Bispecific chimeric antigen receptors targeting cd20 and bcma

Abstract

The present disclosure provides bispecific chimeric antigen receptors targeting CD20 and BCMA. The CARs can comprise an scFv targeting CD20 and an scFv targeting BCMA, a hinge region, a transmembrane domain, a costimulatory region, and a cytoplasmic signaling domain. The chimeric antigen receptors can be used to treat an autoimmune disorder or a cancer.

Description

[0001] Cross-reference to related applications

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

[0003] Incorporate into the sequence list by reference

[0004] The sequence list was submitted as an XML file “11299_011840-WO0_SL.xml”, created on March 26, 2024, and is 168,342 bytes in size. It is incorporated into this paper in its entirety by reference. Technical Field

[0005] This disclosure relates to the field of immunotherapy, and more specifically to a bispecific chimeric antigen receptor (CAR) targeting CD20 and BCMA. Background Technology

[0006] Autoimmune diseases are disorders caused by the immune system's response to the body itself, resulting in damage to its own tissues. These are generally divided into two main 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 mainly involves corticosteroids and immunosuppressants, which greatly affect patients' quality of life and present significant unmet clinical needs (Wang et al., Human autoimmune diseases: a comprehensive update, J. Intern. Med. 2015, 278(4):369-95).

[0007] The etiology of autoimmune diseases remains unclear. In patients, abnormal activation of humoral immunity occurs, leading to the production of large amounts of antibodies against self-antigens. These complexes combine to form pathogenic immune complexes, which then deposit locally and induce an inflammatory response. B cells play a crucial role in the pathogenesis of autoimmune diseases, promoting their development through various mechanisms, including 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 innate immune system cells to produce type I interferon and other pro-inflammatory cytokines, leading to organ damage. Therefore, depletion or removal of lymphocytes has emerged as a potential therapeutic strategy.

[0008] SLE is a prototypical autoimmune disease known to be associated with polyclonal B cell hyperresponsiveness (Dorner et al., Mechanisms of B cell autoimmunity in SLE, Arthritis Res. Ther. 13, 243 (2011)). Therefore, one of the immunological hallmarks of SLE is the production of antinuclear antibodies (ANA), which can mediate SLE pathogenesis by binding to corresponding self-antigens, leading to 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 mainly comes in two types: anti-DNA antibodies and antibodies that recognize RNA-binding proteins (RBPs) (Pisetsky et al., New insights into the role of antinuclear antibodies in systemic lupus erythematosus, Nat. Rev. Rheumatol. 16, 565-579 (2020)). In SLE patients, autoantibodies originate not only from B cells but also from a class of plasma cells called long-lived plasma cells (LLPCs). Anti-DNA antibodies are produced by immature B cells that have transformed into memory B cells and plasmablasts, which maintain high levels of CD19 and CD20 expression on their cell surface. Anti-RBP antibodies are produced by LLPCs, which may lose CD19 and CD20 surface expression but remain positive for B cell maturation antigen (BCMA), a cell surface protein expressed on all mature plasma cells (Dogan et al., B-cell maturation antigen expression across hematologic cancers: a systematic literature review. Blood Cancer J. 10, 73 (2020); Morgan et al., Unraveling B cell trajectories at single cell resolution, Trends Immunol. 43, 210-229 (2022)). Recent studies have shown that CD11c... hi T-bet +A subset of B cells expands in human SLE and acts as precursors to plasma cells that produce autoantibodies. This B cell subset exhibits high expression of CD19 and CD20 and corresponds to autoreactive, age-related mouse B cells (autoreactive B cells or ABC; this term can be used to refer to 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 in Systemic 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)). Besides producing autoantibodies, B cells participate in the pathogenesis of SLE and other autoimmune diseases by secreting cytokines and acting as antigen-presenting cells. Therefore, depleting the B cells of SLE patients could be an effective treatment for this life-threatening disease.

[0009] B cell depletion can be achieved by administering monoclonal antibodies targeting B cell surface markers. While the anti-CD20 antibody rituximab was successful in early open-label trials in SLE, it failed to meet its primary endpoint in two randomized controlled trials (Lee et al., B cell depletion therapies in autoimmune disease: advances and mechanistic insights, Nat. Rev. Drug Discov. 20, 179-199 (2021)). Other antibodies targeting CD19 (obexelimab) have also been tested in SLE. Although patients receiving obexelimab maintained disease inactivity levels after steroid discontinuation in preliminary studies, a phase II clinical trial failed to meet its primary endpoint (Lee et al., B cell depletion therapies in autoimmune disease: advances and mechanistic insights, Nat. Rev. Drug Discov. 20, 179-199 (2021)).

[0010] One promising approach to achieving B-cell exhaustion is adoptive transfer of CAR-T cells. CAR-T cells are genetically engineered T lymphocytes that, in the absence of the major histocompatibility complex (MHC), can recognize specific antigens on target cells, proliferate, and generate cytotoxic immune responses. In a recent study, five patients with refractory SLE received compassionate use of CD19 CAR-T therapy, resulting in deep B-cell exhaustion and drug-free remission, demonstrating that CAR-T cell transfer is feasible, tolerable, and highly effective in SLE (Mackensen et al., Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus, Nat. Med. 28, 2124-2132 (2022)).

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

[0012] This 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 contains three complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3, each having an amino acid sequence that is 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, and wherein V H 1) Contains three CDRs, CDR1, CDR2, and CDR3, each having an amino acid sequence that is 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; 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 contains three complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3, each having an amino acid sequence that is 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, and wherein V H2 contains three CDRs, CDR1, CDR2 and CDR3, which have an amino acid sequence that is 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.

[0013] This 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) anti-BCMA antigen binding region, which includes a light chain variable region (V L 2) and heavy chain variable region (V H 2).

[0014] In one implementation scheme, V L 1 is located in V H The N-terminus of 1. In one implementation, V H 1 is located in V L The N-terminus of 1. In one implementation, V L 2 is located in V H The N-terminus of 2. In one implementation, V H 2 is located in V L The N-terminus of 2.

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

[0016] In some implementations, V L 2 and V H 2 have an amino acid sequence that is about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NO:12 and SEQ ID NO:16.

[0017] 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 an scFv that specifically binds to BCMA.

[0018] A bispecific CAR may further include 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 signal transduction domain.

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

[0020] Transmembrane domains may include transmembrane domains of CD8, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or combinations thereof.

[0021] Co-stimulatory regions may include 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 combinations thereof.

[0022] Cytoplasmic signal transduction domains may include the cytoplasmic signal transduction domains of CD3ζ.

[0023] This disclosure provides a bispecific CAR comprising (or having) 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.

[0024] This disclosure also covers immune cells expressing bispecific CARs. These immune cells can be T cells or natural killer (NK) cells.

[0025] This disclosure provides a nucleic acid encoding a bispecific CAR.

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

[0027] This disclosure provides pharmaceutical compositions comprising a bispecific CAR, immune cells, nucleic acid, or a carrier.

[0028] This disclosure also provides a method for treating an autoimmune disease. The method may include administering immune cells or a pharmaceutical composition to a subject in need.

[0029] Autoimmune diseases can include systemic lupus erythematosus (SLE) (e.g., lupus nephritis), systemic vasculitis, systemic sclerosis, inflammatory myopathies (e.g., polymyositis, dermatomyositis, or inclusion body myositis), systemic scleroderma, multiple sclerosis, myasthenia gravis, myositis autoantibody-driven diseases, or neuromyelitis optica.

[0030] Autoimmune diseases can include polymyositis, dermatomyositis, or inclusion body myositis. Another autoimmune disease is lupus nephritis.

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

[0032] Cancer can be a blood cancer. Cancer can be a B-cell malignancy. Cancer can be Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, and / or multiple myeloma. Cancer can 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 allogeneic or autologous. Attached Figure Description

[0034] Figure 1 The structures of combined chimeric antigen receptors targeting CD20 and BCMA are shown. The CAR structures include 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. Both CAR groups (TOB1-4 and TOBL1-4) contain the V-phase in two scFv sequences. H and V L The four combinations of directions. TOBL1 is also named C-CAR168.

[0035] Figure 2 The expression levels of anti-CD20 and anti-BCMACAR on the surface of T cells were shown.

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

[0037] Figures 4A-4B The expression level of CD137 on the surface of activated CAR-T cells is shown.

[0038] Figures 5A-5B This demonstrates the effect of RTCA assay on CAR-T cells ( Figure 5A TOB1 to TOB4; Figure 5B In vitro cytotoxicity of TOBL1 to TOBL4.

[0039] Figures 6A-6C C-CAR168 exhibited robust potency against CD20+ and BCMA+ cells in vitro. Figure 6A The structures of C-CAR168 (TOBL1 is an anti-CD20 / BCMA CAR), anti-CD20 CAR (C-CAR066), and anti-BCMA CAR (C-CAR088) are shown. Figure 6B The release of IFN-γ was shown after CAR-T cells were co-cultured 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 was demonstrated.

[0040] Figures 7A-7C C-CAR168 exhibits in vitro cytotoxicity against ABC-rich B cells. Figure 7A Production of C-CAR168 CAR-T cells. The figure below shows the CAR positivity rate of C-CAR168 CAR-T cells prepared from peripheral blood of three healthy donors. Figure 7B The distinction between ABC subsets. The figure below shows that after induction of autologous B cell differentiation, the proportion of ABC subsets increased significantly. Figure 7C C-CAR168 induces cell lysis of ABC-rich B cells at different E:T ratios.

