Use of dual-target CAR-T cells in the treatment of B-cell autoimmune diseases

Abstract

Bispecific or bitarget CAR or CAR-T cells can be used to treat and / or prevent autoimmune diseases. Bispecific CARs may target CD19 and BCMA. CARs may have the following loop structure: L-VL, CD19-VH, BCMA-VL, BCMA-VH, CD19-H-TM-C-CD3ζ. BCMA-CD19 CAR-T cells, transduced by chimeric antigen receptors targeting CD19 and BCMA, can completely block the autoantibody-producing pathway, completely covering multiple differentiation stages of B cells, plasmablasts, and plasma cells that produce autoantibodies. As a result, bitarget CAR-T therapies designed based on this concept can target refractory SLE and are expected to achieve faster remission and deeper, more sustained therapeutic effects.

Description

[Technical Field]

[0001] Related Application This application claims priority to Chinese Patent Application No. 202311005978.2 filed on August 10, 2023, and International Application No. PCT / CN2024 / 093376 filed on May 15, 2024. The content of each of these applications is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a computer-readable sequence listing submitted electronically in XML. The sequence listing is entitled "GC012F-500-WO-PCT2.XML", has a size of 24,857 bytes, and was created on August 5, 2024. The sequence listing is incorporated herein by reference in its entirety.

[0003] CAR-T cells can be used for the treatment or prevention of B-cell autoimmune diseases. In specific embodiments, the dual-target or bispecific CAR can be used for treating autoimmune diseases such as systemic lupus erythematosus, myositis, scleroderma or systemic sclerosis (SSc), myasthenia gravis, and nephritis. [Background Art]

[0004] To protect the body from foreign or dangerous substances, the immune system must first identify these foreign or dangerous substances. These substances include bacteria, viruses, parasites (e.g., helminths), some cancer cells, and even transplanted organs and tissues. These substances have molecules that can be recognized by the immune system and stimulate an immune response. These molecules are called antigens. Antigens can be contained inside cells, on the surface of cells (such as bacteria or cancer cells), or in viral components.

[0005] When some white blood cells (B cells and T cells) encounter an antigen, they learn how to attack it and thus protect the body from potentially dangerous antigens. B cells produce antibodies, which are one of the body's main immune defense mechanisms against antigens. Antibodies bind tightly to specific antigens and label them for attack or direct neutralization. The body produces thousands of different types of antibodies. Each antibody is specific to a particular antigen. Cells of the immune system remember specific antigens, and as a result, they can attack them more effectively the next time they encounter them.

[0006] Furthermore, cells in the body's own tissues also possess antigens. Under normal circumstances, the immune system reacts only to foreign or dangerous substances, and does not react to antigens in its own tissues. However, sometimes, immune dysfunction occurs, causing the body to treat its own tissues as foreign and produce antibodies (called autoantibodies) or immune cells that attack its own cells or tissues. This response is called an autoimmune response. It leads to inflammation and tissue damage. This response can cause autoimmune diseases.

[0007] Many autoimmune diseases have been discovered, and these can be systemic, such as systemic lupus erythematosus, which affects the skin, joints, kidneys, and central nervous system. They can also be organic, such as type 1 diabetes, which primarily affects the health of the pancreas. Loss of tolerance to B cells or T cells is often associated with autoimmunity. In fact, human leukocyte antigen (HLA) loci are usually directly associated with an increased risk of autoimmune diseases.

[0008] Currently, immunomodulatory drugs used for autoimmune diseases are broad-spectrum, non-disease-specific, and typically cause side effects such as infections and malignancies. Clearly, most patients do not respond ideally to these treatments, or may not respond at all. Therefore, it is urgent that researchers develop new or re-established drugs through molecular and clinical studies in patients with specific autoimmune diseases, combined with high-throughput integrated database analysis. Furthermore, while personalized drugs and new diagnostic methods complement each other and thus help researchers better analyze the pathogenesis of autoimmune diseases, researchers also need to conduct more patient-centered, relevant clinical trials. Prevention of autoimmune diseases should also be part of early intervention. In this paper, researchers interpret different types of current therapies, such as synthetic drugs, cell therapies, targeted metabolic pathways, and microbiomes, as well as other strategies, and emphasize the dynamic combination of basic research, new technologies, and clinical trials, thus helping to better understand the pathogenesis of autoimmune diseases and develop new therapies.

[0009] While researchers are increasingly recognizing the complexity of autoimmune diseases, current treatments are based solely on a simplified, reductionist understanding of their pathology. In clinical practice, patients are often treated with an escalating, trial-and-error approach, starting with different conventional disease-modifying drugs, such as methotrexate for rheumatoid arthritis, interferon B for multiple sclerosis, and corticosteroids for inflammatory bowel disease and psoriasis. If patients do not respond adequately, researchers upgrade to newer drugs, such as JAK inhibitors or biological agents. All of these drugs have broad-spectrum effects but are not disease-specific and often have specific side effects. Compared to personalized therapies that have entered the field of oncology research, these drugs may lack individualized characteristics. Therefore, to achieve more specific and personalized treatments for patients with autoimmune diseases, researchers need to understand the complexity of individual autoimmune diseases and how the disease manifests in different patients.

[0010] Systemic lupus erythematosus (SLE) is a multi-organ autoimmune inflammatory connective tissue disease with a complex etiology, primarily related to the immune system's misrecognition of endogenous antigens, the emergence of autoreactive B cells, and the production of autoantibodies. The age of onset for SLE is mostly between 20 and 40 years, with a clear gender bias, and a male-to-female ratio of approximately 1:9–12. According to literature statistics, the global prevalence of SLE is approximately 43.7 / 100,000 person-years, with significant regional differences: approximately 3.7–4.9 / 100,000 person-years in North America, approximately 1.5–7.4 / 100,000 person-years in Europe, approximately 15.9 / 100,000 person-years in Central Asia, and approximately 110.82 / 100,000 person-years in tropical Latin America. The prevalence rate in China is approximately 47.53 / 100,000 people / year, and the total number of patients is approximately 700,000.

[0011] Systemic lupus erythematosus (SLE) is an immune-mediated systemic disease that can cause a variety of abnormalities in the skin, kidneys, blood, and musculoskeletal system. The clinical manifestations of SLE vary greatly from person to person and are heterogeneous. Common symptoms of SLE may include joint pain and arthropathy, cheek rash and other rashes, pleurisy or pericarditis, renal or central nervous system involvement, and thrombocytopenia of the vascular system. The symptoms of SLE are variable, and acute relapses and remissions of the disease often occur alternately throughout the course of the illness. The severity of SLE varies greatly, which can pose a significant challenge due to accumulated organ damage and coagulation defects, and can be fatal depending on the severity of organ involvement.

[0012] Treatment status of SLE Currently, for SLE, the recommended basic treatment involves the long-term use of hydroxychloroquine (HCQ) in addition to nonsteroidal anti-inflammatory drugs, low-dose hormones, and immunosuppressants. Cyclophosphamide can be used as an initial treatment for severe SLE or as emergency treatment when other drugs fail. However, a small number of patients still have severe or refractory disease, and their response to the above-mentioned traditional drug therapies is poor, resulting in a poor prognosis. As a result, side effects associated with non-selective immunosuppressants, such as infection and metabolic abnormalities, are gradually increasing with prolonged drug administration. Based on traditional treatment, immunotargeted therapy is also increasingly used in clinical practice. However, traditional immunosuppressants, such as dexamethasone, cyclophosphamide, and anti-CD20 specific antibodies, can eliminate short-lived plasmablasts and plasma cells, but they cannot completely eliminate antibody-secreting cells (ASCs) that secrete autoantibodies, including long-lived plasma cells (LLPCs), which leads to relapsed SLE and therefore cannot achieve significant long-term clinical effects. In summary, improving the treatment of systemic lupus erythematosus is of particular importance.

[0013] Reasons for target selection for CAR-T cell therapy of SLE B cells play a central role in the pathogenesis of SLE through antibody-dependent and non-antibody-dependent functions.

[0014] A lack of self-tolerance during B cell development leads to the development of autoimmunity and the production of autoantibodies. Antinuclear antibodies (ANAs) are autoantibodies against DNA, RNA, proteins, or molecular complexes of these substances in the nucleus and are a major serological marker for SLE. ANAs have long been found in the diagnosis of lupus, and their role in SLE is attracting increasing attention. Firstly, the hematological symptoms of many autoimmune diseases are directly caused by autoantibodies such as Coombs-positive (anti-human globulin or red blood cell) hemolytic anemia and immune platelet deficiency. Secondly, a unique histopathological feature of lupus nephritis is the significant presence of glomerular immune complexes composed of many subtypes of immunoglobulins. In the 1960s, anti-dsDNA antibodies were eluted from the kidneys of lupus nephritis patients. Furthermore, studies have shown that, in addition to anti-dsDNA, various other autoantibodies can be eluted from the kidneys of lupus nephritis patients. Finally, direct evidence that lupus autoantibodies can cause systemic lupus erythematosus arises from neonatal lupus erythematosus (NLE), where passive transmission of maternal autoantibodies across the placenta causes NLE.

[0015] The types of anti-RNA antibodies (ANAs) in SLE patients primarily consist of anti-DNA antibodies and anti-RNA-binding protein (anti-RBP) antibodies. Anti-DNA antibodies are mainly expressed and secreted by naive B cells that have migrated to plasmablasts, and their serum titers change significantly with disease progression, while anti-RBP antibodies are mainly secreted by LLPCs and generally do not change significantly with disease progression. It has been reported that antibody titers in SLE-susceptible mice are mainly maintained by LLPCs. Among these, anti-DNA antibodies can form immune complexes with target antigens, leading to renal deposition, complement activation, and cytokine production. Free anti-DNA antibodies can also cross-bind to receptors in neurons to cause neurological symptoms or directly bind to antigens in the kidneys to produce pathological reactions, while anti-RBP antibodies can also form immune complexes, potentially leading to cytokine production, renal deposition, and complement activation.

[0016] In the early 1990s, it was discovered that B cells not only directly produce pathological autoantibodies in SLE, but also activate lupus-associated T cells via cytokines presented and secreted in response to antigens. First, in MRL / lpr lupus model mice, mice deprived of B cells showed reduced T cell activation and decreased interstitial nephritis, which completely prevented the progression of SLE disease. It is particularly noteworthy that when B cells incapable of secreting immunoglobulins were re-transplanted into the B cell-deprived model mice, inflammatory T cell lesions reappeared in the animals, and the mice became susceptible again, meaning this phenomenon cannot be fully explained by the absence of autoantibodies alone. These studies demonstrate the existence of antibody-independent B cell functions, including antigen presentation and cytokines. Second, in MRL / lpr mice lacking CD11c+ dendritic cells, T cells can still be activated. Nevertheless, when the antigen-presenting ability of B cells is lost at the genetic level, the activation of CD4+ T cells, the differentiation of effector memory T cells and follicular helper T cells (Tfh), and the progression of autoimmunity are inhibited.

[0017] Considering the crucial roles of B cells and plasma cells in the pathogenesis of SLE, current research on targeted therapies for SLE primarily focuses on targets related to B cells and plasma cells, such as the B lymphocyte-stimulating factor BAFF / BLyS, proliferation-inducing ligands APRIL, CD20, CD22, and CD19. Other targets include FcγRIIb, Bruton's tyrosine kinase, proteasome, T cell-related targets, macrophage-related targets, intracellular signaling molecules, costimulators, IgE, and gut microbiota. See Table 1 for details.

[0018] [Table 1]

[0019] Existing B-cell targeted therapies achieve their pharmacological effects by directly targeting B-cell surface markers such as BLyS, APRIL, CD19, CD20, and CD22, and by regulating the activation, proliferation, differentiation, and apoptosis of B cells and / or plasma cells. Belimumab, which targets BLyS, was the first biologic drug approved for the treatment of SLE. Major drugs targeting CD20 include rituximab and ocrelizumab, and both the 2019 EULAR guidelines and the 2020 China SLE diagnostic and treatment guidelines suggest that rituximab can be selected for patients with refractory SLE-related severe thrombocytopenia. However, commercially available targeted therapy products have not achieved significant therapeutic effects or long-term drug remission in SLE. Statistically, 26 relapses / 100 patients were still observed within 12 months or 44 relapses / 100 patients within 24 months after treatment with belimumab. Two randomized trials showed that rituximab was not superior to conventional treatments in non-renal SLE and lupus nephritis. Furthermore, due to long-term medication, patients were in a state of partial immunodeficiency, which increased their risk of infection-related adverse events.

[0020] CD19 is a membrane-surface glycoprotein of the immunoglobulin superfamily with a molecular weight of 95 kDa, and is widely expressed at all stages of B cell development before plasma cell differentiation. Currently, CD19-targeted CAR-T cell therapy is clearly superior to other therapies in the treatment of SLE, mainly due to its favorable therapeutic effect on severe, refractory SLE and its long-term remission in the absence of drugs. CD19-targeted CAR-T therapy has been applied to female patients with severe SLE, and it has been reported that the patients' conditions have been significantly alleviated without apparent side effects. Another study reported five patients with refractory systemic lupus erythematosus whose conditions significantly improved after CD19 CAR-T treatment, and during a 17-month follow-up, there were no relapses, and all patients who achieved remission in the absence of drugs experienced a significant improvement in their quality of life.

[0021] Currently, all CAR-T therapies for SLE on the market are in the development stage. All products approved by IND target CD19, such as Kyverna Therapeutics' KYV-101 and Cabaletta Bio's CABA-201, which have received implicit clinical approval from the FDA for the treatment of lupus nephritis (LN) and active lupus nephritis (active LN) or active SLE without renal involvement. Meanwhile, JW Therapeutics' research drug application for relmacabtagene autoleucel injection for the treatment of moderate to severe refractory systemic lupus erythematosus has received implicit approval from the China Food and Drug Administration. See Table 2 for further details.

[0022] [Table 2]

[0023] Published clinical results indicate that CD19 expression on the surface of plasma cells is almost negative or extremely low. Therefore, while CAR-T therapy against CD19 can reduce autoantibody levels, it does not significantly reduce total IgG antibody levels. In other words, targeting only CD19-positive cells is insufficient to deeply eliminate ASCs, including LLPCs, which contribute to some degree of refractory SLE. Treatment with CAR-T against CD19 in a lupus mouse model also shows that while CAR-T therapy against CD19 can improve lupus symptoms, autoantibodies can only be eliminated by administration in the early stages of the disease. In the later stages of disease progression, B cell elimination alone cannot prevent the continuous production of autoantibodies resulting from LLPC accumulation. To address these issues, we have provided a specific chimeric antigen receptor for biantigens, improved through research, for the effective treatment and / or prevention of systemic lupus erythematosus. Furthermore, the biantigen-specific chimeric antigen receptors disclosed herein can all exhibit favorable therapeutic effects against other B-cell autoimmune diseases involving CD19 and / or BCMA. [Overview of the project]

[0024] In a first embodiment, a bispecific chimeric antigen receptor (CAR) for treating and / or preventing an autoimmune disease is disclosed. The first target of the CAR is CD19, and the second target is BCMA.

[0025] B cell maturation antigen (BCMA) is a type III transmembrane protein containing a cysteine-rich extracellular domain and lacking a signal peptide. It plays a crucial regulatory role in the processes of B cell proliferation, survival, maturation, and differentiation into plasma cells. BCMA is an important surface receptor for plasma cells; its knockout results in a significant reduction in LLPC-dominant plasma cells in mice, and BCMA can promote the long-term survival of LLPCs in the bone marrow. Data show that the expression ratio and abundance of membrane-bound BCMA are significantly higher in plasma cells of SLE patients than in healthy individuals. However, targeting BCMA alone is ineffective in eliminating the large number of pathogenic B cells (CD19-positive to strongly positive, BCMA very weakly positive or negative) and plasmablasts (CD19-positive to weakly positive, BCMA weakly positive to positive) that are activated at specific stages of SLE onset. During the treatment of patients with diffuse large B-cell lymphoma (DLBCL) accompanied by SLE using CAR-T therapy targeting CD19 and BCMA, it was found that simultaneously targeting both antigens could not only reduce ANA to undetectable levels but also significantly reduce total IgG antibody levels. This suggests that this dual-target combination therapy can target ASCs, which produce numerous autoantibodies, and further prevent the production of IgG autoantibodies and relapse.

[0026] According to this disclosure, it has been found that by targeting the combination of CD19 and BCMA, the autoantibody production pathway can be completely blocked, and multiple differentiation stages of B cells, plasmablasts, and plasma cells that produce autoantibodies can be completely covered. Based on this concept, a dual-target CAR-T therapy can be used for B cell-mediated autoimmune diseases, particularly refractory SLE, and is expected to provide faster remission and deeper, more sustained therapeutic effects.

[0027] In some embodiments, bispecific CARs can treat or prevent B cell-mediated autoimmune diseases by killing or inhibiting CD19-positive B cells, plasmablasts, and BCMA-positive plasma cells. The above drugs and preparations have the ability to simultaneously kill CD19-positive B cells, plasmablasts, and BCMA-positive plasma cells. The combination of targeted CD19 and BCMA may not only have a deeper therapeutic effect against B cell-mediated autoimmune diseases but may also reduce the likelihood of relapse of such diseases. The mechanism and principle are shown in Figure 2.

[0028] In one embodiment, a bispecific CAR may be combined with one or more agents. The agents may enhance the efficacy of cells having CAR nucleic acids or polypeptides. The agents may also be for the purpose of mitigating one or more side effects associated with the administration of cells containing CAR nucleic acids or CAR polypeptides. The agents may also be additional agents for the treatment of diseases associated with BCMA and CD19. The agents may be a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

[0029] Drugs can treat autoimmune diseases by killing or inhibiting CD19-positive B cells, plasmablasts, and BCMA-positive plasma cells.

