Application of double-target CAR-T cell in treatment of systemic lupus erythematosus and other B-cell autoimmune diseases

Abstract

The invention belongs to the field of immune cell therapy, and particularly relates to application of a double-target CAR-T cell to treatment of systemic lupus erythematosus and other B-cell autoimmune diseases. A first target point of the chimeric antigen receptor is CD19, a second target point of the chimeric antigen receptor is BCMA, and the chimeric antigen receptor with the Loop structure is specifically the chimeric antigen receptor with the Loop structure, and the structure of the chimeric antigen receptor with the Loop structure is shown as follows: L-VL, CD19-VH, BCMA-VL, BCMA-VH and CD19-H-TM-C-CD3zeta; the BCMA-CD19 CAR-T cell transduced by the chimeric antigen receptor can more comprehensively block the way of generating the autoantibody in the process of targeting CD19 and BCMA, and can more completely cover B cells, plasma mother cells and plasma cells which generate the autoantibody and are in a plurality of differentiation stages; therefore, the double-target CAR-T therapy designed on the basis of the concept can be used for treating refractory SLE, and is expected to obtain quicker relieving and deeper and lasting curative effects.

Description

Technical Field

[0001] The present invention belongs to the field of immune cell therapy, and specifically, relates to the use of a dual-target CAR-T cell in the treatment of systemic lupus erythematosus. It also relates to the use of a dual-target CAR-T cell in the treatment of other B cell autoimmune diseases. Background Art

[0002] Before the immune system can protect the body from foreign or dangerous substances, it must first recognize them. These substances include bacteria, viruses, parasites (such as worms), some cancer cells, and even transplanted organs and tissues. These substances have molecules that the immune system recognizes and stimulates a response from the immune system. These molecules are called antigens. Antigens are parts of cells that can be found inside or on the surface of cells (such as bacteria or cancer cells) or viruses.

[0003] When certain white blood cells (B cells and T cells) encounter an antigen, they learn how to attack it, thereby protecting 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 a specific antigen, marking it for attack or neutralizing it directly. The body produces thousands of different kinds of antibodies. Each antibody is specific to a particular antigen. The cells of the immune system remember specific antigens so that they can attack more effectively the next time they encounter them.

[0004] The cells of a person's own tissues also have antigens. Normally, the immune system only responds to foreign or dangerous substances, not to antigens of one's own tissues. However, sometimes the immune system malfunctions and treats one's own tissues as foreign, producing antibodies (called autoantibodies) or immune cells that attack one's own cells or tissues. This reaction is called an autoimmune reaction. It causes inflammation and tissue damage. This reaction may lead to autoimmune diseases.

[0005] A variety of autoimmune diseases have been discovered. These diseases can be systemic, such as systemic lupus erythematosus, which affects the skin, joints, kidneys and central nervous system; they can also be organ-specific, such as type 1 diabetes, which primarily affects the health of the pancreas. Loss of B-cell or T-cell tolerance is often associated with autoimmunity. In fact, human leukocyte antigen (HLA) loci are often directly associated with an increased risk of susceptibility to autoimmune diseases.

[0006] Current immunomodulatory drugs used for autoimmune diseases are broadly effective, non-disease specific, and often cause side effects such as infection and malignancy; it is clear that most patients do not respond well to these treatments, or even do not respond at all; therefore, researchers are in urgent need of developing new drugs or redirecting drugs by conducting molecular and clinical studies on patients with special autoimmune diseases, combined with high-throughput integrated database analysis. At the same time, personalized drugs can complement new diagnostic methods to help researchers better understand the pathogenesis of autoimmune diseases, and researchers also need to conduct more patient-centered clinical trials; prevention of autoimmune diseases should also be part of early intervention. In this article, researchers interpret different types of current therapies, such as synthetic drugs, cell therapies, strategies targeting metabolic pathways and microbiomes, and also emphasize the use of basic research, new technologies, and clinical trials to dynamically combine to help understand more about the pathogenesis of autoimmune diseases and develop new therapies.

[0007] Researchers are increasingly aware of the complexity of autoimmune diseases, but current treatments are based on a simplistic and reductionist understanding of the pathology; in clinical practice, patients are often treated with a step-up, trial-and-error approach, starting with different conventional disease-modifying drugs, such as methotrexate for rheumatoid arthritis, interferon beta for multiple sclerosis, and corticosteroids for inflammatory bowel disease and psoriasis. If the patient does not respond adequately, researchers will upgrade to new drugs, such as JAK inhibitors or biologics; all types of drugs have one thing in common, that is, they are broadly effective, but they are not disease-specific and often have certain side effects, which may lack personalized characteristics compared to the personalized therapies that have entered the field of oncology research; therefore, in order to achieve more specific and personalized treatment for patients with autoimmune diseases, researchers need to have a deeper understanding of the complexity of individual autoimmune diseases and how the disease manifests in different patients.

[0008] Among them, systemic lupus erythematosus (SLE) is an autoimmune inflammatory connective tissue disease involving multiple organs. Its pathogenesis is complex and is mainly related to the immune system's misidentification of endogenous antigens, the appearance of autoreactive B cells and the production of autoantibodies. The age of onset of SLE is mostly between 20 and 40 years old, and the onset has a clear gender tendency, with the male-female ratio being about 1:9-12. According to literature statistics, the global prevalence of SLE is about 43.7 / 100,000 people / year, and the incidence in different regions varies greatly: about 3.7-4.9 / 100,000 people / year in North America, 1.5-7.4 / 100,000 people / year in Europe, about 15.9 / 100,000 people / year in Central Asia, and about 110.82 / 100,000 people / year in tropical Latin America. The prevalence in China is about 47.53 / 100,000 people / year, with a total number of patients of about 700,000.

[0009] Systemic lupus erythematosus is an immune-mediated systemic disease that causes various abnormalities of the skin, kidneys, blood, and musculoskeletal systems. The clinical symptoms of systemic lupus erythematosus are heterogeneous and vary greatly from person to person. Common manifestations of general symptoms may include joint pain and arthritis, cheek and other rashes, pleurisy or pericarditis, involvement of the kidneys or central nervous system, and blood cell reductions. The manifestations of systemic lupus erythematosus are variable, and the course of the disease is often alternating between acute relapses and remissions. The severity of SLE disease ranges widely and may pose significant challenges due to cumulative organ damage and coagulation defects, and may be fatal depending on the extent of organ involvement.

[0010] Current status of treatment for systemic lupus erythematosus

[0011] At present, for SLE, long-term use of hydroxychloroquine (HCQ) is recommended as the basic treatment. In addition, there are nonsteroidal anti-inflammatory drugs, low-dose hormones and immunosuppressants. Cyclophosphamide can be used as the initial treatment for severe SLE or as a rescue treatment when other drugs are ineffective. However, a small number of critically ill or refractory patients still respond poorly to the above traditional drug treatments and have a poor prognosis. In addition, the adverse reactions related to non-selective immunosuppressants, such as infection and metabolic abnormalities, also gradually increase with the extension of the patient's medication time. On the basis of traditional treatment, immune targeted therapy is also increasingly used in clinical practice. However, although traditional immunosuppressants such as dexamethasone, cyclophosphamide and anti-CD20 specific antibodies can eliminate short-lived plasmablasts and plasma cells, they cannot completely eliminate antibody-secreting cells (ASCs) that secrete autoantibodies, including long-lived plasma cells (LLPCs), which leads to repeated attacks of SLE. Therefore, no long-term significant effects have been achieved in clinical practice. In summary, improvements in the treatment of systemic lupus erythematosus are particularly important.

[0012] Reasons for target selection for CAR-T cell therapy of SLE

[0013] B cells play a central role in the pathogenesis of SLE through both antibody-dependent and antibody-independent functions.

[0014] Numerous evidences indicate that the loss of self-tolerance during B cell development leads to the occurrence of autoimmunity and the production of autoantibodies. Antinuclear antibodies (ANA) are autoantibodies produced against DNA, RNA, protein or molecular complexes of these substances in the cell nucleus and are the main serological markers of systemic lupus erythematosus. Antinuclear antibodies were discovered in the diagnosis of lupus very early, and their role in SLE is now receiving increasing attention. First, the hematological symptoms of many autoimmune diseases are directly driven by autoantibodies, such as Coombs-positive (anti-human globulin or red blood cell) hemolytic anemia and immune platelet deficiency. Second, an inherent histopathological feature of lupus nephritis is the presence of significant glomerular immune complexes composed of multiple subtypes of immunoglobulins. In the 1960s, anti-double-stranded DNA antibodies (anti-dsDNA antibodies) were eluted from the kidneys of lupus nephritis. In addition, studies have shown that a variety of autoantibodies other than anti-dsDNA can be eluted from the kidneys of lupus nephritis. Finally, direct evidence that lupus autoantibodies can lead to systemic inflammation comes from neonatal lupus erythematosus (NLE), in which maternal autoantibodies can cause NLE by passive transfer across the placenta.

[0015] The types of ANA antinuclear antibodies in SLE patients mainly include anti-DNA antibodies and anti-RNA-binding protein antibodies (anti-RBP). Anti-DNA antibodies are mainly expressed and secreted by immature B cells that transition to plasmablasts, and their serum titers change significantly as the disease progresses, while anti-RBP antibodies are mainly secreted by LLPCs and generally do not change significantly with the course of the disease. Reports have shown that the antibody titers of SLE-susceptible mice are mainly maintained by LLPCs. Among them, anti-DNA antibodies can form immune complexes with target antigens, leading to kidney deposition, complement activation, and cytokine production. Free anti-DNA antibodies can also cause neurological symptoms by cross-binding to receptors in neurons or directly bind to antigens in the kidneys to produce pathological reactions, while anti-RBP antibodies can also form immune complexes, leading to cytokine production and possible kidney deposition and complement activation.

