Treatment of autoimmune diseases with engineered immune cells

Abstract

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

Description

Technical Field

[0001] The present invention relates to therapies utilizing engineered T cells expressing chimeric antigen receptors (CAR-T cells), and more particularly, to methods of using CAR-T cells to treat autoimmune diseases. Background Art

[0002] Lupus and rheumatoid arthritis are the two most common autoimmune diseases, affecting approximately 5 million and 14 million people worldwide. Lupus (systemic lupus erythematosus, SLE) affects women of childbearing age. Rheumatoid arthritis (RA) attacks both sexes between the ages of 35 and 50, often leading to disability. SLE and RA are autoimmune diseases for which there is no cure, and symptoms are often not adequately managed with medication. Autoimmune diseases are caused by abnormal activity of the immune system (including B and T cells) against "self" or self-antigens. Current treatments include high doses of corticosteroids for systemic immunosuppression.

[0003] Lupus is characterized by the presence of B cells and 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) targeting CD19, CD20, B cell maturation antigen (BCMA) or BAFF-R. Unfortunately, mAb therapy usually requires weekly intravenous administration, which has a beneficial effect only six weeks after the initial infusion. For some patients, symptoms recur nine months after the infusion.

[0004] For example, rituximab Is an anti-CD20 antibody that targets B cells. It has been shown to be effective in lupus. However, unlike the treatment of tumors, the management of autoimmune diseases requires repeated administration of therapeutic agents, and over time, resistance can develop.

[0005] There is a need for a more reliable and effective therapy for lupus and RA that is well tolerated by patients. Summary of the invention

[0006] The present invention includes methods for treating autoimmune diseases with engineered immune cells, including T cells and natural killer (NK) cells, and compositions for treating autoimmune diseases. The engineered immune cells include chimeric antigen receptors (CARs). The CAR-T cells or CAR-NK cells are administered at a dose much lower than the same CAR-T cells or CAR-NK cells used to treat B cell malignancies.

[0007] In one embodiment, the present invention is a method for treating an autoimmune disease in a patient, the method comprising: administering to the patient a certain amount of a composition comprising engineered immune cells targeting 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 ( syndrome) and multiple sclerosis (MS). In some embodiments, the patient is a human. In some embodiments, the one or more symptoms of the autoimmune disease are selected from the group consisting of: proteinuria, alopecia, elevated IgM and IgG antibody titers, the presence of anti-nucleoprotein 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 targeting CD19 are CAR-T cells expressing anti-CD19 chimeric antigen receptors (CAR). In some embodiments, the engineered immune cells targeting CD19 are CAR-natural killer (NK) cells expressing anti-CD19 chimeric antigen receptors (CAR). In some embodiments, the engineered immune cells targeting CD19 are allogeneic. In some embodiments of the method, the allogeneic immune cells include armored genome modifications. In some embodiments, the armored genome modifications include inactivation of the PDCD1 gene.

[0008] In some embodiments, the anti-CD19 CAR comprises an anti-CD19 scFv, a transmembrane domain, and an intracellular stimulation 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 CD3ζ signaling domain. In some embodiments, the anti-CD19 CAR is encoded by a nucleic acid comprising a coding sequence and a promoter of the anti-CD19 CAR. 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 CRISPR nuclease and nucleic acid targeting nucleic acid (NATNA). In some embodiments, prior to the integration, the nucleic acid encoding the anti-CD19 CAR is delivered to the immune cell by a viral vector.

[0009] In some embodiments, the amount of the composition administered to the patient comprises a dose of engineered immune cells targeting CD19, which 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 comprises 10,000 to 100,000 engineered immune cells targeting CD19. In some embodiments, the amount of the composition administered to the patient comprises 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 comprises about 40,000 engineered immune cells targeting CD19. In some embodiments, the amount of the composition administered to the patient comprises about 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 comprises no more than 600,000 engineered immune cells targeting CD19. In some embodiments, the amount of the composition administered to the patient comprises no more than 10,000 of the engineered immune cells targeting CD19 per kilogram of body weight of the patient.

[0010] In some embodiments, the administration is performed intravenously. In some embodiments, the administration is performed 2-4 times per year. In some embodiments, prior to the administration, the patient undergoes lymphocyte depletion. In some embodiments, the lymphocyte depletion comprises administering a compound selected from the group consisting of cyclophosphamide, fludarabine, azathioprine, methotrexate, mycophenolate, calcineurin inhibitors, and volcosporin. In some embodiments, the lymphocyte depletion comprises administering cyclophosphamide at 60 mg / kg per day for up to 2 days. In some embodiments, the lymphocyte depletion further comprises administering 25 mg / m 2 Fludarabine was administered for up to 5 days.

[0011] In some embodiments, the method further comprises evaluating the patient for improvement in one or more symptoms selected from the group consisting of: proteinuria, alopecia, elevated IgM and IgG antibody titers, the presence of anti-nucleoprotein IgG or IgM in serum, increased B cell counts in plasma, and the presence of skin lesions or discoloration. In some embodiments, the method further comprises increasing the dose of the engineered immune cells targeting CD19 administered to the patient if no improvement is observed.

[0012] 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 cryogen.

[0013] In one embodiment, the present invention is a composition for treating an autoimmune disease, the composition comprising an amount of 1 / 1000 of the engineered immune cells targeting CD19 for treating 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 anti-CD19 chimeric antigen receptors (CAR). In some embodiments, the engineered immune cells targeting CD19 are CAR-natural killer (NK) cells expressing anti-CD19 chimeric antigen receptors (CAR). In some embodiments, the engineered immune cells targeting CD19 are allogeneic. In some embodiments of the composition, the allogeneic immune cells include armored genome modifications. In some embodiments, the armored genome modifications include inactivation of the PDCD1 gene.

[0014] 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 costimulatory domain and a CD3ζ signaling domain.

[0015] 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 the 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 the patient comprises about 40,000 engineered immune cells targeting CD19. In some embodiments, the amount of the composition administered to the patient comprises about 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 comprises no more than 600,000 engineered immune cells targeting CD19. In some embodiments, the amount of the composition administered to the 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 the patient comprises no more than 40,000,000 of the engineered immune cells targeting CD 19. In some embodiments, the amount of the composition administered to the patient comprises no more than 60,000 of the engineered immune cells targeting CD 19 per kilogram of the patient's body weight.

[0016] 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 cryogen.

[0017] In some embodiments, the present invention is a method for treating an autoimmune disease in a patient, the method comprising: administering to the patient a certain amount of a composition comprising an engineered immune cell expressing an anti-CD19 CAR, the anti-CD19 CAR comprising FMC63, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain, wherein the immune cell has been evaluated for in vitro activity against B cells. In some embodiments, the activity against B cells is evaluated in the form of cytotoxicity in co-culture with a composition comprising B cells. In some embodiments, the composition comprising B cells is selected from plasma, PBMC fractions, and B cell fractions. In some embodiments, the co-cultured effector cell: target cell ratio is between 1:10 and 10:1, for example, between 1:8 and 8:1.

[0018] In some embodiments, the activity for B cells is evaluated in the form of a reduction in antibody secretion by 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, PBMC fractions, and B cell fractions. In some embodiments, the reduction in antibody secretion is evaluated by measuring the concentration of IgG specific to autoimmune diseases in a culture comprising B cells. In some embodiments, the culture comprising B cells is selected from plasma, PBMC fractions, and B cell fractions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Depicted is a nucleic acid expression construct encoding an anti-CD19 chimeric antigen receptor (CAR) termed CB-010.

[0020] Figure 2 is used to generate Figure 1 Illustration of the gene editing steps of the CAR-T cell "CB-010" for the CAR construct shown and the resulting CB-010 phenotype.

