Bispecific chimeric antigen receptor targeting CD20 and BCMA

Abstract

The present disclosure provides bispecific chimeric antigen receptors that target CD20 and BCMA. The CAR may comprise an scFv targeting CD20 and an scFv targeting BCMA, a hinge region, a transmembrane domain, a co-stimulatory region, and a cytoplasm signaling domain. The chimeric antigen receptors can be used to treat autoimmune disorders or cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Nos. 63 / 493,495 (filed on March 31, 2023) and 63 / 509,371 (filed on June 21, 2023), each of which is hereby incorporated by reference in its entirety.

[0003] Incorporated by reference into the sequence listing

[0004] The Sequence Listing is submitted as the XML file "11299_011840-WO0_SL.xml", created on March 26, 2024, and is 168,342 bytes in size, which is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates to the field of immunotherapy, and more particularly to bispecific chimeric antigen receptors (CARs) targeting CD20 and BCMA. Background Art

[0006] Autoimmune diseases are disorders caused by the immune system's response to the body itself, resulting in damage to the body's own tissues. These are generally divided into two categories: systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis, and systemic vasculitis; and organ-specific autoimmune diseases, such as autoimmune hepatitis and type 1 diabetes. Most autoimmune diseases are difficult to cure and often require long-term or lifelong medication. Treatment primarily involves corticosteroids and immunosuppressants, which greatly 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).

[0007] The cause of autoimmune diseases is still unclear. In patients, abnormal activation of humoral immunity occurs, leading to the production of large amounts of antibodies against self-antigens. These combine to form pathogenic immune complexes, which then deposit locally and cause an inflammatory response. B cells play an important role in the pathogenesis of autoimmune diseases, promoting the occurrence of autoimmune diseases through various mechanisms such as the production of autoantibodies, the release of cytokines, and the presentation of self-antigens. Autoantibodies, as key factors, can bind to self-antigens to form immune complexes, which can activate cells of the innate immune system to produce type I interferon and other proinflammatory cytokines, leading to organ damage. Therefore, depletion or removal of lymphocytes has become a potential therapeutic strategy.

[0008] SLE is a prototype autoimmune disease, known to be associated with polyclonal B cell hyperreactivity (Dorner et al., Mechanisms of B cell autoimmunity in SLE, Arthritis Res. Ther. 13, 243 (2011)). Therefore, one of the immunological characteristics of SLE is the production of antinuclear antibodies (ANA), which can mediate SLE pathogenesis by binding to corresponding autoantigens, thereby causing immune complex deposition and inducing 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)). ANA is mainly divided into two types: anti-DNA antibodies and antibodies recognizing RNA binding proteins (RBPs) (Pisetsky et al., New insights into the role of antinuclear antibodies insystemic lupus erythematosus, Nat. Rev. Rheumatol. 16, 565-579 (2020)). In SLE patients, the source of autoantibodies includes not only B cells, but also a class of plasma cells called long-lived plasma cells (LLPC). Anti-DNA antibodies are produced by the naive B cells converted into memory B cells and plasmablasts, and the memory B cells and plasmablasts maintain high levels of CD19 and CD20 expression on the cell surface, and anti-RBP antibodies are produced by LLPC, and the LLPC may lose the surface expression of CD19 and CD20, but are positive for B cell maturation antigen (BCMA), BCMA is a cell surface protein (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)) expressed on all mature plasma cells. Recent studies have shown that CD11c hi T-bet +A B cell subset expands in human SLE and serves as a precursor to plasma cells that produce autoantibodies. This B cell subset displays high expression of CD19 and CD20 and corresponds to autoreactive, murine age-related B cells (autoreactive B cells or ABCs; this term can be used to denote human CD11c hi T-bet + B cells) (Jenks et al., Distinct Effector B Cells Induced by Unregulated Toll-like Receptor 7 Contribute to Pathogenic Responses inSystemic Lupus Erythematosus, Immunity 49, 725-739e726 (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 participate in the pathogenesis of SLE and other autoimmune diseases by secreting cytokines and acting as antigen-presenting cells. Therefore, depleting B cells in SLE patients can be an effective therapy for this life-threatening disease.

[0009] B cell depletion can be achieved by administering monoclonal antibodies for B cell surface markers. Although the anti-CD20 antibody rituximab has been successful in the early open label trial of SLE, it failed to reach 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)). Other antibodies targeting CD19 (obexelimab (obexelimab)) have also been tested in SLE. Although in preliminary studies, patients receiving obexelimab maintained disease inactivity levels after discontinuing steroids, Phase II clinical trials failed to reach their primary endpoint (Lee et al., B cell depletion therapies in autoimmune disease:advancesand mechanistic insights,Nat.Rev.Drug Discov.20,179-199(2021)).

[0010] A promising method for achieving B cell exhaustion is the adoptive transfer of CAR-T cells.CAR-T cells are genetically engineered T lymphocytes that, in the absence of major histocompatibility complex (MHC), can identify specific antigens on target cells, proliferate, and produce cytotoxic immune responses. In a recent study, 5 refractory SLE patients received sympathetic use of CD19 CAR-T therapy, resulting in deep depletion of B cells and drug-free remission, indicating that CAR-T cell transfer is feasible, tolerable, and efficient in SLE (Mackensen et al., Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus, Nat. Med. 28, 2124-2132 (2022)).

[0011] There remains an urgent need to develop effective treatments for autoimmune diseases. Summary of the Invention

[0012] The present disclosure provides a bispecific chimeric antigen receptor (CAR), comprising: (i) an anti-CD20 antigen binding region comprising a light chain variable region (V L 1) and heavy chain variable region (V H 1), where V L 1 comprises three complementarity determining regions (CDRs), CDR1, CDR2, and CDR3, which have amino acid sequences approximately 80% to approximately 100% identical to the amino acid sequences shown in SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively, and wherein V H 1 comprises three CDRs, CDR1, CDR2, and CDR3, which have amino acid sequences approximately 80% to approximately 100% identical to the amino acid sequences shown 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), where V L 2 comprises three complementarity determining regions (CDRs), CDR1, CDR2, and CDR3, which have amino acid sequences approximately 80% to approximately 100% identical to the amino acid sequences shown in SEQ ID NO: 134, SEQ ID NO: 136, and SEQ ID NO: 138, respectively, and wherein V H2 comprises three CDRs, CDR1, CDR2, and CDR3, which have amino acid sequences that are approximately 80% to approximately 100% identical to the amino acid sequences shown in SEQ ID NO: 141, SEQ ID NO: 143, and SEQ ID NO: 145, respectively.

[0013] The present disclosure provides a bispecific chimeric antigen receptor (CAR), comprising: (i) an anti-CD20 antigen binding region comprising a light chain variable region (V L 1) and heavy chain variable region (V H 1); 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).

[0014] In one embodiment, V L 1 is located in V H 1. In one embodiment, V H 1 is located in V L 1. In one embodiment, V L 2 is located at V H 2. In one embodiment, V H 2 is located at V L 2's N terminus.

[0015] In certain embodiments, V L 1 and V H 1 has an amino acid sequence that is approximately 80% to approximately 100% identical to the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 8, respectively.

[0016] In certain embodiments, V L 2 and V H 2 have amino acid sequences that are approximately 80% to approximately 100% identical to the amino acid sequences shown in SEQ ID NO: 12 and SEQ ID NO: 16, respectively.

[0017] 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 a scFv that specifically binds to BCMA.

[0018] The bispecific CAR may further comprise one or more of the following: (a) a signal peptide, (b) a hinge region, (c) a transmembrane domain, (d) a co-stimulatory region, and (e) a cytoplasmic signaling domain.

[0019] The hinge region may include a hinge region of IgG4, CD8, CD28, CD137, or a combination thereof.

[0020] The transmembrane domain can 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.

[0021] The co-stimulatory region can include the co-stimulatory 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.

[0022] The cytoplasmic signaling domain may include the cytoplasmic signaling domain of CD3zeta.

[0023] The present disclosure provides a bispecific CAR comprising (or having) an amino acid sequence that is approximately 80% to about 100% identical to the amino acid sequence shown 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.

[0024] The present disclosure also encompasses immune cells expressing bispecific CARs. The immune cells can be T cells or natural killer (NK) cells.

[0025] The present disclosure provides nucleic acids encoding bispecific CARs.

[0026] The present disclosure provides a vector comprising a nucleic acid of the present invention encoding a bispecific CAR.

[0027] The present disclosure provides pharmaceutical compositions comprising bispecific CARs, immune cells, nucleic acids, or vectors.

[0028] The present disclosure also provides a method for treating an autoimmune disorder, which may include administering immune cells or a pharmaceutical composition to a subject in need thereof.

[0029] The autoimmune disorder can be systemic lupus erythematosus (SLE) (e.g., lupus nephritis), systemic vasculitis, systemic sclerosis, inflammatory myopathy (e.g., polymyositis, dermatomyositis, or inclusion body myositis), systemic scleroderma, multiple sclerosis, myasthenia gravis, myositis autoantibody-driven disease, or neuromyelitis optica.

[0030] The autoimmune disorder may be polymyositis, dermatomyositis or inclusion body myositis. The autoimmune disorder may be lupus nephritis.

[0031] The present disclosure also provides a method for treating cancer, which may include administering immune cells or a pharmaceutical composition to a subject in need thereof.

[0032] The cancer may be a hematologic 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 myeloid leukemia, acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0033] Immune cells can be either allogeneic or autologous. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The structure of the combined chimeric antigen receptor targeting CD20 and BCMA is shown. The structure of the CAR includes 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 co-stimulatory region, and a cytoplasmic signaling domain (CD3ζ). TOB1 to TOB4 contain a short IgG4 hinge (12aa) and a CD28 transmembrane domain; TOBL1 to TOBL4 contain a CD8a hinge and a CD8a transmembrane domain. Two groups of CARs (TOB1-4 and TOBL1-4) contain two V sequences in the scFv sequence. H and V L TOBL1 is also named C-CAR168.

[0035] Figure 2 Shown are the expression levels of anti-CD20 and anti-BCMACAR on the surface of T cells.

[0036] Figures 3A-3C The IFN-γ levels secreted by in vitro activated CAR-T cells in the cell culture supernatant are shown. Figure 3A Shown are the levels of IFN-γ secreted by TOB1 to TOB4 CAR-T cells in the cell culture supernatants. Figure 3B Shown are the levels of IFN-γ secreted by TOBL1 to TOBL4 CAR-T cells in the cell culture supernatants. Figure 3CTOBL1 to TOBL4 exhibit high levels of IFN-γ release when co-cultured with target cells naturally expressing CD20 and BCMA. MM.1S is a BCMA-positive multiple myeloma (MM) cell line; RAJI is CD20- and BCMA-positive.

[0037] Figures 4A-4B Shown are the expression levels of CD137 on the surface of activated CAR-T cells.

[0038] Figures 5A-5B The results of RTCA assay for CAR-T cells ( Figure 5A :TOB1 to TOB4; Figure 5B : TOBL1 to TOBL4) in vitro cytotoxicity.

[0039] Figures 6A-6C : C-CAR168 exhibited robust efficacy against CD20+ and BCMA+ cells in vitro. Figure 6A Shown are the structures of C-CAR168 (TOBL1 is an anti-CD20 / BCMA CAR), anti-CD20 CAR (C-CAR066), and anti-BCMA CAR (C-CAR088). Figure 6B Shown is the release of IFN-γ after co-culture of CAR-T cells with CD20-positive and / or BCMA-positive target cells. Figure 6C The cytotoxicity of CAR-T cells targeting CD20 and / or BCMA at different E:T ratios is shown.

[0040] Figures 7A-7C : Cytotoxicity of C-CAR168 against ABC-enriched B cells in vitro. Figure 7A : Generation of C-CAR168 CAR-T cells. The figure below shows the CAR positivity rate of C-CAR168 CAR-T cells prepared from the peripheral blood of three healthy donors. Figure 7B : Differentiation of ABC cells. The figure below shows that after inducing autologous B cell differentiation, the proportion of ABC subsets increased significantly. Figure 7C : Cytolysis of ABC-enriched B cells by C-CAR168 at different E:T ratios.

[0041] Figures 8A-8D : C-CAR168 has no cross-reactivity against the human membrane proteome. Figures 8A-8B : Binding specificity of C-CAR168 scFv-RabFc in membrane protein arrays. Figure 8C :The expressions of ITGB2-ITGAM and ITGB2-ITGAL in 293T cells were detected by flow cytometry. Figure 8D(Left panel): Flow cytometry analysis of the proportion of 4-1BB-positive cells. Figure 8D (Right panel): IFN-γ concentration in the co-culture supernatant was detected by flow cytometry.