[0016] Studies in the early 1990s have found that B cells in SLE not only directly produce pathological autoantibodies, but also activate lupus-related T cells through antigen presentation and secreted cytokines. First, in the MRL / lpr lupus model mice, mice whose B cells were eliminated showed reduced T cell activation and reduced interstitial nephritis, which could completely prevent the progression of SLE. It is particularly noteworthy that this phenomenon cannot be fully explained by the lack of autoantibodies, because when B cells that cannot secrete immunoglobulins are re-implanted into B cell-eliminated model mice, inflammatory T cell foci in the animals will reappear, making the mice susceptible again. These studies all indicate the existence of antibody-independent B cell functions, including antigen presentation and cytokines. Secondly, in MRL / lpr mice lacking CD11c+ dendritic cells, T cells can still be activated; and when the antigen presentation ability of B cells is deleted at the genetic level, the activation of CD4+ T cells, the differentiation of effector memory T and follicular helper T cells (Tfh), and the progression of autoimmunity are blocked.

[0017] In view of the important role of B cells and plasma cells in the pathogenesis of SLE, the current research on targeted therapy of SLE mainly targets B cells and plasma cells, such as B lymphocyte stimulator BAFF / BLyS, proliferation-inducing ligand APRIL, CD20, CD22, CD19, etc. Other targets include FcγRIIb, Bruton's tyrosine kinase, proteasome, T cell-related targets, macrophage-related targets, intracellular signaling molecules, co-stimulatory factors, IgE, and intestinal flora. See Table 1 for details.

[0018] Table 1 Some targeted therapeutic products for SLE that have been marketed and are under development

[0019]

[0020]

[0021] Existing B cell targeted therapies directly target B cell surface markers BLyS, APRIL, CD19, CD20, and CD22, and achieve their pharmacological effects by regulating the activation, proliferation, differentiation, and apoptosis of B cells and / or plasma cells. Among them, Belimumab, which targets BLyS, is the first biological drug approved for the treatment of SLE. The main drugs targeting CD20 are rituximab and ocrelizumab. The 2019 EULAR guidelines and the 2020 Chinese SLE diagnosis and treatment guidelines both recommend that rituximab be used for patients with refractory SLE-related severe thrombocytopenia. However, the targeted therapy products that have been marketed have not achieved significant SLE treatment effects or long-term drug-free remission in clinical practice. In one statistic, after receiving belimumab treatment, 26 relapses / 100 patients within 12 months, or 44 relapses / 100 patients within 24 months were still observed; two randomized trials showed that rituximab was not superior to conventional treatment in non-renal SLE and lupus nephritis. In addition, due to long-term medication, patients have been in a state of partial immunodeficiency, which increases the risk of adverse infectious events.

[0022] CD19 is a membrane surface glycoprotein with a molecular weight of 95kDa in the immunoglobulin superfamily. It is widely expressed in all stages before plasma cell differentiation during B cell development. At present, CD19-targeted CAR-T cell therapy for SLE has significant advantages over other therapies, mainly reflected in its good efficacy for severe and refractory SLE, and can achieve long-term drug-free remission. There are literature reports that after CD19-targeted CAR-T therapy was applied to female patients with severe SLE, the patient's condition was significantly relieved without obvious side effects; other literature reports 5 patients with refractory systemic lupus erythematosus whose condition was significantly improved after CD19 CAR-T treatment. No patient relapsed during the 17-month follow-up, and all achieved drug-free remission, greatly improving the quality of life of patients.

[0023] Currently, CAR-T therapies for SLE indications on the market are all in the development stage. Products that have obtained IND approval are all targeting CD19. For example, Kyverna Therapeutics' KYV-101 and Cabaletta Bio's CABA-201 have obtained FDA's implicit clinical approval for the treatment of lupus nephritis (LN) and active lupus nephritis (active LN) or active systemic lupus erythematosus without renal involvement, respectively. Innovent Biologics' new drug clinical trial application for Rekiolance Injection for the treatment of moderate to severe refractory systemic lupus erythematosus has obtained implicit approval from the China National Medical Products Administration. See Table 2 for details.

[0024] Table 2 IND-approved CAR-T products for SLE-related indications

[0025]

[0026] According to published clinical results, since CD19 on the surface of plasma cells is mostly negative or extremely low in expression, although CAR-T therapy targeting CD19 can reduce the level of autoantibodies, its total IgG antibody level has no significant reduction, that is, targeting CD19-positive cells alone cannot deeply eliminate ASCs including LLPCs that lead to some refractory SLE. CAR-T treatment targeting CD19 in lupus mouse models also shows that although CD19 CAR-T treatment can improve lupus symptoms, autoantibodies can only be eliminated if administered in the early stages of the disease. In the later stages of disease progression, due to the accumulation of LLPCs, simply eliminating B cells cannot prevent the continued production of autoimmune antibodies. In order to solve these problems, the inventors have provided specific chimeric antigen receptors for improved dual antigens to effectively treat and / or prevent systemic lupus erythematosus after research; in addition, for other B cell autoimmune diseases involving CD19 and / or BCMA, the dual antigen-specific chimeric antigen receptors designed by the present invention can all have a good therapeutic effect. Summary of the invention

[0027] In a first aspect of the present invention, the present invention discloses the use of a chimeric antigen receptor of a bispecific antigen in the treatment and / or prevention of autoimmune diseases, wherein the first target of the chimeric antigen receptor is CD19 and the second target is BCMA.

[0028] B cell maturation antigen (BCMA) is a type III transmembrane protein with a cysteine-rich extracellular region without a signal peptide. It plays a key regulatory role in the expansion, survival, maturation and differentiation of B cells into plasma cells. BCMA is an important surface receptor for plasma cells. Its knockout results in a significant reduction in plasma cells, mainly LLPCs, in mice, and BCMA promotes the long-term survival of LLPCs in the bone marrow. Data show that the expression ratio and abundance of membrane-bound BCMA in plasma cells of SLE patients are significantly higher than those in healthy people. However, targeting BCMA alone cannot effectively eliminate a large number of pathogenic B cells (CD19 positive to strong positive, BCMA very weakly positive or negative) and plasmablasts (CD19 positive to weakly positive, BCMA weakly positive to positive) in a specific stage of SLE onset. During the treatment of a patient with diffuse large B-cell lymphoma (DLBCL) accompanied by SLE who received CAR-T therapy targeting CD19 and BCMA, it was found that simultaneous targeting of two antigens not only reduced ANA to an undetectable level, but also significantly reduced the level of total IgG antibodies, suggesting that this dual-target combination therapy can target ASCs that produce a large number of autoantibodies, thereby avoiding the production and recurrence of IgG autoantibodies at a deeper level.

[0029] The present invention found that the combination of targeting CD19 and BCMA can more comprehensively block the pathway for producing autoantibodies, and more completely cover B cells, plasmablasts and plasma cells at multiple differentiation stages that produce autoantibodies. Therefore, the dual-target CAR-T therapy designed based on this concept can target B cell-mediated autoimmune diseases, especially refractory SLE, and is expected to achieve faster relief and deeper and more lasting therapeutic effects.

[0030] In one embodiment of the present invention, the present invention provides the use of a chimeric antigen receptor specific for dual antigens in the preparation of a medicament for treating and / or preventing autoimmune diseases, wherein the first target of the chimeric antigen receptor is CD19 and the second target is BCMA.

[0031] Preferably, the use comprises preparing a medicament or preparation for preventing and / or treating autoimmune diseases.

[0032] In some embodiments, the drug or preparation achieves the treatment of systemic lupus erythematosus by killing or inhibiting CD19-positive B cells, plasmablasts, and BCMA-positive plasma cells. The above-mentioned drugs and preparations have the ability to kill CD19-positive B cells, plasmablasts, and BCMA-positive plasma cells at the same time. The combination of CD19 and BCMA targets can not only have a deeper SLE therapeutic effect, but also has the potential to reduce the possibility of recurrence; its mechanism of action and principle are as follows Figure 2 shown.

[0033] Preferably, the above-mentioned drugs or preparations can be administered in combination with one or more of the following:

[0034] (1) an agent that increases the efficacy of cells containing a CAR nucleic acid or a CAR polypeptide;

[0035] (2) an agent that ameliorates one or more side effects associated with administration of cells containing a CAR nucleic acid or a CAR polypeptide;

[0036] (3) additional agents for treating diseases associated with BCMA and CD19;

[0037] (4) A second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, virotherapy, adjuvant therapy, and any combination thereof.

[0038] The drug achieves the treatment of autoimmune diseases by killing or inhibiting CD19-positive B cells, plasmablasts and BCMA-positive plasma cells.

