A bispecific chimeric antigen receptor targeting CD20 and BCMA

Abstract

The present disclosure provides a bispecific chimeric antigen receptor (CAR) that targets CD20 and BCMA. The CAR may comprise a CD20-targeting scFv, a BCMA-targeting scFv, a hinge region, a transmembrane domain, a costimulatory region, and a cytoplasmic signaling domain. The chimeric antigen receptor can be used to treat autoimmune disorders or cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Nos. 63 / 493,495 (filed March 31, 2023) and 63 / 509,371 (filed June 21, 2023), each of which is incorporated by reference herein in its entirety.

[0002] Incorporation by reference of sequence listing The sequence listing submitted as XML file "11299_011840-WO0_SL.xml", created on March 26, 2024, and 168,342 bytes in size, is hereby incorporated by reference in its entirety.

[0003] Technical Field The present disclosure relates to the field of immunotherapy, and more particularly to bispecific chimeric antigen receptors (CARs) that target CD20 and BCMA. [Background technology]

[0004] Autoimmune diseases are conditions caused by the immune system's response to the body itself, resulting in damage to the body's own tissues. They are typically divided into two major categories: systemic autoimmune diseases (e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis, and systemic vasculitis) and organ-specific autoimmune diseases (autoimmune hepatitis and type 1 diabetes). Most autoimmune diseases are difficult to cure and often require long-term or lifelong medication. Treatments primarily involve corticosteroids and immunosuppressants, which significantly impact patients' quality of life and represent a significant unmet clinical need (Wang et al., Human autoimmune diseases: a comprehensive update, J. Intern. Med. 2015, 278(4):369-95).

[0005] The etiology of autoimmune diseases is unknown. In patients, abnormal activation of humoral immunity occurs, resulting in the production of numerous antibodies against self-antigens. These bind to form pathogenic immune complexes, which then deposit locally and cause inflammatory responses. B cells play an important role in the pathogenesis of autoimmune diseases and promote their development through various mechanisms (e.g., autoantibody production, cytokine release, and autoantigen presentation). Autoantibodies, as an important factor, can bind to self-antigens to form immune complexes, which can activate innate immune system cells to produce type I interferon and other proinflammatory cytokines, resulting in organ damage. Therefore, lymphocyte depletion or removal is a potential treatment strategy.

[0006] SLE is a typical autoimmune disease known to be associated with the hyperactivity of polyclonal B cells (Dorner et al., Mechanisms of B cell autoimmunity in SLE, Arthritis Res. Ther. 13, 243 (2011)). Therefore, one of the immunological hallmarks of SLE is the production of antinuclear antibodies (ANA), which may mediate the pathogenesis of SLE by binding to the respective autoantigens, leading to the deposition of immune complexes and the induction of inflammation and organ damage (e.g., lupus nephritis) (Salmon, JE, Arming T cells against B cells in systemic lupus erythematosus, Nat. Med. 28, 2009-2010 (2022)). There are two main types of ANA: anti-DNA antibodies and antibodies that recognize RNA-binding proteins (RBPs) (Pisetsky et al., New insights into the role of antinuclear antibodies in systemic lupus erythematosus, Nat. Rev. Rheumatol. 16, 565-579 (2020)). In SLE patients, the source of autoantibodies includes not only B cells but also a subset of plasma cells called long-lived plasma cells (LLPCs).Anti-DNA antibodies are produced by memory B cells that maintain high levels of CD19 and CD20 expression on the cell surface and by naive B cells that transition to plasmablasts, whereas anti-RBP antibodies are produced by LLPCs that may lose surface expression of CD19 and CD20 but are positive for B-cell maturation antigen (BCMA), a cell surface protein expressed on all mature plasma cells (Dogan et al., B-cell maturation antigen expression across hematologic cancers: a systematic literature review. Blood Cancer J. 10, 73 (2020); Morgan et al., Unraveling B cell trajectories at single cell resolution, Trends Immunol. 43, 210-229 (2022)). CD11c. hi T-bet + Recent studies have demonstrated that a subset of B cells proliferates in human SLE and functions as a precursor to autoantibody-producing plasma cells. This B cell subset shows high expression of CD19 and CD20 and is classified as autoreactive murine age-associated B cells (autoreactive B cells or ABC; the term is also used for human CD11c). hi T-bet +(Jenks et al., Distinct Effector B Cells Induced by Unregulated Toll-Like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus, Immunity 49, 725-739 e726 (2018); Wang et al., IL-21 Drives Expansion and Plasma Cell Differentiation of Autoreactive CD11c(hi)T-bet(+) B Cells in SLE, Nat. Commun. 9, 1758 (2018)). In addition to producing autoantibodies, B cells also contribute to the pathogenesis of SLE and other autoimmune diseases by secreting cytokines and acting as antigen-presenting cells. Therefore, depleting B cells in SLE patients may be an effective treatment for this life-threatening disease. B cell depletion was achieved by administering monoclonal antibodies against B cell surface markers. The anti-CD20 antibody rituximab was successful in early open-label trials in SLE, but failed to meet its primary endpoint in two randomized controlled trials (Lee et al., B cell depletion therapies in autoimmune disease: advances and mechanistic insights, Nat. Rev. Drug Discov. 20, 179-199 (2021)). Another antibody targeting CD19 (obexelimab) has also been tested in SLE. In early studies, patients receiving obexelimab maintained inactive levels of disease despite steroid discontinuation, but a phase II clinical trial failed to meet its primary endpoint (Lee et al., B cell depletion therapies in autoimmune disease: advances and mechanistic insights, Nat. Rev. Drug Discov. 20, 179-199 (2021)).

[0007] One promising approach to achieving B cell depletion is the adoptive transfer of CAR-T cells. CAR-T cells are genetically engineered T lymphocytes that can recognize specific antigens on target cells, proliferate, and generate cytotoxic immune responses, even in the absence of major histocompatibility complex (MHC) binding. In a recent study, compassionate use of CD19 CAR-T therapy in five patients with refractory SLE resulted in significant B cell depletion and drug-free remission. This suggests that CAR-T cell transplantation is feasible, tolerable, and highly effective in SLE (Mackensen et al., Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus, Nat. Med. 28, 2124-2132 (2022)).

[0008] There remains a pressing need to develop effective methods for treating autoimmune diseases. Summary of the Invention

[0009] The present disclosure provides a bispecific chimeric antigen receptor (CAR) comprising: (i) an anti-CD20 antigen-binding region, wherein the light chain variable region (V L 1) and the heavy chain variable region (V H 1) including V L 1 comprises three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively; H (ii) an anti-CD20 antigen-binding region comprising three CDRs (CDR1, CDR2, and CDR3) having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, respectively; and (ii) an anti-BCMA antigen-binding region comprising a light chain variable region (V L 2) and heavy chain variable region (V H 2) including V L2 comprises three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 134, SEQ ID NO: 136, and SEQ ID NO: 138, respectively; H 2 comprises three CDRs (CDR1, CDR2, and CDR3) having amino acid sequences that are approximately 80% to approximately 100% identical to the amino acid sequences set forth in SEQ ID NO: 141, SEQ ID NO: 143, and SEQ ID NO: 145, respectively.

[0010] The present disclosure provides: (i) a light chain variable region (V L 1) and the heavy chain variable region (V H (i) an anti-CD20 antigen-binding region comprising a light chain variable region (V L 2) and heavy chain variable region (V H 2). An anti-BCMA antigen binding region comprising:

[0011] In one embodiment, V L 1 is V H 1. In one embodiment, V H 1 is V L 1. In one embodiment, V L 2 is V H 2. In one embodiment, V H 2 is V L Located at the N-terminus of 2.

[0012] In certain embodiments, V L 1 and V H 1 has an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 8, respectively.

[0013] In certain embodiments, V L 2 and V H 2 has an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 16, respectively.

[0014] The anti-CD20 antigen-binding region may be a single-chain variable fragment (scFv) that specifically binds to CD20. The anti-BCMA antigen-binding region may be an scFv that specifically binds to BCMA.

[0015] The bispecific CAR may further comprise one or more of: (a) a signal peptide, (b) a hinge region, (c) a transmembrane domain, (d) a costimulatory region, and (e) a cytoplasmic signaling domain.

[0016] The hinge region may comprise the hinge region of IgG4, CD8, CD28, CD137, or a combination thereof.

[0017] The transmembrane domain may include the transmembrane domain of CD8, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0018] The costimulatory region may include a costimulatory region of 4-1BB (CD137), CD28, OX40, CD2, CD7, CD27, CD30, CD40, CD70, CD134, PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0019] The cytoplasmic signaling domain may comprise the cytoplasmic signaling domain of CD3ζ.

[0020] The present disclosure provides bispecific CARs comprising (or having) an amino acid sequence that is about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:26, SEQ ID NO:40, SEQ ID NO:54, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:98, SEQ ID NO:112, or SEQ ID NO:126.

[0021] The present disclosure also encompasses immune cells that express bispecific CARs. The immune cells may be T cells or natural killer (NK) cells.

[0022] The present disclosure provides nucleic acids encoding bispecific CARs.

[0023] The present disclosure provides a vector comprising the present nucleic acid encoding a bispecific CAR.

[0024] The present disclosure provides pharmaceutical compositions comprising the bispecific CAR, immune cells, nucleic acids, or vectors.

[0025] The present disclosure also provides methods of treating an autoimmune disorder. The methods may include administering the immune cells or pharmaceutical compositions to a subject in need thereof.

[0026] The autoimmune disorder may be systemic lupus erythematosus (SLE) (e.g., lupus nephritis), systemic vasculitis, systemic sclerosis, inflammatory myopathy (e.g., polymyositis, dermatomyositis, inclusion body myositis), systemic sclerosis, multiple sclerosis, myasthenia gravis, myositis autoantibody-induced disease, or neuromyelitis optica.

[0027] The autoimmune disorder may be polymyositis, dermatomyositis, or inclusion body myositis. The autoimmune disorder may be lupus nephritis.

[0028] The present disclosure also provides methods of treating cancer. The methods may include administering the immune cells or pharmaceutical compositions to a subject in need thereof.

[0029] The cancer may be a hematological cancer. The cancer may be a B-cell malignancy. The cancer may be Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, and / or multiple myeloma. The cancer may be acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0030] The immune cells may be allogeneic or autologous. [Brief explanation of the drawings]

[0031] [Figure 1] The structure of a composite chimeric antigen receptor targeting CD20 and BCMA is shown. The CAR structure contains a signal peptide (SP), an anti-CD20 scFv (OF), a linker (linker 2), an anti-BCMA scFv (B20), a hinge region, a transmembrane domain, a costimulatory region, and a cytoplasmic signaling domain (CD3ζ). A short IgG4 hinge (12 aa) and a CD28 transmembrane domain are contained in TOB1 to TOB4, and a CD8a hinge and a CD8a transmembrane domain are contained in TOBL1 to TOBL4. Four combinations of VH and VL orientations of the two scFv sequences are contained in two groups of CARs (TOB1-4 and TOBL1-4). TOBL1 is also designated C-CAR168. [Figure 2] Expression levels of anti-CD20 CAR and anti-BCMA CAR on the surface of T cells are shown. [Figure 3A] 1 shows the levels of IFN-γ secreted by in vitro activated CAR-T cells in cell culture supernatants. 1 shows the levels of IFN-γ secreted by TOB1 to TOB4 CAR-T cells in cell culture supernatants. [Figure 3B] 1 shows the levels of IFN-γ secreted by in vitro activated CAR-T cells in cell culture supernatants. 1 shows the levels of IFN-γ secreted by TOBL1 to TOBL4 CAR-T cells in cell culture supernatants. [Figure 3C] Figure 1 shows the levels of IFN-γ secreted by in vitro activated CAR-T cells in cell culture supernatants. TOBL1 to TOBL4 show high levels of IFN-γ release when co-cultured with target cells that naturally express CD20 and BCMA. MM.1S is a BCMA-positive multiple myeloma (MM) cell line, and RAJI is CD20- and BCMA-positive. [Figure 4A] 1 shows the expression level of CD137 on the surface of activated CAR-T cells. [Figure 4B] 1 shows the expression level of CD137 on the surface of activated CAR-T cells. [Figure 5A] The in vitro cytotoxicity of CAR-T cells (Figure 5A: TOB1 to TOB4, Figure 5B: TOBL1 to TOBL4) by RTCA assay is shown. [Figure 5B] The in vitro cytotoxicity of CAR-T cells (Figure 5A: TOB1 to TOB4, Figure 5B: TOBL1 to TOBL4) by RTCA assay is shown. [Figure 6A] C-CAR168 exhibits potent potency against CD20+ and BCMA+ cells in vitro. The structures of C-CAR168 (TOBL1, an anti-CD20 / BCMACAR), anti-CD20CAR (C-CAR066), and anti-BCMACAR (C-CAR088) are shown. [Figure 6B] C-CAR168 exhibits potent potency against CD20+ and BCMA+ cells in vitro, demonstrating the release of IFN-γ after CAR-T cells were co-cultured with CD20-positive and / or BCMA-positive target cells. [Figure 6C] C-CAR168 shows potent potency against CD20+ and BCMA+ cells in vitro. We demonstrate the cytotoxicity of CAR-T cells targeting CD20 and / or BCMA at different E:T ratios. [Figure 7A] Cytotoxicity of C-CAR168 against ABC-enriched B cells in vitro. Generation of C-CAR168 CAR-T cells. The bottom panel shows the CAR-positive rate of C-CAR168 CAR-T cells prepared from peripheral blood of three healthy donors. [Figure 7B] Cytotoxicity of C-CAR168 against ABC-enriched B cells in vitro. ABC differentiation. The bottom panel shows that the proportion of the ABC subpopulation was significantly increased after induction of autologous B cell differentiation. [Figure 7C] Cytolysis of ABC-enriched B cells by C-CAR168 at different E:T ratios. [Figure 8A]C-CAR168 has no cross-reactivity to the human membrane proteome. Binding specificity of C-CAR168 scFv-RabFc on membrane protein arrays. [Figure 8B] C-CAR168 has no cross-reactivity to the human membrane proteome. Binding specificity of C-CAR168 scFv-RabFc on membrane protein arrays. [Figure 8C] C-CAR168 has no cross-reactivity to the human membrane proteome. Flow cytometry detection of ITGB2-ITGAM and ITGB2-ITGAL expression in 293T cells. [Figure 8D] C-CAR168 has no cross-reactivity to the human membrane proteome. Left panel: Flow cytometry detection of the percentage of 4-1BB-positive cells. Right panel: Flow cytometry detection of IFN-γ concentration in the co-culture supernatant. [Figure 9A] In vivo cytotoxicity of C-CAR168 in tumor-bearing mice. C-CAR168 significantly inhibited the growth of A549-CD20 cells in B-NDG tumor-bearing mice. Left panel: Tumor growth curves for each group during the experiment. Right panel: Mean tumor weight in animals from each group on day 42. ***: P<0.001 compared with the vehicle control group. [Figure 9B] In vivo cytotoxicity of C-CAR168 in tumor-bearing mice. C-CAR168 significantly inhibited the growth of human multiple myeloma MM.1S tumor cells in B-NDG tumor-bearing mice. Left panel: Tumor growth curves for each group during the experiment. Right panel: Mean tumor weight in animals from each group on day 28. ***: P<<0.001 compared to the vehicle control group. [Figure 9C] In vivo cytotoxicity of C-CAR168 in tumor-bearing mice. Images of A549-CD20 tumors in animals from each group at day 42. " / " indicates that the animal died. Blank boxes indicate that tumor tissue was not collected. [Figure 9D]In vivo cytotoxicity of C-CAR168 in tumor-bearing mice. Images of MM.1S tumors in animals from each group at day 28. Blank boxes indicate that tumor tissue was not collected. [Figure 9E] In vivo cytotoxicity of C-CAR168 in tumor-bearing mice. C-CAR168 significantly inhibited the growth of K562-CD20-BCMA tumor cells in B-NDG tumor-bearing mice. Tumor growth curves for each group during the experiment. [Figure 9F] In vivo cytotoxicity of C-CAR168 in tumor-bearing mice. C-CAR168 significantly inhibited the growth of K562-CD20-BCMA tumor cells in B-NDG tumor-bearing mice. Survival curves for each group during the experimental period. [Figure 9G] In vivo cytotoxicity of C-CAR168 in tumor-bearing mice. C-CAR168 significantly inhibited the growth of K562-CD20-BCMA tumor cells in B-NDG tumor-bearing mice. Images of tumors from animals in each group on day 28. " / " indicates that the animal died. Blank boxes indicate that tumor tissue was not collected. [Figure 10A] C-CAR168 exhibits potent in vitro potency against autologous B cells from SLE patients. T cells from eight SLE patients were efficiently transduced with a lentiviral vector encoding C-CAR168 to express anti-CD20 / BCMACAR. [Figure 10B] C-CAR168 shows potent in vitro efficacy against autologous B cells from SLE patients. C-CAR168 CAR-T cells generated from SLE patient samples showed potent activity (IFN-γ release) against target cells expressing CD20 and BCMA. K562 is a CD20- and BCMA-negative multiple myeloma cell line, while MM.1S is a BCMA-positive multiple myeloma cell line. [Figure 10C]C-CAR168 exhibits potent in vitro efficacy against autologous B cells from SLE patients. C-CAR168 CAR-T cells generated from SLE patient samples showed potent activity (e.g., IFN-γ release) against pan-B cells isolated from SLE patients. [Figure 10D] C-CAR168 exhibits potent in vitro potency against autologous B cells from SLE patients: PanB cells isolated from SLE patients were recognized and lysed by autologous C-CAR168 cells. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present disclosure provides a chimeric antigen receptor (CAR) that targets both CD20 and BCMA. The CAR may comprise a signal peptide, an anti-CD20 scFv, an anti-BCMA scFv, a hinge region, a transmembrane domain, a costimulatory region, and a cytoplasmic signaling domain. The CAR can be used to treat autoimmune diseases or cancer.