[0042] Figures 9A-9G : In vivo cytotoxicity of C-CAR168 in tumor-bearing mice. Figure 9A C-CAR168 significantly inhibits the growth of A549-CD20 cells in B-NDG tumor-bearing mice. Left: Tumor growth curves for each group during the experimental period; Right: Mean tumor weight for animals in each group on day 42. ***: P < 0.001 compared to the vehicle control group. Figure 9B C-CAR168 significantly inhibited the growth of human multiple myeloma MM.1S tumor cells in B-NDG tumor-bearing mice. Left: Tumor growth curves for each group during the experiment; Right: Mean tumor weight for each group on day 28. ***: P < 0.001 compared to the vehicle control group. Figure 9C : Images of A549-CD20 tumors in animals in each group on day 42. “ / ” indicates animal death. Blank boxes indicate no tumor tissue was collected. Figure 9D : Images of MM.1S tumors in animals in each group on day 28. Open boxes indicate that no tumor tissue was collected. Figures 9E-9G : C-CAR168 significantly inhibited the growth of K562-CD20-BCMA tumor cells in B-NDG tumor-bearing mice. Figure 9E : Tumor growth curves of each group during the experiment. Figure 9F : Survival rate curves of each group during the experimental period. Figure 9G : Images of tumors of animals in each group on day 28. “ / ” indicates animal death. Blank boxes indicate no tumor tissue was collected.

[0043] Figures 10A-10D : C-CAR168 exhibits robust efficacy against autologous B cells from SLE patients in vitro. Figure 10A : T cells from eight SLE patients were successfully transduced with a lentiviral vector encoding C-CAR168 and expressed anti-CD20 / BCMACAR. Figure 10B : C-CAR168 CAR-T cells generated from SLE patient samples showed robust activity (IFN-γ release) against target cells expressing CD20 and BCMA. K562 is negative for both CD20 and BCMA; MM.1S is a BCMA-positive multiple myeloma cell line. Figure 10C : C-CAR168 CAR-T cells generated from SLE patient samples exhibited robust activity (e.g., IFN-γ release) against pan-B cells isolated from SLE patients. Figure 10D: Pan B cells isolated from SLE patients are recognized and lysed by autologous C-CAR168 cells. DETAILED DESCRIPTION

[0044] The present disclosure provides a chimeric antigen receptor (CAR) targeting CD20 and BCMA. CAR may include 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 of the present invention can be used to treat autoimmune diseases or cancer.

[0045] 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 serves as an important receptor for B-cell activating factor (BAFF) and is involved in regulating the differentiation and maturation of B lymphocytes. BCMA is a type III transmembrane protein that is specifically expressed in B cells, particularly plasmablasts and differentiated, mature plasma cells.

[0046] CD20 is a B cell membrane marker, also known as B1, and is a transmembrane glycoprotein encoded by the MS4A gene. 2+ Conductance plays an important role in the development, proliferation, activation, differentiation and malignant transformation of B cells.

[0047] The present disclosure provides a bispecific chimeric antigen receptor (CAR). The bispecific CAR may comprise: (i) an anti-CD20 antigen binding region comprising a light chain variable region (V L 1) and heavy chain variable region (V H 1); 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).

[0048] The bispecific chimeric antigen receptor (CAR) of the present invention may comprise: (i) an anti-CD20 antigen binding region comprising a light chain variable region (V L 1) and heavy chain variable region (V H 1), which has 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: 4 and SEQ ID NO: 8, respectively; and (ii) an anti-BCMA antigen-binding region comprising a light chain variable region (V L 2) and heavy chain variable region (V H2) 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.

[0049] The present disclosure provides a bispecific chimeric antigen receptor (CAR). The bispecific CAR may comprise: (i) an anti-CD20 antigen binding region comprising a light chain variable region (V L 1) and heavy chain variable region (V H 1), 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). V L 1 may comprise three complementarity determining regions (CDRs), CDR1, CDR2, and CDR3, which have 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, or SEQ ID NO: 132, respectively. V H 1 may comprise three CDRs, CDR1, CDR2, and CDR3, which have 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, or SEQ ID NO: 129, respectively. L 2 may comprise three complementarity determining regions (CDRs), CDR1, CDR2, and CDR3, which have 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, or SEQ ID NO: 138, respectively. V H 2 may comprise three CDRs, CDR1, CDR2, and CDR3, which have 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 shown in SEQ ID NO: 141, SEQ ID NO: 143, or SEQ ID NO: 145, respectively.

[0050] In certain embodiments, V L 1 is located in V H 1. In certain embodiments, V H 1 is located in V L1. In certain embodiments, V H 2 is located at V L 2. In certain embodiments, V L 2 is located at V H 2. In one embodiment, V L 1 is located in V H N-terminal end of 1; V L 2 is located at V H 2's N terminus.

[0051] In certain embodiments, V L 1 and V H 1 has an amino acid sequence that is approximately 80% to approximately 100% identical to the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 8, respectively.

[0052] In certain embodiments, V L 2 and V H 2 have amino acid sequences that are approximately 80% to approximately 100% identical to the amino acid sequences shown in SEQ ID NO: 12 and SEQ ID NO: 16, respectively.

[0053] In certain embodiments, the antigen binding region that specifically binds CD20 is located at the N-terminus of the antigen binding region that specifically binds BCMA. In certain embodiments, the antigen binding region that specifically binds BCMA is located at the N-terminus of the antigen binding region that specifically binds CD20.

[0054] The anti-CD20 antigen binding region can be a single-chain variable fragment (scFv) that specifically binds to CD20. The anti-BCMA antigen binding region can be a 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.

[0055] The bispecific CAR may further comprise 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 co-stimulatory region, and (e) a cytoplasmic signaling domain.

[0056] The bispecific CAR of the present invention may comprise, from N-terminus to C-terminus, 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.

[0057] The signal peptide may comprise a signal peptide of CD8, CD28, GM-CSF, CD4, CD137 or a combination thereof (or may be derived from CD8, CD28, GM-CSF, CD4, CD137 or a combination thereof). In one embodiment, the signal peptide is a signal peptide of CD8 (or is derived from CD8).

[0058] 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.

[0059] The hinge region may comprise a hinge region of (or may be derived from) IgG4, CD8, CD28, CD137, or a combination, wild type, or mutant thereof.

[0060] In one embodiment, the hinge region is the hinge region of IgG4 (or is derived from IgG4). 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.

[0061] In one embodiment, the hinge region is the hinge region of CD8a (or is derived from 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.

[0062] The transmembrane domain may comprise the transmembrane domain of (or may be derived from) CD8, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0063] In one embodiment, the transmembrane domain is a transmembrane domain of CD8 (or is derived from 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.

[0064] In one embodiment, the transmembrane domain is the transmembrane domain of CD28 (or is derived from 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: 80.

[0065] The co-stimulatory region may include (or may be derived from) the co-stimulatory 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.

[0066] In one embodiment, the costimulatory region is the costimulatory region of 4-1BB (or is derived from 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 shown in SEQ ID NO: 22.

[0067] The cytoplasmic signaling domain may include the cytoplasmic signaling domain of CD3 zeta (or may be derived from CD3). 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.

[0068] The CAR of the present invention may comprise a V domain of an anti-CD20 antigen binding region. L and V H In one embodiment, the linker (Linker-1) 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: 6.

[0069] The CAR of the present invention may include 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 shown in SEQ ID NO: 10.

[0070] The CAR of the present invention may comprise a V domain of an anti-BCMA antigen binding region. L and V H In one embodiment, the linker (Linker-3) 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: 14.

[0071] In one embodiment, the bispecific CAR comprises, from N-terminus to C-terminus: (a) a light chain variable region (V) having ofatumumab; L 1) and heavy chain variable region (V H 1) anti-CD20 antigen binding region, (ii) light chain variable region (V L 2) and heavy chain variable region (V H 2), (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.

[0072] In one embodiment, the bispecific CAR comprises, from N-terminus to C-terminus: (a) a light chain variable region (V L 1) and heavy chain variable region (V H 1) (having the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 8, respectively), and (ii) having a light chain variable region (V L 2) and heavy chain variable region (V H2) (having the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 16, respectively), (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.

[0073] In one embodiment, the bispecific CAR comprises, from N-terminus to C-terminus: (a) a light chain variable region (V L 1) and heavy chain variable region (V H 1) anti-CD20 antigen binding regions, (ii) light chain variable regions (V L 2) and heavy chain variable region (V H 2), (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.

[0074] In one embodiment, the bispecific CAR comprises, from N-terminus to C-terminus: (a) a light chain variable region (V L 1) and heavy chain variable region (V H 1) (having the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 8, respectively), and (ii) having a light chain variable region (V L 2) and heavy chain variable region (V H 2) (having the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 16, respectively), (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.

[0075] In certain embodiments, V L 1 is located in V H 1. In certain embodiments, V H1 is located in V L 1. In certain embodiments, V H 2 is located at V L 2. In certain embodiments, V L 2 is located at V H 2. In one embodiment, V L 1 is located in V H N-terminal end of 1; V L 2 is located at V H 2's N terminus.

[0076] 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.

[0077] In certain embodiments, the bispecific CAR may have the amino acid sequence shown 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.

[0078] The bispecific CAR of the present invention 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 shown 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.

[0079] The bispecific CAR of the present invention can be encoded by a nucleic acid having a nucleotide sequence shown 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.

[0080] The present disclosure provides immune cells expressing or comprising the bispecific CAR of the present invention. The immune cells can be T cells or natural killer (NK) cells.

[0081] The present disclosure provides an immune cell comprising a vector or nucleic acid encoding a CAR of the present invention (e.g., integrated into its genome). 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.

[0082] The present disclosure also encompasses nucleic acids encoding the chimeric antigen receptors of the invention (e.g., the bispecific CARs of the invention).

[0083] The nucleic acid of the present invention can 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.

[0084] The nucleic acid of the present invention may comprise the nucleotide sequence shown 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.

[0085] The present disclosure provides a vector comprising a nucleic acid of the present invention. 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.

[0086] The present disclosure also provides a pharmaceutical composition comprising a chimeric antigen receptor of the present invention (e.g., a bispecific CAR of the present invention), an immune cell of the present invention, a nucleic acid of the present invention, or a vector of the present invention. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition may be a liquid preparation.

[0087] The pharmaceutical composition may contain a concentration ranging from about 1×10 3 cells / mL to about 1×10 8 cells / mL, or approximately 1×10 4cells / mL to about 1×10 7 cells / mL of the immune cells of the present invention.

[0088] The present disclosure also provides a method for treating an autoimmune disease / disorder. The present disclosure also provides a method for treating cancer. The method may include administering the immune cells of the present invention or the pharmaceutical composition of the present invention to a subject in need thereof.

[0089] Immune cells can be either allogeneic or autologous.

[0090] The autoimmune disorder can be systemic lupus erythematosus (SLE) (e.g., lupus nephritis), systemic sclerosis (SSc), an inflammatory myopathy (e.g., polymyositis, dermatomyositis, or inclusion body myositis), systemic scleroderma, multiple sclerosis, or neuromyelitis optica (NMO).

[0091] The cancer may be a hematologic 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 myeloid leukemia, acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0092] The present disclosure provides a method for preparing immune cells (e.g., CAR-T cells) expressing a chimeric antigen receptor, wherein the method comprises: transducing the nucleic acid molecule of the present invention or the vector of the present invention into an immune cell (e.g., a T cell or a NK cell), thereby obtaining an immune cell (e.g., CAR-T cell) expressing a chimeric antigen receptor.

[0093] The present disclosure provides a chimeric antigen receptor (CAR), wherein the structure of the chimeric antigen receptor can be as shown in Formula I:

[0094] SP-scFv1-linker 2-scFv2-H-TM-C-CSD (I),

[0095] wherein each “-” is independently a connecting 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 domain; 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.

[0096] 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.

[0097] The structure of the anti-CD20 antigen binding region can be shown as follows:

[0098] V H1 -V L1 (A); V L1 -V H1 (B)

[0099] Where V H1 V is the variable region of the heavy chain of the anti-CD20 antibody; L1 is the variable region of the light chain of the anti-CD20 antibody; and “-” is a connecting peptide or peptide bond.

[0100] In one embodiment, the CAR of the present invention has an anti-CD20 antigen binding region (or domain), the structure of which is shown in Formula B.

[0101] In certain embodiments, V L1 The amino acid sequence of is shown in SEQ ID NO: 4, and V H1 The amino acid sequence of is shown in SEQ ID NO:8.

[0102] V L1 and V H1 The peptides may be linked using a connecting peptide (Linker 1 or Linker-1). Linker-1 may have a sequence as shown in SEQ ID NO:6.

[0103] The structure of the anti-BCMA antigen binding region may be shown in Formula C or D below:

[0104] V L2 -V H2 (C); V H2 -V L2 (D)

[0105] Where V L2 V is the variable region of the light chain of the anti-BCMA antibody; H2 is the variable region of the heavy chain of an anti-BCMA antibody; and “-” is a connecting peptide or peptide bond.

[0106] In one embodiment, the CAR of the present invention has an anti-BCMA antigen binding domain, the structure of which is shown in Formula C.

[0107] In certain embodiments, V L2 The amino acid sequence of is shown in SEQ ID NO: 12, and V H2The amino acid sequence of is shown in SEQ ID NO:16.

[0108] V L2 and V H2 The peptides may be linked using a connecting peptide (Linker 3 or Linker-3). Linker-3 may have a sequence as shown in SEQ ID NO: 14.

