Bispecific chimeric antigen receptors targeting BCMA and CD19

Abstract

This disclosure provides a bispecific chimeric antigen receptor targeting BCMA and CD19. The CAR may include a BCMA-targeting scFv, a CD19-targeting scFv, a hinge region, a transmembrane domain, a costimulatory region, and a cytoplasmic signaling domain. The chimeric antigen receptor can be used to treat autoimmune disorders or cancer.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 495,377, filed April 11, 2023, which is incorporated herein by reference in its entirety.

[0002] Inclusion by referencing the sequence list The array list, submitted as an XML file "11299_011843-WO0_SL.xml" created on April 1, 2024, with a size of 175,878 bytes, is incorporated herein by reference in its entirety.

[0003] This disclosure relates to the field of immunotherapy, and more specifically to bispecific chimeric antigen receptors (CARs) that target BCMA and CD19. [Background technology]

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

[0005] The etiology of autoimmune diseases is unknown. In patients, abnormal activation of humoral immunity occurs, leading to the production of numerous antibodies against autoantigens. These bind to form pathogenic immune complexes, which then deposit locally and trigger an inflammatory response. B cells play a crucial role in the pathogenesis of autoimmune diseases, promoting their development through various mechanisms (e.g., production of autoantibodies, release of cytokines, and presentation of autoantigens). Autoantibodies, as key factors, can bind to autoantigens to form immune complexes, which can activate innate immune system cells to produce type I interferons and other pro-inflammatory cytokines, leading to organ damage. Therefore, lymphocyte depletion or removal is a potential treatment strategy.

[0006] SLE is a typical autoimmune disease known to be associated with an overreaction of polyclonal B cells (Dorner et al., Mechanisms of B cell autoimmunity in SLE, Arthritis Res. Ther. 13, 243 (2011)). Therefore, one of the immunological characteristics of SLE is the production of antinuclear antibodies (ANAs), which can mediate the pathogenesis of SLE by binding to their respective autoantigens, leading to the deposition of immune complexes and the induction of inflammation and organ damage (e.g., lupus nephritis) (Salmon, JE, Arming T cells against B cells in systemic lupus erythematosus, Nat. Med. 28, 2009-2010 (2022)). There are two main types of autoantibodies: anti-DNA antibodies and antibodies that recognize RNA-binding proteins (RBPs) (Pisetsky et al., New insights into the role of antinuclear antibodies in systemic lupus erythematosus, Nat. Rev. Rheumatol. 16, 565-579 (2020)). In SLE patients, the source of autoantibodies includes not only B cells but also a subset of plasma cells called long-lived plasma cells (LLPCs).Anti-DNA antibodies are produced by memory B cells that maintain high levels of CD19 and CD20 expression on their cell surface, and by naive B cells that migrate to plasmablasts. Anti-RBP antibodies, on the other hand, are produced by LLPCs that may lose surface expression of CD19 and CD20 but are positive for B cell maturation antigen (BCMA), a cell surface protein expressed in all mature plasma cells (Dogan et al., B-cell maturation antigen expression across hematologic cancers: a systematic literature review. Blood Cancer J.10,73(2020); Morgan et al., Unraveling B cell trajectories at single cell resolution, Trends Immunol.43,210-229(2022)). CD11c. hi T-bet + Recent studies have demonstrated that a B cell subset proliferates in human SLE and functions as a precursor cell for plasma cells that produce autoantibodies. This B cell subset exhibits high expression of CD19 and CD20 and is associated with autoreactive mouse aging-related B cells (autoreactive B cells or ABCs; terminology is human CD11c hi T-bet +(Used to represent B cells) corresponds to (Jenks et al., Distinct Effector B Cells Induced by Unregulated Toll-like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus, Immunity 49, 725-739 e726 (2018); Wang et al., IL-21 drives expansion and plasma cell differentiation of autoreactive CD11c(hi)T-bet(+)B cells in SLE, Nat.Commun. 9, 1758 (2018)). In addition to producing autoantibodies, B cells are also involved in the pathogenesis of SLE and other autoimmune diseases by secreting cytokines and acting as antigen-presenting cells. Therefore, depleting B cells in SLE patients may be an effective treatment for this life-threatening disease.

[0007] B cell depletion was achieved by administering monoclonal antibodies against B cell surface markers. The anti-CD20 antibody rituximab was successful in early open-label trials in SLE, but failed to meet the primary endpoint in two randomized controlled findings (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 (obexerimab) were also tested in SLE. In early studies, patients treated with obexerimab maintained disease inactivation levels despite discontinuation of steroids, but failed to meet the primary endpoint in a Phase II clinical trial (Lee et al., B cell depletion therapies in autoimmune disease: advances and mechanistic insights, Nat. Rev. Drug Discov. 20, 179-199 (2021)).

[0008] One promising approach to achieving B cell depletion is the adoptive transfer of CAR-T cells. CAR-T cells are genetically engineered T lymphocytes that can recognize, proliferate, and generate a cytotoxic immune response against specific antigens on target cells even in the absence of the major histocompatibility complex (MHC). Recent studies have shown that the compassionate use of CD19 CAR-T therapy in five patients with refractory SLE resulted in significant B cell depletion and drug-free remission. This suggests that CAR-T cell transplantation is feasible, tolerable, and highly effective in SLE (Mackensen et al., Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus, Nat. Med. 28, 2124-2132 (2022)).

[0009] There remains an urgent need to develop effective ways to treat autoimmune diseases. [Summary of the Invention]

[0010] The present disclosure provides a bispecific chimeric antigen receptor (CAR) comprising: (i) an anti-BCMA antigen-binding region comprising a light chain variable region (V L L 1) and a heavy chain variable region (V H H 1), wherein V L L 1 has three complementarity-determining regions (CDRs) (CDR1, CDR2, and CDR3) having amino acid sequences that are respectively about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, and V H H 1 has three CDRs (CDR1, CDR2, and CDR3) having amino acid sequences that are respectively about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, an anti-BCMA antigen-binding region; and (ii) an anti-CD19 antigen-binding region comprising a light chain variable region (V L L 2) and a heavy chain variable region (V H H 2), wherein V L2 includes three complementarity-determining regions (CDRs) (CDR1, CDR2, and CDR3) having amino acid sequences that are approximately 80% to 100% identical to the amino acid sequences described in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively. H 2 is an anti-CD19 antigen-binding region comprising three CDRs (CDR1, CDR2, and CDR3) having amino acid sequences that are approximately 80% to 100% identical to the amino acid sequences described in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively.

[0011] This disclosure includes (i) the light chain variable region (V L 1) and heavy chain variable region (V H 1) an anti-BCMA antigen binding region including, and (ii) a light chain variable region (V L 2) and heavy chain variable region (V H The present invention provides a bispecific chimeric antigen receptor (CAR) comprising an anti-CD19 antigen-binding region including (2).

[0012] In one embodiment, V L 1 is V H It is located at the N-terminus of 1. In one embodiment, V H 1 is V L It is located at the N-terminus of 1. In one embodiment, V L 2 is V H It is located at the N-terminus of 2. In one embodiment, V H 2 is V L It is located at the N-terminus of 2.

[0013] In a particular embodiment, V L 1 and V H Compound 1 has an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences described in SEQ ID NO: 24 and SEQ ID NO: 28, respectively.

[0014] In a particular embodiment, V L 2 and V H Compound 2 has an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences described in SEQ ID NO: 32 and SEQ ID NO: 36, respectively.

[0015] The anti-BCMA antigen-binding region may be a single-chain variable fragment (scFv) that specifically binds to BCMA. The anti-CD19 antigen-binding region may be an scFv that specifically binds to CD19.

[0016] The 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 costimulatory region, and (e) a cytoplasmic signaling domain.

[0017] The hinge region may include hinge regions of IgG4, CD8, CD28, CD137, or a combination thereof.

[0018] The transmembrane domain 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.

[0019] The co-stimulatory regions may include the co-stimulatory regions of 4-1BB (CD137), CD28, OX40, CD2, CD7, CD27, CD30, CD40, CD70, CD134, PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.

[0020] The cytoplasmic signaling domain may also include the cytoplasmic signaling domain of CD3ζ.

[0021] This disclosure provides a bispecific CAR containing (or having) an amino acid sequence that is approximately 80% to approximately 100% identical to the amino acid sequences described in SEQ ID NO: 46, SEQ ID NO: 60, SEQ ID NO: 74, SEQ ID NO: 88, SEQ ID NO: 104, SEQ ID NO: 121, SEQ ID NO: 138, or SEQ ID NO: 155.

[0022] This disclosure also includes immune cells expressing bispecific CARs. The immune cells may be T cells or natural killer (NK) cells.

[0023] This disclosure provides nucleic acids that encode bispecific CARs.

[0024] This disclosure provides a vector containing the nucleic acid encoding a bispecific CAR.

[0025] This disclosure provides pharmaceutical compositions comprising bispecific CARs, immune cells, nucleic acids, or vectors.

[0026] This disclosure also provides methods for treating autoimmune disorders. These methods may include administering immune cells or pharmaceutical compositions to subjects in need.

[0027] Autoimmune disorders may include systemic lupus erythematosus (SLE) (e.g., lupus nephritis), systemic vasculitis, systemic sclerosis, inflammatory myopathy (e.g., polymyositis, dermatomyositis, inclusion body myositis), systemic scleroderma, multiple sclerosis, myasthenia gravis, myositis autoantibody-induced disease, or neuromyelitis optica.

[0028] The autoimmune disorder may be polymyositis, dermatomyositis, or inclusion body myositis. The autoimmune disorder may be lupus nephritis.

[0029] 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 of it.

[0030] Cancer may be a blood cancer. Cancer may be a B-cell malignancy. Cancer may be Hodgkin lymphoma, non-Hodgkin lymphoma, leukemia, and / or multiple myeloma. Cancer may be acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0031] Immune cells may be of the same species or of self-derived origin. [Brief explanation of the drawing]

[0032] [Figure 1] The structure of the bispecific chimeric antigen receptor targeting CD19 and BCMA is shown. The structure of the CAR includes a signal peptide (SP), anti-BCMA scFv (B20), linker (linker-2), anti-CD19 scFv (FMC63), hinge region, transmembrane domain, costimulatory region, and cytoplasmic signaling domain (CD3ζ). A short IgG4 hinge (12aa) and CD28 transmembrane domain are included in TB19-1 to TB19-4, while a CD8a (55aa) hinge and CD8a transmembrane domain are included in TB19-L1 to TB19-L4. Four combinations of VH and VL orientations in the two scFv sequences are included in the two groups of CARs (TB19-1-4 and TB19-L1-4). [Figure 2] Figures A and B show the expression levels of anti-BCMA (Figure 2A) and anti-CD19 (Figure 2B) on the surface of T cells. C-CAR088 is an anti-BCMA CAR. C-CAR011 is an anti-CD19 CAR. [Figure 3] A and B show the levels of IFN-γ secreted by in vitro activated anti-BCMA / CD19 CAR-T cells in the cell culture supernatant. A shows the levels of IFN-γ secreted by TB19-1 to TB19-4 and TB19-L1 to TB19-L4 CAR-T cells in the cell culture supernatant. B shows high IFN-γ release by TB19-1 to TB19-4 CAR-T cells. MM.1S is a BCMA-positive (MM) cell line, and RAJI is CD19-positive and BCMA-positive. [Figure 4] A and B show the expression levels of CD137 on the surface of activated CAR-T cells. A: TB19-1 to TB19-4, B: TB19-L1 to TB19-L4. [Figure 5] This demonstrates the in vitro cytotoxic effect of CAR-T cells using an RTCA assay. [Modes for carrying out the invention]

[0033] This disclosure provides a chimeric antigen receptor (CAR) that targets both CD19 and BCMA. The CAR may comprise a signal peptide, an anti-CD19 scFv, an anti-BCMA scFv, a hinge region, a transmembrane domain, a costimulatory region, and a cytoplasmic signaling domain. The CAR can be used to treat autoimmune diseases or cancer.

[0034] This disclosure provides a bispecific chimeric antigen receptor (CAR). The bispecific CAR has (i) a light chain variable region (V L 1) and heavy chain variable region (V H 1) an anti-BCMA antigen binding region including; and (ii) a light chain variable region (V L 2) and heavy chain variable region (V H 2) may include an anti-CD19 antigen binding region.

[0035] This bispecific chimeric antigen receptor (CAR) has a light chain variable region (V) having an amino acid sequence that is approximately 80% to 100%, 85% to 100%, 90% to 100%, or 95% to 100% identical to the amino acid sequences described in SEQ ID NO: 24 and SEQ ID NO: 28, respectively. L 1) and heavy chain variable region (V H (i) an anti-BCMA antigen binding region including (1) an anti-BCMA antigen binding region; and (ii) a light chain variable region (V) having an amino acid sequence that is approximately 80% to approximately 100%, approximately 85% to approximately 100%, approximately 90% to approximately 100%, or approximately 95% to approximately 100% identical to the amino acid sequences described in SEQ ID NO: 32 and SEQ ID NO: 36, respectively. L 2) and heavy chain variable region (V H 2) may include an anti-CD19 antigen binding region.

