYMRIAH®, compared with the experimental allogeneic anti-CD19 CAR-T cell composition CB-010 (source: the above EHA Abstract 12 May 2022). 【Table 2】 【0118】 The administration of allogeneic CAR-T cells has been further demonstrated to result in fewer side effects than the administration of autologous CAR-T cells. Table 3 lists the side effects (each grade 3 or higher: cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and infections) caused by autologous anti-CD19 CAR-T cells YESCARTA®, BREYANZI®, and KYMRIAH®, compared to the experimental allogeneic anti-CD19 CAR-T cell composition CB-010 (source: the above EHA Abstract 12 May 2022). 【Table 3】 【0119】 In some embodiments, the dose of anti-CD19 CAR-expressing cells administered to a human patient to treat an autoimmune disease is about 0.1% (1 / 1000) of the dose of the same CAR-T cells administered to treat a tumor. For example, for the CB-010 allogeneic anti-CD19 CAR-T cells, the dose is about 4×10 7 CAR-T cells compared to 4×10 4 (40,000) CAR-T cells used to treat B-cell non-Hodgkin lymphoma. When expressed as cells per kilogram of body weight, the dose is about 6×10 5 CAR-T cells / kg compared to 6×10 2 (600) allogeneic CAR-T cells / kg used to treat B-cell non-Hodgkin lymphoma. In some embodiments, the patient is administered no more than 600,000 (equivalent to 10 4 cells / kg or less) allogeneic anti-CD19 CAR-expressing cells. 【0120】 In some embodiments, the dose of anti-CD19 CAR-expressing cells administered to a human patient to treat an autoimmune disease is approximately the same as the dose of the same CAR-T cells administered to treat a tumor. For example, for the CB-010 allogeneic anti-CD19 CAR-T cells, the dose is about 4×10 7(40,000,000) total CAR-T cells. When expressed as cells per kilogram of body weight, the dose is approximately 6×10 5 (60,000) allogeneic CAR-T cells / kg. 【0121】 In some embodiments, the invention is a method of treating an autoimmune disease in a patient, the method comprising administering to the patient a composition comprising cells expressing an anti-CD19 protein (e.g., anti-CD19 CAR-T cells or CAR-NK cells) at a dose of about 10,000 to 100,000 cells equivalent to about 100 to 1,000 cells per kilogram of body weight. 【0122】 In some embodiments, the invention is a method of treating an autoimmune disease in a patient, the method comprising administering to the patient a composition comprising 40,000 (equivalent to 600 cells / kg) allogeneic anti-CD19 CAR-T cells. 【0123】 In some embodiments, the invention is a method of treating an autoimmune disease in a patient, the method comprising administering to the patient a composition comprising no more than 600,000 (equivalent to 10 4 cells / kg or less) allogeneic anti-CD19 CAR-T cells. 【0124】 In some embodiments, the invention comprises administering allogeneic anti-CD19 CAR-T cells to the patient at a frequency of 2 to 4 times per year. 【0125】 In some embodiments, the patient is treated with allogeneic anti-CD19 CAR-T cells at a frequency of 2 to 4 or more or less times per year based on the symptom assessment described herein, and the symptom assessment includes blood and urine analysis and visual assessment to detect the progression of the treatment and any side effects. 【0126】 In some embodiments, the therapeutic composition is administered to a patient by a route selected from intravenous, parenteral, intrathecal, topical, and intramuscular. In some embodiments, the administration is by infusion, and the infusion is selected from a single continuous dose, a long-term continuous infusion, and multiple infusions. 