[0030] Autoimmune diseases are autoimmune diseases mediated by B cells. B cell-mediated autoimmune diseases include systemic lupus erythematosus (SLE), glomerulonephritis, e.g., autoimmune chronic kidney disease, lupus nephritis, acute glomerulonephritis, immunonephritis, membranous nephropathy, IgA nephropathy, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV); scleroderma or systemic sclerosis (SSc); myositis or idiopathic inflammatory myositis, including dermatomyositis, polymyositis, immune-mediated necrotizing myopathy (IMNM), anti-synthetic enzyme syndrome, inclusion body myositis, and myositis duplication; multiple sclerosis (MS); inflammatory bowel disease (IBD); rheumatoid arthritis (RA); Sjögren's syndrome (SS); autoimmune hemolytic anemia; neuromyelitis optica (NMO); neuromyelitis optica spectroscopy This includes Lamb's syndrome (NMOSD); idiopathic thrombocytopenic purpura (ITP); systemic autoimmune small vessel vasculitis or polyangiitis associated with antineutrophil cytoplasmic antibodies; Wegener's granulomatosis (GPA), eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss syndrome); pemphigus vulgaris; autoimmune encephalitis; myasthenia gravis; antiphospholipid syndrome; Chagas disease; Graves' disease; polyarteritis nodosa; pulmonary hemorrhagic nephritis syndrome; Kawasaki disease; amyloidosis; monoclonal globulin of unknown significance, POEMS syndrome; Crohn's disease; ulcerative colitis; adult-onset Still's disease; and chronic progressive demyelinating encephalopathy (CIDP).

[0031] In one embodiment, the B cell-mediated autoimmune disease is systemic lupus erythematosus, and in some of the embodiments described above, systemic lupus erythematosus includes moderate to severe refractory systemic lupus erythematosus, lupus nephritis, active lupus nephritis, and active systemic lupus erythematosus without renal lesions.

[0032] In one embodiment, the autoimmune disease mediated by B cells is myositis.

[0033] In one embodiment, the B cell-mediated autoimmune disease is glomerulonephritis, and in some embodiments, glomerulonephritis is at least one of autoimmune chronic kidney disease, lupus nephritis, acute glomerulonephritis, immunonephritis, membranous nephropathy, and IgA nephropathy.

[0034] In one embodiment, the autoimmune disease mediated by B cells is multiple sclerosis.

[0035] In one embodiment, the B cell-mediated autoimmune disease is scleroderma or systemic sclerosis (SSc).

[0036] In one embodiment, the autoimmune disease mediated by B cells is myasthenia gravis.

[0037] A second aspect disclosed herein is the structure of a chimeric antigen receptor. The first antibody or its antigen-binding fragment targeting BCMA, or the second antibody or its antigen-binding fragment targeting CD19, is independently selected from single-chain antibodies such as camel Ig, IgNAR, Fab fragment, Fab' fragment, F(ab')z fragment, F(ab')3 fragment, Fv, scFv, di-scFv, and (scFv)z, microantibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, disulfide-bond-stabilized Fv protein ("dsFv"), and single-domain antibodies (sdAb, nanoantibodies), chimeric antibodies, humanized antibodies, single-domain antibodies, bispecific or multispecific antibodies, binding ligands, or protein domains. The antigen-binding fragment targeting BCMA or CD19 is scFv.

[0038] In one embodiment, the bispecific CAR contains a heavy chain variable region (VH, BCMA) having a complementarity-determining region 1 (HCDR1) including the amino acid sequence of SEQ ID NO: 5, the HCDR2 amino acid sequence of SEQ ID NO: 6, and the HCDR3 amino acid sequence of SEQ ID NO: 7, and a light chain variable region (VL, BCMA) having a complementarity-determining region 1 (LCDR1) including the amino acid sequence of SEQ ID NO: 8, the LCDR2 amino acid sequence of SEQ ID NO: 9, and the LCDR3 amino acid sequence of SEQ ID NO: 10, and an antibody or antigen-binding fragment thereof that targets BCMA.

[0039] In one embodiment, the bispecific CAR contains a CD19-targeting antibody or its antigen-binding fragment, comprising a heavy chain variable region (VH, CD19) having a complementarity-determining region 1 (HCDR1) including the amino acid sequence of SEQ ID NO: 11, the HCDR2 amino acid sequence of SEQ ID NO: 12, and the HCDR3 amino acid sequence of SEQ ID NO: 13, and a light chain variable region (VL, CD19) having a complementarity-determining region 1 (LCDR1) including the amino acid sequence of SEQ ID NO: 14, the LCDR2 amino acid sequence of SEQ ID NO: 15, and the LCDR3 amino acid sequence of SEQ ID NO: 16.

[0040] In one embodiment, the bispecific CAR includes an antibody or antigen-binding fragment thereof that targets BCMA having the above-mentioned CDR (SEQ ID NOs. 5-10), and an antibody or antigen-binding fragment thereof that targets CD19 having the above-mentioned CDR (SEQ ID NOs. 11-16).

[0041] In one embodiment, the bispecific CAR containing VH and BCMA includes an amino acid sequence having at least 95% identity with SEQ ID NO: 1. In another embodiment, VL and BCMA include an amino acid sequence having at least 95% identity with SEQ ID NO: 2.

[0042] In one embodiment, the bispecific CAR containing VH and CD19 includes an amino acid sequence having at least 95% identity with SEQ ID NO: 3. In another embodiment, VL and CD19 include an amino acid sequence having at least 95% identity with SEQ ID NO: 4.

[0043] In some embodiments, the BCMA antigen-binding fragment comprises both SEQ ID NOs: 1 and 2, or sequences having at least 95 percent amino acid identity with them, and / or the CD19 antigen-binding fragment comprises both SEQ ID NOs: 3 and 4, or sequences having at least 95 percent amino acid identity with them.

[0044] In one embodiment, the bispecific CAR may have a structure selected from any one of the following formulas: 1a) L-VL, CD19-VH, BCMA-VL, BCMA-VH, CD19-H-TM-C-CD3ζ; 1b)L-VL, CD19-VL, BCMA-VH, BCMA-VH, CD19-H-TM-C-CD3ζ; 2a) L-VH, CD19-VL, BCMA-VH, BCMA-VL, CD19-H-TM-C-CD3ζ; 2b) L-VH, CD19-VH, BCMA-VL, BCMA-VL, CD19-H-TM-C-CD3ζ; 3a) L-VL, CD19-VH, CD19-H-TM-C-CD3ζ-2A peptide-L-VH, BCMA-VL, BCMA-H-TM'-C-CD3ζ; 3b) L-VH, CD19-VL, CD19-H-TM-C-CD3ζ-2A peptide-L-VH, BCMA-VL, BCMA-H-TM'-C-CD3ζ; 3c) L-VL, CD19-VH, CD19-H-TM-C-CD3ζ-2A peptide-L-VL, BCMA-VH, BCMA-H-TM'-C-CD3ζ; 3d) L-VH, CD19-VL, CD19-H-TM-C-CD3ζ-2A peptide-L-VL, BCMA-VH, BCMA-H-TM'-C-CD3ζ; 4a) L-VH, BCMA-VL, BCMA-H-TM-C-CD3ζ-2A peptide-L-VL, CD19-VH, CD19-H-TM'-C-CD3ζ; 4b) L-VL, BCMA-VH, BCMA-H-TM-C-CD3ζ-2A peptide-L-VL, CD19-VH, CD19-H-TM'-C-CD3ζ; 4c) L-VH, BCMA-VL, BCMA-H-TM-C-CD3ζ-2A peptide-L-VH, CD19-VL, CD19-H-TM'-C-CD3ζ; or 4d) L-VL, BCMA-VH, BCMA-H-TM-C-CD3ζ-2A peptide-L-VH, CD19-VL, CD19-H-TM'-C-CD3ζ.

[0045] In one embodiment, the bispecific CAR has one of the formulas 1a), 2a), 3a), or 4a).

[0046] In the above formulas, 1a), 1b), 2a), and 2b) include loop structures formed by antibodies targeting BCMA and CD19 or their antigen-binding fragments. For example, in formula 1a), VL, CD19-VH, BCMA-VL, BCMA-VH, and CD19 constitute a loop structure, and in formula 2a), VH, CD19-VL, BCMA-VH, BCMA-VL, and CD19 constitute a loop structure.

[0047] Each "-" independently represents a linker peptide or peptide bond.

[0048] L may be absent or present as a signal peptide sequence. L may contain domains derived from the following protein-derived signal peptides: CD8, CD28, GM-CSF, CD4, CD137, or combinations thereof. L may also be a CD8-derived signal peptide. The signal peptide sequence may be the amino acid sequence described in SEQ ID NO: MALPVTALLLPLALLLHAARP.

[0049] VH,BCMA is the variable region of the heavy chain of the anti-BCMA antibody. The sequence of VH,BCMA may include the CDRs described in SEQ ID NOs. 5-7. The sequence of the VH,BCMA antibody heavy chain variable region may be as described in SEQ ID NO. 1.

[0050] VL,BCMA is the variable region of the light chain of the anti-BCMA antibody. The sequence of VL,BCMA may include the CDR described in SEQ ID NOs.8-10. The sequence of the VL,BCMA antibody light chain variable region may be as described in SEQ ID NO.2.

[0051] VH,CD19 is the variable region of the heavy chain of the anti-CD19 antibody. The sequence of VH,CD19 may include the CDRs described in SEQ ID NOs. 11-13. The sequence of the VH,CD19 antibody heavy chain variable region may be as described in SEQ ID NO. 3.

[0052] VL,CD19 is the variable region of the light chain of the anti-CD19 antibody. The sequence of VL,CD19 may include the CDRs described in SEQ ID NOs. 14-16. The sequence of the VL,CD19 antibody light chain variable region is described in SEQ ID NO. 4.

[0053] H is the hinge region, TM is the first transmembrane domain, TM' is the second transmembrane domain, The 2A peptide is selected from P2A, T2A, E2A, or F2A; C is a co-stimulus signaling molecule, CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0054] VH and BCMA are the variable regions of the heavy chain of the anti-BCMA antibody, while VL and BCMA are the variable regions of the light chain of the anti-BCMA antibody. The sequences of the VH and BCMA antibody heavy chain variable regions are as shown in Sequence ID No. 1. QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYYIHWVRQAPGQGLEWIGYINPNSGYTNYAQKFQGRATMTADKSINTAYVELSRLRSDDTAVYFCTRYMWERVTGFFDFWGQGTMVTVSS(Sequence ID 1)

[0055] In the sequence of sequence number 1 above, according to the Kabat numbering system, there are three complementarity determination regions (CRDs) in the heavy chain variable region, which are as follows: HCDR1:DYYIH (Sequence ID 5) HCDR2:YINPNSGYTNYAQKFQG(Sequence ID 6) HCDR3:YMWERVTGFFDF (Sequence ID 7)

[0056] As another example, the VH,BCMA antibody heavy chain variable region includes the amino acid sequence described in SEQ ID NO: 1, or further includes a sequence having one or more amino acid substitutions, deletions, or additions, for example, one, two, or three amino acid substitutions, deletions, or additions.

[0057] In some embodiments, a variant of the VH,BCMA antibody heavy chain variable region has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) with respect to the sequence from which it is derived. In one embodiment, the substitutions are conserved.

[0058] The sequence of the VL, BCMA antibody light chain variable region is as described in Sequence ID No. 2. DIQMTQSPSSVSASVGDRVTITCLASEDISDDLAWYQQKPGKAPKVLVYTTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQTYKFPPTFGGGTKVEIKR (Sequence ID 2).

[0059] In the sequence of sequence number 2 above, according to the Kabat numbering system, there are three complementarity determination regions (CRDs) in the light chain variable region, which are as follows: LCDR1:LASEDISDDLA (Sequence ID 8) LCDR2:TTSSLQS (Sequence ID 9) LCDR3:QQTYKFPPT (Sequence ID 10)

[0060] As another example, the VL,BCMA antibody light chain variable region includes the amino acid sequence described in SEQ ID NO: 2, or further includes a sequence having one or more amino acid substitutions, deletions, or additions, for example, one, two, or three amino acid substitutions, deletions, or additions.

[0061] In some embodiments, the VL, BCMA antibody light chain variable region variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) with respect to the sequence from which it is derived. In one embodiment, the substitutions are conserved.

[0062] VH,CD19 is the variable region of the heavy chain of the anti-CD19 antibody, and VL,CD19 is the variable region of the light chain of the anti-CD19 antibody. The sequence of the VH,CD19 antibody heavy chain variable region is as described in Sequence ID No. 3. EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS; The sequence of the VL, CD19 antibody light chain variable region, is as described in Sequence ID No. 4. DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT.

[0063] In sequence number 3 above, according to the Kabat numbering system, there are three complementarity determination regions (CRDs) in the heavy chain variable region, which are as follows: HCDR1:DYGVS (Sequence ID 11) HCDR2:VIWGSETTYYNSALKS (Sequence ID 12) HCDR3:HYYYGGSYAMDY (Sequence ID 13)

[0064] In the sequence of sequence number 4 above, according to the Kabat numbering system, there are three complementarity determination regions (CRDs) in the light chain variable region, which are as follows: LCDR1:RASQDISKYLN(Sequence ID 14) LCDR2:HTSRLHS(Sequence ID 15) LCDR3:QQGNTLPYT(Sequence ID 16)

[0065] As another example, VH,CD19 includes the amino acid sequence described in Sequence ID No. 3, or further includes a sequence having one or more amino acid substitutions, deletions, or additions, for example, one, two, or three amino acid substitutions, deletions, or additions compared thereto.

[0066] In some embodiments, the VH,CD19 variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) with respect to the sequence from which it is derived. In one embodiment, the substitutions are conserved.

[0067] As another example, VL,CD19 includes the amino acid sequence described in SEQ ID NO: 4, or further includes a sequence having one or more amino acid substitutions, deletions, or additions compared thereto, for example, one, two, or three amino acid substitutions, deletions, or additions.

[0068] In some embodiments, a variant of scFv2 has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99%, or 100% sequence identity with respect to the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) with respect to the sequence from which it is derived. In one embodiment, the substitutions are conserved.

[0069] H is a hinge region, and H may contain domains derived from the following proteins: CD8, CD28, CD137, or combinations thereof. H can be selected from hinge regions derived from CD8. H may contain or consist of the amino acid sequences described in SEQ ID NOs: 17-19: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(Sequence ID 17); Or KPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKP(sequence ID 18); Or SGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (Sequence ID 19).

[0070] TM or TM' is a transmembrane domain (first or second, respectively), and in one example, TM or TM' may be a transmembrane region of a protein domain of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0071] TM or TM' may be a transmembrane region derived from CD8, and the transmembrane region derived from CD8 has the amino acid sequence described in SEQ ID NO: 20.

[0072] IYIWAPLAGTCGVLLLSLVITLYC (Sequence ID 20).

[0073] C may be a co-stimulatory signaling molecule. C may be a co-stimulatory signaling molecule of protein domains such as OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.

[0074] C may be a co-stimulatory signaling molecule derived from 4-1BB. C can be derived from 4-1BB having the amino acid sequence described in SEQ ID NO: 21. KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0075] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ. In one embodiment of use disclosed herein, the CD3ζ signaling region has the amino acid sequence described in SEQ ID NO: 22. RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0076] In another example, the CAR further includes a cell suicide element (e.g., at the C-terminus or N-terminus), where the cell suicide element may be linked to the CAR or L or CD3ζ of the bispecific CAR via T2A.

[0077] In another example, no linker is present, or the linker has the amino acid sequence set forth in SEQ ID NO: 23: GGGGS. In some embodiments, the linker sequence may, in each case, independently be absent, or correspond to SEQ ID NO: 23 or a repeat of SEQ ID NO: 23. In one embodiment, each instance of a linker is independently absent, or repeated 1 to 10 times, 1 to 5 times, 1 to 3 times, or once.

[0078] In another embodiment, in each case, the linker sequence between each of the variable chains of BCMA and / or CD19 may independently be absent, may be SEQ ID NO: 23 or several repeats thereof, and / or may be the amino acid sequence set forth in SEQ ID NO: 24: GSTSGSGKPGSGEGSTKG.

[0079] In one embodiment, the bispecific CAR may comprise the amino acid sequences of SEQ ID NOs: 5-16. In one embodiment, the bispecific CAR may comprise the amino acid sequences of SEQ ID NOs: 5-16, one of SEQ ID NOs: 17-19, and SEQ ID NOs: 20-22. In another embodiment, the bispecific CAR further comprises at least one instance of each of SEQ ID NOs: 23-25. In a specific embodiment, the bispecific CAR comprises SEQ ID NOs: 5-16, 19-22, and 25. In a specific embodiment, the bispecific CAR comprises SEQ ID NOs: 5-16, 19-22, and 25, and at least one instance of SEQ ID NO: 23 and SEQ ID NO: 24. In each of these embodiments, the order of the heavy chain and light chain variable regions of BCMA and CD19 can be adjusted. Exemplary structural orders of BCMA and CD19 heavy chains include V H、BCMA -V L、BCMA -V H、CD19 -V L、CD19 ;V L、BCMA -V H、BCMA -V H、CD19 -V L、CD19 ;V H、BCMA -V L、BCMA -V L、CD19 -V H、CD19 ;V L、BCMA -V H、BCMA -V L、CD19 -V H、CD19 ;V H、CD19 -VL、CD19 -V H、BCMA -V L、BCMA ;V L、CD19 -V H、CD19 -V H、BCMA -V L、BCMA ;V H、CD19 -V L、CD19 -V L、BCMA -V H、BCMA ;V L、CD19 -V H、CD19 -V L、BCMA -V H、BCMA ;V H、CD19 -V L、BCMA -V H、BCMA -V L、CD19 ;V H、CD19 -V H、BCMA -V L、BCMA -V L、CD19 ;V L、CD19 -V L、BCMA -V H、BCMA -V H、CD19 ;V L、CD19 -V H、BCMA -V L、BCMA -V H、CD19 ;V H、BCMA -V L、CD19 -V H、CD19 -V L、BCMA ;V H、BCMA -V H、CD19 -V L、CD19 -V L、BCMA ;V L、BCMA -V L、BCMA -V H、CD19 -V H、CD19 ;or V L、BCMA -V H、BCMA -V L、CD19 -V H、CD19 Examples include, but are not limited to.