[0036] This disclosure provides a bispecific chimeric antigen receptor (CAR). The bispecific CAR has (i) a light chain variable region (V L 1) and heavy chain variable region (V H 1) an anti-BCMA antigen binding region including, and (ii) a light chain variable region (VL 2) and heavy chain variable region (V H 2) May also include an anti-CD19 antigen binding region. L 1 may include three complementarity-determining regions (CDRs) (CDR1, CDR2, and CDR3) having amino acid sequences that are approximately 80% to 100%, 85% to 100%, 90% to 100%, or 95% to 100% identical to the amino acid sequences described in SEQ ID NOs. 2, SEQ ID NOs. 4, and SEQ ID NOs. V H 1 may include three CDRs (CDR1, CDR2, and CDR3) having amino acid sequences that are approximately 80% to 100%, 85% to 100%, 90% to 100%, or 95% to 100% identical to the amino acid sequences described in SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, respectively. L 2 may include three complementarity-determining regions (CDRs) (CDR1, CDR2, and CDR3) having amino acid sequences that are approximately 80% to 100%, 85% to 100%, 90% to 100%, or 95% to 100% identical to the amino acid sequences described in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively. H 2 may include three CDRs (CDR1, CDR2, and CDR3) having amino acid sequences that are approximately 80% to 100%, 85% to 100%, 90% to 100%, or 95% to 100% identical to the amino acid sequences described in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively.

[0037] In a particular embodiment, V L 1 is V H It is located at the N-terminus of 1. In certain embodiments, V H 1 is V L It is located at the N-terminus of 1. In certain embodiments, V H 2 is V L Located at the N-terminus of 2. In certain embodiments, V L 2 is V H It is located at the N-terminus of 2. In one embodiment, V L 1 is V H Located at the N-terminus of 1, V L 2 is V H It is located at the N-terminus of 2.

[0038] In a particular embodiment, V L 1 and V H Compound 1 has an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences described in SEQ ID NO: 24 and SEQ ID NO: 28, respectively.

[0039] In a particular embodiment, V L 2 and V H Compound 2 has an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences described in SEQ ID NO: 32 and SEQ ID NO: 36, respectively.

[0040] In certain 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 CD19.

[0041] The anti-BCMA antigen-binding region may be a single-chain variable fragment (scFv) that specifically binds to BCMA. The anti-CD19 antigen-binding region may be an scFv that specifically binds to CD19. In certain embodiments, the scFv that specifically binds to BCMA is located at the N-terminus of the scFv that specifically binds to CD19. In certain embodiments, the scFv that specifically binds to CD19 is located at the N-terminus of the scFv that specifically binds to BCMA.

[0042] A bispecific CAR further comprises one or more of the following: (a) a signal peptide or SP (or leader sequence), (b) a hinge region, (c) a transmembrane domain, (d) a costimulatory region, and (e) a cytoplasmic signaling domain.

[0043] This bispecific CAR may include a signal peptide, anti-BCMA scFv, anti-BCMA CD19, a hinge region, a transmembrane domain, a costimulatory region, and a cytoplasmic signaling domain from the N-terminus to the C-terminus.

[0044] The signal peptide may include (or be derived from) signal peptides of CD8, CD28, GM-CSF, CD4, CD137, or combinations thereof. In one embodiment, the signal peptide is (or is derived from) the signal peptide of CD8.

[0045] In one embodiment, the signal peptide contains an amino acid sequence that is approximately 80% to 100%, 85% to 100%, 90% to 100%, or 95% to 100% identical to the amino acid sequence described in SEQ ID NO: 22.

[0046] The hinge region may include (or be derived from) IgG4, CD8, CD28, CD137, or a combination thereof, wild-type, or mutant hinge regions.

[0047] In one embodiment, the hinge region is (or derived from) the hinge region of IgG4. In one embodiment, the hinge region contains an amino acid sequence that is approximately 80% to approximately 100%, approximately 85% to approximately 100%, approximately 90% to approximately 100%, or approximately 95% to approximately 100% identical to the amino acid sequence described in SEQ ID NO: 38.

[0048] In one embodiment, the hinge region is the hinge region of CD8a (or derived therefrom). In one embodiment, the hinge region contains an amino acid sequence that is approximately 80% to approximately 100%, approximately 85% to approximately 100%, approximately 90% to approximately 100%, or approximately 95% to approximately 100% identical to the amino acid sequence described in SEQ ID NO: 98.

[0049] The transmembrane domain may include (or be derived from) transmembrane domains of CD8, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or combinations thereof.

[0050] In one embodiment, the transmembrane domain is (or derived from) the transmembrane domain of CD28. In one embodiment, the transmembrane domain contains an amino acid sequence that is approximately 80% to approximately 100%, approximately 85% to approximately 100%, approximately 90% to approximately 100%, or approximately 95% to approximately 100% identical to the amino acid sequence described in SEQ ID NO: 40.

[0051] In one embodiment, the transmembrane domain is (or derived from) the transmembrane domain of CD8a. In one embodiment, the transmembrane domain contains an amino acid sequence that is approximately 80% to approximately 100%, approximately 85% to approximately 100%, approximately 90% to approximately 100%, or approximately 95% to approximately 100% identical to the amino acid sequence described in Sequence ID No. 100.

[0052] The co-stimulatory regions may include (or be derived from) the co-stimulatory regions of 4-1BB (CD137), CD28, OX40, CD2, CD7, CD27, CD30, CD40, CD70, CD134, PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.

[0053] In one embodiment, the co-stimulatory region is (or derived from) the 4-1BB co-stimulatory region. In one embodiment, the co-stimulatory region contains an amino acid sequence that is approximately 80% to 100%, approximately 85% to 100%, approximately 90% to 100%, or approximately 95% to 100% identical to the amino acid sequence described in SEQ ID NO: 42.

[0054] The cytoplasmic signaling domain may include (or be derived from) the cytoplasmic signaling domain of CD3ζ. In one embodiment, the cytoplasmic signaling domain contains an amino acid sequence that is approximately 80% to 100%, approximately 85% to 100%, approximately 90% to 100%, or approximately 95% to 100% identical to the amino acid sequence described in SEQ ID NO: 44.

[0055] This CAR has a V in the anti-BCMA antigen binding region. L and V HA linker (linker-1) may be included between them. In one embodiment, the linker (linker-1) includes an amino acid sequence that is approximately 80% to approximately 100%, approximately 85% to approximately 100%, approximately 90% to approximately 100%, or approximately 95% to approximately 100% identical to the amino acid sequence described in Sequence ID No. 26.

[0056] This CAR may include a linker (linker-2) between the anti-BCMA antigen-binding region and the anti-CD19 antigen-binding region. In one embodiment, the linker (linker-2) includes an amino acid sequence that is approximately 80% to approximately 100%, approximately 85% to approximately 100%, approximately 90% to approximately 100%, or approximately 95% to approximately 100% identical to the amino acid sequence described in SEQ ID NO: 30.

[0057] This CAR has a V anti-CD19 antigen binding region. L and V H A linker (linker-3) may be included between them. In one embodiment, the linker (linker-3) includes an amino acid sequence that is approximately 80% to approximately 100%, approximately 85% to approximately 100%, approximately 90% to approximately 100%, or approximately 95% to approximately 100% identical to the amino acid sequence described in SEQ ID NO: 34.

[0058] In one embodiment, the bispecific CAR has a light chain variable region (V) from the N-terminus to the C-terminus of the BCMA-20 antibody. L 1) and heavy chain variable region (V H (1) an anti-BCMA antigen binding region having (ii) the light chain variable region of the FMC63 antibody (V L 2) and heavy chain variable region (V H (2) comprises an anti-CD19 antigen-binding region having (iii) the amino acid sequence described in SEQ ID NO: 38, (iv) a transmembrane domain having the amino acid sequence described in SEQ ID NO: 40, (v) a costimulatory region having the amino acid sequence described in SEQ ID NO: 42, and (vi) a cytoplasmic signaling domain having the amino acid sequence described in SEQ ID NO: 44.

[0059] In one embodiment, the bispecific CAR has a light chain variable region (V) from the N-terminus to the C-terminus, having the amino acid sequences described in SEQ ID NO: 24 and SEQ ID NO: 28, respectively. L 1) and heavy chain variable region (V H (1) an anti-BCMA antigen binding region having (ii) the amino acid sequences described in SEQ ID NO: 32 and SEQ ID NO: 36 respectively, and (ii) a light chain variable region (V L 2) and heavy chain variable region (V H (2) comprises an anti-CD19 antigen-binding region having (iii) the amino acid sequence described in SEQ ID NO: 38, (iv) a transmembrane domain having the amino acid sequence described in SEQ ID NO: 40, (v) a costimulatory region having the amino acid sequence described in SEQ ID NO: 42, and (vi) a cytoplasmic signaling domain having the amino acid sequence described in SEQ ID NO: 44.

[0060] In one embodiment, the bispecific CAR has a light chain variable region (V) from the N-terminus to the C-terminus of (a) BCMA-20. L 1) and heavy chain variable region (V H (1) an anti-BCMA antigen binding region having (ii) the light chain variable region of the FMC63 antibody (V L 2) and heavy chain variable region (V H (2) comprises an anti-CD19 antigen-binding region having (iii) the amino acid sequence described in SEQ ID NO: 98, (iv) a transmembrane domain having the amino acid sequence described in SEQ ID NO: 100, (v) a costimulatory region having the amino acid sequence described in SEQ ID NO: 42, and (vi) a cytoplasmic signaling domain having the amino acid sequence described in SEQ ID NO: 44.

[0061] In one embodiment, the bispecific CAR has a light chain variable region (V) from the N-terminus to the C-terminus, having the amino acid sequences described in SEQ ID NO: 24 and SEQ ID NO: 28, respectively. L 1) and heavy chain variable region (V H 1) an anti-BCMA antigen binding region having, (ii) each 配列番号32及び配列番号36 Light chain variable region having the amino acid sequence described above (V L 2) and heavy chain variable region (V Han anti-CD19 antigen-binding region having (2), (iii) a hinge region having the amino acid sequence set forth in SEQ ID NO: 98, (iv) a transmembrane domain having the amino acid sequence set forth in SEQ ID NO: 100, (v) a co-stimulatory region having the amino acid sequence set forth in SEQ ID NO: 42, and (vi) a cytoplasmic signaling domain having the amino acid sequence set forth in SEQ ID NO: 44.

[0062] In certain embodiments, V L 1 is located at the N-terminus of V H 1. In certain embodiments, V H 1 is located at the N-terminus of V L 1. In certain embodiments, V H 2 is located at the N-terminus of V L 2. In certain embodiments, V L 2 is located at the N-terminus of V H 2. In one embodiment, V L 1 is located at the N-terminus of V H 1, and V L 2 is located at the N-terminus of V H 2.

[0063] In certain embodiments, the bispecific CAR comprises an amino acid sequence that is about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 46, SEQ ID NO: 60, SEQ ID NO: 74, SEQ ID NO: 88, SEQ ID NO: 104, SEQ ID NO: 121, SEQ ID NO: 138, or SEQ ID NO: 155.

[0064] In certain embodiments, the bispecific CAR may have the amino acid sequence set forth in SEQ ID NO: 46, SEQ ID NO: 60, SEQ ID NO: 74, SEQ ID NO: 88, SEQ ID NO: 104, SEQ ID NO: 121, SEQ ID NO: 138, or SEQ ID NO: 155.

[0065] This bispecific CAR can be encoded in nucleic acids having nucleotide sequences that are approximately 80% to approximately 100%, approximately 85% to approximately 100%, approximately 90% to approximately 100%, or approximately 95% to approximately 100% identical to the nucleotide sequences described in SEQ ID NO: 45, SEQ ID NO: 59, SEQ ID NO: 73, SEQ ID NO: 87, SEQ ID NO: 103, SEQ ID NO: 120, SEQ ID NO: 137, or SEQ ID NO: 154.

[0066] This bispecific CAR can be encoded in nucleic acids having the nucleotide sequence described in SEQ ID NO: 45, SEQ ID NO: 59, SEQ ID NO: 73, SEQ ID NO: 87, SEQ ID NO: 103, SEQ ID NO: 120, SEQ ID NO: 137, or SEQ ID NO: 154.

[0067] This disclosure provides immune cells that express or contain the bispecific CAR. The immune cells may be T cells or natural killer (NK) cells.

[0068] This disclosure provides immune cells comprising a vector or nucleic acid (e.g., one incorporated into its genome) encoding the CAR. The cells may be isolated cells. The cells may be genetically modified cells. The cells may be mammalian cells. In one embodiment, the cells are CAR-T cells and / or CAR-NK cells.