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

[0042] Figure 9A-9G In vivo cytotoxicity of C-CAR168 in tumor-bearing mice. Figure 9A C-CAR168 significantly inhibited the growth of A549-CD20 cells in B-NDG tumor-bearing mice. Left panel: Tumor growth curves for each group during the experiment; Right panel: Mean tumor weight of animals in each group on day 42. ***: P < 0.001 compared to the 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 panel: Tumor growth curves for each group during the experiment; Right panel: Mean tumor weight of animals in each group on day 28. ***: P < 0.001 compared to the control group. Figure 9C Images of A549-CD20 tumors in animals from 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 from each group on day 28. Blank boxes indicate no tumor tissue was collected. Figure 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 for each group during the experiment. Figure 9F Survival rate curves for each group during the experimental period. Figure 9G Images of tumors in animals in each group on day 28. " / " indicates animal death. Blank boxes indicate no tumor tissue was collected.

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

[0044] This disclosure provides a chimeric antigen receptor (CAR) targeting CD20 and BCMA. The CAR may comprise a signal peptide, an anti-CD20 scFv, an anti-BCMA scFv, a hinge region, a transmembrane domain, a co-stimulatory region, and a cytoplasmic signaling domain. The CAR of this 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 acts as an important receptor for B cell activating factor (BAFF) and participates in regulating B lymphocyte differentiation and maturation. BCMA is a type III transmembrane protein specifically expressed in B cells, particularly in plasmablasts and differentiated mature plasma cells.

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

[0047] This disclosure provides a bispecific chimeric antigen receptor (CAR). The bispecific CAR may include: (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) anti-BCMA antigen binding region, which includes 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) 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 shown 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) Each of the amino acid sequences having about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% of the amino acid sequences shown in SEQ ID NO:12 and SEQ ID NO:16, respectively.

[0049] This disclosure provides a bispecific chimeric antigen receptor (CAR). The bispecific CAR may include: (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) anti-BCMA antigen binding regions, which include light chain variable regions (V L 2) and heavy chain variable region (V H 2). V L 1 may contain three complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3, which have approximately 80% to 100%, approximately 85% to 100%, approximately 90% to 100%, or approximately 95% to 100% identical amino acid sequences to those shown in SEQ ID NO:130, SEQ ID NO:131, and SEQ ID NO:132, respectively. H 1 may comprise three CDRs, CDR1, CDR2, and CDR3, each having approximately 80% to 100%, approximately 85% to 100%, approximately 90% to 100%, or approximately 95% to 100% of the amino acid sequences shown in SEQ ID NO:127, SEQ ID NO:128, and SEQ ID NO:129, respectively. V L 2 may contain three complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3, which have approximately 80% to 100%, approximately 85% to 100%, approximately 90% to 100%, or approximately 95% to 100% identical amino acid sequences to those shown in SEQ ID NO:134, SEQ ID NO:136, and SEQ ID NO:138, respectively. V H 2 may contain three CDRs, CDR1, CDR2 and CDR3, which have about 80% to about 100%, about 85% to about 100%, about 90% to about 100% or about 95% to about 100% of the amino acid sequence shown in SEQ ID NO:141, SEQ ID NO:143 and SEQ ID NO:145, respectively.

[0050] In some implementations, V L 1 is located in V H The N-terminus of 1. In some implementations, V H 1 is located in V LThe N-terminus of 1. In some implementations, V H 2 is located in V L The N-terminus of 2. In some implementations, V L 2 is located in V H The N-terminus of 2. In one implementation, V L 1 is located in V H The N-terminus of 1; V L 2 is located in V H The N-terminus of 2.

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

[0052] In some implementations, V L 2 and V H 2 have an amino acid sequence that is about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NO:12 and SEQ ID NO:16.

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

[0054] The anti-CD20 antigen-binding region may be a single-stranded variable fragment (scFv) that specifically binds CD20. The anti-BCMA antigen-binding region may be a scFv that specifically binds BCMA. In some embodiments, the scFv that specifically binds CD20 is located at the N-terminus of the scFv that specifically binds BCMA. In some embodiments, the scFv that specifically binds BCMA is located at the N-terminus of the scFv that specifically binds CD20.

[0055] A bispecific CAR may further include 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 signal transduction domain.

[0056] The bispecific CAR of the present invention may include a signal peptide, anti-CD20 scFv, anti-BCMA scFv, hinge region, transmembrane domain, co-stimulatory region, and cytoplasmic signal transduction domain from the N-terminus to the C-terminus.

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

[0058] In one embodiment, the signal peptide comprises about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% of the amino acid sequence shown in SEQ ID NO:2.

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

[0060] In one embodiment, the hinge region is the hinge region of IgG4 (or derived from IgG4). In one embodiment, the hinge region contains 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:78.

[0061] In one embodiment, the hinge region is the hinge region of CD8a (or derived from CD8a). In one embodiment, the hinge region contains 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:18.

[0062] Transmembrane domains may include transmembrane domains derived from CD8, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or combinations thereof (or may be derived from CD8, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or combinations thereof).

[0063] In one embodiment, the transmembrane domain is a transmembrane domain of CD8 (or derived from CD8). In one embodiment, the transmembrane domain comprises about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% of the amino acid sequence shown in SEQ ID NO:20.

[0064] In one embodiment, the transmembrane domain is a transmembrane domain of CD28 (or derived from CD8). In one embodiment, the transmembrane domain comprises about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% of the amino acid sequence shown in SEQ ID NO:80.

[0065] The co-stimulatory regions may include 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 combinations thereof (or may be derived from 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 combinations thereof).

[0066] In one embodiment, the co-stimulatory region is a 4-1BB co-stimulatory region (or derived from 4-1BB). In one embodiment, the co-stimulatory 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 a CD3ζ cytoplasmic signaling domain (or may be derived from CD3). In one embodiment, the cytoplasmic signaling domain comprises about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% of the amino acid sequence shown in SEQ ID NO:24.

[0068] The CAR of the present invention may include a V region of the anti-CD20 antigen binding domain. L and V H The linker (linker-1) between them. 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 shown in SEQ ID NO:6.

[0069] The CAR of the present invention may include a linker (linker-2) between an anti-CD20 antigen-binding region and an anti-BCMA antigen-binding region. In one embodiment, the linker (linker-2) includes an amino acid sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequence shown in SEQ ID NO:10.

[0070] The CAR of the present invention may include a V region of the anti-BCMA antigen binding domain. L and V H The linker (linker-3) between them. 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 shown in SEQ ID NO:14.

[0071] In one implementation, the bispecific CAR comprises, from the N-terminus to the C-terminus: (a) a light chain variable region (V) having ofatumumab. L 1) and heavy chain variable region (V H 1) the anti-CD20 antigen-binding region, and (ii) the light chain variable region (V) of those possessing BCMA-20 antibodies. L 2) and heavy chain variable region (V H 2) an anti-BCMA antigen binding region, (iii) a hinge region having the amino acid sequence shown in SEQ ID NO:18, (iv) a transmembrane domain having the amino acid sequence shown in SEQ ID NO:20, (v) a co-stimulatory region having the amino acid sequence shown in SEQ ID NO:22, and (vi) a cytoplasmic signal transduction domain having the amino acid sequence shown in SEQ ID NO:24.

[0072] In one implementation, the bispecific CAR comprises, from the N-terminus to the C-terminus,: (a) having a light chain variable region (V... L 1) and heavy chain variable region (V H 1) an anti-CD20 antigen binding region having the amino acid sequences shown in SEQ ID NO:4 and SEQ ID NO:8, respectively, and 2) a light chain variable region having a light chain variable region (V L 2) and heavy chain variable region (V H2) an anti-BCMA antigen binding region having the amino acid sequences shown in SEQ ID NO:12 and SEQ ID NO:16 respectively, (iii) a hinge region having the amino acid sequence shown in SEQ ID NO:18, (iv) a transmembrane domain having the amino acid sequence shown in SEQ ID NO:20, (v) a co-stimulatory region having the amino acid sequence shown in SEQ ID NO:22, and (vi) a cytoplasmic signal transduction domain having the amino acid sequence shown in SEQ ID NO:24.

[0073] In one embodiment, the bispecific CAR comprises, from the N-terminus to the C-terminus,: (a) a light chain variable region (V) having olfamomumab. L 1) and heavy chain variable region (V H 1) the anti-CD20 antigen binding region, and (ii) the light chain variable region (V) of those having BCMA-20. L 2) and heavy chain variable region (V H 2) an anti-BCMA antigen binding region, (iii) a hinge region having the amino acid sequence shown in SEQ ID NO:78, (iv) a transmembrane domain having the amino acid sequence shown in SEQ ID NO:80, (v) a co-stimulatory region having the amino acid sequence shown in SEQ ID NO:22, and (vi) a cytoplasmic signal transduction domain having the amino acid sequence shown in SEQ ID NO:24.