[0021] Figure 3 Shown are the results of in vitro cytotoxicity evaluation of CB-010.

[0022] Figure 4 Shown are the results of in vitro cytotoxicity evaluation of CB-010 against SLE-derived cell fractions and RA-derived cell fractions, respectively.

[0023] Figure 5 Shown are the results of measuring the concentration of autoimmune antibodies in co-cultures of CB-010 with SLE-derived cell fractions and RA-derived cell fractions. DETAILED DESCRIPTION

[0024] definition

[0025] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art. See Sambrook et al., Molecular Cloning, A Laboratory Manual, 4th edition Cold Spring Harbor Lab Press (2012).

[0026] The following definitions are provided to aid in understanding the present disclosure.

[0027] The term "therapeutic benefit" refers to the effect of improving a patient's condition relative to a medical treatment of that condition. The term includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, treatment of cancer can involve, for example, reducing tumor size, reducing tumor invasiveness, reducing tumor growth rate, or preventing metastasis, or prolonging overall survival (OS) or progression-free survival (PFS) of a patient with cancer.

[0028] The terms "pharmaceutically acceptable" and "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse reactions, allergic reactions or other harmful reactions in patients. For example, pharmaceutically and pharmacologically acceptable formulations should meet the standards set forth by the FDA Office of Biological Standards.

[0029] The terms "pharmaceutically acceptable carrier" and "excipient" refer to aqueous solvents (e.g., water, aqueous alcohol solutions, saline solutions, 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, sweeteners, flavoring agents, and dyes. The concentration and pH of the various components in the pharmaceutical composition are adjusted according to the well-known parameters of each component.

[0030] The term "domain" refers to a region of a polypeptide that folds into a specific structure independently of other regions.

[0031] The term "adoptive cells" refers to cells 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.

[0032] The term "cell therapy" refers to the treatment of a disease or condition 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.

[0033] The term "lymphocyte" refers to a white blood cell that is part of the vertebrate immune system. Lymphocytes include T cells, such as CD4 + and / or CD8 +Cytotoxic T cells, α / β T cells, γ / δ 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).

[0034] The terms "effective amount" and "therapeutically effective amount" of a composition (e.g., a cell therapy composition) refer to an amount of the composition sufficient to provide 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 administered alone.

[0035] The terms "peptide", "polypeptide" and "protein" are interchangeable and refer to polymers of amino acids, including natural amino acids and synthetic (non-natural) amino acids, as well as amino acids not found in naturally occurring proteins, such as peptidomimetics and D optical isomers. Polypeptides may be branched or linear and may be interrupted by non-amino acid residues. The terms also encompass amino acid polymers that have been modified by acetylation, disulfide bond formation, glycosylation, lipidation, phosphorylation, cross-linking or conjugation (e.g., with a label). Although a polypeptide need not include the full-length amino acid sequence of a reference molecule, only so much of the reference molecule may be included as needed so that the polypeptide retains its desired activity. For example, the terms "protein" and "polypeptide" encompass polypeptides comprising full-length proteins, fragments thereof, polypeptides with amino acid deletions, additions and substitutions, as 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, as long as the polypeptide retains the desired activity. The determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm such as BLAST, NBLAST, and XBLAST as described in Altschul et al. (1990, J. Mol. Biol. 215:403-410) and available from the National Center for Biotechnology Information (NCBI).

[0036] The terms "CRISPR" (clustered regularly interspaced short palindromic repeats), "CRISPR-Cas" (CRISPR-associated protein) and "CRISPR system" refer to genome editing tools derived from prokaryotic organisms and comprising a nucleic acid guide molecule and a sequence-specific nucleic acid guide endonuclease capable of cleaving the target nucleic acid strand at a site complementary to the sequence in the nucleic acid guide.

[0037] The term "NATNA" (nucleic acid targeting nucleic acid) refers to the nucleic acid guide molecule of the CRISPR system. NATNA can include two nucleic acid targeting polynucleotides ("dual guides"), including CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). NATNA can include a single nucleic acid targeting polynucleotide ("single guide"), which includes crRNA and tracrRNA connected by a fusion region (joint). The crRNA may include a targeting region and an activation region. The tracrRNA may include a region that can hybridize with the activation region of the crRNA. The term "targeting region" refers to a region that can hybridize with a sequence in a target nucleic acid. The term "activation region" refers to a region that interacts with a polypeptide (e.g., a CRISPR nuclease).

[0038] B cells that produce autoantibodies are at least one documented cause 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 active B cells is the 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 malignant B cells expressing CD19 in patients. Attempts to attack autoimmune B cells with CAR-T cells in mouse models have been described in U.S. Application Publication No. US20180264038, Chimeric antigen receptor (CAR) T cells as therapeutic interventions for auto-and alloimmunity, U.S. Application Publication No. US2020078403, Use of chimeric antigen receptor modified cells to treat autoimmune disease, and U.S. Application Publication No. US20200085871, Methods of using cytotoxic T cells for treatment of autoimmune disesases.

[0039] The present invention describes the use of low doses of well-tolerated anti-CD19 allogeneic CAR-T cells to manage symptoms of autoimmune diseases in humans.

[0040] In certain embodiments, the present invention includes adoptive cells and 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). 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.

[0041] In some embodiments, the present invention utilizes T cells isolated from healthy donors. In some embodiments, T cells are obtained from blood samples of healthy donors by leukapheresis. 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 utilizes depletion of CD4+ T cells known to cause symptoms of autoimmune diseases. + In some embodiments, the present invention utilizes a T cell composition that is substantially free of CD4 + T cell composition of T cells.

[0042] In some embodiments, the present invention utilizes natural killer (NK) cells isolated from healthy donors, such as from peripheral blood mononuclear cells (PBMCs), leukapheresis products (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 activity). PLOS ONE, 8(10), e76781.

[0043] In some embodiments, the present invention utilizes NK cells obtained by differentiating human embryonic stem cells (hESC) or induced pluripotent stem cells (iPSC). NK cells differentiated from iPSC are referred to as iNK cells.

[0044] In some embodiments, the NK cells are allogeneic and haplotype matched to the patient in one or more HLA loci, one or more KIR loci, or both.

[0045] In some embodiments, the isolated NK cell composition is depleted of CD3 + In some embodiments, the isolated NK cell composition is enriched for CD56 + In some embodiments, the isolated NK cell composition is enriched for CD45 + In some embodiments, the isolated NK cell composition is enriched for CD56 + / CD45 + In some embodiments, a quality control measurement or characterization step is applied to the isolated NK cell composition, such as determining the CD56+ / CD3 - 、CD45 + / CD3 - Cells, CD56 + / CD45 + or CD56 + / CD45 + / CD3 - In some embodiments, the present invention utilizes a substantially free CD3 + NK cell composition of cells.

[0046] In some embodiments, the isolated lymphocytes are characterized in terms of specificity, frequency and function of each subtype. In some embodiments, the isolated lymphocyte population is enriched for a specific subset of T cells, such as CD8 + 、CD25 + CD62L + See, e.g., Wang et al., Mol. Therapy-Oncolytics (2016) 3:16015. In some embodiments, the isolated NK cell composition is enriched for CD56 + / CD45 + cell.

[0047] 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 performed by flow cytometry to determine whether the composition contains CD56 + / CD45 + The percentage of cells.

[0048] In some embodiments, after isolation, lymphocytes are activated to promote proliferation and differentiation into specialized lymphocytes. For example, T cells can be activated using soluble CD3 / 28 activators or magnetic beads coated with anti-CD3 / anti-CD28 monoclonal antibodies.