[0109] In another embodiment, the chimeric antigen receptor has the structure shown in Formula II below:

[0110] SP-V L1 -V H1 - Connector 2-V L2 -V H2 -H-TM-C-CSD(II)

[0111] In one embodiment, Linker 2 (or Linker-2) has the sequence shown in SEQ ID NO:10.

[0112] In certain embodiments, the anti-CD20 antigen binding region comprises a light chain variable region (V L ), which comprises 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:4.

[0113] In certain embodiments, the anti-CD20 antigen binding region comprises a heavy chain variable region (V H ), which comprises 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:8.

[0114] In certain embodiments, the anti-BCMA antigen binding region comprises a light chain variable region (V L ), which comprises 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.

[0115] In certain embodiments, the anti-BCMA antigen binding region comprises a heavy chain variable region (V H ), which comprises 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.

[0116] The light chain variable region of the anti-CD20 antigen binding region (V L ) may comprise one, two or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, which are respectively identical to those in SEQ ID The CDR1, CDR2 and CDR3 shown at positions 24-34, positions 50-56, and positions 89-97 of NO:4 (CDRs of the light chain variable region of ofatumumab antibody) 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 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.

[0117] The light chain variable region of the anti-CD20 antigen binding region (V L ) may comprise one, two or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, which correspond to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID The CDR1, CDR2 and CDR3 shown in NO:132 (CDRs of the light chain variable region of ofatumumab antibody) 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 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.

[0118] The heavy chain variable region of the anti-CD20 antigen binding region (V H ) may comprise one, two or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, which are respectively as shown in SEQ The CDR1, CDR2 and CDR3 shown at positions 30-35, positions 50-66, and positions 99-111 of ID NO:8 (CDRs of the heavy chain variable region of ofatumumab antibody) 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 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.

[0119] The heavy chain variable region of the anti-CD20 antigen binding region (V H) may comprise one, two or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, which correspond to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID The CDR1, CDR2 and CDR3 shown in NO:129 (CDRs of the heavy chain variable region of ofatumumab antibody) 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 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.

[0120] In certain embodiments, the light chain variable region (V L ) comprises three CDRs, CDR1, CDR2, and CDR3, which are identical to CDR1, CDR2, and CDR3 shown at positions 24-34, 50-56, and 89-97 of SEQ ID NO: 4, respectively (CDRs of the light chain variable region of ofatumumab antibody), and the heavy chain variable region (V H ) comprises three CDRs that are identical to CDR1, CDR2, and CDR3 as shown at positions 30-35, positions 50-66, and positions 99-111 of SEQ ID NO: 8 (CDRs of the heavy chain variable region of the ofatumumab antibody).

[0121] In certain embodiments, the light chain variable region (V L ) comprises three CDRs, CDR1, CDR2, and CDR3, which are identical to CDR1, CDR2, and CDR3 shown in SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively (CDRs of the light chain variable region of ofatumumab antibody), and the heavy chain variable region (V H ) comprises three CDRs, which are identical to CDR1, CDR2 and CDR3 (CDRs of the heavy chain variable region of ofatumumab antibody) as shown in SEQ ID NO: 127, SEQ ID NO: 128 and SEQ ID NO: 129, respectively.

[0122] The light chain variable region of the anti-BCMA antigen binding region (V L) may comprise one, two or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, which are respectively identical to those in SEQ ID The CDR1, CDR2, and CDR3 set forth at positions 24-34, 50-56, and 89-97 of NO: 12 (CDRs of the light chain variable region of the BCMA-20 antibody) 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 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.

[0123] The light chain variable region of the anti-BCMA antigen binding region (V L ) may comprise one, two or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, which correspond to SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID In some embodiments, the CDR1, CDR2, and CDR3 set forth in NO: 138 (CDRs of the light chain variable region of the BCMA-20 antibody) 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 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.

[0124] The heavy chain variable region of the anti-BCMA antigen binding region (V H) may comprise one, two or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, which are respectively identical to those in SEQ ID The CDR1, CDR2, and CDR3 set forth at positions 31-35, positions 50-66, and positions 99-110 of NO: 16 (CDRs of the heavy chain variable region of the BCMA-20 antibody) 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 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.

[0125] The heavy chain variable region of the anti-BCMA antigen binding region (V H ) may comprise one, two or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, which correspond to SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID In some embodiments, the CDR1, CDR2, and CDR3 set forth in NO: 145 (CDRs of the heavy chain variable region of the BCMA-20 antibody) 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 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.

[0126] In certain embodiments, the light chain variable region (V L ) comprises three CDRs, CDR1, CDR2, and CDR3, which are identical to CDR1, CDR2, and CDR3 shown at positions 24-34, positions 50-56, and positions 89-97 of SEQ ID NO: 12, respectively (CDRs of the light chain variable region of the BCMA-20 antibody), and the heavy chain variable region (V H) comprises three CDRs, CDR1, CDR2, and CDR3, which are identical to CDR1, CDR2, and CDR3 as shown in positions 31-35, positions 50-66, and positions 99-110 of SEQ ID NO: 16, respectively (CDRs of the heavy chain variable region of the BCMA-20 antibody).

[0127] In certain embodiments, the light chain variable region of the anti-BCMA antigen-binding region comprises three CDRs, CDR1, CDR2, and CDR3, which correspond to the CDR1, CDR2, and CDR3 shown in SEQ ID NO: 134, SEQ ID NO: 136, and SEQ ID NO: 138, respectively (the light chain variable region (V) of the BCMA-20 antibody). L ) CDR) identical, and the heavy chain variable region (V H ) comprises three CDRs, CDR1, CDR2, and CDR3, which are identical to CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 141, SEQ ID NO: 143, and SEQ ID NO: 145, respectively (CDRs of the heavy chain variable region of the BCMA-20 antibody).

[0128] In certain embodiments, in the CAR of the present invention, the antigen binding domain targeting CD20 comprises a light chain variable domain V derived from ofatumumab antibody. L (SEQ ID NO: 4) and heavy chain variable domain V H (SEQ ID NO:8).

[0129] The light chain variable domain V from ofatumumab (OF) L Can have the following sequence:

[0130] EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQ APRLLIY DASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC Q QRSNWPIT FGQGTRLEIK (SEQ ID NO: 4)

[0131] OF-VL-CDR1: SEQ ID NO: 4, positions 24 to 34. The sequence of OF-VL-CDR1 is: RASQSVSSYLA (SEQ ID NO: 130).

[0132] OF-VL-CDR2: SEQ ID NO: 4, positions 50 to 56. The sequence of OF-VL-CDR2 is: DASNRAT (SEQ ID NO: 131).

[0133] OF-VL-CDR3: SEQ ID NO: 4, positions 89 to 97. The sequence of OF-VL-CDR3 is: QQRSNWPIT (SEQ ID NO: 132).

[0134] The heavy chain variable domain V from ofatumumab H Can have the following sequence:

[0135] EVQLVESGGGLVQPGRSLRLSCAASGFTF NDYAMH WVRQA PGKGLEWVS TISWNSGSIGYADSVKG RFTISRDNAKKSLYLQMN SLRAEDTALYYCAK DIQYGNYYYGMDV WGQGTTVTVSS (SEQ ID NO: 8)

[0136] OF-VH-CDR1: SEQ ID NO: 8, positions 30 to 35. The sequence of OF-VH-CDR1 is: NDYAMH (SEQ ID NO: 127).

[0137] OF-VH-CDR2: SEQ ID NO: 8, positions 50 to 66. The sequence of OF-VH-CDR2 is: TISWNSGSIGYADSVKG (SEQ ID NO: 128).

[0138] OF-VH-CDR3: SEQ ID NO: 8, positions 99 to 111. The sequence of OF-VH-CDR3 is: DIQYGNYYYGMDV (SEQ ID NO: 129).

[0139] In certain embodiments, the antigen binding domain targeting BCMA in the CAR of the present invention comprises a light chain variable domain V derived from the BCMA-20 (B20) antibody. L (SEQ ID NO: 12) and the heavy chain variable domain V H (SEQ ID NO: 16).

[0140] The light chain variable domain V from the BCMA-20 antibody L Can have the following sequence:

[0141] DIQMTQSPSSSLSASVGDRVTITCRASQGISNYLNWYQQKPGK APKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYC MGQTISSYTFGQGTKLEIK(SEQ ID NO:12)

[0142] B20-VL-CDR1: SEQ ID NO: 12, positions 24 to 34. The sequence of B20-VL-CDR1 is: RASQGISNYLN (SEQ ID NO: 134).

[0143] B20-VL-CDR2: SEQ ID NO: 12, positions 50 to 56. The sequence of B20-VL-CDR2 is: YTSNLQS (SEQ ID NO: 136).

[0144] B20-VL-CDR3: SEQ ID NO: 12, positions 89 to 97. The sequence of B20-VL-CDR3 is: MGQTISSYT (SEQ ID NO: 138).

[0145] The heavy chain variable domain V from the BCMA-20 antibody H Can have the following sequence:

[0146] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGADHAIYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVSS(SEQ ID NO:16)

[0147] B20-VH-CDR1: SEQ ID NO: 16, positions 31 to 35. The sequence of B20-VH-CDR1 is: NFDMA (SEQ ID NO: 141).

[0148] B20-VH-CDR2: SEQ ID NO: 16, positions 50 to 66. The sequence of B20-VH-CDR2 is: SITTGADHAIYADSVKG (SEQ ID NO: 143).

[0149] B20-VH-CDR3: SEQ ID NO: 16, positions 99 to 110. The sequence of B20-VH-CDR3 is: HGYYDGYHLFDY (SEQ ID NO: 145).

[0150] The signal peptide may be a CD8 signal peptide having the following sequence:

[0151] MALPVTALLLPLALLLHAARP(SEQ ID NO:2)

[0152] V of anti-CD20 scFv L and V H (or V H and V L ) between the two ends (Linker-1) may have the following sequence: GSTSGGGSGGGSGGGGSS (SEQ ID NO: 6)

[0153] The linker between the anti-CD20 scFv and the anti-BCMA scFv (Linker-2) may have the following sequence: GGGGS (SEQ ID NO: 10)

[0154] Anti-BCMA scFv V L and V H (or V H and V L ) between the two ends (Linker-3) may have the following sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 14)

[0155] 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.

[0156] The hinge region may be derived from CD8a having the following sequence:

[0157] FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO:18)

[0158] The hinge region may be derived from IgG4 having the following sequence:

[0159] ESKYGPPCPPCP (SEQ ID NO: 78)

[0160] The transmembrane domain may be derived from CD8 (CD8TM) having the following sequence: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 20)

[0161] The transmembrane domain may be derived from CD28 (CD28TM) having the following sequence: MFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 80)

[0162] The costimulatory region can be derived from 4-1BB having the following sequence:

[0163] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:22)

[0164] The cytoplasmic signaling domain can be derived from CD3ζ having the following sequence: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 24)

[0165] Chimeric Antigen Receptor (CAR)

[0166] The term "chimeric antigen receptor" or alternatively "CAR" is used interchangeably throughout the text and refers 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 a ζ 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 can also be 4-1BB (i.e., CD137), CD27 and / or CD28 or a fragment of these molecules. In another aspect, CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain comprises a functional signaling domain derived from a stimulatory molecule. CAR may comprise a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain, wherein the intracellular signaling domain comprises a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. Alternatively, CAR may comprise a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain, wherein the intracellular signaling domain comprises two functional signaling domains derived from one or more costimulatory molecules and one functional signaling domain derived from a stimulatory molecule. CAR may also comprise a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain, wherein the intracellular signaling domain comprises at least two functional signaling domains derived from one or more costimulatory molecules and one functional signaling domain derived from a stimulatory molecule. The antigen binding region of CAR may contain any antigen-binding antibody fragment. The antibody fragment may comprise one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or any combination of the foregoing.

[0167] The term "zeta" or alternatively "zeta chain", "CD3-zeta" or "TCR-zeta" can be the protein provided as GenBank Accession No. NP_932170, NP_000725 or XP_011508447; or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.), and the "zeta stimulatory domain" or alternatively "CD3-zeta stimulatory domain" or "TCR-zeta stimulatory domain" can be the amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit the initial signals required for T cell activation.

[0168] Chimeric receptors may refer to non-naturally occurring molecules that can be expressed on the surface of a host cell and comprise an antigen-binding fragment that binds to an antigen. In addition to the antigen-binding fragment, the 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, the chimeric antigen receptor comprises an antigen-binding region (or fragment), a hinge region, a transmembrane domain, and a cytoplasmic signaling domain from N-terminus to C-terminus. In some embodiments, the chimeric antigen receptor further comprises at least one costimulatory region. Therefore, the chimeric antigen receptor may comprise an antigen-binding region (or fragment), a hinge region, a transmembrane domain, a costimulatory region, and a cytoplasmic signaling domain from N-terminus to C-terminus.