[0039] Preferably, the above-mentioned autoimmune disease is preferably a B cell-mediated autoimmune disease. The B cell-mediated autoimmune disease includes systemic lupus erythematosus (SLE), lupus nephritis, multiple sclerosis (MS), inflammatory bowel disease (IBD), rheumatoid arthritis (RA), Sjögren's syndrome (SS), dermatomyositis, autoimmune hemolytic anemia, neuromyelitis optica (NMO), neuromyelitis optica spectrum disease (NMOSD), idiopathic thrombocytopenic purpura (ITP), anti-neutrophil cytoplasmic antibody-associated systemic autoimmune small vessel vasculitis syndrome or polyangiitis, Wegener's granulomatosis (GPA), eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss syndrome), pemphigus vulgaris, idiopathic inflammatory myositis, scleroderma, autoimmune encephalitis, pemphigus vulgaris, myasthenia gravis, antiphospholipid syndrome, Chagas' disease, Graves' disease, polyarteritis nodosa, Goodpasture's syndrome, Kawasaki disease, rapidly progressive glomerulonephritis, immune cell-derived amyloidosis, monoclonal immunoglobulinemia of undetermined significance, POEMS syndrome, Crohn's disease, ulcerative colitis, adult-onset Still's disease, immune nephritis, chronic progressive cortical demyelinating encephalopathy (CIDP), immune-mediated necrotizing myopathy (IMNM).

[0040] In one embodiment of the present invention, the B cell-mediated autoimmune disease is preferably systemic lupus erythematosus. In some embodiments according to the above, 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.

[0041] The second aspect of the present invention discloses the structure of a chimeric antigen receptor, wherein the first antibody or antigen binding fragment thereof targeting BCMA or the second antibody or antigen binding fragment thereof targeting CD19 is independently selected from camel Ig, IgNAR, Fab fragment, Fab' fragment, F(ab')z fragment, F(ab')3 fragment, Fv, single-chain antibody (e.g., scFv, di-scFv, (scFv)z), mini antibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide-stabilized Fv protein ("dsFv") and single domain antibody (sdAb, nano antibody), chimeric antibody, humanized antibody, single domain antibody, bispecific antibody or multispecific antibody, binding ligand or protein domain. The antigen binding fragment targeting BCMA or the antigen binding fragment targeting CD19 is preferably scFv.

[0042] Specifically, selected from any one of the following formulae:

[0043] 1)L-VL,CD19-VH,BCMA-VL,BCMA-VH,CD19-H-TM-C-CD3ζ

[0044] 2)L-VH,CD19-VL,BCMA-VH,BCMA-VL,CD19-H-TM-C-CD3ζ 3)

[0046] L-VL,CD19-VH,CD19-H-TM-C-CD3ζ-2A peptide-L-VH,BCMA-VL,BCMA-H-TM'-C-CD3ζ; 4)

[0048] L-VH, BCMA-VL, BCMA-H-TM-C-CD3ζ-2A peptide-L-VL, CD19-VH, CD19-H-TM'-C-CD3ζ;

[0049] Where:

[0050] In formula 1), VL, CD19-VH, BCMA-VL, BCMA-VH, and CD19 form a loop structure;

[0051] In formula 2), VH, CD19-VL, BCMA-VH, BCMA-VL, and CD19 form a loop structure.

[0052] “-” is independently a connecting peptide or a peptide bond;

[0053] L is none or a signal peptide sequence;

[0054] VH, BCMA is the variable region of the heavy chain of BCMA antibody, and VL, BCMA is the variable region of the light chain of anti-BCMA antibody; the sequence of the variable region of the heavy chain of VH, BCMA antibody is shown in SEQ ID NO: 1, and the sequence of the variable region of the light chain of VL, BCMA antibody is shown in SEQ ID NO: 2;

[0055] 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 variable region of the heavy chain of the VH,CD19 antibody is shown in SEQ ID NO: 3, and the sequence of the variable region of the light chain of the VL,CD19 antibody is shown in SEQ ID NO: 4;

[0056] H is the hinge region;

[0057] TM is the first transmembrane domain;

[0058] TM' is the second transmembrane domain;

[0059] The 2A peptide is selected from P2A, T2A, E2A or F2A;

[0060] C is a co-stimulatory signal molecule;

[0061] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0062] Preferably, the L can be selected from the signal peptides derived from the following proteins: CD8, CD28, GM-CSF, CD4, CD137 or a combination thereof.

[0063] More preferably, L may be a signal peptide derived from CD8.

[0064] VH, BCMA is the variable region of the heavy chain of BCMA antibody, and VL, BCMA is the variable region of the light chain of anti-BCMA antibody; the sequence of the variable region of the heavy chain of VH, BCMA antibody is shown in SEQ ID NO: 1:

[0065] QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYYIHWVRQAPGQGLEWI GYINPNSGYTNYAQKFQGRATMTADKSINTAYVELSRLRSDDTAVYFCTRYM WERVTGFFDFWGQGTMVTVSS(SEQ ID NO: 1)

[0066] In the above sequence SEQ ID NO: 1, according to the Kabat numbering system, three heavy chain variable region complementarity determining regions (CRDs) are included, which are:

[0067] HCDR1:DYYIH

[0068] HCDR2:YINPNSGYTNYAQKFQG

[0069] HCDR3:YMWERVTGFFDF

[0070] As another embodiment, the VH, BCMA antibody heavy chain variable region also includes: a sequence containing the amino acid sequence shown in SEQ ID NO: 1 or having one or more amino acid substitutions, deletions or additions, such as 1, 2 or 3 amino acid substitutions, deletions or additions compared thereto;

[0071] In certain embodiments, the VH, BCMA antibody heavy 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 compared to the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably, the substitutions are conservative substitutions.

[0072] The sequence of the VL, BCMA antibody light chain variable region is shown in SEQ ID NO: 2:

[0073] DIQMTQSPSSVSASVGDRVTITTCLASEDISDDLAWYQQKPGKAPKVLVYT TSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQTYKFPPTFGGGTKVE IKR (SEQ ID NO: 2).

[0074] In the above sequence SEQ ID NO: 2, according to the Kabat numbering system, three complementarity determining regions (CRDs) of the light chain variable region are included, which are:

[0075] LCDR1: LASEDISDDLA

[0076] LCDR2:TTSSLQS

[0077] LCDR3:QQTYKFPPT

[0078] As another embodiment, the VL, BCMA antibody light chain variable region also includes: a sequence containing the amino acid sequence shown in SEQ ID NO: 2 or having one or more amino acid substitutions, deletions or additions, such as 1, 2 or 3 amino acid substitutions, deletions or additions compared thereto;

[0079] In certain 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 compared to the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably, the substitutions are conservative substitutions.

[0080] 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 variable region of the heavy chain of the VH,CD19 antibody is shown in SEQ ID NO: 3:

[0081] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGG SYAMDYWGQGTSVTVSS;

[0082] The sequence of the VL, CD19 antibody light chain variable region is shown in SEQ ID NO: 4:

[0083] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYH TSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKL EIT.

[0084] In the above sequence SEQ ID NO: 3, according to the Kabat numbering system, three heavy chain variable region complementarity determining regions (CRDs) are included, which are:

[0085] HCDR1:DYGVS

[0086] HCDR2:VIWGSETTYYNSALKS

[0087] HCDR3:HYYYGGSYAMDY

[0088] In the above sequence SEQ ID NO: 4, according to the Kabat numbering system, three complementarity determining regions (CRDs) of the light chain variable region are included, which are:

[0089] LCDR1:RASQDISKYLN

[0090] LCDR2:HTSRLHS

[0091] LCDR3:QQGNTLPYT

[0092] As another embodiment, the VH, CD19 also includes: a sequence containing the amino acid sequence shown in SEQ ID NO: 3 or having one or more amino acid substitutions, deletions or additions compared thereto, such as 1, 2 or 3 amino acid substitutions, deletions or additions;

[0093] In certain 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 compared to the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably, the substitutions are conservative substitutions.

[0094] As another embodiment, the VL, CD19 also includes: a sequence containing the amino acid sequence shown in SEQ ID NO: 4 or having one or more amino acid substitutions, deletions or additions compared thereto, such as 1, 2 or 3 amino acid substitutions, deletions or additions;

[0095] In certain embodiments, the 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%, or 100% sequence identity compared to the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably, the substitutions are conservative substitutions.

[0096] H is a hinge region; preferably, H can be selected from the hinge regions of the following proteins: CD8, CD28, CD137 or a combination thereof;

[0097] More preferably, H is selected from the hinge region derived from CD8; said H has an amino acid sequence as shown in SEQ ID NO: 17-19, or consists of:

[0098] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD;

[0099] or KPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKP;

[0100] or SGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

[0101] TM is a transmembrane domain; preferably, in another preferred embodiment, the TM may be a transmembrane region of a protein selected from the group consisting of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or a combination thereof;

[0102] More preferably, the TM is selected from a transmembrane region derived from CD8, and the transmembrane region derived from CD8 has an amino acid sequence as shown in SEQ ID NO: 20:

[0103] IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 20).

[0104] C may be a co-stimulatory signal molecule; preferably, C is a co-stimulatory signal molecule of a protein selected from the group consisting of 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 a combination thereof;

[0105] More preferably, the C is a co-stimulatory signal molecule derived from 4-1BB, and the co-stimulatory signal molecule derived from 4-1BB has an amino acid sequence as shown in SEQ ID NO: 21:

[0106] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0107] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0108] In another preferred example, the CAR (preferably the C-terminus or N-terminus) further includes a cell suicide element; wherein the cell suicide element can be connected to L or CD3ζ of the CAR or the bispecific CAR through T2A.

[0109] The third aspect of the present invention discloses the use of BCMA-CD19 CAR-T cells in the treatment and / or prevention of systemic lupus erythematosus, wherein the BCMA-CD19 CAR-T cells are engineered autologous T cells expressing chimeric antigen receptors as described above on the cell membrane. By collecting the patient's T cells, the CAR gene is transduced using a lentivirus (four-plasmid system packaging), so that the T cells can specifically recognize the BCMA and CD19 antigens expressed on the surface of the target cells.