[0033] B-cell maturation antigen (BCMA) (also known as TNFRSF17 or CD269) is a member of the tumor necrosis factor receptor family. Together with TACI and BAFF-R, it functions as a key receptor for B-cell activating factor (BAFF) and is involved in regulating the differentiation and maturation of B lymphocytes. BCMA is a type III membrane protein that is specifically expressed on B cells, particularly plasmablasts and differentiated mature plasma cells.

[0034] The B cell membrane marker CD20, also known as B1, is a transmembrane glycoprotein encoded by the MS4A gene. 2+ Through the regulation of conductance, it plays an important role in B cell development, proliferation, activation, differentiation, and malignant transformation.

[0035] The present disclosure provides bispecific chimeric antigen receptors (CARs). Bispecific CARs comprise: (i) a light chain variable region (V L 1) and the heavy chain variable region (VH (i) an anti-CD20 antigen-binding region comprising a light chain variable region (V L 2) and heavy chain variable region (V H 2).

[0036] The present bispecific chimeric antigen receptor (CAR) comprises: (i) a light chain variable region (V) having an amino acid sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 8, respectively; L 1) and the heavy chain variable region (V H (ii) an anti-CD20 antigen-binding region comprising: (i) an anti-CD20 antigen-binding region comprising: (i) a light chain variable region (V) having an amino acid sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 16, respectively; L 2) and heavy chain variable region (V H 2).

[0037] The present disclosure provides bispecific chimeric antigen receptors (CARs). Bispecific CARs comprise: (i) a light chain variable region (V L 1) and the heavy chain variable region (V H (i) an anti-CD20 antigen-binding region comprising a light chain variable region (V L 2) and heavy chain variable region (V H 2). L V may contain three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having amino acid sequences that are about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively. HV may contain three CDRs (CDR1, CDR2, and CDR3) having amino acid sequences that are about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, respectively. L V may contain three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having amino acid sequences that are about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 134, SEQ ID NO: 136, and SEQ ID NO: 138, respectively. H 2 may contain three CDRs (CDR1, CDR2, and CDR3) having amino acid sequences that are approximately 80% to 100%, approximately 85% to 100%, approximately 90% to 100%, or approximately 95% to 100% identical to the amino acid sequences set forth in SEQ ID NO: 141, SEQ ID NO: 143, and SEQ ID NO: 145, respectively.

[0038] In certain embodiments, V L 1 is V H 1. In certain embodiments, V H 1 is V L 1. In certain embodiments, V H 2 is V L 2. In certain embodiments, V L 2 is V H 2. In one embodiment, V L 1 is V H Located at the N-terminus of 1, V L 2 is V H Located at the N-terminus of 2.

[0039] In certain embodiments, V L 1 and V H 1 has an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 8, respectively.

[0040] In certain embodiments, V L 2 and V H2 has an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 16, respectively.

[0041] In certain embodiments, the antigen-binding region that specifically binds CD20 is located N-terminal to the antigen-binding region that specifically binds BCMA. In certain embodiments, the antigen-binding region that specifically binds BCMA is located N-terminal to the antigen-binding region that specifically binds CD20.

[0042] The anti-CD20 antigen-binding region may be a single-chain variable fragment (scFv) that specifically binds to CD20. The anti-BCMA antigen-binding region may be an scFv that specifically binds to BCMA. In certain embodiments, the scFv that specifically binds to CD20 is located at the N-terminus of the scFv that specifically binds to BCMA. In certain embodiments, the scFv that specifically binds to BCMA is located at the N-terminus of the scFv that specifically binds to CD20.

[0043] The bispecific CAR further comprises one or more of the following: (a) a signal peptide or SP (or leader sequence), (b) a hinge region, (c) a transmembrane domain, (d) a costimulatory region, and (e) a cytoplasmic signaling domain.

[0044] The bispecific CAR may comprise, from N- to C-terminus, a signal peptide, an anti-CD20scFv, an anti-BCMAscFv, a hinge region, a transmembrane domain, and a costimulatory region, as well as a cytoplasmic signaling domain.

[0045] The signal peptide may comprise (or be derived from) the signal peptide of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof. In one embodiment, the signal peptide is (or is derived from) the signal peptide of CD8.

[0046] In one embodiment, the signal peptide comprises an amino acid sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:2.

[0047] The hinge region may comprise (or be derived from) an IgG4, CD8, CD28, CD137, or a combination, wild-type, or mutant hinge region thereof.

[0048] In one embodiment, the hinge region is (or is derived from) an IgG4 hinge region. In one embodiment, the hinge region comprises an amino acid sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 78.

[0049] In one embodiment, the hinge region is (or is derived from) the hinge region of CD8a. In one embodiment, the hinge region comprises an amino acid sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:18.

[0050] The transmembrane domain may comprise (or be derived from) the transmembrane domain of CD8, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or combinations thereof.

[0051] In one embodiment, the transmembrane domain is (or is derived from) the transmembrane domain of CD8. In one embodiment, the transmembrane domain comprises an amino acid sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:20.

[0052] In one embodiment, the transmembrane domain is (or is derived from) the transmembrane domain of CD28. In one embodiment, the transmembrane domain comprises an amino acid sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:80.

[0053] The costimulatory region may comprise (or be derived from) the costimulatory region of 4-1BB (CD137), CD28, OX40, CD2, CD7, CD27, CD30, CD40, CD70, CD134, PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0054] In one embodiment, the costimulatory region is (or is derived from) the costimulatory region of 4-1BB. In one embodiment, the costimulatory region comprises an amino acid sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:22.

[0055] The cytoplasmic signaling domain may comprise (or be derived from) the cytoplasmic signaling domain of CD3ζ. In one embodiment, the cytoplasmic signaling domain comprises an amino acid sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:24.

[0056] This CAR contains the V of the anti-CD20 antigen-binding region. L and V H A linker (linker 1) may be included between the amino acid sequence set forth in SEQ ID NO:6. In one embodiment, the linker (linker 1) includes an amino acid sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:6.

[0057] The CAR may comprise a linker (linker 2) between the anti-CD20 antigen-binding region and the anti-BCMA antigen-binding region. In one embodiment, the linker (linker 2) comprises an amino acid sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 10.

[0058] This CAR contains the V of the anti-BCMA antigen-binding region. L and V H A linker (linker 3) may be included between the amino acid sequence set forth in SEQ ID NO: 14. In one embodiment, the linker (linker 3) includes an amino acid sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 14.

[0059] In one embodiment, the bispecific CAR comprises, from N-terminus to C-terminus, (a) a light chain variable region (V) of ofatumumab; L 1) and the heavy chain variable region (V H (i) an anti-CD20 antigen-binding region having a light chain variable region (V) of a BCMA-20 antibody; L 2) and heavy chain variable region (V H (iii) a hinge region having the amino acid sequence set forth in SEQ ID NO: 18; (iv) a transmembrane domain having the amino acid sequence set forth in SEQ ID NO: 20; (v) a costimulatory region having the amino acid sequence set forth in SEQ ID NO: 22; and (vi) a cytoplasmic signaling domain having the amino acid sequence set forth in SEQ ID NO: 24.

[0060] In one embodiment, the bispecific CAR comprises, from N- to C-terminus, (a) a light chain variable region (V) having the amino acid sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 8, respectively; L 1) and the heavy chain variable region (V H (ii) an anti-CD20 antigen-binding region having the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 16, respectively; and (iii) a light chain variable region (V L 2) and heavy chain variable region (V H(iii) a hinge region having the amino acid sequence set forth in SEQ ID NO: 18; (iv) a transmembrane domain having the amino acid sequence set forth in SEQ ID NO: 20; (v) a costimulatory region having the amino acid sequence set forth in SEQ ID NO: 22; and (vi) a cytoplasmic signaling domain having the amino acid sequence set forth in SEQ ID NO: 24.

[0061] In one embodiment, the bispecific CAR comprises, from N-terminus to C-terminus, (a) a light chain variable region (V) of ofatumumab; L 1) and the heavy chain variable region (V H (i) an anti-CD20 antigen-binding region having a light chain variable region (V) of that of BCMA-20; L 2) and heavy chain variable region (V H (iii) a hinge region having the amino acid sequence set forth in SEQ ID NO: 78; (iv) a transmembrane domain having the amino acid sequence set forth in SEQ ID NO: 80; (v) a costimulatory region having the amino acid sequence set forth in SEQ ID NO: 22; and (vi) a cytoplasmic signaling domain having the amino acid sequence set forth in SEQ ID NO: 24.

[0062] In one embodiment, the bispecific CAR comprises, from N- to C-terminus, (a) a light chain variable region (V) having the amino acid sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 8, respectively; L 1) and the heavy chain variable region (V H (ii) an anti-CD20 antigen-binding region having the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 16, respectively; and (iii) a light chain variable region (V L 2) and heavy chain variable region (V H (iii) a hinge region having the amino acid sequence set forth in SEQ ID NO: 78; (iv) a transmembrane domain having the amino acid sequence set forth in SEQ ID NO: 80; (v) a costimulatory region having the amino acid sequence set forth in SEQ ID NO: 22; and (vi) a cytoplasmic signaling domain having the amino acid sequence set forth in SEQ ID NO: 24.

[0063] In certain embodiments, V L 1 is VH 1. In certain embodiments, V H 1 is V L 1. In certain embodiments, V H 2 is V L 2. In certain embodiments, V L 2 is V H 2. In one embodiment, V L 1 is V H Located at the N-terminus of 1, V L 2 is V H Located at the N-terminus of 2.

[0064] In certain embodiments, the bispecific CAR comprises an amino acid sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:26, SEQ ID NO:40, SEQ ID NO:54, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:98, SEQ ID NO:112, or SEQ ID NO:126.

[0065] In certain embodiments, the bispecific CAR may have the amino acid sequence set forth in SEQ ID NO:26, SEQ ID NO:40, SEQ ID NO:54, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:98, SEQ ID NO:112, or SEQ ID NO:126.

[0066] The bispecific CAR can be encoded by a nucleic acid having a nucleotide sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:25, SEQ ID NO:39, SEQ ID NO:53, SEQ ID NO:67, SEQ ID NO:83, SEQ ID NO:97, SEQ ID NO:111, or SEQ ID NO:125.

[0067] The bispecific CAR can be encoded by a nucleic acid having the nucleotide sequence set forth in SEQ ID NO:25, SEQ ID NO:39, SEQ ID NO:53, SEQ ID NO:67, SEQ ID NO:83, SEQ ID NO:97, SEQ ID NO:111, or SEQ ID NO:125.

[0068] The present disclosure provides immune cells that express or contain the present bispecific CARs. The immune cells may be T cells or natural killer (NK) cells.

[0069] The present disclosure provides an immune cell comprising a vector or nucleic acid (e.g., integrated into its genome) encoding the CAR. The cell can be an isolated cell. The cell can be a genetically engineered cell. The cell can be a mammalian cell. In one embodiment, the cell is a CAR-T cell and / or a CAR-NK cell.

[0070] The present disclosure also encompasses nucleic acids encoding the subject chimeric antigen receptors (e.g., the subject bispecific CARs).

[0071] The nucleic acid may comprise a nucleotide sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 25, SEQ ID NO: 39, SEQ ID NO: 53, SEQ ID NO: 67, SEQ ID NO: 83, SEQ ID NO: 97, SEQ ID NO: 111, or SEQ ID NO: 125.

[0072] The nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO:25, SEQ ID NO:39, SEQ ID NO:53, SEQ ID NO:67, SEQ ID NO:83, SEQ ID NO:97, SEQ ID NO:111, or SEQ ID NO:125.

[0073] The present disclosure provides a vector comprising the nucleic acid. The vector may comprise DNA or RNA. The vector may be a plasmid, a viral vector, a transposon, or a combination thereof. The vector may comprise a DNA virus or a retroviral vector. The vector may be a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a combination thereof. In one embodiment, the vector is a lentiviral vector.

[0074] The present disclosure provides a pharmaceutical composition comprising the chimeric antigen receptor (e.g., the bispecific CAR), the immune cell, the nucleic acid, or the vector. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition may be a liquid preparation.

[0075] The pharmaceutical composition contains approximately 1 x 10 3 cells / mL ~ approx. 1×10 8 cells / mL, or approximately 1 x 10 4 cells / mL ~ approx. 1×10 7 It may be included at concentrations ranging from 100 cells / mL.

[0076] The present disclosure also provides a method for treating an autoimmune disease / disorder. The present disclosure provides a method for treating cancer. The method may include administering the immune cells or the pharmaceutical composition to a subject in need thereof.

[0077] The immune cells may be allogeneic or autologous.

[0078] The autoimmune disorder may be systemic lupus erythematosus (SLE) (e.g., lupus nephritis), systemic sclerosis (SSc), an inflammatory myopathy (e.g., polymyositis, dermatomyositis, or inclusion body myositis), systemic sclerosis, multiple sclerosis, or neuromyelitis optica (NMO).