[0080] In one embodiment, the bispecific CAR may include the amino acid sequences of SEQ ID NOs: 1-4. In one embodiment, the bispecific CAR may include the amino acid sequences of SEQ ID NOs: 1-4, one of SEQ ID NOs: 17-19, and SEQ ID NOs: 20-22. In one embodiment, the bispecific CAR further includes at least one example of each of SEQ ID NOs: 23-25. In a specific embodiment, the bispecific CAR includes SEQ ID NOs: 1-4, 19-22, and 25. In a very specific embodiment, the bispecific CAR includes at least one example of SEQ ID NOs: 1-4, 19-22, and 25, as well as SEQ ID NOs: 23 and 24.

[0081] In one embodiment, the bispecific CAR includes H having the amino acid sequence of SEQ ID NO: 19, L having the amino acid sequence of SEQ ID NO: 25, VH, BCMA having the amino acid sequence of SEQ ID NO: 1, VL, BCMA having the amino acid sequence of SEQ ID NO: 2, VH, CD19 having the amino acid sequence of SEQ ID NO: 3, VL, CD19 having the amino acid sequence of SEQ ID NO: 4, TM having the amino acid sequence of SEQ ID NO: 20, C having the amino acid sequence of SEQ ID NO: 21, and CD3ζ having the amino acid sequence of SEQ ID NO: 22. The structure of the bispecific CAR may correspond to formula 1a).

[0082] In one embodiment, the bispecific CAR has the amino acid sequence shown in SEQ ID NO: 26. .

[0083] In one embodiment, the bispecific CAR is administered to the subject in one of the following amounts: 0.5±20%×10^5 cells / kg, 1.0±20%×10^5 cells / kg, 2.0±20%×10^5 cells / kg, or 3.0±20%×10^5 cells / kg. In one embodiment, the bispecific CAR is administered to the subject in the following amounts: 0.5×10^5 cells / kg, 1.0×10^5 cells / kg, 2.0×10^5 cells / kg, or 3.0×10^5 cells / kg.

[0084] In one embodiment, a pharmaceutical composition for treating and / or preventing an autoimmune disease is provided, comprising a bispecific CAR as described herein.

[0085] In one embodiment, a method for treating and / or preventing an autoimmune disease is disclosed, comprising administering a bispecific CAR treatment described herein to a subject in need thereof.

[0086] In one embodiment, the use of the bispecific CARs described herein for the treatment and / or prevention of autoimmune diseases is disclosed herein. The use may include the preparation of pharmaceuticals, drugs, or preparations for the treatment and / or prevention of autoimmune diseases.

[0087] A third aspect disclosed herein is BCMA-CD19 CAR-T cells in the treatment and / or prevention of B cell-mediated autoimmune diseases, such as SLE, where BCMA-CD19 CAR-T cells are autologous T cells modified to express the CAR on their cell membrane. Patient T cells were collected and transduced with the CAR gene using a lentivirus (4 plasmid system package), resulting in T cells that could specifically recognize BCMA and CD19 antigens expressed on the surface of target cells.

[0088] In one embodiment, the use of BCMA-CD19 CAR-T cells in the preparation of a drug for treating and / or preventing a B cell-mediated autoimmune disease is provided, wherein the BCMA-CD19 CAR-T cells may be engineered T cells expressing the chimeric antigen receptor on their cell membrane, for example, engineered autologous T cells, or engineered T cells encoding the nucleotide sequence of the chimeric antigen receptor, for example, engineered autologous T cells.

[0089] A fourth aspect discloses nucleic acid molecules encoding bispecific CARs disclosed herein for the treatment of autoimmune diseases. In one embodiment, a recombinant vector for treating and / or preventing autoimmune diseases is disclosed. The vector may comprise bispecific CARs or nucleic acid molecules disclosed herein.

[0090] In one embodiment, the use of a nucleic acid molecule in the preparation of a drug for treating a B cell-mediated autoimmune disease such as systemic lupus erythematosus is disclosed, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the chimeric antigen receptor described above.

[0091] A fifth aspect disclosed herein is the use of a vector in the treatment of a B cell-mediated autoimmune disease, wherein the vector is selected from DNA vectors, RNA vectors, plasmids, liposomes, microparticles, transposon vectors, CRISPR / Cas9 vectors, or viral vectors. In one embodiment, the vector is a lentiviral vector.

[0092] A sixth aspect disclosed herein is the use of BCMA-CD19 CAR-T cells in the preparation of a drug for treating and / or prophylactic a B cell-mediated autoimmune disease such as systemic lupus erythematosus, wherein the BCMA-CD19 CAR-T cells are engineered autologous T cells expressing the chimeric antigen receptor on their cell membrane.

[0093] A seventh aspect disclosed herein is a drug or preparation comprising a chimeric antigen receptor, a nucleic acid molecule, a vector, or BCMA-CD19 CAR-T cells, and optionally, a pharmaceutically acceptable carrier, adjuvant, and / or excipient. The drug or preparation may be used to treat and / or prevent B-cell-mediated autoimmune diseases such as systemic lupus erythematosus.

[0094] Compared to prior art, the subject matter disclosed herein has at least the following obvious advantages and effects.

[0095] BCMA-CD19 CAR-T cells have the ability to simultaneously eliminate B cells (including B progenitor cells) and plasma cells, removing the main source of autoantibodies and achieving a local immune reset. As a result, patients with B cell-mediated autoimmune diseases such as refractory SLE can achieve long-term, sustained remission. [Brief explanation of the drawing]

[0096] [Figure 1A] This is a schematic diagram of an example of the structure of an anti-CD19 / BCMA CAR. [Figure 1B] This is a schematic diagram of an example of the construction of an anti-CD19 / BCMA CAR. [Figure 2] This paper describes the design and mechanism of a BCMA-CD19 CAR for the treatment of SLE or other autoimmune diseases. [Figure 3] This shows CAR expression in BCMA CAR-T cells. [Figure 4] This shows CAR expression in CD19 CAR-T cells. [Figure 5] This shows CAR expression in BCMA-CD19 CAR-T cells of this application. [Figure 6] This shows CAR expression in a blank control that has not been modified for a specific target. [Figure 7A] This shows in vitro differentiation of antibody-secreting cells against different antigens. [Figure 7B] Flow cytometry images showing the expression levels of different cell surface antigens are shown. [Figure 8] This image shows flow cytometry results indicating the number of NT cells after in vitro death. [Figure 9] Flow cytometry showing the number of CD19 CAR-T cells after in vitro death is displayed. [Figure 10] Flow cytometry data showing the number of BCMA CAR-T cells after in vitro death is displayed. [Figure 11] Flow cytometry is shown to indicate the number of BCMA-CD19 dual CAR-T cells after in vitro death. [Figure 12]This paper presents a statistical analysis of flow cytometry results regarding the simultaneous removal of CD19+ and CD19- antibody-secreting cells by BCMA-CD19 dual-target CAR-T receptors. [Figure 13] This study demonstrates the effect of BCMA-CD19 CAR-T on the death of antibody-secreting cells compared to CD19 CAR-T, BCMA CAR-T, and a blank control group. [Figure 14] This histogram shows the number of antibody-secreting cells after treatment with different T cells at different effector-target ratios. [Figure 15] The effects of BCMA-CD19 CAR-T cells on primary B cells at different effector-target ratios are shown compared to the control group (5 hours). [Figure 16] Flow cytometry images of BCMA-CD19 CAR-T cells that kill primary B cells at different effector-target ratios are shown (5 hours). [Figure 17] This shows the levels of IFN-γ cytokine secretion after BCMA-CD19 CAR-T cells killed primary B cells with different effector-target ratios (5 hours). [Figure 18] This shows the levels of IL-2 cytokine secretion after BCMA-CD19 CAR-T cells killed primary B cells with different effector-target ratios (5 hours). [Figure 19] This shows the TNF-α cytokine secretion levels after BCMA-CD19 CAR-T cells killed primary B cells with different effector-target ratios (5 hours). [Figure 20] This shows the levels of IL-10 cytokine secretion after BCMA-CD19 CAR-T cells killed primary B cells with different effector-target ratios (5 hours). [Figure 21] This shows the levels of IL-4 cytokine secretion after BCMA-CD19 CAR-T cells killed primary B cells with different effector-target ratios (5 hours). [Figure 22] This shows the levels of IL-2 cytokine secretion after BCMA-CD19 CAR-T cells killed primary B cells with different effector-target ratios (5 hours). [Figure 23] Flow cytometry images of BCMA-CD19 CAR-T cells that kill antibody-secreting cells at different effector-target ratios are shown (5 hours). [Figure 24] This histogram shows BCMA-CD19 CAR-T cells that kill antibody-secreting cells (including those containing single-target CD19 or BCMA, and those containing dual targets) at different effector-target ratios (5 hours). [Figure 25] This histogram shows cytokine secretion levels after BCMA-CD19 CAR-T cells killed antibody-secreting cells with different effector-target ratios (5 hours). [Figure 26] Gating and flow cytometry images of B cells and ASC cells (including plasma cells CD138+) in peripheral blood samples from SLE patients are shown. [Figure 27A] This image shows flow cytometry data of BCMA-CD19 CAR-T cells that kill B cells in SLE patients in vitro. [Figure 27B] This is a histogram showing the number of B cells after in vitro death by BCMA-CD19 CAR-T cells. [Figure 28A] This image shows flow cytometry data of BCMA-CD19 CAR-T cells that kill ASC cells in SLE patients in vitro. [Figure 28B] This is a histogram showing the number of ASC cells after in vitro death by BCMA-CD19 CAR-T cells. [Figure 29A] This image shows flow cytometry data of BCMA-CD19 CAR-T cells that kill plasma cells in SLE patients in vitro. [Figure 29B] This is a histogram showing the number of plasma cells after in vitro death by BCMA-CD19 CAR-T cells. [Figure 30] This study characterizes CD19 and BCMA expression on B cell subsets from peripheral blood mononuclear cells (PBMCs) of donors with one of the following conditions: Sjögren's syndrome, scleroderma or systemic sclerosis (SSc), myositis, and IgA nephropathy, as well as from healthy donors. [Figure 31A] This paper presents a gating strategy for an in vitro cell death assay using BCMA-CD19 CAR-T cells. [Figure 31B] A typical flow plot of in vitro mortality is shown. [Figure 32] In vitro differentiated plasmablasts and CD19+ cells from a healthy donor are shown. Naive B cells were then differentiated using a proprietary cytokine mixture to drive plasmablast differentiation (BCMA+ B cells). Subsequently, 5 days after differentiation, the cells were co-cultured with BCMA-CD19 Bz CAR-T cells (manufactured using a preclinical manufacturing process) or untransduced T cells (non-targeted) in various effector:target (E:T ratios). Dose-dependent depletion (E:T) by BCMA-CD19 CAR-T cells was observed. [Figure 33] This image shows BCMA-CD19 CAR-T cells co-cultured with in vitro differentiated B cell cultures derived from a healthy donor. Dose-dependent induction of cytokine production associated with T cell activation from BCMA-CD19 CAR-T cells compared to untransduced control T cells. Supernatants were collected from co-cultures of BCMA-CD19 CAR-T cells and healthy in vitro differentiated B cells, aliquoted, and stored for MSD analysis. Before thawing the supernatant, MSD UPLEX plates were coated with antibody linker pairs against the following analytes: IFNγ, IL-2, IL-4, IL-6, IL-10, and TNFα. Samples were thawed, diluted 1:15 in diluent 57, and then incubated on the coated MSD plates. After sample incubation, plates were incubated with sulfotaged secondary antibodies before reading the plates. Analysis was performed using Discovery Workbench software. [Figure 34]This study demonstrates dose-dependent depletion (E:T) of in vitro differentiated plasmablasts and CD19+ cells from Sjögren's syndrome donors by BCMA-CD19 CAR-T cells. Naive B cells were isolated from frozen PBMCs of Sjögren's syndrome patients using a naive B cell isolation kit (Stemcell). After isolation, naive B cells were seeded and differentiated using a cytokine mixture to drive B cell differentiation. Five days after differentiation, differentiated B cells were co-cultured for 24 hours with BCMA-CD19 CAR-T cells or untransduced T cells at effector:target ratios of 1.5:1, 0.75:1, 0.37:1, 0.18:1, and 0.093:1. The co-cultured cells were then stained with the following reagents: (Live / Dead staining, CD20, CD19, CD27, CD38, BCMA, CD3, CD4, CD8, CD69). The death of target-differentiated plasmablasts (live, CD3-, CD27+CD38+) and CD19+ targets (live CD3-, CD27-, CD38+ / -, CD19+CD20+) was evaluated by flow cytometry. The depletion percentage was calculated as 1 - (percentage of targets in experimental wells / average percentage of targets in wells containing only stem cells) * 100. [Figure 35]This study demonstrates that in vitro differentiated plasmablasts and CD19+ cells from scleroderma or systemic sclerosis (SSc) donors exhibit dose-dependent depletion (E:T) by BCMA-CD19 CAR-T cells. Naive B cells were isolated from frozen PBMCs of scleroderma or systemic sclerosis (SSc) patients using a naive B cell isolation kit (Stemcell). After isolation, naive B cells were seeded and differentiated using a cytokine mixture to drive B cell differentiation. Five days after differentiation, differentiated B cells were co-cultured for 24 hours with BCMA-CD19 CAR-T cells or untransduced T cells at effector:target ratios of 1.5:1, 0.75:1, 0.37:1, 0.18:1, and 0.093:1. Next, co-cultured cells were stained with the following reagents: (Live / Dead staining, CD20, CD19, CD27, CD38, BCMA, CD3, CD4, CD8, CD69). The death of target differentiated plasmablasts (live, CD3-, CD27+CD38+) and CD19+ targets (live CD3-, CD27-, CD38+ / -, CD19+CD20+) was evaluated by flow cytometry. The depletion percentage was calculated as 1 - (percentage of targets in experimental wells / average percentage of targets in wells containing only stem cells) * 100. [Figure 36]This study demonstrates dose-dependent depletion (E:T) of in vitro differentiated plasmablasts and CD19+ cells from myositis donors by BCMA-CD19 CAR-T cells. Naive B cells were isolated from frozen myositis patient PBMCs using a naive B cell isolation kit (Stemcell). After isolation, naive B cells were seeded and differentiated using a cytokine mixture to drive B cell differentiation. Five days after differentiation, differentiated B cells were co-cultured for 24 hours with BCMA-CD19 CAR-T cells or untransduced T cells at an effector:target ratio of 1.5:1. The co-cultured cells were then stained with the following reagents: (Live / Dead staining, CD20, CD19, CD27, CD38, BCMA, CD3, CD4, CD8, CD69). The death of target-differentiated plasmablasts (live, CD3-, CD27+CD38+) and CD19+ targets (live CD3-, CD27-, CD38+ / -, CD19+CD20+) was evaluated by flow cytometry. The depletion percentage was calculated as 1 - (percentage of targets in experimental wells / average percentage of targets in wells containing only stem cells) * 100. [Figure 37] This study demonstrates dose-dependent depletion (E:T) of in vitro differentiated plasmablasts and CD19+ cells derived from IgA nephropathy donors by BCMA-CD19 CAR-T cells. Naive B cells were isolated from frozen IgA nephropathy patient PBMCs using a naive B cell isolation kit (Stemcell). After isolation, naive B cells were seeded and differentiated using a proprietary cytokine mixture to drive B cell differentiation. Five days after differentiation, differentiated B cells were co-cultured for 24 hours with BCMA-CD19 CAR-T cells or untransduced T cells at effector:target ratios of 1.5:1, 0.75:1, and 0.37:1. The co-cultured cells were then stained with the following reagents: (Live / Dead staining, CD20, CD19, CD27, CD38, BCMA, CD3, CD4, CD8, CD69). The death of target-differentiated plasmablasts (live, CD3-, CD27+CD38+) and CD19+ targets (live CD3-, CD27-, CD38+ / -, CD19+CD20+) was evaluated by flow cytometry. The depletion percentage was calculated as 1 - (percentage of targets in experimental wells / average percentage of targets in wells containing only stem cells) * 100. [Figure 38] This study demonstrates that BCMA-CD19 CAR-T cells co-cultured with in vitro differentiated B cell cultures derived from Sjögren's syndrome donors exhibit dose-dependent induction of T cell activation and associated cytokine production from BCMA-CD19 CAR-T cells compared to untransduced control T cells. Supernatants were collected from the co-culture of BCMA-CD19 CAR-T cells and Sjögren's syndrome in vitro differentiated B cells, aliquoted, and stored for MSD analysis. Before thawing the supernatant, MSD UPLEX plates were coated with antibody linker pairs against the following analytes: IFNγ, IL-2, IL-4, IL-6, IL-10, and TNFα. Samples were thawed, diluted 1:15 in diluent 57, and then incubated on the coated MSD plates. After sample incubation, plates were incubated with sulfotaged secondary antibodies before reading the plates. Analysis was performed using Discovery Workbench software. [Figure 39] This study demonstrates that BCMA-CD19 CAR-T cells co-cultured with in vitro differentiated B cell cultures derived from scleroderma or systemic sclerosis (SSc) donors exhibit dose-dependent induction of T cell activation and associated cytokine production from BCMA-CD19 CAR-T cells compared to untransduced control T cells. Supernatants were collected from co-cultures of BCMA-CD19 CAR-T cells and in vitro differentiated B cells from scleroderma or systemic sclerosis donors, aliquoted, and stored for MSD analysis. Before thawing the supernatant, MSD UPLEX plates were coated with antibody linker pairs against the following analytes: IFNγ, IL-2, IL-4, IL-6, IL-10, and TNFα. Samples were thawed, diluted 1:15 in diluent 57, and then incubated on the coated MSD plates. After sample incubation, plates were incubated with sulfotaged secondary antibodies before reading the plates. The analysis was performed using Discovery Workbench software. [Figure 40]This study demonstrates that BCMA-CD19 CAR-T cells co-cultured with in vitro differentiated B cell cultures derived from myositis donors exhibit dose-dependent induction of T cell activation and associated cytokine production from BCMA-CD19 CAR-T cells compared to untransduced control T cells. Supernatants were collected from co-cultures of BCMA-CD19 CAR-T cells and myositis in vitro differentiated B cells, aliquoted, and stored for MSD analysis. Before thawing the supernatant, MSD UPLEX plates were coated with antibody linker pairs against the following analytes: IFNγ, IL-2, IL-4, IL-6, IL-10, and TNFα. Samples were thawed, diluted 1:15 in diluent 57, and then incubated on the coated MSD plates. After sample incubation, plates were incubated with sulfotaged secondary antibodies before reading the plates. Analysis was performed using Discovery Workbench software. [Figure 41] This figure shows that BCMA-CD19 CAR-T cells co-cultured with in vitro differentiated B cell cultures derived from IgA nephropathy donors exhibit dose-dependent induction of T cell activation and associated cytokine production from BCMA-CD19 CAR-T cells compared to untransduced control T cells. Supernatants were collected from the co-culture of BCMA-CD19 CAR-T cells and frozen IgA nephropathy in vitro differentiated B cells, aliquoted, and stored for MSD analysis. Before thawing the supernatant, MSD UPLEX plates were coated with antibody linker pairs against the following analytes: IFNγ, IL-2, IL-4, IL-6, IL-10, and TNFα. Samples were thawed, diluted 1:15 in diluent 57, and then incubated on the coated MSD plates. After sample incubation, plates were incubated with sulfotaged secondary antibodies before reading the plates. Analysis was performed using Discovery Workbench software. [Modes for carrying out the invention]

