Bispecific chimeric antigen receptor targeting BCMA-CD19 and uses thereof

Abstract

The present invention provides a bispecific chimeric antigen receptor targeting BCMA-CD19 and its use. The bispecific chimeric antigen receptor comprises an extracellular antigen recognition domain, the extracellular antigen recognition domain comprises an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain, the anti-BCMA extracellular antigen recognition domain comprises a BCMA VH and a BCMA VL, the amino acid sequences of the BCMA VH complementarity determining regions CDR1, CDR2 and CDR3 comprise the amino acid sequences represented by SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the amino acid sequences of the BCMA VL complementarity determining regions CDR1, CDR2 and CDR3 comprise the amino acid sequences represented by SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

Description

[Technical field]

[0001] The present application relates to the field of biomedicine, and in particular to a bispecific chimeric antigen receptor targeting BCMA-CD19 and uses thereof. [Background technology]

[0002] Defined as a malignant proliferation of plasma cells in the bone marrow, multiple myeloma accounts for 1% of all cancer types and is the second most common hematological malignancy. Studies have found that multiple myeloma is more prevalent in people over the age of 60, and its incidence has been steadily increasing in recent years. For most patients, multiple myeloma is incurable and eventually progresses to relapsed / refractory multiple myeloma. Patients with relapsed / refractory multiple myeloma that is ineffective against existing multiple myeloma treatments (such as immunomodulators, proteasome inhibitors, and antibody drugs) have a survival time of only about 13 months.

[0003] B-cell maturation antigen (BCMA) is a transmembrane glycoprotein that belongs to the tumor necrosis factor receptor family. BCMA is highly expressed in multiple myeloma cells, but not in most other cells. Malignant tumor plasma cells usually express higher levels of BCMA than normal plasma cells, and upregulation of BCMA promotes the proliferation of multiple myeloma cancer cells, whereas downregulation of its expression can inhibit the proliferation of multiple myeloma cancer cells.

[0004] Also, multiple myeloma, like B-cell lineage tumors, generally does not express the CD19 molecule, so CD19 is generally not a target for multiple myeloma treatment. However, some literature suggests that mild drug resistance and relapsed multiple myeloma clones also express CD19. + May have a phenotype This suggests that...

[0005] Chimeric antigen receptors (CARs) are the core components of CAR cell therapeutics and may include a targeting moiety (e.g., a moiety that binds to tumor-associated antigens (TAAs)), a hinge region, a transmembrane region, and an intracellular domain. CAR-T cell immunotherapy is considered one of the most promising methods for overcoming tumors. CAR-T cells use genetic engineering methods to make T cells express CAR proteins, which have the ability to recognize unprocessed proteins on the membrane surface independent of antigen presentation, resulting in T cell activation and functional effects. Summary of the Invention [Problem to be solved by the invention]

[0006] In 2021, Bristol-Myers Squibb and Bluebird Bio jointly announced that the U.S. Food and Drug Administration (FDA) has approved a CAR-T cell therapy targeting BCMA (bb2121). This CAR-T cell therapy is for adult patients with relapsed or refractory multiple myeloma after fourth-line treatment (including immunomodulators, proteasome inhibitors and antibody drug therapies). This is the world's first CAR-T cell therapy targeting BCMA. Developing more effective cell therapies targeting BCMA is of practical importance. Effect of the Invention

[0007] This application provides a bispecific chimeric antigen receptor targeting BCMA-CD19 and its use. The inventors constructed multiple expression vectors of bispecific chimeric antigen receptor targeting BCMA-CD19 and prepared bispecific CAR-T cells targeting BCMA-CD19. The inventors also confirmed that BCMA-CD19 bispecific CAR-T cells have good anti-tumor function at the cellular level. [Means for solving the problem]

[0008] an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain, the extracellular antigen recognition domain comprising an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain; A bispecific chimeric antigen receptor targeting BCMA-CD19, wherein the anti-BCMA extracellular antigen recognition domain comprises a BCMA VH and a BCMA VL, the amino acid sequences of the BCMA VH complementarity determining regions CDR1, CDR2 and CDR3 comprise the amino acid sequences represented by SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the amino acid sequences of the BCMA VL complementarity determining regions CDR1, CDR2 and CDR3 comprise the amino acid sequences represented by SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0009] In one embodiment of the bispecific chimeric antigen receptor described above, the anti-CD19 extracellular antigen recognition domain comprises CD19 VH and CD19 VL, the amino acid sequences of the CD19 VH complementarity determining regions CDR1, CDR2 and CDR3 comprise the amino acid sequences represented by SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively, and the amino acid sequences of the CD19 VL complementarity determining regions CDR1, CDR2 and CDR3 comprise the amino acid sequences represented by SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0010] In certain embodiments of the bispecific chimeric antigen receptor described above, the BCMA VH sequence comprises the amino acid sequence represented by SEQ ID NO:13 and the BCMA VL sequence comprises the amino acid sequence represented by SEQ ID NO:14.

[0011] In certain embodiments of the bispecific chimeric antigen receptor described above, the BCMA VH sequence comprises the amino acid sequence represented by SEQ ID NO:15 and the BCMA VL sequence comprises the amino acid sequence represented by SEQ ID NO:16.

[0012] In one embodiment of the bispecific chimeric antigen receptor described above, the CD19 VH sequence comprises the amino acid sequence represented by SEQ ID NO:17, and the CD19 VL sequence comprises the amino acid sequence represented by SEQ ID NO:18.

[0013] In certain embodiments of the bispecific chimeric antigen receptor described above, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor comprises any one structure selected from the group consisting of CD19 VL sequence-first linker sequence-CD19 VH sequence-second linker sequence-BCMA VL sequence-third linker sequence-BCMA VH sequence, BCMA VL sequence-fourth linker sequence-BCMA VH sequence-fifth linker sequence-CD19 VL sequence-sixth linker sequence-CD19 VH sequence, BCMA VL sequence-seventh linker sequence-CD19 VL sequence-eighth linker sequence-CD19 VH sequence-ninth linker sequence-BCMA VH sequence and CD19 VL sequence-tenth linker sequence-BCMA VL sequence-eleventh linker sequence-BCMA VH sequence-twelfth linker sequence-CD19 VH sequence.

[0014] In one embodiment of the bispecific chimeric antigen receptor described above, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor comprises any one structure selected from the group consisting of BCMA VL sequence-7th linker sequence-CD19 VL sequence-8th linker sequence-CD19 VH sequence-9th linker sequence-BCMA VH sequence and CD19 VL sequence-10th linker sequence-BCMA VL sequence-11th linker sequence-BCMA VH sequence-12th linker sequence-CD19 VH sequence.