[0110] In one embodiment, provided is the use of BCMA-CD19 CAR-T cells in the preparation of a medicament for treating and / or preventing systemic lupus erythematosus, wherein the BCMA-CD19 CAR-T cells may be engineered T cells expressing the chimeric antigen receptor as described above on the cell membrane, such as engineered autologous T cells, or engineered T cells encoding a nucleotide sequence of the chimeric antigen receptor as described above, such as engineered autologous T cells.

[0111] The fourth aspect of the present invention provides the use of a nucleic acid molecule in the treatment of systemic lupus erythematosus, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the chimeric antigen receptor described above.

[0112] In one embodiment, the present invention provides the use of a nucleic acid molecule in the preparation of a medicament for treating systemic lupus erythematosus, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the chimeric antigen receptor described above.

[0113] The fifth aspect of the present invention provides the use of a vector in the treatment of systemic lupus erythematosus, characterized in that the vector is selected from a DNA vector, an RNA vector, a plasmid, a liposome, a microparticle, a transposon vector, a CRISPR / Cas9 vector or a viral vector; preferably, the vector is a lentiviral vector.

[0114] The sixth aspect of the present invention discloses a use of BCMA-CD19 CAR-T cells in the preparation of a medicament for treating and / or preventing systemic lupus erythematosus, wherein the BCMA-CD19 CAR-T cells are engineered autologous T cells expressing the above-mentioned chimeric antigen receptor on the cell membrane.

[0115] The seventh aspect of the present invention discloses a drug or preparation, which contains the above-mentioned chimeric antigen receptor, the above-mentioned nucleic acid molecule, the above-mentioned vector, or the above-mentioned BCMA-CD19 CAR-T cell, and optionally, a pharmaceutically acceptable carrier, adjuvant and / or excipient; the drug or preparation can be used to treat and / or prevent systemic lupus erythematosus.

[0116] Compared with the prior art, the present invention has the following significant advantages and effects:

[0117] BCMA-CD19 CAR-T has the potential to simultaneously eliminate B cells (including B precursor cells) and plasma cells, which can eliminate the main source of autoantibodies and achieve local immune reset, enabling refractory SLE patients to achieve long-term and sustained remission. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1A Schematic diagram of the Anti-CD19 / BCMACAR structure; Figure 1BSchematic diagram for Anti-CD19 / BCMA CAR construction;

[0119] Figure 2 The design of BCMA-CD19 CAR and its mechanism of action for the treatment of SLE;

[0120] Figure 3 This is the CAR expression diagram of BCMACAR-T cells;

[0121] Figure 4 CAR expression diagram of CD19 CAR-T cells;

[0122] Figure 5 CAR expression diagram of BCMA-CD19 CAR-T cells for this application;

[0123] Figure 6 This is the CAR expression graph of the blank control without specific target improvement;

[0124] Fig. 7A The diagram is for the in vitro differentiation of antibody secreting cells against different antigens; Figure 7B Flow cytometry graphs showing the expression levels of different cell surface antigens;

[0125] Figure 8-11 This is a flow cytometry graph of the number of cells after different cells kill cells in vitro;

[0126] Fig.12 This is a statistical analysis chart of the flow cytometry results of BCMA-CD19 dual-target CAR-T cells simultaneously clearing CD19+ and CD19- antibody secreting cells;

[0127] Fig.13 This is a graph showing the effect of BCMA-CD19 CAR-T on killing antibody-secreting cells compared with CD19 CAR-T, BCMACAR-T and blank control groups;

[0128] Fig.14 It is a bar graph of the number of antibody-secreting cells after different T cell treatments at different effector-target ratios;

[0129] Fig.15 The figure shows the killing effect of BCMA-CD19 CAR-T cells on primary B cells at different effector-target ratios compared with the control group at 5 hours;

[0130] Fig.16 Flow cytometry of BCMA-CD19 CAR-T cells killing primary B cells (5h) at different effector-target ratios;

[0131] Figure 17-22The graph shows the cytokine secretion level after BCMA-CD19 CAR-T cells kill primary B cells (5h) at different effector-target ratios;

[0132] Fig.23 Flow cytometry of BCMA-CD19 CAR-T cells killing antibody-secreting cells (5h) at different effector-target ratios;

[0133] Fig.24 It is a bar graph of BCMA-CD19 CAR-T cells killing antibody secreting cells under different effector-target ratios (including only single targets CD19, BCMA, and dual targets) (5h);

[0134] Fig.25 The bar graph shows the cytokine secretion level after BCMA-CD19 CAR-T cells killed antibody-secreting cells (5h) at different effector-target ratios;

[0135] Fig.26 The gate and flow cytometry of B cells and ASC cells (including plasma cells CD138+) in peripheral blood samples of SLE patients;

[0136] Fig.27A This is a flow cytometric graph of BCMA-CD19 CAR-T cells killing B cells of SLE patients in vitro; Fig.27B It is a bar graph of the number of B cells after BCMA-CD19 CAR-T cells kill in vitro;

[0137] Fig.28A This is a flow cytometric graph of BCMA-CD19 CAR-T cells killing ASC cells from SLE patients in vitro; Fig.28B It is a bar graph of the number of ASC cells after BCMA-CD19 CAR-T cells kill in vitro;

[0138] Fig.29A This is a flow cytometric graph of BCMA-CD19 CAR-T cells killing plasma cells from SLE patients in vitro; Fig.29B It is a bar graph of the number of plasma cells after BCMA-CD19 CAR-T cells kill in vitro; DETAILED DESCRIPTION

[0139] The present invention is further described below through specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the content of the invention.

[0140] The raw materials and equipment used in the examples are well known to those skilled in the art, and can be purchased or easily obtained or prepared on the market.

[0141] The term "antibody" (Ab) shall include, but is not limited to, immunoglobulins that specifically bind to an antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each H chain comprises a heavy chain variable region (abbreviated herein 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 (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0142] It should be understood that the amino acid names herein are identified by internationally accepted single-letter English letters, and the corresponding three-letter abbreviations of the amino acid names are: 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), and Val (V).

[0143] Antibody preparation

[0144] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy chain and light chain genes of the antibodies of the present invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the antibodies of the present invention.

[0145] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). In addition, these antigen-binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab' fragments can be directly obtained from host cells; Fab' fragments can be chemically coupled to form F(ab')z fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). In addition, Fv, Fab or F(ab')z fragments can also be directly isolated from the culture medium of recombinant host cells. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0146] Conservative substitution

[0147] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the 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 amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family.

[0148] B cell maturation antigen (BCMA)

[0149] BCMA (also known as TNFRF17, BCM or CD269) is a member of the tumor necrosis receptor (TNFR) family and is mainly expressed on terminally differentiated B cells, such as memory B cells and plasma cells. Its ligand is called the B cell activator (BAFF) and proliferation-inducing ligand (APRIL) of the TNF family. BCMA participates in mediating the survival of plasma cells to maintain long-term humoral immunity. The gene of BCMA is encoded on chromosome 16, producing a primary mRNA transcript of 994 nucleotides in length (NCBI accession number NM_001192.2), which encodes a protein of 184 amino acids (NP_001183.2). A second antisense transcript from the BCMA locus has been described, which can play a role in regulating BCMA expression (Laabi Y. et al., Nucleic Acids Res., 1994, 22: 1147-1154). Additional transcript variants of unknown significance have been described (Smirnova AS et al. Mol Immunol., 2008, 45(4): 1179-1183). A second isoform (also called TV4) has been identified (Uniprot identifier Q02223-2).

[0150] However, some patients will still relapse after receiving CAR-T cell therapy targeting BCMA. For these patients with relapse, it is necessary to find another target different from BCMA before continuing treatment.

[0151] CD19

[0152] As used herein, the term "CD19" refers to B lymphocyte antigen CD19, also known as B lymphocyte surface antigen B4 or T cell surface antigen Leu-12, and 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), unless otherwise specified. The amino acid sequence of human CD19 is in NCBI accession number NP_001171569. The term encompasses "full-length", unprocessed human CD19 and any form of human CD19 derived from processing in cells, 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, B cells in early development (i.e., immature B cells), mature B cells and malignant B cells that are terminally differentiated into plasma cells. CD19 is expressed by most pre-B acute lymphoblastic leukemias (ALL), non-Hodgkin's lymphomas, B-cell chronic lymphocytic leukemias (CLL), prolymphocytic leukemias, hairy cell leukemias, common acute lymphocytic leukemias, and some Null-acute lymphoblastic leukemias. The expression of CD19 on plasma cells further suggests that it may be expressed on differentiated B-cell tumors such as multiple myeloma. Therefore, the CD19 antigen is a target for immunotherapy for the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia.

[0153] CD19 is a glycoprotein with a molecular weight of 95kDa, expressed on the surface of pre-B cells and mature B cells, closely related to the transmembrane conduction pathway of B cell Ca++, and has a regulatory effect on the proliferation and differentiation of B cells. CD19 is mainly expressed in normal B cells and cancerous B cells, with high tissue expression specificity, and is a good target for antibody or CAR-T immunotherapy. However, during immunotherapy, the CD19 epitope of B cells is often lost, causing patients to be unresponsive to immunotherapy or relapse.

