Treatment of autoimmune diseases with engineered immune cells targeting BCMA

Abstract

The present invention relates to a method and composition for treating an autoimmune disease with engineered immune cells targeting BCMA, wherein the engineered immune cells targeting BCMA include T cells and natural killer (NK) cells. The engineered immune cells contain an anti-BCMA chimeric antigen receptor (CAR). Methods for producing the engineered cells, methods for administration, and therapeutic regimens are also disclosed.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 499,398, filed on 1 May 2023, and U.S. Provisional Patent Application No. 63 / 584,744, filed on 22 September 2023, both of which are incorporated herein by reference.

[0002] The present invention relates to a therapy using engineered cells (CAR-T cells and CAR-NK cells) that express a chimeric antigen receptor targeting BCMA, and more specifically, to a method for treating autoimmune diseases using CAR-T cells and CAR-NK cells that target BCMA.

[0003] Description of research and development funded by the federal government. none.

[0004] Reference to electronic sequence listings This application includes a sequence listing, which has been submitted electronically in .XML format and is incorporated herein by reference in its entirety. The .XML copy, created on April 22, 2024, is named "CBI050.30.xml" and is 2,404 bytes in size. The sequence listing contained within this .XML file is part of this specification and is incorporated herein by reference in its entirety. [Background technology]

[0005] Lupus and rheumatoid arthritis are two of the most common autoimmune diseases, affecting an estimated 5 million and 14 million people worldwide. Lupus (systemic lupus erythematosus, SLE) develops in women of childbearing age. Rheumatoid arthritis (RA) develops in both sexes between the ages of 35 and 50 and often leads to disability. SLE and RA are autoimmune diseases for which there is no cure, and symptoms are often poorly managed with medication. Autoimmune diseases result from abnormal activity of the immune system, which includes B and T cells being directed against "self" or autoantigens. Current treatments involve high doses of corticosteroids to induce systemic immunosuppression.

[0006] Lupus is characterized by the presence of B cells that possess antibodies against nuclear proteins. Therapies developed for B-cell lymphoma (B-cell depletion therapy) 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 therapy usually requires weekly intravenous administration, and beneficial effects are seen 6 weeks after the first infusion. In some patients, symptoms relapse 9 months after the infusion.

[0007] 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.

[0008] Multiple sclerosis (MS) is another immune system disorder characterized by the systematic destruction of the myelin sheath surrounding nerve fibers, leading to progressive disability. In 2020, an estimated 2.8 million people worldwide had MS, and the prevalence is rising (Walton, C., et al. (2020) Rising prevalence of multiple sclerosis worldwide: Insights from the Atlas of MS, third edition, Mult.Scler., 26(14)1816-1821). B cells play a central role in the pathogenesis of MS by activating and recruiting autoimmune T cells. Several antibody therapies targeting B cells are available for MS. Alemtuzumab targets CD52 and was originally developed to treat chronic lymphocytic leukemia (CLL), a B-cell malignancy. Ocrelizumab targets CD20 and was developed exclusively to treat MS. Rituximab and ofatumumab (both anti-CD20 antibodies) are used off-label in the treatment of MS. Ubrituximab (similarly anti-CD20) and ofatumumab are in clinical studies for approval in MS. Anti-CD19 CAR-T cell therapy has been shown to induce remission in a mouse model of MS, an experimental autoimmune encephalomyelitis. Importantly, CAR-T cells were able to penetrate the CNS and deplete B cells present in the CNS. (Gupta, et al., (2023) CAR-T cell-mediated B cell depletion in central nervous system autoimmunity, Neurology Neuroimmunology and Neuroinflammation, 10:e200080.)

[0009] Anti-CD19 and anti-CD20 antibody therapies are associated with severe side effects resulting from immunosuppression, including progressive multifocal leukoencephalopathy (PML) and hepatitis B reactivation. There is a need for potent and safe therapies for lupus, rheumatoid arthritis, and multiple sclerosis that are well-tolerated by patients.

Summary of the Invention

[0010] In one embodiment, 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 targeting BCMA, 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, neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis, ankylosing spondylitis, pemphigus vulgaris (PV), and multiple sclerosis (MS). In some embodiments, the patient is human. In some embodiments, the one or more symptoms of the autoimmune disease are selected from the group consisting of proteinuria, alopecia, increased IgM and IgG antibody titers, presence of anti-nuclear protein IgG or IgM in serum, increased number of B cells in plasma, complement C3 and C5 levels in serum, and presence of skin lesions or discoloration.

[0011] In some embodiments, the antibody-producing cells are B cells. In some embodiments, the engineered immune cells targeting BCMA are CAR-T cells expressing an anti-BCMA chimeric antigen receptor (CAR). In some embodiments, the engineered immune cells targeting BCMA are CAR-natural killer (NK) cells expressing an anti-BCMA chimeric antigen receptor (CAR). In some embodiments, the cells are homogeneous. In some embodiments, the anti-BCMA CAR comprises an anti-BCMA scFv, a transmembrane domain, and an intracellular stimulatory domain. In some embodiments, the anti-BCMA CAR further comprises a signal peptide and a hinge. In some embodiments, the anti-BCMA CAR comprises an scFv consisting of SEQ ID NO: 1, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the anti-BCMA CAR is encoded by a nucleic acid comprising the coding sequence and promoter of the anti-BCMA CAR. In some embodiments, the nucleic acid is integrated into the genome of the engineered immune cells. In some embodiments, the incorporation of the nucleic acid encoding anti-BCMA CAR is carried out using a CRISPR nuclease and a nucleic acid targeting nucleic acid (NATNA). In some embodiments, prior to incorporation, the nucleic acid encoding anti-BCMA CAR is delivered into immune cells via a viral vector.

[0012] In some embodiments, the amount of the composition administered to the patient comprises an engineered immune cell targeting a certain dose of BCMA, and the dose of the engineered immune cell targeting BCMA is equivalent to 1 / 1000 of the dose used to treat B cell malignancies with the same engineered immune cells targeting BCMA. In some embodiments, the amount of the composition administered to the patient comprises 10,000 to 100,000,000 engineered immune cells targeting BCMA. In some embodiments, the amount of the composition administered to the patient comprises 100 to 1,000,000 engineered immune cells targeting BCMA per kilogram of the patient's body weight. In some embodiments, the amount of the composition administered to the patient comprises approximately 50,000 engineered immune cells targeting BCMA. In some embodiments, the amount of the composition administered to the patient comprises approximately 800 engineered immune cells targeting BCMA per kilogram of the patient's body weight. In some embodiments, the amount of the composition administered to the patient comprises engineered immune cells targeting BCMA that are less than 50,000,000 and more than 50,000. In some embodiments, the amount of the composition administered to the patient comprises engineered immune cells targeting BCMA that are less than 800,000 and more than 800 per kilogram of the patient's body weight.

[0013] 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 comprises administration of a compound selected from the group consisting of cyclophosphamide, fludarabine, azathioprine, methotrexate, mycophenolic acid, calcineurin inhibitors, and voriconazole. In some embodiments, lymphodepletion comprises administering cyclophosphamide at 300 mg / m<00,00001>, 2 per day for up to 3 days. In some embodiments, lymphodepletion comprises administering fludarabine at 30 mg / m 2The method further includes administering the drug for up to three days. 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, presence of antinuclear protein IgG or IgM in serum, complement C3 and C5 levels 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 BCMA-targeted engineered immune cells administered to the patient if no improvement is observed.

[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, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. In some embodiments, the composition further comprises a freezing agent.