[0069] This disclosure also includes nucleic acids encoding the chimeric antigen receptor (e.g., the bispecific CAR).

[0070] This nucleic acid may contain nucleotide sequences that are approximately 80% to 100%, approximately 85% to 100%, approximately 90% to 100%, or approximately 95% to 100% identical to the nucleotide sequences described in SEQ ID NO: 45, SEQ ID NO: 59, SEQ ID NO: 73, SEQ ID NO: 87, SEQ ID NO: 103, SEQ ID NO: 120, SEQ ID NO: 137, or SEQ ID NO: 154.

[0071] This nucleic acid may contain the nucleotide sequence described in SEQ ID NO: 45, SEQ ID NO: 59, SEQ ID NO: 73, SEQ ID NO: 87, SEQ ID NO: 103, SEQ ID NO: 120, SEQ ID NO: 137, or SEQ ID NO: 154.

[0072] The present disclosure provides a vector comprising the present nucleic acid. The vector may comprise DNA or RNA. The vector may be a plasmid, viral vector, transposon, or a combination thereof. The vector may comprise a DNA virus or a retroviral vector. The vector may be a lentiviral vector, adenoviral vector, adeno-associated viral vector, or a combination thereof. In one embodiment, the vector is a lentiviral vector.

[0073] The present disclosure provides a pharmaceutical composition comprising the present chimeric antigen receptor (e.g., the present bispecific CAR), the present immune cell, the present nucleic acid, or the present vector. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition may be a liquid preparation.

[0074] The pharmaceutical composition may comprise the present immune cell at a concentration in the range of about 1×10 3 cells / mL to about 1×10 8 cells / mL, or about 1×10 4 cells / mL to about 1×10 7 cells / mL.

[0075] The present disclosure also provides a method of treating an autoimmune disease / disorder. The present disclosure provides a method of treating cancer. The method may comprise administering the present immune cell or the present pharmaceutical composition to a subject that needs it.

[0076] The immune cell may be allogeneic or autologous.

[0077] The autoimmune disorder may be systemic lupus erythematosus (SLE) (e.g., lupus nephritis), systemic sclerosis (SSc), inflammatory myopathy (e.g., polymyositis, dermatomyositis, or inclusion body myositis), systemic scleroderma, multiple sclerosis, or neuromyelitis optica (NMO).

[0078] Cancer may be a blood cancer. Cancer may be a B-cell malignancy. Cancer may be Hodgkin lymphoma, non-Hodgkin lymphoma, leukemia, and / or multiple myeloma. Cancer may be acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0079] This disclosure provides a method for preparing immune cells (e.g., CAR-T cells) expressing a chimeric antigen receptor, the method comprising introducing the nucleic acid molecule or vector into immune cells (e.g., T cells or NK cells) to obtain immune cells (e.g., CAR-T cells) expressing a chimeric antigen receptor.

[0080] This disclosure provides a chimeric antigen receptor (CAR), the structure of which may be represented by formula I: SP-scFv1-linker2-scFv2-H-TM-C-CSD(I), (In the formula, each "-" is independently a linker peptide or peptide bond; SP is any signal peptide; H is any hinge region; TM is a transmembrane domain; C is a costimulatory region; CSD is a cytoplasmic signaling domain; one of scFv1 and scFv2 is an anti-BCMA antigen-binding region; and the other is an anti-CD19 antigen-binding region.)

[0081] In one embodiment, scFv1 is an anti-BCMA antigen binding region, and scFv2 is an anti-CD19 antigen binding region. In another embodiment, scFv1 is an anti-CD19 antigen binding region, and scFv2 is an anti-BCMA antigen binding region.

[0082] Linker-2 may have the sequence described in Sequence ID No. 30.

[0083] The structure of the anti-BCMA antigen-binding region may be as shown in formula A or B below: V H1 -VL1 (A);V L1 -V H1 (B) (In the formula, V H1 However, it is the variable region of the heavy chain of the anti-BCMA antibody; V L1 (where "-" is the variable region of the anti-BCMA antibody heavy chain; where "-" is a linker peptide or peptide bond).

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

[0085] In a particular embodiment, V L1 The amino acid sequence is shown in SEQ ID NO: 24, V H1 The amino acid sequence is shown in SEQ ID NO: 28.

[0086] V L1 and V H1 This can be linked with a linker peptide (linker 1 or linker-1). Linker-1 may have the sequence described in SEQ ID NO: 26.

[0087] The structure of the anti-CD19 antigen binding region may be as shown in formula C or D below: V L2 -V H2 (C);V H2 -V L2 (D) (In the formula, V L2 However, it is the variable region of the light chain of the anti-CD19 antibody; V H2 (where "-" is the variable region of the anti-CD19 antibody heavy chain; where "-" is a linker peptide or peptide bond).

[0088] In one embodiment, the CAR has an anti-CD19 antigen-binding domain having the structure shown in formula C.

[0089] In a particular embodiment, V L2 The amino acid sequence is shown in Sequence ID No. 32, V H2 The amino acid sequence is shown in SEQ ID NO: 36.

[0090] V L2 and V H2 This can be linked with a linker peptide (linker 3 or linker-3). Linker-3 may have the sequence described in SEQ ID NO: 34.

[0091] In another embodiment, the structure of the chimeric antigen receptor is shown in formula II below: SP-V L1 -V H1 -Linker 2-V L2 -V H2 -H-TM-C-CSD(II) In one embodiment, linker 2 (or linker-2) has the sequence described in sequence number 30.

[0092] In certain embodiments, the anti-BCMA antigen-binding region includes a light chain variable region (VL) containing an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% identical to the amino acid sequence described in Sequence ID No. 24.

[0093] In certain embodiments, the anti-BCMA antigen-binding region includes a heavy chain variable region (VH) containing 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 described in SEQ ID NO: 28.

[0094] In certain embodiments, the anti-CD19 antigen-binding region includes a light chain variable region (VL) containing 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 described in SEQ ID NO: 32.

[0095] In certain embodiments, the anti-CD19 antigen-binding region includes a heavy chain variable region (VH) containing 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 described in SEQ ID NO: 36.

[0096] Light chain variable region of the anti-BCMA antigen binding region (V L) are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least Alternatively, it may include one, two, or three complementarity determination regions (CDRs) (CDR1, CDR2, and CDR3) that are identical by approximately 86%, at least or approximately 87%, at least or approximately 88%, at least or approximately 89%, at least or approximately 90%, at least or approximately 91%, at least or approximately 92%, at least or approximately 93%, at least or approximately 94%, at least or approximately 95%, at least or approximately 96%, at least or approximately 97%, at least or approximately 98%, at least or approximately 99%, or approximately 100%.

[0097] Light chain variable region of the anti-BCMA antigen binding region (V L) are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, and at least or about 8 It may include one, two, or three complementarity determination regions (CDRs) (CDR1, CDR2, and CDR3) that are identical by 6%, 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%.

[0098] Variable heavy chain region of the anti-BCMA antigen binding domain (V H) are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, and at least It may include one, two, or three complementarity determination regions (CDRs) (CDR1, CDR2, and CDR3) that are identical by or approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or approximately 100%.

[0099] Variable heavy chain region of the anti-BCMA antigen binding domain (V H) are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about It may include one, two, or three complementarity determination regions (CDRs) (CDR1, CDR2, and CDR3) that are 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical.

[0100] In certain embodiments, the light chain variable region (V) of the anti-BCMA antigen binding region L The CDRs (CDR1, CDR2, and CDR3) are identical to those described in positions 24-34, 50-56, and 89-97 of SEQ ID NO: 24, and the heavy chain variable region (VH) of the anti-BCMA antigen-binding region includes three CDRs identical to those described in positions 31-35, 50-66, and 99-110 of SEQ ID NO: 28.

[0101] In certain embodiments, the light chain variable region of the anti-BCMA antigen binding region is CDR1, CDR2, and CDR3 (the light chain variable region of the BCMA-20 antibody) as described in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively. LIt contains three CDRs (CDR1, CDR2, and CDR3) identical to those of the CDR of the BCMA-20 antibody, and the heavy chain variable region (VH) of the anti-BCMA antigen binding region contains three CDRs identical to those of CDR1, CDR2, and CDR3 (the heavy chain variable region CDRs of the BCMA-20 antibody) described in SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, respectively.

[0102] The light chain variable region of the anti-CD19 antigen binding region (V L ) are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, and at least or about 85% of the CDR1, CDR2, and CDR3 (CDRs of the light chain variable region of the FMC63 antibody) described at positions 24-34, 50-56, and 89-97 of SEQ ID NO: 32. Or it may include one, two, or three complementarity determination regions (CDRs) (CDR1, CDR2, and CDR3) that are identical by approximately 86%, at least or approximately 87%, at least or approximately 88%, at least or approximately 89%, at least or approximately 90%, at least or approximately 91%, at least or approximately 92%, at least or approximately 93%, at least or approximately 94%, at least or approximately 95%, at least or approximately 96%, at least or approximately 97%, at least or approximately 98%, at least or approximately 99%, or approximately 100%.

[0103] The light chain variable region of the anti-CD19 antigen binding region (V L) are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, and at least or about 8 It may include one, two, or three complementarity determination regions (CDRs) (CDR1, CDR2, and CDR3) that are identical by 6%, 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%.

[0104] The heavy chain variable region of the anti-CD19 antigen binding domain (V H) are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least Alternatively, it may include one, two, or three complementarity determination regions (CDRs) (CDR1, CDR2, and CDR3) that are identical by approximately 86%, at least or approximately 87%, at least or approximately 88%, at least or approximately 89%, at least or approximately 90%, at least or approximately 91%, at least or approximately 92%, at least or approximately 93%, at least or approximately 94%, at least or approximately 95%, at least or approximately 96%, at least or approximately 97%, at least or approximately 98%, at least or approximately 99%, or approximately 100%.

[0105] The heavy chain variable region of the anti-CD19 antigen binding domain (V H) are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, and at least or about 8 It may include one, two, or three complementarity determination regions (CDRs) (CDR1, CDR2, and CDR3) that are identical by 6%, 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%.

[0106] In certain embodiments, the light chain variable region (V) of the anti-CD19 antigen binding region L The heavy chain variable region (VH) of the anti-BCMA antigen-binding region includes three CDRs (CDR1, CDR2, and CDR3) that are identical to CDR1, CDR2, and CDR3 (CDRs of the light chain variable region of the FMC63 antibody) described at positions 24-34, 50-56, and 89-97 of SEQ ID NO: 32, and the heavy chain variable region (VH) of the anti-BCMA antigen-binding region includes three CDRs (CDR1, CDR2, and CDR3) that are identical to CDR1, CDR2, and CDR3 (CDRs of the heavy chain variable region of the FMC63 antibody) described at positions 31-35, 50-65, and 98-109 of SEQ ID NO: 36.

[0107] In certain embodiments, the light chain variable region of the anti-CD19 antigen-binding region includes three CDRs (CDR1, CDR2, and CDR3) identical to those described in SEQ ID NOs. 15, 16, and 17 (CDRs of the light chain variable region (VL) of the FMC63 antibody), and the heavy chain variable region of the anti-BCMA antigen-binding region ( V H) contains three CDRs (CDR1, CDR2, and CDR3) that are identical to those described in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 (CDRs of the heavy chain variable region of the FMC63 antibody).

[0108] In certain embodiments, the antigen-binding domain of this CAR that targets BCMA is derived from the BCMA-20 (B20) antibody, and is a light chain variable domain V L (SEQ ID NO: 24) and heavy chain variable domain V H Includes (Sequence ID 28).

[0109] BCMA-20 antibody-derived light chain variable domain V L It may have the following sequence: [ka]

[0110] B20-VL-CDR1: Sequence ID 24, positions 24-34. The sequence of B20-VL-CDR1 is RASQGISNYLN (Sequence ID 2).

[0111] B20-VL-CDR2: Sequence ID 24, positions 50-56. The sequence for B20-VL-CDR2 is YTSNLQS(Sequence ID 24).

[0112] B20-VL-CDR3: Sequence ID 24, positions 89-97. The sequence of B20-VL-CDR3 is MGQTISSYT (Sequence ID 6).

[0113] BCMA-20 antibody-derived heavy chain variable domain V H It may have the following sequence: [ka] B20-VH-CDR1: Sequence ID 28, positions 31-35. The sequence of B20-VH-CDR1 is NFDMA (Sequence ID 9).

[0114] B20-VH-CDR2: Sequence ID 28, positions 50-66. The sequence of B20-VH-CDR2 is SITTGADHAIYADSVKG (Sequence ID 11).

[0115] B20-VH-CDR3: Sequence ID 28, positions 99-110. The sequence of B20-VH-CDR3 is HGYYDGYHLFDY (Sequence ID 13).