[0169] In some embodiments, the chimeric antigen receptor comprises a hinge region, which can be located between the antigen binding region and the transmembrane domain. The hinge region can contain about 10-200 amino acids, such as 15-150 amino acids, 20-100 amino acids, or 30-60 amino acids. In some embodiments, the hinge region can be about 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 can contain 0-300 amino acids, 2 amino acids to 100 amino acids, 5 amino acids to 80 amino acids, 10 amino acids to 60 amino acids, 10 amino acids to 15 amino acids, 20 amino acids to 80 amino acids, 30 amino acids to 70 amino acids, 40 amino acids to 60 amino acids, 50 amino acids to 60 amino acids, or 30 amino acids to 60 amino acids.

[0170] In some embodiments, the hinge region is a 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 chimeric antigen receptors. In some embodiments, the hinge domain is 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.

[0171] The hinge domain of an antibody (such as an IgG, IgA, IgM, IgE, or IgD antibody) is also suitable for chimeric antigen receptors. In some embodiments, the hinge region is a hinge domain that engages the constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge region is of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge region comprises the hinge domain of the antibody and the CH3 constant region of the antibody. In some embodiments, the hinge region comprises the hinge domain of the antibody and the CH2 constant region and CH3 constant region of the 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 constant region and CH3 constant region of an IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and CH3 constant region of an IgG4 antibody.

[0172] The hinge region can 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, such as a (GlyxSer)n (or (GxS)n) linker, wherein x and n can independently be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or greater.

[0173] Other peptide linkers that can be used in the hinge region of the chimeric receptors described herein are known in the art. See, for example, Wriggers et al. Current Trends in Peptide Science (2005) 80(6):736-746 and PCT Publication WO 2012 / 088461.

[0174] In some embodiments, the chimeric antigen receptor may comprise a transmembrane domain. The transmembrane domain may be of any form known in the art. Suitable transmembrane domains for chimeric antigen receptors may be obtained from naturally occurring proteins. Alternatively, the transmembrane domain may be a synthetic, non-naturally occurring protein segment, for example, a hydrophobic protein segment that is thermodynamically stable in the cell membrane.

[0175] In some embodiments, the transmembrane domain is the transmembrane domain of CD8α. In some embodiments, the transmembrane domain is the transmembrane domain of CD28. In some embodiments, the transmembrane domain is the transmembrane domain of ICOS.

[0176] In some embodiments, chimeric antigen receptor comprises one or more costimulatory regions.Costimulatory region can be at least a portion of a protein that mediates intracellular signal transduction to induce an immune response (such as effector function). The costimulatory region of a chimeric antigen receptor can be from a protein that transduces a signal and regulates a reaction mediated by an immune cell (such as a T cell, a natural killer (NK) cell, a macrophage, a neutrophil, or an eosinophil).

[0177] In some embodiments, the chimeric antigen receptor comprises one or more than one (at least 2, 3, 4 or more) costimulatory domains. In some embodiments, the chimeric antigen receptor comprises more than one costimulatory domain obtained from different proteins. In some embodiments, the chimeric antigen receptor does not comprise a costimulatory domain.

[0178] The example of the costimulatory region for chimeric antigen receptor can be the domain from costimulatory protein, 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, 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 from 4-1BB or ICOS. In some embodiments, the costimulatory region is a fusion domain comprising more than one costimulatory region or more than one costimulatory region. In some embodiments, the costimulatory region is a fusion of the costimulatory region from CD28 and ICOS.

[0179] 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, such as the interaction of an extracellular ligand binding domain with its ligand, to stimulate a cellular response, such as inducing an effector function of the cell (e.g., cytotoxicity).

[0180] Chimeric antigen receptors can be prepared by conventional methods, such as recombinant technology. The method for preparing a chimeric antigen receptor may involve the production of a nucleic acid encoding a polypeptide comprising each domain of a chimeric antigen receptor, including an antigen binding fragment and an optional 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 joined together using recombinant technology. Methods such as PCR amplification or conjugation can be used to join the sequence of each component (e.g., domain) directly or indirectly (e.g., using a nucleic acid sequence encoding a peptide linker) to form a nucleic acid sequence encoding a chimeric antigen receptor. Alternatively, nucleic acids encoding a chimeric antigen receptor can be synthesized. In some embodiments, the nucleic acid is DNA. In other embodiments, the nucleic acid is RNA.

[0181] In one embodiment, the CAR of the present invention comprises, from N-terminus to C-terminus, a signal peptide (also referred to as a leader sequence), an antigen recognition sequence (antigen binding domain), a hinge region, a transmembrane domain, a co-stimulatory region, and a cytoplasmic signaling domain (e.g., a CD3 zeta signaling region (zeta chain portion)).

[0182] Bispecific means that CAR can specifically bind to two different antigens. Bispecific CAR can generate an immune response by binding to one antigen or two antigens.

[0183] As used herein, the terms "CAR-T cells," "CAR-T," "CART," and "CART cells" may refer to T cells expressing the CAR of the present invention that targets both CD20 and BCMA.

[0184] Immune cells expressing chimeric antigen receptors

[0185] The disclosure also provides immune cells expressing the CAR of the present invention. The recognition of target cells having antigens on the cell surface by the antigen-binding fragment of the chimeric antigen receptor can transduce activation signals to the signaling domains (e.g., costimulatory regions and / or cytoplasmic signaling domains) of the chimeric antigen receptor, which can activate effector functions in immune cells expressing chimeric antigen receptors.

[0186] Chimeric antigen receptors can be introduced into suitable immune cells for expression via conventional techniques. In some embodiments, the immune cells are T cells, such as primary T cells or T cell lines. Alternatively, the immune cells can be natural killer (NK) cells, such as established NK cell lines (e.g., NK-92 cells). In some embodiments, the immune cells are cells expressing CD8 (CD8 + ) or CD8 and CD4 (CD8 + / CD4 +In some embodiments, the T cells are T cells of an established T cell line, such as Jurkat cells.

[0187] In some embodiments, immune cell group is derived from a human patient with an autoimmune disorder or cancer (for example, hematopoietic malignancy), for example, derived from bone marrow or PBMC obtained from a patient. In some embodiments, immune cell group is derived from a healthy donor. In some embodiments, immune cell is obtained from a subject to which an immune cell expressing a chimeric antigen receptor is subsequently administered. The immune cell administered to the subject identical to the subject obtained from the cell is referred to as an autologous cell, and the immune cell obtained from a subject who is not to administer the cell can be referred to as an allogeneic cell.

[0188] Desired immune cell types can be expanded within a population of cells obtained by co-culturing the cells with stimulatory molecules, for example, anti-CD3 and anti-CD28 antibodies can be used to expand T cells.

[0189] In order to construct immune cells expressing chimeric antigen receptors as described herein, vectors for stable or transient expression of chimeric antigen receptors can be constructed via conventional methods as described herein and introduced into immune cells. For example, nucleic acids encoding chimeric antigen receptors can be cloned into suitable vectors, such as viral vectors.

[0190] In certain embodiments, immune cells (eg, T cells) are transduced with a lentiviral vector (LV) encoding the CAR of the present invention. Transduced immune cells (eg, T cells) can target CD20 and BCMA, synergistically activate T cells, and induce T cell-mediated immune responses.

[0191] In one embodiment, in the method of the present invention, T cells from an autologous patient (or allogeneic donor) are separated, activated and genetically modified to produce CAR-T cells expressing the CAR of the present invention, and then administered to the patient. CAR-T cells can replicate in vivo, thereby achieving long-term persistence. In addition, CAR-mediated immune responses may be part of an adoptive immunotherapy approach, in which anti-CD20 / BCMA C-T cells trigger an immune response against cells expressing CD20 and / or BCMA.

[0192] In certain embodiments, cells are isolated from mammals (e.g., humans) and genetically modified (i.e., in vitro transduction or transfection) with a vector expressing a CAR disclosed herein. CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be a human. CAR-modified cells can be autologous for the recipient. Alternatively, for the recipient, the cells can be allogeneic, isogenic, or allogeneic.

[0193] The method for preparing the immune cell expressing the chimeric antigen receptor of the present invention can include ex vivo activation and / or amplification immune cells. Activation of immune cells means stimulating immune cells to enter an activated state, in which the cells can be able to play effector functions (e.g., cytotoxicity). The method for activating immune cells will depend on the type of immune cell used to express the chimeric antigen receptor. Amplification of immune cells can involve any method that causes an increase in the number of cells expressing the chimeric antigen receptor, for example, allowing cell proliferation or stimulating cell proliferation. In some embodiments, cells expressing the chimeric receptor as described herein are activated and / or amplified in vitro before being administered to a subject.

[0194] CAR-expressing immune cells can also be used as vaccines and / or in vivo therapies for ex vivo immunization of mammals. In addition to using cell-based vaccines for ex vivo immunization, the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response to antigens in the patient. Preferably, the mammal is a human. For ex vivo immunization, before the cells are administered to a mammal, one or more of the following can be performed in vitro: i) amplifying cells, ii) introducing nucleic acids encoding CAR into cells, and / or iii) freezing cells.

[0195] carrier

[0196] The present disclosure provides a nucleic acid encoding the CAR of the present invention. The present disclosure also provides a vector comprising the nucleic acid of the present invention.

[0197] Vectors include, but are not limited to, plasmids, phagemids, phage derivatives, viruses, and cosmids.

[0198] The vector can be a viral vector. Viruses suitable for use as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In certain embodiments, the vector of the present invention is a retroviral vector, such as a lentiviral vector. In some embodiments, the vector used to express the chimeric antigen receptor is a retrovirus. In some embodiments, the vector used to express the chimeric antigen receptor is a lentivirus. In some embodiments, the vector used to express the chimeric antigen receptor is an adeno-associated virus.

[0199] A variety of promoters can be used to express chimeric receptors, including but not limited to cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as Rous sarcoma virus (Rous sarcoma virus) LTR, HIV-LTR, HTLV-1LTR, Moloney murine leukemia virus (Maloney 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, elongation factor 1-alpha (EF1-alpha) promoter with or without EF1-alpha intron. Additional promoters for expressing chimeric receptors include any constitutively active promoter in immune cells. Alternatively, any regulatable promoter (e.g., inducible promoter) can be used so that its expression can be regulated in immune cells.

[0200] The vector can be introduced into cells, such as mammalian, bacterial, yeast or insect cells, by any method in the art. For example, the vector can be transferred into the cells by physical, chemical or biological methods.

[0201] Physical methods for introducing polynucleotides into cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0202] Biological methods for introducing polynucleotides 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.

[0203] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0204] In some embodiments, the vector (nucleic acid) encoding the chimeric antigen receptor is a DNA vector and 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, which can be electroporated into immune cells.

[0205] Any vector comprising a nucleic acid encoding a chimeric antigen receptor as described herein is also within the scope of the present disclosure. Such a vector can be delivered to a host cell such as an immune cell by a suitable method. Methods for delivering a vector to an immune cell are well known in the art and can include DNA, RNA or transposon electroporation, transfection reagents (such as liposomes or nanoparticles) to deliver DNA, RNA or transposon; DNA, RNA or transposon or protein is delivered by mechanical deformation (see, for example, Sharei et al. PNAS (2013) 110 (6): 2082-2087); or viral transduction. In some embodiments, the vector for expressing the chimeric receptor is delivered to the cell by viral transduction.

[0206] In the example where a viral vector is used to introduce a vector encoding a chimeric antigen receptor into a host cell, viral particles capable of infecting immune cells and carrying the vector can be produced by any method known in the art. Viral particles are harvested from the cell culture supernatant and isolated and / or purified before contacting the viral particles with the immune cells.

[0207] Pharmaceutical composition

[0208] The present disclosure provides a pharmaceutical composition comprising the immune cells of the present invention, the CAR of the present invention, the nucleic acid of the present invention, or the vector of the present invention. The pharmaceutical composition of the present invention may further comprise 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 1×10 4 -1×10 7 cells / mL.

[0209] As will be appreciated by those skilled in the art, an effective amount will vary, depending on the particular disorder being treated, the severity of the disorder, individual patient parameters (including age, physical condition, size, sex, and weight), duration of treatment, the nature of concurrent therapy (if any), the specific route of administration, and similar factors within the knowledge and expertise of the healthcare professional. In some embodiments, an effective amount alleviates, relieves, improves, increases, reduces the symptoms of a disease or disorder in a subject, or slows the progression of a disease or disorder in a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0210] Pharmaceutically acceptable carriers, including buffers, are well known in the art and may include phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or nonionic surfactants. See, for example, Remington: The Science and Practice of Pharmacy 20th edition (2000) Lippincott Williams and Wilkins, ed. KE Hoover.

[0211] The pharmaceutical composition of the present invention can be delivered to cells by contacting the cells with the pharmaceutical composition of the present invention.

[0212] The pharmaceutical compositions of the present invention can be delivered / administered to a subject by any route, including but not limited to intravenous, intracerebroventricular (ICV) injection, intracisternal injection or infusion, oral, transdermal, ocular, intraperitoneal, subcutaneous, implant, sublingual, subcutaneous, intramuscular, rectal, mucosal, ocular, intrathecal, intraarticular, intraarterial, subarachnoid, bronchial and lymphatic administration. The pharmaceutical compositions of the present invention can be administered parenterally or systemically. The compositions of the present invention can be applied topically. The pharmaceutical compositions can be formulated for intravenous administration.