[0154] Chimeric Antigen Receptor (CAR)

[0155] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including a costimulatory molecule. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0156] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a joint may be incorporated. As used herein, the term "joint" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The joint may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0157] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen binding domain targeting BCMA (or BCMA and CD19). When the CAR of the present invention is expressed in T cells, it is possible to perform antigen recognition based on antigen binding specificity. When it is combined with its associated antigen, the ability to kill CD19-positive B cells, plasmablasts, and BCMA-positive plasma cells, etc. The antigen binding domain is preferably fused with one or more intracellular domains from a costimulatory molecule and a ζ chain. Preferably, the antigen binding domain is fused with the intracellular domain of a combination of a 4-1BB signaling domain and a CD3ζ signaling domain.

[0158] As used herein, "antigen binding domain" and "single-chain antibody fragment" all refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments with antigen binding activity. Fv antibodies contain the variable region of the antibody heavy chain and the variable region of the light chain, but no constant region, and are the smallest antibody fragments with all antigen binding sites. Generally, Fv antibodies also include a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding. The antigen binding domain is usually scFv (single-chain variable fragment). The size of scFv is generally 1 / 6 of a complete antibody. A single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain. As a preferred embodiment of the present invention, the antigen binding domain comprises an antibody that specifically recognizes BCMA, and optionally, the antigen binding domain also comprises an antibody that specifically recognizes CD19, preferably a single-chain antibody.

[0159] For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some examples, a transmembrane domain can be selected, or modified by amino acid replacement to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0160] The intracellular domain in the CAR of the present invention includes the signaling domain of 4-1BB and the signaling domain of CD3ζ.

[0161] Preferably, the CAR of the present invention also includes a cell suicide element.

[0162] Bispecific CAR targeting CD19 and BCMA

[0163] Bispecificity means that the same CAR can specifically bind to and immune recognize two different antigens, and CAR can produce an immune response by binding to any one antigen.

[0164] In another preferred embodiment, the bispecific CAR targeting CD19 and BCMA is as described in the first aspect of the present invention.

[0165] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen binding domain targeting CD19 and BCMA, including anti-CD19 scFv and anti-BCMA scFv.

[0166] In another preferred embodiment, the present invention provides a bispecific chimeric antigen receptor for CD19 and BCMA antigens. The structural components of the 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, wherein the CD19 scFv and the BCMA scFv are connected by a short peptide segment (G4S) xN. The CAR structure targeting both CD19 and BCMA is as described in the second aspect of the present invention.

[0167] In another preferred embodiment, the present invention optimizes the sequence of the BCMA scFv, and the BCMA scFv (SscFv) has high affinity and good specificity for BCMA, and can specifically target the full-length antigen and extracellular region of BCMA.

[0168] In a preferred embodiment of the present invention, (G4S)x3 is used to connect CD19scFv and BCMAscFv, at which time the activity and lethality of CAR are optimal.

[0169] The present invention uses a CAR that targets CD19 and BCMA with dual specificity. Compared with a CAR that targets a single antigen, the affinity is significantly enhanced, the activity of immune cells is significantly increased, and there is a synergistic effect, as demonstrated in the following examples. In addition, due to the uneven expression levels of CD19 and BCMA in B cells and plasma cells, the dual-targeted CAR-T treatment range is wider. CAR-immune cells that simultaneously target CD19 and BCMA can reduce the possibility of antigen escape caused by downregulation or loss of a single surface antigen. In addition, the combination of targeting CD19 and BCMA can more comprehensively block the pathway for producing autoantibodies, and more completely cover B cells, plasmablasts, and plasma cells that produce autoantibodies at multiple stages of differentiation; the combination of CD19 and BCMA targets can not only have a deeper SLE therapeutic effect, but also have the potential to reduce the possibility of recurrence.

[0170] Chimeric Antigen Receptor T Cells (CAR-T Cells)

[0171] As used herein, the terms "CAR-T cells", "CAR-T", and "CAR-T cells of the present invention" include the BCMA-CD19 CAR-T cells included in the third aspect of the present invention.

[0172] Suicide gene switch

[0173] In order to further control adverse effects such as non-tumor targeting and cytokine release syndrome of CAR-T cells, the CART cells in the present invention are all equipped with a suicide gene switch, which can effectively eliminate CAR-T cells in the body under the action of exogenous drugs and block unknown or uncontrollable long-term toxicity to ensure the safety of patients.

[0174] The suicide switch used in the present invention can be herpes simplex virus thymidine kinase (the herpes symplexvirus thymidine kinase, HSV-TK), inducible caspase9 (inducible caspase9, iCasp9), CD20, mutant human thymidylate kinase (mutated human thymidylate kinase, mTMPK), etc. In comparison, HSV-TK, iCasp9 and CD20 have the same ability to clear CAR-cells, but iCasp9 and CD20 are cleared more quickly, and HSV-TK is cleared more slowly.

[0175] The iCasp9 suicide switch contains the FKBP12-F36V domain, which can be connected to caspase 9 through a flexible linker, which does not contain a recruitment domain. FKBP12-F36V contains an FKBP domain with phenylalanine replacing valine at the 36th amino acid residue. It has high selectivity and subnanomolar affinity and can bind to dimerization synthetic ligands, such as the otherwise inert small molecule AP1903. When small molecules are added, they can promote their dimerization, thereby inducing cell apoptosis, while having no effect on normal cells that do not carry the suicide switch.

[0176] The inducible safety switch caspase9 (iCasp9) uses human caspase9 fused to FK506 binding protein (FKBP), which can be induced to form dimers using chemical inducers (AP1903 / Rimiducid, Bellicum Pharmaceutical), leading to apoptosis of cells expressing the fusion protein.

[0177] Although CD19 and BCMA are highly expressed in tumor cells, they are also expressed in normal B cells. The engineered immune cells of the present invention will attack normal B cells in vivo.

[0178] How to control the safety of CAR-cells has always been an urgent problem to be solved. Adding a safety switch to CAR-cells is the safest way to terminate the activity of CAR-cells. After CAR-cells produce severe toxicity (CRS / neurotoxicity) or after the patient achieves long-term sustained remission, the inducible iCasp9 safety switch controls the clearance of CAR-cells.

[0179] Carrier

[0180] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a known vector comprising the gene, or by directly isolating from cells and tissues comprising the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0181] The present invention also provides vectors into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0182] In brief summary, the expression cassette or nucleic acid sequence of the present invention is usually operably connected to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0183] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0184] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into such vectors, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0185] Further, expression vector can be provided to cell in the form of viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. Generally, suitable vectors are included in at least one organism that works on the origin of replication, promoter sequence, convenient restriction enzyme sites and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193).

[0186] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0187] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that when an element is inverted or moved relative to another, the promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it is shown that individual elements can work cooperatively or independently to start transcription.

[0188] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0189] In order to evaluate the expression of CAR polypeptides or parts thereof, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population seeking to be transfected or infected by a viral vector. In other aspects, selectable markers may be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences so that they can be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0190] Reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of regulatory sequences. Generally, reporter gene is the following gene: it is not present in or expressed by a receptor organism or tissue, and it encodes a polypeptide whose expression is clearly indicated by some easily detectable properties such as enzymatic activity. After DNA has been introduced into the receptor cell, the expression of the reporter gene is measured at the appropriate time. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein (for example, Ui-Tei et al., 2000FEBS Letters479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, the construct with a minimum of 5 flanking regions showing the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be connected to a reporter gene and used to evaluate the ability of a reagent to regulate promoter-driven transcription.

[0191] Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0192] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0193] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0194] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0195] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid preparations to introduce nucleic acid into a host cell (in vitro, ex vivo or in vivo). On the other hand, the nucleic acid can be associated with a lipid. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome through a connecting molecule associated with both the liposome and the oligonucleotide, trapped in the liposome, compounded with the liposome, dispersed in a solution comprising lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles or compounded with micelles, or otherwise associated with lipids. The lipids, lipid / DNA or lipid / expression vectors associated with the composition are not limited to any specific structure in the solution. For example, they can be present in a bilayer structure, as micelles or have a "collapsed (collapsed)" structure. They can also be simply dispersed in a solution, possibly forming aggregates of size or shape inhomogeneity. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0196] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0197] Drugs, preparations and compositions thereof

[0198] The present invention provides a preparation containing the CAR-T cells of the present invention and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the preparation is a liquid preparation. Preferably, the preparation is an injection. Preferably, the concentration of the CAR-T cells in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0199] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

[0200] In certain embodiments, the pharmaceutical composition of the present invention comprises: the modified immune cell or immune cell composition of the present invention.

[0201] The isolated nucleic acid molecules, vectors, host cells, modified immune cells or immune cell compositions of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injection solution. In addition, the sterile injection solution can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCI), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0202] The isolated nucleic acid molecules, vectors, host cells, modified immune cells or immune cell compositions of the present invention can be applied by any suitable method known in the art, including but not limited to oral, oral, sublingual, eyeball, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (such as, powders, ointments or drops), or nasal routes. However, for many therapeutic uses, preferred routes of administration / modes are parenteral administration (such as intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). It will be appreciated by those skilled in the art that routes of administration and / or modes will change according to the intended purpose. In certain embodiments, isolated nucleic acid molecules, nucleic acid constructs, vectors, host cells, modified immune cells or immune cell compositions of the present invention are administered by intravenous injection or push injection.