[0116] In certain embodiments, the antigen-binding domain of this CAR that targets CD19 is the light chain variable domain V derived from the FMC63 antibody. L (SEQ ID NO: 32) and heavy chain variable domain V H Includes (Sequence ID 36).

[0117] Light chain variable domain V derived from FMC63 antibody L It may have the following sequence: [ka] FMC63-VL-CDR1: Sequence ID 32, positions 24-34. The sequence of FMC63-VL-CDR1 is RASQDISKYLN (Sequence ID 15).

[0118] FMC63-VL-CDR2: Sequence ID 32, positions 50-56. The sequence for FMC63-VL-CDR2 is HTSRLHS (Sequence ID 16).

[0119] FMC63-VL-CDR3: Sequence ID 32, positions 89-97. The sequence of FMC63-VL-CDR3 is QQGNTLPYT (Sequence ID 17).

[0120] FMC63 antibody-derived heavy chain variable domain V H It may have the following sequence: [ka]

[0121] FMC63-VH-CDR1: Sequence ID 36, positions 31-35. The sequence for FMC63-VH-CDR1 is DYGVS (Sequence ID 18).

[0122] FMC63-VH-CDR2: Sequence ID 36, positions 50-66. The sequence for FMC63-VL-CDR2 is VIWGSETTYYNSALKS (Sequence ID 19).

[0123] FMC63-VH-CDR3: Sequence ID 36, positions 98-109. The sequence of FMC63-VH-CDR3 is HYYYGGSYAMDY (Sequence ID 20).

[0124] The signal peptide may be a CD8 signal peptide having the following sequence: MALPVTALLLPLALLLHAARP (Sequence ID 22) V of anti-BCMA scFv (linker-1) L and V H (or V H and V L The linker between them may have the following sequence: GGGGSGGGGSGGGGS (Sequence ID 26) The linker between anti-BCMA scFv and anti-CD19 scFv (linker-2) may have the following sequence: GGGGS (sequence number 30) V of anti-CD19 scFv (linker-3) L and V H (or V H and V L The linker between them may have the following sequence: GGGGSGGGGSGGGGS (Sequence ID 34) The hinge region between the extracellular domain (antigen-binding domain) and the transmembrane domain may originate from IgG4, CD8 (CD8a), CD28, CD137, or a combination thereof.

[0125] The hinge region may originate from IgG4 having the following sequence: ESKYGPPCPPCP(Sequence ID 38) The hinge region may originate from CD8a having the following arrangement: FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (Sequence ID 98) The transmembrane domain may be derived from CD28 (CD28™) having the following sequence: MFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 40).

[0126] The transmembrane domain may be derived from CD8 (CD8™) having the following sequence: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 100).

[0127] The co-stimulatory region may originate from 4-1BB having the following sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (Sequence ID 42) The cytoplasmic signaling domain may be derived from CD3ζ having the following sequence:RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 44).

[0128] Chimeric antigen receptor (CAR) The terms “chimeric antigen receptor” or “CAR” are used interchangeably throughout and refer, for example, to a recombinant polypeptide construct comprising 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 zeta chain associated with the T cell receptor complex. In one embodiment, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule. The co-stimulatory molecule may be 4-1BB (i.e., CD137), CD27, and / or CD28, or fragments of those molecules. In another embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from the stimulating molecule. CAR may comprise a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. Alternatively, CAR may comprise a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. CAR may also comprise a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. The antigen-binding region of CAR may contain any antigen-binding antibody fragment. The antibody fragment may comprise one or more CDRs, a variable region (or part thereof), a constant region (or part thereof), or any combination of the above.

[0129] The terms “zeta” or, instead, “zeta chain,” “CD3 zeta,” or “TCR zeta” refer to the protein provided as GenBank accession numbers NP_932170, NP_000725, or XP_011508447; or equivalent residues from non-human species (e.g., mouse, rodent, monkey, ape, etc.); and the “zeta-stimulating domain” or “CD3 zeta-stimulating domain” or “TCR zeta-stimulating domain” refer to amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally transmit the initial signals necessary for T cell activation.

[0130] A chimeric receptor may refer to a non-native 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, the chimeric receptor may further include one or more of the following: a hinge region, a transmembrane domain, at least one costimulatory region, and a cytoplasmic signaling domain. In some embodiments, the chimeric antigen receptor includes, from the N-terminus to the C-terminus, an antigen-binding region (or fragment), a hinge region, a transmembrane domain, and a cytoplasmic signaling domain. In some embodiments, the chimeric antigen receptor further includes at least one costimulatory region. Thus, the chimeric antigen receptor may include, from the N-terminus to the C-terminus, an antigen-binding region (or fragment), a hinge region, a transmembrane domain, a costimulatory region, and a cytoplasmic signaling domain.

[0131] In some embodiments, the chimeric antigen receptor includes a hinge region which may be located between the antigen-binding domain and the transmembrane domain. The hinge region may contain about 10 to 200 amino acids (e.g., 15 to 150 amino acids, 20 to 100 amino acids, or 30 to 60 amino acids). In some embodiments, the hinge region may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid lengths. The hinge region is configured to contain 0 to 300 amino acids, 2 to 100 amino acids, 5 to 80 amino acids, 10 to 60 amino acids, 10 to 15 amino acids, 20 to 80 amino acids, 30 to 70 amino acids, 40 to 60 amino acids, 50 to 60 amino acids, or 30 to 60 amino acids.

[0132] In some embodiments, the hinge region is a naturally occurring hinge domain of a protein. Any hinge domain of any protein known in the art to contain a hinge domain is suitable for use in a chimeric antigen receptor. In some embodiments, the hinge domain is of CD8α or CD28α. In some embodiments, the hinge domain is a part 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.

[0133] The hinge domain of an antibody (e.g., IgG, IgA, IgM, IgE, or IgD) is also suitable for use in chimeric antigen receptors. In some embodiments, the hinge region is a hinge domain that binds the constant domains CH1 and CH2 of the antibody. In some embodiments, the hinge region comprises the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge region comprises the hinge domain of the antibody and the CH3 constant region of the antibody. In some embodiments, the hinge region comprises the hinge domain of the antibody and the CH2 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 comprises the hinge region of the IgG4 antibody and the CH2 and CH3 constant regions. In some embodiments, the hinge region includes the hinge region of the IgG4 antibody and the constant CH3 region.

[0134] The hinge region may be a peptide that does not exist in nature. In some embodiments, the hinge region between the extracellular antigen-binding domain and the transmembrane domain is a peptide linker (e.g., a (GlyxSer)n (or (GxS)n) linker), where x and n are independently integers from 3 to 12 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more).

[0135] Additional peptide linkers that may be used in the hinge region of the chimeric receptors described herein are known in the art. See, for example, Wriggers et al. Current Trends in Peptide Science (2005) 80(6):736-746 and PCT Publication WO2012 / 088461.

[0136] In some embodiments, the chimeric antigen receptor may include a transmembrane domain. The transmembrane domain can take any form known in the art. A transmembrane domain suitable for use in the chimeric antigen receptor may be obtained from a naturally occurring protein. Alternatively, the transmembrane domain may be a synthetic, non-naturally occurring protein segment (e.g., a thermodynamically stable hydrophobic protein segment within the cell membrane).

[0137] In some embodiments, the transmembrane domain is CD8α. In some embodiments, the transmembrane domain is CD28. In some embodiments, the transmembrane domain is ICOS.

[0138] In some embodiments, the chimeric antigen receptor comprises one or more costimulatory regions. The costimulatory regions may be at least a portion of proteins that mediate signal transduction within a cell to induce an immune response (e.g., effector function). The costimulatory regions of the chimeric antigen receptor may originate from proteins that transmit signals and modulate immune cell-mediated responses (e.g., T cells, natural killer (NK) cells, macrophages, neutrophils, and eosinophils).

[0139] In some embodiments, the chimeric antigen receptor includes one or more (at least two, three, four, or more) costimulatory regions. In some embodiments, the chimeric antigen receptor includes multiple costimulatory regions derived from different proteins. In some embodiments, the chimeric antigen receptor does not include costimulatory regions.

[0140] Examples of co-stimulatory regions used in chimeric antigen receptors may be domains of the co-stimulatory protein (including, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, Cd40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, 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 containing multiple co-stimulatory regions or a portion of multiple co-stimulatory regions. In some embodiments, the co-stimulatory region is a fusion of co-stimulatory regions from CD28 and ICOS.

[0141] In some embodiments, the chimeric antigen receptor includes a cytoplasmic signaling domain. Any cytoplasmic signaling domain may be used in the chimeric antigen receptors described herein. The cytoplasmic signaling domain may transmit signals (e.g., interaction of an extracellular ligand-binding domain with its ligand (e.g., cytotoxicity)) to stimulate a cellular response (e.g., induce effector function of the cell).

[0142] Chimeric antigen receptors can be prepared by conventional methods (e.g., recombinant techniques). Methods for preparing chimeric antigen receptors may include generating nucleic acids encoding polypeptides containing each of the domains of the chimeric antigen receptor (e.g., an antigen-binding fragment, optionally a hinge region, a transmembrane domain, at least one costimulatory region, and a cytoplasmic signaling domain). In some embodiments, the nucleic acids encoding each component of the chimeric antigen receptor are linked using recombinant techniques. Nucleic acid sequences encoding the chimeric antigen receptor can be formed by directly or indirectly linking the sequences of the components (e.g., domains) (e.g., using a nucleic acid sequence encoding a peptide linker) using methods such as PCR amplification or ligation. Alternatively, nucleic acids encoding the chimeric antigen receptor can be synthesized. In some embodiments, the nucleic acid is DNA. In other embodiments, the nucleic acid is RNA.

[0143] In one embodiment, the CAR includes a signal peptide (also called a leader sequence), an antigen recognition sequence (antigen-binding domain), a hinge region, a transmembrane domain, a costimulatory region, and a cytoplasmic signaling domain (e.g., a CD3ζ signaling region (ζ chain portion)) from the N-terminus to the C-terminus.

[0144] 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.

[0145] As used herein, the terms “CAR-T cell,” “CAR-T,” “CART,” and “CART cell” may refer to T cells expressing this CAR that targets both BCMA and CD19.

[0146] Immune cells that express chimeric antigen receptors This disclosure provides immune cells expressing the CAR. When a target cell having an antigen(s) on its cell surface is recognized by the antigen-binding fragment of the chimeric antigen receptor, an activation signal is transmitted to the signaling domain(s) of the chimeric antigen receptor (e.g., the co-stimulatory region and / or cytoplasmic signaling domain), which can activate effector function within the immune cell expressing the chimeric antigen receptor.

[0147] Chimeric antigen receptors can be introduced into immune cells suitable for expression by conventional techniques. In some embodiments, the immune cells are T cells (e.g., primary T cells or T cell lines). Alternatively, the immune cells may be natural killer (NK) cells (e.g., established NK cell lines (e.g., NK-92 cells)). In some embodiments, the immune cells are CD8 cells (CD8 + ) or CD8 and CD4 (CD8 + / CD4 + These are T cells that express ). In some embodiments, the T cells are T cells from an established T cell line (e.g., Jurkat cells).

[0148] Primary T cells can be obtained from any source (e.g., peripheral blood mononuclear cells (PBMCs), bone marrow, tissue (e.g., spleen, lymph nodes, thymus), or tumor). In some embodiments, the population of immune cells originates from a human patient with autoimmune disorder or cancer (e.g., hematopoietic malignancies) (e.g., bone marrow or PBMCs obtained from the patient). In some embodiments, the population of immune cells originates from a healthy donor. In some embodiments, the immune cells are obtained from a target to which immune cells expressing chimeric antigen receptors are subsequently administered. Immune cells administered to the same target from which the cells were obtained are called autologous cells, while immune cells obtained from a different target to which the cells are administered are sometimes called allogeneic cells.

[0149] Desired types of immune cells can be proliferated within a cell population obtained by co-incubating cells with stimulating molecules. For example, anti-CD3 antibodies and anti-CD28 antibodies can be used to proliferate T cells.

[0150] 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 by conventional methods described herein and introduced into immune cells. For example, the nucleic acid encoding the chimeric antigen receptor can be cloned into a suitable vector (e.g., a viral vector).

[0151] In certain embodiments, a lentiviral vector (LV) encoding the CAR is introduced into immune cells (e.g., T cells). The introduced immune cells (e.g., T cells) target CD19 and BCMA, synergistically activating T cells and inducing a T cell-mediated immune response.

[0152] In one embodiment, the method involves isolating, activating, and genetically modifying T cells from the patient (or an allogeneic donor) to generate CAR-T cells expressing the CAR, which are then administered to the patient. The CAR-T cells replicate in vivo, which can lead to long-term persistence. Furthermore, the CAR-mediated immune response may be part of an adoptive immunotherapy approach in which anti-BCMA / CD19 CAR-T cells induce an immune response against cells expressing BCMA and / or CD19.