[0079] The cancer may be a hematological cancer. The cancer may be a B-cell malignancy. The cancer may be Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, and / or multiple myeloma. The cancer may be acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0080] The present disclosure provides a method for preparing an immune cell (e.g., a CAR-T cell) that expresses a chimeric antigen receptor, the method comprising introducing the nucleic acid molecule or the vector into an immune cell (e.g., a T cell or an NK cell), thereby obtaining the immune cell (e.g., a CAR-T cell) that expresses the chimeric antigen receptor.

[0081] The present disclosure provides a chimeric antigen receptor (CAR), the structure of which may be represented by Formula I: [ka] (wherein each "-" is independently a linker peptide or a peptide bond; SP is an optional signal peptide; H is an optional hinge region; TM is a transmembrane domain; C is a costimulatory region; CSD is a cytoplasmic signaling domain; one of scFv1 and scFv2 is an anti-CD20 antigen-binding region; and the other is an anti-BCMA antigen-binding region).

[0082] In one embodiment, scFv1 is an anti-CD20 antigen-binding region and scFv2 is an anti-BCMA antigen-binding region. In another embodiment, scFv1 is an anti-BCMA antigen-binding region and scFv2 is an anti-CD20 antigen-binding region.

[0083] The structure of the anti-CD20 antigen-binding region may be as shown in Formula A or B below: [ka] (In the formula, V H1 is an anti-CD20 antibody heavy chain variable region; V L1 is an anti-CD20 antibody light chain variable region; and "-" is a linker peptide or peptide bond).

[0084] In one embodiment, the CAR has an anti-CD20 antigen-binding region (or domain) having the structure shown in Formula B.

[0085] In certain embodiments, V L1The amino acid sequence of V is shown in SEQ ID NO: 4. H1 The amino acid sequence of is shown in SEQ ID NO:8.

[0086] V L1 and V H1 may be linked by a linker peptide (Linker 1 or Linker 1). Linker 1 may have the sequence set forth in SEQ ID NO:6.

[0087] The structure of the anti-BCMA antigen binding region may be as shown in Formula C or D below: [ka] (In the formula, V L2 is an anti-BCMA antibody light chain variable region; V H2 is an anti-BCMA antibody heavy chain variable region; and "-" is a linker peptide or peptide bond).

[0088] In one embodiment, the CAR has an anti-BCMA antigen binding domain of the structure shown in formula C.

[0089] In certain embodiments, V L2 The amino acid sequence of V is shown in SEQ ID NO: 12. H2 The amino acid sequence of is shown in SEQ ID NO:16.

[0090] V L2 and V H2 may be linked by a linker peptide (linker 3 or linker 3). Linker 3 may have the sequence set forth in SEQ ID NO:14.

[0091] In another embodiment, the structure of the chimeric antigen receptor is shown below in Formula II: [ka]

[0092] In one embodiment, linker 2 (or linker 2) has the sequence set forth in SEQ ID NO:10.

[0093] In certain embodiments, the anti-CD20 antigen-binding region comprises a light chain variable region (V) comprising an amino acid sequence at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:4. L ) is included.

[0094] In certain embodiments, the anti-CD20 antigen-binding region comprises a heavy chain variable region (V) comprising an amino acid sequence at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:8. H ) is included.

[0095] In certain embodiments, the anti-BCMA antigen-binding region comprises a light chain variable region (V) comprising an amino acid sequence that is at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 12. L ) is included.

[0096] In certain embodiments, the anti-BCMA antigen-binding region comprises a heavy chain variable region (V) comprising an amino acid sequence that is at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 16. H ) is included.

[0097] The light chain variable region (V) of the anti-CD20 antigen-binding regionL ) are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 99%, at least or about 99%, at least or about 100%, at least or about 101%, at least or about 102%, at least or about 103%, at least or about 104%, at least or about 105%, at least or about 106%, at least or about 107%, at least or about 108%, at least or about 109%, at least or about 1109%, at least or about 1111%, at least or about 112%, at least or about 113%, at least or about 114%, at least or about 115%, at least or about 116%, at least or about 117%, at least or about 118%, at least or about 119%, at least or about 120%, at least or about 121%, at least or about 122%, at least or about 123%, at least or about 124%, at least or about 125%, at least or about 126%, at least or about 127%, at least or about 128%, at least or about 129%, at least or about 130%, at least or about 131%, at least or about 132%, at least or about 133%, at least or about 134%, at least or about 135%, at least or about 136%, at least or about 137%, at least or about 138%, at least or about 139%, at least or about 140%, at least or about 141%, at least or about 142%, at least or about 14 or may comprise one, two, or three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) that are about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical.

[0098] The light chain variable region (V) of the anti-CD20 antigen-binding region L) are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 99%, at least or about 99%, at least or about 99%, at least or about 100%, at least or about 101%, at least or about 102%, at least or about 103%, at least or about 104%, at least or about 105%, at least or about 106%, at least or about 107%, at least or about 108%, at least or about 109%, at least or about 1109%, at least or about 1111%, at least or about 1121%, at least or about 113%, at least or about 114%, at least or about 115%, at least or about 116%, at least or about 117%, at least or about 118%, at least or about 119%, at least or about 120%, at least or about 1212%, at least or about 1221, at least or about 123%, at least or about 124%, at least or about 125%, at least or about 126%, at least or about 127%, at least or about 128%, at least or about 129%, at least or about 130%, at least or about 1313, at least or about 1324, at least or about 1325, at least or about 133%, at least or about 134%, at least or about 135%, at least or about 136%, at least or about 137%, at least or about 138%, at least or about 139%, at least or about 140%, at least may comprise one, two, or three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) that are about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical.

[0099] The heavy chain variable region (V H) are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 99%, at least or about 99%, at least or about 100%, at least or about 101%, at least or about 102%, at least or about 103%, at least or about 104%, at least or about 105%, at least or about 106%, at least or about 107%, at least or about 108%, at least or about 109%, at least or about 1 ... or may comprise one, two, or three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) that are about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical.

[0100] The heavy chain variable region (V L) are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 99%, at least or about 99%, at least or about 99%, at least or about 100%, at least or about 101%, at least or about 102%, at least or about 103%, at least or about 104%, at least or about 105%, at least or about 106%, at least or about 107%, at least or about 108%, at least or about 109%, at least or about 1109%, at least or about 1111%, at least or about 112%, at least or about 113%, at least or about 114%, at least or about 115%, at least or about 116%, at least or about 117%, at least or about 118%, at least or about 119%, at least or about 120%, at least or about 1211%, at least or about 1212%, at least or about 1213%, at least or about 1214%, at least or about 1215%, at least or about 1216%, at least or about 1217%, at least or about 1218%, at least or about 1219%, at least or about 1221%, at least or about 1222%, at least or about 1223%, at least or about 1224%, at least or about 1225%, at least or about 1226%, at least or about 1227, at least or about 1228, at least or about 1229, at least or about 1231, at least or about 12 may comprise one, two, or three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) that are about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical.

[0101] In certain embodiments, the light chain variable region (V L ) contain three CDR1s, CDR2s, and CDR3s identical to the CDR1s, CDR2s, and CDR3s (CDRs of the light chain variable region of the ofatumumab antibody) set forth at positions 24 to 34, 50 to 56, and 89 to 97 of SEQ ID NO: 4, respectively, and contain a heavy chain variable region (V H ) contain three CDRs identical to CDR1, CDR2, and CDR3 (CDRs of the heavy chain variable region of the ofatumumab antibody) set forth at positions 30 to 35, positions 50 to 66, and positions 99 to 111 of SEQ ID NO: 8, respectively.

[0102] In certain embodiments, the light chain variable region (V L) contain three CDRs (CDR1, CDR2, and CDR3) identical to the CDR1, CDR2, and CDR3 (CDRs of the light chain variable region of the ofatumumab antibody) set forth in SEQ ID NOs: 130, 131, and 132, respectively, and contain a heavy chain variable region (V H ) contain three CDRs identical to CDR1, CDR2, and CDR3 (CDRs of the heavy chain variable region of the ofatumumab antibody) set forth in SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, respectively.

[0103] The light chain variable region (V L ) are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 99%, at least or about 99%, at least or about 100%, at least or about 101%, at least or about 102%, at least or about 103%, at least or about 104%, at least or about 105%, at least or about 106%, at least or about 107%, at least or about 108%, at least or about 109%, at least or about 1109%, at least or about 1111%, at least or about 1122, at least or about 113%, at least or about 114%, at least or about 115%, at least or about 116%, at least or about 117%, at least or about 118%, at least or about 119%, at least or about 129%, at least or about 121 or may comprise one, two, or three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) that are about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical.

[0104] The light chain variable region (V L) are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 99%, at least or about 99%, at least or about 99%, at least or about 100%, at least or about 101%, at least or about 102%, at least or about 103%, at least or about 104%, at least or about 105%, at least or about 106%, at least or about 107%, at least or about 108%, at least or about 109%, at least or about 1109%, at least or about 1111%, at least or about 112%, at least or about 113%, at least or about 114%, at least or about 115%, at least or about 116%, at least or about 117%, at least or about 118 may comprise one, two, or three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) that are about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical.

[0105] The heavy chain variable region (V H) are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 9 ... The complementary determining regions (CDRs) may comprise one, two, or three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) that are at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical to each other.

[0106] The heavy chain variable region (V H) are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 99%, at least or about 99%, at least or about 99%, at least or about 100%, at least or about 101%, at least or about 102%, at least or about 103%, at least or about 104%, at least or about 105%, at least or about 106%, at least or about 107%, at least or about 108%, at least or about 109%, at least or about 1109%, at least or about 1111%, at least or about 112%, at least or about 113%, at least or about 114%, at least or about 115%, at least or about 116%, at least or about 117%, at least or about 118%, at least or about 119%, at least or about 120%, at least or about 1211%, at least or about 122%, at least or about 123%, at least or about 124%, at least or about 125%, at least or about 126%, at least or about 127%, at least or about 128%, at least or about 129%, at least or about 130%, at least or about 131 may comprise one, two, or three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) that are about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical.

[0107] In certain embodiments, the light chain variable region (V L ) contain three CDRs (CDR1, CDR2, and CDR3) identical to CDR1, CDR2, and CDR3 (CDRs of the light chain variable region of the BCMA-20 antibody) set forth at positions 24 to 34, 50 to 56, and 89 to 97 of SEQ ID NO: 12, respectively, and contain a heavy chain variable region (V H ) contain three CDRs (CDR1, CDR2, and CDR3) that are identical to CDR1, CDR2, and CDR3 (CDRs of the heavy chain variable region of the BCMA-20 antibody) set forth at positions 31 to 35, positions 50 to 66, and positions 99 to 110 of SEQ ID NO: 16, respectively.

[0108] In certain embodiments, the light chain variable region of the anti-BCMA antigen binding region comprises CDR1, CDR2, and CDR3 (light chain variable region of the BCMA-20 antibody (V)) set forth in SEQ ID NO: 134, SEQ ID NO: 136, and SEQ ID NO: 138, respectively. L)) and the heavy chain variable region (V H ) contain three CDRs (CDR1, CDR2, and CDR3) identical to the CDR1, CDR2, and CDR3 (CDRs of the heavy chain variable region of the BCMA-20 antibody) set forth in SEQ ID NO: 141, SEQ ID NO: 143, and SEQ ID NO: 145, respectively.

[0109] In certain embodiments, in the CAR, the antigen-binding domain targeting CD20 is a light chain variable domain V derived from the ofatumumab antibody. L (SEQ ID NO: 4) and heavy chain variable domain V H (SEQ ID NO: 8).

[0110] Light chain variable domain V derived from ofatumumab (OF) antibody L may have the following sequence: [ka]

[0111] OF-VL-CDR1: SEQ ID NO: 4, positions 24 to 34. The sequence of OF-VL-CDR1 is RASQSVSSYLA (SEQ ID NO: 130).

[0112] OF-VL-CDR2: SEQ ID NO: 4, positions 50 to 56. The sequence of OF-VL-CDR2 is DASNRAT (SEQ ID NO: 131).

[0113] OF-VL-CDR3: SEQ ID NO: 4, positions 89 to 97. The sequence of OF-VL-CDR3 is QQRSNWPIT (SEQ ID NO: 132).

[0114] Heavy chain variable domain V from ofatumumab antibody H may have the following sequence: [ka]

[0115] OF-VH-CDR1: SEQ ID NO: 8, positions 30 to 35. The sequence of OF-VH-CDR1 is NDYAMH (SEQ ID NO: 127).

[0116] OF-VH-CDR2: SEQ ID NO: 8, positions 50 to 66. The sequence of OF-VH-CDR2 is TISWNSGSIGYADSVKG (SEQ ID NO: 128).

[0117] OF-VH-CDR3: SEQ ID NO: 8, positions 99 to 111. The sequence of OF-VH-CDR3 is DIQYGNYYYGMDV (SEQ ID NO: 129).

[0118] In certain embodiments, the BCMA-targeting antigen-binding domain of the CAR comprises a light chain variable domain V derived from the BCMA-20 (B20) antibody. L (SEQ ID NO: 12) and heavy chain variable domain V H (SEQ ID NO: 16).

[0119] Light chain variable domain V from BCMA-20 antibody L may have the following sequence: DIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCMGQTISSYTFGQGTKLEIK (SEQ ID NO: 12).

[0120] B20-VL-CDR1: SEQ ID NO: 12, positions 24 to 34. The sequence of B20-VL-CDR1 is RASQGISNYLN (SEQ ID NO: 134).

[0121] B20-VL-CDR2: SEQ ID NO: 12, positions 50 to 56. The sequence of B20-VL-CDR2 is YTSNLQS (SEQ ID NO: 136).

[0122] B20-VL-CDR3: SEQ ID NO: 12, positions 89 to 97. The sequence of B20-VL-CDR3 is MGQTISSYT (SEQ ID NO: 138).

[0123] Heavy chain variable domain V from BCMA-20 antibody H may have the following sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGADHAIYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVSS (SEQ ID NO: 16).

[0124] B20-VH-CDR1: SEQ ID NO: 16, positions 31 to 35. The sequence of B20-VH-CDR1 is NFDMA (SEQ ID NO: 141).

[0125] B20-VH-CDR2: SEQ ID NO: 16, positions 50 to 66. The sequence of B20-VH-CDR2 is SITTGADHAIYADSVKG (SEQ ID NO: 143).

[0126] B20-VH-CDR3: SEQ ID NO: 16, positions 99 to 110. The sequence of B20-VH-CDR3 is HGYYDGYHLFDY (SEQ ID NO: 145).

[0127] The signal peptide may be the signal peptide of CD8, which has the following sequence: MALPVTALLLPLALLLHAARP (SEQ ID NO: 2).

[0128] V of anti-CD20 scFv (linker 1) L and V H (or V H and V L ) may have the following sequence: GSTSGGGSGGGSGGGGSS (SEQ ID NO: 6).

[0129] The linker between the anti-CD20 scFv and the anti-BCMA scFv (linker 2) may have the following sequence: GGGGS (SEQ ID NO: 10).

[0130] V of anti-BCMA scFv (linker 3) L and V H (or VH and V L ) may have the following sequence: GGGGSGGGSGGGGS (SEQ ID NO: 14).

[0131] The hinge region between the extracellular region (antigen-binding domain) and the transmembrane domain may be derived from IgG4, CD8 (CD8a), CD28, CD137, or a combination thereof.

[0132] The hinge region may be derived from CD8a having the following sequence: FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 18).