[0015] In certain embodiments of the bispecific chimeric antigen receptor described above, the first linker sequence, the second linker sequence, the third linker sequence, the fourth linker sequence, the fifth linker sequence, the sixth linker sequence, the seventh linker sequence, the eighth linker sequence, the ninth linker sequence, the tenth linker sequence, the eleventh linker sequence and the twelfth linker sequence are each independently selected from one or more of SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0016] In certain embodiments of the bispecific chimeric antigen receptor described above, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor comprises the amino acid sequence represented by SEQ ID NO: 19 or SEQ ID NO: 20.

[0017] In certain embodiments of the bispecific chimeric antigen receptor described above, The hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84 and CD8α, optionally, the amino acids of the hinge region are derived from CD8α, and further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence set forth in SEQ ID NO:21.

[0018] In certain embodiments of the bispecific chimeric antigen receptor described above, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40 and Fc70, optionally, the amino acids of the transmembrane region are derived from CD8α, and further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence set forth in SEQ ID NO:22.

[0019] In certain embodiments of the bispecific chimeric antigen receptor described above, the intracellular domain comprises an intracellular signaling region, and optionally, the intracellular domain further comprises a costimulatory signaling region.

[0020] In certain embodiments of the bispecific chimeric antigen receptor described above, the intracellular signaling region is derived from one or more of CD3zeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, FcRgamma, FcRbeta, CD66d, DAP10, DAP12, and Syk, optionally, the intracellular signaling region is derived from CD3zeta, and further optionally, the amino acid sequence of the intracellular signaling region comprises the amino acid sequence set forth in SEQ ID NO:23.

[0021] In certain embodiments of the bispecific chimeric antigen receptors described above, the costimulatory signaling region is derived from one or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88; optionally, the costimulatory signaling region is derived from CD28 or 4-1BB; and further optionally, the amino acid sequence of the costimulatory signaling region comprises the amino acid sequence set forth in SEQ ID NO:24.

[0022] In certain embodiments of the bispecific chimeric antigen receptor described above, the bispecific chimeric antigen receptor further comprises a guide peptide located N-terminal to the amino acid sequence of the chimeric antigen receptor, optionally, the guide peptide is derived from CD8α, and further optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence set forth in SEQ ID NO:25.

[0023] In certain embodiments of the bispecific chimeric antigen receptor described above, the bispecific chimeric antigen receptor comprises the amino acid sequence represented by SEQ ID NO:28 or SEQ ID NO:29.

[0024] The present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the above-described bispecific chimeric antigen receptor.

[0025] In certain embodiments of the isolated nucleic acid molecule, the nucleotide sequence encoding the bispecific chimeric antigen receptor is 1) a nucleotide sequence encoding the BCMA VH amino acid sequence represented by SEQ ID NO: 13, represented by SEQ ID NO: 37, and a nucleotide sequence encoding the BCMA VL amino acid sequence represented by SEQ ID NO: 14, represented by SEQ ID NO: 38, and / or 2) It comprises a nucleotide sequence represented by SEQ ID NO:39, which encodes the CD19 VH amino acid sequence represented by SEQ ID NO:17, and a nucleotide sequence represented by SEQ ID NO:40, which encodes the CD19 VL amino acid sequence represented by SEQ ID NO:18.

[0026] The present application also provides a vector comprising the above-described isolated nucleic acid molecule.

[0027] In certain embodiments of the above vectors, the vector is an expression vector, in certain embodiments the vector is a viral vector, and in certain embodiments the vector is a lentiviral vector.

[0028] The present application also provides an engineered immune effector cell comprising the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned vector.

[0029] In certain embodiments of the engineered immune effector cells described above, the engineered immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells.

[0030] In certain embodiments of the engineered immune effector cells described above, said engineered immune effector cells are T lymphocytes, and optionally the source of said T lymphocytes is autologous or allogeneic T lymphocytes.

[0031] The present application also provides a pharmaceutical composition comprising the engineered immune effector cells described above and a pharma- ceutically acceptable adjuvant.

[0032] In certain embodiments of the above pharmaceutical composition, the pharma- ceutically acceptable adjuvant comprises a protectant.

[0033] In certain embodiments of the above pharmaceutical composition, the pharma- ceutically acceptable adjuvant comprises a cell cryopreservation solution.

[0034] In certain embodiments, the pharmaceutical composition is an intravenous injection.

[0035] The application also provides the use of said chimeric antigen receptor, said isolated nucleic acid molecule, said vector, or said engineered immune effector cell in the preparation of a medicament for use in the treatment of a disease or condition associated with expression of BCMA.

[0036] In certain embodiments of the above uses, the disease or condition associated with BCMA expression is cancer, optionally, the cancer is multiple myeloma, further optionally, the cancer is refractory or relapsed multiple myeloma.

[0037] In certain embodiments of the above uses, the disease or condition associated with BCMA expression is an autoimmune disease.

[0038] In certain embodiments of the above uses, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.

[0039] The present application also provides a method of treating a disease or condition associated with BCMA expression, comprising administering to a subject in need thereof an effective amount of the engineered immune effector cells or the pharmaceutical composition described above.

[0040] In certain embodiments of the above methods, the disease or condition associated with BCMA expression is cancer, optionally, the cancer is multiple myeloma, and further optionally, the cancer is refractory or relapsed multiple myeloma.

[0041] In certain embodiments of the above methods, the disease or condition associated with BCMA expression may be an autoimmune disease.

[0042] In certain embodiments of the above methods, the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.

[0043] In certain embodiments of the above methods, the method of administration is intravenous injection.

[0044] In certain embodiments of the above methods, the method of administration is administering an effective amount of said engineered immune effector cells or said pharmaceutical composition to the subject in a single injection.

[0045] In one embodiment of the above method, the effective amount of the engineered immune effector cells or the pharmaceutical composition is 1×10 5 ~1×10 7 cells / kg.

[0046] The present application also provides an engineered immune effector cell as described above or a pharmaceutical composition as described above for use in treating a disease or condition associated with expression of BCMA.

[0047] In certain embodiments of said engineered immune effector cell or said pharmaceutical composition, said disease or condition associated with BCMA expression is cancer, optionally, said cancer is multiple myeloma, further optionally, said cancer is refractory or relapsed multiple myeloma.

[0048] In certain embodiments of said engineered immune effector cells or said pharmaceutical compositions, said disease or condition associated with expression of BCMA may be an autoimmune disease.