[0153] In certain embodiments, cells are isolated from a mammal (e.g., human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. CAR-modified cells can be administered to a mammalian receptor to provide a therapeutic effect. The mammalian receptor may be human. CAR-modified cells may be autologous to the recipient. Alternatively, the cells may be homogeneous, syngeneic, or heterogeneous to the recipient.

[0154] Methods for preparing immune cells expressing the chimeric antigen receptor may include activating and / or proliferating the immune cells ex vivo. Activating immune cells means stimulating them to an activated state, which may enable them to exert effector functions (e.g., cytotoxicity). The method for activating immune cells will vary depending on the type of immune cell used to express the chimeric antigen receptor. Proliferation of immune cells may include any method that results in an increase in the number of cells expressing the chimeric antigen receptor (e.g., enabling or stimulating cell proliferation). In some embodiments, the cells expressing the chimeric receptor described herein are activated and / or proliferated ex vivo before administration to a subject.

[0155] CAR-expressing immune cells can also function as vaccines for ex vivo immunization and / or in vivo therapy in mammals. In addition to the use of cell-based vaccines in ex vivo immunization, this disclosure also provides compositions and methods for in vivo immunization to induce an antigen-directed immune response in a patient. Preferably, the mammal is human. With respect to ex vivo immunization, one or more of the following may occur in vitro before administering the cells to a mammal: i) cell proliferation, ii) introduction of nucleic acids encoding the CAR into the cells, and / or iii) cryopreservation of the cells.

[0156] vector This disclosure provides nucleic acids that encode the CAR. This disclosure also provides vectors containing the nucleic acids.

[0157] Vectors include, but are not limited to, plasmids, phagemids, phage derivatives, viruses, and cosmids.

[0158] The vector may be a viral vector. Useful viruses as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In certain embodiments, the vector is a retroviral vector (e.g., a lentiviral vector). In some embodiments, the vector for the expression of a chimeric antigen receptor is a retrovirus. In some embodiments, the vector for the expression of a chimeric antigen receptor is a lentivirus. In some embodiments, the vector for the expression of a chimeric antigen receptor is an adeno-associated virus.

[0159] Various promoters (e.g., cytomegalovirus (CMV) intermediate early promoter, viral LTRs (e.g., Roussarcoma virus LTR), HIV-LTR, HTLV-1 LTR, Maloney's mouse leukemia virus (MMLV) LTR, myeloproliferative sarcoma virus (MPSV) LTR, splenic fociforming virus (SFFV) LTR, simian virus 40 (SV40) early promoter, herpes simplex TK virus promoter, and elongation factor 1-α (EF1-α) promoter with or without the EF1-α intron) can be used for chimeric receptor expression. Additional promoters for chimeric receptor expression include constitutively active promoters within immune cells. Alternatively, any moduloable promoter (e.g., inducible promoter) can also be used to regulate its expression within immune cells.

[0160] The vector can be introduced into cells (e.g., mammalian cells, bacterial cells, yeast cells, or insect cells) by any method in the art. For example, the vector can be introduced into cells by physical, chemical, or biological means.

[0161] Physical methods for introducing polynucleotides into cells include calcium phosphate precipitation, lipofection, particle impact, microinjection, and electroporation. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0162] Biological methods for introducing target polynucleotides into cells include the use of DNA vectors and RNA vectors. Viral vectors can be derived from retroviruses, lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others.

[0163] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems (e.g., polymer complexes, nanocapsules, microspheres, beads) and lipid-based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as a delivery medium in vitro and in vivo is liposomes (e.g., artificial membrane vesicles).

[0164] In some embodiments, the vector (nucleic acid) encoding the chimeric antigen receptor is a DNA vector that can be electroporated into immune cells (see, e.g., Till, et al. Blood (2012) 119(17):3940-3950). In some embodiments, the vector (nucleic acid) encoding the chimeric antigen receptor is an RNA molecule that can be electroporated into immune cells.

[0165] Any vector comprising nucleic acids encoding chimeric antigen receptors as described herein is within the scope of this disclosure. Such vectors can be delivered to host cells (e.g., immune cells) by appropriate means. Methods for delivering vectors to immune cells are well known in the art and may include electroporation of DNA, RNA, or transposons; transfection reagents (e.g., liposomes or nanoparticles for delivering DNA, RNA, or transposons); delivery of DNA, RNA, or transposons or proteins by mechanical deformation (see, for example, Sharei et al. PNAS (2013) 110(6):2082-2087); or viral transduction. In some embodiments, vectors for the expression of chimeric receptors are delivered to cells by viral transduction.

[0166] In cases where a vector encoding a chimeric antigen receptor is introduced into host cells using a viral vector, the vector can infect immune cells, and the viral particles carrying the vector can be prepared by any method known in the art. The viral particles can be collected from the cell culture supernatant and isolated and / or purified before contacting the viral particles with immune cells.

[0167] Pharmaceutical composition This disclosure provides a pharmaceutical composition comprising the immune cells, the CAR, the nucleic acid, or the vector. The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the preparation is a liquid preparation. In one embodiment, the concentration of immune cells (e.g., CAR-T cells) in the preparation is 1 × 10⁻⁶ 3 ~1 × 10 8 cells / mL, or 1 × 10⁶ 4 ~1 × 10 7 The concentration is cells / mL.

[0168] The effective dose varies depending on the specific condition being treated, the severity of the condition, the parameters of the individual patient (e.g., age, physical condition, size, sex, weight), the duration of treatment, the nature of any concomitant therapy, the specific route of administration, and similar factors within the scope of the knowledge and opinion of the healthcare professional, as is recognized by those skilled in the art. In some embodiments, the effective dose alleviates, reduces, improves, enhances, reduces, or slows the progression of the disease or disability. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0169] Pharmaceutically acceptable carriers (e.g., buffers) are well known in the art and may include phosphoric acid, citrate, and other organic acids; antioxidants (e.g., ascorbic acid and methionine); preservatives; low molecular weight polypeptides; proteins (e.g., serum albumin, gelatin, or immunoglobulin); 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 Ed. (2000) Lippincott Williams and Wilkins, Ed. KE. Hoover.

[0170] This pharmaceutical composition can be delivered to cells by bringing the cells into contact with the pharmaceutical composition.

[0171] This pharmaceutical composition may be delivered / administered to the target by any route (e.g., but not limited to intravenous, intraventricular (ICV) injection, intracisional injection or infusion, oral, transdermal, intraocular, intraperitoneal, subcutaneous, implant, sublingual, subcutaneous, intramuscular, rectal, mucosal, intraocular, intrathecal, intra-articular, intra-arterial, subarachnoid, bronchial, and lymphatic administration). This pharmaceutical composition may be administered parenterally or systemically. This composition may be administered topically. This pharmaceutical composition may be formulated for intravenous administration.

[0172] The composition may be administered by any convenient method (e.g., by aerosol inhalation, injection, ingestion, blood transfusion, implantation, or transplantation). The composition may be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intramedullarily, intramuscularly, intravenously (iv) or intraperitoneally. In one embodiment, the composition is administered to the subject (e.g., the patient) by intradermal or subcutaneous injection. In another embodiment, the composition is administered by intravenous injection. The composition may be injected directly into a tumor, lymph node, or site of injury.

[0173] These immune cells or pharmaceutical compositions may be delivered / administered to a target by intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration (e.g., injection or infusion).

[0174] This pharmaceutical composition may be administered in a manner appropriate to the disease to be treated (or prevented). The dosage and frequency of administration will be determined by factors such as the patient's condition, as well as the type and severity of the patient's disease, although the appropriate dosage may be determined through clinical trials.

[0175] Where indicated as “effective dose,” “therapeutic effective dose,” or “therapeutic dose,” the exact amount of the composition to be administered can be determined by a physician, taking into account the patient's age, weight, tumor size, degree of infection or metastasis, and individual differences in condition. If the pharmaceutical composition contains immune cells, 10 4 ~10 9 pieces / kg body weight, or 10 5 ~10 6 The drug may be administered in doses of cells / kg body weight (including all integer values ​​within these ranges). The composition may also be administered multiple times in these doses. Cells can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al, New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be easily determined by a person skilled in the art of medicine by monitoring the patient for signs of the disease and adjusting treatment accordingly.

[0176] The dosage of the above treatment to be administered to the patient may vary depending on the exact nature of the condition being treated and the recipient of the treatment. Scaling of the dosage to be administered to the patient can be carried out in accordance with established practices in the art. In one embodiment, 1 × 10 6 ~1 × 10 10 Individual immune cells (e.g., CAR-T cells) can be administered to the patient, for example, by intravenous infusion after each procedure or at each stage of the procedure.

[0177] Conditions that require treatment This CAR, immune cell, or pharmaceutical composition may be used to treat autoimmune diseases / disorders or to treat cancer or tumors.

[0178] In certain embodiments, the anti-BCMA / CD19 bispecific CAR may target both B cells and plasma cells, thereby reducing / eradicating autoimmune antibodies. In certain embodiments, the anti-BCMA / CD19 bispecific CAR may reduce / deplete B cells, plasmablasts, and / or long-lived plasma cells (LLPCs) to reduce / eradicate autoantibody production.

[0179] This disclosure provides a method for treating autoimmune diseases / disorders. The method may include administering CARs, immune cells, or pharmaceutical compositions to a subject in need.

[0180] Autoimmune disorders may include: systemic lupus erythematosus (SLE), lupus nephritis (LN), systemic sclerosis (SSc), CREST syndrome (calcification, Raynaud's syndrome, esophageal motility disorders, finger sclerosis, and telangiectasia), ocular clonus, inflammatory myopathy (e.g., polymyositis, dermatomyositis, and inclusion body myositis), myositis autoantibody-induced diseases, systemic sclerosis, primary biliary cirrhosis, celiac disease (e.g., gluten-induced bowel disease), herpetiform dermatitis, Miller-Fischer syndrome, acute motor axonal neuropathy (AMAN), multifocal motor neuropathy with conduction block, autoimmune hepatitis, and anti-inflammatory drugs. Lipid syndrome, Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, rheumatoid arthritis, chronic autoimmune hepatitis, scleromyositis, myasthenia gravis (MG), Lambert-Eaton myasthenic syndrome, Hashimoto's thyroiditis, Graves' disease, paraneoplastic cerebellar degeneration, Stiffperson syndrome, limbic encephalitis, Isaacs syndrome, Sydenham's chorea, childhood autoimmune streptococcal infection-associated neuropsychiatric disorders (PANDAS), encephalitis, type 1 diabetes, neuromyelitis optica (NMO), chronic inflammatory bowel disease, Hashimoto's disease, organ transplant rejection, and / or neuromyelitis optica spectrum disorder (NMOSD).

[0181] Autoimmune disorders may include pernicious anemia, Addison's disease, psoriasis, inflammatory bowel disease (IBD), psoriatic arthritis, Sjögren's syndrome, lupus erythematosus (e.g., discoid lupus erythematosus, drug-induced lupus erythematosus, and neonatal lupus erythematosus), multiple sclerosis, and / or reactive arthritis.

[0182] Autoimmune disorders may include: polymyositis, dermatomyositis, polyendocrine insufficiency, Schmidt syndrome, autoimmune uveitis, adrenal inflammatory bowel disease, thyroiditis, autoimmune thyroid disease, gastric mucosal atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, presenile dementia, demyelinating diseases, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, alopecia areata, bullous pemphigoid, scleroderma, progressive systemic sclerosis, adult-onset diabetes (e.g., type II diabetes), autoimmune infertility in men and women, ankylosing spondylitis, ulcerative colitis, Crohn's disease, sprue, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, juvenile Rheumatoid arthritis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture syndrome, Chagas disease, sarcoidosis, rheumatic fever, asthma, recurrent miscarriage, antiphospholipid syndrome, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird lung, allergic diseases, allergic encephalomyelitis, toxic epidermal necrolysis, alopecia, Alport syndrome, alveolitis, allergic alveolitis, fibrous alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndromeSyndrome, eczema, lymphomatous granulomatosis, Behçet's disease, Kaplan syndrome, Kawasaki disease, dengue fever, endocarditis, endocardial fibrosis, endophthalmitis, persistent erythema elevata, fetal erythroblastosis, eosinophilic fasciitis, Scholmann syndrome, Felty syndrome, filariasis, cyclitis, chronic cyclitis, metachronous cyclitis, Fuchs cyclitis, IgA nephropathy, Henoch-Schönlein purpura, graft-versus-host disease, transplant rejection, human immunodeficiency virus infection, echovirus Infections, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Hodgkin lymphoma and non-Hodgkin lymphoma, renal cell carcinoma, multiple myeloma, Eaton-Lambert syndrome, relapsing polychondritis, malignant melanoma, cryoglobulinemia, Waldenström macroglobulinemia, Epstein-Barr virus infection, mumps, Evans syndrome, and / or autoimmune gonadal dysfunction.