[0049] In some embodiments, the present invention is a method for treating an autoimmune disease in a patient, the method comprising administering to the patient a composition comprising immune cells expressing 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.

[0050] In some embodiments, the protein targeting CD19 is an anti-CD19 T cell receptor. In some embodiments, the anti-CD19 T cell receptor is a chimeric antigen receptor (CAR). In some embodiments, the immune cell is a CAR-T cell or a CAR-NK cell.

[0051] In some embodiments, the CAR comprises an extracellular domain comprising a CD19 binding region, a transmembrane domain, and one or more intracellular co-activation (costimulatory) and activation (stimulatory) domains.

[0052] 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 camelid single domain antibody (V HH ) heavy chain (V H ) or a light chain (V L ) of the variable part. These fragments can be derived from monoclonal antibodies. Single-chain variable fragments (scFv) have the ability to bind CD19. ScFv comprises the Fv region of the immunoglobulin heavy chain (H chain) and light chain (L chain) connected by a spacer sequence. In some embodiments, the scFv binding to CD19 is FMC63, see Nicholson et al., (1997) Construction and characterization of a functional CD19specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma, Mol. Immunol. 34: 1157.

[0053] In some embodiments, the transmembrane domain of CAR is derived from a membrane-bound protein or a transmembrane protein. For example, the transmembrane domain of CAR can be the following transmembrane domain: T cell receptor α-chain or β-chain, CD3-ζ chain, CD28, CD3-ε 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 a CD8 transmembrane domain. In some embodiments, the transmembrane domain of CAR is a CD8A transmembrane domain.

[0054] The intracellular signaling domain of CAR is responsible for activating one or more effector functions of immune cells expressing CAR. In some embodiments, the intracellular signaling domain of CAR comprises a portion or the entire sequence of: CD3-ζ chain, CD3-ε chain, CD2, CD28, CD27, OX40 / CD134, 4-1BB / CD137, ICOS / CD278, IL-2Rβ / CD122, IL-2Rα / CD132, DAP10, DAP12, DNAM1, TLR1, TLR2, TLR4, TLR5, TLR6, MyD88, CD40 or a combination thereof. In some embodiments, the intracellular domain of CAR consists of 4-1BB and CD3ζ chain.

[0055] In some embodiments, CAR comprises a hinge domain. In some embodiments, the hinge domain of CAR is a CD8 hinge domain. In some embodiments, the hinge domain of CAR is a CD8A hinge domain.

[0056] Figure 1 An exemplary anti-CD19 CAR is shown in Figure 1. The CAR comprises a signal sequence, an anti-CD19 scFv, a CD8 hinge domain, a transmembrane domain, 4-1BB, and a CD3-ζ intracellular domain.

[0057] In some embodiments, CAR is a fully human protein or is humanized to reduce the immunogenicity of human patients. In some embodiments, the nucleic acid sequence encoding CAR is optimized for codon usage in human cells.

[0058] The nucleic acid encoding 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 translating CAR, and in some embodiments, the cDNA sequence further includes a non-translated element that improves, for example, the stability of CARmRNA or the translation rate.

[0059] In some embodiments, cells (T cells, natural killer (NK) cells, iNK cells, CAR-T cells or CAR-NK cells) for treating autoimmune diseases further include genomic modifications that achieve armoring of cells to protect them from the attack of the immune system of patients with autoimmune diseases. In some embodiments, armoring modifications include protecting the host from being recognized by cytotoxic T cells. Cytotoxic T cells recognize MHC class I antigens. MHC class I molecules include beta-2 microglobulin (B2M) associated with the heavy chain of HLA-I proteins (selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G) on the cell surface. The B2M / HLA-I complex on the surface of allogeneic cells is cytotoxic CD8+ T cells recognize, and if HLA-I is recognized as non-self, the allogeneic cells are killed by T cells. In some embodiments, the cells of the invention comprise armored genome modifications comprising disruption of the B2M gene and thus disruption of MHC class I antigen recognition and cytotoxic T cell attack.

[0060] In some embodiments, the armored genome modification comprises the destruction of the recognition of the host's NK cells. NK cells recognize cells without MHC-I proteins as "missing self" and kill such cells. NK cells are inhibited by HLA-I molecules, which include HLA-E, a HLA-I protein with minimal polymorphism. In some embodiments, the cells of the present invention comprise a first armored genome modification, which comprises the destruction of the B2M gene and thus the destruction of MHC class I antigen recognition and cytotoxic T cell attack, and further comprises a second armored genome modification, which comprises the insertion of an HLA-E gene fused to a beta-2-microglobulin (B2M) gene and thus the expression of an HLA-E / B2M construct and protects the cell from NK cell attack. See, e.g., Gornalusse et al. (2017) HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells, Nat. Biotechnol. (2017) 35:765-772.

[0061] In some embodiments, the armor modification comprises transcriptional silencing or disruption of 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. Application Publication US20150017136 for engineering allogeneic and highly active T cells for immunotherapy (Methods for engineering allogeneic and highly active T cell for immunotherapy).

[0062] In some embodiments, patients receiving treatment with immune cells expressing proteins targeting CD19 are monitored to assess the clinical manifestations of autoimmune diseases. Symptoms are expected to be alleviated by treatment as described herein. In some embodiments, before administering immune cells expressing proteins targeting CD19, the clinical manifestations of the patient's autoimmune diseases are assessed. In some embodiments, after administering T cells expressing proteins targeting CD19 for the first time, the patient is assessed hourly, daily, weekly or monthly. In some embodiments, the patient is assessed in conjunction with daily, weekly or monthly administration regimens of immune cells expressing proteins targeting CD19.

[0063] In some embodiments, clinical manifestations of autoimmune diseases include one or more of the following: proteinuria, alopecia, organomegaly, the presence of high cell glomeruli, IgG tissue deposition, IgM and IgG antibody titers in serum and IgG or IgM antinuclear antibodies, an increase in the total number or concentration of B cells in plasma, and the presence of skin lesions or discoloration. Therefore, the clinical manifestations of the patient's autoimmune disease are assessed by one or more of urinalysis, blood analysis (including total blood cell count), and physical examination.

[0064] 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.

[0065] In some embodiments, the patient's immune cells (such as T cells, NK cells, CAR-T cells or CAR-NK cells) expressing proteins targeting CD19 are evaluated for presence and relative quantity. In some embodiments, the presence and relative quantity of cells are evaluated by one or more methods selected from the following: flow cytometry, ELISA, fluorescence microscopy, fluorescence in situ hybridization (FISH), PCR and RT-PCR, which are intended to detect the presence of proteins targeting CD19, genes encoding proteins targeting CD19 or mRNA encoding proteins targeting CD19, respectively.

[0066] In some embodiments, anti-CD19 CAR is encoded by a nucleic acid construct introduced into a cell (T cell, natural killer (NK) cell or iNK cell) for treating an autoimmune disease. In some embodiments, an anti-CD19 CAR expression construct comprises a coding sequence and a promoter of a CAR targeting CD19.

[0067] In some embodiments, the CAR expression construct targeting CD19 is introduced by an expression vector or an RNA encoding a CAR protein targeting CD19. In some embodiments, the target cell is contacted with a nucleic acid encoding a CAR targeting CD19 in vitro, in vivo or ex vivo.