[0074] In one implementation, the bispecific CAR comprises, from the N-terminus to the C-terminus,: (a) having a light chain variable region (V... L 1) and heavy chain variable region (V H 1) an anti-CD20 antigen binding region having the amino acid sequences shown in SEQ ID NO:4 and SEQ ID NO:8, respectively, and 2) a light chain variable region having a light chain variable region (V L 2) and heavy chain variable region (V H 2) an anti-BCMA antigen binding region having the amino acid sequences shown in SEQ ID NO:12 and SEQ ID NO:16 respectively, (iii) a hinge region having the amino acid sequence shown in SEQ ID NO:78, (iv) a transmembrane domain having the amino acid sequence shown in SEQ ID NO:80, (v) a co-stimulatory region having the amino acid sequence shown in SEQ ID NO:22, and (vi) a cytoplasmic signal transduction domain having the amino acid sequence shown in SEQ ID NO:24.

[0075] In some implementations, V L 1 is located in V H The N-terminus of 1. In some implementations, V H1 is located in V L The N-terminus of 1. In some implementations, V H 2 is located in V L The N-terminus of 2. In some implementations, V L 2 is located in V H The N-terminus of 2. In one implementation, V L 1 is located in V H The N-terminus of 1; V L 2 is located in V H The N-terminus of 2.

[0076] In some embodiments, the bispecific CAR comprises about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% of 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.

[0077] In some 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 about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% of the same nucleotide sequence as 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 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.

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

[0081] This disclosure provides an immune cell comprising a vector or nucleic acid encoding the CAR of the present invention (e.g., integrated into its genome). The cell may be an isolated cell. The cell may be a genetically engineered cell. The cell may be a mammalian cell. In one embodiment, the cell is a CAR-T cell and / or a CAR-NK cell.

[0082] This disclosure also covers nucleic acids encoding the chimeric antigen receptor of the present invention (e.g., the bispecific CAR of the present invention).

[0083] The nucleic acid of the present invention may comprise about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% of 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.

[0084] The nucleic acids of the present invention may comprise nucleotide sequences 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] This disclosure provides a vector comprising the 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 adenovirus vector, an adeno-associated virus vector, or a combination thereof. In one embodiment, the vector is a lentiviral vector.

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

[0087] The pharmaceutical composition may contain a concentration range of about 1 × 10⁻⁶. 3 Cells / mL to approximately 1×10⁻¹ 8 cells / mL, or approximately 1×10⁻⁶ 4Cells / mL to approximately 1×10⁻¹ 7 The immune cells of the present invention are expressed as cells / mL.

[0088] This disclosure also provides a method for treating autoimmune diseases / conditions. This disclosure 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.

[0089] Immune cells can be allogeneic or autologous.

[0090] Autoimmune diseases can include systemic lupus erythematosus (SLE) (e.g., lupus nephritis), systemic sclerosis (SSc), inflammatory myopathies (e.g., polymyositis, dermatomyositis, or inclusion body myositis), systemic scleroderma, multiple sclerosis, or neuromyelitis optica (NMO).

[0091] Cancer can be a blood cancer. Cancer can be a B-cell malignancy. Cancer can be Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, and / or multiple myeloma. Cancer can 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] This disclosure provides a method for preparing immune cells (e.g., CAR-T cells) expressing chimeric antigen receptors, wherein the method includes: transducing the nucleic acid molecule of the present invention or the vector of the present invention into immune cells (e.g., T cells or NK cells) to obtain immune cells (e.g., CAR-T cells) expressing chimeric antigen receptors.

[0093] This 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-Connector 2-scFv2-H-TM-C-CSD(I),

[0095] In this context, each "-" represents a linking peptide or peptide bond independently; SP is an optional signal peptide; H is an optional hinge region; TM is a transmembrane domain; C is a co-stimulatory region; CSD is a cytoplasmic signal transduction 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 in formula A or B as follows:

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

[0099] Where V H1 For the variable region of the heavy chain of the anti-CD20 antibody; V L1 This is the variable region of the light chain of the anti-CD20 antibody; and "-" represents a linking peptide or peptide bond.

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

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

[0102] V L1 and V H1 Linking can be performed using a linker peptide (linker 1 or linker-1). Linker-1 may have the sequence shown in SEQ ID NO:6.

[0103] The structure of the anti-BCMA antigen-binding region can be represented by formula C or D as follows:

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

[0105] Where V L2 For the variable region of the light chain of the anti-BCMA antibody; V H2 This represents the variable region of the heavy chain of the anti-BCMA antibody; and "-" indicates a linking 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 some implementations, V L2 The amino acid sequence is shown in SEQ ID NO:12, and V H2The amino acid sequence is shown in SEQ ID NO:16.

[0108] V L2 and V H2 It can be linked using a linker peptide (linker 3 or linker-3). Linker-3 may have the sequence shown in SEQ ID NO:14.

[0109] In another embodiment, the structure of the chimeric antigen receptor is 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, connector 2 (or connector-2) has the sequence shown in SEQ ID NO:10.

[0112] In some implementations, the anti-CD20 antigen binding region includes a light chain variable region (V... L ), which contains 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 shown in SEQ IDNO:4.

[0113] In some implementations, the anti-CD20 antigen binding region includes a heavy chain variable region (V... H The amino acid sequence comprising 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% of the amino acid sequence shown in SEQ IDNO:8.

[0114] In some implementations, the anti-BCMA antigen binding region includes a light chain variable region (V... L The amino acid sequence comprising 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% of the amino acid sequence shown in SEQ IDNO:12.

[0115] In some implementations, the anti-BCMA antigen-binding region includes a heavy chain variable region (V). H The amino acid sequence comprising 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% of the amino acid sequence shown in SEQ IDNO:16.

[0116] The light chain variable region (V) of the anti-CD20 antigen binding region L It may contain one, two, or three complementary determination regions (CDRs), CDR1, CDR2, and CDR3, which are respectively associated with, as shown in SEQ ID NO. The CDR1, CDR2, and CDR3 (CDRs of the light chain variable region of the olfamom antibody) shown at positions 24-34, 50-56, and 89-97 of NO:4 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 (V) of the anti-CD20 antigen binding region L It may contain one, two, or three complementary determination regions (CDRs), CDR1, CDR2, and CDR3, which are respectively associated with, for example, SEQ ID NO:130, SEQ ID NO:131, and SEQ ID NO:132. The CDR1, CDR2, and CDR3 (CDRs of the light chain variable region of the oflamb monoclonal antibody) shown in NO:132 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] Heavy chain variable region (V) of the anti-CD20 antigen binding region H It may contain one, two, or three complementary determination regions (CDRs), CDR1, CDR2, and CDR3, which are respectively associated with, as shown in SEQ The CDR1, CDR2, and CDR3 (CDRs of the heavy chain variable region of the oflamb monoclonal antibody) shown at positions 30-35, 50-66, and 99-111 of IDNO:8 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] Heavy chain variable region (V) of the anti-CD20 antigen binding region HIt may contain one, two, or three complementary determination regions (CDRs), CDR1, CDR2, and CDR3, which are respectively associated with, for example, SEQ ID NO:127, SEQ ID NO:128, and SEQ ID NO:129. The CDR1, CDR2, and CDR3 (CDRs of the heavy chain variable region of the olfamom antibody) shown in NO:129 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 some implementations, the light chain variable region (V) of the anti-CD20 antigen binding region L It contains three CDRs, CDR1, CDR2, and CDR3, which are identical to CDR1, CDR2, and CDR3 (the CDRs of the light chain variable region of the olfamom antibody) shown at positions 24-34, 50-56, and 89-97 of SEQ ID NO:4, respectively, and the heavy chain variable region (V) of the anti-CD20 antigen binding region. H It contains three CDRs, which are the same as CDR1, CDR2 and CDR3 (CDRs of the heavy chain variable region of the olfamom antibody) shown in positions 30-35, 50-66 and 99-111 of SEQ ID NO:8.

[0121] In some implementations, the light chain variable region (V) of the anti-CD20 antigen binding region L It contains three CDRs, CDR1, CDR2, and CDR3, which are identical to CDR1, CDR2, and CDR3 (the CDRs of the light chain variable region of the oflamb monoclonal antibody) shown in SEQ ID NO:130, SEQ ID NO:131, and SEQ ID NO:132, respectively, and the heavy chain variable region (V) of the anti-CD20 antigen binding region. H It contains three CDRs, which are the same as CDR1, CDR2 and CDR3 (CDRs of the heavy chain variable region of the olfamom antibody) shown in SEQ ID NO:127, SEQ ID NO:128 and SEQ ID NO:129 respectively.

[0122] The light chain variable region (V) of the anti-BCMA antigen-binding region LIt may contain one, two, or three complementary determination regions (CDRs), CDR1, CDR2, and CDR3, which are respectively associated with, as shown in SEQ ID NO. The CDR1, CDR2, and CDR3 (CDRs of the light chain variable region of the BCMA-20 antibody) shown at positions 24-34, 50-56, and 89-97 of NO:12 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 (V) of the anti-BCMA antigen-binding region L It may contain one, two, or three complementary determination regions (CDRs), CDR1, CDR2, and CDR3, which are respectively associated with, for example, SEQ ID NO:134, SEQ ID NO:136, and SEQ ID NO:137. The CDR1, CDR2, and CDR3 (CDRs of the light chain variable region of the BCMA-20 antibody) shown in NO:138 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 (V) of the anti-BCMA antigen-binding region HIt may contain one, two, or three complementary determination regions (CDRs), CDR1, CDR2, and CDR3, which are respectively associated with, as shown in SEQ ID NO. The CDR1, CDR2, and CDR3 (CDRs of the heavy chain variable region of the BCMA-20 antibody) shown at positions 31-35, 50-66, and 99-110 of NO:16 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 (V) of the anti-BCMA antigen-binding region H It may contain one, two, or three complementary determination regions (CDRs), CDR1, CDR2, and CDR3, which are respectively associated with, for example, SEQ ID NO:141, SEQ ID NO:143, and SEQ ID NO:144. The CDR1, CDR2, and CDR3 (CDRs of the heavy chain variable region of the BCMA-20 antibody) shown in NO:145 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 some implementations, the light chain variable region (V) of the anti-BCMA antigen-binding region L It contains three CDRs, CDR1, CDR2, and CDR3, which are identical to CDR1, CDR2, and CDR3 (the CDRs of the light chain variable region of the BCMA-20 antibody) shown at positions 24-34, 50-56, and 89-97 of SEQ ID NO:12, respectively, and the heavy chain variable region (V) of the anti-BCMA antigen binding region... HIt contains three CDRs, CDR1, CDR2 and CDR3, which are the same as CDR1, CDR2 and CDR3 (CDRs of the heavy chain variable region of the BCMA-20 antibody) shown at positions 31-35, 50-66 and 99-110 of SEQ ID NO:16.