[0049] In certain embodiments of said engineered immune effector cells or said pharmaceutical composition, said autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia. [Brief description of the drawings]

[0050] [Figure 1] FIG. 1 shows a schematic structural diagram of various BCMA-CD19 bispecific CARs, CD19 CARs and BCMA CARs in Example 1 of the present application. [Figure 2A] FIG. 2A shows CAR expression of two naive CARs (i.e., CD19-2A-BCMA and BCMA-2A-CD19) linked by the self-cleaving polypeptide T2A on the surface of CAR-T cells (day 6 post-infection) in Example 2 of the present application, the left panel of FIG. 2A is the CD19-2A-BCMA group, the middle panel of FIG. 2A is the BCMA-2A-CD19 group, and the right panel of FIG. 2A is the UTD group (T cells without CAR transduction). [Figure 2B] Figures 2B to 2E show CAR expression on the surface of four bispecific CAR-T cells (i.e., Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells and Loop BCMA-CD19 cells) in Example 2 of the present application (6 days after infection), where Figure 2B is the Tan CD19-BCMA group, Figure 2C is the Tan BCMA-CD19 group, Figure 2D is the Loop CD19-BCMA group, and Figure 2E is the Loop BCMA-CD19 cell group. [Diagram 3]Figures 3A-3C show cytokine release of various BCMA-CD19 bispecific CARs, CD19 CARs and BCMA CARs after activation by positive target cells in Example 4 of the present application, with Figure 3A showing the release of IL-2 in each group, Figure 3B showing the release of IFN-γ in each group, and Figure 3C showing the release of TNF-α in each group. In particular, Figures 3A, 3B and 3C each include two dotted boxes, in which the five bars in the first dotted box in Figures 3A, 3B and 3C represent, from left to right, the cytokine release of BCMA cells after activation by UTD cells, TanCD19-BCMA cells, TanBCMA-CD19 cells, LoopCD19-BCMA cells, LoopBCMA-CD19 cells and K562-BCMA cells, and in the second dotted box in Figures 3A, 3B and 3C represent, from left to right, the cytokine release of CD19 cells after activation by UTD cells, TanCD19-BCMA cells, TanBCMA-CD19 cells, LoopCD19-BCMA cells, LoopBCMA-CD19 cells and K562-CD19 cells. [Figure 4] Figures 4A to 4C show the killing effects of various BCMA-CD19 bispecific CARs, CD19 CARs and BCMA CAR cells against different target cells in Example 5 of the present application, where Figure 4A is the NALM6 group, which are CD19+BCMA target cells, Figure 4B is the MM.1S group, which are BCMA+CD19 target cells, and Figure 4C is the NALM6-KO CD19 group, which is a negative target cell control that does not express BCMA but has CD19 knocked out. [Diagram 5] Figures 5A to 5D show the sustained proliferation of CAR-T cells in each group after multiple antigen stimulations in Example 6 of the present application, where Figure 5A shows the proliferation of CD3+ cells stimulated with MM.1S cells, Figure 5B shows the proliferation of CD3+ cells stimulated with NALM6 cells, Figure 5C shows the proliferation of CAR+ cells stimulated with MM.1S cells, and Figure 5D shows the proliferation of CAR+ cells stimulated with NALM6 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] The embodiments of the present invention will be described with reference to the following specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the disclosure of this specification.

[0052] This application is further described below. In the present invention, unless otherwise specified, scientific and technical terms used herein have the meaning commonly understood by those skilled in the art. Furthermore, the terms and experimental procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology and immunology used herein are terms and routine procedures widely used in the corresponding fields. In order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0053] In this application, "chimeric antigen receptor (CAR)" is the core component of CAR cell therapy and may include an extracellular antigen recognition domain (e.g., a portion that binds to a tumor-associated antigen (TAA), a hinge region, a transmembrane region, and an intracellular domain). CAR-T (chimeric antigen receptor T) cell immunotherapy is considered one of the most promising methods for overcoming tumors. CAR-T cells use a genetic engineering method to make T cells express CAR proteins, which have the ability to recognize unprocessed proteins on the membrane surface independent of antigen presentation, resulting in T cell activation and functional effects.

[0054] In this application, "extracellular antigen recognition domain" refers to antigen recognition domain (ARD). The reason why CAR cell therapy products (such as CAR-T cells) can specifically recognize and / or bind target antigens expressed by tumor cells depends on the extracellular antigen recognition domain. Until now, antigen recognition domains have been derived from single chain variable fragments of antibodies (abbreviated as scFv) or from receptor-ligand interactions, TCR mimics, variable lymphocyte receptors (VLR). So far, the most common source is the scFv segment of an antibody. The scFv comprises an antibody heavy chain variable region (VH region) and an antibody light chain variable region (VL region), which are linked by a peptide chain such as the 18 amino acid linker sequence GSTSGSGKPGSGEGSTKG. An scFv antibody targeting two or more targets comprises VH and VL regions targeting different targets, and the different regions are linked directly or indirectly via a linking sequence in any of the following configurations: target 1 VL-target 1 VH-target 2 VL-target 2 VH, target 2 VL-target 2 VH-target 1 VL-target 1 VH, target 1 VL-target 2 VL-target 2 VH-target 1 VH, target 2 VL-target 1 VL-target 1 VH-target 2 VH, target 2 VL-target 1 VL-target 1 VH-target 2 VH, where "-" indicates linkage via a linking sequence.

[0055] In this application, "specific recognition and / or binding" refers to the recognition and / or binding between a CAR and a specific target, and indicates that the CAR binds to this target with higher affinity, avidity, greater ease and / or longer duration than the CAR binds to other targets.

[0056] In this application, "hinge region" refers to the linker segment between the extracellular antigen recognition domain and the transmembrane domain. This region allows CAR to recognize antigens by providing a range of activity to the antigen recognition domain. Currently used hinge regions are mainly derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84 and CD8α. In addition, typical hinge regions also contain several residues that are involved in CAR dimerization and contribute to enhanced antigen sensitivity.

[0057] In this application, "transmembrane region" refers to the transmembrane domain that connects the intracellular and extracellular components of the CAR structure. Different transmembrane domains may affect the expression and stability of CAR to some extent, but they are not directly involved in signal transduction, but improve downstream signal transduction by interaction. The transmembrane region may be derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40 and Fc70.

[0058] In this application, the "intracellular domain" includes an intracellular signaling region, and may further include a costimulatory signaling region.

[0059] In the present application, an "intracellular signaling region" refers to the activation of at least one normal effector function of an immune effector cell that is responsible for the expression of a CAR. The intracellular signaling region may be derived from one or more of CD3zeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, FcRgamma, FcRbeta, CD66d, DAP10, DAP12, and Syk.