[0097] This disclosure is further illustrated by specific embodiments, and it should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosed subject matter.

[0098] The raw materials and equipment used in the examples are well known to those skilled in the art, and are all commercially available, readily available, or can be prepared.

[0099] The term “antibody” (Ab) refers to an immunoglobulin, or an antigen-binding moiety thereof, that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (hereinafter abbreviated as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (hereinafter abbreviated as VL) and a light chain constant region. The light chain constant region comprises the constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are incorporated into more conserved regions called framework regions (FRs). Each VH or VL comprises three CDRs and four FRs, which are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0100] In this specification, amino acid names are identified by a single letter commonly used worldwide, and it should be understood that the corresponding abbreviations for the three letters of an amino acid name are as follows: Ala(A), Arg(R), Asn(N), Asp(D), Cys(C), Gln(Q), Glu(E), Gly(G), His(H), Ile(I), Leu(L), Lys(K), Met(M), Phe(F), Pro(P), Ser(S), Thr(T), Trp(W), Tyr(Y), Val(V).

[0101] Antibody preparation The antibodies disclosed herein can be prepared by various methods known in the art, such as genetic engineering recombination techniques. For example, the DNA molecules encoding the heavy and light chain genes of the antibodies disclosed herein are obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies disclosed herein.

[0102] The antigen-binding fragments disclosed herein can be obtained by hydrolyzing intact antibody molecules (Morimoto et al., Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Furthermore, these antigen-binding fragments can also be directly produced by recombinant host cells (summarized in Hudson, Curr. Opin. Immunol. 11:548-557 (1999) and Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells. Fab' fragments can be chemically bound to form F(ab')z fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). Furthermore, Fv, Fab, or F(ab')z fragments can also be isolated directly from recombinant host cell cultures. Those skilled in the art are well aware of other techniques for preparing these antigen-binding fragments.

[0103] conservative substitution As used herein, the term “conservative substitution” means an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide, including its amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with similar side chains, for example, substitutions of residues that are physically or functionally similar to the corresponding amino acid residue (similar size, shape, charge, chemical properties, e.g., ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid and glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferable to substitute the corresponding amino acid residue with another amino acid residue from the same side chain family.

[0104] B cell maturation antigen (BCMA) BCMA (also known as TNFRF17, BCM, or CD269) is a member of the tumor necrosis factor receptor (TNFR) family and is primarily expressed on terminally differentiated B cells such as memory B cells and plasma cells. Its ligands are called B-cell activator (BAFF) and proliferation-inducing ligand (APRIL) of the TNF family. BCMA is involved in maintaining long-term humoral immunity by mediating plasma cell survival. The BCMA gene is encoded on chromosome 16 and yields a 994-nucleotide primary mRNA transcript (NCBI accession number NM_001192.2) encoding a 184-amino acid protein (NP_001183.2). A second antisense transcript from the BCMA locus has been described, which may play a role in regulating BCMA expression (Laabi Y. et al., Nucleic Acids Res., 1994, 22:1147-1154). Another transcription variant of undetermined importance has been described (Smirnova AS et al., Mol Immunol., 2008, 45(4):1179~1183). A second isoform (also known as TV4) has been identified (Uniprot identifier Q02223-2).

[0105] However, some patients still experience a relapse process after receiving CAR-T cell therapy targeting BCMA. For these relapsed patients, it is necessary to find a target other than BCMA before continuing treatment.

[0106] CD19 As used herein, the term “CD19” refers to the B lymphocyte antigen CD19, also known as B lymphocyte surface antigen B4 or T cell surface antigen Leu-12, and unless otherwise specified, includes any natural CD19 of any vertebrate origin, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The NCBI accession number for the amino acid sequence of human CD19 is NP_001171569. The term includes “full-length” unprocessed human CD19 and any form of human CD19 derived from intracellular processing, as long as the antibodies reported herein bind to it. CD19 is a structurally unique cell surface receptor expressed on the surface of human B cells, including, but not limited to, pre-B cells, early-developing B cells (i.e., immature B cells), mature B cells that have terminally differentiated into plasma cells, and malignant B cells. CD19 is expressed in most pre-B acute lymphoblastic leukemias (ALL), non-Hodgkin lymphomas, B-cell chronic lymphoblastic leukemias (CLL), lymphoblastic leukemias, hairy cell leukemias, common acute lymphoblastic leukemias, and some null acute lymphoblastic leukemias. CD19 expression on plasma cells further suggests that CD19 may be expressed in differentiated B-cell tumors such as multiple myeloma. Therefore, the CD19 antigen is a target for immunotherapy in non-Hodgkin lymphomas, chronic lymphoblastic leukemias, and / or acute lymphoblastic leukemias.

[0107] CD19 is a 95 kD glycoprotein expressed on the membrane surface of pre- and mature B cells. It is closely involved in the transmembrane conduction pathway of Ca++ in B cells and can regulate B cell proliferation and differentiation. CD19 is mainly expressed in normal and cancerous B cells, has high tissue expression specificity, and is a good target for antibody or CAR-T immunotherapy. However, loss of CD19 epitopes on B cells often occurs during immunotherapy, which can cause patients to be unable to respond to immunotherapy or to experience relapse.

[0108] CAR (Chimera Antigen Receptor) The chimeric antigen receptors (CARs) of this disclosure comprise an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain comprises a target-specific binding element (also called an antigen-binding domain). The intracellular domain comprises a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain that contains costimulatory molecules. Costimulatory molecules are cell surface molecules required by lymphocytes to respond effectively to antigens, rather than antigen receptors or their ligands.

[0109] Linkers can be incorporated between the extracellular domain and the transmembrane domain of a CAR, or between the cytoplasmic domain and the transmembrane domain of a CAR. As used herein, the term “linker” generally refers to any oligopeptide or polypeptide that acts to link the transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain. Linkers may consist of 0 to 300 amino acids, 2 to 100 amino acids, and 3 to 50 amino acids.

[0110] In one embodiment, the extracellular domain of the CAR provided herein includes an antigen-binding domain that targets BCMA (or BCMA and CD19). When the CAR disclosed herein is expressed in T cells, it can recognize antigens based on antigen-binding specificity. When it binds to its associated antigen, it can kill CD19-positive B cells, plasmablasts, and BCMA-positive plasma cells. The antigen-binding domain can be fused with an intracellular domain derived from one or more costimulatory molecules and zeta chains. The antigen-binding domain can be fused with an intracellular domain of a combination of a 4-1BB signaling domain and a CD3ζ zeta signaling domain.

[0111] As used herein, both “antigen-binding domain” and “single-chain antibody fragment” refer to a Fab fragment, Fab' fragment, F(ab')2 fragment, or a single Fv fragment having antigen-binding activity. An Fv antibody contains a variable heavy chain region and a variable light chain region, but lacks a constant region and has the smallest antibody fragment of all antigen-binding sites. Generally, an Fv antibody further contains a polypeptide linker between the VH and VL domains to form the structure necessary for antigen binding. The antigen-binding domain is typically an scFv (single-chain variable fragment). The size of an scFv is generally 1 / 6 the size of a complete antibody. A single-chain antibody may be an amino acid chain sequence encoded by a nucleotide chain. In another embodiment, the antigen-binding domain contains an antibody that specifically recognizes BCMA, and optionally, the antigen-binding domain further contains an antibody that specifically recognizes CD19. In some embodiments, the antigen-binding domain is a single-chain variable fragment.

[0112] Regarding the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that naturally associates with one of the domains in the CAR is used. In some examples, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domain binding to the transmembrane domain of the same or different surface membrane proteins, thus minimizing interaction with other members of the receptor complex.

[0113] The intracellular domains in the disclosed CAR include a 4-1BB signaling domain and a CD3ζ signaling domain.

[0114] The CARs disclosed herein also include cell suicide elements.

[0115] Bispecific CAR targeting CD19 and BCMA Bispecificity means that the same CAR can specifically bind to and be recognized by two different antigens, and that binding to either antigen can trigger an immune response. A bispecific CAR is a dual-target CAR.

[0116] In another example, a bispecific CAR targeting CD19 and BCMA is described in the first aspect of this disclosure.

[0117] In one embodiment, the extracellular domain of the CAR provided by this disclosure includes antigen-binding domains that target CD19 and BCMA, including anti-CD19 scFv and anti-BCMA scFv.

[0118] In another example, the present disclosure provides a bispecific CAR for CD19 and BCMA antigens. The structural components of a CAR targeting both CD19 and BCMA may include a signal peptide, an anti-CD19 scFv, an anti-BCMA scFv, a hinge region, a transmembrane region, and an intracellular T cell signaling region, where the CD19 scFv and BCMA scFv are linked by a short peptide segment (G4S)xN. The CAR structure targeting both CD19 and BCMA is as described in a second aspect of the present disclosure.

[0119] In another example, the BCMA scFv sequence has been optimized in this disclosure, and BCMA scFv(S scFv) has high affinity and good specificity for BCMA, and can specifically target the full-length BCMA antigen and extracellular region.

[0120] In one embodiment, CD19scFv and BCMAscFv are linked using (G4S)x3, and at this point, the activity and lethality of the CAR are optimal.

[0121] Compared to CARs targeting a single antigen, the CD19 and BCMA-targeting bispecific CARs disclosed herein exhibit significantly enhanced affinity and provide a synergistic effect, with significantly increased immune cell activity, as demonstrated in the following examples. Furthermore, due to the heterogeneous expression levels of CD19 and BCMA in B cells and plasma cells, bispecific CAR-Ts have a broader therapeutic range. CAR-T immune cells that simultaneously target CD19 and BCMA can reduce the likelihood of antigen escape caused by the downregulation or deletion of a single surface antigen. Additionally, targeting the CD19 and BCMA combination allows for more comprehensive blockade of the autoantibody production pathway, thereby encompassing multiple differentiation stages of B cells, plasmablasts, and plasma cells that more completely produce autoantibodies. The CD19 and BCMA targeting combination not only has a deeper therapeutic effect against SLE but also has the potential to reduce the likelihood of relapse.

[0122] In one embodiment, the bispecific CAR includes an amino acid sequence represented by SEQ ID NO: 26.

[0123] Chimeric antigen receptor T cells (CAR-T cells) As used herein, the terms “CAR-T cells,” “CAR-T cells,” and “CAR-T cells of the Disclosure” include BCMA-CD19 CAR-T cells as included in the third aspect of the Disclosure.

[0124] Suicide gene switch To further control non-tumor targeting and cytokine release syndrome of CAR-T cells, all CAR cells in this disclosure are equipped with suicide gene switches, which can effectively eliminate CAR-T cells in vivo under the action of exogenous drugs, blocking unknown or uncontrollable long-term toxicity and ensuring patient safety.

[0125] The suicide switches used in this disclosure may include herpes simplex virus thymidine kinase (HSV-TK), inducible caspase 9 (iCasp9), CD20, and mutant human thymidylate kinase (mTMPK). Compared to each other, HSV-TK, iCasp9, and CD20 have the same elimination ability against CAR-T cells, but iCasp9 and CD20 have a faster elimination rate, while HSV-TK has a slower elimination rate.

[0126] The iCasp9 suicide switch contains the FKBP12-F36V domain, which can be linked to cysteine ​​aspartate protease 9 via a flexible linker, the latter not containing a recruitment domain. FKBP12-F36V contains the FKBP domain, with phenylalanine replaced by valine at the 36th amino acid residue. It has high selectivity and sub-nanomolecular affinity and can form ligands in combination with dimerization of other inactive small molecules such as AP1903. Upon addition of small molecules, they can promote their dimerization and thus induce cell apoptosis, but are ineffective in normal cells that do not possess a suicide switch.

[0127] Inducible safe switch caspase 9 (iCasp9) can fuse with FK506-binding protein (FKBP) using human caspase 9, and as a result, can be induced to dimerize by a chemical inducer (AP1903 / Rimiducid, Bellicum Pharmaceutical), leading to apoptosis in cells expressing the fusion protein.

[0128] CD19 and BCMA are highly expressed in tumor cells, but they are also expressed in normal B cells, and the manipulated immune cells of this disclosure attack normal B cells in vivo.

[0129] Controlling the safety of CAR-T cells has always been an urgent issue. Adding a safety switch to CAR-T cells is the safest way to terminate their activity. The inducible iCasp9 safety switch controls the removal of CAR-T cells after they become severely toxic (CRS / neurotoxic) or after a patient has achieved long-term continuous remission.

[0130] vector Nucleic acid sequences encoding a desired molecule can be obtained by recombinant methods known in the art, such as screening a library from cells expressing the gene, obtaining the gene from a vector known to contain the gene, or directly isolating the gene from cells and tissues containing the gene using standard techniques. Optionally, the gene of interest can be produced synthetically.

[0131] This disclosure further provides vectors into which the expression cassette of this disclosure has been inserted. Retroviral vectors, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they enable the long-term and stable integration of the transgene and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses, such as mouse leucoviruses, because they can be transduced into non-proliferating cells such as hepatocytes. They also have the advantage of lower immunogenicity.

[0132] In short, the expression cassette or nucleic acid sequence described herein is typically operably ligated to a promoter and incorporated into an expression vector. The vector is suitable for replicating and incorporating eukaryotic cells. A typical cloning vector includes transcriptional and translational terminators, an initiation sequence, and a promoter that can be used to regulate the expression of a desired nucleic acid sequence.

[0133] The expression constructs disclosed herein may also be used in nucleic acid immunotherapy and gene therapy by using standard gene delivery protocols. Methods of gene delivery are well known in the art. See, for example, U.S. Patents 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the herein disclosure provides gene therapy vectors.

[0134] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into such vectors, including but not limited to plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.

[0135] Furthermore, expression vectors can be delivered to cells in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other manuals on virology and molecular biology. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector includes an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selection markers (e.g., International Publication Nos. 01 / 96584, 01 / 29058, and U.S. Patent No. 6,326,193).

[0136] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. Recombinant viruses can then be isolated and delivered to target cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. Many adenovirus vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0137] Additional promoter elements, such as enhancers, can modulate the frequency of transcription initiation. Generally, these are located in a 30–110 bp region upstream of the initiation site, although many promoters have recently been shown to also contain functional elements downstream of the initiation site. The spacing between promoter elements is often flexible to maintain promoter function when one element is reversed or moved relative to another. In thymidine kinase promoters, the spacing between promoter elements can be widened to 50 bp before activity begins to decline. Depending on the promoter, individual elements can act to initiate transcription cooperatively or independently.

[0138] An example of a suitable promoter is the pre-early cytomegalovirus (CMV) promoter sequence. A promoter sequence is a potent constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence functionally linked to it. Another example of a suitable promoter is elongation factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to the Simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) terminal repeat sequence (LTR) promoter, MoMuLV promoter, trileukovirus promoter, Epstein-Barr virus pre-early promoter, Roussarcoma virus promoter, and human gene promoters including but not limited to actin promoters, myosin promoters, heme promoters, and creatine kinase promoters. Furthermore, this disclosure should not be limited to the application of constitutive promoters. Inducible promoters are also considered part of the present invention. The use of inductive promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to the inductive promoter when such expression is desired, or turn it off when such expression is undesirable. Examples of inductive promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0139] To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into cells may also contain either or both a selection marker gene and / or a reporter gene to identify and select expressing cells from a population of cells to be transfected or infected by the viral vector. In other embodiments, the selectable marker may be supported on a single fragment of DNA and used in a co-transfection procedure. Both the selection marker and reporter genes may be flanked by appropriate regulatory sequences so that they can be expressed in host cells. Useful selection markers include, for example, antibiotic resistance genes such as neo.