[0068] 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). Suitable vectors are non-replicative in target cells. In some embodiments, the vector is selected from SV40, EBV, HSV or BPV, or is 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 a regulatory sequence, including a transcription activator binding sequence, a transcription repressor binding sequence, an enhancer, an intron, etc. In some embodiments, the viral vector supplies a constitutive promoter or an inducible promoter. In some embodiments, the promoter is selected from EF1α, PGK1, MND, Ubc, CAG, CaMKIIa and β-actin promoter. In some embodiments, the promoter is selected from SV40 early and late promoters, cytomegalovirus (CMV) immediate early promoter and Rous sarcoma (Rous sarcoma) virus long terminal repeat sequence (RSV-LTR) promoter, mouse mammary tumor virus long terminal repeat sequence (MMTV-LTR) promoter, beta-interferon promoter, hsp70 promoter and EF-1α promoter. In some embodiments, the promoter is EF-1α promoter. In some embodiments, the promoter is MND promoter.

[0069] In some embodiments, the viral vector supplies a transcription terminator or a polyadenylation signal. In some embodiments, the transcription terminator or polyadenylation signal is a BGH transcription terminator and polyadenylation signal.

[0070] In some embodiments, the vector is a plasmid selected from a prokaryotic plasmid, a eukaryotic plasmid, and a shuttle plasmid.

[0071] In some embodiments, the expression vector comprises one or more selection markers. In some embodiments, the selection marker is an antibiotic resistance gene or other negative selection marker. In some embodiments, the selection marker comprises a protein whose mRNA is transcribed together with the CAR mRNA targeting CD19 and the polycistronic transcript is cut before translation.

[0072] In some embodiments, the expression vector comprises a polyadenylation site. In some embodiments, the polyadenylation site is the SV-40 polyadenylation site.

[0073] In some embodiments, the coding sequence of the CAR targeting CD19 is introduced into the cell by a viral vector (e.g., AAV vector (AAV6) or any other suitable viral vector capable of delivering enough payload). In some embodiments, in order to promote homologous recombination, the coding sequence is connected to the homology arms of 5' (upstream or left) and 3' (downstream or right) of the insertion site in the desired insertion site in the genome. In some embodiments, the length of the homology arm is about 500bp. In some embodiments, the sequence of the CAR encoding targeting CD19 is cloned into a viral vector plasmid together with the homology arm. Plasmid is used to package the sequence into a virus.

[0074] Figure 1 An exemplary nucleic acid construct is shown in . In addition to the CAR coding region, the construct comprises an EF1α promoter, a left homology arm (LHA) and a right homology arm (RHA).

[0075] In some embodiments, cells (T cells, natural killer (NK) cells or iNK cells) are contacted with a viral vector so that the genetic material delivered by the vector is integrated into the genome of the target cell and then expressed in the cell or on the cell surface. Transduced and transfected cell expression can be tested using methods well known in the art such as fluorescence activated cell sorting (FACS), microfluidic-based screening, ELISA or protein blotting (Western blot) .

[0076] In some embodiments, the coding sequence of a CAR targeting CD19 is introduced into cells (T cells, natural killer (NK) cells, or iNK cells) as a "naked" nucleic acid by electroporation as described, for example, in U.S. Pat. No. 6,410,319.

[0077] In some embodiments, the engineered 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 in a single molecule).

[0078] In some embodiments, NATNA is selected from the embodiments described in U.S. Patent No. 9,260,752. Briefly, NATNA may comprise a spacer extension, a spacer, a minimal CRISPR repeat sequence, a single guide connector, a minimal tracrRNA, a 3' tracrRNA sequence, and a tracrRNA extension in a 5' to 3' order. In some cases, the nucleic acid targeting nucleic acid may comprise a tracrRNA extension, a 3' tracrRNA sequence, a minimal tracrRNA, a single guide connector, a minimal CRISPR repeat sequence, a spacer, and a spacer extension in any order.

[0079] In some embodiments, the guide nucleic acid targeting nucleic acid may comprise a single guide NATNA. NATNA comprises a spacer sequence that can be engineered to hybridize with a target nucleic acid sequence. NATNA further comprises a CRISPR repeat sequence that comprises a sequence that can hybridize with a tracrRNA sequence. Optionally, NATNA may have a spacer extension and a tracrRNA extension. These elements may include elements that can contribute to the stability of NATNA. CRISPR repeat sequences and tracrRNA sequences may interact to form a double-stranded structure of base pairing. The structure may promote the binding of endonucleases to NATNA.

[0080] In some embodiments, the single-guide NATNA comprises a spacer sequence positioned at the 5' position of the first duplex, the first duplex comprising a hybridization region between the minimum CRISPR repeat sequence and the minimum tracrRNA sequence. The first duplex may be interrupted by a protrusion. The protrusion promotes the recruitment of endonucleases to the NATNA. The protrusion may be followed by a first stem comprising a joint connecting the minimum CRISPR repeat sequence and the minimum tracrRNA sequence. The last pair of nucleotides at the 3' end of the first duplex may be connected to a second joint connecting the first duplex to the mid-tracrRNA. The mid-tracrRNA may comprise one or more additional hairpins.

[0081] In some embodiments, the NATNA may comprise a dual-guide nucleic acid structure. The dual-guide NATNA comprises a spacer extension, a spacer, a minimal CRISPR repeat sequence, 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, and the minimal tracrRNA sequence comprises a 5'tracrRNA sequence, and the dual-guide NATNA can hybridize through the minimal CRISPR repeat sequence and the minimal tracrRNA sequence.

[0082] In some embodiments, NATNA is an engineered guide RNA (CRISPR hybrid RDNA or chRDNA) comprising one or more DNA residues. In some embodiments, NATNA is selected from the embodiments described in U.S. Patent No. 9,650,617. In brief, some chRDNA used with the type II CRISPR system can be composed of two chains forming a secondary structure, the secondary structure including an activation region consisting of an upper duplex region, a lower duplex region, a protrusion, a targeting region, a connection, and one or more hairpins. The nucleotide sequence immediately downstream of the targeting region may contain DNA and RNA in different proportions. Other chRDNAs may be single guide D (R) NAs used with the type II CRISPR system, the system comprising a targeting region and an activation region, the activation region consisting of a lower duplex region, an upper duplex region, a fusion region, a protrusion, a connection, and one or more hairpins. The nucleotide sequence immediately downstream of the targeting region may contain DNA and RNA in different proportions. For example, the targeting region may contain DNA or a mixture of DNA and RNA, and the activation region may contain RNA or a mixture of DNA and RNA.

[0083] 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 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 capable of propagation in the cell. In some embodiments, the gene encoding the NATNA guide is inserted into a plasmid capable of propagation in the cell.

[0084] In some embodiments, the components of the CRISPR system (i.e., the nucleic acid-guided endonuclease and the NATNA guide) are introduced into the cell in the form of RNA, such as mRNA encoding the nucleic acid-guided endonuclease and the NATNA guide.

[0085] In some embodiments, the components of the CRISPR system (i.e., nucleic acid-guided endonucleases and NATNA guides) are introduced into the cell as preassembled nucleoprotein complexes. In some embodiments, the components of the CRISPR system (i.e., nucleic acid-guided endonucleases and NATNA guides) are introduced into the cell by any combination of different means, such as the endonuclease is introduced as DNA via a plasmid containing a gene encoding the endonuclease, and the guide is introduced in its final format as RNA (or RNA containing DNA nucleotides).

[0086] In some embodiments, components of the CRISPR system (ie, nucleic acids encoding nucleic acid-guided endonucleases and NATNA guides) are introduced into cells by electroporation.

[0087] In some embodiments, components of the CRISPR system (i.e., nucleic acids encoding nucleic acid-guided endonucleases) are introduced into cells in the form of mRNA as described, for example, in U.S. Pat. No. 10,584,352 by electroporation with viral pseudotransduction as described herein.