[0203] The pharmaceutical composition of the present invention may include a "therapeutically effective amount" or a "prophylactic effective amount" of an isolated nucleic acid molecule, a nucleic acid construct, a vector, a host cell, a modified immune cell or an immune cell composition of the present invention. A "prophylactic effective amount" refers to an amount sufficient to prevent, prevent or delay the occurrence of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent a disease and its complications in a patient who already has a disease. The therapeutically effective amount of an isolated nucleic acid molecule, a nucleic acid construct, a vector, a host cell, a modified immune cell or an immune cell composition of the present invention may vary depending on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0204] In the present invention, the dosage regimen can be adjusted to obtain the best desired response (e.g., therapeutic or preventive response). For example, the dosage can be administered once, multiple times over a period of time, or the dosage can be reduced or increased in proportion to the urgency of the treatment situation.

[0205] Therapeutic applications

[0206] The therapeutic application of the present invention includes collecting T cells from patients, using lentivirus (four-plasmid system packaging) to transduce the CAR gene to obtain BCMA-CD19 CAR-T cells, so that the BCMA-CD19 CAR-T cells can specifically recognize target cells with BCMA and CD19 antigens expressed on the cell surface, thereby performing therapeutic applications.

[0207] Therefore, the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the following steps: administering the CAR-T cell of the present invention to the mammal.

[0208] In one embodiment, the present invention includes a type of cell therapy that separates the patient's autologous T cells (or allogeneic donors), activates and genetically modifies them to produce CAR-T cells, and then injects them into the same patient. This method has a very low probability of graft-versus-host disease, and the antigen is recognized by the T cells in an MHC-free manner.

[0209] In one embodiment, the CAR-T cells of the present invention can undergo a robust in vivo T cell expansion and can sustain an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which the CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR. For example, anti-BCMA and / or CD19 CAR-T cells cause a specific immune response against cells expressing BCMA and / or CD19.

[0210] Although the data disclosed herein specifically disclose lentiviral vectors comprising anti-BCMA and / or CD19 scFv, hinge and transmembrane regions, and 4-1BB / CD28 and CD3ζ signaling domains, the present invention should be construed to include any number of variations to each of the construct components.

[0211] Treatable SLE includes moderate to severe refractory SLE, lupus nephritis, active SLE, and active SLE without renal involvement.

[0212] For ex vivo immunization, at least one of the following occurs in vitro prior to administering the cells into a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR into the cells, and / or iii) cryopreserving the cells.

[0213] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from mammals (preferably humans) and genetically modified (i.e., in vitro transduction or transfection) with vectors expressing CAR disclosed herein. CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0214] The present invention provides a method for treating a tumor, comprising administering a therapeutically effective amount of the CAR-modified T cells of the present invention to a subject in need thereof.

[0215] Currently, the planned number of patients to be enrolled in the single-arm clinical trial of BCMA-CD19 CAR-T for SLE is 18. The inclusion criteria are detailed in NCT05858684. Three doses are planned to be used, namely DL-1: 0.5±20%×10^5 / kg, DL1: 1±20%×10^5 / kg, DL2: 2±20%×10^5 / kg DL3: 3±20%×10^5 / kg.

[0216] The CAR-modified T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell groups. Simply put, the pharmaceutical composition of the present invention may include a target cell group as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such a composition may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The composition of the present invention is preferably formulated for intravenous administration.

[0217] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease-although the appropriate dosage can be determined by clinical trials.

[0218] Example

[0219] Example 1: In vitro differentiation of antibody secreting cells

[0220] Day0-Day3: After isolating B cells, take an appropriate amount to count and detect the cell phenotype by flow cytometry. The remaining cells were resuspended to a density of 0.5M / mL with freshly prepared B cell basal medium (IMDM+Glutamax+10% FBS), and then transferred to a 24-well plate with a feeder cell layer (irradiated L cells expressing human CD40L, 0.125M / well), and cytokines IL-2 (20U / mL), IL-21 (50ng / mL) and antibodies F(ab')2goat anti-human IgM and IgG (10ug / mL) were added at their respective concentrations for culture;

[0221] Day3-Day6: B cells cultured on Day 3 were transferred from the well plate, and appropriate amount was taken for counting and cell phenotype was detected by flow cytometry. After counting, the supernatant was discarded by centrifugation at 400g for 5min, and then resuspended in B cell basal medium (IMDM+Glutamax+10% FBS) to a density of 0.2M / mL, and cytokines IL-2 (20U / mL), IL-21 (50ng / mL), and 1x final concentration of hybridoma growth supplement HyMAX were added. TM Continue culturing with Hybridoma Fusion & Cloning Supplement, Lipid Mixture 1, and MEM amino acids solution;

[0222] Day6-Day13: When the cells are cultured to Day6, take an appropriate amount of cells for counting and detect the phenotype by flow cytometry. After counting, centrifuge at 400g for 5min to discard the supernatant, resuspend the cells to a density of 0.5M / mL with B cell basal medium (IMDM+Glutamax+10% FBS), and transfer them to a 6-well plate with bone marrow stromal cells (irradiated M2-10B4 cells, 0.2M / well), add 4mL to each well, and add cytokines IL-6 (10ng / mL), IL-21 (50ng / mL), IFN-α (100U / mL) at their respective concentrations, and 1x final concentration of hybridoma growth additive HyMAX TM Hybridoma Fusion & Cloning Supplement, lipid mixture LipidMixture 1 and amino acid solution MEM amino acids solution were further cultured; half of the medium was changed on Day 10, and the culture was continued until Day 12-Day 13 for use in in vitro target cell killing experiments.

[0223] Example 2: CAR expression flow staining

[0224] Take part of the cells in culture and mix them evenly. Take 1×10 5 ~5×10 5 The cells were centrifuged at 300 × g for 5 minutes and the supernatant was discarded;

[0225] Adjust the concentration of BCMA-his antigen to 10 μg / mL (1:10 dilution) with FACS buffer, take 100 μL of the prepared primary antibody to resuspend the cell sample, and stain at 4°C for 50 min;

[0226] After staining, wash once with FACS buffer;

[0227] Dilute Anti-His Tag-PE, Rabbit AntiMouse FMC63 scFv MonoclonalAntibody Alexa Fluor 647 (Clone: ​​R19M), BV421-anti-CD3, PE-Cy7 anti-CD4, APC-H7anti-CD8 at 1:100, prepare the secondary antibody mixture, take 100 μL of each resuspended cell sample, mix well, and stain at 4°C in the dark for 25 min;

[0228] After staining, wash twice with FACS buffer, resuspend with 7-AAD buffer diluted 1:300, and detect on the machine. Figure 3-6As shown (wherein the CD19 scFv and BCMA scFv in CD19 CAR-T and BCMACAR-T are the same as the corresponding protein sequences in BCMA-CD19-L1 dual-target CAR-T), it can be seen that the surface of BCMA-CD19 CAR-T cells of the present application can achieve efficient expression of antibodies against CD19 and BCMA.

[0229] Example 3: Flow cytometry staining of antibody secreting cells

[0230] Take part of the cells in culture and mix them evenly. Take 1×10 5 ~5×10 5 The cells were centrifuged at 300 × g for 5 min and the supernatant was discarded;

[0231] Take a clean 1.5ml EP tube, add an appropriate amount of FACS buffer according to the sample volume, and then add the following antibodies at a ratio of 1:100: APC-anti-CD19, PE-anti-BCMA, BUV395-anti-CD27, FITC-anti-CD38, BV421-anti-CD138, BV605-anti-CD20, and mix thoroughly to obtain the antibody staining solution;

[0232] The antibody-secreting cells in culture were taken out on Day 0, Day 3, Day 6, Day 10, Day 13, and Day 14, respectively. After mixing, an appropriate amount of cells were taken, centrifuged at 400 g for 5 min, and the supernatant was discarded.

[0233] Resuspend with the prepared staining solution and stain for 25 min at 4°C in the dark; wash once with FACS buffer, centrifuge at 400g for 5 min, and discard the supernatant.

[0234] Add FACS buffer and wash once more, centrifuge at 400 g for 5 min, and discard the supernatant;

[0235] Resuspend with FACS buffer and immediately test on the machine. The absolute number changes of different cell subsets at different days of differentiation and the expression and percentage changes of CD19, BCMA, CD20, CD27, CD38, CD138 on the cell surface are shown in the figure. Fig. 7A As shown, Figure 7B The expression levels of different cell surface antigens and the protein expression on the cell surface during the 14-day differentiation cycle were displayed. The proportion of plasma cells with double positive CD38 and CD138 was 82% at the end of differentiation, and the proportion of antibody-secreting cells with double positive CD38 and CD27 was 94.3% at the end of differentiation. At the end of differentiation, 31.5% of the cells were CD19 negative. It can be seen that at the end of differentiation, we obtained antibody-secreting cells with higher purity, and a certain proportion of these cells were CD19 negative.

[0236] After the antibody-secreting cells that had completed in vitro differentiation were killed for 5 hours in vitro with NT, CD19 CAR-T, BCMACAR-T, and BCMA-CD19 dual-target CAR-T, they were subjected to flow cytometry to detect the absolute number of residual live antibody-secreting cells, thereby detecting the killing ability of the three CAR-Ts on total antibody-secreting cells. The results are as follows: Figure 8-12 As shown, it can be seen that the BCMA-CD19 dual-target CAR-T can simultaneously eliminate CD19+ and CD19- antibody-secreting cells.

[0237] Example 4: Antibody Secreting Cell ELISPOT

[0238] Day 0: (Aseptic operation required)

[0239] Take out the Millipore 96-well PVDF ELISPOT plate, add 25 μL 70% ethanol to each well and treat at room temperature for 1 min;

[0240] Wash the plate twice with PBS, adding 200 μL to each well each time. After the last wash, discard the liquid and gently dry it on sterile paper.