[0140] Reporter genes are used to identify potential transfected cells and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene encoding a polypeptide that is either absent from or expressed by the recipient organism or tissue, and whose expression is manifested by several readily detectable characteristics, such as enzymatic activity. After the DNA is introduced into the recipient cell, the expression of the reporter gene is determined at an appropriate time. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or are commercially available. Generally, a construct having at least five adjacent regions exhibiting the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be ligated to the reporter gene and used to evaluate the ability of reagents to regulate promoter-driven transcription.

[0141] Methods for introducing genes into cells and methods for expressing genes in cells are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0142] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle impact, microinjection, and electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the field. See, for example, Sambrook et al. (2001, molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Polynucleotides can be introduced into host cells using calcium phosphate transfection.

[0143] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, are the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0144] Chemical means for introducing polynucleotides into host cells include colloidal dispersions, such as polymer complexes, nanocapsules, microspheres, and beads, as well as lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane sacs).

[0145] When nonviral delivery systems are used, an exemplary delivery vehicle is a liposome. Lipid preparations can be used to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with lipids. Lipid-associated nucleic acids may be encapsulated within the aqueous interior of a liposome, dispersed in the lipid bilayer of a liposome, bound to a liposome via linking molecules associated with both the liposome and oligonucleotides, captured by a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in micelles or complexed with micelles, or otherwise associated with lipids. The lipids, lipid / DNA, or lipid / expression vectors involved in the composition are not limited to any particular structure in solution. For example, they may exist in a bilayer structure as micelles or have a "disintegrated" structure. They may also be readily dispersed in solution and may form aggregates with heterogeneous size or shape. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include naturally occurring lipid droplets in the cytoplasm, as well as compounds such as long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0146] In one embodiment, the vector is a lentiviral vector.

[0147] Drugs, preparations thereof, and compositions This disclosure provides a preparation comprising the CAR-T cells of this disclosure and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the preparation is a liquid preparation. In one embodiment, the preparation is an injection. The concentration of CAR-T cells in the preparation is 1 × 10⁻⁶. 3 ~1 × 10 8 Cells / mL range, or 1 × 10⁶ 4 ~1 × 10 7 This is within the range of cells / mL.

[0148] In one embodiment, the formulation may include a buffer such as neutral buffered saline or sulfate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran or mannitol; proteins: polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The preparations of this disclosure can be formulated for intravenous administration.

[0149] In certain embodiments, the pharmaceutical compositions of this disclosure include modified immune cells or immune cell compositions disclosed herein.

[0150] The isolated nucleic acid molecules, vectors, host cells, modified immune cells, or immune cell compositions of this disclosure can be prepared into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, lozenges, suppositories, injections (including injections, sterile powders for injection, and concentrated solutions for injection), inhalants, and sprays. The dosage form depends in part on the intended mode of administration and therapeutic use. The pharmaceutical compositions of this disclosure should be sterile and stable under production and storage conditions. An example of a dosage form is injection. Such injections may be sterile injection solutions. Sterile injection solutions may also be prepared into sterile lyophilized powders (e.g., vacuum-dried or lyophilized) for convenience of storage and use. Such sterile, freeze-dried powders can be dispersed before use in suitable carriers such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffer solutions (e.g., phosphate buffer), Ringer's solution, and any combination thereof.

[0151] The isolated nucleic acid molecules, vectors, host cells, modified immune cells, or immune cell compositions of this disclosure may be administered by any suitable method known in the art, including but not limited to oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, cisternal, inguinal, intravesical, topical (e.g., powder, ointment, or drops) or nasal routes. However, for many therapeutic purposes, the route / mode of administration may be parenteral (e.g., intravenous injection or infusion, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or mode of administration may vary depending on the intended purpose. In some embodiments, the isolated nucleic acid molecules, nucleic acid constructs, vectors, host cells, modified immune cells, or immune cell compositions of this disclosure are administered by intravenous injection or infusion.

[0152] The pharmaceutical compositions of this disclosure may comprise a therapeutically effective or preventively effective amount of the isolated nucleic acid molecules, nucleic acid constructs, vectors, host cells, modified immune cells, or immune cell compositions of this disclosure. “Preventively effective amount” means an amount sufficient to prevent, halt, or delay the onset of a disease. “Therapeutally effective amount” means an amount sufficient to cure, or at least partially prevent, a disease and its complications in patients already suffering from the disease. The therapeutically effective amount of the isolated nucleic acid molecules, nucleic acid constructs, vectors, host cells, modified immune cells, or immune cell compositions of this disclosure may vary according to the following factors: the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general circumstances such as age, weight, and sex, the mode of drug administration, and other treatments applied concurrently.

[0153] In this disclosure, the administration regimen can be modified to obtain the best desired response (e.g., a therapeutic or prophylactic response). For example, the drug may be administered as a single dose or multiple doses over a period of time, or the dose may be reduced or increased in proportion to the urgency of the treatment situation.

[0154] therapeutic use The therapeutic applications of this disclosure include collecting a patient's T cells, transducing the CAR gene with a lentivirus (4 plasmid system package) to obtain BCMA-CD19 CAR-T cells, enabling the BCMA-CD19 CAR-T cells to specifically recognize target cells having BCMA and CD19 antigens expressed on their cell surface, thereby carrying out the therapeutic applications.

[0155] Accordingly, the present disclosure further provides a method for stimulating a T cell-mediated immune response against a target cell population or tissue in a mammal, comprising administering the CAR-T cells of the present disclosure to a subject (e.g., a mammal, a human) in need of treatment. The subject has or may be suspected of having an autoimmune disease and / or cancer. A person suspected of having an autoimmune disease may exhibit symptoms generally associated with the disease. Suspicion of disease in an individual may also be determined based on a physical examination of the subject by a qualified professional, the results of one or more gene and / or protein assays that may indicate a disease state, and / or one or more risk factors of the subject. In some cases, the autoimmune disease is a B cell-mediated autoimmune disease.

[0156] In one embodiment, the disclosure includes a type of cell therapy in which the patient's own T cells (or xenodont cells) are isolated, activated, and genetically modified to produce CAR-T cells, and then the cells are injected into the same patient. In this way, the probability of graft-versus-host disease is extremely low, and antigens are recognized by T cells without MHC restriction.

[0157] In one embodiment, the CAR-T cells of this disclosure can undergo stable T cell proliferation in vivo and can persist for a long period of time. Furthermore, a CAR-T mediated immune response may be part of an adoptive immunotherapy step, where the CAR-T modified T cells induce an immune response specific to the antigen-binding domain in the CAR. For example, CAR-T cells resistant to BCMA and / or CD19 will evoke a specific immune response against cells expressing BCMA and / or CD19.

[0158] The data disclosed herein specifically disclose lentiviral vectors comprising anti-BCMA and / or CD19scFv, hinge and transmembrane regions, and 4-1BB / CD28 and CD3ζ signaling domains, but this disclosure should be interpreted as including any number of variations for each of the components of the construct.

[0159] Drugs can treat autoimmune diseases by killing or inhibiting CD19-positive B cells, plasmablasts, and BCMA-positive plasma cells. B cells are associated with many autoimmune diseases. Autoreactive B cells and hyperactive or hyperstimulated B cells play a role in autoimmune diseases. In some cases, autoimmune diseases may have abnormal B cell maturation and / or autoantibody production. B cell-mediated autoimmune diseases are characterized by a loss of self-tolerance during B cell development. Therefore, B cells are important to the pathology of autoimmune diseases. Examples of B cell-mediated autoimmune diseases are discussed below. Each of these diseases may be treated by administering the compositions, formulations, or pharmaceutical compositions disclosed herein to subjects in need of treatment. Subjects may have or be suspected of having an autoimmune disease.

[0160] Autoimmune diseases can be autoimmune diseases mediated by B cells. B cell-mediated autoimmune diseases include systemic lupus erythematosus (SLE), glomerulonephritis, e.g., autoimmune chronic kidney disease, lupus nephritis, acute glomerulonephritis, immunonephritis, membranous nephropathy, IgA nephropathy, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV); scleroderma or systemic sclerosis (SSc); myositis or idiopathic inflammatory myositis, including dermatomyositis, polymyositis, immune-mediated necrotizing myopathy (IMNM), anti-synthetic enzyme syndrome, inclusion body myositis, and multiple myositis; multiple sclerosis (MS); inflammatory bowel disease (IBD); rheumatoid arthritis (RA); Sjögren's syndrome (SS); autoimmune hemolytic anemia; neuromyelitis optica (NMO); neuromyelitis optica spectroscopy This includes Lamb's syndrome (NMOSD); idiopathic thrombocytopenic purpura (ITP); systemic autoimmune small vessel vasculitis or polyangiitis associated with antineutrophil cytoplasmic antibodies; Wegener's granulomatosis (GPA), eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss syndrome); pemphigus vulgaris; autoimmune encephalitis; myasthenia gravis; antiphospholipid syndrome; Chagas disease; Graves' disease; polyarteritis nodosa; pulmonary hemorrhagic nephritis syndrome; Kawasaki disease; amyloidosis; monoclonal globulin of unknown significance, POEMS syndrome; Crohn's disease; ulcerative colitis; adult-onset Still's disease; and chronic progressive demyelinating encephalopathy (CIDP).

[0161] As mentioned above, these diseases are B-cell autoimmune diseases. Many of these diseases result from the deposition of immune complexes in the affected tissue, inflammation, or other attacks by autoantibodies that damage cells / tissues. Some of the pathologies of the B-cell mediated autoimmune diseases described above will be discussed further below.

[0162] As mentioned above, SLE is an autoimmune disease that can affect multiple organs. SLE is the most common type of lupus. The symptoms of SLE are heterogeneous, affecting multiple different organs, and the severity varies from patient to patient. SLE includes moderate to severe refractory SLE, lupus nephritis, active lupus nephritis, and active systemic lupus erythematosus without renal involvement. Refractory (or severe) SLE may include patients who have received one or more prior drug therapies for an autoimmune disease but have not responded to or have had a poor response to drug therapy.

[0163] SLE is characterized by an immune response to endogenous nuclei (e.g., nucleic acids and nuclear proteins) and cytoplasmic material. The production of autoantibodies (e.g., ANA) is characteristic of SLE and is driven by autoreactive B cells. The immune response activated by autoantibodies can lead to inflammation. Therefore, B cells play a crucial role in the pathogenesis of SLE, and SLE may be treated and / or prevented with the compositions, formulations, or pharmaceutical compositions disclosed herein.

[0164] Glomerulonephritis (GN) refers to the pathogenesis involved in autoimmune chronic kidney disease (CKD). Autoimmune CKD is associated with the production of autoantibodies by B cells and can include membranous nephropathy, IgA nephropathy, and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). GN is inflammation of the glomeruli of the kidney. CKD refers to a decrease or inability of the kidney to filter blood, which can result from antibody-associated damage to the glomeruli. Lupus nephritis (described below) is a type of GN caused by SLE; that is, LN is a secondary effect of SLE. Membranous nephropathy (described below) is another type of GN that can occur as a secondary effect of SLE or as a primary effect (e.g., without associated diseases such as SLE). GN involves a loss of tolerance and adaptive immune response to autoantigens. In particular, immune complexes are directed towards IgA and / or IgG circulating in the blood. For example, IgA nephropathy is characterized by IgA autoantibodies deposited in the glomeruli. Autoantibodies are a characteristic feature of the disease pathogenesis of GN. Therefore, GN can be prevented and / or treated by depleting B cells using the compositions, formulations, or pharmaceutical compositions disclosed herein.

[0165] Lupus nephritis (LN) is a common consequence of SLE. ANA can form immune complexes that can deposit in the glomeruli or react with the glomerular basement membrane of the kidney. Lupus nephritis is associated with a higher morbidity and can progress to end-stage renal disease. Therefore, preventing or reducing the progression of LN is important. Since LN itself is a progression of SLE, it is also a B-cell mediated autoimmune disease.

[0166] Membranous nephropathy (MN) is an autoimmune disease characterized by thickening of the glomerular capillary walls due to immune complex deposition. MN occurs in all regions and ethnic groups, with a rising prevalence among East Asian populations, accounting for approximately 25% of all major nephrotic syndromes (GNs) in China. The annual incidence of MN is estimated at 10–12 cases per million in North America and 2–17 cases per million in Europe. The disease affects individuals of all ages, with a mean age of diagnosis of 50–60 years and a 2:1 male-to-female ratio for unknown reasons. MN is the most common cause of idiopathic nephrotic syndrome in non-diabetic adults worldwide, accounting for 20–37% of most kidney biopsy series, increasing to as high as 58% in adults over 65 years of age. MN is rare in children. The natural course of untreated MN has been reported to have a spontaneous complete remission rate of 20–30% and a 10-year kidney survival rate of 60–80%. In patients with persistent nephrotic syndrome, renal failure develops in 40-50% of cases over a 10-year period. These patients are also at high risk of life-threatening thromboembolic and cardiovascular events. Antibodies against PLA2R are specific to MN and are found in approximately 70% of adult patients with the disease. Porphyrocytes are targets of circulating autoantibodies and are likely a major source of autoantigens. Several additional antigens have been identified, including thrombospondin type 1 domain-containing 7A (THSD7A), which accounts for less than 5% of MN cases. The current standard of treatment for MN includes treatment with one or more of the following: prednisolone, cyclophosphamide, calcineurin inhibitors, and / or rituximab. However, none of these treatments deplete antibody-producing B cells.

[0167] Multiple sclerosis (MS) is a chronic disease that can be caused by autoantibodies that attack the central nervous system, particularly myelin in nerve fibers. There are four main types of MS: relapsing-remitting MS, secondary progressive MS, primary progressive MS, and progressive-relapsing MS. Each form is typically defined by the frequency and severity of symptoms associated with attacks. MS can affect vision, cognition, mood, muscle movement, and bladder control. B cells may be involved in the pathogenesis of MS by (i) driving antigen presentation to T cells and the autoproliferation of brain-homing T cells (possibly by memory B cells), (ii) producing pro-inflammatory cytokines and chemokines that propagate inflammation, (iii) producing soluble toxic factors that contribute to oligodendrocyte and neuronal damage, (iv) contributing to the formation of ectopic lymphoid aggregates in the meninges, and (v) providing a reservoir for Epstein-Barr (EBV) virus infection (see Comi et al., Ann. Neurol, Vol. 89(1), pp. 13-23, January 2021). B cell depletion has been successful in the treatment of patients with relapsing MS and primary progressive MS. Ibid. Therefore, the CAR compositions, formulations, or pharmaceutical compositions disclosed herein may be used to treat or prevent MS in patients who have or are suspected of having MS.

[0168] Myositis (idiopathic inflammatory muscle disease) refers to a group of autoimmune diseases characterized by weakness, swelling, pain, and / or inflammation of skeletal muscles. The most common forms of myositis are polymyositis and dermatomyositis. Other forms include immune-mediated necrotizing myopathy (IMNM), antisynthesis syndrome, inclusion body myositis, and multiple myositis. Dermatomyositis primarily affects the skin, muscles, joints, and lungs. Skin lesions are generally associated with the disease and may precede muscle symptoms. Polymyositis is associated with muscle weakness and is primarily diagnosed by excluding other diseases. The involvement of B cells in myositis is based on the presence of autoantibodies produced by B cells. Myositis is usually associated with myositis-specific autoantibodies (MSAs) or myositis-associated autoantibodies (MAAs). BAFF is involved in the production of these autoantibodies in patients with myositis. Therefore, in patients with or suspected of having myositis, B cell depletion by one of the compositions, formulations, or pharmaceutical compositions disclosed herein is desirable for treating and / or preventing the disease.

[0169] Myasthenia gravis (MG) affects the neuromuscular junction of skeletal muscle. It typically involves the muscles of the eyes, throat, and limbs, manifesting as muscle weakness. MG is an autoimmune disease in which autoantibodies against one or more of the following are found in the majority of patients: nicotinic acetylcholine receptors, muscle-specific kinases, and lipoprotein-related proteins 4. The production of autoantibodies indicates the role of B cells in the pathology of MG (see Yi et al., Muscle Nerve, vol. 57(2), pp. 172-184, February 2018).

[0170] Scleroderma, or systemic sclerosis (SSc), is a connective tissue disorder that can be focal or diffuse (e.g., systemic). It typically manifests as thickening of the skin. ANA is present in about 90% of cases. Autoantibodies against centromeres, SCL70, and RNA polymerase III are also prevalent in the majority of cases. Systemic sclerosis can affect multiple organs, including the skin, gastrointestinal tract, lungs, kidneys, skeletal muscle, and pericardium. Activated B cells produce autoantibodies that directly contribute to the disease. For in vitro immunization, at least one of the following should be performed in vitro before administering cells to mammals: i) growing the cells, ii) introducing CAR-encoding nucleic acids into the cells, and / or iii) cryopreserving the cells.

[0171] Amyloidosis is a disease in which amyloid deposits accumulate in human organs (e.g., the heart, brain, kidneys, stomach, intestines, and spleen). There are three types of amyloidosis. Light chain (AL) amyloidosis occurs when plasma cells produce an excess of abnormal light chain proteins that accumulate in tissues and organs. Plasma cells can be targeted by bispecific CARs disclosed herein. Other forms of amyloidosis include serum amyloid A protein (AA) amyloidosis and transthyretin (ATTR) amyloidosis.