[0213] The use of the composition of the present invention can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, blood transfusion, implantation or transplantation. The composition can be administered subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (iv) injection or intraperitoneally to the patient. 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 iv injection. The composition can be directly injected into a tumor, lymph node or condition site.

[0214] The immune cells or pharmaceutical compositions of the present invention can be delivered / administered to a subject via intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration, such as by injection or infusion.

[0215] The pharmaceutical composition of the present invention can be used in a manner suitable for the disease to be treated (or prevented). Although appropriate dosage can be determined by clinical trials, the quantity and frequency of use will be determined by factors such as the patient's condition and the type and severity of the patient's disease.

[0216] When "effective amount", "therapeutically effective amount" or "therapeutic amount" is indicated, the exact amount of the composition to be administered can be determined by the physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). Pharmaceutical compositions containing immune cells can be 10 4 to 10 9 cells / kg body weight or 10 5 to 10 6 The composition can also be administered at multiple doses of cells / kg body weight (including all integer values ​​within these ranges). The composition can also be administered multiple times at these doses. Cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a particular patient can be easily determined by a technician in the medical field by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0217] The dosage of the above treatment administered to the patient may vary depending on the specific nature of the condition being treated and the recipient of the treatment. The dosage administered to the patient may be adjusted according to practices generally recognized in the art. In one embodiment, for each treatment or each course of treatment, 1×10 6 to 1×10 10 immune cells (e.g., CAR-T cells).

[0218] Condition to be treated

[0219] The CAR, immune cells or pharmaceutical compositions of the present invention can be used to treat autoimmune diseases / disorders, or to treat cancer or tumors.

[0220] In certain embodiments, the anti-CD20 / BCMA bispecific CAR targeting B cells and plasma cells of the present invention can reduce / eliminate autoimmune antibodies. In certain embodiments, the anti-CD20 / BCMA bispecific CAR of the present invention can reduce / exhaust B cells, plasmablasts and / or long-lived plasma cells (LLPC) to reduce / eliminate autoantibody production.

[0221] 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.

[0222] The autoimmune disorder can be systemic lupus erythematosus (SLE), lupus nephritis (LN), systemic sclerosis (SSc), CREST syndrome (calcinosis, Raynaud's syndrome, esophageal dysmotility, scleroderma, and telangiectasia), opsoclonus, inflammatory myopathies (e.g., polymyositis, dermatomyositis, and inclusion body myositis), myositis autoantibody-driven diseases, systemic scleroderma, 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, antiphospholipid syndrome, Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, 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's chorea, pediatric autoimmune neuropsychiatric disorders associated with streptococci (PANDAS), encephalitis, type 1 diabetes, neuromyelitis optica (NMO), chronic inflammatory bowel disease, Hashimoto's disease, organ transplant rejection, and / or neuromyelitis optica spectrum disorder (NMOSD).

[0223] The autoimmune disorder may be pernicious anemia, Addison's disease, psoriasis, inflammatory bowel disease (IBD), psoriatic arthritis, Sjögren's syndrome ( syndrome), lupus erythematosus (e.g., discoid lupus erythematosus, drug-induced lupus erythematosus, and neonatal lupus erythematosus), multiple sclerosis, and / or reactive arthritis.

[0224] The autoimmune disorder can be polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, adrenalitis, thyroiditis, autoimmune thyroid disease, gastric atrophy, chronic hepatitis, lupus hepatitis, atherosclerosis, Alzheimer's disease, demyelinating disease, 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 2 diabetes), autoimmune infertility in men and women, ankylosing spondylitis, ulcerative colitis, Crohn's disease, disease), sprue, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, juvenile-onset 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 syndrome), alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behçet's disease, Caplan's syndrome, Kawasaki's disease, dengue fever, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema raised, erythrocytosis fetalis, eosinophilic maskitis, Shulman's syndrome, Felty's syndrome syndrome), filariasis, cyclitis, chronic cyclitis, metachronous cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpurapurpura), graft-versus-host disease, transplant rejection, human immunodeficiency virus infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, postvaccination syndrome, congenital rubella infection, Hodgkin and non-Hodgkin lymphoma, renal cell carcinoma, multiple myeloma, Eaton-Lambert syndrome, relapsing polychondritis, malignant melanoma, cryoglobulinemia, Waldenstrom's macroglobulemia, Epstein-Barr virus infection, mumps, Evan's syndrome, and / or autoimmune gonadal failure.

[0225] 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-onset diabetes), multiple sclerosis, scleroderma, ankylosing spondylitis, sarcoidosis, pemphigus vulgaris, pemphigoid, psoriasis, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, Behcet's syndrome, Reiter's disease, Berger's disease, and leukemia. disease), dermatomyositis, polymyositis, antineutrophil cytoplasmic antibody-associated vasculitis (e.g., granulomatosis with polyangiitis (also known as Wegener's granulomatosis), microscopic polyangiitis, and Chargar-Strauss syndrome), scleroderma, Sjögren's syndrome, anti-glomerular basement membrane disease (including 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.

[0226] Autoimmune diseases include inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, sprue, autoimmune arthritis, rheumatoid arthritis, type I 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, atopic dermatitis, graft-versus-host disease, acute or chronic immune disorders associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, Graves' disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schönlein purpura, renal microscopic vasculitis, chronic active hepatitis, and grape Meningitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington disease, Parkinson disease, Alzheimer disease, stroke, primary biliary cirrhosis, hemolytic anemia, polyglandular deficiency syndrome type I and II, Schmidt syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthropathy, arthropathy, Reiter disease, psoriatic arthropathy, chlamydia, Yersinia, and Salmonella-associated arthropathy (yersinia and salmonella associated arthropathy), spondyloarthropathies, atherosclerotic disease / 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 diseaseDisease), chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, acquired immunodeficiency syndrome, acquired immunodeficiency-related diseases, hepatitis C, shared transformation immunodeficiency (shared transformation hypogammaglobulinemia), dilated cardiomyopathy, fibrosing lung disease, cryptogenic fibrosing alveolitis, postinflammatory interstitial lung disease, interstitial pneumonia, connective tissue disease-related interstitial lung disease, mixed connective tissue disease-related lung disease, systemic sclerosis-related interstitial lung disease, rheumatoid arthritis-related interstitial lung disease, systemic lupus erythematosus-related lung disease, dermatomyositis / polymyositis-related lung disease, Sjögren's syndrome-related lung disease, ankylosing spondylitis-related lung disease, vasculitic diffuse lung disease, hemosiderosis-related lung disease, drug-induced interstitial lung disease, radiation-induced fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrate lung disease, postinfectious interstitial lung disease Gouty arthritis, autoimmune hepatitis, autoimmune hepatitis type 1 (classic autoimmune or lupus-like hepatitis), autoimmune hepatitis type 2 (anti-LKM antibody hepatitis), autoimmune hypoglycemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthritis, primary sclerosing cholangitis, idiopathic leukopenia, autoimmune neutropenia, kidney disease (NOS), glomerulonephritis, renal microscopic vasculitis, discoid lupus, lupus erythematosus, idiopathic or NOS male infertility, sperm autoimmunity, multiple sclerosis (all subtypes), insulin-dependent diabetes mellitus, sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestations of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease disease), systemic sclerosis, Takayasu disease / arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goiter, autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxedema, lenticular uveitis, primary vasculitis, vitiligo, allergic rhinitis (pollen allergy), anaphylaxis, pet allergy, latex allergy, drug allergy, allergic rhinoconjunctivitis, eosinophilic esophagitis, eosinophilic syndrome, eosinophilic gastroenteritis, cutaneous lupus erythematosus, eosinophilic esophagitis, eosinophilic syndrome, and eosinophilic gastroenteritis.

[0227] An autoimmune disorder can be an inflammatory muscle disease. Inflammatory myopathies are 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 myopathies are polymyositis, which affects skeletal muscles (the type involved in body movement) on both sides of the body; dermatomyositis, which causes progressive muscle weakness; inclusion body myositis, which is characterized by slow, progressive muscle weakness, muscle atrophy, and muscle loss; and necrotizing autoimmune myopathy, which involves muscle weakness in the upper and lower body.

[0228] In another embodiment, the autoimmune disease is an autoimmune disease caused by overexpression of B cells (such as lupus erythematosus).

[0229] The present disclosure also encompasses a method of treating cancer. The method may include administering a CAR, immune cell, or pharmaceutical composition to a subject in need thereof.

[0230] The present disclosure provides chimeric antigen receptors for use in treating CD20-positive diseases, such as B-cell lymphomas.

[0231] The cancer may be a BCMA-positive malignancy. The cancer may be multiple myeloma (MM) or plasma cell leukemia.

[0232] The cancer can be a hematologic cancer. The cancer can be a plasma cell malignancy. The cancer can be a B cell malignancy. The B cell malignancy can be acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B cell acute lymphoblastic leukemia (B-ALL), B cell leukemia, or B cell lymphoma.

[0233] The cancer may be Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia and / or multiple myeloma (MM).

[0234] The cancer can 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.

[0235] Diseases that can be treated using the CAR of the present invention, immune cells or pharmaceutical compositions include CD20 positive tumors and diseases such as those caused by excessive B cells (such as autoimmune diseases, such as lupus erythematosus, etc.).CD20 positive tumors can include CD20 positive non-solid tumors (such as hematological cancers, such as leukemia and lymphoma) or solid tumors.Tumors or cancers treated using the CAR of the present invention, immune cells or pharmaceutical compositions include but are not limited to cancer, blastoma and sarcoma and leukemia or lymphatic malignancies, benign and malignant tumors and malignant tumors, such as sarcoma, cancer, gastric cancer, gastric cancer peritoneal metastasis, liver cancer, kidney cancer, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumors, bladder tumors, non-small cell lung cancer (NSCLC), glioma, endometrial cancer and melanoma.Including adult tumors / cancer and childhood tumors / cancer.

[0236] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or blood-borne) cancers include leukemias, such as acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0237] Cancer can be a solid tumor. Solid tumors can be benign or malignant. Different types of solid tumors are named according to the cell type that forms them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcomas, myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, pancreatic cancer, and ovarian cancer.

[0238] Reagent test kit

[0239] Kits for using the CAR, immune cells, nucleic acids, vectors, or pharmaceutical compositions of the present invention are also within the scope of the present disclosure. Such kits may include one or more containers comprising the CAR, immune cells, nucleic acids, vectors, or pharmaceutical compositions of the present invention.

[0240] In some embodiments, the kit may include instructions for use in any of the methods described herein. The included instructions may include a description of administering the pharmaceutical composition to a subject to achieve the desired 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 a description of administering the pharmaceutical composition to a subject in need of treatment.

[0241] Instructions for use of a pharmaceutical composition typically include information regarding the dosage, dosing schedule, and route of administration for the intended treatment.The container may be a unit dose, bulk package (eg, multi-dose package), or sub-unit dose.

[0242] The kits provided herein are all in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, soft packaging, etc.

[0243] The following examples of specific aspects of implementing the present disclosure are provided for illustrative purposes only and are not intended to limit the scope of the disclosure in any way.

[0244] Example 1 Construction of anti-CD20 / BCMACAR

[0245] We generated eight bispecific CARs in which the order of anti-CD20 scFv and anti-BCMA scFv was the same (i.e., anti-CD20 scFv (“OF”) followed by anti-BCMA scFv (“B20”)), but V H / V L The sequences are different and have different hinge regions and / or transmembrane domains: TOB1-4 and TOBL1-4, of which TOBL1 is C-CAR168 ( Figure 1 ).

[0246] Anti-CD20 / BCMACAR-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 previously coated with CD3 monoclonal antibody (OKT3) and fibronectin (TAKARA). The culture medium was GT-551 cell culture medium containing 1% human albumin and 300U / mL recombinant human interleukin 2 (IL-2). On day 3, activated PBMCs were transduced with a lentiviral vector encoding anti-CD20 / BCMACAR.

[0247] Figure 2The expression levels of anti-CD20 and anti-BCMACAR on the surface of T cells are shown. The expression level of anti-BCMACAR was detected by flow cytometry using BCMA-Fc fusion protein; the expression level of anti-CD20 CAR was detected by flow cytometry using an antibody specific for OF scFv.

[0248] Example 2 In vitro antigen-specific activation of anti-CD20 / BCMACAR-T cells

[0249] Anti-CD20 / BCMACAR-T antigen-specific activation is assessed by measuring IFN-γ release and CD137 expression when CAR-T cells are co-cultured with target cells.Target cells (" T ") include 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.Effect cells (" E ") are anti-CD20 / BCMACAR-T cells.

[0250] PBMCs were isolated from venous blood of healthy donors by density gradient centrifugation. On day 0, PBMCs were activated in cell culture flasks previously coated with CD3 monoclonal antibody (OKT3) and fibronectin (TAKARA). The culture medium consisted of GT-551 cell culture medium supplemented with 1% human albumin and 300 U / mL recombinant human interleukin-2 (IL-2). On day 3, the activated PBMCs were transduced with a lentiviral vector encoding an anti-CD20 / BCMACAR. Starting on day 6, CAR-T cells could be removed for activity assays.