[0088] In some embodiments, the coding sequence of the CAR targeting CD19 is inserted into the double-strand break in the genome of the cell (T cell, natural killer (NK) cell or iNK cell). In some embodiments, the introduction of the coding sequence occurs simultaneously with the inactivation of another gene by inserting the CAR gene (gene knockout and simultaneous 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.

[0089] In some embodiments, the (anti-CD19) CAR-T cells targeting CD19 are allogeneic. Allogeneic CAR-T cells may include armored modifications that protect allogeneic cells from the patient's (recipient's) immune system attack. In some embodiments, the armored modifications include transcriptional silencing or destruction of one or more immune checkpoint genes. In some embodiments, the checkpoint gene is PDCD1 (encoding PD-1 protein).

[0090] Programmed cell death protein 1 (PD-1, encoded by the gene PDCD1) (also known as CD279) is a cell surface receptor that plays an important role in down-regulating the immune system and promoting self-tolerance by inhibiting 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 its other ligand PD-L2 (also known as CD273). PD-1 prevents autoimmunity by promoting programmed cell death (apoptosis) of antigen-specific T cells in lymph nodes while reducing apoptosis in anti-inflammatory suppressive T cells (regulatory T cells). Through these mechanisms, the PD-1 binding of PD-L1 inhibits the immune system, thereby preventing autoimmune disorders, and preventing the immune system from killing cancer cells. Therefore, mutation or knockout of PD-1 expression (e.g., by destroying the PDCD1 gene) can be beneficial in T cell therapy.

[0091] In some embodiments, an endonuclease of a nucleic acid chain in a target sequence of a gene to be destroyed is used to specifically cut the immune checkpoint gene. The chain cutting performed by a sequence-specific endonuclease results in a nucleic acid chain break that can be repaired by non-homologous end joining (NHEJ). NHEJ is an imperfect repair process that may result in direct reconnection, but more generally results in the deletion, insertion or substitution of one or more nucleotides in the target sequence. Such deletion, insertion or substitution of one or more nucleotides in the target sequence may result in missense or nonsense mutations in the protein coding sequence, and eliminates the production of any protein or causes the production of non-functional protein.

[0092] In some embodiments, the armor modification comprises targeted cutting and repair of the PDCD1 gene, resulting in gene inactivation. In some embodiments, the PDCD1 gene is destroyed by cutting the PDCD1 locus in exon 2 of the PDCD1 gene on human chromosome 2 by CRISPR-Cas endonucleases (e.g., Cas9) and guide polynucleotides (NATNA). In some embodiments, the guide polynucleotide (NATNA) is a CRISPR hybrid RNA-DNA polynucleotide (chRDNA).

[0093] In some embodiments, the activity of anti-CD19 CAR-T cells against B cells is evaluated. In some embodiments, the activity of anti-CD19 CAR-T cells against B cells derived from patients diagnosed with an autoimmune disease is evaluated.

[0094] In some embodiments, the activity of anti-CD19 CAR-T cells against B cells is assessed in vitro as cytotoxicity against B cells.

[0095] In some embodiments, the in vitro assessment of the cytotoxic properties of anti-CD19 CAR-T cells utilizes target cells or target cell lines. In some embodiments, the target cells are primary cells obtained from human blood samples. In some embodiments, human samples are from patients diagnosed with autoimmune diseases. In some embodiments, human samples are control samples obtained from subjects without autoimmune diseases. In some embodiments, the sample is processed to extract blood fractions, such as peripheral blood mononuclear cells (PBMCs), B cells, or non-B cells.

[0096] In some embodiments, the target cell is an established lymphocyte cell line. In some embodiments, the target cell is an established B cell line. In some embodiments, the target cell is a lymphoid tumor cell line of an established B cell tumor cell line.

[0097] In some embodiments, the expression of CD19 in the target cells is confirmed before evaluating the cytotoxicity of anti-CD19 CAR-T cells. In some embodiments, the expression of CD19 is confirmed by a method selected from the following: flow cytometry with anti-CD19 antibodies, staining with a marker-conjugated anti-CD19 antibody, fluorescent in situ hybridization, Western blot, or any other method known in the art for detecting the expression of proteins on the cell surface.

[0098] In some embodiments, the cytotoxicity of anti-CD19 CAR-T cells is evaluated in the form of 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 comprising or consisting of B cells. The co-culture can be established with different effector: target ratios (E: T ratios). In some embodiments, the E: T ratio is in the range of about 0.1: 1 (1: 10) to about 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 following E: T ratios are evaluated: 0.125: 1 (1: 8), 0.25: 1 (1: 4), 0.5: 1 (1: 2), 1: 1, 2: 1, 4: 1, 8: 1.

[0099] In some embodiments, the cell-permeable fluorescent dye (e.g., CellTrace TM luciferase (CTV), Thermo Fisher Scientific, Carlsbad, Calif.) in conjunction with a reactive dye to label target cells to detect cell lysis by 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 can also be used to determine cell lysis by incorporating a reactive dye and measuring an increase in fluorescence or by using cells containing a fluorescent reporter gene and measuring a decrease in fluorescence. Impedance-based systems such as the xCELLigence system (Agilent, Santa Clara, Calif.) in Santa Clara, Calif.) can also provide dynamic real-time monitoring of cell lysis.

[0100] In some embodiments, control experiments are performed to assess the lysis of anti-CD19 CAR-T cells to a cell population consisting of non-B cells. In some embodiments, control experiments are performed to assess the lysis of anti-CD19 CAR-T cells to a cell population comprising both B cells and non-B cells (e.g., PBMC).

[0101] In some embodiments, lysis of B cells by anti-CD19 CAR-T cells is compared in primary cell samples from autoimmune patients and primary cell samples from subjects without autoimmune disease.

[0102] In some embodiments, an anti-CD19 CAR-T cell population that achieves the highest percentage of B cell lysis is selected for administration to a patient with an autoimmune disease. In some embodiments, an anti-CD19 CAR-T cell population that achieves a high percentage of B cell lysis but has low non-B cell lysis is selected for administration to a patient with an autoimmune disease.

[0103] In some embodiments, the activity of anti-CD19 CAR-T cells against B cells is evaluated in vitro in the form of a reduction in B cell autoantibody secretion. In some embodiments, autoantibody secretion is evaluated by co-culturing anti-CD19 CAR-T cells (effectors, E) with a cell population comprising B cells (targets, T). In some embodiments, the E:T ratio of co-culture is in the range of about 1:10 to about 10:1. In some embodiments, the E:T ratio of co-culture is about 1:1. In some embodiments, autoantibodies in co-culture supernatants are qualitatively or quantitatively evaluated. Autoantibodies can be evaluated in the form of total IgG in the supernatant. Specific types of autoantibodies (e.g., anti-dsDNA IgG specific to SLE) can be detected using antibody-based or antibody-conjugate-based assays (such as Western blots or ELISAs) and similar secondary antibody-based methods and colorimetric methods, chemiluminescent methods, or fluorescent detection methods. Anti-dsDNA antibodies can also be detected using the Farr radioimmunoassay, which measures radiolabeled dsDNA bound to the anti-dsDNA antibodies, or using the Crithidia luciliae indirect immunofluorescence test (CLIFT).

[0104] In some embodiments, the present invention includes a composition comprising cells (T cells, natural killer (NK) cells or iNK cells) expressing proteins targeting CD19. In some embodiments, the composition includes cytotoxic CAR-T cells or CAR-NK cells expressing anti-CD19 chimeric antigen receptors (CAR). In some embodiments, the composition includes cells and one or more pharmaceutically acceptable excipients. Exemplary excipients include, but are not limited to, carbohydrates, inorganic salts, antimicrobials, antioxidants, surfactants, buffers, acids, bases and combinations thereof. Excipients suitable for injectable compositions include water, alcohols, polyols, glycerol, vegetable oils, phospholipids and surfactants. Carbohydrates (such as sugars), derived sugars (such as sugar alcohols), 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, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, inositol, etc. Excipients may also include inorganic salts or buffers such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium dihydrogen phosphate, sodium dihydrogen phosphate, and combinations thereof.