[0172] Ex vivo procedures are well known in the art and are fully described below. Briefly, cells are isolated from a mammal (e.g., human) and genetically modified with a vector expressing a CAR disclosed herein (i.e., transduced or transfected in vitro). CAR-T modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient may be human, and the CAR-T modified cells may be self to the recipient. Optionally, the cells may be allogeneic, syngeneic, or heterogeneic to the recipient.

[0173] This disclosure provides a method for treating a tumor, comprising administering a therapeutically effective dose of the CAR-T modified T cells of this disclosure to a subject in need thereof.

[0174] The CAR-containing formulations, compositions, or pharmaceutical compositions disclosed herein may be administered in doses of 0.5 ± 20% × 10^5 cells / kg, 1 ± 20% × 10^5 cells / kg, 2 ± 20% × 10^5 cells / kg, or 3 ± 20% × 10^5 cells / kg. A single-arm clinical trial for SLE has been initiated. See NCT05858684 for detailed registration criteria. In one embodiment, the doses are 0.5 × 10^5 cells / kg, 1 × 10^5 cells / kg, 2 × 10^5 cells / kg, or 3 × 10^5 cells / kg. The above doses may be administered to patients who have or are suspected of having an autoimmune disease. The autoimmune disease may be a B-cell mediated autoimmune disease.

[0175] The CAR-T modified T cells of this disclosure may be administered alone or as part of a pharmaceutical composition in combination with diluents and / or other components such as IL-2, IL-17, or other cytokines or cell populations. In short, the pharmaceutical compositions of this disclosure may include, for example, one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients, along with the target cell populations described herein. Such compositions may include buffers, e.g., neutral buffered saline, phosphate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins: polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In one embodiment, the compositions of this disclosure are formulated for intravenous administration.

[0176] The pharmaceutical compositions of this disclosure can be administered in a manner appropriate to the disease being treated (or prevented). The number and frequency of administrations will be determined by factors such as the patient's condition and the type and severity of the patient's disease, but the appropriate dose can be determined by clinical trials. [Examples]

[0177] Example 1: Differentiation of antibody-secreting cells in vitro Days 0-3: After isolating B cells, an appropriate number of cells were counted and their cellular phenotype was detected by flow cytometry. The remaining cells were resuspended to a density of 0.5 M / mL in freshly prepared B cell basal medium (IMDM + Glutamax + 10% FBS), and then transferred to a 24-well plate covered with a feeder cell layer (irradiated L cells expressing human CD40L, 0.125 M / well). Cytokines IL-2 (20 U / mL) and IL-21 (50 ng / mL), and antibodies F(ab')2 goat anti-human IgM and IgG (10 μg / mL) were added according to their respective concentrations.

[0178] Days 3-6: B cells cultured up to day 3 were transferred from the plates, an appropriate amount of cells were counted, and the cell phenotype was detected by flow cytometry. After counting, the cells were centrifuged at 400g for 5 minutes, the supernatant was discarded, and the cells were then resuspended in B cell basal medium (IMDM + Glutamax + 10% FBS) to a density of 0.2 M / mL. Simultaneously, cytokines IL-2 (20 U / mL) and IL-21 (50 ng / mL), as well as the hybridoma proliferation additive HyMAX® Hybridoma Fusion & Cloning Supplement at a final concentration of 1×, lipid mixture 1, and MEM amino acid solution were added for continuous culture.

[0179] Days 6-13: After culturing up to day 6, an appropriate amount of cells were counted and their phenotype was detected by flow cytometry. After counting, the cells were centrifuged at 400g for 5 minutes, the supernatant was discarded, and the cells were then resuspended in B cell basal medium (IMDM + Glutamax + 10% FBS) to a density of 0.5 M / mL. These cells were then transferred at a rate of 4 mL per well to a 6-well plate lined with bone marrow stromal cells (irradiated M2-10B4 cells, 0.2 M / well). Simultaneously, cytokines IL-6 (10 ng / mL), IL-21 (50 ng / mL), and IFN-α (100 U / mL) were added according to their respective concentrations, as well as the hybridoma proliferation additive HyMAX® Hybridoma Fusion & Cloning Supplement, Lipid Mixture 1, and MEM amino acid solution for further culture. On day 10, half of the culture medium was replaced, and cultivation was continued until day 12-13. The cells were then used in an in vitro cell death assay for target cells.

[0180] Example 2: Flow cytometry staining for CAR expression Take some cells from the culture, mix them well, and mix 1 × 10 5 ~5×10 5 Individual cells were collected, centrifuged at 300 x g for 5 minutes, and the supernatant was discarded.

[0181] The concentration of BCMA-his antigen was adjusted to 10 μg / mL (1:10 dilution) with FACS buffer. 100 μL of the prepared primary antibody was taken and the cell sample was resuspended. The system was allowed to stand at 4°C for 50 minutes before staining.

[0182] After staining was complete, the cells were washed once with FACS buffer.

[0183] A mixture of secondary antibodies was prepared by diluting anti-His tag-PE, rabbit anti-mouse FMC63 scFv monoclonal antibody Alexa Fluor 647 (clone: ​​R19M), BV 421-anti-CD3, PE-Cy7 anti-CD4, and APC-H7 anti-CD8 in a 1:100 ratio. Then, 100 μL of this mixture was taken and the cell sample was resuspended in it. The system was thoroughly mixed, and then allowed to stand in the dark at 4°C for 25 minutes for staining.

[0184] After staining was complete, the cells were washed twice with FACS buffer, resuspended in 7-AAD buffer diluted 1:300, and detected by machine. The results are shown in Figures 3-6 (in the figures, the CD19 scFv and BCMA scFv in CD19 CAR-T and BCMA CAR-T cells are the same as the corresponding protein sequences in BCMA-CD19-L1 dual-target CAR-T cells). This application demonstrates that high levels of expression of both anti-CD19 and anti-BCMA antibodies can be achieved on the surface of BCMA-CD19 CAR-T cells.

[0185] Example 3: Flow cytometry staining of antibody-secreting cells Take some cells from the culture, mix them well, and mix 1 × 10 5 ~5×10 5 Individual cells were collected, centrifuged at 300 x g for 5 minutes, and the supernatant was discarded.

[0186] A clean 1.5 mL EP tube was taken, an appropriate amount of FACS buffer was added according to the sample size, and then the following antibodies: APC-anti-CD19, PE-anti-BCMA, BUV 395-anti-CD27, FITC-anti-CD38, BV 421-anti-CD138, and BV 605-anti-CD20 were added in a 1:100 ratio, and these were thoroughly mixed to obtain the antibody staining solution.

[0187] Antibody-secreting cells were removed from the culture on days 0, 3, 6, 10, 13, and 14, mixed thoroughly, and an appropriate amount of cells was collected. The cells were then centrifuged at 400g for 5 minutes, and the supernatant was discarded.

[0188] The cells were resuspended in the prepared staining solution, stained in the dark at 4°C for 25 minutes, washed once with FACS buffer, centrifuged at 400g for 5 minutes, and the supernatant was discarded.

[0189] The cells were washed again with FACS buffer, centrifuged at 400g for 5 minutes, and the supernatant was discarded.

[0190] Cells were resuspended in FACS buffer and immediately tested by machine. Figure 7A shows the changes in the absolute number of different cell subsets at different differentiation days, and the changes in the expression levels and percentages of CD19, BCMA, CD20, CD27, CD38, and CD138 on the cell surface. Figure 7B shows the expression levels of different antigens on the cell surface and protein expression on the cell surface during a 14-day differentiation cycle. At the end of differentiation, the percentage of CD38 and CD138-positive plasma cells was 82%, and the percentage of CD38 and CD27-positive antibody-secreting cells was 94.3%. At the end of differentiation, 31.5% of the cells were CD19-negative. After differentiation, high-purity antibody-secreting cells were obtained, and it was found that a certain percentage of these cells were CD19-negative.

[0191] Antibody-secreting cells differentiated in vitro were killed for 5 hours using NT, CD19 CAR-T, BCMA CAR-T, and BCMA-CD19 dual-target CAR-T. Flow cytometry experiments were then performed to detect the absolute number of remaining viable antibody-secreting cells after killing, and the cell-killing ability of the three types of CAR-T against all antibody-secreting cells was detected. The results are shown in Figures 8 to 12, and these figures show that BCMA-CD19 dual-target CAR-T can simultaneously eliminate CD19+ antibody-secreting cells and CD19- antibody-secreting cells.

[0192] Example 4: ELISPOT of antibody-secreting cells Day 0: (Aseptic technique required) A Millipore 96-well PVDF ELISPOT plate was removed, and 25 μL of 70% ethanol was added to each well. The plate was then treated at room temperature for 1 minute.

[0193] The plates were washed twice with PBS (200 μL / well each time), the washing buffer was discarded after the final wash, and the plates were gently tapped on sterile paper to dry.

[0194] 50 μL of diluted coated antibody or specific antigen was added to each well, and the system was incubated overnight at 4°C.

[0195] Day 1: (Aseptic technique required) The coated antibody or antigen was removed from the plate, and the plate was washed three times with sterile PBS, 200 μL / well each time. After the final wash, the wash buffer was discarded, and the plate was dried by gently tapping it on sterile paper.

[0196] 200 μL of blocking buffer (1×) was added to each well, and the mixture was blocked at room temperature for at least 1 hour.

[0197] The blocking buffer was removed (without washing the plate), and the prepared cells from each group were added, followed by 100 μL of cell suspension in each well.

[0198] After adding the cells, gently tap the edges of the plate to spread the cells evenly, then place the plate in an incubator and incubate at 37°C and 5% CO2 for 16-20 hours (be careful not to move the plate during incubation).

[0199] Day 2: (Aseptic techniques are not required) The cells in the plate were removed, and the plate was washed twice with PBS to completely remove the cells, with each wash being 200 μL / well.

[0200] The plate was washed five times with washing buffer, 250 μL per well each time.

[0201] 100 μL of diluted detection antibody was added to each well, and the system was incubated at room temperature for 2 hours or overnight at 4°C.

[0202] The detection antibody was removed, and the plate was washed five times with washing buffer, 250 μL per well each time.

[0203] 100 μL of diluted conjugate was added to each well, and the system was incubated in the dark at room temperature for 1 hour.

[0204] The conjugate was removed, and the plate was washed five times with washing buffer, 250 μL per well each time.

[0205] 100 μL of freshly prepared AEC colorimetric solution was added to each well, and the system was incubated in the dark at room temperature for 30 minutes until a clear spot appeared.

[0206] The color-developing solution was removed, and the color development was stopped each time with 50 μL of deionized water per well.

[0207] The excess liquid was removed from the plate, and the plate was left to dry completely at room temperature in a dark place.

[0208] Spots were counted using an ELISPOT reader. In vitro differentiated antibody-secreting cells were killed in vitro for 5 hours with NT, CD19 CAR-T, BCMA CAR-T, and BCMA-CD19 dual-target CAR-T cells. Then, an ELISPOT experiment was performed to detect the absolute number of cells capable of secreting IgG among the remaining cells, thereby detecting the cell-killing ability of the three CAR-T cells against all antibody-secreting cells. At the end of the experiment, the experiment was photographed and analyzed with a CTL enzyme-coupled fluorescence reader. Photographs showing the absolute number of spots are shown in Figure 8, and the statistical analysis of the absolute number of spots is as follows. The results showed that the BCMA-CD19 dual-target CAR-T group had the fewest remaining total antibody-secreting cells. The results are shown in Figure 8. As can be seen from the results in Figure 8, BCMA-CD19 CAR-T cells were able to eliminate more antibody-secreting cells than CD19-CAR-T cells and BCMA-CAR-T cells, and have a stronger cell-killing ability.

[0209] Example 5: In vitro assay for the death of B cells and antibody-secreting cells The concentrations of primary B cells and in vitro differentiated antibody-secreting cells isolated from PBMCs were 4 × 10⁻⁶, respectively. 5 cells / mL and 5 × 10 5 The concentration was cells / mL.

[0210] Effector cells were prepared, and the concentration of CAR-positive cells was increased to 12 × 10⁻⁶. 5 cells / mL and 5 × 10 5The cells were adjusted to cells / mL, and CAR-positive cells were sequentially diluted according to the planned effector to target (E / T) ratio.

[0211] A clean 96-well plate was taken and marked for experimental groups. Three parallel wells were set for each group, 100 μL of target cell suspension and 100 μL of effector cell suspension were sequentially added to the correspondingly marked wells, bringing the total volume to 200 μL.

[0212] Effector cells and target cells were co-incubated in a 37°C incubator for 5 hours.

[0213] After 5 hours of incubation, the cells were centrifuged at 300 g for 5 minutes, and two 50 μL samples were collected from each well for CBA detection.

[0214] Thereafter, 1 μL of FITC-anti-CD3, 1 μL of APC-anti-CD19, and 1 μL of PE-anti-BCMA antibody were added to each original cell well, the system was mixed uniformly, and stained at 4°C for 25 minutes. After staining, the cells were washed once with FACS buffer, then resuspended in 120 μL of DAPI diluent (1:4800 V / V), stained at room temperature in the dark for 5 minutes, and detected immediately by instrument.

[0215] Samples with equal absolute volumes were collected from each well to analyze the absolute number of viable B cells and antibody-secreting cells in the remaining samples.

[0216] Data processing: The percentage of specific killing of ASCs was calculated according to the following formula: CAR-T cell killing percentage (%) = (CD3 in target cell group - DAPI - Absolute number of cells - absolute number of CD3 - DAPI - cells in the co-incubation group of CAR-T and target cells) / absolute number of CD3-DAPI- cells in the target cell group.

[0217] The percentage of specific B cell death was calculated according to the following formula: CAR-T cell death percentage (%) = (CD19 in the target cell population) + Absolute number of DAPI cells, CAR-T cells, and CD19 in the target cell co-incubation group + (absolute number of DAPI cells) / CD19 in target cell population + The absolute number of DAPI cells. The results are shown in Tables 3-5 and Figures 9-20. From the results in Tables 3-5 and Figures 9-20, BCMA-CD19 CAR-T cells have a stronger ability to kill primary B cells and antibody-secreting cells. The cytokine secretion levels after death in primary B cells and antibody-secreting cells were significantly lower than those of CD19-CAR-T and BCMA-CAR-T cells.

[0218] The experiment shown in Figure 10 was conducted as follows: B cells were isolated and purified from PBMCs and incubated in vitro with BCMA-CD19 dual-target CAR-T cells for 5 hours. The absolute number of remaining viable B cells was then detected, and the ability of BCMA-CD19 dual-target CAR-T cells to kill B cells in vitro was statistically analyzed. In the figure, RD-012C20221001 (BCMA-CD19-L1) is a BCMA-CD19 dual-target CAR-T cell, and RD-012C20221001-NT is a T cell control from the same donor source. Refer to Figure 10 for specific cell death values ​​for each group. The results show that BCMA-CD19 dual-target CAR-T cells can effectively eliminate primary B cells in vitro.

[0219] [Table 3] E / T = Effector to Target Ratio

[0220] [Table 4] E / T = Effector to Target Ratio

[0221] Table 3 and Figures 12-16 show cytokine secretion levels after primary B cells were killed by BCMA-CD19 dual-target CAR-T cells (5 hours).

[0222] The cytokine release results showed that a certain amount of IFN-r, which is related to the efficiency of CAR-T cell death, and a small amount of the cytokine IL-2, which is related to proliferation, were indeed released during the death process, while other factors were released in smaller amounts. This cytokine release is similar to that which has killed tumor cells in the past, and the cytokine secretion pattern suggests that it may not be entirely different from existing CRS data in the human body when used to treat SLE, which has certain implications for its safety.

[0223] [Table 5] E / T = Effector to Target Ratio

[0224] Table 4 and Figure 19 show the situation 5 hours after the death of antibody-secreting cells by BCMA-CD19 dual-target CAR-T cells.

[0225] [Table 6] E / T = Effector to Target Ratio

[0226] Table 5 and Figure 20 show the cytokine secretion levels of BCMA-CD19 dual-target CAR-T cells 5 hours after antibody-secreting cells were killed.

[0227] Example 6: Detection of cytokines in the supernatant Solution preparation: Preparation of mixed beads: 808 μL each of IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ beads, totaling 4.848 mL, were added. 19.392 mL of assay diluent was then added while shaking, and the mixture was homogeneously mixed to obtain a total volume of mixed beads of 24.24 mL.

[0228] Sample dilution: The sample was diluted 1:1 with assay diluent, and the total volume of the diluted sample was 50 µL per test.

[0229] Standard preparation: 2 mL of assay diluent was added to the standard powder, and the mixture was left to stand at room temperature for 15 minutes, and used as the top standard. Thereafter, the standard was serially diluted 2-fold with assay diluent, and the final dilution was used as the assay diluent.

[0230] Operational steps: Take a 96-well U-bottom plate, add 50 µL of mixed beads + 50 µL of standard / sample + 50 µL of human TH1-TH2 PE detection reagent in sequence, shake the plate for 5 minutes, and leave it to stand in the dark at room temperature for 3 hours.

[0231] 100 µL of washing buffer was added, the system was mixed well, centrifuged at 300 g for 5 minutes, and the supernatant was discarded.

[0232] The beads were resuspended in 100 µL of washing buffer.

[0233] The system was shaken for 2 minutes, and loaded into the instrument with a sample volume of 50 µL.

[0234] Data analysis The coefficient of determination R² of the linear equation of the calibration curve is 0.99 or more. For the results of cytokine concentration, cytokine concentration was analyzed by FCAP Array™ (BD).

[0235] Example 7: Depletion of B cells in peripheral blood samples from SLE patients Peripheral blood samples were collected from SLE patients, and diluted 1:1 with DPBS for later use.