[0241] Add 50 μL of diluted coating antibody or specific antigen to each well and incubate overnight at 4°C;

[0242] Day 1: (Aseptic operation required)

[0243] Remove the coated antibody or antigen in the well plate and wash it three times with sterile PBS, 200 μL per well each time; after the last wash, discard the liquid and gently dry it on sterile paper;

[0244] Add 200uL blocking buffer (1X) to each well and block at room temperature for at least 1h;

[0245] Remove the blocking solution (no need to wash the plate), add the prepared cells of each group, and add 100 μL of cell suspension to each well;

[0246] After adding the cells, gently tap the edge of the plate to spread the cells evenly and place in an incubator at 37°C and 5% CO. 2 Incubate for 16 to 20 hours (be careful not to move the plate during the incubation period);

[0247] Day 2: (No need for sterile operation)

[0248] Remove the cells from the well plate and wash twice with PBS to completely remove the cells, 200 μL per well each time;

[0249] Wash the plate 5 times with washbuffer, 250 μL per well each time;

[0250] Add 100uL of diluted detection antibody to each well and incubate at room temperature for 2h or at 4℃ overnight;

[0251] Remove the detection antibody and wash the plate 5 times with wash buffer, 250 μL per well each time;

[0252] Add 100 μL of diluted conjugate to each well and incubate at room temperature in the dark for 1 h;

[0253] To remove the conjugate, wash the plate five times with wash buffer, 250 μL per well each time;

[0254] Add 100 μL of freshly prepared AEC color development solution to each well and incubate at room temperature in the dark for 30 min until clear spots appear;

[0255] Remove the color development solution and stop the color development with deionized water, 50 μL per well each time;

[0256] Remove excess liquid from the well plate and place it in a dark place at room temperature to dry thoroughly;

[0257] Use ELISPOT reader to count the number of spots. After the antibody-secreting cells that have completed in vitro differentiation are killed in vitro for 5 hours using NT, CD19CAR-T, BCMACAR-T and BCMA-CD19 dual-target CAR-T, they are subjected to ELISPOT experiment again to detect the absolute number of cells that can secrete IgG among the residual cells killed, thereby detecting the killing ability of the three CAR-Ts on the total antibody-secreting cells. At the end of the experiment, the CTL enzyme-linked fluorescence plate reader was used to take pictures and analyze the experiment. The photo of the absolute number of spots is shown in the figure, and the statistical analysis of the absolute number of spots is as follows. It can be seen from the results that the BCMA-CD19 dual-target CAR-T group has the least residual total antibody-secreting cells. The results are as follows Figure 8 As shown, from Figure 8 Judging from the results, BCMA-CD19 CAR-T cells can clear more antibody-secreting cells than CD19-CAR-T and BCMA-CAR-T, and have stronger killing ability.

[0258] Example 5: In vitro killing experiment of B cells and antibody secreting cells

[0259] Primary B cells isolated from PBMC and antibody-secreting cells induced and differentiated in vitro were cultured at a concentration of 4 × 10 5 / mL and 5×10 5 Pieces / mL;

[0260] Prepare effector cells and adjust the concentration of CAR-positive cells to 12 × 10 5 / mL and 5×105 / mL, and CAR-positive cells were diluted in sequence according to the planned E / T ratio;

[0261] Take a clean 96-well plate, mark the experimental groups, set up 3 parallel wells for each group, and add 100 μL of target cell and effector cell suspensions to the corresponding marked wells, respectively, with a total volume of 200 μL;

[0262] Effector cells and target cells were co-incubated in a 37°C incubator for 5 h;

[0263] After 5 h of incubation, centrifuge at 300 g for 5 min, and take 50 μL x 2 samples from each well for CBA detection;

[0264] Then add 1 μL FITC-anti-CD3, 1 μL APC-anti-CD19, and 1 μL PE-anti-BCMA antibodies to each well of the original cell wells, mix well, and stain for 25 min at 4°C in the dark. After staining, wash once with FACS buffer, resuspend the cells with 120 μL DAPI diluent (1:4800 v / v), stain for 5 min at room temperature in the dark, and immediately detect on the machine.

[0265] An equal absolute volume of sample was collected from each well for analysis of the absolute number of live B cells and antibody-secreting cells in the residual sample.

[0266] Data processing:

[0267] The percentage of ASC-specific killing was calculated as follows: CAR-T cell killing percentage % = (target cell group CD3 - DAPI - Absolute cell count – CD3 - DAPI - absolute cell number) / target cell group CD3 - DAPI - The absolute number of cells.

[0268] The percentage of B cell specific killing was calculated as follows: CAR-T cell killing percentage % = (target cell group CD19 + DAPI - Absolute cell count – CD19 in CAR-T and target cell co-culture group + DAPI - absolute cell number) / target cell group CD19 + DAPI - The absolute number of cells. The results are shown in Table 3-5. Figure 9-20 As shown in Table 3-5, Figure 9-20From the results, BCMA-CD19CAR-T cells have a stronger ability to kill primary B cells and antibody-secreting cells; the level of cytokine secretion after killing primary B cells and antibody-secreting cells is significantly lower than that of CD19-CAR-T and BCMA-CAR-T.

[0269] in Fig.10 The experiment shown is: B cells were isolated and purified from PBMC, co-incubated with BCMA-CD19 dual-target CAR-T cells in vitro for 5 hours, and then the absolute number of residual live B cells was detected, and the ability of BCMA-CD19 dual-target CAR-T 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. The specific killing values ​​of each group are shown in Fig.10 The results show that BCMA-CD19 dual-target CAR-T can effectively eliminate primary B cells in vitro.

[0270]

[0271] Table 3 Specific values ​​of cytokine secretion levels after BCMA-CD19 CAR-T cells killed primary B cells (5h)

[0272]

[0273] Table 3 and Figure 12-16 Shows the cytokine secretion levels after BCMA-CD19 dual-target CAR-T cells kill primary B cells (5h).

[0274] The results of cytokine release show that a certain amount of IFN-r related to the killing efficacy of CAR-T cells and a small amount of IL-2, a cytokine related to proliferation, are indeed released during the killing process, and other factors are released in smaller amounts. This cytokine release is similar to the previous killing of tumor cells, suggesting that the cytokine secretion pattern when used to treat SLE may not be significantly different from the existing CRS data in humans, which has a certain indication of its safety.

[0275] Table 4 Specific values ​​of BCMA-CD19 CAR-T cells killing antibody secreting cells (5h)

[0276]

[0277] Table 4 and Fig.19 Shows the situation after BCMA-CD19 dual-target CAR-T cells kill antibody-secreting cells (5h).

[0278] Table 5 Specific values ​​of cytokine secretion levels after BCMA-CD19 CAR-T cells killed antibody-secreting cells (5h)

[0279]

[0280] Table 5 and Fig. 20 Shows the cytokine secretion levels after BCMA-CD19 dual-target CAR-T cells kill antibody-secreting cells (5h).

[0281] Example 6: Detection of cytokines in supernatant

[0282] Solution preparation:

[0283] Prepare mix beads: add 808 μL of IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ beads, totaling 4.848 mL, vortex and add 19.392 mL of assay diluent to mix, and prepare mix beads with a total volume of 24.24 mL;

[0284] Dilute the sample: dilute with assay diluent 1:1, the total sample volume after dilution is 50μL / test;

[0285] Prepare standard: add 2mL assay diluent to standard powder and leave at room temperature for 15 minutes as top standard. Then dilute the standard by 2 times with assay diluent, with the last one being assay diluent.

[0286] Steps:

[0287] Take a 96-well U-bottom plate, add 50μL mixed beads + 50μL standards / samples + 50μL HumanTH1-TH2 PE Detection Reagent in sequence, shake for 5 minutes, and place at room temperature away from light for 3 hours;

[0288] Add 100 μL wash buffer and mix well, centrifuge at 300 g for 5 min, and discard the supernatant;

[0289] Resuspend beads in 100 μL wash buffer;

[0290] Oscillate for 2 min, load on the machine, and load 50 μL;

[0291] Data analysis

[0292] Standard curve linear equation determination coefficient R2 ≥ 0.99; Cytokine concentration results Cytokine concentration application FCAPArray TM (BD) analysis;

[0293] Example 7: B cell killing in peripheral blood samples of SLE patients

[0294] Peripheral blood samples of SLE patients were obtained and diluted with DPBS at a ratio of 1:1 for later use;

[0295] Take the Ficoll separation solution at room temperature, and add Ficoll and diluted peripheral blood sample in a volume ratio of 1:3;

[0296] Adjust the centrifuge speed to 4 and 3 respectively, the centrifugal speed to 1000g, and centrifuge for 20 minutes at room temperature;

[0297] Use a pipette to aspirate the PBMC cells in the buffy coat, count them, and centrifuge at 400 g for 8 min to collect the cells, which are the target cells;

[0298] Resuspend the target cells in 1640 complete medium at a concentration of 5 × 105 cells / mL;

[0299] According to the E:T ratio of the experimental plan, the effector T cells were serially diluted;

[0300] Take a clean 96-well U-bottom plate, add 100ul of target cells and effector cells respectively, mark the plate and put it into a 37-degree carbon dioxide incubator for 5 hours;

[0301] At the end of incubation, take the supernatant and keep it as a sample;

[0302] The remaining cells were centrifuged and added to FACS buffer: each 100ul FACS buffer contained 1ul of the following antibodies: APC-anti-CD38, BV421-anti-CD138, BUV395-anti-CD27, BV510-anti-CD45, FITC-anti-CD3 / CD14, Pe-Cy7-antiCD19, BV786-anti-IgD. After mixing, stain at 4 degrees in the dark for 30 minutes;

[0303] Wash twice with FACS buffer, and resuspend in FACS buffer diluted with DAPI at 1:4800 for the third time for detection. Collect equal absolute volume of samples in each well to analyze the absolute number of live B cells and antibody-secreting cells in the residual samples. The results are shown in Figure 21-24 shown; Fig.21Non-ASC refers to non-antibody secreting cells, ASC refers to CD38 and CD27 double positive antibody secreting cells, CD138 positive ASC is plasma cell (PC), CD19-PC refers to CD19 negative plasma cells. Subsequent killing experiments are analyzed based on the above gates.