[0183] Autoimmune diseases 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 diabetes), multiple sclerosis, scleroderma, ankylosing spondylitis, sarcoid syndrome, pemphigus vulgaris, bullous pemphigoid, psoriasis, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, Behçet's syndrome, and Reiter's disease. Diseases, Buerger's disease, dermatomyositis, polymyositis, antineutrophil cytoplasmic antibody-associated vasculitis (e.g., granulomatosis with polyangiitis (also known as Wegener's granulomatosis), microscopic polyangiitis, and Churg-Strauss syndrome), scleroderma, Sjögren's syndrome, anti-glomerular basement membrane disease (e.g., Goodpasture 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.

[0184] Autoimmune diseases include: inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, sprue, autoimmune arthritis, rheumatoid arthritis, type 1 diabetes, multiple sclerosis, graft-versus-host disease after bone marrow transplantation, osteoarthritis, juvenile chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus, thyroiditis, asthma, psoriasis, scleroderma, atopic dermatitis, graft-versus-host disease, acute or chronic immune disorders associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, and Graves' disease. Diseases, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schönlein purpura, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, septic syndrome, cachexia, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's disease, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, polyglandular dysfunction type I and polyglandular dysfunction type II, Schmidt syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthropathy, arthropathy, Reiter's disease Psoriatic arthropathy, Chlamydia, Yersinia and Salmonella-associated arthropathy, spondyloarthropathy, atherosclerosis / pulsal sclerosis, atopic allergy, food allergy, autoimmune bullous disease, 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, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, idiopathic autoimmune hepatitis, acquired immunodeficiency syndrome, acquired immunodeficiency-related disease, hepatitis C, unclassifiable immunodeficiency (unclassifiable hypogastric disease) (Lumputh globulinemia), dilated cardiomyopathy, fibrous lung disease, idiopathic fibrous 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 disease-related lung disease, ankylosing spondylitis-related lung disease, vasculitic diffuse lung disease, hemosiderosis-related lung disease, drug-induced interstitial lung disease, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocyte-infiltrating lung disease, post-infectious interstitial lung disease,Gouty arthritis, autoimmune hepatitis, type 1 autoimmune hepatitis (classical autoimmune hepatitis or lupoid hepatitis), type 2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune hypoglycemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disorders associated with organ transplantation, chronic immune disorders associated with organ transplantation, osteoarthritis, primary sclerosing cholangitis, idiopathic leukopenia, idiopathic leukopenia, renal disease NOS, glomerulonephritis, microscopic vasculitis of the kidney, discoid lupus, lupus erythematosus, male infertility (idiopathic or NOS), sperm autoimmunity, multiple sclerosis (all subtypes), insulin-dependent diabetes mellitus, sympathetic ophthalmitis, pulmonary hypertension due to connective tissue disease, Goodpasture - Syndrome, pulmonary symptoms of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Takayasu's arteritis / arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goiterous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxedema, lens-induced uveitis, primary vasculitis, vitiligo, allergic rhinitis (pollen allergy), anaphylaxis, pet allergy, latex allergy, drug allergy, allergic rhinoconjunctivitis, eosinophilic esophagitis, eosinophilic syndrome, eosinophilic gastroenteritis, cutaneous lupus erythematosus, eosinophilic esophagitis, eosinophilic syndrome, and eosinophilic gastroenteritis.

[0185] Autoimmune disorders can be inflammatory muscle diseases. Inflammatory myopathy is a group of diseases that include chronic muscle inflammation, muscle weakness, and in some cases, muscle pain. The four main types of chronic or long-term inflammatory myopathy are: polymyositis (affecting the skeletal muscles on both sides of the body, the type involved in body movement); dermatomyositis (causing progressive muscle weakness); inclusion body myositis (characterized by slowly progressing muscle weakness and muscle atrophy and loss); and necrotizing autoimmune myopathy (with muscle weakness in the upper and lower body).

[0186] In another embodiment, the autoimmune disease is an autoimmune disease caused by the overexpression of B cells (e.g., lupus erythematosus).

[0187] This disclosure also includes methods for treating cancer. These methods may include administering CARs, immune cells, or pharmaceutical compositions to subjects in need.

[0188] This disclosure provides a chimeric antigen receptor for treating CD19-positive diseases (e.g., B-cell lymphoma).

[0189] This cancer may be a BCMA-positive malignant tumor. This cancer may be multiple myeloma (MM) or plasma cell leukemia.

[0190] Cancer may be a blood cancer. Cancer may be a plasma cell malignancy. Cancer may be a B cell malignancy. B cell malignancies may be acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), B cell leukemia, or B cell lymphoma.

[0191] The cancer may be Hodgkin lymphoma, non-Hodgkin lymphoma, leukemia, and / or multiple myeloma (MM).

[0192] The cancer may be acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0193] Diseases that can be treated with this CAR, immune cells, or pharmaceutical composition include CD19-positive tumors and diseases, such as those caused by an excess of B cells (e.g., autoimmune diseases, such as systemic lupus erythematosus). CD19-positive tumors may include CD19-positive non-solid tumors (e.g., hematological malignancies, such as leukemia and lymphoma) or solid tumors. Tumors or cancers to be treated with this CAR, immune cells, or pharmaceutical composition include, but are not limited to, carcinomas, blastomas, sarcomas, leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors (e.g., sarcomas, carcinomas, gastric cancer, peritoneal metastases 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 cancer, adrenal tumors, bladder tumors, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, and melanoma). This includes adult tumors / cancers and pediatric tumors / cancers.

[0194] Blood cancers are cancers of the blood or bone marrow. Examples of blood cancers (or hematogenous cancers) include leukemia (e.g., acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, 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 lymphoma (low-grade and high-grade), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia).

[0195] Cancer can be a solid tumor. Solid tumors can be benign or malignant. Different types of solid tumors are named based on the type of cells that form them (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors (e.g., sarcomas and carcinomas) include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, pancreatic cancer, and ovarian cancer.

[0196] kit Kits for using the CAR, immune cells, nucleic acids, vectors, or pharmaceutical compositions 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.

[0197] In some embodiments, the kit may include instructions for use in any of the methods described herein. The included instructions may include instructions for administering the pharmaceutical composition to a subject to achieve the intended activity in the subject. The kit may further include instructions for selecting a subject suitable for treatment based on the identification of whether the subject requires treatment. In some embodiments, the instructions for use include instructions for administering the pharmaceutical composition to a subject requiring treatment.

[0198] Instructions for use of a pharmaceutical composition generally include information regarding the dosage, administration schedule, and route of administration for the intended treatment. Containers may be unit doses, multi-dose packages (e.g., multi-dose packages) or sub-unit doses.

[0199] The kits provided herein are contained in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, and flexible packaging.

[0200] The following examples of specific ways of implementing this disclosure are provided for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0201] Example 1: Construction of an anti-BCMA / CD19 CAR The inventors have anti-BCMA scFv and anti-CD19 scFv in the same order (i.e., anti-BCMA scFv ("B20") followed by anti-CD19 ("FMC63")), but V H / V L Eight bispecific CARs were prepared with different sequences and distinct hinge regions and / or transmembrane domains: TB19-1 to TB19-4 and TB19-L1 to TB19-L4 (Figure 1).

[0202] Anti-BCMA / CD19 CAR-T cells were prepared using apheresis therapy from healthy donors. Specifically, PBMCs were separated from the venous blood of healthy donors by density gradient centrifugation. On day 0, PBMCs were activated in a cell culture flask pre-coated with CD3 monoclonal antibody (OKT3) and Retronectin (TAKARA). The medium was GT-551 cell culture medium containing 1% human albumin and 300 U / mL recombinant human interleukin 2 (IL-2). On day 3, a lentiviral vector encoding anti-BCMA / CD19 CAR was introduced into the activated PBMCs.

[0203] Figures 2A - 2B show the expression levels of anti-BCMA and anti-CD19 on the T cell surface. The expression level of anti-BCMA CAR was detected by flow cytometry using BCMA-Fc fusion protein; the expression level of anti-CD19 CAR was detected by flow cytometry using Protein L.

[0204] Example 2 In vitro antigen-specific activation of anti-BCMA / CD19 CAR-T cells When CAR-T cells were co-cultured with target cells, the antigen-specific activation of anti-BCMA / CD19 CAR-T was evaluated by assaying IFN-γ release and CD137 expression. Target cells ("T") included CD19-positive A549-CD19 tumor cells, BCMA-positive A549-BCMA tumor cells, CD19 and BCMA double-positive A549-CD19-BCMA tumor cells, Raji cells, MM.1S cells, and double-negative A549 tumor cells. Effector cells (E) were anti-BCMA / CD19 CAR-T cells.

[0205] PBMCs were isolated from venous blood of healthy donors by density gradient centrifugation. On day 0, PBMCs were activated in cell culture flasks pre-coated with CD3 monoclonal antibody (OKT3) and retronectin (TAKARA). The culture medium was GT-551 cell culture medium containing 1% human albumin and 300 U / mL recombinant human interleukin-2 (IL-2). On day 3, activated PBMCs were introduced into a lentiviral vector encoding anti-BCMA / CD19 CAR. Starting from day 6, CAR-T cells could be harvested for activity assays.

[0206] IFN-γ release was measured using CAR-T cells cultured for 7 days. 1 × 10⁶ 5 Each CAR-T cell was cultured for 18 hours in 200 μl of culture medium at a 1:1 E:T ratio with CD19-positive A549-CD19 tumor cells, BCMA-positive A549-BCMA tumor cells, CD19 and BCMA double-positive A549-CD19-BCMA tumor cells, double-negative A549 tumor cells, or no tumor cells (NT). Next, the levels of IFN-γ secreted into the cell culture supernatant were measured by ELISA.

[0207] CD137 expression levels were measured using CAR-T cells cultured for 7 days. 1 × 10⁶ 5 Each CAR-T cell was cultured for 18 hours in 200 μl of culture medium at a 1:1 E:T ratio with CD19-positive A549-CD19 tumor cells, BCMA-positive A549-BCMA tumor cells, CD19 and BCMA double-positive A549-CD19-BCMA tumor cells, double-negative A549 tumor cells, or no tumor cells. Next, the expression level of CD137 on the surface of the CAR-T cells was measured by flow cytometry.

[0208] The results of IFN-γ release are shown in Figures 3A and 3B. After co-culturing CAR-T cells with A549 cells expressing CD19 and / or BCMA antigens, anti-CD19 / BCMA CAR-T cells (TB19-1 to 4 and TB19-L1 to L4) can recognize CD19 single-positive BCMA and CD19 / BCMA double-positive target cells, as well as high levels of IFN-γ release. TB19-1 to TB19-4 showed high IFN-γ release when co-culturing with CD19 / BCMA-positive target cells.

[0209] Flow cytometry results showed that anti-CD19 / BCMA CAR-T cells were activated by various CD19 / BCMA single-positive or double-positive cells, upregulating CD137 expression levels (Figures 4A-4B).

[0210] Example 3: In vitro cytotoxicity of anti-CD19 / BCMA CAR-T cells Anti-BCMA / CD19 CAR-T cells were co-cultured with target cells at E:T ratios of 0:1, 1:1, 2:1, and 4:1, respectively. Cytotoxicity of CAR-T cells against target cells was evaluated using real-time cell analysis (RTCA) label-free technology.

[0211] The results show that anti-BCMA / CD19 CAR-T cells effectively killed BCMA / CD19 single-positive or double-positive tumor cells (A549-CD19, A549-BCMA, A549-BCMA-CD19) in vitro, but had no effect on A549 cells that did not express CD19 or BCMA (Figure 5).

[0212] Example 4: Cytotoxicity of anti-BCMA / CD19 CAR-T cells against autoreactive B cells in vitro. Recent studies have shown that in patients with systemic lupus erythematosus (SLE), CD11c hi T-bet +The proportion of B cell subsets is significantly increased and has been shown to be closely related to the production of autoantibodies and the clinical symptoms of patients. Autoantibodies are characteristic of reactive B cells (see below: 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). These subset of cells are also called aging-related B cells (ABCs) because they become concentrated with age in the peripheral blood of several animal models of autoimmune diseases and in patients with rheumatoid arthritis (see below: 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).

[0213] TLR7 activation plays a crucial 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 aging-related B cells (ABCs). ABCs are B cells with the potential to recognize self-antigens and produce autoantibodies that can target and damage the body's own tissues. Wang et al., Nature Communications, (2018) 9:1758.

[0214] To confirm that anti-BCMA / CD19 CAR-T cells also possess the ability to remove ABCs, the inventors prepared CAR-T cells from the peripheral blood of healthy human donors. The inventors also isolated autologous B cells from PBMCs of healthy donors, induced differentiation in vitro to obtain ABC-enriched autologous B cells, and then performed cytotoxicity experiments using these as target cells. After co-culture for 2-4 hours, the cytotoxicity of anti-BCMA / CD19 CAR-T cells derived from different donors was assayed compared to control T cells without CAR transduction.