[0015] In one embodiment, the present invention relates to a composition for treating an autoimmune disease, comprising engineered immune cells targeting BCMA in an amount equivalent to 1 / 1000 of the dose used to treat B-cell malignancies with engineered immune cells targeting the same BCMA. In some embodiments, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), type 1 diabetes mellitus (T1D), Sjögren's syndrome, pemphigus vulgaris (PV), and multiple sclerosis (MS). In some embodiments, the engineered immune cells targeting BCMA are CAR-T cells expressing an anti-BCMA chimeric antigen receptor (CAR). In some embodiments, the engineered immune cells targeting BCMA are CAR-natural killer (NK) cells expressing an anti-BCMA chimeric antigen receptor (CAR). In some embodiments, the cells are homogeneous. In some embodiments, the anti-BCMA CAR comprises an anti-BCMA scFv, a transmembrane domain, and an intracellular stimulatory domain. In some embodiments, the anti-BCMA CAR further comprises a signal peptide and a hinge. In some embodiments, the anti-BCMA CAR comprises an scFv consisting of SED number 1, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the composition comprises 10,000 to 100,000 BCMA-targeting engineered immune cells. In some embodiments, the amount of composition administered to a patient comprises 100 to 1,000 BCMA-targeting engineered immune cells per kilogram of the patient's body weight. In some embodiments, the amount of composition administered to a patient comprises about 50,000 BCMA-targeting engineered immune cells. In some embodiments, the amount of composition administered to a patient comprises about 800 BCMA-targeting engineered immune cells per kilogram of the patient's body weight. In some embodiments, the amount of composition administered to a patient comprises fewer than 50,000,000 BCMA-targeting engineered immune cells.In some embodiments, the amount of composition administered to the patient contains fewer than 80,000 BCMA-targeted engineered immune cells per kilogram of the patient's body weight. In some embodiments, the composition further comprises one or more pharmaceutically acceptable excipients. In some embodiments, one or more excipients are selected from the group consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. In some embodiments, the composition further comprises a cryotherapy agent. [Brief explanation of the drawing]

[0016] [Figure 1] An example of a nucleic acid expression construct encoding an anti-BCMA chimeric antigen receptor (CAR) is shown. [Figure 2] This shows an example of a protective mechanism that shields CAR-T cells from attack by the patient's immune system. [Figure 3] The results of the in vitro cytotoxicity evaluation of anti-BCMA CAR-T cells against SLE-derived cell fractions are shown. [Figure 4] This shows the measurement of total or autoimmune antibody concentrations in co-cultures of anti-BCMA CAR-T cells and SLE-derived cell fractions. [Modes for carrying out the invention]

[0017] definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. (Sambrook et al., Molecular Cloning, A Laboratory Manual, 4) th See Ed. Cold, Spring, Harbor Lab Press (2012).

[0018] The following definitions are provided to aid in understanding this disclosure. 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 signs or symptoms of the disease. For example, cancer treatment may include, for instance, a reduction in tumor size, a reduction in tumor invasiveness, a reduction in tumor growth rate, or prevention of metastasis, or an extension of overall survival (OS) or progression-free survival (PFS) in patients with cancer.

[0019] The terms "pharmaceutically acceptable" and "pharmacologically acceptable" refer to molecular entities and compositions that do not cause adverse, allergic, or other adverse reactions in patients. For example, pharmaceutically and pharmacologically acceptable preparations should meet standards set forth by the FDA Office of Biological Standards. The terms “pharmaceutically acceptable carrier” and “excipient” refer to aqueous solvents (e.g., water, aqueous solutions of alcohol, physiological saline, sodium chloride, Ringer’s solution, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters), as well as dispersions, coatings, surfactants, gels, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption retarders, stabilizers, binders, disintegrants, lubricants, sweeteners, flavoring agents, and colorants. The concentrations and pH of the various components in a pharmaceutical composition are adjusted according to well-known parameters for each component.

[0020] The term "domain" refers to a region within a polypeptide that is folded into a specific structure independently of other regions.

[0021] The term "adoptive cells" refers to cells that can be genetically modified for use in cell therapy. Examples of adoptive cells include macrophages, as well as lymphocytes, including T cells and natural killer (NK) cells.

[0022] 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 therapies that use 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.

[0023] The term "lymphocyte" refers to white blood cells that are part of the vertebrate immune system. Lymphocytes include T cells, such as CD4 cells. + and / or CD8 + Lymphocytes include 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), such as dendritic cells. Lymphocytes also include tumor-infiltrating lymphocytes (TILs).

[0024] The terms “effective dose” and “therapeutic effective dose” for a composition, such as a cell therapy composition, refer to the amount of the composition sufficient to produce the desired response in the patient to whom the composition is administered. In the context of administering a combination of therapeutic compounds, the effective dose of each therapeutic compound in the combination may differ from the effective dose of each therapeutic compound administered individually.

[0025] The terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to polymers of amino acids, including natural and synthetic (unnatural) amino acids, as well as amino acids not found in naturally occurring proteins, such as peptide mimes and D optical isomers. Polypeptides can be branched or linear and may be interrupted by non-amino acid residues. The terms also encompass amino acid polymers modified via acetylation, disulfide bond formation, glycosylation, lipidation, phosphorylation, crosslinking, or conjugation (e.g., labeling). Polypeptides do not need to contain the full-length amino acid sequence of a reference molecule, but may contain only as much of the reference molecule as is necessary for the polypeptide to retain its desired activity. For example, polypeptides containing full-length proteins, their fragments, and polypeptides with amino acid deletions, additions, and substitutions are encompassed by the terms “protein” and “polypeptide” as long as the desired activity is retained. For example, polypeptides having 95%, 90%, 80%, 70%, or less sequence identity with a reference polypeptide are included as long as the desired activity is retained by the polypeptide. The determination of percentage identity between two nucleotide or amino acid sequences can be achieved using mathematical algorithms, such as 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).

[0026] The terms "CRISPR" (clustered, regularly arranged short palindromic repeats), "CRISPR-Cas" (CRISPR-related protein), and "CRISPR system" refer to genome editing tools originating from prokaryotes, comprising a nucleic acid guide molecule and a sequence-specific nucleic acid-guided endonuclease capable of cleaving a target nucleic acid strand at a site complementary to the sequence in the nucleic acid guide.

[0027] The term "NATNA" (Nucleic Acid Targeting Nucleic Acid) refers to the nucleic acid guide molecule of the CRISPR system. A NATNA can consist of two nucleic acid targeting polynucleotides ("dual guides"), including CRISPR RNA (crRNA) and transactivated CRISPR RNA (tracrRNA). A NATNA can also consist of a single nucleic acid targeting polynucleotide ("single guide"), including crRNA and tracrRNA linked by a fusion region (linker). crRNA may contain a targeting region and an activation region. tracrRNA may contain 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 the target nucleic acid. The term "activation region" refers to a region that interacts with a polypeptide, such as a CRISPR nuclease.

[0028] B cells that produce autoantibodies are the cause of at least one demonstrated autoimmune disease, such as lupus (SLE and other forms of lupus), rheumatoid arthritis (RA), type 1 diabetes (T1D), Sjögren's syndrome, and multiple sclerosis (MS).

[0029] Common characteristics of active B cells include the surface expression of CD19, CD20, and CD52. Anti-CD19, anti-CD20, and anti-CD52 antibodies, such as tafacitamab, rituximab, ofatumumab, alemtuzimab, and others, have been successfully used to treat B-cell malignancies. In addition, CD19-targeted cytotoxic T cells, including autologous and allogeneic CAR-T cells, 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 U.S. Patent Application Publication 2018 / 0264038, "Chimeric antigen receptor (CAR) T cells as therapeutic interventions for auto- and alloimmunity," U.S. Patent Application Publication 2020 / 078403, "Use of chimeric antigen receptor modified cells to treat autoimmune disease," and U.S. Patent Application Publication 2020 / 0085871, "Methods of using cytotoxic T cells for treatment of autoimmune diseases."

[0030] More recently, attempts have been made to develop CAR-T therapies exclusively for autoimmune symptoms. Chimeric autoantibody receptors (CAARs) specifically target autoimmune B cell receptors (BCRs) on the surface of autoimmune B cells. In vitro proof-of-concept experiments have demonstrated the autoantigen-dependent cytotoxicity of CAAR-T cells against autoimmune B cells. See Ellebrecht, et al. (2016) Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease, Science 353:179-184 and Zhang, et al. (2021) In vitro elimination of autoreactive B cells from rheumatoid arthritis patients by universal chimeric antigen receptor T cells, Ann Rheum Dis.;80:176-184. While this approach offers the potential for precise elimination of autoimmune cells in each autoimmune disease, it lacks the universality of more conventional B cell targets, such as CD19 and CD20.

[0031] However, existing anti-CD19 and anti-CD20 antibody therapies are associated with severe side effects resulting from immunosuppression. Treating autoimmune diseases requires long-term, or even lifelong, administration of therapy to alleviate the patient's symptoms while maintaining their quality of life. For this reason, therapeutic agents for autoimmune diseases must be effective at low doses and have minimal side effects.