[0304] SLE samples were co-incubated with BCMA-CD19 dual-target CAR-T cells and control NT cells at a certain ratio for 5 h, and then the absolute number and percentage of B cells, antibody-secreting cells and plasma cells in the samples after incubation were detected. The B cell flow cytometry and absolute number statistics were as follows: Fig.27A The changes of B cells in SLE samples after the killing was completed, and the absolute number of residual cells was statistically shown as follows: Fig.27B It can be seen that BCMA-CD19 dual-target CAR-T can significantly kill B cells in SLE samples.

[0305] The flow cytometry and absolute number of ASC cells were analyzed. Fig.28A The changes in antibody-secreting cells in SLE samples after the killing was completed, and the absolute number of residual cells was statistically shown as follows Fig.28B It can be seen that BCMA-CD19 dual-target CAR-T can significantly kill antibody-secreting cells in SLE samples.

[0306] Flow cytometry and absolute number of plasma cells were performed. Fig.29A The changes in plasma cells in SLE samples after the killing was completed, and the absolute number of residual cells was statistically shown as follows Fig.29B It can be seen that BCMA-CD19 dual-target CAR-T can significantly kill plasma cells in SLE samples.

[0307] Figure 22-24 The results showed that BCMA-CD19 CAR-T cells can kill B cells and ASC cells (including plasma cells CD138+) of SLE patients in vitro.

[0308] At this point, those skilled in the art should recognize that, although multiple exemplary embodiments of the present invention have been shown and described in detail herein, many other variations or modifications that conform to the principles of the present invention can still be directly determined or derived based on the content disclosed in the present invention without departing from the spirit and scope of the present invention. Therefore, the scope of the present invention should be understood and recognized as covering all these other variations or modifications.

Claims

1. Use of a bispecific chimeric antigen receptor in the preparation of a medicament for treating and / or preventing an autoimmune disease, characterized in that: The first target of the chimeric antigen receptor is CD19, and the second target is BCMA.

2. Use of a bispecific chimeric antigen receptor in combination with one or more of the following in the preparation of a pharmaceutical combination for treating and / or preventing autoimmune diseases: (1) an agent that increases the efficacy of cells containing a CAR nucleic acid or a CAR polypeptide; (2) an agent that ameliorates one or more side effects associated with administration of cells containing a CAR nucleic acid or a CAR polypeptide; (3) Additional agents for treating diseases associated with BCMA and CD19.

3. The use according to claim 1 or 2, characterized in that The treatment and / or prevention also includes use in combination with a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, virotherapy, adjuvant therapy, and any combination thereof.

4. The use according to claim 3, characterized in that The drug achieves the treatment of autoimmune diseases by killing or inhibiting CD19-positive B cells, plasmablasts and BCMA-positive plasma cells.

5. The use according to claim 4, characterized in that The autoimmune disease is preferably a B cell-mediated autoimmune disease.

6. The use according to claim 5, characterized in that The B cell-mediated autoimmune diseases include systemic lupus erythematosus, lupus nephritis, multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, Sjögren's syndrome, dermatomyositis, autoimmune hemolytic anemia, neuromyelitis optica, neuromyelitis optica spectrum disease, idiopathic thrombocytopenic purpura, anti-neutrophil cytoplasmic antibody-associated systemic autoimmune small vessel vasculitis syndrome or polyangiitis, Wegener's granulomatosis, eosinophilic granulomatosis with polyangiitis, pemphigus vulgaris, idiopathic Inflammatory myositis, scleroderma, autoimmune encephalitis, pemphigus vulgaris, myasthenia gravis, antiphospholipid syndrome, Chagas' disease, Graves' disease, polyarteritis nodosa, Goodpasture's syndrome, Kawasaki disease, rapidly progressive glomerulonephritis, immune cell-derived amyloidosis, monoclonal gammaglobulinemia of undetermined significance, POEMS syndrome, Crohn's disease, ulcerative colitis, adult-onset Still's disease, immune nephritis, chronic progressive cortical demyelinating encephalopathy, or immune-mediated necrotizing myopathy.

7. The use according to claim 6, characterized in that The autoimmune disease is preferably systemic lupus erythematosus; 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.

8. The use according to claim 1, characterized in that The first antibody or antigen-binding fragment thereof targeting BCMA or the second antibody or antigen-binding fragment thereof targeting CD19 is independently selected from camel Ig, IgNAR, Fab fragment, Fab' fragment, F(ab')z fragment, F(ab')3 fragment, Fv, single-chain antibody (e.g., scFv, di-scFv, (scFv)z), miniantibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, disulfide-stabilized Fv proteins ("dsFv") and single domain antibodies (sdAb, nanoantibodies), chimeric antibodies, humanized antibodies, single domain antibodies, bispecific antibodies or multispecific antibodies, binding ligands or protein domains.

9. The use according to claim 8, characterized in that The antigen-binding fragment targeting BCMA or the antigen-binding fragment targeting CD19 is preferably scFv.

10. The use according to claim 9, characterized in that The structure of the chimeric antigen receptor is selected from any one of the following formulae: 1)L-VL,CD19-VH,BCMA-VL,BCMA-VH,CD19-H-TM-C-CD3ζ 2)L-VH,CD19-VL,BCMA-VH,BCMA-VL,CD19-H-TM-C-CD3ζ 3) L-VL,CD19-VH,CD19-H-TM-C-CD3ζ-2A peptide-L-VH,BCMA-VL,BCMA-H-TM'-C-CD3ζ; 4) L-VH, BCMA-VL, BCMA-H-TM-C-CD3ζ-2A peptide-L-VL, CD19-VH, CD19-H-TM'-C-CD3ζ; Where: In formula 1), VL, CD19-VH, BCMA-VL, BCMA-VH, and CD19 form a loop structure; In formula 2), VH, CD19-VL, BCMA-VH, BCMA-VL, and CD19 form a loop structure. "-" is independently a connecting peptide or a peptide bond; L is none or a signal peptide sequence; VH, BCMA is the variable region of the heavy chain of BCMA antibody, and VL, BCMA is the variable region of the light chain of anti-BCMA antibody; the sequence of the variable region of the heavy chain of VH, BCMA antibody is shown in SEQ ID NO: 1, and the sequence of the variable region of the light chain of VL, BCMA antibody is shown in SEQ ID NO: 2; 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 variable region of the heavy chain of the VH,CD19 antibody is shown in SEQ ID NO: 3, and the sequence of the variable region of the light chain of the VL,CD19 antibody is shown in SEQ ID NO: 4; 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-stimulatory signal molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

11. The use according to claim 10, characterized in that The VH, BCMA comprises HCDR1 of the amino acid sequence of SEQ ID NO: 5, HCDR2 of the amino acid sequence of SEQ ID NO: 6, and HCDR3 of the amino acid sequence of SEQ ID NO: 7; The VL, BCMA comprises LCDR1 of the amino acid sequence of SEQ ID NO:8, LCDR2 of the amino acid sequence of SEQ ID NO:9, and LCDR3 of the amino acid sequence of SEQ ID NO:

10.

12. The use according to claim 11, characterized in that The VH, CD19 comprises HCDR1 of the amino acid sequence of SEQ ID NO: 11, HCDR2 of the amino acid sequence of SEQ ID NO: 12, and HCDR3 of the amino acid sequence of SEQ ID NO: 13;. The VL, CD19 comprises LCDR1 of the amino acid sequence of SEQ ID NO:14, LCDR2 of the amino acid sequence of SEQ ID NO:15, and LCDR3 of the amino acid sequence of SEQ ID NO:

16.

13. Use of a nucleic acid molecule in the preparation of a drug for treating systemic lupus erythematosus, characterized in that: The nucleic acid molecule comprises a nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 10-12.

14. Use of a recombinant vector in the preparation of a medicament for treating systemic lupus erythematosus, characterized in that: The recombinant vector comprises the chimeric antigen receptor according to any one of claims 10 to 12, or the nucleic acid molecule according to claim 13.

15. The use according to claim 14, characterized in that The recombinant vector includes 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; Preferably, the vector is a lentiviral vector.

16. Use of BCMA-CD19 CAR-T cells in the preparation of a medicament for treating and / or preventing systemic lupus erythematosus, characterized in that: The BCMA-CD19 CAR-T cells are engineered autologous T cells that express the chimeric antigen receptor according to any one of claims 10 to 12 on their cell membranes.

17. A medicine or preparation, characterized in that The drug or preparation contains the chimeric antigen receptor according to any one of claims 10 to 12, the nucleic acid molecule according to claim 13, the recombinant vector according to any one of claims 14 to 15, or the BCMA-CD19 CAR-T cell according to claim 16, and optionally a pharmaceutically acceptable carrier, adjuvant or excipient.

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