[0105] In some embodiments, the composition further comprises an antimicrobial agent for preventing or preventing microbial growth. In some embodiments, the antimicrobial agent is selected from benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimerosal, and combinations thereof.

[0106] 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, sodium metabisulfite, and combinations thereof.

[0107] 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); phosphatidylethanolamines; fatty acids and fatty esters; steroids, such as cholesterol.

[0108] In some embodiments, the composition further comprises a cryogen, such as 3% to 12% dimethyl sulfoxide (DMSO) or 1% to 5% human albumin.

[0109] The number of adoptive cells (such as T cells, NK cells, CAR-T cells or CAR-NK cells) in the composition will vary depending on many factors, but optimally will be a therapeutically effective dose per vial.

[0110] The minimum or optimal therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the composition to determine which amount produces a reduction in autoimmune disease symptoms.

[0111] The maximum or optimal therapeutically effective dose can be determined experimentally by repeated administration of decreasing amounts of the composition to determine which amount produces a reduction in autoimmune disease symptoms while producing no or an acceptable level of undesirable side effects.

[0112] The present invention includes the step of administering to a patient a composition comprising immune cells (T cells, NK cells or iNK cells) expressing a protein targeting CD19.

[0113] In some embodiments, before administering immune cells, the patient is subjected to lymphocyte depletion pretreatment to reduce any immune system attack against the administered immune cells. In some embodiments, the patient is pretreated with immunosuppressants known to be safe and effective for autoimmune diseases, see, for example, Fava A. and Petri, M. (2019) Systemic lupus erythematosus: diagnosis and clinical management (Systemic lupus erythematosus: diagnosis and clinical management) Journal of Autoimmunity (J. Autoimmun.) 96: 1-13.

[0114] In some embodiments, the immunosuppressant is cyclophosphamide, an alkylating agent that has a history of use in lupus patients and is known to deplete T and B cells.

[0115] In some embodiments, the immunosuppressant is azathioprine, a purine analog with a history of use in lupus patients.

[0116] 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.

[0117] In some embodiments, the immunosuppressant is mycophenolate, an agent that depletes guanosine nucleotides and has a history of use in lupus patients and is known to inhibit T and B cell proliferation.

[0118] In some embodiments, the immunosuppressant is a calcineurin inhibitor (eg, cyclosporine) that has a history of use in lupus patients and is known to reduce T cell activity.

[0119] In some embodiments, lymphocyte depletion comprises a cyclophosphamide regimen. In some embodiments, lymphocyte depletion comprises administering cyclophosphamide at 60 mg / kg per day for 2 days.

[0120] In some embodiments, lymphocyte depletion further comprises a fludarabine regimen. In some embodiments, lymphocyte depletion comprises 25 mg / m 2 Fludarabine was administered for 5 days.

[0121] At the end of lymphocyte depletion pretreatment, patients were given

[0122] Including not more than 600,000 (equivalent to not more than 10 4 / kg) of immune cells expressing anti-CD19 CAR. In some embodiments, 40,000 (equivalent to 600 / kg) anti-CD19 allogeneic CAR-T cells are administered to the patient.

[0123] The dose of CD19-targeted cells (such as anti-CD19 CAR-T cells and CAR-NK cells) required to treat autoimmune diseases is significantly 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 is significantly lower than the dose of autologous CAR-T or CAR-NK cells. Table 1 lists the doses of autologous anti-CD19 CAR-T cells compared to the experimental allogeneic anti-CD19 CAR-T cell composition CB-010. and Compared with the relative dose of , CB-010 produced greater overall response and complete response in patients (Source: European Hematology Association (EHA) Abstract, CB-010 Clinical Program Update, May 12, 2022).

[0124] Table 1: Dose comparison between allogeneic and autologous CAR-T cell therapy

[0125]

[0126] Source: Package insert

[0127] Mackensen et al. have used 10 6 cells / kg (4×10 7 -9×10 7Remission was achieved in SLE patients treated with autologous anti-CD19 CAR-T cells at a dose of 100 cells / mL. 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 1 / 10-1 / 2 of the dose used to treat B cell malignancies with autologous CAR-T cells (Table 1).

[0128] It has also been demonstrated that administration of allogeneic CAR-T cells produces a better response in patients than administration of autologous CAR-T cells. Table 2 lists the results of autologous anti-CD19 CAR-T cells compared to the experimental allogeneic anti-CD19 CAR-T cell composition CB-010. and Efficacy produced (measured as overall response (ORR) and complete response (CR)) (Source: EHA Abstract, May 12, 2022, supra).

[0129] Table 2: Efficacy of allogeneic and autologous CAR-T cell therapy

[0130]

[0131] It has been further demonstrated that administration of allogeneic CAR-T cells produces fewer side effects than administration of autologous CAR-T cells. Table 3 lists the side effects of autologous anti-CD19 CAR-T cells compared to the experimental allogeneic anti-CD19 CAR-T cell composition CB-010. and Adverse events (≥ Grade 3 for each of the following: cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and infections) (Source: EHA Abstract May 12, 2022, supra).

[0132] Table 3: Safety of allogeneic and autologous CAR-T cell therapy

[0133]

[0134] 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 CB-010 allogeneic anti-CD19 CAR-T cells, compared to 4×10 7 Compared with CAR-T cells, the dose is about 4×10 4 (40,000) CAR-T cells. Expressed as cells per kg body weight, this is comparable to the 6×10 CAR-T cells used to treat B-cell non-Hodgkin lymphoma. 5 CAR-T cells / kg, compared with a dose of approximately 6×10 2 (600) allogeneic CAR-T cells / kg. In some embodiments, no more than 600,000 (equivalent to no more than 10 4 / kg) of allogeneic anti-CD19 CAR-expressing cells.

[0135] In some embodiments, the dose of anti-CD19 CAR-expressing cells administered to a human patient to treat an autoimmune disease is about the same as the dose of the same CAR-T cells administered to treat a tumor. For example, for CB-010 allogeneic anti-CD19 CAR-T cells, the dose is about 4×10 7 (40,000,000) total CAR-T cells. Expressed as cells per kg body weight, the dose is approximately 6×10 5 (60,000) allogeneic CAR-T cells / kg.

[0136] 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 cells expressing an anti-CD19 protein (such as anti-CD19 CAR-T cells or CAR-NK cells) at a dose of 10,000-100,000 cells (equivalent to about 100-1000 cells per kilogram of body weight).

[0137] 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 40,000 (equivalent to 600 / kg) anti-CD19 allogeneic CAR-T cells.

[0138] 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 no more than 600,000 (equivalent to no more than 10 4 / kg) of anti-CD19 allogeneic CAR-T cells.

[0139] In some embodiments, the present invention comprises administering anti-CD19 allogeneic CAR-T cells to a patient at a frequency of 2-4 times per year.

[0140] In some embodiments, patients are treated with anti-CD19 allogeneic CAR-T cells more or less frequently than 2-4 times per year based on symptom assessments (including blood and urine analysis) and visual assessments described herein to detect treatment progress and any side effects.

[0141] 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, the administration is by infusion, and the infusion is selected from a single sustained dose, extended continuous infusion, and multiple infusions.