【Example】 【0127】 Example 1. (Prediction) Administer allogeneic anti-CD19 CAR-T cells to measurably reduce lupus symptoms In this example, a human patient is subjected to one or more of urinalysis, blood analysis (including total blood cell count), and physical evaluation, and hereinafter, if one or more of proteinuria, alopecia, organ enlargement, presence of hypercellular glomeruli, IgG tissue deposition, IgM and IgG antibody titers, and presence of IgG or IgM antinuclear antibodies in serum, increase in the total number or concentration of B cells in plasma, and presence of skin lesions or discoloration are present, the patient is diagnosed with lupus. 【0128】 The patient receives lymphodepleting pre-treatment consisting of cyclophosphamide at 60 mg / kg per day for 2 days and fludarabine at 25 mg / m 2 per day for 5 days. 【0129】 At the end of the lymphodepleting pre-treatment, the patient is administered a composition containing 40,000 (equivalent to 600 cells / kg) allogeneic anti-CD19 CAR-T cells. 【0130】 Starting one week after administration, the patient is evaluated by one or more of urinalysis, blood analysis (including total blood cell count), and physical evaluation to detect a decrease in any of the previously existing symptoms of lupus selected from proteinuria, alopecia, organ enlargement, presence of hypercellular glomeruli, IgG tissue deposition, IgM and IgG antibody titers, and presence of IgG or IgM antinuclear antibodies in serum, increase in the total number or concentration of B cells in plasma, and presence of skin lesions or discoloration. 【0131】 The total number or concentration of B cells in plasma is evaluated by flow cytometry. IgG or IgM antinuclear antibodies in serum are evaluated by ELISA. 【0132】 Also, the patient is evaluated for the presence (persistence) of anti-CD19 allogeneic CAR-T cells. These cells are detected by flow cytometry, ELISA, fluorescence microscopy, fluorescence in situ hybridization (FISH), PCR, and RT-PCR, which aim to detect the presence of a CAR targeting CD19, a gene encoding the CAR, or an mRNA encoding the CAR. 【0133】 If a decrease in symptoms is not observed, the patient is administered another dose or a larger dose of anti-CD19 allogeneic CAR-T cells. If a low number of anti-CD19 allogeneic CAR-T cells are detected in the patient's circulation, or if anti-CD19 allogeneic CAR-T cells are not detected in the patient's circulation, the patient is administered another dose or a larger dose of anti-CD19 allogeneic CAR-T cells. 【0134】 Example 2. CB-010: Allogeneic anti-CD19 CAR-T cells An allogeneic anti-CD19 CAR-T cell with PD-1 inactivation, designated CB-010 (Figure 2), was developed for relapsed / refractory B-cell non-Hodgkin lymphoma. (See Abstract for European Hematology Association (EHA), 12 May 2022 CB-010 Clinical Program Update). The structure of the CAR is shown in Figure 1. 【0135】 Briefly, CB-010 cells were generated from T cells obtained by leukapheresis of healthy donor blood samples. The CRISPR Cas9 endonuclease (CRISPR hybrid RNA-DNA guide) with chRDNA was used for genome editing. The anti-CD19 CAR transgene (Figure 1) was delivered via an AAV vector and inserted into the T cell receptor alpha chain (TRAC) locus on chromosome 14. Additionally, the PDCD1 gene on chromosome 2 was disrupted using Cas9 / chRDNA, resulting in the suppression of PD-1 expression. 【0136】 Example 3. Specific lysis of B cells by anti-CD19 CAR-T cells (CB-010) In this example, anti-CD19 CAR-T cells (allogeneic anti-CD19 CAR-T cells with PDCD1 gene inactivation, designated CB-010 as described in the Abstract for European Hematology Association (EHA), 12 May 2022 CB-010 Clinical Program Update) were co-cultured with cell fractions obtained from blood samples of autoimmune patients or isolated non-diseased B cells. As a control, donor-matched T cells with an inactivated TRAC locus but no CAR insertion (TRAC KO) were used. Briefly, the targets were labeled with CTV to distinguish them from effector cells. Non-diseased B cells were co-cultured at the following E:T ratios: 8:1, 4:1, 2:1, 1:1, 0.5:1, 0.25:1, 0.125:1, 0:1. Cell fractions from autoimmune patients were co-cultured at the following E:T ratios: 0.5:1, 0.25:1, 0.125:1, 