[0236] Ficoll separation medium at room temperature was taken, and Ficoll and the diluted peripheral blood sample were added at a volume ratio of 1:3.

[0237] The acceleration and deceleration of the centrifuge were adjusted to 4 and 3 respectively, the centrifugation speed was 1000 g, and the system was centrifuged at room temperature for 20 minutes.

[0238] PBMC cells in the buffy coat were pipetted, counted, and centrifuged at 400g for 8 minutes to collect the cells, which were the target cells.

[0239] Target cells, 5 × 10 5 The cells were resuspended in 1640 complete medium at a concentration of cells / mL.

[0240] Effector T cells were gradient-diluted according to the E:T ratio of the experimental design.

[0241] A clean 96-well U-bottom plate was taken, 100 μl each of target cells and effector cells were added, the plate was labeled, and incubated in a 37°C carbon dioxide incubator for 5 hours.

[0242] After incubation, the supernatant was taken and used as the preserved sample.

[0243] The remaining cells were centrifuged and FACS buffer was added. Each 100 μl of FACS buffer contained 1 μl of each of the following antibodies: APC-anti-CD38, BV 421-anti-CD138, BUV 395-anti-CD27, BV 510-anti-CD45, FITC-anti-CD3 / CD14, Pe-Cy 7-anti-CD19, and BV 786-anti-IgD. After mixing, the system was stained in the dark at 4°C for 30 minutes.

[0244] The samples were washed twice with FACS buffer, resuspended a third time in 1:4800 diluted DAPI FACS buffer, and loaded into the machine for detection. Samples of equal absolute volume were collected from each well, and the absolute number of viable B cells and antibody-secreting cells in the remaining samples was analyzed. The results are shown in Figures 21-24. In Figure 21, non-ASC refers to non-antibody-secreting cells, ASC refers to antibody-secreting cells that are positive for CD38 and CD27, CD138-positive ASC refers to plasma cells (PCs), and CD19-PC refers to CD19-negative plasma cells. Subsequent cell death experiments were all analyzed based on the gating described above.

[0245] SLE samples were co-incubated for 5 hours with BCMA-CD19 dual-target CAR-T cells and control NT cells according to a specific ratio. The absolute number and percentage changes in B cells, antibody-secreting cells, and plasma cells in the samples after incubation were then detected. Flow cytometry images and statistical absolute numbers of B cells are shown in Figure 23. Statistical changes in B cells in SLE samples and the absolute number of remaining cells after death are also shown in Figure 23. It can be seen that BCMA-CD19 dual-target CAR-T cells can significantly kill B cells in SLE samples.

[0246] Figure 23 shows flow cytometry images and statistical absolute numbers of ASC cells. Figure 23B shows the changes in antibody-secreting cells in SLE samples and the statistical absolute number of remaining cells after cell death. It can be seen that BCMA-CD19 dual-target CAR-T receptors can significantly kill antibody-secreting cells in SLE samples.

[0247] Flow cytometry images and the statistical absolute number of plasma cells are shown in Figure 24. Changes in plasma cells and the statistical absolute number of residual cells in the SLE sample are shown in Figure 24 below. It can be seen that BCMA-CD19 dual-target CAR-T can significantly kill plasma cells in the SLE sample.

[0248] The results in Figures 22-24 demonstrate that BCMA-CD19 CAR-T cells can kill B cells and ASC cells (including CD138+ plasma cells) in SLE patients in vitro.

[0249] Those skilled in the art should recognize that, although several exemplary embodiments of the Disclosure have been shown and described in detail herein, many other variations or modifications in line with the principles of the Disclosure can be determined or inferred directly from the Disclosure without departing from the spirit and scope of the Disclosure. Therefore, the scope of the Disclosure should be understood and deemed to encompass all such other variations or modifications.

[0250] Example 8: BCMA and CD19 antigen density in B cell subsets across Sjögren's syndrome, scleroderma or systemic sclerosis (SSc), myositis, IgA nephropathy, and healthy donors.

[0251] In Examples 8-13, a bispecific CAR with a loop structure (e.g., formula 1a)) was used. The ability to deplete BCMA+ and CD19+ cells depends on the antigen density on the cell surface. Fresh or frozen peripheral blood mononuclear cells (PBMCs) from donors with Sjögren's syndrome, scleroderma or systemic sclerosis (SSc), or myositis or IgA nephropathy, as well as from healthy donors, were evaluated for CD19 and BCMA antigen density.

[0252] As shown in Figure 30, target expression of CD19 and BCMA is broadly similar across B cell subsets and disease indications, with the exception of the elevated expression observed in myositis patients.

[0253] Example 9: A representative gating strategy for an in vitro BCMA-CD19 CAR-T cell death assay.

[0254] All PMBC Sjögren's syndrome, scleroderma or systemic sclerosis (SSc), myositis, IgA nephropathy, and healthy donors were counted and seeded for flow cytometry staining. After thawing, cells were stained with the following antibodies (Live / Dead staining, CD20, dumpgate-CD3, CD14, CD16, and CD56, CD19, CD27, CD38, IgD, CD138). All cells shown in Figure 30 are lymphocytes defined by forward scatter / lateral scatter (FSC / SSC), live cells, dumpgate-negative, CD20+ / -, and CD19+ / low, DN2 cells (IgD-, CD27-), memory B cells (CD27+IgD-), naive B cells (IgD+CD27-), and NCSM (non-class-switched memory, IgD+CD27).

[0255] The gating strategy shown in Figure 31A illustrates how target cells for both arms of CAR-T cells were identified for depletion rate calculations after co-culture with BCMA-CD19 CAR-T cells. In vitro death of BCMA+ and CD19+ targets is shown in Figure 31B.

[0256] Example 10: BCMA-CD19 CAR-T cells deplete in vitro differentiated BCMA+ targets and CD19 targets from healthy donors.

[0257] The BCMA-CD19 CAR-T cell research lot was prepared from fresh peripheral blood of individual healthy donors participating in the AstraZeneca blood donation program. Briefly, PBMCs from healthy donors were collected from the blood by Ficol gradient centrifugation, and CD4 and CD8-positive T cells were concentrated from the leukocyte fraction. The isolated T cells were then activated, transduced with the BCMA-CD19 BZ lentiviral vector, grown in culture flasks, washed, harvested, and frozen in cryopreservation medium.

[0258] Naive B cells were isolated from frozen healthy donor PBMCs using a naive B cell isolation kit (Stemcell). After isolation, the naive B cells were seeded and differentiated using a proprietary cytokine mixture to drive B cell differentiation. Five days after differentiation, the differentiated B cells were co-cultured for 24 hours with BCMA-CD19 CAR-T cells or untransduced T cells at effector:target ratios of 1.5:1, 0.75:1, 0.37:1, 0.18:1, 0.093:1, and 0.0461:1. The co-cultured cells were then stained with the following reagents: (Live / Dead staining, CD20, CD19, CD27, CD38, BCMA, CD3, CD4, CD8, CD69). The death of differentiated plasmablasts (live, CD3-, CD27+CD38+) and CD19+ targets (live CD3-, CD27-, CD38+ / -, CD19+CD20+) was evaluated by flow cytometry. The depletion percentage was expressed as 1 - (percentage of targets in the experimental well / average percentage of targets in the well containing only stem cells). * It was calculated using 100.

[0259] As shown in Figure 32, the bispecific CAR was able to efficiently deplete BCMA+ and CD19+ targets in healthy controls. Dose-dependent depletion (E:T) by BCMA-CD19 CAR-T cells was observed.

[0260] Example 11: Co-culture of BCMA-CD19 CAR-T cells with in vitro differentiated BCMA+ and CD19+ targets derived from healthy donor PBMCs drives dose-dependent secretion of cytokines associated with T cell activation.

[0261] After 24 hours of co-culture of differentiated B cells and BCMA-CD19 CAR-T cells, the supernatant was collected, divided, and stored at -80°C for subsequent Meso Scale Discover (MSD) analysis. BCMA-CD19 CAR-T cells demonstrated dose-dependent secretion of IFNγ, IL-2, IL-4, IL-6, IL-10, and TNFα in response to targets derived from a healthy donor, as seen in Figure 33. This indicates target-specific activation of CAR T cells.

[0262] Example 12: BCMA-CD19 CAR-T cells deplete in vitro differentiated BCMA+ targets and CD19 targets from Sjögren's syndrome, scleroderma or systemic sclerosis (SSc), myositis donors, and IgA nephropathy donors.

[0263] Primary human naive B cells (IgD+CD27-selected) were isolated from frozen Sjögren's syndrome, scleroderma or systemic sclerosis (SSc), myositis, and patient PBMCs. The naive B cells were then differentiated using a proprietary cytokine mixture to drive plasmablast differentiation (BCMA+ B cells). Five days after differentiation, the cells were co-cultured with BCMA-CD19 Bz CAR T cells (manufactured using a preclinical manufacturing process) or untransduced T cells (non-targeted) at various effector:target ratios. BCMA-CD19 CAR-T cells were able to similarly deplete BCMA+ and CD19+ targets in Sjögren's syndrome (Figure 34), scleroderma or systemic sclerosis (Figure 35), myositis (Figure 36), and IgA nephropathy (Figure 37), as observed in healthy controls.

[0264] Example 13: Co-culture of BCMA-CD19 CAR-T cells with in vitro differentiated BCMA+ and CD19+ targets derived from donors with Sjögren's syndrome, scleroderma, myositis, and IgA nephropathy similarly drives dose-dependent secretion of cytokines associated with T cell activation.

[0265] After 24 hours of co-culture of differentiated B cells and BCMA-CD19 CAR-T cells, the supernatant was collected, divided, and stored at -80°C for subsequent MSD analysis. BCMA-CD19 CAR-T cells showed dose-dependent secretion of IFNγ, IL-2, IL-4, IL-6, IL-10, and TNFα in response to targets derived from Sjögren's syndrome (Figure 38), scleroderma or systemic sclerosis (Figure 39), myositis (Figure 40), and IgA nephropathy (Figure 41).

Claims

1. A bispecific chimeric antigen receptor (CAR) for treating and / or preventing autoimmune diseases, characterized in that the first target of the CAR is CD19 and the second target is BCMA.

2. The following medications: (1) Agents that enhance the efficacy of cells containing CAR nucleic acids or CAR polypeptides, (2) Agents for improving one or more adverse effects associated with the administration of cells containing CAR nucleic acids or CAR polypeptides, and / or (3) The bispecific CAR according to claim 1, in combination with one or more additional agents for treating diseases related to BCMA and CD19.

3. A bispecific CAR according to claim 1 or 2, wherein the antibody targeting BCMA or its antigen-binding fragment, or the antibody targeting CD19 or its antigen-binding fragment, is independently selected from single-chain antibodies, microantibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, disulfide-bond-stabilized Fv protein ("dsFv"), and single-domain antibodies (sdAb, nanoantibodies), chimeric antibodies, humanized antibodies, single-domain antibodies, bispecific antibodies or multispecific antibodies, binding ligands, or protein domains, wherein the bispecific CAR according to claim 1 or 2.

4. The bispecific CAR according to any one of claims 1 to 3, wherein the antigen-binding fragment targeting BCMA or the antigen-binding fragment targeting CD19 is scFv.

5. The antibody targeting BCMA or its antigen-binding fragment comprises a heavy chain variable region (VH, BCMA) including a complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 5, an HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and an HCDR3 containing the amino acid sequence of SEQ ID NO:

7. A bispecific CAR according to any one of claims 1 to 4, comprising a complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 8, an LCDR2 containing the amino acid sequence of SEQ ID NO: 9, and a light chain variable region (VL, BCMA) containing the amino acid sequence of SEQ ID NO:

10.

6. The antibody or antigen-binding fragment targeting CD19 comprises a complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 11, a heavy chain variable region (VH, CD19) containing the amino acid sequence of SEQ ID NO: 12, and a heavy chain variable region (VH, CD19) containing the amino acid sequence of SEQ ID NO:

13. A bispecific CAR according to any one of claims 1 to 4, comprising a complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 14, an LCDR2 containing the amino acid sequence of SEQ ID NO: 15, and a light chain variable region (VL, CD19) containing the amino acid sequence of SEQ ID NO:

16.

7. The antibody or antigen-binding fragment thereof that targets the BCMA, A heavy chain variable region (VH, BCMA) containing complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 5, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 7, It includes a complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 8, an LCDR2 containing the amino acid sequence of SEQ ID NO: 9, and a light chain variable region (VL, BCMA) containing the amino acid sequence of SEQ ID NO: 10, The antibody or antigen-binding fragment that targets CD19, A heavy chain variable region (VH, CD19) containing complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 11, HCDR2 containing the amino acid sequence of SEQ ID NO: 12, and HCDR3 containing the amino acid sequence of SEQ ID NO: 13, A bispecific CAR according to any one of claims 1 to 4, comprising a complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 14, an LCDR2 containing the amino acid sequence of SEQ ID NO: 15, and a light chain variable region (VL, CD19) containing the amino acid sequence of SEQ ID NO:

16.

8. The bispecific CAR according to any one of claims 1 to 7, wherein the VH and BCMA include an amino acid sequence having at least 95% identity with SEQ ID NO:

1.

9. The bispecific CAR according to any one of claims 1 to 8, wherein the VL and BCMA include an amino acid sequence having at least 95% identity with SEQ ID NO:

2.

10. The bispecific CAR according to any one of claims 1 to 9, wherein the VH, CD19 comprises an amino acid sequence having at least 95% identity with SEQ ID NO:

3.

11. The bispecific CAR according to any one of claims 1 to 10, wherein the VL, CD19 comprises an amino acid sequence having at least 95% identity with SEQ ID NO:

4.

12. The bispecific CAR according to any one of claims 1 to 11, wherein the VH and BCMA include amino acid sequences having at least 95% sequence identity with SEQ ID NO: 1, and the VL and BCMA include amino acid sequences having at least 95% sequence identity with SEQ ID NO:

2.

13. The bispecific CAR according to any one of claims 1 to 12, wherein the VH, CD19 region includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3, and the VL, CD19 region includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

4.

14. The bispecific CAR according to any one of claims 1 to 13, wherein the VH and BCMA include the amino acid sequence of SEQ ID NO: 1, and the VL and BCMA include the amino acid sequence of SEQ ID NO:

2.

15. The bispecific CAR according to any one of claims 1 to 14, wherein VH, CD19 comprises the amino acid sequence of SEQ ID NO: 3, and VL, CD19 comprises the amino acid sequence of SEQ ID NO:

4.

16. The structure of the bispecific CAR is selected from one of the following formulas: 1a) L-VL, CD19-VH, BCMA-VL, BCMA-VH, CD19-H-TM-C-CD3ζ; 1b) L-VL, CD19-VL, BCMA-VH, BCMA-VH, CD19-H-TM-C-CD3ζ; 2a) L-VH, CD19-VL, BCMA-VH, BCMA-VL, CD19-H-TM-C-CD3ζ; 2b) L-VH, CD19-VH, BCMA-VL, BCMA-VL, CD19-H-TM-C-CD3ζ; 3a) L-VL, CD19-VH, CD19-H-TM-C-CD3ζ-2A peptide-L-VH, BCMA-VL, BCMA-H-TM'-C-CD3ζ; 3b) L-VH, CD19-VL, CD19-H-TM-C-CD3ζ-2A peptide-L-VH, BCMA-VL, BCMA-H-TM'-C-CD3ζ; 3c) L-VL, CD19-VH, CD19-H-TM-C-CD3ζ-2A peptide-L-VL, BCMA-VH, BCMA-H-TM'-C-CD3ζ; 3d) L-VH, CD19-VL, CD19-H-TM-C-CD3ζ-2A peptide-L-VL, BCMA-VH, BCMA-H-TM'-C-CD3ζ; 4a) L-VH, BCMA-VL, BCMA-H-TM-C-CD3ζ-2A peptide-L-VL, CD19-VH, CD19-H-TM'-C-CD3ζ; 4b) L-VL, BCMA-VH, BCMA-H-TM-C-CD3ζ-2A peptide-L-VL, CD19-VH, CD19-H-TM'-C-CD3ζ; 4c) L-VH, BCMA-VL, BCMA-H-TM-C-CD3ζ-2A peptide-L-VH, CD19-VL, CD19-H-TM'-C-CD3ζ; 4d) L-VL, BCMA-VH, BCMA-H-TM-C-CD3ζ-2A peptide-L-VH, CD19-VL, CD19-H-TM'-C-CD3ζ; During the ceremony, Each of 1a), 1b), 2a), and 2b) includes a loop structure formed by an antibody or antigen-binding fragment targeting BCMA and CD19, The "-" symbol independently represents a linker peptide or peptide bond. L is either absent or is a signal peptide sequence. VH and BCMA are the variable heavy chain regions of the anti-BCMA antibody, and VL and BCMA are the variable light chain regions of the anti-BCMA antibody. VH, CD19 is the variable region of the heavy chain of the anti-CD19 antibody, and VL, CD19 is the variable region of the light chain of the anti-CD19 antibody. H is the hinge region, TM is the first transmembrane domain, TM' is the second transmembrane domain, The 2A peptide is selected from P2A, T2A, E2A, or F2A. C is a co-stimulus signaling molecule, The bispecific CAR according to any one of claims 1 to 15, wherein CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

17. The bispecific CAR according to claim 16, wherein L comprises a domain derived from one or more of CD8, CD28, GM-CSF, CD4, and CD137.

18. The bispecific CAR according to claim 16, wherein L comprises a domain derived from CD8.

19. The bispecific CAR according to claim 16, wherein L comprises the amino acid sequence of SEQ ID NO:

25.