[0215] In vitro ABC differentiation PBMCs from healthy donors are isolated by gradient centrifugation using a Ficol and cryopreserved. On the day of ABC differentiation, panB cells are first isolated from the thawed PBMCs using a human B cell isolation kit (Miltenyi Biotec; negative selection, e.g., labeling and depleting non-B cells) according to the manufacturer's instructions. Next, the B cells are seeded into a 96-well plate 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 medium is changed daily by replenishing with complete medium and stimulation cocktails. ABC induction is confirmed by FACS analysis. Antibodies for FACS staining include live / dead staining, anti-human CD19, CD38, CD27, IgD, CD11c, CD21, and T-bet.

[0216] Cytotoxicity assay After differentiation, anti-BCMA / CD19 CAR or non-transduced (NT) T cells are co-cultured with ABC-enriched B cells at the desired E:T ratio. After 24 hours, the cells are stained with LIVE / DEAD Fixable Aqua Dead Cell Stain (Invitrogen), and their viability is determined along with anti-CD19 and anti-CD3 antibodies to distinguish between B cells and T cells. Cytotoxicity is assessed by the viability of CD19 antibodies. +This is determined by the depletion of a certain percentage of cells. B cell lysis is calculated using the following formula: lysis rate (%) = (1 - (surviving CD19 + cell fraction in anti-BCMA / CD19 CAR co-culture / surviving CD19 + cell fraction in UT co-culture)) × 100. See also: Lin et al., Preclinical evaluation of CD8+ anti-BCMA mRNA CAR T-cells for treatment of multiple myeloma. Leukemia. 2021, 35(3):752-763.

[0217] Example 5: Inhibitory effect of anti-BCMA / CD19 CAR-T cells on mouse tumor cells We will evaluate the in vivo cytotoxic effects of anti-BCMA / CD19 CAR-T cells on CD19 or BCMA mono-positive cells in a mouse subcutaneous tumor model established using tumor cell lines expressing either CD19 (A549-CD19) or BCMA (MM.1S).

[0218] Female B-NDG mice aged 6-8 weeks were subcutaneously inoculated with A549-CD19 (CD19+) or MM.1S (BCMA+) cells. The average tumor volume was 100 mm². 3 When it reaches this stage, 3-5 × 10⁻¹⁶ anti-BCMA / CD19 CAR-T cells are produced. 6 The drug is administered via the tail vein at a dose of one CAR-T cell per mouse.

[0219] Specifically, female B-NDG(NOD.Cg-Prkdc scid Il2rg tm1Vst / Vst) Mouse, 5x10 6 A549-CD19 cells were subcutaneously inoculated into animals. The average tumor volume was approximately 100 mm². 3 When this is reached, 20 animals are selected and randomly divided into two groups of 10 each (vehicle control group vs. anti-BCMA / CD19 CAR group). Vehicle control or anti-BCMA / CD19 CAR T cells (3 × 10) 6 A single dose of CAR-T cells (animal) is administered to mice via tail vein injection.

[0220] The in vivo effect of anti-BCMA / CD19 CAR on BCMA single-positive target cells was evaluated, and female B-NDG (NOD.CB17-Prkdc scid Il2rg tm1 / Bcgen) 20 mice, 5 x 10 6 Individual MM.1S cells / mouse were subcutaneously inoculated. The average tumor volume was approximately 100 mm². 3 When the target is reached, 15 mice are selected and randomly divided into three groups of 5 mice each (one vehicle control group versus two anti-BCMA / CD19 CAR groups). Each mouse receives a single dose via tail vein injection. For anti-BCMA / CD19 CAR, the dose is 5 × 10⁶. 6 CAR-T cells / animals.

[0221] To evaluate the in vivo antitumor effect of anti-BCMA / CD19 CAR, 65 female B-NDG (NOD.CB17-Prkdc) were used. scid Il2rg tm1 / Bcgen) Mouse, 1x10 6 K562-CD19-BCMA cells were subcutaneously inoculated into animals. The average tumor volume was approximately 100 mm². 3 When this is reached, 50 mice are selected and randomly assigned to 5 groups (vehicle control group, T cell control group, anti-BCMA / CD19 CAR low-dose group (1 × 10)). 6 (1 CAR-T cells / mouse), medium dose group (5 × 10⁶) 6 (10 CAR-T cells / mouse), and high-dose group (10 × 10) 6 The CAR-T cells were divided into individual mice. The T cell control group was injected with non-transduced T cells from the same donor as the anti-BCMA / CD19 CAR group, and the dose was matched to the total T cell count of the high-dose anti-BCMA / CD19 CAR group. Each mouse received one dose via tail vein injection.

[0222] Example 6: Activity of anti-BCMA / CD19 CAR against autologous B cells derived from SLE patients To study CAR-T therapy for the treatment of autoimmune diseases (e.g., SLE), the inventors evaluate the efficiency of CAR-T cells in depleting autoreactive B cells. The inventors also study the effectiveness of CAR-T cells on remission and survival in the lupus model.

[0223] Efficiency of anti-BCMA / CD19 CAR in removing panB cells from lupus patients in vitro Collect 10 - 15 mL of peripheral blood samples from 8 SLE patients. The patients have different activities and autoantibody profiles, show different organ damages (preferably patients with lupus nephritis), and receive different treatments, so as to represent the heterogeneous nature of lupus patients. Recently, patients administered with B cell depletion antibodies are excluded.

[0224] In each sample, a part of the blood is used to isolate T cells for CAR-T production, and the remaining blood is used to isolate panB cells as targets for the cell lysis assay. T cells isolated from SLE patients are introduced with a lentiviral vector encoding anti-BCMA / CD19 CAR, and the expression of the CAR is tested.

[0225] Anti-BCMA / CD19 CAR-T cells or non-transduced (NT) T cells generated from patient samples are co-cultured with target cell lines expressing CD19 or / and BCMA, and the IFN-γ level is evaluated by ELISA (enzyme-linked immunosorbent assay).

[0226] PanB cells isolated from samples of 8 patients are co-cultured with autologous anti-BCMA / CD19 CAR-T cells or non-transduced (NT) T cells at the desired E:T (effector to target) ratio. After 24 hours, the co-culture supernatant is collected for ELISA to evaluate the IFN-γ level. Cytotoxicity is determined by fluorescence-activated cell sorting (FACS) and calculation of the depletion of the percentage of viable CD19+ panB cells. The cell lysis of B cells is calculated by the following formula: Lysis rate (%) = (1 - (fraction of viable CD19+ cells in anti-BCMA / CD19 CAR-T co-culture / fraction of viable CD19+ cells in UT co-culture)) × 100.

[0227] Efficiency of CAR-T to remove ABCs from lupus patients in vitro This study will investigate the efficiency of CAR-T receptors in vitro in removing ABCs, an essential subset of pathogenic B cells derived from lupus patients.

[0228] Blood samples or PBMCs from lupus patients will be processed for ABC differentiation and CAR-T production, as well as functional analysis.

[0229] The study will confirm the efficiency with which CAR-T cells deplete ABCs derived from lupus patients in vitro, under in vitro conditions.

[0230] Efficiency of CAR-T in vivo in depleting B cells and therapeutic efficacy Using a humanized mouse model of SLE, we will evaluate in vivo the efficiency of CAR-T cells in depleting B cells and their therapeutic efficacy. CD34 + Stem cell-humanized mice will be obtained. Two or more mice will be killed and their spleens collected using sterile techniques. Subsequently, T cells will be isolated from the spleens for CAR-T cell generation. The remaining mice will be used to induce the development of lupus disease, and if the induction is successful, the mice will be divided into groups to receive either CAR-T or control treatment (e.g., non-transduced T cells). Blood samples will be taken periodically from the mice to monitor the persistence of CAR-T cells and the efficiency of B cell depletion by FACS (e.g., ABC). Serum samples will be used to measure the titers of various autoantibodies. Urine samples will also be collected periodically to measure the level of proteinuria. At the end of the study, or if animals die early (presumably in the control group), tissues will be collected for histology (e.g., to examine the deposition of immune complexes in the kidneys and the severity of nephritis). The presence of B cells or plasma cells in affected tissues will also be examined. Survival curves will be generated compared to the effect of CAR-T on control treatment.

[0231] Table 1 shows the structures of the anti-BCMA / CD19 CARs, TB19-1 to TB19-4 and TB19-L1 to TB19-L4. [Table 1] [Table 2] [Table 3]

[0232] FMC63-V L -CDR1:RASQDISKYLN(Sequence ID 15) FMC63-V L -CDR2: HTSRLHS (Sequence ID 16) FMC63-VL-CDR3: QQGNTLPYT (Sequence ID 17)

[0233] FMC63-VH-CDR1: DYGVS (Sequence ID 18) FMC63-VH-CDR2:VIWGSETTYYNSALKS (Sequence ID 19) FMC63-VH-CDR3:HYYYGGSYAMDY (Sequence ID 20)

[0234] TN-B20-19-1(TB19-1) CD8a SP nucleic acid sequence (63nt) ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (Sequence No. 21)

[0235] CD8a SP amino acid sequence: [ka]

[0236] B20V L Nucleic acid sequence (321nt) Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgca gtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag (SEQ ID NO: 23)

[0237] B20 V L amino acid sequence [ka]

[0238] Linker-1 nucleic acid sequence (45nt) Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(Sequence No. 25)

[0239] Linker-1 amino acid sequence [ka]

[0240] B20 V H Nucleic acid sequence (363nt) Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccga ctccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc (Sequence number 27)

[0241] B20 V H amino acid sequence [ka]

[0242] Linker-2 nucleic acid sequence (15nt) GGAGGTGGTGGATCC (Sequence ID 29)

[0243] Linker-2 amino acid sequence [ka]

[0244] FMC63 V L Nucleic acid sequence (321nt) Gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattaca ctcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcaca (SEQ ID NO: 31)

[0245] FMC63 V L amino acid sequence [ka]

[0246] Linker-3 nucleic acid sequence (45nt) Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(Sequence 33)

[0247] Linker-3 amino acid sequence [ka]

[0248] FMC63 V H Nucleic acid sequence (360nt) Gaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcag ctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctca (SEQ ID NO: 35)

[0249] FMC63 V H amino acid sequence [ka]

[0250] IgG4 hinge nucleic acid sequence (36nt) GAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCT(Sequence No. 37)

[0251] IgG4 hinge amino acid sequence [ka]

[0252] CD28 TM (84nt) ATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTG (Sequence No. 39)

[0253] CD28™ amino acid sequence [ka]

[0254] 4-1BB costimulatory region nucleic acid sequence (126nt) Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg (SEQ ID NO: 41)

[0255] 4-1BB costimulatory region nucleic acid sequence [ka]

[0256] CD3z cytoplasmic signaling domain nucleic acid sequence (336nt) Agagtgaagttcagcaggagcgcagacgccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgtttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcct gtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa (SEQ ID NO: 43)

[0257] CD3z cytoplasmic signaling domain nucleic acid sequence [ka]

[0258] TB19-1 nucleic acid sequence (2118nt):

[0259] TB19-1 amino acid sequence:

Chemical formula

[0260] TN-B20-19-2 (TB19-2) CD8a SP nucleic acid sequence ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (SEQ ID NO: 47)

[0261] B20 V H Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc (SEQ ID NO: 48)

[0262] Linker-1 Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct (SEQ ID NO: 49)

[0263] B20 V L Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag (SEQ ID NO: 50)

[0264] Linker-2 GGAGGTGGTGGATCC (SEQ ID NO: 51)

[0265] FMC63 V L Gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcaca (SEQ ID NO: 52)

[0266] Linker-3 Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct (SEQ ID NO: 53)

[0267] FMC63 V H Gaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcag ctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctca (SEQ ID NO: 54)

[0268] Hinge GAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCT(Sequence ID 55)

[0269] CD28 TM ATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTG (Sequence No. 56)

[0270] 4-1BB costimulation area AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 57)

[0271] CD3z cytoplasmic signaling domain CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGGAAGAACCCCCAGGAAGCCTGTATAACGAACTGCAGAAAACAAGATGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGGCGGGCAAGGCCACGACGGGCCGTGATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG (sequence number 58)

[0272] TB19-2(2118nt)

[0273] TB19-2 Amino Acid Sequence

Chem.