[0133] The hinge region may be derived from IgG4 having the following sequence: ESKYGPPCPPCP (SEQ ID NO: 78).

[0134] The transmembrane domain may be derived from CD8 (CD8™) having the following sequence: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 20).

[0135] The transmembrane domain may be derived from CD28 (CD28TM) having the following sequence: MFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 80).

[0136] The costimulatory region may be derived from 4-1BB having the following sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 22).

[0137] The cytoplasmic signaling domain may be derived from CD3ζ, which has the following sequence: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 24).

[0138] Chimeric antigen receptor (CAR) The terms "chimeric antigen receptor" or "CAR" are used interchangeably throughout and refer to a recombinant polypeptide construct comprising, for example, an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain. Lee et al., Clin. Cancer Res. (2012) 18(10):2780; Jensen et al., Immunol Rev. (2014) 257(1):127. In one embodiment, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule. The costimulatory molecule may be 4-1BB (i.e., CD137), CD27, and / or CD28, or fragments of these molecules. In another aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. A CAR may comprise a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. Alternatively, a CAR may comprise a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. A CAR may also comprise a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. The antigen-binding region of a CAR may contain any antigen-binding antibody fragment. The antibody fragment may comprise one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or any combination of the above.

[0139] The term "zeta" or alternatively "zeta chain," "CD3 zeta," or "TCR zeta" can refer to the protein provided as GenBank Accession No. NP_932170, NP_000725, or XP_011508447; or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.), and a "zeta stimulatory domain" or "CD3 zeta stimulatory domain" or "TCR zeta stimulatory domain" can be amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally transmit the initial signal required for T cell activation.

[0140] A chimeric receptor may refer to a non-naturally occurring molecule that can be expressed on the surface of a host cell and comprises an antigen-binding fragment that binds to an antigen. In addition to the antigen-binding fragment, a chimeric receptor may further comprise one or more of a hinge region, a transmembrane domain, at least one costimulatory region, and a cytoplasmic signaling domain. In some embodiments, a chimeric antigen receptor comprises, from N-terminus to C-terminus, an antigen-binding region (or fragment), a hinge region, a transmembrane domain, and a cytoplasmic signaling domain. In some embodiments, a chimeric antigen receptor further comprises at least one costimulatory region. Thus, a chimeric antigen receptor may comprise, from N-terminus to C-terminus, an antigen-binding region (or fragment), a hinge region, a transmembrane domain, a costimulatory region, and a cytoplasmic signaling domain.

[0141] In some embodiments, the chimeric antigen receptor comprises a hinge region that may be located between the antigen-binding region and the transmembrane domain. The hinge region may contain approximately 10 to 200 amino acids (e.g., 15 to 150 amino acids, 20 to 100 amino acids, or 30 to 60 amino acids). In some embodiments, the hinge region may be approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids in length. The hinge region may contain 0 to 300 amino acids, 2 to 100 amino acids, 5 to 80 amino acids, 10 to 60 amino acids, 10 to 15 amino acids, 20 to 80 amino acids, 30 to 70 amino acids, 40 to 60 amino acids, 50 to 60 amino acids, or 30 to 60 amino acids.

[0142] In some embodiments, the hinge region is the hinge domain of a naturally occurring protein. The hinge domain of any protein known in the art to contain a hinge domain is suitable for use in a chimeric antigen receptor. In some embodiments, the hinge domain is that of CD8α or CD28α. In some embodiments, the hinge domain is a portion of the hinge domain of CD8α, for example, a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α or CD28α.

[0143] Hinge domains of antibodies (e.g., IgG, IgA, IgM, IgE, or IgD) are also adapted for use in chimeric antigen receptors. In some embodiments, the hinge region is the hinge domain connecting the constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge region comprises the hinge domain of an antibody and one or more constant regions of an antibody. In some embodiments, the hinge region comprises the hinge domain of an antibody and the CH3 constant region of an antibody. In some embodiments, the hinge region comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of an antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and CH3 constant region of an IgG4 antibody.

[0144] The hinge region may be a non-naturally occurring peptide. In some embodiments, the hinge region between the extracellular antigen-binding domain and the transmembrane domain is a peptide linker (e.g., a (GlyxSer)n (or (GxS)n) linker), where x and n can independently be integers between 3 and 12 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more).

[0145] Additional peptide linkers that can be used in the hinge region of the chimeric receptors described herein are known in the art, see, e.g., Wriggers et al. Current Trends in Peptide Science (2005) 80(6):736-746 and PCT Publication WO2012 / 088461.

[0146] In some embodiments, a chimeric antigen receptor may comprise a transmembrane domain. The transmembrane domain may take any form known in the art. A transmembrane domain suitable for use in a chimeric antigen receptor may be obtained from a naturally occurring protein. Alternatively, the transmembrane domain may be a synthetic, non-naturally occurring protein segment (e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane).

[0147] In some embodiments, the transmembrane domain is that of CD8α. In some embodiments, the transmembrane domain is that of CD28. In some embodiments, the transmembrane domain is that of ICOS.

[0148] In some embodiments, the chimeric antigen receptor comprises one or more costimulatory regions. A costimulatory region may be at least a portion of a protein that mediates signal transduction within a cell to induce an immune response (e.g., effector function). The costimulatory region of a chimeric antigen receptor may be derived from a protein that transmits a signal and regulates responses mediated by immune cells (e.g., T cells, natural killer (NK) cells, macrophages, neutrophils, and eosinophils).

[0149] In some embodiments, the chimeric antigen receptor comprises one or more (at least, two, three, four, or more) costimulatory regions. In some embodiments, the chimeric antigen receptor comprises multiple costimulatory regions obtained from different proteins. In some embodiments, the chimeric antigen receptor does not comprise a costimulatory region.

[0150] Examples of costimulatory regions used in chimeric antigen receptors can be domains of costimulatory proteins (including, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, Cd40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3). In some embodiments, the costimulatory region is derived from 4-1BB, CD28, or ICOS. In some embodiments, the costimulatory region is derived from CD28, and the chimeric antigen receptor includes a second costimulatory region derived from 4-1BB or ICOS. In some embodiments, the costimulatory region is a fusion domain comprising multiple costimulatory regions or portions of multiple costimulatory regions. In some embodiments, the costimulatory region is a fusion of the costimulatory regions from CD28 and ICOS.

[0151] In some embodiments, the chimeric antigen receptor comprises a cytoplasmic signaling domain. Any cytoplasmic signaling domain can be used in the chimeric antigen receptors described herein. The cytoplasmic signaling domain can transmit a signal (e.g., the interaction of the extracellular ligand-binding domain with its ligand (e.g., cytotoxicity)) to stimulate a cellular response (e.g., induce a cellular effector function).

[0152] Chimeric antigen receptors can be prepared by conventional methods (e.g., recombinant techniques). Methods for preparing chimeric antigen receptors may include generating nucleic acids encoding polypeptides comprising each of the domains of a chimeric antigen receptor (e.g., an antigen-binding fragment, optionally a hinge region, a transmembrane domain, at least one costimulatory region, and a cytoplasmic signaling domain). In some embodiments, nucleic acids encoding each component of a chimeric antigen receptor are combined using recombinant techniques. Methods such as PCR amplification and ligation can be used to directly or indirectly combine the sequences of the components (e.g., domains) (e.g., using a nucleic acid sequence encoding a peptide linker) to form a nucleic acid sequence encoding a chimeric antigen receptor. Alternatively, a nucleic acid encoding a chimeric antigen receptor can be synthesized. In some embodiments, the nucleic acid is DNA. In other embodiments, the nucleic acid is RNA.

[0153] In one embodiment, the CAR comprises, from N- to C-terminus, a signal peptide (also called a leader sequence), an antigen recognition sequence (antigen binding domain), a hinge region, a transmembrane domain, a costimulatory region, and a cytoplasmic signaling domain (e.g., a CD3 zeta signaling region (zeta chain portion)).

[0154] Bispecificity means that the CAR can specifically bind to two different antigens. A bispecific CAR can generate an immune response by binding to one antigen or both antigens.

[0155] As used herein, the terms "CAR-T cells," "CAR-T," "CART," and "CART cells" can refer to T cells expressing the present CAR that targets both CD20 and BCMA.

[0156] Immune cells expressing chimeric antigen receptors The present disclosure provides immune cells expressing the present CARs. Upon recognition of a target cell bearing an antigen(s) on its surface by the antigen-binding fragment of the chimeric antigen receptor, an activation signal is transmitted to the signaling domain(s) of the chimeric antigen receptor (e.g., the costimulatory region and / or the cytoplasmic signaling domain), which can activate effector functions in the immune cell expressing the chimeric antigen receptor.

[0157] The chimeric antigen receptor can be introduced into suitable immune cells for expression by conventional techniques. In some embodiments, the immune cells are T cells (e.g., primary T cells or T cell lines). Alternatively, the immune cells can be natural killer (NK) cells (e.g., established NK cell lines (e.g., NK-92 cells)). In some embodiments, the immune cells are CD8 (CD8 + ) or CD8 and CD4 (CD8 + / CD4 + In some embodiments, the T cells are T cells of an established T cell line (e.g., Jurkat cells).

[0158] Primary T cells can be obtained from any source (e.g., peripheral blood mononuclear cells (PBMCs), bone marrow, tissue (e.g., spleen, lymph nodes, thymus), or tumor. In some embodiments, the population of immune cells is derived from a human patient (e.g., bone marrow or PBMCs obtained from the patient) with an autoimmune disorder or cancer (e.g., a hematopoietic malignancy). In some embodiments, the population of immune cells is derived from a healthy donor. In some embodiments, the immune cells are obtained from a subject to whom the immune cells expressing the chimeric antigen receptor are subsequently administered. Immune cells administered to the same subject from which the cells were obtained are referred to as autologous cells, while immune cells obtained from a subject different from the subject to whom the cells are administered may be referred to as allogeneic cells.

[0159] Desired types of immune cells can be expanded within the resulting cell population by co-incubating the cells with stimulatory molecules, for example, anti-CD3 and anti-CD28 antibodies can be used to expand T cells.

[0160] To construct immune cells expressing the chimeric antigen receptor described herein, a vector for stable or transient expression of the chimeric antigen receptor can be constructed by conventional methods described herein and introduced into immune cells. For example, a nucleic acid encoding the chimeric antigen receptor can be cloned into an appropriate vector (e.g., a viral vector).

[0161] In certain embodiments, immune cells (e.g., T cells) are transduced with a lentiviral vector (LV) encoding the CAR, which targets CD20 and BCMA, synergistically activating the T cells and inducing a T cell-mediated immune response.

[0162] In one embodiment, the method involves isolating, activating, and genetically modifying T cells from an autologous patient (or allogeneic donor) to generate CAR-T cells expressing the CAR, which are then administered to the patient. CAR-T cells can replicate in vivo, resulting in long-term persistence. Furthermore, the CAR-mediated immune response can be part of an adoptive immunotherapy approach, in which anti-CD20 / BCMA CAR-T cells induce an immune response against cells expressing CD20 and / or BCMA.

[0163] In certain embodiments, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic effect. The mammalian recipient may be human. The CAR-modified cells can be autologous to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic to the recipient.

[0164] Methods for preparing immune cells expressing the present chimeric antigen receptor may include activating and / or expanding the immune cells ex vivo. Activating immune cells means stimulating the immune cells to an activated state, allowing the cells to exert effector function (e.g., cytotoxicity). The method for activating immune cells will vary depending on the type of immune cells used to express the chimeric antigen receptor. Expanding immune cells may include any method that results in an increase in the number of cells expressing the chimeric antigen receptor (e.g., allowing the cells to grow or stimulating the growth of the cells). In some embodiments, cells expressing the chimeric receptors described herein are activated and / or expanded ex vivo prior to administration to a subject.

[0165] CAR-expressing immune cells can also function as vaccines for ex vivo immunization and / or in vivo therapy in mammals. In addition to the use of cell-based vaccines in ex vivo immunization, the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient. Preferably, the mammal is a human. With respect to ex vivo immunization, one or more of the following can occur in vitro before administering the cells to a mammal: i) expansion of the cells, ii) introduction of a nucleic acid encoding a CAR into the cells, and / or iii) cryopreservation of the cells.

[0166] vector The present disclosure provides nucleic acids encoding the CAR. The present disclosure also provides vectors comprising the nucleic acids.

[0167] Vectors include, but are not limited to, plasmids, phagemids, phage derivatives, viruses, and cosmids.

[0168] The vector may be a viral vector. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In certain embodiments, the vector is a retroviral vector (e.g., a lentiviral vector). In some embodiments, the vector for expression of a chimeric antigen receptor is a retrovirus. In some embodiments, the vector for expression of a chimeric antigen receptor is a lentivirus. In some embodiments, the vector for expression of a chimeric antigen receptor is an adeno-associated virus.

[0169] Various promoters can be used for the expression of chimeric receptors (e.g., but not limited to, the cytomegalovirus (CMV) intermediate-early promoter, viral LTRs (e.g., Rous sarcoma virus LTR), HIV-LTR, HTLV-1 LTR, Moloney murine leukemia virus (MMLV) LTR, myeloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, simian virus 40 (SV40) early promoter, herpes simplex TK virus promoter, and the elongation factor 1-α (EF1-α) promoter with or without the EF1-α intron). Additional promoters for the expression of chimeric receptors include constitutively active promoters in immune cells. Alternatively, any regulatable promoter (e.g., an inducible promoter) can be used so that its expression can be regulated in immune cells.

[0170] The vector can be introduced into a cell (e.g., a mammalian cell, a bacterial cell, a yeast cell, or an insect cell) by any method known in the art, for example, the vector can be introduced into the cell by physical, chemical, or biological means.

[0171] Physical methods for introducing polynucleotides into cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. See, e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0172] Biological methods for introducing a polynucleotide of interest into cells include the use of DNA and RNA vectors. Viral vectors can be derived from retroviruses, lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others.

[0173] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems (e.g., macromolecule complexes, nanocapsules, microspheres, beads) and lipid-based systems (e.g., 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., artificial membrane vesicle).

[0174] In some embodiments, the vector (nucleic acid) encoding the chimeric antigen receptor is a DNA vector that can be electroporated into immune cells (see, e.g., Till, et al. Blood (2012) 119(17):3940-3950). In some embodiments, the vector (nucleic acid) encoding the chimeric antigen receptor is an RNA molecule that can be electroporated into immune cells.

[0175] Any vector containing a nucleic acid encoding a chimeric antigen receptor described herein is within the scope of the present disclosure. Such vectors can be delivered to host cells (e.g., immune cells) using appropriate methods. Methods for delivering vectors to immune cells are well known in the art and may include electroporation of DNA, RNA, or transposons, transfection reagents (e.g., liposomes or nanoparticles that deliver DNA, RNA, or transposons), delivery of DNA, RNA, or transposons or proteins by mechanical deformation (see, for example, Sharei et al. PNAS (2013) 110(6):2082-2087), or viral transduction. In some embodiments, vectors for expressing chimeric receptors are delivered to cells by viral transduction.

[0176] In an example where a vector encoding a chimeric antigen receptor is introduced into host cells using a viral vector, viral particles capable of infecting immune cells and carrying the vector can be produced by any method known in the art. The viral particles can be harvested from cell culture supernatant and isolated and / or purified before contacting the viral particles with immune cells.