[0142] Examples

[0143] Example 1. (Prophetic) Administration of anti-CD19 allogeneic CAR-T cells to measurably alleviate lupus symptoms

[0144] In this example, the human patient is subjected to one or more of urinalysis, blood analysis (including total blood cell count), physical evaluation, and is diagnosed as having lupus if one or more of the following is present: proteinuria, alopecia, organomegaly, the presence of hypercellular glomeruli, IgG tissue deposits, IgM and IgG antibody titers in serum and IgG or IgM antinuclear antibodies, an increased total number or concentration of B cells in plasma, and the presence of skin lesions or discoloration.

[0145] Patients received cyclophosphamide 60 mg / kg daily for 2 days and 25 mg / m 2 Pretreatment with lymphodepletion consisting of fludarabine for 5 days.

[0146] At the end of lymphodepleting pretreatment, the patient was administered a composition comprising 40,000 (equivalent to 600 / kg) anti-CD19 allogeneic CAR-T cells.

[0147] Beginning one week after administration, patients are evaluated by one or more of urinalysis, blood analysis (including total blood cell count), and physical assessment to detect any reduction in pre-existing lupus symptoms selected from the following: proteinuria, alopecia, organomegaly, the presence of tall cellular glomeruli, IgG tissue deposits, IgM and IgG antibody titers in serum and IgG or IgM antinuclear antibodies, an increase in the total number or concentration of B cells in plasma, and the presence of skin lesions or discoloration.

[0148] The total number or concentration of B cells in plasma was assessed by flow cytometry, and IgG or IgM antinuclear antibodies in serum were assessed by ELISA.

[0149] Patients were also evaluated for the presence (persistence) of anti-CD19 allogeneic CAR-T cells. These cells were detected by flow cytometry, ELISA, fluorescence microscopy, fluorescence in situ hybridization (FISH), PCR, and RT-PCR to detect the presence of CAR targeting CD19, genes encoding CAR, or mRNA encoding CAR.

[0150] If no symptom reduction is observed, another dose or greater dose of anti-CD19 allogeneic CAR-T cells is administered to the patient. If low amounts of anti-CD19 allogeneic CAR-T cells or no anti-CD19 allogeneic CAR-T cells are detected in the patient's circulation, another dose or greater dose of anti-CD19 allogeneic CAR-T cells is administered to the patient.

[0151] Example 2. CB-010: Allogeneic anti-CD19 CAR-T cells

[0152] Allogeneic anti-CD19 CAR-T cells with PD-1 inactivation, called CB-010 ( Figure 2 ) is being developed for relapsed / refractory B-cell non-Hodgkin's lymphoma. (See European Hematology Association (EHA) Abstract, CB-010 Clinical Program Update, May 12, 2022). Figure 1 The structure of CAR is shown in FIG.

[0153] Briefly, CB-010 cells are generated from T cells obtained by leukapheresis of healthy donor blood samples. CRISPR Cas9 endonuclease with chRDNA (CRISPR hybrid RNA-DNA guide) is used for genome editing. Anti-CD19 CAR transgene ( Figure 1 ) was delivered by an AAV vector and inserted into the T cell receptor alpha chain (TRAC) locus on chromosome 14. Additionally, Cas9 / chRDNA was used to disrupt the PDCD1 gene on chromosome 2, resulting in abrogation of PD-1 expression.

[0154] Example 3. Specific lysis of B cells by anti-CD19 CAR-T cells (CB-010)

[0155] In this example, anti-CD19 CAR-T cells (allogeneic anti-CD19 CAR-T cells with PDCD1 gene inactivation referred to as CB-010 described in the European Hematology Association (EHA) abstract, May 12, 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 target was labeled with CTV to distinguish it 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. Autoimmune patient-derived cell fractions 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. 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)) for cytotoxicity measurement by flow cytometry (iQue Screener Plus, Intellicyt, Albuquerque, NM). Cytotoxicity was determined by gating on the viable cell population within the CTV-labeled target cell population or within the B cell and non-B cell populations of the CTV-labeled target cells. For each well, specific lysis was calculated using the following equation: Specific lysis = 1 - (% of live target cells in co-culture sample / % of live target cells in target only sample). Specific lysis curves for the different samples were then generated, and the area under the curve (AUC) measurements for specific lysis were determined for the different populations and conditions.

[0156] Figure 3 and Figure 4 The results are shown in . Figure 3 The results of the in vitro cytotoxicity evaluation of CB-010 allogeneic anti-CD19 CAR-T cells are shown. Specific lysis of PBMCs, B cells and non-B cells from systemic lupus erythematosus (SLE) derived cell fractions by CB-010 at different E:T ratios is shown. Figure 4The results of the in vitro cytotoxicity assessment of CB-010 for SLE derived cell fractions and rheumatoid arthritis (RA) derived cell fractions are shown. Cytotoxicity is expressed as the area under the curve (AUC) measurement results of the specific lysis of PBMC, B cells and non-B cells from SLE patients and RA patients by CB-010. Data represent 4 independent donors (2 SLE patient derived PBMCs and 2 RA patient derived PBMCs). Error bars represent mean ± SD. By paired t test between CB-010 and TRAC KO co-culture conditions, ns (not significant) represents p>0.05, and ** represents p≤0.01.

[0157] Example 4. Reduction of B cell autoantibody secretion in the presence of anti-CD19 CAR-T cells (CB-010).

[0158] In this example, CB-010 allogeneic anti-CD19 CAR-T cells are co-cultured with cell fractions obtained from blood samples of autoimmune patients or non-diseased B cells separated. As a control, the target is also cultured alone or co-cultured with donor-matched T cells with inactivated TRAC loci but without CAR insertion (TRAC KO). Non-diseased B cells are co-cultured with effector cells with an E:T ratio of 1:1, and autoimmune derived cell fractions are co-cultured with effector cells with an E:T ratio of 1:4, to illustrate that B cells are only part of PBMC. In the presence of ODN2006 (a CpG oligonucleotide that strongly activates B cells by TLR9 activation), the co-culture is maintained for 6 days. After 6 days, supernatant is collected from the co-culture. The concentrations of total IgG and anti-dsDNA IgG in the co-culture supernatants were measured using ELISA kits specific for total IgG detection (Invitrogen, Carlsbad, Cal.) or anti-dsDNA IgG detection (Abnova, Taipei City, Taiwan, China). Figure 5 The results of the measurement results of the concentration of autoimmune antibodies in the co-culture of CB-010 with SLE-derived cell fractions and RA-derived cell fractions are shown in Figure . The data represent 6 independent donors (2 isolated healthy B cells, 2 SLE patient-derived PBMCs, and 2 RA patient-derived PBMCs). Error bars represent mean ± SD. By paired t tests between only target and CB-010 co-culture conditions, ns (not significant) represents p>0.05, * represents p≤0.05, *** represents p≤0.001, and **** represents p≤0.0001.

[0159] 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 may be made within the scope of the present invention. Therefore, the scope of the present invention should not be limited by the examples described herein, but by the following claims.

Claims

1. A method for treating an autoimmune disease in a patient, the method comprising: An amount of a composition comprising engineered immune cells targeted to CD19 is administered to the patient, thereby ameliorating 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 (T1D), Sjögren's syndrome ( syndrome) and multiple sclerosis (MS).

3. The method of claim 1, wherein the patient is a human.

4. The method of claim 1, wherein the one or more symptoms of the autoimmune disease are selected from the group consisting of proteinuria, alopecia, elevated IgM and IgG antibody titers, the presence of anti-nucleoprotein IgG or IgM in serum, increased B cell counts in plasma, and the presence of skin lesions or discoloration.

5. The method of claim 1, wherein the antibody-producing cells are B cells.

6. The method of claim 1, wherein the engineered immune cells targeting CD19 are CAR-T cells expressing anti-CD19 chimeric antigen receptor (CAR).