[0060] In the present application, the "costimulatory signaling region" is present because, in addition to stimulation by an antigen-specific signal, many immune effector cells require co-stimulation to promote cell proliferation, differentiation and survival, as well as effector function of activated cells. In some examples, the CAR may further comprise one or more co-stimulatory signaling regions, which may be derived from one or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88.

[0061] In this application, "isolated" generally means something obtained from nature by artificial means. When an "isolated" substance or component exists in nature, the natural environment in which it is located may have been altered, or the substance may have been separated from its natural environment, or both. For example, when a non-isolated polynucleotide or polypeptide exists in a living animal, the polynucleotide or polypeptide of high purity isolated from this natural state is called an isolated polynucleotide or polypeptide. "Isolated" does not exclude substances that have been artificially or synthetically obtained from their natural state by artificial means, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0062] In this application, "guide peptide" refers to a short peptide preceding an extracellular antigen recognition domain (e.g., scFv sequence), whose function is to guide recombinant proteins synthesized within cells to be exported out of the cells. Commonly used guide peptides include human CD8α signal peptide or human GM-CSF receptor α signal peptide.

[0063] In this application, one of the important factors that determine the therapeutic effect of CAR immune cells is the selection of tumor target antigen. In this application, "BCMA" refers to B cell maturation antigen, which is a member of the tumor necrosis factor receptor superfamily. Human BCMA is almost exclusively expressed on plasma cells and multiple myeloma cells. BCMA can be a suitable tumor antigen target for immunotherapeutic agents against multiple myeloma. However, because the specific antigens on the surface of multiple myeloma cells are heterogeneous, the selection of the antigen target is not necessarily single. By selecting the appropriate target, the antitumor activity of CAR-T cells can be optimized. The "CD19" molecule is currently the main target for treating hematological tumors derived from B lymphocytes, and has also been a focus of CAR-T cell therapy research. Most malignant tumor cells derived from B cells express the CD19 molecule on their surface. Multiple myeloma does not generally express the CD19 molecule, as B cell lineage tumors do. Therefore, CD19 is not usually used as a target for the treatment of multiple myeloma. However, some literature studies have shown that a small number of drug-resistant and relapsed multiple myeloma clones also express CD19 + This suggests that the phenotype is contained. When using this dual-targeting CAR-T product, as long as one tumor antigen target is recognized, CAR-T cells can be activated to prevent tumor antigen leakage. Compared with preparing CAR immune cells that target different targets and using them together, dual-targeting CAR immune cells have the following advantages: 1. Fewer immune cells are required, it is easy to prepare, and it can save costs; 2. From the viewpoint of administration, the safety and operability of administration of one product is much higher than that of administration of two products.

[0064] In the present application, a "linker sequence" generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links any structure / region of the chimeric antigen receptor of the present invention. The linker sequence may be composed of different amino acid residues (e.g., glycine and serine) to allow adjacent protein domains to move freely relative to each other. A longer linker sequence may be used when it is desired to ensure that two adjacent domains do not spatially interfere with each other.

[0065] In this application, an "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides of any length, deoxyribonucleotides or ribonucleotides, that can be isolated from their natural environment or a synthetic analogue.

[0066] In this application, when CAR gene introduction / transfection and target gene expression are performed, the gene introduction / transfection method mainly includes viral and non-viral methods, such as gamma retrovirus vector, lentivirus vector, adenovirus-associated virus vector, plasmid DNA-dependent vector, transposon-dependent gene introduction and mRNA-mediated gene introduction.

[0067] A "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and which can express the protein. A vector can transform, transduce, or transfect a host cell so that the genetic material element carried by the vector can be expressed in the host cell. For example, vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages such as lambda phage or M13 phage, and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papilloma viruses (such as SV40). A vector may include various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also include an origin of replication. Vectors may also contain components that aid in cell entry, such as, but not limited to, viral particles, liposomes, or protein coats. "Transposon" refers to a discontinuous DNA segment that has the ability to move between chromosomal sites and carry genetic information, such as the Sleeping Beauty SB system and the PB system derived from lepidopteran insects. In some instances, mRNA can also be transduced into T cells by electrotransduction.

[0068] In the present application, "immune effector cells" generally refers to cells involved in immune responses, for example, cells that promote immune effector responses. Immune effector cells can be selected from one or more of the following groups: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T lymphocytes differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-Ts), NK cells differentiated from induced pluripotent stem cells (iPSC-NKs), and embryonic stem cells.

[0069] In this application, "pharmaceutical composition" generally refers to a pharmaceutical composition suitable for administration to a patient, which may include immune effector cells as described in this application, and may further include one or more pharmaceutically acceptable adjuvants, such as, for example, carriers, protective agents, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives. In some examples, the pharmaceutically acceptable adjuvants include protective agents, such as cell cryopreservation solutions. In some examples, the pharmaceutical composition of the present application is a cell suspension or cryopreserved cells thereof.

[0070] In this application, a "subject" generally refers to a human or non-human animal, including, but not limited to, a mouse, rat, cat, dog, rabbit, horse, pig, cow, sheep, or monkey.

[0071] In this application, "comprising" generally refers to the inclusion of the explicitly specified features but not to the exclusion of other elements.

[0072] In this application, "about" generally refers to a range of variation above or below a specified value that would be acceptable to one of ordinary skill in the art, for example, a variation within ±0.5% to 10%, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% above or below the specified value.

[0073] Chimeric antigen receptor, nucleic acid, vector, immune effector cell, pharmaceutical composition In one aspect, the present application provides a method for the detection of an antibody comprising: an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain, wherein said extracellular antigen recognition domain comprises an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain; The present invention provides a bispecific chimeric antigen receptor that targets BCMA-CD19, wherein the anti-BCMA extracellular antigen recognition domain comprises a BCMA VH and a BCMA VL, the amino acid sequences of the BCMA VH complementarity determining regions CDR1, CDR2 and CDR3 comprise the amino acid sequences represented by SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the amino acid sequences of the BCMA VL complementarity determining regions CDR1, CDR2 and CDR3 comprise the amino acid sequences represented by SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0074] In antibodies, common rules for dividing CDRs include Kabat, AbM, Chothia, Contact and IMGT. These rules are well known to those skilled in the art. When applying the website that implements these rules, input VH and VL sequences and select corresponding rules to obtain CDR sequences based on different rules. In this application, IMGT rules are used to divide CDRs. However, those skilled in the art should understand that the scope of protection of this application encompasses the combination of CDR sequences obtained by analyzing using different rules.