[0127] In some embodiments, the light chain variable region of the anti-BCMA antigen binding region comprises three CDRs, CDR1, CDR2, and CDR3, which are respectively associated with CDR1, CDR2, and CDR3 (light chain variable regions of BCMA-20 antibodies) as shown in SEQ ID NO:134, SEQ ID NO:136, and SEQ ID NO:138. L The CDR of the anti-BCMA antigen binding region is the same, and the heavy chain variable region (V) of the anti-BCMA antigen binding region is the same. H It contains three CDRs, CDR1, CDR2 and CDR3, which are the same as CDR1, CDR2 and CDR3 (CDRs of the heavy chain variable region of the BCMA-20 antibody) shown in SEQ ID NO:141, SEQ ID NO:143 and SEQ ID NO:145, respectively.

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

[0129] V light chain variable domain derived from OF antibody L It can have the following sequences:

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

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

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

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

[0134] Heavy chain variable domain V derived from ofamumab antibody H It can have the following sequences:

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

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

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

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

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

[0140] Light chain variable domain V derived from BCMA-20 antibody L It can have the following sequences:

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

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

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

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

[0145] Heavy chain variable domain V derived from BCMA-20 antibody H It can have the following sequences:

[0146] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGADHAIYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVSS(SEQ ID NO:16)

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

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

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

[0150] The signal peptide can be a CD8 signal peptide with the following sequence:

[0151] MALPVTALLLPLALLLHAARP(SEQ ID NO:2)

[0152] V anti-CD20 scFv L and V H (or V) H and V L The connector (connector-1) between the two terminals can have the following sequence: GSTSGGGSGGGSGGGGSS (SEQ ID NO:6)

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

[0154] V anti-BCMA scFv L and V H (or V) H and V L The connector (connector-3) between the two terminals can have the following sequence: GGGGSGGGGSGGGGS (SEQ ID NO:14)

[0155] The hinge region between the extracellular region (antigen-binding domain) and the transmembrane domain can be derived from IgG4, CD8 (CD8a), CD28, CD137, or a combination thereof.

[0156] The hinge region can be derived from CD8a with the following sequence:

[0157] FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO:18)

[0158] The hinge region can originate from IgG4 with the following sequence:

[0159] ESKYGPPCPPCP (SEQ ID NO:78)

[0160] The transmembrane domain can originate from CD8 (CD8TM) with the following sequence: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:20)

[0161] The transmembrane domain can originate from CD28 (CD28TM) with the following sequence: MFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:80)

[0162] The co-stimulatory region can originate from 4-1BB with the following sequence:

[0163] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:22)

[0164] Cytoplasmic signaling domains can originate from CD3ζ with the following sequence: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:24)

[0165] Chimeric antigen receptor (CAR)

[0166] The terms “chimeric antigen receptor” or alternatively “CAR” are used interchangeably throughout the text and refer to a recombinant polypeptide construct comprising, for example, an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain. (Lee et al., Clin. Cancer Res. (2012) 18(10): 2780; Jensen et al., Immunol Rev. (2014) 257(1): 127.) In one embodiment, the stimulating 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 co-stimulating molecule. The co-stimulating molecule may also be 4-1BB (i.e., CD137), CD27, and / or CD28, or fragments of these molecules. In another aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain comprising a functional signaling domain derived from the stimulating molecule. CARs can comprise chimeric fusion proteins containing an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain. The intracellular signal transduction domain includes a functional signal transduction domain derived from a co-stimulatory molecule and a functional signal transduction domain derived from a stimulatory molecule. Alternatively, a CAR can comprise chimeric fusion proteins containing an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain. The intracellular signal transduction domain includes two functional signal transduction domains derived from one or more co-stimulatory molecules and one functional signal transduction domain derived from a stimulatory molecule. CARs can also comprise chimeric fusion proteins containing an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain. The intracellular signal transduction domain includes at least two functional signal transduction domains derived from one or more co-stimulatory molecules and one functional signal transduction domain derived from a stimulatory molecule. The antigen-binding region of a CAR can contain any antigen-binding antibody fragment. The antibody fragment can contain one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or any combination thereof.

[0167] The term “ζ” or alternatives to “ζ chain,” “CD3-ζ,” or “TCR-ζ” may be a protein provided with GenBank accession numbers NP_932170, NP_000725, or XP_011508447; or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.), and “ζ-stimulatory domain” or alternatives to “CD3-ζ-stimulatory domain” or “TCR-ζ-stimulatory domain” may be an amino acid residue from the cytoplasmic domain of the ζ chain, which is sufficient to functionally deliver the initial signal required for T cell activation.

[0168] A chimeric receptor can refer to a non-naturally occurring molecule that can be expressed on the surface of a host cell and contains an antigen-binding fragment that binds to an antigen. In addition to the antigen-binding fragment, a chimeric receptor may further include one or more of a hinge region, a transmembrane domain, at least one co-stimulatory region, and a cytoplasmic signaling domain. In some embodiments, the chimeric antigen receptor includes an antigen-binding region (or fragment), a hinge region, a transmembrane domain, and a cytoplasmic signaling domain from its N-terminus to its C-terminus. In some embodiments, the chimeric antigen receptor further includes at least one co-stimulatory region. Therefore, a chimeric antigen receptor may include an antigen-binding region (or fragment), a hinge region, a transmembrane domain, a co-stimulatory region, and a cytoplasmic signaling domain from its N-terminus to its C-terminus.

[0169] In some embodiments, the chimeric antigen receptor includes a hinge region located between the antigen-binding region and the transmembrane domain. The hinge region may contain approximately 10-200 amino acids, such as 15-150, 20-100, or 30-60 amino acids. In some embodiments, the length of the hinge region may be approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids. The hinge region may 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. Hinge domains of any protein known in the art to contain a hinge domain are suitable for 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] Hinge domains of antibodies (such as IgG, IgA, IgM, IgE, or IgD antibodies) are also suitable for chimeric antigen receptors. In some embodiments, the hinge region is a hinge domain that binds to the constant CH1 and CH2 domains of the antibody. In some embodiments, the hinge region is of the antibody and includes the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge region includes the hinge domain of the antibody and the CH3 constant region of the antibody. In some embodiments, the hinge region includes the hinge domain of the antibody as well as the CH2 and CH3 constant regions 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 includes the hinge region of an IgG4 antibody as well as the CH2 and CH3 constant regions. In some embodiments, the hinge region includes the hinge region of an IgG4 antibody and the CH3 constant region.

[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, where x and n can be independently integers 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 receptor described herein are known in the art. See, for example, Wriggers et al. Current Trends in Peptide Science (2005) 80(6):736-746 and PCTP Publication WO 2012 / 088461.

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

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

[0176] In some embodiments, the chimeric antigen receptor includes one or more co-stimulatory regions. The co-stimulatory region may be at least a portion of a protein that mediates intracellular signal transduction to induce an immune response, such as effector function. The co-stimulatory region of the chimeric antigen receptor may originate from a protein that transduces signals and regulates responses mediated by immune cells, such as T cells, natural killer (NK) cells, macrophages, neutrophils, or eosinophils.

[0177] In some embodiments, the chimeric antigen receptor includes one or more (at least two, three, four or more) co-stimulatory regions. In some embodiments, the chimeric antigen receptor includes more than one co-stimulatory region derived from different proteins. In some embodiments, the chimeric antigen receptor does not include any co-stimulatory regions.

[0178] Examples of co-stimulatory regions for chimeric antigen receptors can be domains derived from co-stimulatory proteins, including but not limited to CD27, CD28, 4-1BB, OX40, CD30, Cd40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3. In some embodiments, the co-stimulatory region is derived from 4-1BB, CD28, or ICOS. In some embodiments, the co-stimulatory region is derived from CD28, and the chimeric antigen receptor includes a second co-stimulatory region derived from 4-1BB or ICOS. In some embodiments, the co-stimulatory region is a fusion domain comprising more than one co-stimulatory region or portions thereof. In some embodiments, the co-stimulatory region is a fusion of co-stimulatory regions derived from CD28 and ICOS.

[0179] In some embodiments, the chimeric antigen receptor includes a cytoplasmic signaling domain. Any cytoplasmic signaling domain may be used in the chimeric antigen receptor described herein. The cytoplasmic signaling domain can transmit signals, such as the interaction between an extracellular ligand-binding domain and its ligand, to stimulate cellular responses, such as inducing effector functions of the cell (e.g., cytotoxicity).