[0251] 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 culture medium at an E:T ratio of 1:1 for 18 hours. The secretion level of IFN-γ in the cell culture supernatant was then measured by ELISA.

[0252] The expression level of CD137 was determined using CAR-T cells cultured for 7 days. 5CAR-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 in 200 μl of culture medium at an E:T ratio of 1:1 for 18 h. The expression level of CD137 on the surface of CAR-T cells was then detected by flow cytometry.

[0253] IFNγ release results Figures 3A-3C and Figure 6B As shown. After co-culturing CAR-T cells with A549 cells expressing CD20 and / or BCMA antigens, anti-CD20 CAR-T (C-CAR066) cells can specifically recognize CD20 single-positive or CD20 / BCMA double-positive target cells and release IFN-γ. Similarly, anti-BCMACAR-T (C-CAR088) cells can specifically recognize BCMA single-positive or CD20 / BCMA double-positive target cells to release IFN-γ. Only anti-CD20 / BCMACAR-T (TOB1-4 and TOBL1-4, of which TOBL1 is C-CAR168) cells can recognize CD20 single-positive, BCMA single-positive, and CD20 / BCMA double-positive target cells, and release high levels of IFN-γ. TOB1 to TOB4 CAR-T cells exhibit high IFN-γ release when co-cultured with CD20-positive target cells, but show lower reactivity to BCMA single-positive target cells. TOBL1 to TOBL4 CAR-T cells exhibited high IFN-γ release when co-cultured with CD20-positive and BCMA-positive target cells. TOBL1 to TOBL4 CAR-T cells exhibited high IFN-γ release when co-cultured with target cells that naturally express CD20 and BCMA.

[0254] Flow cytometry results showed that anti-CD20 / BCMA CAR-T cells were activated by a variety of CD20 / BCMA single-positive or double-positive cells and upregulated the expression level of CD137 ( Figure 4A and 4B ).

[0255] Example 3 Anti-CD20 / BCMACAR-T cell cytotoxicity in vitro

[0256] Anti-CD20 / BCMACAR-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. Real-time cell analysis (RTCA) was used to assess the cytotoxicity of CAR-T cells against target cells.

[0257] The results showed that anti-CD20 / BCMACAR-T cells effectively killed CD20 / BCMA single-positive or double-positive tumor cells (A549-CD20+, A549-BCMA+, A549-BCMA+CD20+) in vitro, but had no effect on A549 cells that did not express CD20 or BCMA ( Figures 5A-5B 、 Figure 6C Their killing abilities were comparable to those of anti-CD20 and anti-BCMA monospecific CAR-T cells, and were dose-dependent ( Figures 5A-5B , Figure 6C TOBL1 to TOBL4 CAR-T cells ( Figure 5B ) showed high cytotoxicity against CD20-positive and BCMA-positive target cells. TOB1 to TOB4 CAR-T cells ( Figure 5A ) exhibited lower cytotoxicity against BCMA single-positive target cells (compared to anti-BCMACAR (C-CAR088)).

[0258] Example 4 In vitro cytotoxicity of anti-CD20 / BCMACAR-T cells against autoreactive B cells

[0259] Recent studies have shown that CD11c hi T-bet + The proportion of B cell subsets increased significantly and was closely related to the production of autoantibodies and the clinical manifestations of patients. Autoantibodies are a characteristic of reactive B cells (see Distinct Effector B Cells Induced by Unregulated Toll-like Receptor7Contribute to Pathogenic Responses in Systemic Lupus Erythematosus, Immunity, 2018, 16; 49(4): 725-739.e6. IL-21drives expansion and plasma cell differentiation of autoreactive CD11chiT-bet+B cells in SLE, Nat. Commun. 2018; 9(1): 1758). In some autoimmune disease animal models and the peripheral blood of rheumatoid arthritis patients, this cell subset is enriched with age, so they are also called age-related B cells (ABC) (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).

[0260] TLR7 activation plays a role in the accumulation of autoreactive B cells and the production of autoantibodies in autoimmune diseases. One of the consequences of abnormal TLR7 activation is the accumulation of autoreactive B cells or age-related B cells (ABC). ABC is a B cell that recognizes self-antigens and has the potential to produce autoantibodies that can target and damage the body's own tissues. Wang et al., Nature Communications, (2018) 9: 1758.

[0261] To verify that anti-CD20 / BCMACAR-T cells also have the ability to eliminate ABC, 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 PBMCs of healthy donors and induced their differentiation in vitro to obtain autologous B cells rich in ABC, which were then used as target cells for cytotoxicity experiments. After 2 to 4 hours of co-culture, C-CAR168 CAR-T cells from different donors showed significant cytotoxic effects on autologous B cells rich in ABC at different E:T ratios compared with control T cells that were not transduced with CAR ( Figures 7A-7C ).

[0262] C-CAR168 can simultaneously target CD20+ B cells and BCMA+ plasma cells, which can provide a longer duration of response in autoimmune diseases. The results showed that C-CAR168 CAR-T cells can effectively eliminate ABC cells.

[0263] In vitro ABC differentiation

[0264] PBMCs from healthy donors were isolated by gradient centrifugation using Ficoll and cryopreserved. On the day of ABC differentiation, pan-B 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. B cells were then inoculated into 96-well plates containing 200 μl 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 replaced daily by supplementing complete medium and stimulation mixture. The induction of ABC was confirmed by FACS analysis. Antibodies for FACS staining included live / dead dyes, anti-human CD19, CD38, CD27, IgD, CD11c, CD21, and T-bet.

[0265] Cytotoxicity assay

[0266] After differentiation, ABC-enriched B cells were co-cultured with C-CAR168 or non-transduced (NT) T cells at the indicated E:T ratios. After 24 hours, cells were stained with LIVE / DEAD Fixable Aqua Dead Cell Stain (Invitrogen) to determine their viability, and anti-CD19 and anti-CD3 antibodies were used to distinguish B cells from T cells. Cytotoxicity was determined by live CD19 + The percentage of live CD19 cells in the C-CAR168 co-culture was determined by the depletion of B cells. The cytolysis of B cells was calculated by the following formula: Lysis percentage (%) = (1-(live CD19 cells in the C-CAR168 co-culture) + Live CD19 in cell fraction / UT co-cultures + Cell fraction)) × 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.

[0267] Example 5 Inhibitory Effect of Anti-CD20 / BCMACAR-T Cells on Mouse Tumor Cells

[0268] C-CAR168 effectively inhibits the growth of CD20 single-positive and BCMA single-positive tumor cells in tumor-bearing mice

[0269] The in vivo cytotoxic effect of anti-CD20 / BCMA CART-T cells on CD20 or BCMA single-positive cells was evaluated by using a mouse subcutaneous tumor model established using tumor cell lines expressing CD20 (A549-CD20) or BCMA (MM.1S).

[0270] Female B-NDG mice aged 6-8 weeks were subcutaneously inoculated with A549-CD20 (CD20+) or MM.1S (BCMA+) cells. When the average tumor volume reached 100 mm 3 When 3~5×10 6 C-CAR168 CAR-T cells were administered via the tail vein at a dose of 100 CAR-T cells / mouse. Tumor volume in mice treated with C-CAR168 CAR-T cells continued to decrease during the experiment. 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.

[0271] Specific method: Female B-NDG (NOD.Cg-Prkdc scid Il2rg tm1Vst / Vst) mice were subcutaneously inoculated with 5×10 6 A549-CD20 cells / animal. When the average tumor volume reached approximately 100 mm 3 At the time of the experiment, 20 animals were randomly divided into two groups (vehicle control group and C-CAR168 group), with 10 animals in each group. A single dose of vehicle control or C-CAR168 (3×10 6 After administration, the mean tumor volume of the vehicle control group continued to increase, reaching 494.16±31.5 mm on day 42. 3 The average tumor weight was 0.254 ± 0.025 g. The average tumor volume of the C-CAR168 group began to decrease from day 10. By day 42, the average tumor volume was 10.02 ± 7.04 mm 3 ( Figure 9A , left panel), and the tumor weight was 0.013±0.01 g, which was significantly different from that of the vehicle control group (P<0.001) ( Figure 9A (right panel). The tumor growth inhibition rate calculated based on tumor weight was 94.88%. The results showed that C-CAR168 can significantly inhibit the growth of CD20-positive target cells in vivo.

[0272] 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, 20 female B-NDG (NOD.CB17-Prkdc scid Il2rg tm1 / Bcgen) mice were subcutaneously inoculated with 5×10 6 MM.1S cells / animal. When the average tumor volume reached about 100mm 3 At the time of the experiment, 15 animals were randomly divided into three groups (one vehicle control group and two C-CAR168 groups), with 5 animals in each group. Each mouse was given once by tail vein injection. For C-CAR168, the dose was 5×10 6 After administration, the average tumor volume of the vehicle control group continued to increase, reaching 2220.86±117.35mm on day 28. 3 The tumor weight was 2.409±0.216g. The average tumor volume of C-CAR168-1 and C-CAR168-2 groups decreased from day 10 ( Figure 9B , left panel). By day 28, the average tumor volume was 109.2 ± 88.92 mm 3 and 9.07±5.58mm 3 , and the tumor weights were 0.041±0.034g and 0.003±0.002g ( Figure 9B , right), which was 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 a single intravenous administration of 5×10 6 The C-CAR168 CAR-T cells / mouse were well tolerated in B-NDG tumor-bearing mice, and C-CAR168 significantly inhibited the growth of BCMA-positive target cells in vivo.

[0273] C-CAR168 effectively inhibits the growth of CD20 and BCMA double-positive tumor cells in tumor-bearing mice

[0274] To evaluate the anti-tumor effect of C-CAR168 in vivo, 65 female B-NDG (NOD.CB17-Prkdc scid Il2rg tm1 / Bcgen) mice were subcutaneously inoculated with 1×10 6 K562-CD20-BCMA cells / animal. When the average tumor volume reached approximately 100 mm 3 At the same time, 50 animals were selected and randomly divided into 5 groups: vehicle control group, T cell control group, C-CAR168 low-dose group (1×10 6CAR-T cells / mouse), medium-dose group (5×10 6 CAR-T cells / mouse) and high-dose group (10×10 6 The T cell control group was injected with untransduced T cells from the same donor as C-CAR168, and the dose was consistent with the total T cell number in the C-CAR168 high-dose group. Each mouse was dosed once via tail vein injection.

[0275] During the experiment, the mean tumor volumes of animals in the vehicle control group and the T cell control group continued to increase, and on day 17, the mean tumor volumes were 2628.78 ± 117.32 mm, respectively. 3 and 2536.23±97.80mm 3 The tumor volume of the C-CAR168 low-dose group continued to increase, although after 10 days of administration, the tumor volume was significantly lower than that of the vehicle control group and the T cell control group. The average tumor volume of the C-CAR168 medium-dose group and the high-dose group began to decline on the 6th day. The tumors in the C-CAR168 low-dose, medium-dose and high-dose groups showed a dose-dependent decrease. On the 17th day, the tumor growth inhibition rates were 55.47%, 97.75% and 98.01%, respectively. On the 28th day, no tumor tissue was observed in the C-CAR168 medium-dose group and the high-dose group ( Figure 9E ). Figure 9F The survival curves of each group during the experimental period are shown. Although all animals in the vehicle control group and the T cell control group died around day 17, all mice in the C-CAR168 medium-dose group and the high-dose group survived.

[0276] In summary, a single intravenous administration of 1×10 6 , 5×10 6 or 10×10 6 C-CAR168 CAR-T cells / mouse were 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.

[0277] Example 6 Antigen Specificity of Anti-CD20 / BCMACAR

[0278] In membrane protein arrays, full-length cDNA sequences of human membrane proteins are genetically engineered into expression vectors, transiently transfected into HEK293T cells, and arranged into arrays using microfluidics or chip printing techniques. It is a high-throughput screening technique used to study the interaction between test substances and membrane proteins.

[0279] To examine the affinity and specificity of anti-CD20 / BCMACAR, we used membrane protein array analysis to assess the risk of off-target binding between the antigen-binding domain of C-CAR168 and 5220 human cell membrane proteins.

[0280] The chimeric rabbit monoclonal antibody C-CAR168 scFv-RabFc was generated by linking anti-CD20 scFv (e.g., derived from ofatumumab mAb) and anti-BCMA scFv (e.g., derived from BCMA-20 mAb) to the rabbit IgG Fc region in frame. The chimeric antibody was added to an array of HEK293T cells transiently transfected with 5220 membrane protein at a concentration of 20 μg / mL. Flow cytometry results showed that C-CAR168 scFv-RabFc tightly bound to human CD20 and BCMA ( Figure 8A The average fluorescence intensity of C-CAR168 scFv-RabFc binding to CD20 and BCMA in flow cytometry was approximately 60 times and 110 times that of the negative control group, respectively ( Figure 8B In addition to CD20 and BCMA, C-CAR168 scFv-RabFc also showed specific binding to FCGR1A ( Figure 8B ), and the average fluorescence intensity was 2.5 times that of the negative control group. This is mainly due to the binding between FCGR1A and the rabbit-derived Fc of the recombinant protein, so there is no relevant risk in clinical application. C-CAR168 scFv-RabFc binds weakly to ITGB2-ITGAM and ITGB2-ITGAL heterodimers, and the average fluorescence intensity is 2 to 3 times that of the negative control group. For other proteins found in the preliminary screening (MPZ, F11R, CLEC2B, and MC2R), the average fluorescence intensity of binding to C-CAR168 scFv-RabFc does not change with concentration. At concentrations of 20 μg / mL and 5 μg / mL, it did not exceed 2 times that of the negative control group, so the possibility of specific binding of these proteins to C-CAR168 scFv is low or extremely small.