[0177] Pharmaceutical Composition The present disclosure provides a pharmaceutical composition comprising the immune cells, the CAR, the nucleic acid, or the vector. The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the preparation is a liquid preparation. In one embodiment, the concentration of immune cells (e.g., CAR-T cells) in the preparation is 1×10 3 ~1×10 8 cells / mL, or 1 x 10 4 ~1×10 7 cells / mL.

[0178] The effective amount will vary, as those skilled in the art will recognize, depending on the particular condition being treated, the severity of the condition, individual patient parameters (e.g., age, physical condition, size, sex, weight), duration of treatment, the nature of concomitant therapy (if any), the particular route of administration, and similar factors within the knowledge and belief of a medical professional. In some embodiments, an effective amount alleviates, relieves, ameliorates, improves, or reduces the subject's symptoms or slows the progression of a disease or disorder. In some embodiments, the subject is a mammal. In some embodiments, the subject is human.

[0179] Pharmaceutically acceptable carriers (e.g., buffers) are well known in the art and may include phosphate, citric acid, and other organic acids; antioxidants (e.g., ascorbic acid and methionine); preservatives; low molecular weight polypeptides; proteins (e.g., serum albumin, gelatin, or immunoglobulins); amino acids; hydrophobic polymers; monosaccharides, disaccharides, and other carbohydrates; metal complexes; and / or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K.E. Hoover.

[0180] The pharmaceutical composition can be delivered to a cell by contacting the cell with the pharmaceutical composition.

[0181] The pharmaceutical composition can be delivered / administered to a subject by any route (e.g., but not limited to, intravenous, intracerebroventricular (ICV) injection, intracisternal injection or infusion, oral, transdermal, intraocular, intraperitoneal, subcutaneous, implant, sublingual, subcutaneous, intramuscular, rectal, mucosal, intraocular, intrathecal, intra-articular, intra-arterial, intrathecal, bronchial, and lymphatic administration). The pharmaceutical composition can be administered parenterally or systemically. The composition can be administered topically. The pharmaceutical composition can be formulated for intravenous administration.

[0182] Administration of the composition can be carried out by any convenient method (e.g., by aerosol inhalation, injection, ingestion, transfusion, implant, or transplant). The composition can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously (i.v.), or intraperitoneally. In one embodiment, the composition is administered to a subject (e.g., a patient) by intradermal or subcutaneous injection. In another embodiment, the composition is administered by intravenous injection. The composition can be injected directly into a tumor, lymph node, or lesion site.

[0183] The immune cells or pharmaceutical compositions may be delivered / administered to a subject by intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration (e.g., injection or infusion).

[0184] The pharmaceutical composition can be administered in a manner appropriate for the disease to be treated (or prevented). The dosage and frequency of administration will be determined by such factors as the patient's condition and the type and severity of the patient's disease, and the appropriate dosage can be determined by clinical trials.

[0185] When an "effective amount," "therapeutically effective amount," or "therapeutic amount" is indicated, the exact amount of the composition to be administered can be determined by a physician taking into account the age, weight, tumor size, degree of infection or metastasis, and individual differences in condition of the patient (subject). 4 ~10 9 Pieces / kg body weight, or 10 5 ~10 6 The compositions may be administered at a dosage of 100 cells / kg body weight (including all integer values ​​within these ranges). The compositions may also be administered multiple times at these dosages. The cells may be administered by injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0186] Dosages of the above treatments to be administered to a patient may vary depending on the exact nature of the condition being treated and the recipient of the treatment. Scaling of dosages for administration to a patient can be performed according to art-recognized practices. In one embodiment, 1 x 10 6 ~1×10 10 The immune cells (e.g., CAR-T cells) can be administered to the patient using, for example, intravenous infusion for each treatment or course of treatment.

[0187] Condition to be treated The CAR, immune cell, or pharmaceutical composition can be used to treat an autoimmune disease / disorder, or to treat a cancer or tumor.

[0188] In certain embodiments, the present anti-CD20 / BCMA bispecific CARs may target both B cells and plasma cells, thereby reducing / eradicating autoimmune antibodies. In certain embodiments, the present anti-CD20 / BCMA bispecific CARs may reduce / deplete B cells, plasmablasts, and / or long-lived plasma cells (LLPCs) to reduce / eradicate autoantibody production.

[0189] The present disclosure provides a method for treating an autoimmune disease / disorder. The method may include administering a CAR, an immune cell, or a pharmaceutical composition to a subject in need thereof.

[0190] The autoimmune disorder may be: systemic lupus erythematosus (SLE), lupus nephritis (LN), systemic sclerosis (SSc), CREST syndrome (calcinosis, Raynaud's syndrome, esophageal motility disorders, digital sclerosis, and telangiectasia), opsoclonus, inflammatory myopathies (e.g., polymyositis, dermatomyositis, and inclusion body myositis), myositis autoantibody-induced disease, systemic sclerosis, primary biliary cirrhosis, celiac disease (e.g., gluten-sensitive enteropathy), dermatitis herpetiformis, Miller-Fisher syndrome, acute motor axonal neuropathy (AMAN), multifocal motor neuropathy with conduction block, autoimmune hepatitis, anti-inflammatory drugs (ART), ... phospholipid syndrome, Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, rheumatoid arthritis, chronic autoimmune hepatitis, scleromyositis, myasthenia gravis (MG), Lambert-Eaton myasthenic syndrome, Hashimoto's thyroiditis, Graves' disease, paraneoplastic cerebellar degeneration, stiff-person syndrome, limbic encephalitis, Isaacs syndrome, Sydenham chorea, childhood autoimmune neuropsychiatric disorder associated with streptococcal infection (PANDAS), encephalitis, type 1 diabetes, neuromyelitis optica (NMO), chronic inflammatory bowel disease, Hashimoto's thyroiditis, organ transplant rejection, and / or neuromyelitis optica spectrum disorder (NMOSD).

[0191] The autoimmune disorder may be pernicious anemia, Addison's disease, psoriasis, inflammatory bowel disease (IBD), psoriatic arthritis, Sjogren's syndrome, lupus erythematosus (e.g., discoid lupus erythematosus, drug-induced lupus erythematosus, and neonatal lupus erythematosus), multiple sclerosis, and / or reactive arthritis.

[0192] The autoimmune disorder may be: polymyositis, dermatomyositis, polyendocrine deficiency, Schmidt's syndrome, autoimmune uveitis, adrenalitis, thyroiditis, autoimmune thyroid disease, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, presenile dementia, demyelinating diseases, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, alopecia areata, pemphigoid, scleroderma, progressive systemic sclerosis, adult-onset diabetes (e.g., type II diabetes), male and female autoimmune infertility, ankylosing spondylitis, ulcerative colitis, Crohn's disease, sprue, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, juvenile Rheumatoid arthritis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent miscarriage, antiphospholipid syndrome, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird fancier's lung, allergic diseases, allergic encephalomyelitis, toxic epidermal necrolysis, alopecia, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reactions, leprosy, malaria, leishmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sumpter's syndromesyndrome), eczema, lymphomatoid granulomatosis, Behçet's disease, Kaplan's syndrome, Kawasaki disease, dengue fever, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum, erythroblastosis fetalis, eosinophilic fasciitis, Schorman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, metachronous cyclitis, Fuchs' cyclitis, IgA nephropathy, Henoch-Schönlein purpura, graft-versus-host disease, transplant rejection, human immunodeficiency virus infection, echovirus Infectious diseases, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Hodgkin's lymphoma and non-Hodgkin's lymphoma, renal cell carcinoma, multiple myeloma, Eaton-Lambert syndrome, relapsing polychondritis, malignant melanoma, cryoglobulinemia, Waldenström's macroglobulinemia, Epstein-Barr virus infection, mumps, Evans syndrome, and / or autoimmune hypogonadism.

[0193] Autoimmune diseases also include, for example, acute disseminated encephalomyelitis, alopecia areata, antiphospholipid syndrome, autoimmune hepatitis, autoimmune myocarditis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune uveitis, inflammatory bowel disease (Crohn's disease, ulcerative colitis), type I diabetes (e.g., juvenile diabetes), multiple sclerosis, scleroderma, ankylosing spondylitis, sarcoid, pemphigus vulgaris, pemphigoid, psoriasis, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, Behcet's syndrome, Reiter's syndrome, disease, Buerger's disease, dermatomyositis, polymyositis, antineutrophil cytoplasmic antibody-associated vasculitis (e.g., granulomatosis with polyangiitis (also known as Wegener's granulomatosis), microscopic polyangiitis, and Churg-Strauss syndrome), scleroderma, Sjögren's syndrome, anti-glomerular basement membrane disease (e.g., Goodpasture's syndrome), dilated cardiomyopathy, primary biliary cirrhosis, thyroiditis (e.g., Hashimoto's thyroiditis, Graves' disease), transverse myelitis, allergies, arthritis, fibromyalgia, fibromatosis, lupus, vitiligo, and Guillain-Barré syndrome.

[0194] Autoimmune diseases include: inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, sprue, autoimmune arthritis, rheumatoid arthritis, type 1 diabetes, multiple sclerosis, graft-versus-host disease after bone marrow transplantation, osteoarthritis, juvenile chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, insulin-dependent diabetes mellitus, thyroiditis, asthma, psoriasis, scleroderma dermatitis, atopic dermatitis, graft-versus-host disease, acute or chronic immune disorders associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, Graves' disease Disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schönlein purpura, microscopic vasculitis of the kidney, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, septic syndrome, cachexia, acquired immune deficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, polyglandular deficiency type I syndrome and polyglandular deficiency type II syndrome, Schmidt's syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthropathy, arthropathy, Reiter's disease , psoriatic arthropathy, Chlamydia-, Yersinia- and Salmonella-associated arthropathy, spondyloarthropathies, atherosclerosis / arteriosclerosis, atopic allergies, food allergies, autoimmune bullous diseases, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune hemolytic anemia, Coombs-positive hemolytic anemia, acquired pernicious anemia, juvenile pernicious anemia, myalgic encephalitis / Royal Free disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, idiopathic autoimmune hepatitis, acquired immunodeficiency syndrome, acquired immunodeficiency-associated diseases, hepatitis C, common variable immunodeficiency (common variable hypogastric syndrome) globulinemia), dilated cardiomyopathy, fibrotic lung disease, idiopathic fibrosing alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonia, connective tissue disease-associated interstitial lung disease, mixed connective tissue disease-associated lung disease, systemic sclerosis-associated interstitial lung disease, rheumatoid arthritis-associated interstitial lung disease, systemic lupus erythematosus-associated lung disease, dermatomyositis / polymyositis-associated lung disease, Sjogren's disease-associated lung disease, ankylosing spondylitis-associated lung disease, vasculitic diffuse lung disease, hemosiderosis-associated lung disease, drug-induced interstitial lung disease, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrate lung disease, post-infectious interstitial lung disease,Gouty arthritis, autoimmune hepatitis, type 1 autoimmune hepatitis (classical autoimmune hepatitis or lupoid hepatitis), type 2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune hypoglycemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disorders associated with organ transplantation, chronic immune disorders associated with organ transplantation, osteoarthritis, primary sclerosing cholangitis, idiopathic leukopenia, idiopathic leukopenia, renal disease NOS, glomerulonephritis, microscopic vasculitis of the kidney, discoid lupus, lupus erythematosus, male infertility (idiopathic or NOS), sperm autoimmunity, multiple sclerosis (all subtypes), insulin-dependent diabetes mellitus, sympathetic ophthalmia, pulmonary hypertension due to connective tissue disease, Goodpaschaft's disease syndrome, pulmonary manifestations of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Takayasu's disease / arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxedema, lens-induced uveitis, primary vasculitis, vitiligo, allergic rhinitis (pollen allergy), anaphylaxis, pet allergy, latex allergy, drug allergy, allergic rhinoconjunctivitis, eosinophilic esophagitis, hypereosinophilic syndrome, eosinophilic gastroenteritis, cutaneous lupus erythematosus, eosinophilic esophagitis, hypereosinophilic syndrome, and eosinophilic gastroenteritis.

[0195] The autoimmune disorder may be an inflammatory myopathy. Inflammatory myopathy is a group of diseases that involve chronic muscle inflammation, muscle weakness, and in some cases, muscle pain. The four main types of chronic or long-term inflammatory myopathy are: polymyositis (affecting skeletal muscles (the type involved in body movement) on both sides of the body); dermatomyositis (causing progressive muscle weakness); inclusion body myositis (characterized by slowly progressive muscle weakness and muscle atrophy and loss); and necrotizing autoimmune myopathy (associated with muscle weakness in both the upper and lower body).

[0196] In another embodiment, the autoimmune disease is an autoimmune disease caused by overexpression of B cells (eg, lupus erythematosus).

[0197] The present disclosure also includes methods of treating cancer. The methods can include administering a CAR, immune cell, or pharmaceutical composition to a subject in need thereof.

[0198] The present disclosure provides chimeric antigen receptors for treating CD20-positive diseases (e.g., B-cell lymphoma).

[0199] The cancer may be a BCMA-positive malignancy. The cancer may be multiple myeloma (MM) or plasma cell leukemia.

[0200] The cancer may be a hematological cancer. The cancer may be a plasma cell malignancy. The cancer may be a B-cell malignancy. The B-cell malignancy may be acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), B-cell leukemia, or B-cell lymphoma.

[0201] The cancer may be Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, and / or multiple myeloma (MM).

[0202] The cancer may be acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0203] Diseases that can be treated using the present CARs, immune cells, or pharmaceutical compositions include CD20-positive tumors and diseases (e.g., those caused by an excess of B cells, e.g., autoimmune diseases such as systemic lupus erythematosus). CD20-positive tumors may include CD20-positive non-solid tumors (e.g., blood cancers, e.g., leukemia and lymphoma) or solid tumors. Tumors or cancers to be treated with the present CARs, immune cells, or pharmaceutical compositions include, but are not limited to, carcinomas, blastomas, sarcomas, leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors (e.g., sarcomas, carcinomas, gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, kidney cancer, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumors, bladder tumors, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, and melanoma). Includes adult tumors / cancers and pediatric tumors / cancers.

[0204] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological cancers (or hematogenous cancers) include leukemias (e.g., acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute myelogenous leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemias (e.g., chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (low-grade and high-grade), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia).

[0205] The cancer may be a solid tumor. Solid tumors may be benign or malignant. Various types of solid tumors are named based on the type of cells that form them (e.g., sarcoma, carcinoma, and lymphoma). Examples of solid tumors (e.g., sarcoma and carcinoma) include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, pancreatic cancer, and ovarian cancer.

[0206] kit Kits for using the present CAR, immune cells, nucleic acids, vectors, or pharmaceutical compositions are also within the scope of the present disclosure. Such kits may include one or more containers containing the present CAR, immune cells, nucleic acids, vectors, or pharmaceutical compositions.

[0207] In some embodiments, the kit may include instructions for use in any of the methods described herein. The included instructions may include instructions for administering the pharmaceutical composition to a subject to achieve the intended activity in the subject. The kit may further include instructions for selecting a subject suitable for treatment based on identifying whether the subject is in need of treatment. In some embodiments, the instructions include instructions for administering the pharmaceutical composition to a subject in need of treatment.

[0208] Instructions for use of a pharmaceutical composition generally include information regarding dosage, administration schedule, and route of administration for the intended treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses.

[0209] The kits provided herein are in suitable packaging, including but not limited to vials, bottles, jars, flexible packaging, and the like.

[0210] The following examples of specific embodiments for carrying out the present disclosure are offered for illustrative purposes only and are not intended to limit the scope of the disclosure in any way.