[0180] Chimeric antigen receptors can be prepared using conventional methods, such as recombinant techniques. Methods for preparing chimeric antigen receptors may involve generating nucleic acids encoding polypeptides comprising each domain of the chimeric antigen receptor, including an antigen-binding fragment and optionally a hinge region, a transmembrane domain, at least one co-stimulatory region, and a cytoplasmic signaling domain. In some embodiments, recombinant techniques are used to concatenate the nucleic acids encoding each component of the chimeric antigen receptor together. The sequences of each component (e.g., domains) can be concatenated directly or indirectly (e.g., using nucleic acid sequences encoding peptide linkers) using methods such as PCR amplification or ligation to form a nucleic acid sequence encoding the chimeric antigen receptor. Alternatively, the nucleic acid encoding the 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 the N-terminus to the C-terminus, a signal peptide (also known as a leader sequence), an antigen recognition sequence (antigen-binding domain), a hinge region, a transmembrane domain, a co-stimulatory region, and a cytoplasmic signal transduction domain (e.g., the CD3ζ signal transduction region (ζ chain portion)).

[0182] Bispecificity means that a CAR can specifically bind to two different antigens. A bispecific CAR can generate an immune response by binding to one or both antigens.

[0183] As used herein, the terms “CAR-T cell,” “CAR-T,” “CART,” and “CART cell” can refer to T cells that express the CAR of the present invention that targets both CD20 and BCMA.

[0184] Immune cells expressing chimeric antigen receptors

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

[0186] Chimeric antigen receptors can be introduced into suitable immune cells for expression using 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 CD8-expressing cells. + ) or CD8 and CD4 (CD8 + / CD4 +The T cells are T cells from an established T cell line, such as Jurkat cells. In some implementations, the T cells are T cells from an established T cell line.

[0187] Primary T cells can be obtained from any source, such as peripheral blood mononuclear cells (PBMCs), bone marrow, spleen, lymph nodes, thymus, or tumor tissue. In some embodiments, the immune cell population is derived from a patient with an autoimmune disease or cancer (e.g., a hematopoietic malignancy), for example, from bone marrow or PBMCs obtained from the patient. In some embodiments, the immune cell population is derived from a healthy donor. In some embodiments, the immune cells are obtained from a subject who will subsequently receive immune cells expressing chimeric antigen receptors. Immune cells administered to the same subject from whom the cells were obtained are referred to as autologous cells, while immune cells obtained from a subject who is not the subject from whom the cells will be received may be referred to as allogeneic cells.

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

[0189] To construct immune cells expressing the chimeric antigen receptor described herein, vectors for stable or transient expression of the chimeric antigen receptor can be constructed using conventional methods as described herein and introduced into immune cells. For example, the nucleic acid encoding the chimeric antigen receptor can be cloned into a suitable vector, such as a viral vector.

[0190] In some embodiments, immune cells (e.g., T cells) are transduced using a lentiviral vector (LV) encoding the CAR of the present invention. The transduced immune cells (e.g., T cells) can target CD20 and BCMA, synergistically activating T cells and inducing a T cell-mediated immune response.

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

[0192] In some embodiments, cells are isolated from mammals (e.g., humans) and genetically modified (i.e., transduced or transfected in vitro) using a vector expressing the CAR disclosed herein. The CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefits. The mammalian recipient can be human. The CAR-modified cells can be autologous to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or allogeneic to the recipient.

[0193] Methods for preparing immune cells expressing the chimeric antigen receptor of the present invention may include in vitro activation and / or expansion of immune cells. Activation of immune cells means stimulating immune cells into an activated state in which the cells may be able to perform effector functions (e.g., cytotoxicity). The method of activating immune cells will depend on the type of immune cells used to express the chimeric antigen receptor. Expanding immune cells may involve any method that results in an increase in the number of cells expressing the chimeric antigen receptor, such as allowing cell proliferation or stimulating cell proliferation. In some embodiments, cells expressing the chimeric receptor described herein are activated and / or expanded in vitro prior to administration to a subject.

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

[0195] carrier

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

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

[0198] The vector can be a viral vector. Viruses suitable as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In some embodiments, the vector of the present invention is a retroviral vector, such as a lentiviral vector. In some embodiments, the vector for expressing a chimeric antigen receptor is a retrovirus. In some embodiments, the vector for expressing a chimeric antigen receptor is a lentivirus. In some embodiments, the vector for expressing a 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 the cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeloproliferative sarcoma virus (MPSV) LTR, spleen focal formation virus (SFFV) LTR, simian virus 40 (SV40) early promoter, herpes simplex virus (TK) promoter, and extension factor 1-α (EF1-α) promoter with or without the EF1-α intron. Additional promoters for expressing chimeric receptors include any constitutively active promoter in immune cells. Alternatively, any tunable promoter (e.g., an inducible promoter) can be used such that its expression can be modulated within 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 cells by physical, chemical, or biological methods.

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

[0202] Biological approaches to 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 used as a delivery medium 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 that can be electroporated into immune cells (see, for example, Till et al. Blood (2012) 119(17):3940-3950). In some embodiments, the vector (nucleic acid) encoding the chimeric antigen receptor is an RNA molecule that can be electroporated into immune cells.

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

[0206] In instances where a viral vector encoding a chimeric antigen receptor is introduced into host cells, the 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 cell culture supernatant and isolated and / or purified before being brought into contact with immune cells.

[0207] Pharmaceutical Composition

[0208] This disclosure provides a pharmaceutical composition comprising the immune cells, CARs, nucleic acids, or vectors of the present invention. The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable loads, diluents, or excipients. In one embodiment, the formulation is a liquid formulation. In one embodiment, the concentration of immune cells (e.g., CAR-T cells) in the formulation is 1 × 10⁻⁶. 3 -1×10 8 cells / mL, or 1×10⁻⁶ 4 -1×10 7 Cells / mL.

[0209] As those skilled in the art will recognize, the effective dose varies depending on the specific disease being treated, the severity of the disease, individual patient parameters (including age, physical condition, body size, sex, and weight), duration of treatment, the nature of any concurrent therapies, the specific route of administration, and similar factors within the knowledge and expertise of the healthcare professional. In some embodiments, the effective dose alleviates, relieves, improves, enhances, reduces, or delays the progression of the subject's disease or condition. 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 contain phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives; low molecular weight peptides; 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. KEHoover.

[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 subjects via any route, including but not limited to intravenous, intraventricular (ICV) injection, intracisional injection or infusion, oral, transdermal, ocular, intraperitoneal, subcutaneous, implantation, sublingual, subcutaneous, intramuscular, rectal, mucosal, ocular, intrathecal, intra-articular, intra-articular, 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 administered topically. The pharmaceutical compositions can be formulated for intravenous administration.

[0213] The compositions of the present invention can be administered in any convenient manner, including by aerosol inhalation, injection, ingestion, blood transfusion, implantation, or transplantation. The compositions can be administered to a patient subcutaneously, intradermally, intratumorally, intranodularly, intramedullaryly, intramuscularly, intravenously (iv), or intraperitoneally. In one embodiment, the composition is administered to a subject (e.g., a patient) via intradermal or subcutaneous injection. In another embodiment, the composition is administered via intravenous injection. The compositions can be injected directly into the tumor, lymph node, or lesion site.

[0214] The immune cell or pharmaceutical composition 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 compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). Although the appropriate dosage can be determined through clinical trials, the amount and frequency of administration will be determined based on factors such as the patient's condition and the type and severity of the patient's disease.

[0216] When referring to "effective dose," "therapeutic effective dose," or "therapeutic dose," the precise amount of the composition administered can be determined by the physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the patient's (subject's) condition. Pharmaceutical compositions containing immune cells can be 10... 4 Up to 10 9 Cells / kg body weight or 10 5 Up to 10 6 The composition is administered at a dose of cells per kg body weight (inclusive of all integer values ​​within these ranges). These doses may also be administered multiple times. 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 readily determined by a medical professional by monitoring the patient's signs of disease and adjusting treatment accordingly.

[0217] The dosage of the above treatment administered to a 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 can be adjusted according to recognized practices in the art. In one embodiment, for each treatment or each course of treatment, 1 × 10⁻⁶ may be administered to the patient via, for example, intravenous infusion. 6 Up to 1×10 10 Immune cells (e.g., CAR-T cells).

[0218] Diseases awaiting treatment

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

[0220] In some embodiments, the anti-CD20 / BCMA bispecific CAR of the present invention targets B cells and plasma cells, which can reduce / eliminate autoantibodies. In some embodiments, the anti-CD20 / BCMA bispecific CAR of the present invention can reduce / deplete B cells, plasmablasts, and / or long-lived plasma cells (LLPCs) to reduce / eliminate autoantibody production.

[0221] This disclosure provides a method for treating an autoimmune disease / condition. The method may include administering a CAR, immune cells, or pharmaceutical composition to a subject in need.