0.0625:1, 0.03125:1, 0.015625:1, 0.0078125:1, 0:1. The co-cultures were maintained for 24 hours, after which the co-cultures were stained with B cell marker-specific antibodies (e.g., CD19 or CD20) and a viability dye (e.g., propidium iodide (PI)), and cytotoxicity measurements were performed via flow cytometry (iQue Screener Plus, Intellicyt, Albuquerque, N.M.). Cytotoxicity was determined by gating on the live cell population within the CTV-labeled target cell population, or the live cell population within the B cell and non-B cell populations of CTV-labeled target cells. Specific lysis was calculated for each well using the following equation. Specific lysis = 1 - (% of live target cells in co-culture sample / % of live target cells in target-only sample). Next, specific lysis curves were generated for different samples, and the area under the curve (AUC) measurements of specific lysis were determined for different populations and conditions. 【0137】 The results are shown in FIGS. 3 and 4. FIG. 3 shows the results of the in vitro cytotoxicity evaluation of CB-010 allogeneic anti-CD19 CAR-T cells. Specific lysis of PBMC, B cells, and non-B cells from cell fractions derived from systemic lupus erythematosus (SLE) at various E:T ratios with CB-010 is shown. FIG. 4 separately shows the results of the in vitro cytotoxicity evaluation of CB-010 for cell fractions derived from SLE and rheumatoid arthritis (RA). Cytotoxicity is represented as the area under the curve (AUC) measurement of specific lysis for PBMC, B cells, and non-B cells from SLE patients and RA patients by CB-010. The data represent four independent donors (PBMC from two SLE patients and PBMC from two RA patients). Error bars represent mean ± SD. By a paired t-test between CB-010 and TRAC KO co-culture conditions, ns (not significant) indicates p > 0.05, and ** indicates p ≤ 0.01. 【0138】 Example 4. Decrease in autoantibody secretion by B cells in the presence of anti-CD19 CAR-T cells (CB-010). In this example, CB-010 allogeneic anti-CD19 CAR-T cells were co-cultured with cell fractions obtained from blood samples of autoimmune patients or isolated non-diseased B cells. Also, as a control, the target was cultured alone or co-cultured with donor-matched T cells (TRAC KO) that have an inactivated TRAC locus but no CAR insertion. Non-diseased B cells were co-cultured with effector cells at an E:T ratio of 1:1, and cell fractions derived from autoimmunity were co-cultured with effector cells at an E:T ratio of 1:4, taking into account that B cells are a very small part of PBMCs. The co-cultures were maintained for 6 days in the presence of ODN2006, a CpG oligonucleotide that strongly activates B cells via TLR9 activation. After 6 days, the supernatant was recovered from the co-cultures. Total IgG and anti-dsDNA IgG concentrations were measured in the co-culture supernatants using ELISA kits specific for total IgG detection (Invitrogen, Carlsbad, Cal.) or anti-dsDNA IgG detection (Abnova, Taipei City, Taiwan). The results are shown in FIG. 5 as the measurement of autoantibody concentrations in co-cultures of CB-010 with cell fractions derived from SLE and cell fractions derived from RA. The data represent 6 independent donors (2 isolated healthy B cells, 2 PBMCs from SLE patients, and 2 PBMCs from RA patients). Error bars represent mean ± SD. By a paired t-test between the target-only and CB-010 co-culture conditions, ns (not significant) indicates p > 0.05, * indicates p ≤ 0.05, *** indicates p ≤ 0.001, and **** indicates p ≤ 0.0001. 【0139】 Although the present invention has been described in detail with reference to specific examples, it will be apparent to those skilled in the art that various modifications can be made within the scope of the present invention. Therefore, the scope of the present invention should be limited not by the examples described herein but by the claims presented below.