[0211] Example 1 Construction of an anti-CD20 / BCMA CAR We have anti-CD20 scFv and anti-BCMA scFv in the same order (i.e., anti-CD20 scFv (“OF”) followed by anti-BCMA scFv (“B20”)), but H / V L Eight bispecific CARs were prepared that differed in the order of the nucleotides, the hinge region, and / or the transmembrane domain: TOB1-4 and TOBL1-4 (TOBL1 is C-CAR168) (Figure 1).

[0212] Anti-CD20 / BCMA CAR-T cells were prepared using apheresis from healthy donors. Specifically, PBMCs were isolated from venous blood of healthy donors by density gradient centrifugation. On day 0, PBMCs were activated in cell culture flasks pre-coated with CD3 monoclonal antibody (OKT3) and Retronectin (TAKARA). The culture medium was GT-551 cell culture medium containing 1% human albumin and 300 U / mL recombinant human interleukin-2 (IL-2). On day 3, activated PBMCs were transduced with a lentiviral vector encoding the anti-CD20 / BCMA CAR.

[0213] Figure 2 shows the expression levels of anti-CD20 CAR and anti-BCMA CAR on the surface of T cells. The expression level of anti-BCMA CAR was detected using a BCMA-Fc fusion protein; the expression level of anti-CD20 CAR was detected, followed by flow cytometry using an antibody specific for OF scFv.

[0214] Example 2: Antigen-specific activation of anti-CD20 / BCMA CAR-T cells in vitro Antigen-specific activation of anti-CD20 / BCMA CAR-T cells was assessed by assaying IFN-γ release and CD137 expression when co-cultured with target cells. Target cells ("T") included CD20-positive A549-CD20+ tumor cells, BCMA-positive A549-BCMA+ tumor cells, CD20 and BCMA double-positive A549-CD20+BCMA+ tumor cells, Raji cells, MM.1S cells, and double-negative A549 tumor cells. Effector cells (E) were anti-CD20 / BCMA CAR-T cells.

[0215] PBMCs were isolated from venous blood of healthy donors by density gradient centrifugation. On day 0, PBMCs were activated in cell culture flasks pre-coated with CD3 monoclonal antibody (OKT3) and Retronectin (TAKARA). The culture medium was GT-551 cell culture medium containing 1% human albumin and 300 U / mL recombinant human interleukin-2 (IL-2). On day 3, activated PBMCs were transduced with a lentiviral vector encoding an anti-CD20 / BCMA CAR. Starting on day 6, CAR-T cells could be harvested for activity assays.

[0216] IFNγ release was measured using CAR-T cells cultured for 7 days. 5 CAR-T cells were cultured with CD20-positive A549-CD20+ tumor cells, BCMA-positive A549-BCMA+ tumor cells, CD20 and BCMA double-positive A549-CD20+BCMA+ tumor cells, double-negative A549 tumor cells, or no tumor cells (NT) in 200 μl of medium at an E:T ratio of 1:1 for 18 hours. The level of IFN-γ secreted into the cell culture supernatant was then measured by ELISA.

[0217] The CD137 expression level was measured using CAR-T cells cultured for 7 days. 5 CAR-T cells were cultured with CD20-positive A549-CD20+ tumor cells, BCMA-positive A549-BCMA+ tumor cells, CD20 and BCMA double-positive A549-CD20+BCMA+ tumor cells, double-negative A549 tumor cells, or no tumor cells at a 1:1 E:T ratio in 200 μl of medium for 18 hours. CD137 expression levels on the CAR-T cell surface were then measured by flow cytometry.

[0218] The IFNγ release results are shown in Figures 3A-3C and 6B. After coculture of CAR-T cells with A549 cells expressing CD20 and / or BCMA antigens, anti-CD20 CAR-T (C-CAR066) cells could specifically recognize CD20 single-positive or CD20 / BCMA double-positive target cells and release IFNγ. Similarly, anti-BCMA CAR-T (C-CAR088) cells could specifically recognize BCMA single-positive or CD20 / BCMA double-positive target cells to release IFNγ. Only anti-CD20 / BCMA CAR-T (TOB1-4 and TOBL1-4 (TOBL1 is C-CAR168)) cells could recognize CD20 single-positive, BCMA single-positive, and CD20 / BCMA double-positive target cells and release high levels of IFNγ. TOB1-TOB4 CAR-T cells showed high IFN-γ release when co-cultured with CD20-positive target cells, but low reactivity to BCMA-single-positive target cells. TOBL1-TOBL4 CAR-T cells showed high IFN-γ release when co-cultured with CD20-positive and BCMA-positive target cells. TOBL1-TOBL4 CAR-T cells showed high IFN-γ release when co-cultured with target cells that naturally express CD20 and BCMA.

[0219] Flow cytometry results showed that anti-CD20 / BCMA CAR-T cells activated by various CD20 / BCMA single- or double-positive cells upregulated the expression levels of CD137 (Figures 4A and 4B).

[0220] Example 3 In vitro cytotoxicity of anti-CD20 / BCMA CAR-T cells Anti-CD20 / BCMA CAR-T cells were co-cultured with target cells at E:T ratios of 0:1, 0.25:1, 0.5:1, 1:1, 2:1, and 4:1, respectively. Real-time cell analysis (RTCA) label-free technology was used to assess the cytotoxicity of CAR-T cells against target cells.

[0221] The results show that anti-CD20 / BCMA CAR-T cells effectively killed CD20 / BCMA single- or double-positive tumor cells (A549-CD20+, A549-BCMA+, and A549-BCMA+CD20+) in vitro, but not A549 cells, which do not express CD20 or BCMA (Figures 5A-5B and 6C). These killing capacities were comparable to those of anti-CD20 and anti-BCMA monospecific CAR-T cells, and all were dose-dependent (Figures 5A-5B and 6C). TOBL1-TOBL4 CAR-T cells (Figure 5B) showed high cytotoxicity against CD20- and BCMA-positive target cells. TOB1-TOB4 CAR-T cells (Figure 5A) showed reduced cytotoxicity against BCMA-single-positive target cells (compared to the anti-BCMA CAR, C-CAR088).

[0222] Example 4 Cytotoxicity of anti-CD20 / BCMA CAR-T cells against autoreactive B cells in vitro Recent studies have shown that CD11c is expressed in patients with systemic lupus erythematosus (SLE). hi T-bet +The proportion of B cell subsets has been shown to be significantly increased and closely correlated with autoantibody production and patient clinical symptoms. Autoantibodies are a hallmark of reactive B cells (see: Distinct Effector B Cells Induced by Unregulated Toll-like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus, Immunity, 2018, 16;49(4):725-739. e6. IL-21 drives expansion and plasma cell differentiation of autoreactive CD11chi T-bet+ B cells in SLE, Nat. Commun. 2018;9(1):1758). This subset of cells is also called age-associated B cells (ABC) because it enriches with age in the peripheral blood of several animal models of autoimmune diseases and patients with rheumatoid arthritis (see: Toll-like receptor 7 (TLR7)-driven accumulation of a novel CD11c + B-cell population is important for the development of autoimmunity.Blood,2011;118(5):1305-15.A B-cell subset uniquely responsive to innate stimuli accumulates in aged mice,Blood,2011;118(5):1294-304).

[0223] TLR7 activation plays an important role in the accumulation of autoreactive B cells and the production of autoantibodies in autoimmune diseases. One consequence of abnormal TLR7 activation is the accumulation of autoreactive B cells, or age-associated B cells (ABCs). ABCs are B cells that have the potential to recognize self-antigens and produce autoantibodies, which can target and damage the body's own tissues. (Wang et al., Nature Communications, (2018) 9:1758)

[0224] To confirm that anti-CD20 / BCMA CAR-T cells also have the ability to eliminate ABCs, we prepared C-CAR168 (TOBL1) CAR-T cells from the peripheral blood of three healthy human donors (HD10, HD11, and HD12). We also isolated autologous B cells from the healthy donors' PBMCs and induced their differentiation in vitro to obtain ABC-enriched autologous B cells, which were then used as target cells for cytotoxicity experiments. After 2 to 4 hours of coculture, C-CAR168 CAR-T cells from different donors showed clear cytotoxic effects against ABC-enriched autologous B cells at different E:T ratios compared to control T cells without CAR transduction (Figures 7A-7C).

[0225] C-CAR168 can target both CD20+ B cells and BCMA+ plasma cells and provide excellent response duration in autoimmune diseases. The results show that C-CAR168 CAR-T cells can efficiently eliminate ABC cells.

[0226] In vitro ABC differentiation PBMCs from healthy donors were isolated by Ficoll gradient centrifugation and cryopreserved. On the day of ABC differentiation, panB cells were first isolated from thawed PBMCs using a human B cell isolation kit (Miltenyi Biotec; negative selection, e.g., non-B cells were labeled and depleted) according to the manufacturer's instructions. Next, B cells were seeded into 96-well plates containing 200 μl of RPMI complete medium and stimulated for 3 days with TLR7 ligand R848, CD40L, BAFF, IL-2, goat anti-human IgA+IgG+IgM (H+L), IL-21, and IFN-γ. The cell culture medium was changed daily by replenishing the complete medium and stimulation cocktail. ABC induction was confirmed by FACS analysis. Antibodies used for FACS staining included live / dead staining, anti-human CD19, CD38, CD27, IgD, CD11c, CD21, and T-bet.

[0227] Cytotoxicity assay After differentiation, ABC-enriched B cells were co-cultured with C-CAR168 or non-transduced (NT) T cells at the indicated E:T ratio. After 24 hours, cells were stained with LIVE / DEAD Fixable Aqua Dead Cell Stain (Invitrogen) to determine viability with anti-CD19 and anti-CD3 antibodies to distinguish B and T cells. Cytotoxicity was assessed by the quantification of viable CD19 and T cells. + The percentage of depleted B cells was determined. B cell lysis was calculated using the following formula: lysis rate (%) = (1 - (viable CD19 of C-CAR168 coculture)). + Viable CD19 in cell fraction / UT co-cultures + Cell fraction) x 100. See: Lin et al., Preclinical evaluation of CD8+ anti-BCMA mRNA CAR T-cells for treatment of multiple myeloma. Leukemia. 2021, 35(3):752-763.

[0228] Example 5 Inhibitory effect of anti-CD20 / BCMA CAR-T cells on mouse tumor cells C-CAR 168 effectively inhibited the growth of CD20 single-positive and BCMA single-positive tumor cells in tumor-bearing mice. The in vivo cytotoxic effects of anti-CD20 / BCMA CAR-T cells on CD20- or BCMA-single-positive cells were evaluated in a mouse subcutaneous tumor model established using tumor cell lines expressing either CD20 (A549-CD20) or BCMA (MM.1S).

[0229] Six to eight week old female B-NDG mice were subcutaneously inoculated with A549-CD20 (CD20+) or MM.1S (BCMA+) cells. The mean tumor volume was 100 mm. 3 When the target cell number reached 3–5 × 10 C-CAR168 CAR-T cells, 6The tumors were administered via the tail vein at a dose of 10 CAR-T cells per mouse. During the experiment, tumor volume in mice treated with C-CAR168 CAR-T cells continued to decrease. At the end of the experiment, tumor weight in the C-CAR168 group was significantly lower than that in the vehicle control group. C-CAR168 cells exhibited potent cytotoxicity against CD20- and BCMA-positive target cells in vivo.

[0230] Specifically, female B-NDG (NOD.Cg-Prkdc scid Il2rg tm1Vst / Vst) mice, 5 × 10 6 A549-CD20 cells / animal were inoculated subcutaneously. The average tumor volume was approximately 100 mm 3 When the number of animals reached 10, 20 animals were selected and randomly divided into two groups (vehicle control group vs. C-CAR168 group), with 10 animals in each group. 6 Mice were administered a single dose of CAR-T cells (per animal) via tail vein injection. After administration, the mean tumor volume in the vehicle control group continued to increase, reaching 494.16 ± 31.5 mm at day 42. 3 The mean tumor volume in the C-CAR168 group began to decrease from day 10. By day 42, the mean tumor volume was 10.02 ± 7.04 mm. 3 (Figure 9A, left panel). The tumor weight was 0.013±0.01 g, which was significantly different from the vehicle control group (P<0.001) (Figure 9A, right panel). The tumor growth inhibition rate calculated based on the tumor weight was 94.88%. The results indicate that C-CAR168 can significantly inhibit the growth of CD20-positive target cells in vivo.

[0231] To evaluate the in vivo effect of C-CAR168 on BCMA single-positive target cells and compare the in vivo efficacy of different batches of C-CAR168, female B-NDG (NOD.CB17-Prkdc) mice were used. scid Il2rg tm1 / Bcgen) mice, 5 × 10 6MM.1S cells were subcutaneously inoculated per mouse. The average tumor volume was approximately 100 mm. 3 When the dose reached 100 mg / kg, 15 mice were selected and randomly divided into three groups (one vehicle control group vs. two C-CAR168 groups), with five mice in each group. Each mouse was dosed once via tail vein injection. For C-CAR168, the dosage was 5 × 10 6 After administration, the mean tumor volume in the vehicle control group continued to increase and reached 2220.86 ± 117.35 mm at day 28. 3 The tumor weight reached 2.409 ± 0.216 g. The mean tumor volumes in the C-CAR168-1 and C-CAR168-2 groups began to decrease from day 10 (Figure 9B, left panel). By day 28, the mean tumor volumes were 109.2 ± 88.92 mm, respectively. 3 and 9.07±5.58mm 3 The tumor weights were 0.041±0.034 g and 0.003±0.002 g, respectively (FIG. 9B, right panel), which were significantly different from the vehicle control group (P<0.001, P<0.001). The tumor growth inhibition rates calculated based on tumor weight were 98.30% and 99.88%, respectively. There was no significant difference between the two batches of C-CAR168. The results showed that 5×10 6 We demonstrated that a single intravenous administration of C-CAR168 CAR-T cells / mouse was well tolerated in B-NDG tumor-bearing mice, and C-CAR168 could significantly inhibit the growth of BCMA-positive target cells in vivo.

[0232] C-CAR168 effectively inhibited the growth of CD20 and BCMA double-positive tumor cells in tumor-bearing mice. To evaluate the in vivo antitumor efficacy of C-CAR168, 65 female B-NDG (NOD.CB17-Prkdc) rats were used. scid Il2rg tm1 / Bcgen) mice, 1 × 10 6 K562-CD20-BCMA cells / animal were inoculated subcutaneously. The mean tumor volume was approximately 100 mm. 3When the target population reached 100, 50 mice were selected and randomly divided into 5 groups: vehicle control group, T cell control group, and C-CAR168 low-dose group (1 × 10 6 CAR-T cells / mouse), medium dose group (5 × 10 6 CAR-T cells / mouse), and the high-dose group (10 × 10 6 The control group received untransduced T cells from the same donor as the C-CAR168 group, and the dose matched the total T cell count in the high-dose C-CAR168 group. Each mouse received a single dose via tail vein injection.