[0222] Autoimmune diseases can include systemic lupus erythematosus (SLE), lupus nephritis (LN), systemic sclerosis (SSc), CREST syndrome (calcification, Raynaud's syndrome, esophageal motility disorder, scleroderma, and telangiectasia), oculoclonus, 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), herpetic dermatitis, Miller-Fisher syndrome, acute motor axonal neuropathy (AMAN), multifocal motor neuropathy with conduction block, autoimmune hepatitis, antiphospholipid syndrome, Wegener's granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome. Myasthenia gravis (MG), rheumatoid arthritis, chronic autoimmune hepatitis, sclerosing myositis, myasthenia gravis, Lambert-Eaton myasthenic syndrome, Hashimoto's thyroiditis, Graves' disease, paraneoplastic cerebellar degeneration, stiff-person syndrome, limbic encephalitis, Isaacs syndrome, Sydenham's chorea, PANDAS (paraneoplastic neuropsychiatric disorders of childhood), 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] Autoimmune diseases can include pernicious anemia, Addison's disease, psoriasis, inflammatory bowel disease (IBD), psoriatic arthritis, and Sjögren's syndrome. Lupus syndrome (e.g., discoid lupus erythematosus, drug-induced lupus erythematosus, and neonatal lupus erythematosus), multiple sclerosis, and / or reactive arthritis.

[0224] Autoimmune diseases can include polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, adrenalitis, thyroiditis, autoimmune thyroid disease, gastric atrophy, chronic hepatitis, lupus-like hepatitis, atherosclerosis, Alzheimer's disease, demyelinating diseases, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenic purpura, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, alopecia areata, bullous pemphigoid, scleroderma, progressive systemic sclerosis, adult-onset diabetes mellitus (e.g., type II diabetes), male and female autoimmune infertility, ankylosing spondylitis, ulcerative colitis, and Crohn's disease. Diseases including stomatitis, inflammatory diarrhea, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, juvenile rheumatoid arthritis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent miscarriage, antiphospholipid syndrome, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, allergic diseases, allergic encephalomyelitis, toxic epidermal necrolysis, alopecia, and Alport's syndrome. Syndrome, alveolitis, allergic alveolitis, fibrotic alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosa, transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis, Takayasu's sarteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granuloma, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue fever, endocarditis, endocardial myocardial fibrosis, endophthalmitis, erythema protuberans, fetal polycythemia, eosinophilic facial mask, Shulman's syndrome, Felty's syndrome Syndrome, filariasis, cyclitis, chronic cyclitis, metachronous cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura(purpura), graft-versus-host disease, transplant rejection, human immunodeficiency virus infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Hodgkin's and non-Hodgkin's 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 1 diabetes (e.g., juvenile-onset diabetes), multiple sclerosis, scleroderma, ankylosing spondylitis, sarcoidosis, pemphigus vulgaris, bullous pemphigoid, psoriasis, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, Behcet's syndrome, Reiter's disease, and Berger's disease. Diseases including dermatomyositis, polymyositis, antineutrophil cytoplasmic antibody-associated vasculitis (e.g., granulomatous polyangiitis (also known as Wegener's granulomatosis), microscopic polyangiitis, and Chag-Strauss syndrome), scleroderma, Sjögren's syndrome, antiglomerular basement membrane disease (including Goodpasser 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, stomatitis, autoimmune arthritis, rheumatoid arthritis, type 1 diabetes, multiple sclerosis, graft-versus-host disease after bone marrow transplantation, osteoarthritis, juvenile chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, insulin-dependent diabetes mellitus, thyroiditis, asthma, psoriasis, scleroderma, atopic dermatitis, graft-versus-host disease, acute or chronic immune diseases associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, Graves' disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, allergic purpura, renal microvasculitis, chronic active hepatitis, and staphylococcal vasculitis. Meningitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's disease, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, type I and type II polyendocrine disorders, Schmidt syndrome, adult (acute) respiratory distress syndrome, alopecia areata, alopecia areata, seronegative arthropathy, arthropathy, Reiter's disease, psoriatic arthropathy, chlamydia, Yersinia and Salmonella-associated arthropathy (Yersinia) and salmonella associated arthropathy), spondyloarthritis, atherosclerosis / arteriosclerosis, atopic allergy, food allergy, autoimmune bullous diseases, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, linear IgA disease, autoimmune hemolytic anemia, Coombs-positive hemolytic anemia, acquired pernicious anemia, juvenile pernicious anemia, myalgic encephalitis / Royal Free disease.Diseases, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, acquired immunodeficiency syndrome, acquired immunodeficiency-related diseases, hepatitis C, co-transfer immunodeficiency (co-transfer hypogammaglobulinemia), dilated cardiomyopathy, fibrotic lung disease, cryptogenic fibrotic alveolitis, post-inflammatory 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 fibrosis, obliterative bronchiolitis, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, post-infectious interstitial lung disease. Serous lung disease, gouty arthritis, autoimmune hepatitis, type 1 autoimmune hepatitis (classical autoimmune or lupus-like hepatitis), type 2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune hypoglycemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, organ transplant-related acute immune diseases, organ transplant-related chronic immune diseases, osteoarthritis, primary sclerosing cholangitis, idiopathic leukopenia, autoimmune neutropenia, NOS nephropathy, glomerulonephritis, renal microvasculitis, discoid lupus, systemic lupus erythematosus, idiopathic or NOS male infertility, sperm autoimmune disease, multiple sclerosis (all subtypes), insulin-dependent diabetes mellitus, sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasser syndrome, pulmonary manifestations of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease. Diseases including: systemic sclerosis, angina / arteritis, autoimmune thrombocytopenic purpura, idiopathic thrombocytopenic purpura, autoimmune thyroid disease, hyperthyroidism, goiter, autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxedema, lens-derived 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] Autoimmune diseases can be inflammatory muscle diseases. Inflammatory myopathy is a group of diseases involving chronic muscle inflammation, muscle weakness, and (in some cases) muscle pain. The four main types of chronic or long-term inflammatory myopathy are polymyositis, which affects the skeletal muscles (the type involved in body movement) on both sides of the body; dermatomyositis, which leads to progressive muscle weakness; inclusion body myositis, characterized by slow, progressive muscle weakness, muscle atrophy, and muscle loss; and necrotizing autoimmune myopathy, which involves muscle weakness in both the upper and lower body.

[0228] In another implementation, autoimmune diseases are autoimmune diseases caused by the overexpression of B cells (such as lupus).

[0229] This disclosure also covers a method for treating cancer. The method may include administering a CAR, immune cells, or a pharmaceutical composition to a subject in need.

[0230] This disclosure provides a chimeric antigen receptor for the treatment of CD20-positive diseases such as B-cell lymphoma.

[0231] Cancer can be a BCMA-positive malignant tumor. Cancer can be multiple myeloma (MM) or plasma cell leukemia.

[0232] Cancer can be a blood cancer. Cancer can be a plasma cell malignancy. Cancer can be a B-cell malignancy. B-cell malignancies can include acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), B-cell leukemia, or B-cell lymphoma.

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

[0234] Cancer can 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.

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

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

[0237] Cancer can be a solid tumor. Solid tumors can be benign or malignant. Different types of solid tumors are named according to the type of cells that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcoma and carcinoma, include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, pancreatic cancer, and ovarian cancer.

[0238] Reagent test kit

[0239] Kits for use with the CAR, immune cells, nucleic acids, vectors, or pharmaceutical compositions of the present invention are also within the scope of this disclosure. Such kits may include one or more containers containing 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 with any of the methods described herein. The included instructions may include a description of administering the pharmaceutical composition to a subject to achieve the intended activity in the subject. The kit may further include a description of selecting suitable subjects for treatment based on determining whether the subject requires treatment. In some embodiments, the instructions include a description of administering the pharmaceutical composition to a subject who requires treatment.

[0241] Instructions for use of a pharmaceutical composition typically include information on the intended therapeutic dose, dosing schedule, and route of administration. Containers may be unit doses, bulk packaging (e.g., multi-dose packaging), or subunit doses.

[0242] All reagent kits provided in this article are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, and flexible packaging.

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

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

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

[0246] Anti-CD20 / BCMACAR-T cells were prepared using apheresis of 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 medium containing 1% human serum albumin and 300 U / 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 were shown. The expression level of anti-BCMACAR was detected by flow cytometry using the BCMA-Fc fusion protein; the expression level of anti-CD20 CAR was detected by flow cytometry using an antibody specific to OF scFv.

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

[0249] Antigen-specific activation of anti-CD20 / BCMACAR-T cells was assessed by measuring IFN-γ release and CD137 expression when CAR-T cells were co-cultured with target cells. Target cells (“T”) included CD20-positive A549-CD20+ tumor cells, BCMA-positive A549-BCMA+ tumor cells, CD20 and BCMA double-positive A549-CD20+BCMA+ tumor cells, Raji cells, MM.1S cells, and double-negative A549 tumor cells. Effector cells (“E”) were anti-CD20 / BCMACAR-T cells.

[0250] PBMCs were isolated from venous blood of healthy donors using 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 medium containing 1% human serum albumin and 300 U / mL recombinant human interleukin-2 (IL-2). On day 3, activated PBMCs were transduced with a lentiviral vector encoding anti-CD20 / BCMACAR. From day 6 onwards, CAR-T cells could be harvested for activity assays.

[0251] IFNγ release was measured using CAR-T cells cultured for 7 days. 1×10 5 CAR-T cells were cultured in 200 μl of medium at a 1:1 E:T ratio 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 tumor-free cells (NT) for 18 h. The secretion level of IFN-γ in the cell culture supernatant was then detected by ELISA.