[0032] B-cell maturation antigens (BCMAs) are attractive targets for treating autoimmune diseases. CD19 and CD20 surface antigens are expressed throughout the lymphoid lineage. In contrast, BCMAs are surface antigens present only on mature B cells, including germinal center B cells, and are far more robustly present on antibody-producing B cells, memory cells, and plasma cells; see Dogan, et al., (2020) B-cell maturation antigen expression across hematologic cancers: a systematic literature review, Blood Cancer Journal, 10:73. For this reason, BCMA-targeted drugs deplete mature antibody-secreting (including autoantibody-secreting) cells while leaving immature B cells in the bone marrow. In some embodiments, BCMA-targeted drugs are used at lower doses compared to similarly designed CD19 or CD20-targeted drugs.

[0033] To enable even lower doses, engineered immune cells, including autologous or allogeneic anti-BCMA CAR-T cells or CAR-NK cells, can be armed or disguised against the patient's immune system. Limiting the destruction of CAR-T or CAR-NK cells by the host immune system ensures that lower doses of cells yield therapeutic effects. In some embodiments, BCMA-targeting cells armed against the patient's immune system are used at lower doses compared to similarly designed BCMA-targeting cells without the arming modification.

[0034] The autoimmune disease treatments disclosed herein include anti-BCMA allogeneic CAR-T cells or CAR-NK cells that exhibit good tolerability at low doses, and these anti-BCMA allogeneic CAR-T cells or CAR-NK cells are armed against the patient's immune system by disrupting the beta-2 microglobulin (B2M) gene and inserting a B2M-HLA-E peptide fusion into the B2M locus (Figure 2).

[0035] In some embodiments, the present invention includes adoptive cells and the use of adoptive cells for treating or alleviating autoimmune diseases, the autoimmune diseases including lupus, rheumatoid arthritis, type 1 diabetes (T1D), Sjögren's syndrome, and multiple sclerosis (MS). 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.

[0036] In some embodiments, the present invention uses T cells isolated from a healthy donor. In some embodiments, T cells are obtained from a blood sample of a healthy donor via leukocyte removal. Techniques for isolating lymphocytes are well known in the art; see, for example, Smith, JW (1997) Apheresis techniques and cellular immunomodulation, Ther. Apher. 1:203-206. In some embodiments, the present invention uses CD4, which is known to contribute to the symptoms of autoimmune diseases. + The present invention uses a T cell composition in which T cells (T helper cells) are depleted. In some embodiments, the present invention uses CD4 + A T cell composition that substantially does not contain T cells is used.

[0037] In some embodiments, the present invention uses natural killer (NK) cells isolated from a healthy donor, e.g., from peripheral blood mononuclear cells (PBMCs), peripheral blood stem cells (PBSCs), bone marrow, or umbilical cord blood, by methods well known in the art. See, e.g., 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.

[0038] 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.

[0039] 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.

[0040] 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 CD45 +The cells are concentrated. In some embodiments, the isolated cell NK composition is CD56 + / CD45 + The cells are concentrated. 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 is, for example, CD56 in the composition. + / CD3 - CD45 + / CD3 - cells, CD56 + / CD45 + , or CD56 + / CD45 + / CD3 - Determine the percentage of CD3. In some embodiments, the present invention determines the percentage of CD3 + An NK cell composition that is substantially free of cells is used.

[0041] In some embodiments, 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, e.g., CD8 + CD25 + , or CD62L + It is concentrated with respect to CD56. For example, see Wang et al., Mol. Therapy-Oncolytics (2016) 3:16015. In some embodiments, the isolated NK cell composition is CD56 + / CD45 + It is concentrated in the cells.

[0042] In some embodiments, a quality control measurement or characterization step is applied to the cell-containing composition. In some embodiments, the quality control measurement or characterization step is applied to the CD56 in the composition. + / CD45 + The percentage of cells is determined by flow cytometry.

[0043] In some embodiments, after isolation, lymphocytes are activated to promote proliferation and differentiation into specific lymphocytes. For example, T cells may be activated using a soluble CD3 / 28 activator or magnetic beads coated with an anti-CD3 / anti-CD28 monoclonal antibody.

[0044] In some embodiments, the present invention relates to a method for treating an autoimmune disease in a patient, comprising administering to the patient a composition comprising immune cells expressing a BCMA-targeting protein. In some embodiments, the immune cells are selected from T cells, natural killer (NK) cells, and iNK cells. In some embodiments, the immune cells are selected from CAR-T cells and CAR-NK cells.

[0045] In some embodiments, the protein targeting BCMA is an anti-BCMA T cell receptor. In some embodiments, the anti-BCMA T cell receptor is a chimeric antigen receptor (CAR). In some embodiments, the immune cells are CAR-T cells or CAR-NK cells.

[0046] In some embodiments, the CAR comprises an extracellular domain including a BCMA-binding region, a transmembrane domain, and one or more intracellular co-activation (co-stimulation) and activation (stimulation) domains.

[0047] In some embodiments, the BCMA binding region of CAR is derived from a monoclonal antibody. In some embodiments, the BCMA binding region is derived from a single-chain variable fragment (scFv) or a camel single-domain antibody (V HH The variable part of the heavy chain (V H ) fragment or variable part of the light chain (V LThe fragments include BCMA. These fragments may be derived from monoclonal antibodies. The single-chain variable fragment (scFv) has the ability to bind to BCMA. The scFv consists of Fv regions of immunoglobulin heavy chains (H chains) and light chains (L chains) linked via a spacer sequence. In some embodiments, BCMA-binding scFv, BCMA-targeting CAR-T cells, and CAR-NK cells are described in U.S. Patents 10,927,182, 11,021,542, 11,142,583, and 11,299,549. In some embodiments, the BCMA-binding scFv is Sequence ID No. 6 of U.S. Patent 10,927,182.

[0048] In some embodiments, the transmembrane domain of CAR is derived from a membrane-bound or transmembrane protein. For example, the transmembrane domain of CAR may be the transmembrane domain of the T cell receptor alpha 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 CAR is the CD8 transmembrane domain. In some embodiments, the transmembrane domain of CAR is the CD8A transmembrane domain.

[0049] The intracellular signaling domain of CAR is involved in the activation of one or more effector functions of immune cells expressing CAR. In some embodiments, the intracellular signaling domain of CAR includes a portion or all of the sequences of 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 combinations thereof. In some embodiments, the intracellular domain of CAR consists of 4-1BB and CD3 zeta chain.

[0050] In some embodiments, the CAR includes 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.

[0051] An exemplary anti-BCMA chimeric antigen receptor (CAR) construct is shown in Figure 1. The CAR contains a signal sequence (SS), anti-BCMA scFv, CD8 hinge domain, CD8 transmembrane domain (TM), and 4-1BB and CD3 zeta intracellular domains. Expression is driven by the MND promoter. The construct is inserted into the cellular genome with the assistance of two adjacent homology arms (HA).

[0052] In some embodiments, CARs are either fully human proteins or humanized to reduce immunogenicity in human patients. In some embodiments, the nucleic acid sequences encoding the CARs are optimized for codon use in human cells.

[0053] The nucleic acid encoding the CAR can be introduced into the cell as a genomic DNA sequence or a cDNA sequence. The cDNA sequence includes an open reading frame for CAR translation and, in some embodiments, further includes untranslated elements that improve, for example, the stability or translation rate of the CAR mRNA.

[0054] In some embodiments, cells used to treat autoimmune diseases (T cells, natural killer (NK) cells, iNK cells, CAR-T cells, or CAR-NK cells) further include genomic modifications that result in the cells being armed against attack by the immune system of the recipient autoimmune disease patient. In some embodiments, the arming modifications include protection from recognition by the host's cytotoxic T cells. Cytotoxic T cells recognize MHC class I antigens. MHC class I molecules consist of beta-2 microglobulin (B2M) associated with the heavy chain of the 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 associated with cytotoxic CD8 + If HLA-I is recognized by T cells and recognized as non-self, the allogeneic cell is killed by the T cell. In some embodiments, the cells of the present invention include armed genome modifications that include disruption of the B2M gene, and therefore disruption of MHC class I antigen recognition and cytotoxic T cell attack.

[0055] In some embodiments, armed genome modification involves disruption of recognition by host NK cells. NK cells recognize cells lacking MHC-I protein as "self-loss" and kill such cells. NK cells are inhibited by HLA-I molecules, including HLA-E, which is the least polymorphic HLA-I protein. In some embodiments, the cells of the present invention include a first armed genome modification comprising disruption of the B2M gene and thus disruption of MHC class I antigen recognition and cytotoxic T cell attack, and further includes a second armed genome modification comprising insertion of an HLA-E gene fused to the beta-2 microglobulin (B2M) gene and thus expression of an HLA-E / B2M construct, thereby arming the cells against NK cell attack. For example, see Gornalusse et al., (2017) HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells, Nat. Biotechnol. (2017) 35:765-772.