7. The method of claim 1, wherein the engineered immune cells targeting CD19 are CAR-natural killer (NK) cells expressing anti-CD19 chimeric antigen receptor (CAR).

8. The method of claim 1, wherein the engineered immune cells targeting CD19 are allogeneic.

9. The method of claim 8, wherein the allogeneic immune cells comprise armored genome modifications.

10. The method of claim 9, wherein the armored genome modification comprises inactivation of the PDCD1 gene.

11. The method of claim 6, wherein the anti-CD19 CAR comprises an anti-CD19 scFv, a transmembrane domain, and an intracellular stimulatory domain.

12. The method of claim 11, wherein the anti-CD19 CAR further comprises a signal peptide and a hinge.

13. The method of claim 6, wherein the anti-CD19 CAR comprises FMC63, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain.

14. The method of claim 6, wherein the anti-CD19 CAR is encoded by a nucleic acid comprising a coding sequence and a promoter of the anti-CD19 CAR.

15. The method of claim 14, wherein the nucleic acid is integrated into the genome of the engineered immune cell.

16. The method of claim 15, wherein the integration of the nucleic acid encoding the anti-CD19 CAR is performed using CRISPR nuclease and nucleic acid targeting nucleic acid (NATNA).

17. The method of claim 15, wherein prior to the integration, the nucleic acid encoding the anti-CD19 CAR is delivered to the immune cell via a viral vector.

18. The method of claim 1, wherein the amount of the composition administered to the patient comprises a dose of engineered immune cells targeting CD19 that is equivalent to 1 / 1000 of the dose used to treat B cell malignancies with the engineered immune cells targeting CD19.

19. The method of claim 1, wherein the amount of the composition administered to the patient comprises 10,000 to 100,000,000 of the engineered immune cells targeting CD19.

20. The method of claim 1, wherein the amount of the composition administered to the patient comprises 100 to 1,000,000 of the engineered immune cells targeting CD19 per kilogram of the patient's body weight.

21. The method of claim 1, wherein the amount of the composition administered to the patient comprises about 40,000 of the engineered immune cells targeted to CD19.

22. The method of claim 1, wherein the amount of the composition administered to the patient comprises about 600 of the engineered immune cells targeting CD19 per kilogram of the patient's body weight.

23. The method of claim 1, wherein the amount of the composition administered to the patient comprises no more than 600,000 of the engineered immune cells targeted to CD19.

24. The method of claim 1, wherein the amount of the composition administered to the patient comprises no more than 10,000 of the engineered immune cells targeting CD19 per kilogram of the patient's body weight.

25. The method of claim 1, wherein the administering is performed intravenously.

26. The method of claim 1, wherein the administering is performed 2-4 times per year.

27. The method of claim 1, wherein prior to said administering, said patient undergoes lymphocyte depletion.

28. The method of claim 27, wherein the lymphocyte depletion comprises administering a compound selected from the group consisting of cyclophosphamide, fludarabine, azathioprine, methotrexate, mycophenolate, calcineurin inhibitors, and volcosporin.

29. The method of claim 28, wherein the lymphocyte depletion comprises administering cyclophosphamide at 60 mg / kg daily for up to 2 days.

30. The method of claim 29, wherein the lymphocyte depletion further comprises administering 25 mg / m 2 Fludarabine was administered for up to 5 days.

31. The method of claim 1, further comprising assessing the patient for improvement of one or more symptoms selected from the group consisting of proteinuria, alopecia, elevated IgM and IgG antibody titers, presence of anti-nucleoprotein IgG or IgM in serum, increased B cell counts in plasma, and presence of skin lesions or discoloration.

32. The method of claim 31, further comprising increasing the dose of the engineered immune cells targeting CD-19 administered to the patient if no improvement is observed.

33. The method of claim 1, wherein the composition further comprises one or more pharmaceutically acceptable excipients.

34. The method of claim 33, wherein 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.

35. The method of claim 1, wherein the composition further comprises a cryogen.

36. A composition for treating an autoimmune disease, the composition comprising an amount of engineered immune cells targeting CD19 equivalent to 1 / 1000 of the dose used to treat B-cell malignancies using engineered immune cells targeting CD19.

37. The composition of claim 36, wherein 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).

38. The composition of claim 36, wherein the engineered immune cells targeting CD19 are CAR-T cells expressing anti-CD19 chimeric antigen receptor (CAR).

39. The composition of claim 36, wherein the engineered immune cells targeting CD19 are CAR-natural killer (NK) cells expressing anti-CD19 chimeric antigen receptor (CAR).

40. The composition of claim 36, wherein the engineered immune cells targeting CD19 are allogeneic.

41. The composition of claim 36, wherein the allogeneic immune cells comprise an armored genome modification.

42. The composition of claim 41, wherein the armored genome modification comprises inactivation of the PDCD1 gene.

43. The composition of claim 38, wherein the anti-CD19 CAR comprises an anti-CD19 scFv, a transmembrane domain, and an intracellular stimulatory domain.

44. The composition of claim 43, wherein the anti-CD19 CAR further comprises a signal peptide and a hinge.

45. The composition of claim 36, wherein the anti-CD19 CAR comprises FMC63, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 ζ signaling domain.

46. ​​The composition of claim 36, comprising 10,000 to 100,000 of the engineered immune cells targeting CD19.

47. The composition of claim 36, wherein the amount of the composition administered to the patient comprises 100 to 1,000 of the engineered immune cells targeting CD19 per kilogram of the patient's body weight.

48. The composition of claim 36, wherein the amount of the composition administered to the patient comprises about 40,000 of the engineered immune cells targeted to CD19.

49. The composition of claim 36, wherein the amount of the composition administered to the patient comprises about 600 engineered immune cells targeting CD19 per kilogram of the patient's body weight.

50. The composition of claim 36, wherein the amount of the composition administered to the patient comprises no more than 600,000 of the engineered immune cells targeted to CD19.

51. The composition of claim 36, wherein the amount of the composition administered to the patient comprises no more than 10,000 of the engineered immune cells targeting CD19 per kilogram of the patient's body weight.

52. The composition of claim 36, further comprising one or more pharmaceutically acceptable excipients.

53. The composition of claim 52, wherein 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.

54. The composition of claim 36, further comprising a cryogen.

55. A method of treating an autoimmune disease in a patient, the method comprising: The patient is administered an amount of a composition comprising engineered immune cells expressing an anti-CD19 CAR comprising FMC63, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain, wherein the immune cells have been evaluated for in vitro activity against B cells.

56. The method of claim 55, wherein the activity against B cells is assessed in the form of cytotoxicity in co-culture with a composition comprising B cells.

57. The method of claim 55, wherein the composition comprising B cells is selected from plasma, a PBMC fraction, and a B cell fraction.

58. The method of claim 55, wherein the co-cultured effector cell:target cell ratio is between 1:10 and 10:

1.

59. The method of claim 55, wherein the co-cultured effector cell:target cell ratio is between 1:8 and 8:

1.

60. The method of claim 55, wherein the activity against B cells is assessed as a reduction in antibody secretion by the B cells.

61. The method of claim 60, wherein the reduction in antibody secretion is assessed by measuring total IgG concentration in a culture comprising B cells.

62. The method of claim 61, wherein the culture comprising B cells is selected from plasma, a PBMC fraction, and a B cell fraction.

63. The method of claim 60, wherein the reduction in antibody secretion is assessed by measuring the concentration of IgG characteristic of an autoimmune disease in a culture comprising B cells.

64. The method of claim 63, wherein the culture comprising B cells is selected from plasma, a PBMC fraction, and a B cell fraction.

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