[0252] The expression level of CD137 was measured using CAR-T cells cultured for 7 days. 1×10⁻⁶ cells were used. 5CAR-T cells were cultured in 200 μl of medium at a 1:1 E:T ratio for 18 h 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 tumor-free cells. The expression level of CD137 on the surface of CAR-T cells was then detected by flow cytometry.

[0253] IFNγ release results are as follows 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 specifically recognized CD20 single-positive or CD20 / BCMA double-positive target cells and released IFN-γ. Similarly, anti-BCMACAR-T (C-CAR088) cells specifically recognized BCMA single-positive or CD20 / BCMA double-positive target cells to release IFN-γ. Only anti-CD20 / BCMACAR-T (TOB1-4 and TOBL1-4, where TOBL1 is C-CAR168) cells recognized CD20 single-positive, BCMA single-positive, and CD20 / BCMA double-positive target cells and released high levels of IFN-γ. TOB1 to TOB4 CAR-T cells showed high IFN-γ release when co-cultured with CD20-positive target cells, but showed low responsiveness 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.

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

[0255] Example 3: In vitro cytotoxicity against CD20 / BCMACAR-T cells

[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. The cytotoxicity of CAR-T cells to target cells was assessed using real-time cell analysis (RTCA) label-free technology.

[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 do not express CD20 or BCMA. Figures 5A-5B , Figure 6C Their killing ability is comparable to that of anti-CD20 and anti-BCMA single-specific CAR-T cells, and both are dose-dependent. Figures 5A-5B , Figure 6C TOBL1 to TOBL4 CAR-T cells ( Figure 5B TOB1 to TOB4 CAR-T cells exhibit high cytotoxicity against CD20-positive and BCMA-positive target cells. Figure 5A It 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 is present in patients with systemic lupus erythematosus (SLE). hi T-bet + The proportion of B cell subsets is significantly increased and 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 Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus, Immunity, 2018, 16; 49(4):725-739.e6.IL-21 drives expansion and plasma cell differentiation of autoreactive CD11chiT-bet+B cells in SLE, Nat. Commun. 2018; 9(1):1758). In some animal models of autoimmune diseases and in the peripheral blood of patients with rheumatoid arthritis, this cell subset is enriched with age, and therefore 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 consequence of abnormal TLR7 activation is the accumulation of autoreactive B cells, or age-related B cells (ABCs). ABCs are B cells that recognize self-antigens and have 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 possess the ability to eliminate ABC, we prepared C-CAR168 (TOBL1) CAR-T cells from peripheral blood of three healthy human donors (HD10, HD11, and HD12). We also isolated autologous B cells from healthy donor PBMCs and induced their differentiation in vitro to obtain ABC-rich autologous B cells, which were then used as target cells for cytotoxicity experiments. After co-culturing for 2 to 4 hours, C-CAR168 CAR-T cells from different donors, at different E:T ratios, exhibited significant cytotoxic effects against ABC-rich autologous B cells compared to control T cells that had not undergone CAR transduction. 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 indicate that C-CAR168 CAR-T cells can effectively eliminate ABC cells.

[0263] In vitro ABC differentiation

[0264] PBMCs from healthy donors were isolated using Ficoll gradient centrifugation 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 seeded in 96-well plates containing 200 μl of RPMI complete medium and stimulated for 3 days with TLR7 ligand R848, CD40L, BAFF, IL-2, goat anti-human IgA+IgG+IgM (H+L), IL-21, and IFN-γ. The cell culture medium was changed daily by replenishing the complete medium and stimulation mixture. ABC induction was confirmed by FACS analysis. Antibodies used for FACS staining included live / dead stains, anti-human CD19, CD38, CD27, IgD, CD11c, CD21, and T-bet.

[0265] Cytotoxicity assay

[0266] Following differentiation, ABC-rich B cells were co-cultured with C-CAR168 or untransduced (NT) T cells at an indicated E:T ratio. After 24 hours, cell viability was determined by staining with LIVE / DEAD Fixable Aqua Dead Cell Stain (Invitrogen), and B cells and T cells were distinguished using anti-CD19 and anti-CD3 antibodies. Cytotoxicity was assessed via live CD19. + The percentage of cell depletion is used to determine lysis. B cell lysis is calculated using the following formula: Percentage of lysis (%) = (1 - (C - live CD19 cells in CAR168 co-culture)) + Cell fraction / UT coculture of live CD19 + 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 inhibited the growth of CD20-positive and BCMA-positive tumor cells in tumor-bearing mice.

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

[0270] Six- to eight-week-old female B-NDG mice were subcutaneously inoculated with A549-CD20 (CD20+) or MM.1S (BCMA+) cells. When the average tumor volume reached 100 mm², the tumor cells were inoculated into the target cells. 3 At that time, 3 to 5 × 10 6 C-CAR168 CAR-T cells were administered via tail vein at a dose of 1 CAR-T cell / mouse. During the experiment, tumor volume in mice treated with C-CAR168 CAR-T cells continued to decrease. At the end of the experiment, the tumor weight in the C-CAR168 group was significantly lower than that in the vector control group. C-CAR168 cells exhibited strong cytotoxicity against CD20-positive and BCMA-positive target cells in vivo.

[0271] Specific method: Female B-NDG (NOD.Cg-Prkdc) scid Il2rg tm1Vst / Vst) mice subcutaneously injected 5×10 6 A549-CD20 cells / animal. When the average tumor volume reaches approximately 100 mm. 3 Twenty animals were selected and randomly divided into two groups (vector control group and C-CAR168 group), with 10 animals in each group. Mice were administered a single dose of either the vector control or C-CAR168 (3 × 10⁻⁶) via tail vein injection. 6 (CAR-T cells / animal). Following administration, the mean tumor volume in the vector 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 in 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 figure), and the tumor weight was 0.013±0.01g, which was significantly different from the control group (P<0.001). Figure 9A (See right figure). The tumor growth inhibition rate calculated based on tumor weight was 94.88%. The results indicate that C-CAR168 can significantly inhibit the growth of CD20-positive target cells in vivo.

[0272] To evaluate the in vivo effects of C-CAR168 on BCMA single-positive target cells and to compare the in vivo efficacy of different batches of C-CAR168, 20 female B-NDG (NOD.CB17-Prkdc) mice were used. scid Il2rg tm1 / Bcgen) mice subcutaneously injected 5×10 6 MM.1S cells / animal. When the average tumor volume reaches approximately 100 mm. 3 Fifteen animals were selected and randomly divided into three groups (one vector control group and two C-CAR168 groups), with five animals in each group. Each mouse was administered the drug once via tail vein injection. For C-CAR168, the dose was 5 × 10⁻⁶. 6 CAR-T cells / animal. Following administration, the mean tumor volume in the vector control group continued to increase, reaching 2220.86 ± 117.35 mm on day 28. 3 The tumor weight was 2.409 ± 0.216 g. The mean tumor volume in both the C-CAR168-1 and C-CAR168-2 groups began to decrease from day 10. Figure 9B (Left figure). By day 28, the average tumor volume was 109.2 ± 88.92 mm. 3 and 9.07±5.58mm 3 The tumor weights were 0.041±0.034g and 0.003±0.002g, respectively. Figure 9B (See right figure) The tumor growth inhibition rates, calculated based on tumor weight, were 98.30% and 99.88%, respectively, compared to the control group (P<0.001, P<0.001). There was no significant difference between the two batches of C-CAR168. The results showed that a single intravenous administration of 5×10 6 C-CAR168 CAR-T cells / mouse were well tolerated in B-NDG tumor-bearing mice, and C-CAR168 significantly inhibited the growth of BCMA-positive target cells in vivo.

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

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

[0275] During the experiment, the mean tumor volume of animals in both the vector control group and the T cell control group continued to increase, and the mean tumor volume on day 17 was 2628.78 ± 117.32 mm. 3 and 2536.23±97.80mm 3 Tumor volume continued to increase in the low-dose C-CAR168 group, although it was significantly lower than that in the mediator control and T-cell control groups after 10 days of administration. The mean tumor volume in the medium-dose and high-dose C-CAR168 groups began to decrease on day 6. Tumor volume in the low-dose, medium-dose, and high-dose C-CAR168 groups all showed dose-dependent reduction, with tumor growth inhibition rates of 55.47%, 97.75%, and 98.01% on day 17, respectively. On day 28, no tumor tissue was observed in either the medium-dose or high-dose C-CAR168 groups. Figure 9E ). Figure 9F Survival curves for each group during the experimental period are shown. Although all animals in the vector control group and the T cell control group died around day 17, all mice in the medium-dose and high-dose C-CAR168 groups 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, the full-length cDNA sequences of human membrane proteins are constructed into expression vectors using genetic engineering methods, then transiently transfected into HEK293T cells, and arranged into arrays using microfluidic or microarray printing technologies. This is a high-throughput screening technique for studying the interactions 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] A chimeric rabbit monoclonal antibody, C-CAR168 scFv-RabFc, was generated by co-linking anti-CD20 scFv (e.g., derived from oflambumab mAb) and anti-BCMA scFv (e.g., derived from BCMA-20 mAb) with the Fc region of rabbit IgG. The chimeric antibody was added at a concentration of 20 μg / mL to an array of HEK293T cells transiently transfected with 5220 membrane proteins. Flow cytometry results showed that C-CAR168 scFv-RabFc bound tightly to human CD20 and BCMA. Figure 8A During flow cytometry, the mean fluorescence intensity of C-CAR168 scFv-RabFc binding to CD20 and BCMA 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 exhibits specific binding to FCGR1A. Figure 8B The average fluorescence intensity of C-CAR168 scFv-RabFc was 2.5 times that of the negative control group. This is mainly due to the binding between FCGR1A and the rabbit Fc of the recombinant protein, and therefore there is no associated risk in clinical application. C-CAR168 scFv-RabFc bound weakly to the ITGB2-ITGAM and ITGB2-ITGAL heterodimers, and the average fluorescence intensity was 2 to 3 times that of the negative control group. For other proteins identified in the initial screening (MPZ, F11R, CLEC2B, and MC2R), the average fluorescence intensity bound to C-CAR168 scFv-RabFc did not change with concentration. At concentrations of 20 μg / mL and 5 μg / mL, the fluorescence intensity did not exceed twice that of the negative control group, therefore the likelihood of these proteins specifically binding to C-CAR168 scFv is low or minimal.