[0056] An example of armament is shown in Figure 2. The T cell receptor alpha constant (TRAC) gene was knocked out (KO) to prevent the expression of the TCR alpha chain and the assembly of the cell surface TCR α / β heterodimer complex, thereby reducing potential graft-versus-host disease (GvHD). An anti-BCMA CAR expression cassette was inserted into the TRAC locus. The beta-2 microglobulin (B2M) gene was knocked out to reduce host T cell-mediated rejection by preventing the cell surface expression of major and non-major histocompatibility complex (MHC) class I antigens. An expression cassette of a fusion protein combining B2M and human leukocyte antigen class 1E (B2M-HLA-E peptide) was inserted into the B2M locus to inhibit host NK cell-mediated rejection. (Garner, E., Degagne, E., et al., EA BCMA-specific allogeneic CAR-T cell therapy (CB-011) genome engineered to express an HLA-E fusion transgene to prevent immune cell rejection, Poster LB009, American Association for Cancer Research (AACR) Annual Meeting, April 10, 2022).

[0057] In some embodiments, the modification involves transcriptionally silencing or disrupting one or more immune checkpoint genes. In some embodiments, 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.

[0058] In some embodiments, patients receiving treatment with immune cells expressing BCMA-targeting proteins are monitored to assess clinical signs of autoimmune disease. Symptoms are expected to decrease with the treatment described herein. In some embodiments, patients are evaluated for clinical signs of autoimmune disease prior to administration of immune cells expressing BCMA-targeting proteins. In some embodiments, patients are evaluated hourly, daily, weekly, or monthly after the first administration of T cells or NK cells expressing BCMA-targeting proteins. In some embodiments, patients are evaluated in connection with daily, weekly, or monthly administration regimens of immune cells expressing BCMA-targeting proteins.

[0059] In some embodiments, the clinical signs of autoimmune disease include proteinuria, alopecia, organ enlargement, presence of hypercellular glomeruli, IgG tissue deposition, IgM and IgG antibody titers, and IgG or IgM antinuclear antibodies in serum and CD3 in plasma. + CD8 + This includes one or more of the following: an increase in the total number or concentration of cells, an increase in the total number or concentration of B cells in plasma, complement C3 levels, complement C4 levels, and the presence of skin lesions or discoloration. Therefore, patients are evaluated for clinical signs of autoimmune disease by one or more of the following: urine analysis, blood analysis (including total blood count), and physical assessment. In some embodiments, the total number or concentration of B cells in plasma is assessed by flow cytometry. In some embodiments, the presence of IgG or IgM antinuclear antibodies in serum is assessed by ELISA.

[0060] In some embodiments, the patient is evaluated for the presence and relative number of immune cells expressing BCMA-targeting proteins, such as T cells, NK cells, CAR-T cells, or CAR-NK cells. In some embodiments, the presence and relative number of cells are evaluated by one or more methods selected from flow cytometry, ELISA, fluorescence microscopy, fluorescence insight hybridization (FISH), PCR, and RT-PCR, each of which aims to detect the presence of BCMA-targeting proteins, genes encoding BCMA-targeting proteins, or mRNA encoding BCMA-targeting proteins.

[0061] In some embodiments, anti-BCMA CARs are encoded by nucleic acid constructs introduced into cells (T cells, natural killer (NK) cells, or iNK cells) used to treat autoimmune diseases. In some embodiments, the anti-BCMA CAR expression construct comprises a coding sequence and promoter of a BCMA-targeting CAR.

[0062] In some embodiments, BCMA-targeting CAR expression constructs are introduced via an expression vector or RNA encoding a BCMA-targeting CAR protein. In some embodiments, target cells come into contact with the nucleic acid encoding the BCMA-targeting CAR in vitro, in vivo, or ex vivo.

[0063] In some embodiments, the vector is a viral vector (e.g., a retroviral vector, an adenovirus vector, an adeno-associated virus vector, or a lentiviral vector). A preferred vector is non-replicating in target cells. 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 transcription activator-binding sequences, transcription repressor-binding sequences, enhancers, and introns. In some embodiments, the viral vector supplies a constitutive or inductive 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) very early promoter, and the Roussarcoma virus long-terminal repeat (RSV-LTR) promoter, the mouse mammary cancer 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.

[0064] In some embodiments, the viral vector provides a transcription terminator or a polyadenylation signal. In some embodiments, the transcription terminator or polyadenylation signal is a BGH transcription terminator and a polyadenylation signal. In some embodiments, the vector is a plasmid selected from prokaryotic plasmids, eukaryotic plasmids, and shuttle plasmids.

[0065] In some embodiments, the expression vector includes one or more selection markers. In some embodiments, the selection markers are antibiotic resistance genes or other negative selection markers. In some embodiments, the selection markers include a protein having mRNA that is transcribed together with CAR mRNA targeting BCMA, and the polycistronic transcript is cleaved before translation.

[0066] In some embodiments, the expression vector includes a polyadenylation site. In some embodiments, the polyadenylation site is an SV-40 polyadenylation site.

[0067] In some embodiments, the coding sequence for a BCMA-targeting CAR is introduced into the cell via a viral vector, such as an AAV vector (AAV6), or any other suitable viral vector capable of delivering a suitable payload. In some embodiments, to facilitate homologous recombination, the coding sequence is bound to homologous arms located at the 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 homologous arms are approximately 500 bp long. In some embodiments, the sequence encoding the BCMA-targeting CAR, together with the homologous arms, is cloned into a viral vector plasmid. The plasmid is used to package the sequence within the virus.

[0068] In some embodiments, cells (T cells, natural killer (NK) cells, or iNK cells) come into contact with a viral vector, thereby incorporating the genetic material delivered by the vector into the genome of the target cell, and subsequently being expressed in or on the cell surface. Transduced and transfected cells can be tested for transgene expression using methods well known in the art, such as fluorescence-activated cell sorting (FACS), microfluidic technology-based screening, ELISA, or Western blotting.

[0069] In some embodiments, the coding sequence of a BCMA-targeting CAR is introduced into a cell (T cell, natural killer (NK) cell, or iNK cell) as a "naked" nucleic acid by electroporation, for example, as described in U.S. Patent No. 6,410,319.

[0070] In some embodiments, the manipulated CRISPR system is introduced into a cell (T cell, natural killer (NK) cell, or iNK cell). 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 tracrRNA, crRNA, or a single guide RNA incorporating elements of tracrRNA and crRNA into 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 may include, in the order of 5' to 3', 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 cases, a nucleic acid targeting a nucleic acid may include, 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 the nucleic acid may include a single guide NATNA. The NATNA includes a spacer sequence that can be manipulated to hybridize to the target nucleic acid sequence. The NATNA further includes a CRISPR repeat that can hybridize to a tracrRNA sequence. Optionally, the NATNA may have spacer extensions and tracrRNA extensions. These elements may include elements that can contribute to the stability of the NATNA. The CRISPR repeats and tracrRNA sequence can interact to form a double-stranded structure at the base pair. This structure can facilitate the binding of an endonuclease to the NATNA.

[0073] In some embodiments, a single guide NATNA includes a spacer sequence located at 5' of a first double helix containing a region for hybridization between minimal CRISPR repeats and a minimal tracrRNA sequence. The first double helix may be interrupted by a bulge, which facilitates the recruitment of endonucleases to the NATNA. Following the bulge, there may be a first stem containing a linker connecting the minimal CRISPR repeats and the minimal tracrRNA sequence. The nucleotides in the last pair at the 3' end of the first double helix may be linked to a second linker connecting the first double helix to an intermediate tracrRNA. The intermediate tracrRNA may contain one or more additional hairpins.

[0074] In some embodiments, NATNA may include a dual-guide nucleic acid structure. The dual-guide NATNA includes 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 includes a 3' CRISPR repeat sequence, the minimal tracrRNA sequence includes 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, NATNA is an engineered guide RNA (CRISPR hybrid RDNA or chRDNA) containing one or more DNA residues. In some embodiments, NATNA is selected from embodiments described in U.S. Patent No. 9,650,617. Briefly, some chRDNA for use with type II CRISPR systems may consist of two strands forming a secondary structure comprising an upper double-stranded region, a lower double-stranded region, a bulge, a targeting region, a nexus, and an activation region consisting of one or more hairpins. The nucleotide sequence immediately downstream of the targeting region may contain varying proportions of DNA and RNA. Other chRDNA may be a single guide D(R)NA for use with type II CRISPR systems, which comprises a targeting region and an activation region consisting of a lower double-stranded region, an upper double-stranded region, a fusion region, a bulge, a nexus, and one or more hairpins. The nucleotide sequence immediately downstream of the targeting region may contain varying proportions of DNA and RNA. For example, the targeting region may include DNA or a mixture of DNA and RNA, and the activation region may include RNA or a mixture of DNA and RNA.