[0233] During the experiment, the mean tumor volumes in the vehicle control and T cell control animals continued to increase, and by day 17, the mean tumor volumes were 2628.78 ± 117.32 mm, respectively. 3 and 2536.23±97.80mm 3 The tumor volume in the low-dose C-CAR168 group continued to increase, but 10 days after administration, tumor volume was significantly lower than that in the vehicle control and T cell control groups. The mean tumor volume in the medium- and high-dose C-CAR168 groups began to decrease on day 6. The low-, medium-, and high-dose C-CAR168 groups showed dose-dependent tumor reduction, with tumor growth inhibition rates of 55.47%, 97.75%, and 98.01%, respectively, on day 17. Tumor tissue was observed in the medium- and high-dose C-CAR168 groups on day 28 (Figure 9E). Figure 9F shows the survival curves for each group during the experimental period. All animals in the vehicle control and T cell control groups died around day 17, while all mice in the medium- and high-dose C-CAR168 groups survived.

[0234] In summary, 1×10 6 , 5×10 6 , or 10×10 6 A single intravenous administration of C-CAR168 CAR-T cells / mouse was well tolerated in B-NDG tumor-bearing mice, and C-CAR168 significantly inhibited the growth of K562-CD20-BCMA tumor cells in a dose-dependent manner.

[0235] Example 6 Antigen specificity of anti-CD20 / BCMA CAR In membrane protein arrays, full-length cDNA sequences of human membrane proteins are constructed into expression vectors using genetic engineering methods, which are then transiently transfected into HEK293T cells and arrayed using microfluidic or chip printing techniques. This is a high-throughput screening technique for studying the interactions between test substances and membrane proteins.

[0236] To test the affinity and specificity of the anti-CD20 / BCMA CAR, we used a membrane protein array assay to assess the risk of off-target binding between the antigen-binding domain of C-CAR168 and 5220 human cell membrane proteins.

[0237] The chimeric rabbit monoclonal antibody C-CAR168 scFv-RabFc was linked in-frame with the rabbit IgG Fc region to generate anti-CD20 scFv (e.g., derived from ofatumumab mAb) and anti-BCMA scFv (e.g., derived from BCMA-20 mAb). The chimeric antibody was added at a concentration of 20 μg / mL to HEK293T cell arrays transiently transfected with 5220 membrane proteins. Flow cytometry results showed that C-CAR168 scFv-RabFc strongly bound to human CD20 and BCMA (Figure 8A). The mean fluorescence intensity of binding to CD20 and BCMA in flow cytometry was approximately 60-fold and 110-fold higher than that of the negative control group, respectively (Figure 8B). In addition to CD20 and BCMA, C-CAR168 scFv-RabFc showed specific binding to FCGR1A (Figure 8B), with a mean fluorescence intensity 2.5-fold higher than that of the negative control. This is primarily due to binding between FCGR1A and the rabbit-derived Fc of the recombinant protein, and therefore poses no associated risk in clinical applications. C-CAR168 scFv-RabFc also showed weak binding to ITGB2-ITGAM and ITGB2-ITGAL heterodimers, with mean fluorescence intensities 2-3-fold higher than those of the negative control. For other proteins discovered in preliminary screening (MPZ, F11R, CLEC2B, and MC2R), the mean fluorescence intensity binding to C-CAR168 scFv-RabFc did not change with concentration. At concentrations of 20 μg / mL and 5 μg / mL, it did not exceed 2-fold higher than the negative control, indicating that these proteins are unlikely to specifically bind to C-CAR168 scFv or are minimally resistant to it.

[0238] To test whether ITGB2-ITGAM and ITGB2-ITGAL heterodimers expressed on the cell membrane could be recognized by C-CAR168 CAR-T cells to activate downstream events, C-CAR168 was cocultured with 293T cells transfected with ITGB2-ITGAM or ITGB2-ITGAL. Expression of CD137 on C-CAR168 CAR-T cells and levels of IFN-γ, TNF-α, IL-2, and other cytokines in the cell culture supernatant were assessed. 293T cells transfected with an empty vector were used as a negative control, and 293T cells transfected with CD20 and BCMA were used as a positive control.

[0239] CD137 (4-1BB) is a cell surface marker for antigen-specific activation of T cells. Antigen-specific activation of CAR-T cells can be assessed by detecting upregulation of CD137 expression on the cell surface. Experiments revealed that after co-culture of three batches of C-CAR168 cells with cells expressing CD20 and BCMA, the percentage of 4-1BB-positive cells increased compared to non-transduced T cells ("NT"). After co-culture with those cells expressing ITGB2-ITGAM and ITGB2-ITGAL, the percentage of 4-1BB-positive cells was not significantly different from that of the non-transduced T cell group ("NT") (Figure 8D, left panel). This indicated that C-CAR168 does not specifically bind to ITGB2-ITGAM or ITGB2-ITGAL in vitro.

[0240] Cytokines in the cell culture supernatant were assayed, and the results showed that C-CAR168 CAR-T cells secreted high levels of IFN-γ when cocultured with cells expressing CD20 or BCMA. When cocultured with cells expressing ITGB2-ITGAM or ITGB2-ITGAL, the IFN-γ concentration in the supernatant did not increase significantly compared with untransfected T cells (Figure 8D, right panel). The results further demonstrated that C-CAR168 did not specifically recognize ITGB2-ITGAM or ITGB2-ITGAL in vitro.

[0241] In summary, the results of membrane protein array and in vitro coculture indicate that the antigen-binding domain of C-CAR168 strongly binds to human CD20 and BCMA and has no other nonspecific binding sites. The membrane protein array study confirmed that C-CAR168 has no cross-reactivity with the membrane proteome except for weak binding to two heterocomplexes.

[0242] Example 7 C-CAR168 shows potent efficacy against autologous B cells in SLE patients To study CAR-T therapy for the treatment of autoimmune diseases (e.g., SLE), we evaluated the efficiency of CAR-T cells to deplete autoreactive B cells. We also study the effectiveness of CAR-T cells on remission and survival in lupus models.

[0243] Efficiency of C-CAR168 in vitro depletion of panB cells from lupus patients We collected 10-15 mL peripheral blood samples from eight SLE patients. The patients had different activity and autoantibody profiles, showed different organ damage (preferably patients with lupus nephritis), and had received different treatments, representing the heterogeneous nature of lupus patients. Patients who had recently received B-cell-depleting antibodies were excluded.

[0244] For each sample, a portion of the blood was used to isolate T cells for CAR-T production, and the remaining blood was used to isolate pan-B cells as targets for cytolytic assays. T cells isolated from eight SLE patients were transduced with a lentiviral vector encoding C-CAR168 and tested for CAR expression. T cell samples from eight SLE patient samples were efficiently transduced and expanded sufficiently for functional assays (Figure 10A).

[0245] Target cell lines expressing CD20 and / or BCMA were cocultured with C-CAR168 CAR-T cells or non-transduced (NT) T cells generated from eight patient samples. K562 is a CD20- and BCMA-negative multiple myeloma cell line, while MM.1S is a BCMA-positive multiple myeloma cell line. After 24 hours, coculture supernatants were collected and IFN-γ levels were assessed by ELISA (enzyme-linked immunosorbent assay). Results from one representative sample of eight patients are shown in Figure 10B. Thus, C-CAR168 cells generated from the SLE patient sample demonstrated potent activity against target cells expressing CD20 and BCMA.

[0246] PanB cells isolated from eight patient samples were cocultured with autologous C-CAR168 CAR-T cells or non-transduced (NT) T cells at the indicated E:T (effector to target) ratio. After 24 hours, coculture supernatants were collected for ELISA to assess IFN-γ levels. Cytotoxicity was determined by fluorescence-activated cell sorting (FACS) and calculation of the percentage of viable CD19+ panB cells depleted. B cell cytolysis was calculated using the following formula: lysis rate (%) = (1 - (viable CD19+ cell fraction in C-CAR168 coculture / viable CD19+ cell fraction in UT coculture)) × 100. Results from a representative sample of eight patients are shown in Figures 10C and 10D. PanB cells isolated from eight SLE patient samples were recognized and lysed by autologous C-CAR168 cells. The results confirmed the in vitro efficacy of C-CAR168 CAR-T cells in depleting peripheral B cells from lupus patients.

[0247] In vitro efficiency of CAR-T for the clearance of ABCs from lupus patients The efficacy of CAR-T in vitro to eliminate ABC, an essential subset of pathogenic B cells from lupus patients, will be studied.

[0248] Blood samples or PBMCs from lupus patients will be processed for ABC differentiation and CAR-T production and functional analysis.

[0249] The study will confirm the efficiency of CAR-T cells in depleting ABCs from lupus patients outside the body in an in vitro setting.

[0250] In vivo B cell depletion efficiency and therapeutic efficacy of CAR-T A humanized mouse model of SLE will be used to evaluate the efficiency of CAR-T in depleting B cells and its therapeutic efficacy in vivo. + Stem cell-humanized mice will be obtained. Two or more mice will be sacrificed and their spleens will be collected using aseptic techniques. T cells will then be isolated from the spleens for CAR-T cell generation. The remaining mice will be used to induce lupus disease development. If induction is successful, the mice will be divided into groups receiving CAR-T or control treatment (e.g., non-transduced T cells). Blood samples will be obtained periodically from the mice to monitor the persistence of CAR-T cells and the efficiency of B cell depletion (e.g., ABC) by FACS. Serum samples will be used to measure the titers of various autoantibodies. Urine samples will also be collected periodically to measure the level of proteinuria. At the end of the study, or if an animal dies early (perhaps in the control group), tissues will be collected for histology (e.g., to examine immune complex deposition in the kidney and the severity of nephritis). The presence of B cells or plasma cells in affected tissues will also be examined. Survival curves will be generated comparing the effects of CAR-T to control treatment.

[0251] References Bhoj,VGet al.Persistence of long-lived plasma cells and humoral immunity in individuals responding to CD19-directed CAR T-cell therapy.Blood 128,360-370(2016). Taubmann et al.Long term safety and efficacy of CAR-T cell treatment in refractory systemic lupus erythematosus-data from the first seven patients.Annals of the Rheumatic Diseases,OP0141,page 93(2023). Qin,C.,Tian,DS.,Zhou,LQ.et al.Anti-BCMA CAR T-cell therapy CT103A in relapsed or refractory AQP4-IgG seropositive neuromyelitis optica spectrum disorders:phase 1 trial interim results.Sig Transduct Target Ther 8,5(2023). Tai et al.Role of B-cell-activating factor in adhesion and growth of human multiple myeloma cells in the bone marrow microenvironment.Cancer research.2006;66(13):6675-82. Pavlasova et al.(2020).The regulation and function of CD20: an “enigma” of B-cell biology and targeted therapy.Haematologica,105(6),1494-1506. Krumbholz et al.B cells and antibodies in multiple sclerosis pathogenesis and therapy[J].Nature reviews Neurology,2012,8(11):613-23. Parker et al.,Single-Cell Analyses Identify Brain Mural Cells Expressing CD19 as Potential Off-Tumor Targets for CAR-T Immunotherapies.Cell.2020 Oct 1;183(1):126-142.e17. Qu et al.Phase 1 study of C-CAR088,a novel humanized anti-BCMA CAR T-cell therapy in relapsed / refractory multiple myeloma.J Immunother Cancer.2022 Sep;10(9):e005145.

[0252] The structures of the anti-CD20 / BCMA CARs, TOB1-4, and TOBL1-4, are shown in Table 1. [Table 1]

[0253] array: TN-OF-B20-L1 (TOBL1, or C-CAR168) CD8a SP nucleic acid sequence (63 nt) atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg (SEQ ID NO: 1)

[0254] CD8a SP amino acid sequence: [ka]

[0255] OF V L Nucleic acid sequence (321 nt) GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGC CACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA (SEQ ID NO: 3)

[0256] OF V L Amino acid sequence: [ka]

[0257] Linker 1 nucleic acid sequence (54 nt) GGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGC (SEQ ID NO: 5)

[0258] Linker 1 amino acid sequence: [ka]

[0259] OF V H Nucleic acid sequence (366nt) GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGAC TCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 7)

[0260] OF V H Amino acid sequence: [ka]

[0261] Linker 2 nucleic acid sequence (15 nt) GGAGGTGGTGGATCC (SEQ ID NO: 9)

[0262] Linker 2 amino acid sequence: [ka]

[0263] B20 V L Nucleic acid sequence (321 nt) Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgca gtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag (SEQ ID NO: 11)

[0264] B20 V L Amino acid sequence: [ka]

[0265] Linker 3 nucleic acid sequence (45 nt) Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct (SEQ ID NO: 13)

[0266] Linker 3 amino acid sequence: [ka]

[0267] B20 V H Nucleic acid sequence (363 nt) Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccga ctccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc (Sequence number 15)

[0268] B20 V H Amino acid sequence: [ka]

[0269] CD8a hinge nucleic acid sequence (165 nt) Ttcgtgccggtcttcctgccagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat (SEQ ID NO: 17)

[0270] CD8a hinge amino acid sequence: [ka]

[0271] CD8a TM nucleic acid sequence (72 nt) Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc (SEQ ID NO: 19)

[0272] CD8a TM amino acid sequence: [ka]

[0273] 4-1BB nucleic acid sequence (126 nt) Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg (SEQ ID NO: 21)

[0274] 4-1BB amino acid sequence: [ka]

[0275] CD3z nucleic acid sequence (336 nt) Agagtgaagttcagcaggagcgcagacgccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgtttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcct gtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa (SEQ ID NO: 23)

[0276] CD3z amino acid sequence: [ka]

[0277] TOBL1 nucleic acid sequence (2247 nt)

[0278] TOBL1 amino acid sequence: [ka]

[0279] TN-OF-B20-L2(TOBL2) CD8a SP nucleic acid sequence (63 nt) Atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg (SEQ ID NO: 27)

[0280] V H Nucleic acid sequence GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACT CTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 28)

[0281] Linker 1 nucleic acid sequence GGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGC (SEQ ID NO: 29)

[0282] V L Nucleic acid sequence GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO: 30)

[0283] Linker 2 nucleic acid sequence GGAGGTGGTGGATCC(SEQ ID NO: 31)

[0284] BCMA-20 scFv(729nt): B20 V L Nucleic acid sequence(321nt) gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag(SEQ ID NO: 32)

[0285] Linker 3 nucleic acid sequence(45nt) ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(SEQ ID NO: 33)

[0286] B20 V H Nucleic acid sequence (363 nt) gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc (SEQ ID NO: 34)

[0287] CD8a hinge nucleic acid sequence (165 nt) Ttcgtgccggtcttcctgccagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat (SEQ ID NO: 35)

[0288] CD8a TM nucleic acid sequence (72 nt) Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc (SEQ ID NO: 36)

[0289] 4-1BB nucleic acid sequence (126 nt) Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggagagatggctgtagctgccgatttccagaagaagaaggaggatgtgaactg (sequence number 37)

[0290] CD3z nucleic acid sequence(336nt) Agagtgaagttcagcaggagcgcagacgccccccgcgtaccagcagggccagaaccagctcatataacgagctcaatctaggacgaagagaggagtacgatgtttggacaagagagacgtggccgggaccctgagatgggggggaaaagccgaaggaagaacctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtagcagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa(sequence number 38)

[0291] TOBL2 nucleic acid sequence(2247nt)

[0292] TOBL2 amino acid sequence: [ka]

[0293] TN-OF-B20-L3(TOBL3) CD8a SP (63nt) nucleic acid sequence Atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg (SEQ ID NO: 41)

[0294] V H Nucleic acid sequence GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACT CTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 42)

[0295] Linker 1 nucleic acid sequence GGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGC (SEQ ID NO: 43)

[0296] V L Nucleic acid sequence GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO: 44)

[0297] Linker 2 nucleic acid sequence GGAGGTGGTGGATCC(SEQ ID NO: 45)