[0281] To test whether the 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. The expression of CD137 on C-CAR168 CAR-T cells and the levels of IFN-γ, TNF-α, IL-2, and other cytokines in the cell culture supernatant were measured. 293T cells transfected with the empty vector served as a negative control, and 293T cells transfected with CD20 and BCMA served as a positive control.

[0282] CD137 (4-1BB) is a cell surface marker of antigen-specific activation of T cells. The upregulation of CD137 expression on the cell surface can be used to assess antigen-specific activation of CAR-T cells. Experiments showed that after co-culturing three batches of C-CAR168 cells with cells expressing CD20 and BCMA, the proportion of 4-1BB-positive cells increased compared to untransduced T cells (“NT”). After co-culturing with cells expressing ITGB2-ITGAM and ITGB2-ITGAL, the proportion of 4-1BB-positive cells did not differ significantly from the untransduced T cell group (“NT”). Figure 8D (Left figure) This indicates that C-CAR168 binds nonspecifically 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 to untransduced T cells. Figure 8D (See right figure). The results further show that C-CAR168 nonspecifically recognizes ITGB2-ITGAM and ITGB2-ITGAL in vitro.

[0284] In summary, membrane protein array and in vitro co-culture results show that the antigen-binding domain of C-CAR168 binds tightly to human CD20 and BCMA, with no other non-specific binding sites. Membrane protein array studies revealed that, apart from weak binding to the two heterogeneous complexes, C-CAR168 exhibits no cross-reactivity with the membrane proteome.

[0285] Example 7: C-CAR168 demonstrated robust potency against autologous B cells derived from SLE patients.

[0286] To investigate CAR-T therapy for treating autoimmune diseases such as SLE, we evaluated the efficiency of CAR-T cells in depleting autoreactive B cells. We will also investigate the efficacy of CAR-T cells in improving remission and survival in a lupus model.

[0287] C-CAR168's efficiency in in vitro elimination of pan-B cells in lupus patients

[0288] Peripheral blood samples (10–15 mL) were collected from eight SLE patients. The patients exhibited varying activity levels, autoantibody profiles, organ damage (preferably lupus nephritis), and treatments, reflecting the heterogeneity of lupus patients. Patients who had recently received B-cell depletion antibodies were excluded.

[0289] For each sample, a portion of the blood was used to isolate T cells for CAR-T cell production, and the remaining blood was used to isolate pan-B cells as a target for a cell lysis assay. T cells isolated from eight SLE patients were transduced using a lentiviral vector encoding C-CAR168 and tested for CAR expression. T cell samples from eight SLE patients were successfully transduced and well-amplified for functional assays. Figure 10A ).

[0290] C-CAR168 CAR-T cells or untransduced (NT) T cells derived from eight patient samples were co-cultured with target cell lines expressing CD20 and / or BCMA. K562 was negative for both CD20 and BCMA; MM.1S was 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 a representative sample of eight patients are shown. Therefore, C-CAR168 cells derived from SLE patient samples exhibit robust activity against target cells expressing CD20 and BCMA.

[0291] Isolated pan-B cells from eight patient samples were co-cultured with autologous C-CAR168 CAR-T cells or untransduced (NT) T cells at an indicated E:T (effectant to target) ratio. After 24 hours, the co-culture supernatant was collected for ELISA to assess IFN-γ levels. Cytotoxicity was determined by fluorescence activated cell sorting (FACS) and calculation of the percentage of viable CD19+ pan-B cells exhausted. B cell lysis was calculated using the following formula: Percentage of lysis (%) = (1 - (fraction of viable CD19+ cells in C-CAR168 co-culture / fraction of viable CD19+ cells in UT co-culture)) × 100. Figure 10C and 10D Results from a representative sample of eight patients are presented. Pan-B cells isolated from eight 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 from lupus patients in vitro.

[0292] The efficiency of CAR-T in clearing ABC from lupus patients in vitro

[0293] We will investigate the efficiency of CAR-T in eliminating ABC (the 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, CAR-T production, and functional analysis.

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

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

[0297] We will use a humanized SLE mouse model to evaluate the efficiency and therapeutic efficacy of CAR-T depleted B cells in vivo. We will obtain CD34. + Humanized mice derived from stem cells. Two or more mice are sacrificed, and spleens are harvested using aseptic techniques. T cells are then isolated from the spleen for CAR-T production. The remaining mice will be used to induce lupus disease flare-ups, and after successful induction, the mice will be divided into groups to receive either CAR-T or control therapy (e.g., untransduced T cells). Blood samples will be collected periodically from the mice to monitor CAR-T cell persistence and the efficiency of B cell depletion (including ABCs) via FACS. Serum samples will be used to measure titers of various autoantibodies. Urine samples will also be routinely collected to measure proteinuria levels. At the end of the study, or if animals die early (presumably in the control group), tissues will be collected for histological examination, e.g., to examine for immune complex deposition in the kidneys and the severity of nephritis. The presence of B cells or plasma cells in diseased tissues will also be examined. Survival curves will be generated to compare the efficacy of CAR-T versus control therapy.

[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 CellsExpressing CD19 as Potential Off-Tumor Targets for CAR-TImmunotherapies.Cell.2020Oct1;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 ImmunotherCancer.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 (63nt)

[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 (54nt)

[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] CD8a™ nucleic acid sequence (72nt)

[0350] Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc(SEQ ID NO:19)

[0351] CD8a™ amino acid sequence:

[0352] IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:20)

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

[0354] Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg(SEQ ID NO:21)

[0355] 4-1BB amino acid sequence:

[0356] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:22)

[0357] CD3z nucleic acid sequence (336nt)

[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 (63 nt)

[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-20scFv(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] CD8a™ nucleic acid sequence (72nt)

[0386] Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc(SEQ ID NO:36)

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

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

[0389] CD3z nucleic acid sequence (336nt)

[0390] Agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa(SEQ ID NO:38)

[0391] TOBL2 nucleic acid sequence (2247nt)

[0392]

[0393] TOBL2 amino acid sequence:

[0394] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGSTSGGGSGGGSGGGGSSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCMGQTISSYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGADHAIYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQID NO:40)

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

[0396] CD8aSP (63nt) 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-20scFv(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] CD8a™ nucleic acid sequence (72nt)

[0416] Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc(SEQ ID NO:50)

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

[0418] Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg(SEQ ID NO:51)

[0419] CD3z nucleic acid sequence (336nt)

[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 (63nt)

[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-20scFv(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] CD8a™ nucleic acid sequence (72nt)

[0446] Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc(SEQ ID NO:64)

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

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

[0449] CD3z nucleic acid sequence (336nt)

[0450] Agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa(SEQ ID NO:66)

[0451] TOBL4 nucleic acid sequence (2247nt)

[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 (36nt)

[0473] GAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCT(SEQ ID NO:77)

[0474] IgG4 hinge amino acid sequence:

[0475] ESKYGPPCPPCP(SEQ ID NO:78)

[0476] CD28TM nucleic acid sequence (84nt)

[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 (2130nt)

[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™ 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 (2130nt)

[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™ 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 (2130nt)

[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™ 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 (2130nt)

[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 this invention is not limited to what has been specifically shown and described above. Those skilled in the art will recognize that suitable alternatives exist for the examples of materials, configurations, constructions, and dimensions depicted. Numerous references, including patents and various publications, have been cited and discussed in the description of this invention. Such citations and discussions are solely for the purpose of clarifying the description of the invention and do not imply 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. Variations, modifications, and other implementations of the content described herein will be apparent to those skilled in the art without departing from the spirit and scope of the invention. Although certain embodiments of the invention have been shown and described, those skilled in the art will understand that changes and modifications can be made without departing from the spirit and scope of the invention. The content set forth in the foregoing description and drawings is provided by way of illustration only and is not intended to be limiting.

Claims

1. A bispecific chimeric antigen receptor (CAR) comprising an anti-CD20 antigen-binding region and an anti-BCMA antigen-binding region, wherein the bispecific CAR comprises an amino acid sequence as shown in SEQ ID NO:

26.

2. An immune cell comprising the bispecific CAR as described in claim 1.

3. The immune cell of claim 2, wherein the immune cell is a T cell or a natural killer (NK) cell.

4. A nucleic acid sequence encoding the bispecific CAR as described in claim 1.

5. A vector comprising the nucleic acid sequence as described in claim 4.

6. A pharmaceutical composition comprising the immune cells as described in claim 2.

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