[0076] In some embodiments, components of the CRISPR system are introduced into cells in the form of nucleic acids. In some embodiments, components of the CRISPR system are introduced into cells in the form of DNA encoding nucleic acid-guided endonucleases and NATNA guides. 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 that can grow in cells. In some embodiments, the gene encoding the NATNA guide is inserted into a plasmid that can grow in cells.

[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 RNA, such as mRNA, that encodes 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 nuclear protein 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 by 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 components of the CRISPR system, namely the nucleic acid-guided endonuclease and the nucleic acid encoding the NATNA guide, are introduced into the cell via electroporation.

[0080] In some embodiments, the components of the CRISPR system, namely the nucleic acids encoding nucleic acid-guided endonucleases, are introduced into the cell in the form of mRNA, for example, as described in U.S. Patent No. 10,584,352, and introduced via electroporation of viral pseudotransduction, as described therein.

[0081] In some embodiments, the coding sequence of a BCMA-targeting CAR 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 occurs concurrently 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 inactivated gene are TRAC, CBLB, PDCD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, and 2B4. In some embodiments, the BCMA-targeting CAR sequence is inserted into the T cell receptor alpha (TRAC) gene.

[0082] In some embodiments, anti-BCMA CAR-T cells are evaluated for their activity against B cells before administration to patients. In some embodiments, anti-BCMA CAR-T cells are evaluated for their activity against B cells derived from patients diagnosed with autoimmune diseases.

[0083] In some embodiments, the activity of anti-BCMA CAR-T cells against B cells is evaluated in vitro as cytotoxicity against B cells derived from patients diagnosed with autoimmune diseases.

[0084] In some embodiments, in vitro evaluation of the cytotoxic properties of anti-BCMA CAR-T cells is performed using target cells or target cell lines. 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 blood fractions, e.g., peripheral blood mononuclear cells (PBMCs), B cells, or non-B cells. In some embodiments, B cells are identified as cells expressing CD19 and / or CD20.

[0085] In some embodiments, the target cells are established lymphoid cell lines. In some embodiments, the target cells are established B cell lines. In some embodiments, the target cells are established lymphoid tumor cell lines of B cell tumor cells.

[0086] In some embodiments, BCMA expression in target cells is confirmed before evaluating the cytotoxicity of anti-BCMA CAR-T cells. In some embodiments, BCMA expression is confirmed by flow cytometry with an anti-BCMA antibody, staining with a labeled conjugate anti-BCMA antibody, fluorescence insight hybridization, Western blotting, or any other method known in the art for detecting protein expression on the cell surface.

[0087] In some embodiments, the cytotoxicity of anti-BCMA CAR-T cells is evaluated as B cell lysis in vitro. B cell lysis can be evaluated by co-culturing anti-BCMA CAR-T cells (effector cells or effectors) with a cell population containing or consisting of B cells. Co-culturing can be established with different effector:target ratios (E:T ratios). In some embodiments, the E:T ratio is in the range of approximately 0.1:1 (1:10) to approximately 10:1. In some embodiments, two or more E:T ratios within a 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, and 8:1 are evaluated.

[0088] 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, Cal.) 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. Time-lapse imaging may also be used to determine cell lysis, which is determined either by incorporating a viability dye and measuring an increase in fluorescence, or by using cells containing a fluorescent reporter and measuring a decrease in fluorescence. Impedance-based systems, such as the Agilent xCELLigence system, can also provide dynamic real-time monitoring of cell lysis.

[0089] In some embodiments, control experiments are performed to evaluate the lysis of a cell population consisting of non-B cells by anti-BCMA CAR-T cells. In some embodiments, control experiments are performed to evaluate the lysis of a cell population (e.g., PBMCs) containing both B cells and non-B cells by anti-BCMA CAR-T cells. In some embodiments, B cell lysis by anti-BCMA CAR-T cells is compared in primary cell samples from autoimmune patients and primary cell samples from subjects without autoimmune diseases.

[0090] In some embodiments, an anti-BCMA CAR-T cell population that yields the highest percentage of B cell lysis is selected for administration to patients with autoimmune diseases. In some embodiments, an anti-BCMA CAR-T cell population that yields a high percentage of B cell lysis but has low non-B cell lysis is selected for administration to patients with autoimmune diseases.

[0091] In some embodiments, the activity of anti-BCMA CAR-T cells against B cells is assessed in vitro as a decrease in autoantibody secretion by B cells. In some embodiments, the autoantibody is anti-DNA IgG. In some embodiments, autoantibody secretion is assessed by co-culturing anti-BCMA CAR-T cells (effectors, E) with a cell population containing B cells (targets, T). In some embodiments, the co-culturing is at an E:T ratio in the range of about 1:10 to about 10:1. In some embodiments, the co-culturing is at an E:T ratio of about 1:1. In some embodiments, autoantibodies in the co-culturing supernatant are assessed qualitatively or quantitatively. Autoantibodies may be assessed as total IgG in the supernatant. Autoantibodies of specific species (e.g., anti-dsDNA IgG characteristic of SLE) may be detected by antibody-based or antibody conjugate-based assays, e.g., Western blotting or ELISA, and similar secondary antibody-based methods using colorimetric, chemiluminescent, or fluorescence detection methods. Anti-dsDNA antibodies can also be detected using a radioisotope immunoassay that measures radiolabeled dsDNA bound to the anti-dsDNA antibody, or using the Crithidia luciliae indirect immunofluorescence assay (CLIFT).

[0092] In some embodiments, the present invention comprises a composition comprising cells (T cells, natural killer (NK) cells, or iNK cells) expressing a BCMA-targeting protein. In some embodiments, the composition comprises cytotoxic CAR-T cells or CAR-NK cells expressing an anti-BCMA 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, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Suitable excipients for injectable compositions include water, alcohols, polyols, glycerin, vegetable oils, phospholipids, and surfactants. Carbohydrates, such as sugars, derivatized sugars, such as alditol, aldonic acid, 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, melegitose, maltodextrin, dextran, and starch; and algitols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosylsorbitol, and myo-inositol. Excipients may 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.

[0093] In some embodiments, the composition further comprises an antimicrobial agent for preventing or inhibiting microbial growth. In some embodiments, the antimicrobial agent is selected from benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercury nitrate, thimerosal, and combinations thereof.

[0094] In some embodiments, the composition further comprises antioxidants added to prevent lymphocyte degradation. In some embodiments, the antioxidants are selected from ascorbyl palmitate, butylhydroxyanisole, butylhydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium disulfite, and combinations thereof.

[0095] 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, and steroids, such as cholesterol.

[0096] In some embodiments, the composition further comprises a freezing agent, such as 3% to 12% dimethyl sulfoxide (DMSO) or 1% to 5% human albumin.

[0097] 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 is optimally the therapeutically effective dose per vial.

[0098] The minimum or optimal therapeutically effective dose can be experimentally determined by repeatedly administering gradually increasing doses of the composition to determine which amount results in a reduction in the symptoms of the autoimmune disease.

[0099] The maximum or optimal therapeutically effective dose can be experimentally determined by repeatedly administering tapered doses of the composition to determine which dose results in a reduction of symptoms of the autoimmune disease without causing undesirable side effects, or with an acceptable degree of undesirable side effects.

[0100] 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 that targets BCMA.

[0101] In some embodiments, prior to the administration of immune cells, the patient receives lymphocyte depletion pretreatment to reduce any immune system attack against the administered immune cells.

[0102] In some embodiments, patients are pre-treated with immunosuppressants 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.

[0103] In some embodiments, the immunosuppressant is cyclophosphamide, which is an alkylating agent with a history of use in lupus patients and is known to deplete T and B cells.

[0104] In some embodiments, the immunosuppressant is azathioprine, which is a purine analog with a history of use in lupus patients.