[0281] To test whether ITGB2-ITGAM and ITGB2-ITGAL heterodimers expressed on the cell membrane can be recognized by C-CAR168 CAR-T cells to activate downstream events, C-CAR168 was co-cultured with 293T cells transfected with ITGB2-ITGAM or ITGB2-ITGAL. CD137 expression on C-CAR168 CAR-T cells and levels of IFN-γ, TNF-α, IL-2, and other cytokines in the cell culture supernatant were measured. 293T cells transfected with an empty vector served as a negative control, and 293T cells transfected with CD20 and BCMA served as positive controls.

[0282] 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 the upregulation of CD137 expression on the cell surface. The experiment found that after three batches of C-CAR168 cells were co-cultured with cells expressing CD20 and BCMA, the proportion of 4-1BB-positive cells increased compared with non-transduced T cells ("NT"). After co-culture with those cells expressing ITGB2-ITGAM and ITGB2-ITGAL, the proportion of 4-1BB-positive cells was not significantly different from that of the non-transduced T cell group ("NT") ( Figure 8D , left panel), indicating that C-CAR168 does not specifically bind to ITGB2-ITGAM or ITGB2-ITGAL in vitro.

[0283] Cytokines in the cell culture supernatant were measured, and the results showed that C-CAR168 CAR-T cells secreted high levels of IFN-γ when co-cultured with cells expressing CD20 or BCMA. When co-cultured with cells expressing ITGB2-ITGAM or ITGB2-ITGAL, the IFN-γ concentration in the supernatant was not significantly increased compared with untransduced T cells ( Figure 8D , right). The results further showed that C-CAR168 nonspecifically recognized ITGB2-ITGAM and ITGB2-ITGAL in vitro.

[0284] In summary, membrane protein array and in vitro co-culture results showed that the antigen binding domain of C-CAR168 tightly binds to human CD20 and BCMA, with no other nonspecific binding sites. Membrane protein array studies found that, except for weak binding to two heterogeneous complexes, C-CAR168 had no cross-reactivity with the membrane proteome.

[0285] Example 7C - CAR168 exhibits robust efficacy against autologous B cells from SLE patients

[0286] To investigate CAR-T therapy for the treatment of autoimmune diseases such as SLE, we are evaluating the efficiency of CAR-T cells in depleting autoreactive B cells. We will also investigate the efficacy of CAR-T cells in inducing remission and survival in lupus models.

[0287] The efficiency of C-CAR168 in depleting pan-B cells from lupus patients in vitro

[0288] Peripheral blood samples (10-15 mL) were collected from eight patients with SLE. The patients varied in activity and autoantibody profiles, organ damage (preferably lupus nephritis), and prior treatment, reflecting the heterogeneity of lupus patients. Patients who had recently received B-cell depleting antibodies were excluded.

[0289] For each sample, part of the blood was used to separate the T cells for CAR-T production, and the remaining blood was used to separate pan-B cells as targets for cell lysis 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 successfully transduced and well amplified for functional assays ( Figure 10A ).

[0290] C-CAR168 CAR-T cells or non-transduced (NT) T cells generated from 8 patient samples were co-cultured with target cell lines expressing CD20 and / or BCMA. K562 is negative for both CD20 and BCMA; MM.1S is a BCMA-positive multiple myeloma cell line. After 24 hours, the co-culture supernatant was collected for ELISA (enzyme-linked immunosorbent assay) to assess IFN-γ levels. Figure 10B Results from one representative sample of eight patients are shown. Thus, C-CAR168 cells generated from SLE patient samples exhibited robust activity against target cells expressing CD20 and BCMA.

[0291] The pan B cells separated from 8 patient samples were co-cultured with autologous C-CAR168CAR-T cells or untransduced (NT) T cells with the indicated E:T (effector and target) ratio. After 24 hours, the co-culture supernatant was collected and ELISA was performed to assess IFN-γ levels. Cytotoxicity was determined by fluorescence activated cell sorting (FACS) and calculation of the depletion percentage of live CD19+ pan B cells. The cytolysis of B cells was calculated by the following formula: lysis percentage (%)=(1-(live CD19+ cell fraction / UT co-culture in C-CAR168 co-culture))×100. Figure 10C and 10D Results from a representative sample of 8 patients are shown. Pan B cells isolated from 8 SLE patient samples were recognized and lysed by autologous C-CAR168 cells. The results confirm the efficiency of C-CAR168 CAR-T cells in depleting peripheral B cells in lupus patients in vitro.

[0292] Efficiency of CAR-T in clearing ABC in lupus patients in vitro

[0293] We will study the efficiency of CAR-T in eliminating ABC (basic subset of pathogenic B cells) from lupus patients in vitro.

[0294] Blood samples or PBMCs from lupus patients will be processed for ABC differentiation and CAR-T generation and functional analysis.

[0295] The study will confirm the efficiency of CAR-T cells in depleting ABCs in lupus patients in vitro.

[0296] CAR-T B cell depletion efficiency and in vivo therapeutic efficacy

[0297] The efficiency of CAR-T in depleting B cells and its therapeutic efficacy will be evaluated in vivo using a humanized SLE mouse model. + Stem cell humanized mice. Two or more mice will be sacrificed and their spleens will be harvested using sterile techniques. T cells will then be isolated from the spleen for CAR-T production. The remaining mice will be used to induce the onset of lupus disease, and after successful induction, the mice will be divided into several groups to receive CAR-T or control treatment (e.g., untransduced T cells). Blood samples will be obtained from the mice regularly to monitor the persistence of CAR-T cells and the efficiency of B cell depletion (including ABC) by FACS. Serum samples will be used to measure the titers of various autoantibodies. Urine samples will also be routinely collected to measure proteinuria levels. At the end of the study, or if the animal dies early (presumably in the control group), tissues will be collected for histological examination, for example, to examine the deposition of immune complexes in the kidneys and the severity of nephritis. The presence of B cells or plasma cells in the diseased tissue will also be examined. Survival curves will be generated to compare the effects of CAR-T and control treatments.

[0298] References

[0299] Bhoj,VGet al.Persistence of long-lived plasma cells and humoralimmunity in individuals responding to CD19-directed CAR T-cell therapy.Blood128,360-370(2016).

[0300] Taubmann et al.Long term safety and efficacy of CAR-T cell treatmentin refractory systemic lupus erythematosus-data from the first sevenpatients.Annals of the Rheumatic Diseases,OP0141,page93(2023).

[0301] Qin,C.,Tian,DS.,Zhou,LQ.et al.Anti-BCMA CART-cell therapy CT103A inrelapsed or refractory AQP4-IgG seropositive neuromyelitis optica spectrumdisorders:phase 1trial interim results.Sig Transduct Target Ther 8,5(2023).

[0302] Tai et al.Role of B-cell-activating factor in adhesion and growth ofhuman multiple myeloma cells in the bone marrow microenvironment.Cancerresearch.2006;66(13):6675-82.

[0303] Pavlasova et al.(2020).The regulation and function of CD20:an″enigma"of B-cell biology and targeted therapy.Haematologica,105(6),1494-1506.

[0304] Krumbholz et al.B cells and antibodies in multiple sclerosispathogenesis and therapy[J].Nature reviews Neurology,2012,8(11):613-23.

[0305] 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.

[0306] Quctal. 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.

[0307] The structures of anti-CD20 / BCMA CAR, TOB1-4, and TOBL1-4 are shown in Table 1.

[0308] Table 1

[0309]

[0310]

[0311] Sequence:

[0312] TN-OF-B20-L1 (TOBL1 or C-CAR168)

[0313] CD8aSP nucleic acid sequence (63 nt)

[0314] atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg(SEQ ID NO:1)

[0315] CD8a SP amino acid sequence:

[0316] MALPVTALLLPLALLLHAARP(SEQ ID NO:2)

[0317] OFV L Nucleic acid sequence (321nt)

[0318] GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO:3)

[0319] OF V L Amino acid sequence:

[0320] EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQ APRLLIY DASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC Q QRSNWPIT FGQGTRLEIK(SEQ ID NO:4)

[0321] Linker-1 nucleic acid sequence (54 nt)

[0322] GGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTG GGGGCGGCAGCAGC(SEQ ID NO:5)

[0323] Linker-1 amino acid sequence:

[0324] GSTSGGGSGGGSGGGGSS(SEQ ID NO:6)

[0325] OFV H Nucleic acid sequence (366nt)

[0326] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ IDNO:7)

[0327] OF V H Amino acid sequence:

[0328] EVQLVESGGGLVQPGRSLRLSCAASGFTF NDYAMH WVRQA PGKGLEWVS TISWNSGSIGYADSVKG RFTISRDNAKKSLYLQMN SLRAEDTALYYCAK DIQYGNYYYGMDV WGQGTTVTVSS(SEQ ID NO:8)

[0329] Linker-2 nucleic acid sequence (15nt)

[0330] GGAGGTGGTGGATCC(SEQ ID NO:9)

[0331] Linker-2 amino acid sequence:

[0332] GGGGS(SEQ ID NO:10)

[0333] B20V L Nucleic acid sequence (321nt)

[0334] Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag(SEQ ID NO:11)

[0335] B20 V L Amino acid sequence:

[0336] DIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKP GKAPKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFA TYYCMGQTISSYTFGQGTKLEIK(SEQ ID NO:12)

[0337] Linker-3 nucleic acid sequence (45nt)

[0338] Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(SEQ ID NO:13)

[0339] Linker-3 amino acid sequence:

[0340] GGGGSGGGGSGGGGS(SEQ ID NO:14)

[0341] B20V H Nucleic acid sequence (363nt)

[0342] Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc(SEQ IDNO:15)

[0343] B20 V H Amino acid sequence:

[0344] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGADHAIYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVSS(SEQ ID NO:16)

[0345] CD8a hinge nucleic acid sequence (165nt)

[0346] Ttcgtgccggtcttcctgccagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat(SEQ ID NO:17)

[0347] CD8a hinge amino acid sequence:

[0348] FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO:18)

[0349] CD8aTM nucleic acid sequence (72 nt)

[0350] Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc(SEQ ID NO:19)

[0351] CD8a TM amino acid sequence:

[0352] IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:20)

[0353] 4-1BB nucleic acid sequence (126 nt)

[0354] Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg(SEQ ID NO:21)

[0355] 4-1BB amino acid sequence:

[0356] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:22)

[0357] CD3z nucleic acid sequence (336 nt)

[0358] Agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa(SEQ ID NO:23)

[0359] CD3z amino acid sequence:

[0360] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:24)

[0361] TOBL1 nucleic acid sequence (2247nt)

[0362]

[0363] TOBL1 Amino acid sequence:

[0364] MALPVTALLLPLALLLHAARPEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCMGQTISSYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGADHAIYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:26)

[0365] TN-OF-B20-L2(TOBL2)

[0366] CD8aSP nucleic acid sequence (63nt)

[0367] Atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg(SEQID NO:27)

[0368] OF V H Nucleic acid sequence

[0369] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ IDNO:28)

[0370] Linker-1 nucleic acid sequence

[0371] GGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTG GGGGCGGCAGCAGC(SEQ ID NO:29)

[0372] OF V L Nucleic acid sequence

[0373] GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO:30)

[0374] Linker-2 nucleic acid sequence

[0375] GGAGGTGGTGGATCC(SEQ ID NO:31)

[0376] BCMA-20 scFv (729nt):

[0377] B20V L Nucleic acid sequence (321nt)

[0378] gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag(SEQ ID NO:32)

[0379] Linker-3 nucleic acid sequence (45nt)

[0380] ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(SEQ ID NO:33)

[0381] B20V H Nucleic acid sequence (363nt)

[0382] gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc(SEQ IDNO:34)

[0383] CD8a hinge nucleic acid sequence (165nt)

[0384] Ttcgtgccggtcttcctgccagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggg gcgcagtgcacacgagggggctggacttcgcctgtgat(SEQ ID NO:35)

[0385] CD8aTM nucleic acid sequence (72 nt)

[0386] Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc(SEQ ID NO:36)

[0387] 4-1BB nucleic acid sequence (126 nt)

[0388] Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactac tcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg(SEQ ID NO:37)

[0389] CD3z nucleic acid sequence (336 nt)

[0390] Agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa(SEQ ID NO:38)

[0391] TOBL2 nucleic acid sequence (2247 nt)

[0392]

[0393] TOBL2 amino acid sequence:

[0394] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGSTSGGGSGGGSGGGGSSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCMGQTISSYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGADHAIYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQID NO:40)