[0298] BCMA-20 scFv (729 nt): B20 V H Nucleic acid sequence (363 nt) Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc(SEQ ID NO: 46)

[0299] Linker 3 nucleic acid sequence (45 nt) Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct (SEQ ID NO: 47)

[0300] B20 V L Nucleic acid sequence (321 nt) Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag (SEQ ID NO: 48)

[0301] CD8a hinge nucleic acid sequence (165 nt) Ttcgtgccggtcttcctgccagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat (SEQ ID NO: 49)

[0302] CD8a TM nucleic acid sequence (72 nt) Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc (SEQ ID NO: 50)

[0303] <照 4-1BB nucleic acid sequence (12,6 nt) Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggagagatggctgtagctgccgatttccagaagaagaaggaggatgtgaactg (sequence number 51)

[0304] CD3z nucleic acid sequence(336nt) Agagtgaagttcagcaggagcgcagacgccccccgcgtaccagcagggccagaaccagctcatataacgagctcaatctaggacgaagagaggagtacgatgtttggacaagagagacgtggccgggaccctgagatggggggaaagccgaaggaagaacctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtaacgccaccaaggacacctacgacgccaccttcacatgcaggccctgcccctcgctaa(sequence number 52)

[0305] TOBL3 nucleic acid sequence

[0306] TOBL3 amino acid sequence: [ka]

[0307] TN-OF-B20-L4(TOBL4) CD8a SP nucleic acid sequence (63 nt) Atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg (SEQ ID NO: 55)

[0308] V L Nucleic acid sequence GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGC CACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA (SEQ ID NO: 56)

[0309] Linker 1 nucleic acid sequence GGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGC (SEQ ID NO: 57)

[0310] V H Nucleic acid sequence GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 58)

[0311] Linker 2 nucleic acid sequence GGAGGTGGTGGATCC (SEQ ID NO: 59)

[0312] BCMA-20 scFv(729nt): B20 V H Nucleic acid sequence (363nt) Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc (SEQ ID NO: 60)

[0313] Linker 3 nucleic acid sequence (45nt) Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct (SEQ ID NO: 61)

[0314] B20 V L Nucleic acid sequence (321 nt) Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag (SEQ ID NO: 62)

[0315] CD8a hinge nucleic acid sequence (165 nt) Ttcgtgccggtcttcctgccagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat (SEQ ID NO: 63)

[0316] CD8a TM nucleic acid sequence (72 nt) Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc (SEQ ID NO: 64)

[0317] 4-1BB nucleic acid sequence (126 nt) Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggagagatggctgtagctgccgatttccagaagaagaaggagggatgtgaactg (sequence number 65)

[0318] CD3z nucleic acid sequence(336nt) Agagtgaagttcagcaggagcgcagacgcccccgtaccagcagggccagaaccagctcatataacgagctcaatctaggacgaagagaggagtacgatgtttggacaagagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaacctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtagcagccaccaaggacacctacgacgccaccttcacatgcaggccctgcccctcgctaa(sequence number 66)

[0319] TOBL4 nucleic acid sequence(2247nt)

[0320] TOBL4 amino acid sequence: [ka]

[0321] TN-OF-B20-1(TOB1) CD8a SP nucleic acid sequence ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (SEQ ID NO: 69)

[0322] V L Nucleic acid sequence GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGC CACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA (SEQ ID NO: 70)

[0323] Linker 1 nucleic acid sequence GGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGC (SEQ ID NO: 71)

[0324] V H Nucleic acid sequence GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 72)

[0325] Linker 2 nucleic acid sequence GGAGGTGGTGGATCC (SEQ ID NO: 73)

[0326] B20 V H Nucleic acid sequence Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc (SEQ ID NO: 74)

[0327] Linker 3 nucleic acid sequence ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct (SEQ ID NO: 75)

[0328] B20 V L Nucleic acid sequence Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgca gtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag (SEQ ID NO: 76)

[0329] IgG4 hinge nucleic acid sequence (36 nt) GAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCT (SEQ ID NO: 77)

[0330] IgG4 hinge amino acid sequence: [ka]

[0331] CD28 TM nucleic acid sequence (84 nt) ATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTG (SEQ ID NO: 79)

[0332] CD28 TM amino acid sequence: [ka]

[0333] 4-1BB nucleic acid sequence AAACGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(sequence number 81)

[0334] CD3z nucleic acid sequence CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGGAAGAACCCCCAGGAAGCCTGTATAACGAACTGCAGAAAACAAGATGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGGCGGGCAAGGCCACGACGGGCCGTGATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG (sequence number 82)

[0335] TOB1 nucleic acid sequence (2130nt)

[0336] TOB1 amino acid sequence: [ka]

[0337] TN-OF-B20-2(TOB2) CD8a SP nucleic acid sequence ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (SEQ ID NO: 85)

[0338] V L Nucleic acid sequence GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGC CACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA (SEQ ID NO: 86)

[0339] Linker 1 nucleic acid sequence GGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGC (SEQ ID NO: 87)

[0340] V H Nucleic acid sequence GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 88)

[0341] Linker 2 nucleic acid sequence GGAGGTGGTGGATCC (SEQ ID NO: 89)

[0342] B20 V L Nucleic acid sequence Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag (SEQ ID NO: 90)

[0343] Linker 3 nucleic acid sequence ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct (SEQ ID NO: 91)

[0344] B20 V H Nucleic acid sequence Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccga ctccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc (Sequence number 92)

[0345] Hinge nucleic acid sequence GAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCT (SEQ ID NO: 93)

[0346] CD28 TM nucleic acid sequence ATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTG (SEQ ID NO: 94)

[0347] 4-1BB nucleic acid sequence AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 95)

[0348] CD3z nucleic acid sequence CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGGAAGAACCCCCAGGAAGCCTGTATAACGAACTGCAGAAAACAAGATGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGGCGGGCAAGGCCACGACGGGCCGTGATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG (sequence number 96)

[0349] TOB2 nucleic acid sequence (2130nt)

[0350] TOB2 amino acid sequence: [ka]

[0351] TN-OF-B20-3(TOB3) CD8a SP nucleic acid sequence ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (SEQ ID NO: 99)

[0352] V H Nucleic acid sequence GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACT CTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 100)

[0353] Linker 1 nucleic acid sequence GGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGC (SEQ ID NO: 101)

[0354] V L Nucleic acid sequence GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO: 102)

[0355] Linker 2 nucleic acid sequence GGAGGTGGTGGATCC(SEQ ID NO: 103)

[0356] B20 V L Nucleic acid sequence Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag(SEQ ID NO: 104)

[0357] Linker 3 nucleic acid sequence ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(SEQ ID NO: 105)

[0358] B20 V H Nucleic acid sequence Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaac ttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgac tccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc (Sequence number 106)

[0359] Hinge nucleic acid sequence GAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCT (SEQ ID NO: 107)

[0360] CD28 TM nucleic acid sequence ATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTG (SEQ ID NO: 108)

[0361] 4-1BB nucleic acid sequence AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 109)

[0362] CD3z nucleic acid sequence CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGGAAGAACCCCCAGGAAGCCTGTATAACGAACTGCAGAAAACAAGATGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGGCGGGCAAGGCCACGACGGGCCGTGATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG (sequence number 110)

[0363] TOB3 nucleic acid sequence (2130nt)

[0364] TOB3 amino acid sequence: [ka]

[0365] TN-OF-B20-4(TOB4) CD8a SP nucleic acid sequence ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (SEQ ID NO: 113)

[0366] V H Nucleic acid sequence GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACT CTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 114)

[0367] Linker 1 nucleic acid sequence GGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGC (SEQ ID NO: 115)

[0368] V L Nucleic acid sequence GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO: 116)

[0369] Linker 2 nucleic acid sequence GGAGGTGGTGGATCC(SEQ ID NO: 117)

[0370] B20 V H Nucleic acid sequence Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc(SEQ ID NO: 118)

[0371] Linker 3 nucleic acid sequence ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(SEQ ID NO: 119)

[0372] B20 V L Nucleic acid sequence Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcag tccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag (SEQ ID NO: 120)

[0373] Hinge nucleic acid sequence GAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCT (SEQ ID NO: 121)

[0374] CD28 TM nucleic acid sequence ATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTG (SEQ ID NO: 122)

[0375] 4-1BB nucleic acid sequence AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 123)

[0376] CD3z nucleic acid sequence CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGCAGAAGGGAAGAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGGAAGAACCCCCAGGAAGCCTGTATAACGAACTGCAGAAAACAAGATGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGGCGGGCAAGGCCACGACGGGCCGTGATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG (sequence number 124)

[0377] TOB4 nucleic acid sequence (2130nt)

[0378] TOB4 amino acid sequence: [ka] (SEQ ID NO: 126)

[0379] OF-V H -CDR1: NDYAMH (SEQ ID NO: 127)

[0380] OF-V H - CDR2: TISWNSGSIGYADSVKG (SEQ ID NO: 128)

[0381] OF-V H - CDR3: DIQYGNYYYGMDV (SEQ ID NO: 129)

[0382] OF-V L - CDR1: RASQSVSSYLA (SEQ ID NO: 130)

[0383] OF-V L -CDR2: DASNRAT (SEQ ID NO: 131)

[0384] OF-V L -CDR3: QQRSNWPIT (SEQ ID NO: 132)

[0385] [Table 2] [Table 3]

[0386] The scope of the present invention is not limited by what has been specifically shown and described above. Those skilled in the art will recognize that there are suitable alternatives to the illustrated examples of materials, configurations, structures, and dimensions. Numerous references (e.g., patents and various publications) have been cited and discussed in the description of the present invention. Citation and discussion of such references is provided solely to clarify the description of the present invention and does not constitute an admission that any reference is prior art to the invention described herein. All references cited and described herein are incorporated herein by reference in their entirety. Variations, modifications, and other implementations of what is described herein will occur to those skilled in the art without departing from the spirit and scope of the present invention. While specific embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that changes and modifications can be made without departing from the spirit and scope of the present invention. The matter set forth in the foregoing description and accompanying drawings is offered by way of example only, and not as a limitation.

Claims

1. A bispecific chimeric antigen receptor (CAR), comprising: (i) an anti-CD20 antigen-binding region, comprising a light chain variable region (V Ｌ 1) and a heavy chain variable region (V Ｈ 1), V Ｌ V1 comprises three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NOs: 130, 131, and 132, respectively; Ｈ the anti-CD20 antigen-binding region, wherein said CDR1 comprises three CDRs (CDR1, CDR2, and CDR3) having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, respectively; and (ii) an anti-BCMA antigen-binding region, comprising a light chain variable region (V Ｌ 2) and the heavy chain variable region (V Ｈ 2), including V Ｌ 2 comprises three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NOs: 134, 136, and 138, respectively; Ｈ 2 comprises the anti-BCMA antigen binding region, wherein SEQ ID NO: 141, SEQ ID NO: 143, and SEQ ID NO: 145 comprise three CDRs (CDR1, CDR2, and CDR3) having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 141, SEQ ID NO: 143, and SEQ ID NO: 145, respectively.

2. A bispecific chimeric antigen receptor (CAR), comprising: (i) light chain variable region (V Ｌ 1) and a heavy chain variable region (V Ｈ 1); and an anti-CD20 antigen-binding region comprising: (ii) a light chain variable region (V Ｌ 2) and the heavy chain variable region (V Ｈ 2).

3. V Ｌ 1 is V Ｈ 3. The bispecific CAR of claim 1 or 2, wherein the nucleotide sequence is located at the N-terminus of nucleotide 1.

4. V Ｌ 2 is V Ｈ The bispecific CAR of claim 1 or 2, wherein the nucleotide sequence is located at the N-terminus of nucleotide 2.

5. V Ｈ 1 is V Ｌ 3. The bispecific CAR of claim 1 or 2, wherein the nucleotide sequence is located at the N-terminus of nucleotide 1.

6. V Ｈ 2 is V Ｌ The bispecific CAR of claim 1 or 2, wherein the nucleotide sequence is located at the N-terminus of nucleotide 2.

7. V Ｌ 1 and V Ｈ The bispecific CAR according to any one of claims 1 to 6, wherein SEQ ID NO: 1 has an amino acid sequence that is about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 8, respectively.

8. V Ｌ 2 and V Ｈ 8. The bispecific CAR according to any one of claims 1 to 7, wherein SEQ ID NO: 1 and SEQ ID NO: 2 have amino acid sequences that are about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 16, respectively.

9. The bispecific CAR according to any one of claims 1 to 8, wherein the anti-CD20 antigen-binding region is a single-chain variable fragment (scFv) that specifically binds to CD20, and the anti-BCMA antigen-binding region is an scFv that specifically binds to BCMA.

10. The bispecific CAR of any one of claims 1 to 9, further comprising one or more of the following: (a) a signal peptide, (b) the hinge region; (c) a transmembrane domain; (d) co-stimulatory area, and (e) Cytoplasmic signaling domain.

11. The bispecific CAR of claim 10, wherein the costimulatory region comprises a costimulatory region of 4-1BB (CD137), CD28, OX40, CD2, CD7, CD27, CD30, CD40, CD70, CD134, PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

12. The bispecific CAR of claim 10 or 11, wherein said cytoplasmic signaling domain comprises the cytoplasmic signaling domain of CD3ζ.

13. The bispecific CAR according to any of claims 10 to 12, wherein the hinge region comprises a hinge region of IgG4, CD8, CD28, CD137, or a combination thereof.

14. The bispecific CAR according to any one of claims 10 to 13, wherein the transmembrane domain comprises a transmembrane domain of CD8, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

15. The bispecific CAR according to any one of claims 1 to 14, comprising an amino acid sequence that is about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:26, SEQ ID NO:40, SEQ ID NO:54, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:98, SEQ ID NO:112, or SEQ ID NO:

126.

16. An immune cell expressing the bispecific CAR according to any one of claims 1 to 15.

17. The immune cell of claim 16, wherein the immune cell is a T cell or a natural killer (NK) cell.

18. A nucleic acid encoding the bispecific CAR according to any one of claims 1 to 15.

19. A vector comprising the nucleic acid of claim 18.

20. A pharmaceutical composition comprising the bispecific CAR according to any one of claims 1 to 15, the immune cell according to claim 16 or 17, the nucleic acid according to claim 18, or the vector according to claim 19.

21. 21. A method of treating an autoimmune disorder, the method comprising administering to a subject in need thereof an immune cell according to claim 16 or 17, or a pharmaceutical composition according to claim 20.

22. 22. The method of claim 21, wherein the autoimmune disorder is systemic lupus erythematosus (SLE), systemic sclerosis, inflammatory myopathy, systemic sclerosis, multiple sclerosis, myasthenia gravis, myositis autoantibody-induced disease, or neuromyelitis optica.

23. 23. The method of claim 22, wherein the inflammatory myopathy is polymyositis, dermatomyositis, or inclusion body myositis.

24. 23. The method of claim 22, wherein the SLE is lupus nephritis.

25. 21. A method of treating cancer, the method comprising administering to a subject in need thereof an immune cell of claim 16 or 17, or a pharmaceutical composition of claim 20.

26. 26. The method of claim 25, wherein the cancer is a hematological cancer.

27. 26. The method of claim 25, wherein the cancer is a B-cell malignancy.

28. 26. The method of claim 25, wherein the cancer is Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, and / or multiple myeloma.

29. 26. The method of claim 25, wherein the cancer is acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

30. The method of any of claims 21 to 29, wherein the immune cells are allogeneic or autologous.

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