[0075] In some examples, the anti-CD19 extracellular antigen recognition domain comprises CD19 VH and CD19 VL, the amino acid sequences of the CD19 VH complementarity determining regions CDR1, CDR2 and CDR3 comprise the amino acid sequences represented by SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively, and the amino acid sequences of the CD19 VL complementarity determining regions CDR1, CDR2 and CDR3 comprise the amino acid sequences represented by SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0076] In some examples, the BCMA VH sequence comprises the amino acid sequence represented by SEQ ID NO:13 and the BCMA VL sequence comprises the amino acid sequence represented by SEQ ID NO:14.

[0077] In some examples, the BCMA VH sequence comprises the amino acid sequence represented by SEQ ID NO:15 and the BCMA VL sequence comprises the amino acid sequence represented by SEQ ID NO:16.

[0078] In some examples, the CD19 VH sequence comprises the amino acid sequence represented by SEQ ID NO:17 and the CD19 VL sequence comprises the amino acid sequence represented by SEQ ID NO:18.

[0079] In some examples, the present application also includes substitution, deletion, addition and / or insertion of one or more amino acids in the amino acid sequence of any one of the above bispecific chimeric antigen receptors, and activity equivalent to any one of the above chimeric antigen receptors. Those skilled in the art know that amino acids in the FR regions in the VH and VL sequences can be substituted so that the CDR regions of the modified antibody can retain a suitable antigen binding site during the humanization process. Therefore, the present application necessarily includes different amino acid sequences obtained by humanizing the FR regions in the VH and VL sequences based on the above CDRs. Furthermore, those skilled in the art know that, if necessary, 1%, 2%, 3%, or 10% or less of the amino acid sequences in the CDRs can be substituted, deleted, added and / or inserted to ensure that the CDR regions of the modified antibody can retain a suitable antigen binding site during the humanization process, which are also included in the present application.

[0080] In some examples, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor comprises any one of the structures selected from the group consisting of CD19 VL sequence-first linker sequence-CD19 VH sequence-second linker sequence-BCMA VL sequence-third linker sequence-BCMA VH sequence, BCMA VL sequence-fourth linker sequence-BCMA VH sequence-fifth linker sequence-CD19 VL sequence-sixth linker sequence-CD19 VH sequence, BCMA VL sequence-seventh linker sequence-CD19 VL sequence-eighth linker sequence-CD19 VH sequence-ninth linker sequence-BCMA VH sequence, and CD19 VL sequence-tenth linker sequence-BCMA VL sequence-eleventh linker sequence-BCMA VH sequence-twelfth linker sequence-CD19 VH sequence.

[0081] In some examples, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor comprises any one of the structures selected from the group consisting of BCMA VL sequence-7th linker sequence-CD19 VL sequence-8th linker sequence-CD19 VH sequence-9th linker sequence-BCMA VH sequence and CD19 VL sequence-10th linker sequence-BCMA VL sequence-11th linker sequence-BCMA VH sequence-12th linker sequence-CD19 VH sequence.

[0082] In some examples, the first linker sequence, the second linker sequence, the third linker sequence, the fourth linker sequence, the fifth linker sequence, the sixth linker sequence, the seventh linker sequence, the eighth linker sequence, the ninth linker sequence, the tenth linker sequence, the eleventh linker sequence and the twelfth linker sequence are each independently selected from one or more of SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0083] In some examples, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor comprises the amino acid sequence represented by SEQ ID NO:19 or SEQ ID NO:20.

[0084] In some examples, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84 and CD8α, optionally, the amino acid sequence of the hinge region is derived from CD8α, and further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence set forth in SEQ ID NO:21.

[0085] In some examples, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40 and Fc70, and optionally, the amino acid sequence of the transmembrane region is derived from CD8α, and further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence set forth in SEQ ID NO:22.

[0086] In some examples, the intracellular domain comprises an intracellular signaling region, optionally, the intracellular domain further comprises a costimulatory signaling region, and optionally, the intracellular signaling region is derived from one or more of CD3zeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, FcRgamma, FcRbeta, CD66d, DAP10, DAP12, and Syk, and optionally, the intracellular signaling region is derived from CD3zeta, for example, the amino acid sequence of the intracellular signaling region comprises the amino acid sequence represented by SEQ ID NO:23.

[0087] In some examples, the costimulatory signaling region is derived from one or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88, optionally, the costimulatory signaling region is derived from CD28 or 4-1BB, and further optionally, the amino acid sequence of the costimulatory signaling region comprises the amino acid sequence set forth in SEQ ID NO:24.

[0088] In some examples, the bispecific chimeric antigen receptor further comprises a guide peptide located N-terminal to the amino acid sequence of the chimeric antigen receptor, optionally, the guide peptide is derived from CD8α, and further optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence set forth in SEQ ID NO:25.

[0089] In some examples, the bispecific chimeric antigen receptor comprises the amino acid sequence represented by SEQ ID NO:28 or SEQ ID NO:29.

[0090] In another aspect, the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific chimeric antigen receptor described above.

[0091] In some instances, the nucleotide sequence encoding the bispecific chimeric antigen receptor comprises 1) a nucleotide sequence encoding the BCMA VH amino acid sequence represented by SEQ ID NO: 13, represented by SEQ ID NO: 37, and a nucleotide sequence encoding the BCMA VL amino acid sequence represented by SEQ ID NO: 14, represented by SEQ ID NO: 38, and / or 2) It comprises a nucleotide sequence represented by SEQ ID NO:39, which encodes the CD19 VH amino acid sequence represented by SEQ ID NO:17, and a nucleotide sequence represented by SEQ ID NO:40, which encodes the CD19 VL amino acid sequence represented by SEQ ID NO:18.

[0092] In another aspect, the present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.Vector includes plasmid, phagemid, cosmid, yeast artificial chromosome (YAC), artificial chromosome such as bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC), phage such as lambda phage or M13 phage, and animal virus.Types of animal virus used as vector include retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), poxvirus, baculovirus, papilloma virus and papilloma virus (such as SV40).

[0093] In some examples, the vector is an expression vector, optionally, the vector is a viral vector, and further optionally, the vector is a lentiviral vector.

[0094] In another aspect, the present application also provides an engineered immune effector cell comprising the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned vector.

[0095] In some examples, the engineered immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells.

[0096] In some instances, the engineered immune effector cells are T lymphocytes, and optionally, the source of the T lymphocytes is autologous or allogeneic T lymphocytes.

[0097] In some instances, the chimeric antigen receptors described in the present application can be or are expressed on the surface of engineered immune effector cells.