[0105] In some embodiments, the immunosuppressant is methotrexate, an antimetabolite that has a history of use in lupus patients and is known to suppress pro-inflammatory T cells. In some embodiments, the immunosuppressant is mycophenolic acid, which is a drug known to deplete guanoside nucleotides, have a history of use in lupus patients, and inhibit the proliferation of T and B cells.

[0106] In some embodiments, the immunosuppressant is a calcineurin inhibitor (e.g., volcosporine) that has a history of use in lupus patients and is known to reduce T cell activity.

[0107] In some embodiments, anti-BCMA CAR-T cells or anti-BCMA CAR-NK cells have the arming modifications described herein. In the case of armed cells, the lymphocyte depletion regimen is milder, i.e., uses lower doses of lymphocyte depletion agents compared to the lymphocyte depletion regimen used for unarmed CAR-T cells or CAR-NK cells. In some embodiments, lymphocyte depletion involves administering cyclophosphamide at a dose of less than 60 mg / kg per day for two days and fludarabine at 25 mg / m² per day. 2 This includes administering a dose of less than 5 days.

[0108] In some embodiments, lymphocyte depletion is treated with cyclophosphamide at a dose of 300 mg / m² per day. 2 Administer at this dose for 3 days, and add fludarabine at 30 mg / m² per day. 2 This includes administering a dose for 3 days. In some embodiments, lymphocyte depletion includes the simultaneous administration of cyclophosphamide and fludarabine. In some embodiments, lymphocyte depletion includes the sequential administration of cyclophosphamide and fludarabine.

[0109] In some embodiments, a lower dose of the lympho-depleting agent is used. In some embodiments, the lower dose is used when the standard dose is not tolerable by patients with autoimmune diseases. In some embodiments, the lower dose is used when a high ratio of disease modification is observed with the standard dose.

[0110] At the end of pre-treatment for lymphocyte depletion, the patient's lymphocyte count was less than 50,000,000 (8 × 10⁶). 4The patient is administered a composition containing immune cells expressing anti-BCMA protein at a rate of less than 1 cell / kg. In some embodiments, the patient is administered 50,000 anti-BCMA allogeneic CAR-T cells (equivalent to 800 cells / kg).

[0111] The dose of BCMA-targeting cells (e.g., anti-BCMA CAR-T cells and CAR-NK cells) required to treat autoimmune diseases is substantially lower than the dose of CAR-T or CAR-NK cells required to treat tumors. In addition, the dose of allogeneic CAR-T or CAR-NK cells required to achieve a therapeutic effect on tumors can be lower than the dose of autologous CAR-T or CAR-NK cells. Table 1 lists the doses of ABECMA® and CARVYKTI®, which are autologous anti-BCMA CAR-T cell therapies, compared to CB-011, an experimental allogeneic anti-BCMA CAR-T cell therapy (see Garner, E., Degagne, E., et al., A BCMA-specific allogeneic CAR-T cell therapy (CB-011) genome engineered to express an HLA-E fusion transgene to prevent immune cell rejection, Poster LB009, American Association for Cancer Research (AACR) Annual Meeting, April 10, 2022). [Table 1]

[0112] In some embodiments, the dose of anti-BCMA CAR-T cells or CAR-NK cells for a human patient is approximately 0.1% (1 / 1000) of the dose of anti-BCMA CAR-expressing cells compared to the dose of the same CAR-T cells administered to treat the tumor. For example, for CB-011, an allogeneic anti-BCMA CAR-T cell, the dose is 5 × 10⁶, which is used to treat multiple myeloma. 7Compared to (50,000,000) CAR-T cells or CAR-NK cells, 5 × 10 4 (50,000) ~ 5 × 10 8 This is (500,000,000) CAR-T cells or CAR-NK cells. When expressed in terms of cells per kilogram of body weight, the dose used to treat multiple myeloma is 8 × 10⁶ 5 Compared to individual CAR-T cells / kg, 8 × 10 2 (800) cells / kg ~ 8 × 10 6 It is an allogeneic CAR-T (or CAR-NK) with (8,000,000) cells / kg.

[0113] In some embodiments, the patient has fewer than 500,000,000 (8 × 10 6 The patient is administered allogeneic anti-BCMA CAR-expressing cells (equivalent to less than 1 cell / kg).

[0114] In some embodiments, the patient is administered at least 50,000 (equivalent to at least 800 cells / kg) of allogeneic anti-BCMA CAR-expressing cells.

[0115] In some embodiments, the present invention includes administering anti-BCMA allogeneic CAR-T cells or CAR-NK cells to a patient at a frequency of 2 to 4 times per year. In some embodiments, patients are treated with anti-BCMA allogeneic CAR-T cells at a frequency of more than 2 to 4 times per year or less, based on the symptom assessment described herein, which includes blood and urine analysis, as well as visual assessment, to detect the progression of treatment, as well as the progression of the disease and any side effects.

[0116] In some embodiments, the therapeutic composition is administered to the patient by a route selected from intravenous, parenteral, intrathecal, topical, and intramuscular. In some embodiments, administration is by infusion, which can be selected from a single sustained dose, a long-term continuous infusion, and multiple infusions. [Examples]

[0117] Example 1. CB-011: Allogeneic anti-BCMA CAR-T cells We developed armed allogeneic anti-BCMA CAR-T cells, called CB-011, for the treatment of multiple myeloma. (See Garner, E., Degagne, E., et al., A BCMA-specific allogeneic CAR-T cell therapy (CB-011) genome engineered to express an HLA-E fusion transgene to prevent immune cell rejection, Poster LB009, American Association for Cancer Research (AACR) Annual Meeting, April 10, 2022.)

[0118] In short, CB-011 cells were generated from T cells obtained by leukocyte apheresis from healthy donor blood samples. A CRISPR Cas12a endonuclease with chRDNA (CRISPR hybrid RNA-DNA guide) was used for genome editing. An anti-BCMA 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, a fusion of beta-2 microglobulin (B2M) and HLA-E sequences was inserted into the B2M locus using Cas12a / chRDNA, which resulted in suppression of B2M expression and B2M-HLA-E peptide fusion expression on the cell surface.

[0119] Example 2. Specific lysis of B cells by (expected) anti-BCMA CAR-T cells (CB-011). In this example, anti-BCMA CAR-T cells (Example 1) are co-cultured with cell fractions obtained from patients with autoimmune diseases and control samples from individuals without autoimmune diseases. Depending on the nature of the autoimmune disease, patient B cells may be obtained from plasma or bone marrow (lupus or other signs), cerebrospinal fluid (MS), or synovial fluid (RA). As a control, donor-matched T cells (TRAC KO) that have an inactivated TRAC locus but no CAR insertion are used. Target cells are labeled with CTV to distinguish them from effector cells. Non-disease B cell controls were co-cultured with CB-011 at the following E:T ratios: 8:1, 4:1, 2:1, 1:1, 0.5:1, 0.25:1, 0.125:1, and 0:1. Cell fractions derived 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, and 0:1. The co-cultures were maintained for 24 hours, after which cytotoxicity was measured by flow cytometry (iQue Screener Plus, Intellicyt, Albuquerque, NM) after staining with B cell marker-specific antibodies (e.g., CD19 or CD20) and viability dyes (e.g., propidium iodide (PI)). Cytotoxicity was determined by gating the viable cell populations within the CTV-labeled target cell population or within the B cell and non-B cell populations of the CTV-labeled target cells. Specific lysis was calculated for each well using the following equation. Specific lysis = 1 - (percentage of surviving target cells in co-culture sample / percentage of surviving target cells in target-only sample). Then, specific lysis curves are generated for different samples, and the area under the curve (AUC) for specific lysis is determined for different populations and conditions.

[0120] Example 3. Reduction in autoantibody secretion by B cells in the presence of (hypothetical) anti-BCMA CAR-T cells (CB-011). In this example, allogeneic anti-BCMA CAR-T cells, CB-011 (Example 1), are co-cultured with cell fractions obtained from patients with autoimmune diseases and control samples from individuals without autoimmune diseases. Depending on the nature of the autoimmune disease, patient B cells may be obtained from plasma or bone marrow (lupus or other signs), cerebrospinal fluid (MS), or synovial fluid (RA). As a control, target cells are cultured alone or co-cultured with donor-matched T cells (TRAC KO) that have an inactivated TRAC locus but no CAR insertion. Non-disease B cell controls are co-cultured with effector cells in a 1:1 E:T ratio, and the autoimmune-derived cell fraction is co-cultured with effector cells in a 1:4 E:T ratio, taking into consideration that only B cells constitute the PBMC fraction. The co-culture is maintained for 6 days in the presence of ODN2006, a CpG oligonucleotide that strongly activates B cells via TLR9 activation. Six days later, the supernatant is collected from the co-culture. Total IgG and anti-dsDNA IgG concentrations are measured in the co-culture supernatant using an ELISA kit specific for total IgG detection (Invitrogen, Carlsbad, Cal.) or anti-dsDNA IgG detection (Abnova, Taipei City, Taiwan). The measured autoimmune antibody concentrations in the co-culture of CB-011 cells from SLE-derived cell fractions and RA-derived cell fractions are compared.