[0395] TN-OF-B20-L3(TOBL3)

[0396] CD8aSP (63 nt) nucleic acid sequence

[0397] Atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg(SEQID NO:41)

[0398] OF V H Nucleic acid sequence

[0399] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ IDNO:42)

[0400] Linker-1 nucleic acid sequence

[0401] GGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTG GGGGCGGCAGCAGC(SEQ ID NO:43)

[0402] OF V L Nucleic acid sequence

[0403] GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO:44)

[0404] Linker-2 nucleic acid sequence

[0405] GGAGGTGGTGGATCC(SEQ ID NO:45)

[0406] BCMA-20 scFv (729nt):

[0407] B20V H Nucleic acid sequence (363nt)

[0408] Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc(SEQ IDNO:46)

[0409] Linker-3 nucleic acid sequence (45nt)

[0410] Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(SEQ ID NO:47)

[0411] B20V L Nucleic acid sequence (321nt)

[0412] Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag(SEQ ID NO:48)

[0413] CD8a hinge nucleic acid sequence (165nt)

[0414] Ttcgtgccggtcttcctgccagcgaagcccaccacgacgccagcgccgcgaccaccaacacc ggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggg gcgcagtgcacacgagggggctggacttcgcctgtgat(SEQ ID NO:49)

[0415] CD8aTM nucleic acid sequence (72 nt)

[0416] Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc(SEQ ID NO:50)

[0417] 4-1BB nucleic acid sequence (126 nt)

[0418] Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg(SEQ ID NO:51)

[0419] CD3z nucleic acid sequence (336 nt)

[0420] Agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa(SEQ ID NO:52)

[0421] TOBL3 nucleic acid sequence

[0422]

[0423] TOBL3 amino acid sequence:

[0424] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGSTSGGGSGGGSGGGGSSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGADHAIYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCMGQTISSYTFGQGTKLEIKFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQID NO:54)

[0425] TN-OF-B20-L4(TOBL4)

[0426] CD8aSP nucleic acid sequence (63 nt)

[0427] Atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg(SEQID NO:55)

[0428] OF V L Nucleic acid sequence

[0429] GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO:56)

[0430] Linker-1 nucleic acid sequence

[0431] GGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTG GGGGCGGCAGCAGC(SEQ ID NO:57)

[0432] OF V H Nucleic acid sequence

[0433] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ IDNO:58)

[0434] Linker-2 nucleic acid sequence

[0435] GGAGGTGGTGGATCC(SEQ ID NO:59)

[0436] BCMA-20 scFv (729nt):

[0437] B20V H Nucleic acid sequence (363nt)

[0438] Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc(SEQ IDNO:60)

[0439] Linker-3 nucleic acid sequence (45nt)

[0440] Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(SEQ ID NO:61)

[0441] B20V L Nucleic acid sequence (321nt)

[0442] Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag(SEQ ID NO:62)

[0443] CD8a hinge nucleic acid sequence (165nt)

[0444] Ttcgtgccggtcttcctgccagcgaagcccaccacgacgccagcgccgcgaccaccaacacc ggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggg gcgcagtgcacacgagggggctggacttcgcctgtgat(SEQ ID NO:63)

[0445] CD8aTM nucleic acid sequence (72 nt)

[0446] Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc(SEQ ID NO:64)

[0447] 4-1BB nucleic acid sequence (126 nt)

[0448] Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactac tcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg(SEQ ID NO:65)

[0449] CD3z nucleic acid sequence (336 nt)

[0450] Agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa(SEQ ID NO:66)

[0451] TOBL4 nucleic acid sequence (2247 nt)

[0452]

[0453] TOBL4 amino acid sequence:

[0454] MALPVTALLLPLALLLHAARPEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGADHAIYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCMGQTISSYTFGQGTKLEIKFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQID NO:68)

[0455] TN-OF-B20-1(TOB1)

[0456] CD8a SP nucleic acid sequence

[0457] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTT GCTGCTCCACGCCGCCAGGCCG(SEQID NO:69)

[0458] OF V L Nucleic acid sequence

[0459] GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO:70)

[0460] Linker-1 nucleic acid sequence

[0461] GGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTG GGGGCGGCAGCAGC(SEQ ID NO:71)

[0462] OF V H Nucleic acid sequence

[0463] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ IDNO:72)

[0464] Linker-2 nucleic acid sequence

[0465] GGAGGTGGTGGATCC(SEQ ID NO:73)

[0466] B20 V H Nucleic acid sequence

[0467] Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc(SEQ IDNO:74)

[0468] Linker-3 nucleic acid sequence

[0469] ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(SEQ ID NO:75)

[0470] B20 V L Nucleic acid sequence

[0471] Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag(SEQ ID NO:76)

[0472] IgG4 hinge nucleic acid sequence (36 nt)

[0473] GAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCT(SEQ ID NO:77)

[0474] IgG4 hinge amino acid sequence:

[0475] ESKYGPPCPPCP(SEQ ID NO:78)

[0476] CD28TM nucleic acid sequence (84 nt)

[0477] ATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTG(SEQ ID NO:79)

[0478] CD28™ amino acid sequence:

[0479] MFWVLVVVGGVLACYSLLVTVAFIIFWV(SEQ ID NO:80)

[0480] 4-1BB nucleic acid sequence

[0481] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO:81)

[0482] CD3z nucleic acid sequence

[0483] CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG(SEQ ID NO:82)

[0484] TOB1 nucleic acid sequence (2130 nt)

[0485]

[0486] TOB1 amino acid sequence:

[0487] MALPVTALLLPLALLLHAARPEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGADHAIYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCMGQTISSYTFGQGTKLEIKESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:84)

[0488] TN-OF-B20-2(TOB2)

[0489] CD8a SP nucleic acid sequence

[0490] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG(SEQID NO:85)

[0491] OF V L Nucleic acid sequence

[0492] GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO:86)

[0493] Linker-1 nucleic acid sequence

[0494] GGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGC(SEQ ID NO:87)

[0495] OF V H Nucleic acid sequence

[0496] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ IDNO:88)

[0497] Linker-2 nucleic acid sequence

[0498] GGAGGTGGTGGATCC(SEQ ID NO:89)

[0499] B20 V L Nucleic acid sequence

[0500] Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag(SEQ ID NO:90)

[0501] Linker-3 nucleic acid sequence

[0502] ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(SEQ ID NO:91)

[0503] B20 V H Nucleic acid sequence

[0504] Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc(SEQ IDNO:92)

[0505] Hinge nucleic acid sequence

[0506] GAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCT(SEQ ID NO:93)

[0507] CD28 TM nucleic acid sequence

[0508] ATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTG(SEQ ID NO:94)

[0509] 4-1BB nucleic acid sequence

[0510] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO:95)

[0511] CD3z nucleic acid sequence

[0512] CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG(SEQ ID NO:96)

[0513] TOB2 nucleic acid sequence (2130 nt)

[0514]

[0515] TOB2 amino acid sequence:

[0516] MALPVTALLLPLALLLHAARPEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCMGQTISSYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGADHAIYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVSSESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:98)

[0517] TN-OF-B20-3(TOB3)

[0518] CD8a SP nucleic acid sequence

[0519] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG(SEQID NO:99)

[0520] OF V H Nucleic acid sequence

[0521] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ IDNO:100)

[0522] Linker-1 nucleic acid sequence

[0523] GGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGC(SEQ ID NO:101)

[0524] OF V L Nucleic acid sequence

[0525] GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO:102)

[0526] Linker-2 nucleic acid sequence

[0527] GGAGGTGGTGGATCC(SEQ ID NO:103)

[0528] B20 V L Nucleic acid sequence

[0529] Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag(SEQ ID NO:104)

[0530] Linker-3 nucleic acid sequence

[0531] ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(SEQ ID NO:105)

[0532] B20 V H Nucleic acid sequence

[0533] Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc(SEQ IDNO:106)

[0534] Hinge nucleic acid sequence

[0535] GAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCT(SEQ ID NO:107)

[0536] CD28 TM nucleic acid sequence

[0537] ATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTG(SEQ ID NO:108)

[0538] 4-1BB nucleic acid sequence

[0539] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO:109)

[0540] CD3z nucleic acid sequence

[0541] CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG(SEQ ID NO:110)

[0542] TOB3 nucleic acid sequence (2130 nt)

[0543]

[0544] TOB3 amino acid sequence:

[0545] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGSTSGGGSGGGSGGGGSSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCMGQTISSYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGADHAIYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVSSESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:112)

[0546] TN-OF-B20-4(TOB4)

[0547] CD8a SP nucleic acid sequence

[0548] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG(SEQID NO:113)

[0549] OF V H Nucleic acid sequence

[0550] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ IDNO:114)

[0551] Linker-1 nucleic acid sequence

[0552] GGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGC(SEQ ID NO:115)

[0553] OF V L Nucleic acid sequence

[0554] GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA(SEQ ID NO:116)

[0555] Linker-2 nucleic acid sequence

[0556] GGAGGTGGTGGATCC(SEQ ID NO:117)

[0557] B20 V H Nucleic acid sequence

[0558] Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc(SEQ IDNO:118)

[0559] Linker-3 nucleic acid sequence

[0560] ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(SEQ ID NO:119)

[0561] B20 V L Nucleic acid sequence

[0562] Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag(SEQ ID NO:120)

[0563] Hinge nucleic acid sequence

[0564] GAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCT(SEQ ID NO:121)

[0565] CD28 TM nucleic acid sequence

[0566] ATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTG(SEQ ID NO:122)

[0567] 4-1BB nucleic acid sequence

[0568] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO:123)

[0569] CD3z nucleic acid sequence

[0570] CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG(SEQ ID NO:124)

[0571] TOB4 nucleic acid sequence (2130 nt)

[0572]

[0573] TOB4 amino acid sequence:

[0574] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGSTSGGGSGGGSGGGGSSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGADHAIYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCMGQTISSYTFGQGTKLEIKESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:126)

[0575] OF-V H -CDR1: NDYAMH(SEQ ID NO:127)

[0576] OF-V H -CDR2: TISWNSGSIGYADSVKG(SEQ ID NO:128)

[0577] OF-V H -CDR3: DIQYGNYYYGMDV(SEQ ID NO:129)

[0578] OF-V L -CDR1: RASQSVSSYLA (SEQ ID NO: 130)

[0579] OF-V L -CDR2: DASNRAT (SEQ ID NO: 131)

[0580] OF-V L -CDR3: QQRSNWPIT (SEQ ID NO: 132)

[0581] BCMA-20 V L

[0582]

[0583] BCMA-20 V H

[0584]

[0585]

[0586] The scope of the present invention is not limited by what is specifically shown and described above. Those skilled in the art will recognize that there are suitable alternatives to the examples of materials, configurations, constructions and sizes depicted. Many references, including patents and various publications, are cited and discussed in the description of the present invention. The citation and discussion of such references are merely for the purpose of illustrating the description of the present invention and do not admit that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety. Without departing from the spirit and scope of the present invention, those of ordinary skill in the art may conceive of variations, modifications and other embodiments of the contents described herein. Although certain embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that changes and modifications may be made without departing from the spirit and scope of the present invention. The contents set forth in the foregoing description and the accompanying drawings are provided by way of illustration only and are not intended to be limiting.

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 heavy chain variable region (V H 1), where V L 1 comprises three complementarity determining regions (CDRs), CDR1, CDR2, and CDR3, which have amino acid sequences that are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively, and wherein V H 1 comprises three CDRs, CDR1, CDR2, and CDR3, which have 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), where V L 2 comprises three complementarity determining regions (CDRs), CDR1, CDR2, and CDR3, which have amino acid sequences approximately 80% to approximately 100% identical to the amino acid sequences shown in SEQ ID NO: 134, SEQ ID NO: 136, and SEQ ID NO: 138, respectively, and wherein V H 2 comprises three CDRs, CDR1, CDR2, and CDR3, which have amino acid sequences that are approximately 80% to approximately 100% identical to the amino acid sequences shown in SEQ ID NO: 141, SEQ ID NO: 143, and SEQ ID NO: 145, respectively.

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

3. The bispecific CAR of claim 1 or 2, wherein V L 1 is located in V H 1's N terminus.

4. The bispecific CAR of claim 1 or 2, wherein V L 2 is located at V H 2's N terminus.

5. The bispecific CAR of claim 1 or 2, wherein V H 1 is located in V L 1's N terminus.

6. The bispecific CAR of claim 1 or 2, wherein V H 2 is located at V L 2's N terminus.

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

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

9. The bispecific CAR of 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 wherein the anti-BCMA antigen binding region is a scFv that specifically binds to BCMA.

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

11. The bispecific CAR of claim 10, wherein the costimulatory region comprises 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 the cytoplasmic signaling domain comprises the cytoplasmic signaling domain zeta of CD3.

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

14. The bispecific CAR of any one of claims 10-13, 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 of any one of claims 1-14, comprising an amino acid sequence that is about 80% to about 100% identical to the amino acid sequence shown 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 of any one of claims 1-15.

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

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

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

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

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

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

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

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

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

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

29. 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 one of claims 21-29, wherein the immune cells are allogeneic or autologous.

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