Claims

[Claim 1] A pharmaceutical composition for treating autoimmune diseases in human patients, A pharmaceutical composition comprising engineered allogeneic CAR-T cells targeting CD19, wherein the anti-CD19-19CAR comprises the scFv of FMC63, the CD8 hinge region, the CD8 transmembrane domain, the 4-1BB or CD28 costimulatory domain, and the CD3 zeta signaling domain, and the CAR-T cells further comprise an inactivated PDCD1 gene. [Claim 2] The pharmaceutical composition according to claim 1, wherein the manipulated allogeneic CAR-T cells targeting CD19 further comprise an inactivated TRAC gene. [Claim 3] The pharmaceutical composition according to claim 1 or 2, wherein the CAR-T cells are derived from primary T cells of a healthy human donor. [Claim 4] The pharmaceutical composition according to claim 1 or 2, wherein the autoimmune disease is selected from systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), type 1 diabetes mellitus (T1D), Sjögren's syndrome, and multiple sclerosis (MS). [Claim 5] The pharmaceutical composition according to claim 1 or 2, wherein the anti-CD19 CAR is encoded by nucleic acid incorporated into the T cell genome. [Claim 6] The pharmaceutical composition according to claim 5, wherein the nucleic acid is incorporated into the TRAC gene. [Claim 7] The pharmaceutical composition according to claim 5, wherein the integration of the nucleic acid encoding the anti-CD19 CAR into the T cell genome is carried out using a CRISPR nuclease and a nucleic acid targeting nucleic acid (NATNA). [Claim 8] The pharmaceutical composition according to claim 7, wherein, prior to the aforementioned integration, the nucleic acid encoding the anti-CD19 CAR is delivered into the T cell via a viral vector. [Claim 9] The pharmaceutical composition according to claim 1 or 2, wherein the composition comprising the CD19-targeted engineered allogeneic CAR-T cells is administered in a dose equivalent to 1 / 1000 of the dose used to treat B-cell malignancies with the CD19-targeted engineered allogeneic CAR-T cells. [Claim 10] The pharmaceutical composition according to claim 1 or 2, wherein the composition comprising the CD19-targeted engineered allogeneic CAR-T cells is administered in a dose of 10,000 to 100,000,000 of the CD19-targeted engineered allogeneic CAR-T cells. [Claim 11] The pharmaceutical composition according to claim 1 or 2, wherein the patient has previously received lymphocyte depletion treatment with a compound selected from cyclophosphamide, fludarabine, azathioprine, methotrexate, mycophenolic acid, calcineurin inhibitors, and volcosporine. [Claim 12] The aforementioned patient received lymphocyte depletion treatment with cyclophosphamide at a dose of 60 mg / kg per day for up to two days, and 25 mg / m² per day. 2 The method according to claim 11, wherein the patient has received up to five days of further treatment with fludarabine. [Claim 13] The pharmaceutical composition according to claim 1 or 2, wherein the treatment alleviates one or more symptoms of an autoimmune disease selected from proteinuria, alopecia, increased IgM and IgG antibody titers, the presence of antinuclear protein IgG or IgM in the serum, an increased B cell count in the plasma, and the presence of skin lesions or discoloration. [Claim 14] The pharmaceutical composition according to claim 1 or 2, further comprising one or more pharmaceutically acceptable excipients. [Claim 15] A pharmaceutical composition for treating autoimmune diseases in human patients, A pharmaceutical composition comprising engineered allogeneic CAR-T cells targeting CD19, wherein the anti-CD19 CAR comprises the scFv of FMC63, the CD8 hinge, the CD8 transmembrane domain, the 4-1BB or CD28 costimulatory domain, and the CD3 zeta signaling domain, and the CAR-T cells further comprise an inactivated PDCD1 gene and an inactivated TRAC gene, wherein the CAR-T cells have been evaluated for their in vitro activity against B cells. [Claim 16] The pharmaceutical composition according to claim 15, wherein the activity against B cells is evaluated as cytotoxicity in co-culture with a B cell composition selected from plasma, PBMC fraction, and B cell fraction. [Claim 17] The pharmaceutical composition according to claim 16, wherein the co-culture has an effector cell:target cell ratio of 1:10 to 10:

1. [Claim 18] The pharmaceutical composition according to claim 15, wherein the activity on B cells is evaluated as a reduction in antibody secretion by B cells. [Claim 19] The pharmaceutical composition according to claim 18, wherein the reduction in antibody secretion is evaluated by measuring the IgG concentration in a culture containing B cells selected from plasma, PBMC fraction, and B cell fraction.

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