20. The bispecific CAR according to claim 16, wherein H comprises a domain derived from one or more of CD8, CD28, and CD137.

21. The bispecific CAR according to claim 16, wherein H comprises one of the amino acid sequences of SEQ ID NOs: 17 to 19.

22. The bispecific CAR according to claim 21, wherein H comprises the amino acid sequence of SEQ ID NO:

19.

23. The bispecific CAR according to claim 16, wherein TM or TM' includes a domain derived from one or more of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

24. The bispecific CAR according to claim 16, wherein TM or TM' includes a domain derived from CD8.

25. The bispecific CAR according to claim 16, wherein TM or TM' comprises the amino acid sequence of SEQ ID NO:

20.

26. The bispecific CAR according to claim 16, wherein C comprises a domain derived from one or more of the following: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, and TLR2.

27. The bispecific CAR according to claim 16, wherein C comprises a domain derived from one or more of 4-1BB.

28. The bispecific CAR according to claim 16, wherein C comprises the amino acid sequence of SEQ ID NO:

21.

29. The bispecific CAR according to claim 16, wherein CD3ζ contains the amino acid sequence of SEQ ID NO:

22.

30. The bispecific CAR according to claim 16, wherein in at least one case, "-" includes one or more amino acid sequences of SEQ ID NO:

23.

31. The bispecific CAR according to claim 16, wherein in at least one case, "-" includes the amino acid sequence of SEQ ID NO:

24.

32. H contains the amino acid sequence of SEQ ID NO: 19, L contains the amino acid sequence of SEQ ID NO: 25, VH and BCMA contain the amino acid sequence of SEQ ID NO:

1. VL and BCMA contain the amino acid sequence of SEQ ID NO:

2. VH, CD19 contains the amino acid sequence of SEQ ID NO: 3, VL, CD19 contains the amino acid sequence of SEQ ID NO: 4, TM contains the amino acid sequence of SEQ ID NO: 20, C contains the amino acid sequence of SEQ ID NO: 21, The bispecific CAR according to claim 16, wherein CD3ζ contains the amino acid sequence of SEQ ID NO:

22.

33. The bispecific CAR according to claim 33, wherein the bispecific CAR has the structure according to formula 1a).

34. A bispecific CAR containing the amino acid sequence of SEQ ID NO: 26 for the treatment and / or prevention of autoimmune diseases.

35. The bispecific CAR according to any one of claims 1 to 34, wherein the bispecific CAR is administered in an amount of 0.5 ± 20% × 10^5 cells / kg.

36. The bispecific CAR according to any one of claims 1 to 34, wherein the bispecific CAR is administered in an amount of 1.0 ± 20% × 10^5 cells / kg.

37. The bispecific CAR according to any one of claims 1 to 34, wherein the bispecific CAR is administered in an amount of 2.0 ± 20% × 10^5 cells / kg.

38. The bispecific CAR according to any one of claims 1 to 34, wherein the bispecific CAR is administered in an amount of 3.0 ± 20% × 10^5 cells / kg.

39. The bispecific CAR according to any one of claims 1 to 34, wherein the bispecific CAR is administered in an amount of 0.5 × 10^5 cells / kg, 1.0 × 10^5 cells / kg, 2.0 × 10^5 cells / kg, or 3.0 × 10^5 cells / kg.

40. The bispecific CAR according to any one of claims 1 to 39, further comprising using the treatment and / or prevention in combination with a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

41. The bispecific CAR according to any one of claims 1 to 40, wherein the bispecific CAR kills or inhibits CD19-positive B cells, plasmablasts, and BCMA-positive plasma cells.

42. The bispecific CAR according to any one of claims 1 to 41, wherein the autoimmune disease is a B cell-mediated autoimmune disease.

43. B-cell-mediated autoimmune diseases include systemic lupus erythematosus (SLE), glomerulonephritis, such as autoimmune chronic kidney disease, lupus nephritis, acute glomerulonephritis, immunonephritis, membranous nephropathy, IgA nephropathy, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV); scleroderma or systemic sclerosis (SSc); myositis or idiopathic inflammatory myositis, including dermatomyositis, polymyositis, immune-mediated necrotizing myopathy (IMNM), anti-synthesis syndrome, inclusion body myositis, and myositis duplication; multiple sclerosis (MS); inflammatory bowel disease (IBD); rheumatoid arthritis (RA); Sjögren's syndrome (SS); autoimmune hemolytic anemia; neuromyelitis optica (NMO); neuromyelitis optica spectrum disorder (NMOSD); and idiopathic hemolytic anemia. The bispecific CAR according to claim 42, comprising one or more of the following: thracnopenic purpura (ITP); systemic autoimmune small vessel vasculitis syndrome or polyangiitis associated with antineutrophil cytoplasmic antibodies; Wegener's granulomatosis (GPA), eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss syndrome); pemphigus vulgaris; autoimmune encephalitis; pemphigus vulgaris; myasthenia gravis; antiphospholipid antibody syndrome; Chagas disease; Graves' disease; polyarteritis nodosa; pulmonary hemorrhagic nephritis syndrome; Kawasaki disease, amyloidosis; monoclonal globulin of unknown significance, POEMS syndrome; Crohn's disease; ulcerative colitis; adult-onset Still's disease; and chronic progressive demyelinating encephalopathy (CIDP).

44. The bispecific CAR according to any one of claims 1 to 43, wherein the autoimmune disease is systemic lupus erythematosus, and systemic lupus erythematosus includes moderate to severe refractory systemic lupus erythematosus, lupus nephritis, active lupus nephritis, and active systemic lupus erythematosus without renal involvement.

45. The bispecific CAR according to any one of claims 1 to 43, wherein the autoimmune disease is myositis.

46. The bispecific CAR according to any one of claims 1 to 43, wherein the autoimmune disease is glomerulonephritis.

47. The bispecific CAR according to claim 46, wherein the glomerulonephritis is at least one of autoimmune chronic kidney disease, lupus nephritis, acute glomerulonephritis, immunonephritis, membranous nephropathy, and IgA nephropathy.

48. The bispecific CAR according to any one of claims 1 to 43 or 46 to 47, wherein the autoimmune disease is membranous nephropathy.

49. The bispecific CAR according to any one of claims 1 to 43 or 46 to 47, wherein the autoimmune disease is IgA nephropathy.

50. The bispecific CAR according to any one of claims 1 to 43, wherein the autoimmune disease is multiple sclerosis.

51. The bispecific CAR according to any one of claims 1 to 43, wherein the autoimmune disease is scleroderma or systemic sclerosis (SSc).

52. The bispecific CAR according to any one of claims 1 to 43, wherein the autoimmune disease is myasthenia gravis.

53. A pharmaceutical composition comprising a bispecific CAR according to any one of claims 1 to 34 for the treatment and / or prevention of autoimmune diseases.

54. The pharmaceutical composition according to claim 53, wherein the bispecific CAR is administered in an amount of 0.5 ± 20% × 10^5 cells / kg.

55. The pharmaceutical composition according to claim 53, wherein the bispecific CAR is administered in an amount of 1.0 ± 20% × 10^5 cells / kg.

56. The pharmaceutical composition according to claim 53, wherein the bispecific CAR is administered in an amount of 2.0 ± 20% × 10^5 cells / kg.

57. The pharmaceutical composition according to claim 53, wherein the bispecific CAR is administered in an amount of 3.0 ± 20% × 10^5 cells / kg.

58. The pharmaceutical composition according to claim 53, wherein the bispecific CAR is administered in an amount of 0.5 × 10^5 cells / kg, 1.0 × 10^5 cells / kg, 2.0 × 10^5 cells / kg, or 3.0 × 10^5 cells / kg.

59. The pharmaceutical composition according to any one of claims 53 to 58, further comprising using the treatment and / or prevention in combination with a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

60. The pharmaceutical composition according to any one of claims 53 to 59, wherein the bispecific CAR kills or inhibits CD19-positive B cells, plasmablasts, and BCMA-positive plasma cells.

61. The pharmaceutical composition according to any one of claims 53 to 60, wherein the autoimmune disease is a B cell-mediated autoimmune disease.

62. B-cell-mediated autoimmune diseases include systemic lupus erythematosus (SLE), glomerulonephritis, such as autoimmune chronic kidney disease, lupus nephritis, acute glomerulonephritis, immunonephritis, membranous nephropathy, IgA nephropathy, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV); scleroderma or systemic sclerosis (SSc); myositis or idiopathic inflammatory myositis, including dermatomyositis, polymyositis, immune-mediated necrotizing myopathy (IMNM), anti-synthesis syndrome, inclusion body myositis, and myositis duplication; multiple sclerosis (MS); inflammatory bowel disease (IBD); rheumatoid arthritis (RA); Sjögren's syndrome (SS); autoimmune hemolytic anemia; neuromyelitis optica (NMO); neuromyelitis optica spectrum disorder (NMOSD); idiopathic The pharmaceutical composition according to claim 61, comprising one or more of the following: thrombocytopenic purpura (ITP); systemic autoimmune small vessel vasculitis syndrome or polyangiitis associated with antineutrophil cytoplasmic antibodies; Wegener's granulomatosis (GPA), eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss syndrome); pemphigus vulgaris; autoimmune encephalitis; pemphigus vulgaris; myasthenia gravis; antiphospholipid antibody syndrome; Chagas disease; Graves' disease; polyarteritis nodosa; pulmonary hemorrhagic nephritis syndrome; Kawasaki disease, amyloidosis; monoclonal globulin of unknown significance, POEMS syndrome; Crohn's disease; ulcerative colitis; adult-onset Still's disease; and chronic progressive demyelinating encephalopathy (CIDP).

63. The pharmaceutical composition according to any one of claims 53 to 62, wherein the autoimmune disease is systemic lupus erythematosus, and the systemic lupus erythematosus includes moderate to severe refractory systemic lupus erythematosus, lupus nephritis, active lupus nephritis, and active systemic lupus erythematosus without renal involvement.

64. The pharmaceutical composition according to any one of claims 53 to 62, wherein the autoimmune disease is myositis.

65. The pharmaceutical composition according to any one of claims 53 to 62, wherein the autoimmune disease is glomerulonephritis.

66. The pharmaceutical composition according to claim 65, wherein the glomerulonephritis is at least one of autoimmune chronic kidney disease, lupus nephritis, acute glomerulonephritis, immunonephritis, membranous nephropathy, and IgA nephropathy.

67. The pharmaceutical composition according to any one of claims 53 to 62 or 66, wherein the autoimmune disease is membranous nephropathy.

68. The pharmaceutical composition according to any one of claims 53 to 62 or 66, wherein the autoimmune disease is IgA nephropathy.

69. The pharmaceutical composition according to any one of claims 53 to 62, wherein the autoimmune disease is multiple sclerosis.

70. The pharmaceutical composition according to any one of claims 53 to 62, wherein the autoimmune disease is scleroderma or systemic sclerosis (SSc).

71. The pharmaceutical composition according to any one of claims 53 to 62, wherein the autoimmune disease is myasthenia gravis.

72. A method for treating and / or preventing an autoimmune disease, comprising administering a bispecific CAR according to any one of claims 1 to 34 to a subject in need of such treatment.

73. The method according to claim 72, wherein the bispecific CAR is administered in an amount of 0.5 ± 20% × 10^5 cells / kg.

74. The method according to claim 72, wherein the bispecific CAR is administered in an amount of 1.0 ± 20% × 10^5 cells / kg.

75. The method according to claim 72, wherein the bispecific CAR is administered in an amount of 2.0 ± 20% × 10^5 cells / kg.

76. The method according to claim 72, wherein the bispecific CAR is administered in an amount of 3.0 ± 20% × 10^5 cells / kg.

77. The method according to claim 72, wherein the bispecific CAR is administered in an amount of 0.5 × 10^5 cells / kg, 1.0 × 10^5 cells / kg, 2.0 × 10^5 cells / kg, or 3.0 × 10^5 cells / kg.

78. The method according to any one of claims 72 to 77, further comprising using the treatment and / or prevention in combination with a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

79. The method according to any one of claims 72 to 78, wherein the bispecific CAR kills or inhibits CD19-positive B cells, plasmablasts, and BCMA-positive plasma cells.

80. The method according to any one of claims 72 to 79, wherein the autoimmune disease is a B cell-mediated autoimmune disease.

81. B-cell-mediated autoimmune diseases include systemic lupus erythematosus (SLE), glomerulonephritis, such as autoimmune chronic kidney disease, lupus nephritis, acute glomerulonephritis, immunonephritis, membranous nephropathy, IgA nephropathy, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV); scleroderma or systemic sclerosis (SSc); myositis or idiopathic inflammatory myositis, including dermatomyositis, polymyositis, immune-mediated necrotizing myopathy (IMNM), anti-synthesis syndrome, inclusion body myositis, and multiple myositis; multiple sclerosis (MS); inflammatory bowel disease (IBD); rheumatoid arthritis (RA); Sjögren's syndrome (SS); autoimmune hemolytic anemia; neuromyelitis optica (NMO); neuromyelitis optica spectrum disorder (NMOSD); and The method according to claim 80, comprising one or more of the following: thrombocytopenic purpura (ITP); systemic autoimmune small vessel vasculitis syndrome or polyangiitis associated with antineutrophil cytoplasmic antibodies; Wegener's granulomatosis (GPA), eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss syndrome); pemphigus vulgaris; autoimmune encephalitis; pemphigus vulgaris; myasthenia gravis; antiphospholipid antibody syndrome; Chagas disease; Graves' disease; polyarteritis nodosa; pulmonary hemorrhagic nephritis syndrome; Kawasaki disease, amyloidosis; monoclonal globulin of unknown significance, POEMS syndrome; Crohn's disease; ulcerative colitis; adult-onset Still's disease; and chronic progressive demyelinating encephalopathy (CIDP).

82. The method according to any one of claims 72 to 81, wherein the autoimmune disease is systemic lupus erythematosus, and the systemic lupus erythematosus includes moderate to severe refractory systemic lupus erythematosus, lupus nephritis, active lupus nephritis, and active systemic lupus erythematosus without renal involvement.

83. The method according to any one of claims 72 to 81, wherein the autoimmune disease is myositis.

84. The method according to any one of claims 72 to 81, wherein the autoimmune disease is glomerulonephritis.

85. The method according to claim 84, wherein the glomerulonephritis is at least one of autoimmune chronic kidney disease, lupus nephritis, acute glomerulonephritis, immunonephritis, membranous nephropathy, and IgA nephropathy.

86. The method according to any one of claims 72 to 81 or 85, wherein the autoimmune disease is membranous nephropathy.

87. The method according to any one of claims 72 to 81 or 85, wherein the autoimmune disease is IgA nephropathy.

88. The method according to any one of claims 72 to 81, wherein the autoimmune disease is multiple sclerosis.

89. The method according to any one of claims 72 to 81, wherein the autoimmune disease is scleroderma or systemic sclerosis (SSc).

90. The method according to any one of claims 72 to 81, wherein the autoimmune disease is myasthenia gravis.

91. A nucleic acid molecule for treating an autoimmune disease, comprising a nucleotide sequence encoding a bispecific CAR according to any one of claims 1 to 34.

92. A recombinant vector for treating and / or preventing autoimmune diseases, comprising a bispecific CAR according to any one of claims 1 to 34 or a nucleic acid molecule according to claim 91.

93. The recombinant vector according to claim 92, wherein the recombinant vector comprises or is selected from a DNA vector, an RNA vector, a plasmid, a liposome, a particle, a transposon vector, a CRISPR / Cas9 vector, a lentiviral vector, or a viral vector.

94. Cells for treating and / or preventing autoimmune diseases, which are engineered autologous T cells expressing the bispecific CAR described in any one of claims 1 to 34.

95. The nucleic acid according to claim 91, the recombinant vector according to claim 92 or 93, or the cell according to claim 94, wherein the autoimmune disease is a B cell-mediated autoimmune disease.

96. B-cell-mediated autoimmune diseases include systemic lupus erythematosus (SLE), glomerulonephritis, such as autoimmune chronic kidney disease, lupus nephritis, acute glomerulonephritis, immunonephritis, membranous nephropathy, IgA nephropathy, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV); scleroderma or systemic sclerosis (SSc); myositis or idiopathic inflammatory myositis, including dermatomyositis, polymyositis, immune-mediated necrotizing myopathy (IMNM), anti-synthesis syndrome, inclusion body myositis, and myositis duplication; multiple sclerosis (MS); inflammatory bowel disease (IBD); rheumatoid arthritis (RA); Sjögren's syndrome (SS); autoimmune hemolytic anemia; neuromyelitis optica (NMO); neuromyelitis optica spectrum disorder (NMOSD); and idiopathic thrombocytopenia. The nucleic acid, recombinant vector, or cell according to claim 95, comprising one or more of the following: purpura minor (ITP); systemic autoimmune small vessel vasculitis syndrome or polyangiitis associated with antineutrophil cytoplasmic antibodies; Wegener's granulomatosis (GPA), eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss syndrome); pemphigus vulgaris; autoimmune encephalitis; pemphigus vulgaris; myasthenia gravis; antiphospholipid antibody syndrome; Chagas disease; Graves' disease; polyarteritis nodosa; pulmonary hemorrhagic nephritis syndrome; Kawasaki disease, amyloidosis; monoclonal globulin of unknown significance, POEMS syndrome; Crohn's disease; ulcerative colitis; adult-onset Still's disease; and chronic progressive demyelinating cortical encephalopathy (CIDP).

97. A drug or preparation comprising a bispecific CAR according to any one of claims 1 to 34, a nucleic acid molecule according to claim 91, a recombinant vector according to claim 92 or 93, or a cell according to claim 94, and optionally a pharmaceutically acceptable carrier, adjuvant, or excipient.

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