[0098] In another aspect, the present application also provides a pharmaceutical composition comprising the engineered immune effector cells described above and a pharma- ceutically acceptable adjuvant, the pharma-ceutically acceptable adjuvant comprising one or more of a carrier, a protectant, a stabilizer, and a diluent.

[0099] In some instances, the pharma- ceutically acceptable adjuvant comprises a protective agent, such as a cell cryopreservation solution.

[0100] In some instances, the pharmaceutical composition is a cell suspension or a cryopreserved cell thereof.

[0101] In some instances, the pharmaceutical composition is administered intravenously.

[0102] Preparation method In another aspect, the present application also provides a method of preparing an engineered immune effector cell, comprising transducing an immune effector cell with a vector described in the present application.

[0103] In some examples, the engineered immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells.

[0104] In some instances, the engineered immune effector cells are T lymphocytes, and optionally, the source of the T lymphocytes is autologous or allogeneic T lymphocytes.

[0105] use In another aspect, the present application also provides the use of a bispecific chimeric antigen receptor, isolated nucleic acid molecule, vector and / or engineered immune effector cell described in the present application in the preparation of a medicament for use in the treatment of a disease or condition associated with expression of BCMA.

[0106] In some instances, the disease or condition associated with expression of BCMA is cancer, optionally, the cancer is multiple myeloma, and further optionally, the cancer is refractory or relapsed multiple myeloma.

[0107] In some instances, the disease or condition associated with expression of BCMA may be an autoimmune disease.

[0108] In some instances, the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.

[0109] In another aspect, the present application also provides a method of treating a disease or condition associated with BCMA expression comprising administering to a subject in need thereof an effective amount of a chimeric antigen receptor, isolated nucleic acid molecule, vector and / or engineered immune effector cell described in the present application.

[0110] In some instances, the disease or condition associated with expression of BCMA is cancer, optionally, the cancer is multiple myeloma, and further optionally, the cancer is refractory or relapsed multiple myeloma.

[0111] In some instances, the disease or condition associated with expression of BCMA may be an autoimmune disease.

[0112] In some instances, the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.

[0113] In some examples, the administration can be performed in different ways, such as intravenous administration, intratumoral administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, topical administration, or intradermal administration. For example, the administration can be administered to a subject by intravenous injection. In some examples, an effective amount of the engineered immune effector cells or pharmaceutical composition can be administered to a subject in a single dose or in divided doses over a period of time, for example, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months.

[0114] In some instances, dosages may vary for different indications, and dosages may also vary depending on the severity of the patient's condition. 5 CAR positive T cells / kg~1×10 7 It may be CAR-positive T cells / kg, for example, 1×10 5 CAR positive T cells / kg~1×10 6 CAR positive T cells / kg, 1×10 6CAR positive T cells / kg~1×10 7 CAR positive T cells / kg, 0.5×10 6 CAR positive T cells / kg, 0.6×10 6 CAR positive T cells / kg, 0.7×10 6 CAR positive T cells / kg, 0.8×10 6 CAR positive T cells / kg, 0.9×10 6 CAR positive T cells / kg, 1.0×10 6 CAR positive T cells / kg, 1.1×10 6 CAR positive T cells / kg, 1.2×10 6 CAR positive T cells / kg, 1.3×10 6 CAR positive T cells / kg, 1.4×10 6 CAR positive T cells / kg, 1.5×10 6 CAR positive T cells / kg, 1.6×10 6 CAR positive T cells / kg, 1.7×10 6 CAR positive T cells / kg, 1.8×10 6 CAR positive T cells / kg, 1.9×10 6 CAR positive T cells / kg, 2.0×10 6 It may be CAR-positive T cells / kg.

[0115] In some examples, subjects may include humans and non-human animals, for example, but not limited to, mice, rats, cats, dogs, horses, pigs, cows, sheep, rabbits, or monkeys.

[0116] In another aspect, the present application also provides a chimeric antigen receptor, an isolated nucleic acid molecule, a vector and / or an engineered immune effector cell for use in treating a disease or condition associated with expression of BCMA.

[0117] In some instances, the disease or condition associated with expression of BCMA may include a non-solid tumor, and optionally, the non-solid tumor is a hematological tumor.

[0118] In some instances, the disease or condition associated with expression of BCMA may include multiple myeloma.

[0119] In some instances, the multiple myeloma is relapsed or refractory multiple myeloma.

[0120] Without wishing to be bound by any theory, the following examples are described only to illustrate the chimeric antigen receptor, engineered immune effector cells, preparation methods and uses of the present application, and are not used to limit the scope thereof. The examples do not include detailed descriptions of conventional methods such as how to construct vectors and plasmids, how to insert genes encoding proteins into such vectors and plasmids, or how to introduce plasmids into host cells. Such methods are well known to those skilled in the art and are described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press. EXAMPLES

[0121] Example 1 Obtaining BCMA-CD19 bispecific CAR-T cells To obtain a superior bispecific CAR structure, a BCMA-specific humanized antibody (whose VH amino acid sequence is represented by SEQ ID NO: 13, whose VH nucleotide sequence is represented by SEQ ID NO: 37, whose VL amino acid sequence is represented by SEQ ID NO: 14, whose VL nucleotide sequence is represented by SEQ ID NO: 38, whose VH CDR1, CDR2 and CDR3 amino acid sequences are represented by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and whose VL CDR1, CDR2 and CDR3 amino acid sequences are represented by SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively) was combined with a CD19-specific antibody (whose VH amino acid sequence is represented by SEQ ID NO: 17, whose VH nucleotide sequence is represented by SEQ ID NO: 39, whose VL amino acid sequence is represented by SEQ ID NO: 18, whose VL nucleotide sequence is represented by SEQ ID NO: 40, whose VH CDR1, CDR2 and CDR3 amino acid sequences are represented by SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively, and whose VL CDR1, CDR2 and CDR3 amino acid sequences are represented by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively). The amino acid sequences of CDR1, CDR2 and CDR3 are represented by SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively. Considering the order of the targets BCMA and CD19 and the change in the order of VH and VL in the extracellular antigen recognition domain of each target, there are multiple options for designing the structure of BCMA-CD19 bispecific CAR. We chose to screen the candidate BCMA-CD19 bispecific CAR structures on the second generation CAR structures.

[0122] Six BCMA-CD19 bispecific CAR structures shown in Figure 1 were tried, with the BCMA CAR structure and the CD19 CAR structure used as controls. In each CAR structure, the following structures were used in this example: CD8α guide chain was used as the signal peptide (represented by SEQ ID NO:25), CD8α structure was used as the hinge region (represented by SEQ ID NO:21) and the transmembrane region (represented by SEQ ID NO:22), 4-1BB was used as the intracellular costimulatory signal (represented by SEQ ID NO:24), and CD3ζ was used as the T cell activation signal (represented by SEQ ID NO:23).