[0121] Example 4. Administer (hypothetical) anti-BCMA allogeneic CAR-T cells to measurably reduce lupus symptoms. In this embodiment, a human patient is subjected to one or more of the following: urine analysis, blood analysis (including total blood count), and physical evaluation. If one or more of the following are present, lupus is diagnosed: proteinuria, alopecia, organ enlargement, presence of hypercellular glomeruli, IgG tissue deposition, IgM and IgG antibody titers, and IgG or IgM antinuclear antibodies in the serum, an increase in the total number or concentration of B cells in the plasma, and the presence of skin lesions or discoloration.

[0122] The patient receives 300 mg / m² per day. 2 Cyclophosphamide for 3 days, and 30 mg / m² per day. 2The patient receives a three-day lymphocyte depletion pretreatment consisting of fludarabine, either concurrently or consecutively.

[0123] At the end of pre-treatment for lymphocyte depletion, the patient is administered a composition containing a minimum of 50,000 anti-BCMA allogeneic CAR-T cells, CB-011 (equivalent to 800 cells / kg).

[0124] Starting one week after administration, patients are evaluated by one or more of the following: urine analysis, blood analysis (including total blood count), and physical assessment, to detect a reduction in any of the previously present symptoms of lupus, which include proteinuria, alopecia, organ enlargement, presence of hypercellular glomeruli, IgG tissue deposition, IgM and IgG antibody titers, as well as serum IgG or IgM antinuclear antibodies and plasma CD3 + CD8 + Selected from an increase in the total number or concentration of cells, an increase in the total number or concentration of B cells in plasma, complement C3 levels, complement C4 levels, and the presence of skin lesions or discoloration.

[0125] 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.

[0126] The levels of major complement proteins are evaluated using commercially available tests, such as the functional hemolysis test CH50 or CH100.

[0127] Furthermore, the presence (persistence) of CB-011, an anti-BCMA allogeneic CAR-T cell, will be evaluated in patients. These cells will be detected by flow cytometry, ELISA, fluorescence microscopy, fluorescence insight hybridization (FISH), PCR, ddPCR, or RT-PCR, with the aim of detecting the presence of BCMA-targeting CARs, CAR-coding genes, or CAR-coding mRNA.

[0128] If no reduction in symptoms is observed, the patient should be administered a different or higher dose of CB-011, an anti-BCMA allogeneic CAR-T cell. If a low number of anti-BCMA allogeneic CAR-T cells, CB-011, are detected in the patient's circulation, or if anti-BCMA allogeneic CAR-T cells, CB-011, are not detected in the patient's circulation, the patient should be administered a different or higher dose of anti-BCMA allogeneic CAR-T cells, CB-011.

[0129] Alternatively, if a low number of anti-BCMA allogeneic CAR-T cells, CB-011, are detected in the patient's circulation, or if anti-BCMA allogeneic CAR-T cells, CB-011, are not detected in the patient's circulation, the patient should be administered a different or higher dose of lymphocyte depletion regimen and another dose of anti-BCMA allogeneic CAR-T cells, CB-011.

[0130] Example 5. Specific lysis of B cells by anti-BCMA CAR-T cells. In this example, anti-BCMA CAR-T cells were co-cultured with cell fractions obtained from blood samples of autoimmune patients or isolated non-disease B cells. As control effector cell samples, donor-matched T cells (TRAC KO) possessing an inactivated TRAC locus but lacking anti-BCMA CAR insertion were used. Briefly, the target was labeled with CTV to distinguish them from effector cells. Non-disease 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, and 0:1. Cells derived 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, and 0:1. Co-cultures were maintained for 24 hours, and then stained with antibodies specific to B cell markers (CD19 or CD20) and the viability dye propidium iodide (PI). Cytotoxicity was measured via flow cytometry (iQue Screener Plus, Intellicyt, Albuquerque, NM). Cytotoxicity was determined by gating the viable cell populations within the CTV-labeled target cell population or within the B cell and non-B cell populations of the CTV-labeled target cells. Specific lysis was calculated for each well using the following equation: Specific lysis = 1 - (% of viable target cells in the co-culture sample / % of viable target cells in the target-only sample). Then, specific lysis curves were generated for different samples, and the area under the curve (AUC) of specific lysis was calculated.

[0131] Figure 3 shows the results of in vitro cytotoxicity evaluation of anti-BCMA CAR-T cells (CB-011, Example 1) in a cell fraction derived from SLE. Cytotoxicity is expressed as the area under the curve (AUC) of specific lysis of PBMCs, B cells, and non-B cells from SLE patients using the allogeneic anti-BCMA CAR-T cell CB-011. The data represent four independent donors (PBMCs derived from four SLE patients). Error bars represent mean ± SD. In the paired t-test between CB-010 and TRAC KO co-culture conditions, ns (not significant) indicates p>0.05, and ** indicates p≦0.01.

[0132] Example 6. Reduction in autoantibody secretion by SLE cells in the presence of anti-BCMA CAR-T cells. In this example, allogeneic anti-BCMA CAR-T cells, CB-011 (Example 1), were co-cultured with cell fractions obtained from blood samples of autoimmune patients or isolated non-disease B cells. As a control, the target was cultured alone or co-cultured with donor-matched T cells (TRAC KO) that possessed an inactivated TRAC locus but lacked anti-BCMA CAR insertion. Non-disease B cells were co-cultured with effector cells in a 1:1 E:T ratio, and the autoimmune-derived cell fraction was co-cultured with effector cells in a 1:4 E:T ratio to account for the B cell fraction being PBMC. The co-cultures were maintained for 7 days in the presence of ODN2006, a CpG oligonucleotide that strongly activates B cells via TLR9 activation. After 7 days, the supernatant was collected from the co-cultures. Total IgG and anti-dsDNA IgG concentrations were measured in the co-culture supernatant using an ELISA kit specific for the detection of total IgG (Invitrogen) or anti-dsDNA IgG (Abnova). The results are shown in Figure 4 as measurements of total or autoimmune antibody concentrations in co-cultures of CB-011 and SLE-derived cell fractions. The data represent four independent donors (PBMCs from four SLE patients). Error bars represent mean ± SD. By paired t-tests between TRAC KO and CB-011 co-culture conditions, ** indicates p ≤ 0.01 and *** indicates p ≤ 0.001.

[0133] Anti-BCMA scFv (SEQ ID NO: 1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGYIIPYNDATKYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARYNYDGYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSEILTQSPATLSLSPGERATLSCRASQSISDYLHWQQKPGQAPRLLIYYASQSITGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFPPTFGGGTKVEIK

[0134] Literature Dogan,et al.,(2020)B-cell maturation antigen expression across hematologic cancers:a systematic literature review,Blood cancer Journal,10:73. Gornalusse et al.,(2017)HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells,Nat.Biotechnol.(2017)35:765-772. Zhang et al.,(2021)Treatment of systemic lupus erythematosus using BCMA-CD19 compound CAR,Stem Cell Reviews and Reports,17:2120-2123. Kansal et al.,(2019)Sustained B cell depletion by CD-19 targeted Car-T cells is a highly effective treatment for murine lupus,Science Trans.Med.,11 eeav1648. Jin et al.,(2021)Therapeutic efficacy of anti-CD19 CAR-T cells in a mouse model of systemic lupus erythematosus,Cellular and Molecular Immun.,18(6):1896-1903. DiLillo et al.,(2008)Maintenance of long-lived plasma cells and serological memory despite mature and memory B cell depletion during CD20 immunotherapy in mice,J Immunol.,180(1):361-371. Khodadadi,et al.,(2015)Bortezomib plus continuous B cell depletion results in sustained plasma cell depletion and amelioration of lupus nephritis in NZB / W F1 mice,PLOS One 10:e0135081. Garner,E.,Degagne,E.,et al.,A BCMA-specific allogeneic CAR-T cell therapy(CB-011)genome engineered to express an HLA-E fusion transgene to prevent immune cell rejection,Poster LB009,American Association for Cancer Research(AACR)Annual Meeting,April 10,2022. Qin,et al.,(2023)Anti-BCMA CAR-T cell therapy CT103A in relapsed or refractory AQP4-IgG seropositive neuromyelitis optica spectrum disorders:phase 1 trial interim results,Signal Transduction and Targeted Therapy,8:5. Gupta, et al., (2023) CAR-T cell-mediated B cell depletion in central nervous system autoimmunity, Neurology Neuroimmunology and Neuroinflammation, 10:e200080. Ellebrecht, et al. (2016) Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease.Science 353:179-184. Zhang,et al.,(2021)In vitro elimination of autoreactive B cells from rheumatoid arthritis patients by universal chimeric antigen receptor T cells.Ann Rheum Dis.;80:176-184.