[0274] TN-B20-19-3 (TB19-3) CD8a SP ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (SEQ ID NO: 61)

[0275] B20 V H Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc (SEQ ID NO: 62)

[0276] Linker-1 Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct (SEQ ID NO: 63)

[0277] B20 V L Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag(SEQ ID NO: 64)

[0278] Linker-2 GGAGGTGGTGGATCC(SEQ ID NO: 65)

[0279] FMC63 V H Gaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctca(SEQ ID NO: 66)

[0280] Linker-3 Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(SEQ ID NO: 67)

[0281] FMC63 V L Gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattaca ctcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcaca (SEQ ID NO: 68)

[0282] Hinge GAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCT(Sequence No. 69)

[0283] CD28 TM TGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTG(Sequence No. 70)

[0284] 4-1BB costimulation area AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 71)

[0285] CD3z cytoplasmic signaling domain CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGGAAGAACCCCCAGGAAGCCTGTATAACGAACTGCAGAAAACAAGATGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGGCGGGCAAGGCCACGACGGGCCGTGATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG (sequence number 72)

[0286] TB19-3(2118nt)

[0287] TB19-3 amino acid sequence

Chem.

[0288] TN-B20-19-4 (TB19-4) CD8a SP ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (SEQ ID NO: 75)

[0289] B20 V L Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag (SEQ ID NO: 76)

[0290] Linker-1 Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct (SEQ ID NO: 77)

[0291] B20 V H Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc(SEQ ID NO: 78)

[0292] Linker-2 GGAGGTGGTGGATCC(SEQ ID NO: 79)

[0293] FMC63 V H Gaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctca(SEQ ID NO: 80)

[0294] Linker-3 Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(Sequence No. 81)

[0295] FMC63 V L Gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattaca ctcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcaca (SEQ ID NO: 82)

[0296] Hinge GAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCT(Sequence No. 83)

[0297] CD28 TM ATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTG (Sequence No. 84)

[0298] 4-1BB costimulation area AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 85)

[0299] CD3z cytoplasmic signaling domain CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGGAAGAACCCCCAGGAAGCCTGTATAACGAACTGCAGAAAACAAGATGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGGCGGGCAAGGCCACGACGGGCCGTGATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG (sequence number 86)

[0300] TB19-4(2118nt)

[0301] TB19-4 amino acid sequence

Chemical formula

[0302] TN-B20-19-L1 (TB19-L1) CD8a SP (63nt) ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (SEQ ID NO: 89)

[0303] B20 V L (321nt) Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag (SEQ ID NO: 90)

[0304] Linker-1 (45nt) Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct (SEQ ID NO: 91)

[0305] B20 V H Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc(SEQ ID NO: 92)

[0306] Linker-2 (15 nt) GGAGGTGGTGGATCC(SEQ ID NO: 93)

[0307] FMC63 V L (321 nt) Gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcaca(SEQ ID NO: 94)

[0308] Linker-3 (45 nt) Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(SEQ ID NO: 95)

[0309] FMC63 V H (360nt) Gaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcag ctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctca (SEQ ID NO: 96)

[0310] CD8a hinge (165nt) Ttcgtgccggtcttcctgccagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat (SEQ ID NO: 97)

[0311] CD8a Hinge Amino Acid Sequence [ka]

[0312] CD8a TM(72nt) Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc(Sequence ID 99)

[0313] CD8a™ amino acid sequence [ka]

[0314] 4-1BB costimulatory region (126nt) Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg (SEQ ID NO: 101)

[0315] CD3z cytoplasmic signaling domain (336nt) Agagtgaagttcagcaggagcgcagacgccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgtttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctg tacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa (SEQ ID NO: 102)

[0316] TB19-L1(2235nt)

[0317] TB19-L1 amino acid sequence [ka]

[0318] TN-B20-19-L2 (TB19-L2) CD8a SP(63nt) ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (Sequence No. 105)

[0319] B20 V H Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaac ttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgac tccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc (Sequence number 106)

[0320] Linker-1 (45nt) ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(L107)

[0321] Linker-1 amino acid sequence GGGGSGGGGSGGGGS (Sequence No. 108)

[0322] B20 V L(321nt) Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcag tccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag (SEQ ID NO: 109)

[0323] Linker-2 (15nt) GGAGGTGGTGGATCC (Sequence ID 110)

[0324] Linker-2 amino acid sequence: GGGGS (Sequence ID 111)

[0325] FMC63 V L (321nt) Gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacac tcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcaca (SEQ ID NO: 112)

[0326] Linker-3 (45nt) Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(Sequence No. 113)

[0327] Linker-3 amino acid sequence GGGGSGGGGSGGGGS (Sequence No. 114)

[0328] FMC63 V H (360nt) Gaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcag ctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctca (SEQ ID NO: 115)

[0329] CD8a hinge (165nt) Ttcgtgccggtcttcctgccagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat (SEQ ID NO: 116)

[0330] CD8a TM(72nt) Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc(Sequence ID 117)

[0331] 4-1BB costimulatory region (126 nt) Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg (SEQ ID NO: 118)

[0332] CD3z cytoplasmic signaling domain (336 nt) Agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa (SEQ ID NO: 119)

[0333] TB19-L2 (2235 nt)

[0334] TB19-L2 amino acid sequence [ka]

[0335] TN-B20-19-L3 (TB19-L3) CD8a SP(63nt) ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (Sequence No. 122)

[0336] B20 V H Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaac ttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgac tccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc (Sequence number 123)

[0337] Linker-1 (45nt) ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(Sequence No. 124)

[0338] Linker-1 amino acid sequence: GGGGSGGGGSGGGGS (Sequence No. 125)

[0339] B20 V L(321 nt) Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcagtccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag (SEQ ID NO: 126)

[0340] Linker-2 (15 nt) GGAGGTGGTGGATCC (SEQ ID NO: 127)

[0341] Linker-2 amino acid sequence GGGGS (SEQ ID NO: 128)

[0342] FMC63 V H (360 nt) Gaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctca (SEQ ID NO: 129)

[0343] Linker-3 (45nt) Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(Sequence No. 130)

[0344] Linker-3 amino acid sequence GGGGSGGGGSGGGGS (Sequence No. 131)

[0345] FMC63 V L (321nt) Gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacac tcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcaca (SEQ ID NO: 132)

[0346] CD8a hinge (165nt) Ttcgtgccggtcttcctgccagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgaggggggctggacttcgcctgtgat (SEQ ID NO: 133)

[0347] CD8a TM(72nt) Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc(Sequence ID 134)

[0348] 4-1BB costimulatory region (126nt) Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg (SEQ ID NO: 135)

[0349] CD3z cytoplasmic signaling domain (336 nt) Agagtgaagttcagcaggagcgcagacgccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgtttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctg tacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa (SEQ ID NO: 136)

[0350] TB19-L3(2235nt)

[0351] TB19-L3 amino acid sequence [ka]

[0352] TN-B20-19-L4 (TB19-L4) CD8a SP(63nt) ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (Sequence No. 139)

[0353] B20 V L (321nt) Gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgggtgaccatcacctgccgggcctcccagggcatctccaactacctgaactggtaccagcagaagcccggcaaggcccccaagcccctgatctactacacctccaacctgcag tccggcgtgccctcccggttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgcatgggccagaccatctcctcctacaccttcggccagggcaccaagctggagatcaag (SEQ ID NO: 140)

[0354] Linker-1 (45nt) ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(Sequence No. 141)

[0355] Linker-1 amino acid sequence: GGGGSGGGGSGGGGS (Sequence No. 142)

[0356] B20 V H Gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccctgcggctgtcctgcgccgcctccggcttcaccttctccaacttcgacatggcctgggtgcggcaggcccccggcaagggcctggtgtgggtgtcctccatcaccaccggcgccgaccacgccatctacgccgactccgtgaagggccggttcaccatctcccgggacaacgccaagaacaccctgtacctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgtgcggcacggctactacgacggctaccacctgttcgactactggggccagggcaccctggtgaccgtgtcctcc(SEQ ID NO: 143)

[0357] Linker-2 (15 nt) GGAGGTGGTGGATCC(SEQ ID NO: 144)

[0358] Linker-2 amino acid sequence GGGGS(SEQ ID NO: 145)

[0359] FMC63 V H (360 nt) Gaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctca(SEQ ID NO: 146)

[0360] Linker-3 (45nt) Ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct(Sequence No. 147)

[0361] Linker-3 amino acid sequence GGGGSGGGGSGGGGS (Sequence No. 148)

[0362] FMC63 V L (321nt) Gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacac tcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcaca (SEQ ID NO: 149)

[0363] CD8a hinge (165nt) Ttcgtgccggtcttcctgccagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat (SEQ ID NO: 150)

[0364] CD8a TM(72nt) Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc(Sequence ID 151)

[0365] 4-1BB costimulatory region (126nt) Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg (SEQ ID NO: 152)

[0366] CD3z cytoplasmic signaling domain (336nt) Agagtgaagttcagcaggagcgcagacgccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgtttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctg tacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaa (SEQ ID NO: 153)

[0367] TB19-L4(2235nt)

[0368] TB19-L4 amino acid sequence [ka]

[0369] The scope of the present invention is not limited to what is specifically shown and described above. Those skilled in the art will understand that there are suitable alternatives to the examples of materials, composition, structure, and dimensions shown. Numerous references (e.g., patents and various publications) are cited and discussed in the description of the present invention. Such citations and discussions of references are provided solely to clarify the description of the present invention and do not constitute an admission that any reference is prior art to the present invention as described herein. All references cited and discussed herein are incorporated herein by reference in their entirety. Variations, modifications, and other implementations described herein will be conducive to those skilled in the art without departing from the spirit and scope of the present invention. While specific embodiments of the present invention are shown and described, it will be apparent to those skilled in the art that changes and modifications may be made without departing from the spirit and scope of the present invention. Matters described above and in the accompanying drawings are provided only as examples and not as limitations.

Claims

1. A bispecific chimeric antigen receptor (CAR), (i) Anti-BCMA antigen binding region, light chain variable region (V Ｌ 1) and heavy chain variable region (V Ｈ 1) including V Ｌ 1 includes 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 described in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, and V Ｈ 1 comprises the anti-CD20 antigen binding region, which includes 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 described in SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13; and (ii) Anti-CD19 antigen binding region, light chain variable region (V Ｌ 2) and heavy chain variable region (V Ｈ 2) including V Ｌ 2 comprises 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 described in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and V Ｈ The bispecific chimeric antigen receptor comprising the anti-BCMA antigen-binding region, wherein 2 comprises three CDRs (CDR1, CDR2, and CDR3) each having an amino acid sequence approximately 80% to 100% identical to the amino acid sequences described in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:

20.

2. A bispecific chimeric antigen receptor (CAR), (i) An anti-BCMA antigen-binding region comprising a light chain variable region (V Ｌ 1) and a heavy chain variable region (V Ｈ 1); and (ii) Light chain variable region (V Ｌ 2) and heavy chain variable region (V Ｈ The bispecific chimeric antigen receptor comprising an anti-CD19 antigen-binding region including (2).

3. V Ｌ 1 is V Ｈ A bispecific CAR according to claim 1 or 2, located at the N-terminus of 1.

4. V Ｌ 2 is V Ｈ A bispecific CAR according to claim 1 or 2, located at the N-terminus of 2.

5. V Ｈ 1 is V Ｌ A bispecific CAR according to claim 1 or 2, located at the N-terminus of 1.

6. V Ｈ 2 is V Ｌ A bispecific CAR according to claim 1 or 2, located at the N-terminus of 2.

7. V Ｌ 1 and V Ｈ The bispecific CAR according to any one of claims 1 to 6, wherein each of the amino acids has an amino acid sequence that is approximately 80% to approximately 100% identical to the amino acid sequences described in SEQ ID NO: 24 and SEQ ID NO:

28.

8. V Ｌ 2 and V Ｈ The bispecific CAR according to any one of claims 1 to 7, wherein each of the two amino acids has an amino acid sequence that is approximately 80% to approximately 100% identical to the amino acid sequences described in SEQ ID NO: 32 and SEQ ID NO:

36.

9. The bispecific CAR according to any one of claims 1 to 8, wherein the anti-BCMA antigen-binding region is a single-chain variable fragment (scFv) that specifically binds to BCMA, and the anti-CD19 antigen-binding region is an scFv that specifically binds to CD19.

10. The bispecificity CAR according to any one of claims 1 to 9 further comprises one or more of the following: (a) signal peptide, (b) Hinge region, (c) Transmembrane domain, (d) co-stimulation area, and (e) Cytoplasmic signaling domain.

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

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

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

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

15. A bispecific CAR according to any one of claims 1 to 14, comprising an amino acid sequence that is approximately 80% to approximately 100% identical to the amino acid sequence described in SEQ ID NO: 46, SEQ ID NO: 60, SEQ ID NO: 74, SEQ ID NO: 88, SEQ ID NO: 104, SEQ ID NO: 121, SEQ ID NO: 138, or SEQ ID NO:

155.

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

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

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

19. A vector comprising the nucleic acid described in claim 18.

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

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

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

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

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

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

26. The method according to claim 25, wherein the cancer is a blood cancer.

27. The method according to claim 25, wherein the cancer is a B-cell malignant tumor.

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

29. The method according to claim 25, wherein the cancer is 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.

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

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