[0123] 1. Construction of lentiviral vector Six BCMA-CD19 bispecific CAR structures shown in Figure 1, as well as BCMA CAR structure and CD19 CAR structure as controls, were artificially synthesized. The amino acid sequences of Tan CD19-BCMA structure, Tan BCMA-CD19 structure, Loop CD19-BCMA structure, Loop BCMA-CD19 structure, CD19-2A-BCMA structure, BCMA-2A-CD19 structure, CD19 CAR structure and BCMA CAR structure are represented by SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively. The following description is a simple summary of the main identification parts in the CAR structure, and does not fully show the signal peptide, linker sequence, hinge region, transmembrane region, intracellular costimulatory signal region, and T cell activation signal region. The order of each structure is shown in Figure 1. SP represents a signal peptide region, Hinge represents a hinge region, TM represents a transmembrane region, and the linker sequence is present between VL and VH.

[0124] 1)Tan CD19-BCMA structure: CD19 VL-CD19 VH-BCMA VL-BCMA VH 2)Tan BCMA-CD19 structure: BCMA VL-BCMA VH-CD19 VL-CD19 VH 3)Loop CD19-BCMA structure: BCMA VL-CD19 VL-CD19 VH-BCMA VH 4)Loop BCMA-CD19 structure: CD19 VL-BCMA VL-BCMA VH-CD19 VH 5) CD19-2A-BCMA structure: CD19 CAR-T2A-BCMA CAR (The complete CD19 CAR structure and the BCMA CAR structure were found before and after the self-cleaving polypeptide T2A in this structure, respectively. The complete CAR structure refers to the same signal peptide, hinge region, transmembrane region, intracellular costimulatory signal region, and T cell activation signal region as the other groups.) 6) BCMA-2A-CD19 structure: BCMA CAR-T2A-CD19 CAR (The complete BCMA CAR and CD19 CAR structures were observed before and after the self-cleaving polypeptide T2A, respectively. The complete CAR structure refers to the same signal peptide, hinge region, transmembrane region, intracellular costimulatory signal region and T cell activation signal region as the other groups). 7)CD19CAR structure:CD19 VL-CD19 VH 8)BCMA CAR structure: BCMA VL-CD19 VH

[0125] The above six BCMA-CD19 bispecific CAR constructs, one CD19 CAR construct and one BCMA CAR construct were each constructed into a modified empty lentiviral vector (manufacturer: SBI Corporation, catalog number: CD500-CD800, conventional resistance modification was performed as described in Example 1 of WO 2021 / 121227) to obtain a CAR expression vector, and then the CAR expression vector and three packaging plasmids were combined and transfected into 293T cells to obtain a functional lentiviral vector after harvesting and purification. The three packaging plasmids were PMD2.0G (purchased from Biovector Corporation, catalog number: Biovector 012259), pMDLg / pRRE (purchased from Biovector Corporation, catalog number: Biovector 012251) and pRSV-Rev (purchased from Biovector Corporation, catalog number: Biovector 012253), respectively.

[0126] 2. Preparation of six matched BCMA-CD19 bispecific CAR-T cells, one CD19 CAR-T cell and one BCMA CAR-T cell by lentiviral transduction Transduction experiments were performed according to conventional methods known to those skilled in the art. The transduction steps are briefly described below.

[0127] 1) T cell selection Peripheral blood mononuclear cells (PBMCs) were isolated from the subject's apheresis cells, and T cells were then sorted from the PBMC cells.

[0128] 2) T cell activation Resuspend the isolated T cells in complete lymphocyte culture medium (X-VIVO15 medium + 5% FBS + 300 IU / ml IL-2 or X-VIVO15 medium + 5% FBS + 5 ng / ml IL-15 + 10 ng / ml IL-7) to a final concentration of (1–2) × 10 6 cells / ml and add 5-10 μL of CD3 / CD28 stimulating magnetic beads. Mix the mixture well and place in an incubator at 37 °C with 5% CO 2 The cells were cultured for more than 24 hours under the above culture conditions.

[0129] 3) Lentiviral transduction of T cells The activated cultured T cells were removed, polybrene was added to a final concentration of 8 μg / ml, and mixed well. The lentiviral vector was slowly added at MOI=2. After mixing well, the mixture was placed in a centrifuge and centrifuged at 1500 rpm for 1.5 hours. It was then placed in an incubator at 37°C with 5% CO. 2 The cells were cultured for more than 24 hours under the above culture conditions.

[0130] 4) Expansion culture of transduced T cells The transduced cells were harvested and the cell density was adjusted to (0.5-1) x 10 for use in the following examples. 6 The cells / ml were monitored. After infecting T cells with lentivirus containing Tan CD19-BCMA structure, Tan BCMA-CD19 structure, Loop CD19-BCMA structure, Loop BCMA-CD19 structure, CD19-2A-BCMA structure, BCMA-2A-CD19 structure, CD19 CAR and BCMA CAR, the resulting T cells were named Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells, CD19-2A-BCMA cells, BCMA-2A-CD19 cells, CD19 cells and BCMA cells, respectively. Then, the six bispecific CAR structures were screened at the cellular level to determine the characteristics of each CAR structure and select the superior bispecific CAR structure.

[0131] Example 2 Detection of CAR molecules expressed on the surface of BCMA-CD19 bispecific CAR-T cells The CAR protein molecules expressed on the surface of the six bispecific CAR-T cells obtained in Example 1 were detected. The six BCMA-CD19 bispecific CAR-T cells obtained in Example 1 and UTD cells (T cells not transduced with CAR) were stained with PE fluorescent-labeled CD19 antigen (manufacturer: ACRO Biosystems, catalog number: CD9-HP2H3) and FITC fluorescent-labeled BCMA antigen (manufacturer: ACRO Biosystems, catalog number: BCA-HF254), and the detection and analysis of the CAR molecule positive rate by flow cytometry were performed. First, as shown in FIG. 2A, it was found that for CD19-2A-BCMA cells and BCMA-2A-CD19 cells, 6 days after T cell infection, the CAR molecule linked to the back of T2A was not sufficiently expressed, and therefore, the two CAR structures in CD19-2A-BCMA cells and BCMA-2A-CD19 cells were first eliminated. As shown in Figure 2B, Figure 2C, Figure 2D and Figure 2E, 6 days after T cell infection, the CAR expression rates of the other four bispecific CAR-T cells, Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells and Loop BCMA-CD19 cells, were 42.98%, 66.71%, 53.46% and 61.63%, respectively (based on the results after BCMA antigen staining, the reasons of which will be described in detail in Example 3).