[0135] Although the present invention is described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various modifications can be made within the scope of the invention. Therefore, the scope of the invention should be limited not by the embodiments described herein, but by the claims presented below.

Claims

1. A method for treating autoimmune diseases in patients, A method comprising administering to the patient a certain amount of a composition comprising engineered immune cells that target BCMA, thereby improving one or more symptoms of the autoimmune disease in the patient.

2. The method according to claim 1, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), type 1 diabetes mellitus (T1D), Sjögren's syndrome, neuromyelitis optica syndrome (NMOSD), myasthenia gravis, ankylosing spondylitis, pemphigus vulgaris (PV), and multiple sclerosis (MS).

3. The method according to claim 1, wherein the patient is a human being.

4. The method according to claim 1, wherein one or more of the symptoms of the autoimmune disease are selected from the group consisting of proteinuria, alopecia, increased IgM and IgG antibody titers, presence of antinuclear protein IgG or IgM in serum, increased B cell count in plasma, increased complement C3 and C5 levels in serum, and the presence of skin lesions or discoloration.

5. The method according to claim 1, wherein the antibody-producing cell is a B cell.

6. The method according to claim 1, wherein the manipulated immune cells targeting BCMA are CAR-T cells expressing an anti-BCMA chimeric antigen receptor (CAR).

7. The method according to claim 1, wherein the engineered immune cells targeting BCMA are CAR-natural killer (NK) cells expressing an anti-BCMA chimeric antigen receptor (CAR).

8. The method according to claim 1, wherein the manipulated immune cells targeting the BCMA are of the same species.

9. The method according to claim 6, wherein the anti-BCMA CAR comprises an anti-BCMA scFv, a transmembrane domain, and an intracellular stimulation domain.

10. The method according to claim 9, wherein the anti-BCMA CAR further comprises a signal peptide and a hinge.

11. The method according to claim 6, wherein the anti-BCMA CAR comprises scFv consisting of SEQ ID NO: 1, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

12. The method according to claim 6, wherein the anti-BCMA CAR is encoded by a nucleic acid comprising the coding sequence and promoter of the anti-BCMA CAR.

13. The method according to claim 12, wherein the nucleic acid is incorporated into the genome of the manipulated immune cells.

14. The method according to claim 13, wherein the incorporation of the nucleic acid encoding the anti-BCMA CAR is carried out using a CRISPR nuclease and a nucleic acid targeting nucleic acid (NATNA).

15. The method according to claim 13, wherein, prior to the incorporation, the nucleic acid encoding the anti-BCMA CAR is delivered into the immune cell via a viral vector.

16. The method according to claim 1, wherein the amount of the composition administered to the patient comprises a certain dose of BCMA-targeting engineered immune cells, the dose of the BCMA-targeting engineered immune cells being equivalent to 1 / 1000 of a dose used to treat a B-cell malignancy with the same BCMA-targeting engineered immune cells.

17. The method according to claim 1, wherein the amount of the composition administered to the patient comprises 10,000 to 100,000,000 engineered immune cells targeting the BCMA.

18. The method according to claim 1, wherein the amount of the composition administered to the patient comprises 100 to 1,000,000 BCMA-targeting engineered immune cells per kilogram of the patient's body weight.

19. The method according to claim 1, wherein the amount of the composition administered to the patient comprises about 50,000 engineered immune cells targeting the BCMA.

20. The method according to claim 1, wherein the amount of the composition administered to the patient comprises about 800 BCMA-targeting engineered immune cells per kilogram of the patient's body weight.

21. The method according to claim 1, wherein the amount of the composition administered to the patient comprises engineered immune cells targeting BCMA, wherein the amount is less than 50,000,000 and 50,000 or more.

22. The method according to claim 1, wherein the amount of the composition administered to the patient comprises engineered immune cells targeting BCMA, wherein the amount is less than 800,000 and 800 or more per kilogram of the patient's body weight.

23. The method according to claim 1, wherein the administration is performed intravenously.

24. The method according to claim 1, wherein the administration is performed two to four times per year.

25. The method according to claim 1, wherein the patient undergoes lymphocyte depletion before administering the aforementioned drug.

26. The method according to claim 25, wherein the lymphocyte depletion comprises the administration of a compound selected from the group consisting of cyclophosphamide, fludarabine, azathioprine, methotrexate, mycophenolic acid, calcineurin inhibitors, and volcosporine.

27. The aforementioned lymphocyte depletion was treated with cyclophosphamide at a rate of 300 mg / m² per day. ２ The method according to claim 26, comprising administering the drug for a maximum of three days.

28. The aforementioned lymphocyte depletion was treated with fludarabine at a rate of 30 mg / m² per day. ２ The method according to claim 27, further comprising administering the drug for a maximum of three days.

29. The method according to claim 1, further comprising evaluating the patient for improvement in one or more symptoms selected from the group consisting of proteinuria, alopecia, increased IgM and IgG antibody titers, presence of antinuclear protein IgG or IgM in serum, serum complement C3 and C5 levels, increased B cell count in plasma, and the presence of skin lesions or discoloration.

30. The method according to claim 29, further comprising increasing the dose of the BCMA-targeting engineered immune cells administered to the patient if no improvement is observed.

31. The method according to claim 1, wherein the composition further comprises one or more pharmaceutically acceptable excipients.

32. The method according to claim 31, wherein 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.

33. The method according to claim 1, wherein the composition further comprises a freezing agent.

34. A composition for treating an autoimmune disease, comprising engineered immune cells targeting BCMA in an amount equivalent to 1 / 1000 of the dose used to treat B-cell malignancies with the same engineered immune cells targeting BCMA.

35. The composition according to claim 34, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), type 1 diabetes mellitus (T1D), Sjögren's syndrome, pemphigus vulgaris (PV), and multiple sclerosis (MS).

36. The composition according to claim 34, wherein the manipulated immune cells targeting BCMA are CAR-T cells expressing an anti-BCMA chimeric antigen receptor (CAR).

37. The composition according to claim 34, wherein the engineered immune cells targeting BCMA are CAR-natural killer (NK) cells expressing an anti-BCMA chimeric antigen receptor (CAR).

38. The composition according to claim 34, wherein the manipulated immune cells that target the BCMA are of the same species.

39. The composition according to claim 38, wherein the anti-BCMA CAR comprises an anti-BCMA scFv, a transmembrane domain, and an intracellular stimulating domain.

40. The composition according to claim 38, wherein the anti-BCMA CAR further comprises a signal peptide and a hinge.

41. The composition according to claim 34, wherein the anti-BCMA CAR comprises an scFv consisting of SED number 1, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

42. The composition according to claim 34, comprising 10,000 to 100,000 engineered immune cells targeting the BCMA.

43. The composition according to claim 34, wherein the amount of the composition administered to the patient comprises 100 to 1,000 BCMA-targeting engineered immune cells per kilogram of the patient's body weight.

44. The composition according to claim 34, wherein the amount of the composition administered to the patient comprises about 50,000 engineered immune cells targeting the BCMA.

45. The composition according to claim 34, wherein the amount of the composition administered to the patient comprises about 800 BCMA-targeting engineered immune cells per kilogram of the patient's body weight.

46. The composition according to claim 34, wherein the amount of the composition administered to the patient comprises less than 50,000,000 engineered immune cells targeting the BCMA.

47. The composition according to claim 34, wherein the amount of the composition administered to the patient comprises fewer than 80,000 BCMA-targeting engineered immune cells per kilogram of the patient's body weight.

48. The composition according to claim 34, further comprising one or more pharmaceutically acceptable excipients.

49. The composition according to claim 48, wherein 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.

50. The composition according to claim 48, further comprising a freezing agent.

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