[0132] Example 3 Detection of CAR molecules on the surface of BCMA-CD19 bispecific CAR-T cells The present inventors tried single staining (single staining means staining bispecific CAR-T cells with only FITC fluorescent-labeled BCMA antigen or PE fluorescent-labeled CD19 antigen) and co-staining (co-staining means staining bispecific CAR-T cells with both FITC fluorescent-labeled BCMA antigen and PE fluorescent-labeled CD19 antigen) of the bispecific CAR-T cells obtained in Example 1 11 days after T cell infection to select the staining method. Since the extracellular portion of BCMA and the extracellular portion of CD19 were constructed on one scFv and always co-expressed, theoretically, whether the bispecific CAR-T cells are stained by single staining method or co-staining method, the difference in the results should be within the error range. However, as shown in Table 1 (especially the grayscale part), it was found that the selection of different antigens for staining has a significant effect on the calculation of CAR+ cells of bispecific CAR-T cells.

[0133] [Table 1]

[0134] Taking the characteristics of the percentage of CAR+ cells in Tan BCMA-CD19 cells as an example, when BCMA antigen was used for single staining and BCMA antigen + CD19 antigen was used for co-staining, the percentages of CAR+ detected via BCMA antigen were all within the error range. However, when CD19 antigen was used for single staining and BCMA antigen + CD19 antigen was used for co-staining, the values ​​detected via CD19 antigen fluctuated greatly beyond the error range, and the percentages of CAR+ cells detected via CD19 antigen were significantly lower than the percentages of CAR+ cells detected via BCMA antigen. On the other hand, when CD19 antigen was used for detection, a significant difference was also observed between the percentages of CAR+ cells detected by single staining and the percentages of CAR+ cells detected by co-staining. The above observations were not limited by the relative positions of the BCMA extracellular antigen recognition domain and the CD19 extracellular antigen recognition domain in the CAR structure. Compared with the CD19 antigen, the BCMA antigen is smaller, which is thought to be easier to overcome the steric hindrance problem in the bispecific CAR structure binding to the CAR protein. Therefore, in the subsequent experiments of this application, the data of detection via BCMA antigen was used to characterize the positive rate of BCMA-CD19 bispecific CARs. Of course, the data of detection via BCMA antigen in the co-staining method could also characterize the positive rate of BCMA-CD19 bispecific CARs, but the single staining method was simpler.

[0135] Example 4 Cytokine release experiments in BCMA-CD19 bispecific CAR-T cells Cytokine release experiment: The four bispecific CAR-T cells (Tan CD19-BCMA, Tan BCMA-CD19, Loop CD19-BCMA, Loop BCMA-CD19), BCMA CAR-T cells, CD19 CAR-T cells and UTD cells obtained in Example 1 were co-cultured with target cells in X-VIVO15 medium at an effector-target ratio of 1:1 for 24 hours, and then the concentrations of IL-2, IFN-γ and TNF-α in the cell supernatant were detected by ELISA method. K562 is a double negative target cell of BCMA and CD19, K562-BCMA is a positive target cell that exogenously expresses BCMA but does not express CD19, and K562-CD19 is a positive target cell that exogenously expresses CD19 but does not express BCMA. K562-BCMA cells did not express CD19 antigen, so they were not used to detect CD19 CAR-T cells. Similarly, K562-CD19 cells did not express the BCMA antigen and were therefore not used to detect BCMA CAR-T cells (Figures 3A, 3B, and 3C, the K562-BCMA and K562-CD19 groups are each missing one bar).

[0136] The experimental results of the release of cytokines IL-2, IFN-γ and TNF-α are shown in Figures 3A, 3B and 3C, respectively. The arrows in Figures 3A, 3B and 3C indicate CAR-T cells with low levels of cytokine release. As can be seen from Figures 3A, 3B and 3C, the levels of various cytokines released by bispecific CARs of Loop CD19-BCMA structure and Loop BCMA-CD19 structure were generally higher than those of Tan CD19-BCMA structure and Tan BCMA-CD19 structure. In particular, as can be seen from the cytokine release levels in Tan CD19-BCMA structure and Tan BCMA-CD19 structure, when the scFv far from the cell membrane in Tan CD19-BCMA structure and Tan BCMA-CD19 structure was stimulated by antigen, the levels of cytokines released by CAR-T cells were relatively low.

[0137] Example 5 Cell killing experiments in BCMA-CD19 bispecific CAR-T cells Cell killing experiment: The four bispecific CAR-T cells (Tan CD19-BCMA, Tan BCMA-CD19, Loop CD19-BCMA, Loop BCMA-CD19), BCMA CAR-T cells, CD19 CAR-T cells and UTD cells obtained in Example 1 were co-cultured with target cells at different effector-target ratios (0:1, 1:1, 3:1 or 9:1) in X-VIVO15 medium for 4 hours, respectively. Then, the target cell killing rate was detected by detecting the activity of luciferase stably expressed in the target cells. NALM6 (human acute lymphoblastic leukemia cells) were CD19+BCMA - The target cells, MM.1S (human multiple myeloma cells), are BCMA+CD19 - Target cells, NALM6 - KO CD19 was a negative target cell control in which CD19 was knocked out and BCMA was not expressed. The cell killing results are shown in Figure 4A-C. As shown in Figure 4A, Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells and CD19 CAR-T cells all had good killing effects on the positive target cells NALM6 that endogenously express CD19, while BCMA CAR-T cells and UTD cells had no killing effect on the positive target cells NALM6 that endogenously express CD19. As shown in Figure 4B, Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells and BCMA CAR-T cells all had good killing effects on MM.1S positive target cells that endogenously express BCMA, while CD19 CAR-T cells and UTD cells had no killing effect on MM.1S positive target cells that endogenously express BCMA. As shown in Figure 4C, all cells had no killing effect on the negative target cell control in which CD19 was knocked out and BCMA was not expressed.

[0138] Example 6 Sustained expansion of BCMA-CD19 bispecific CAR-T cells Antigen stimulation can activate CAR-T cells and induce CAR-T cell proliferation, but sustained activation of T cells leads to cell exhaustion. The proliferation and effector function of exhausted T cells are reduced. After multiple antigen stimulation experiments, the sustained proliferation of BCMA-CD19 bispecific CAR-T cells was verified by detecting the proliferation of CD3+ cells (i.e., T cell proliferation), proliferation of CAR+ cells, and the percentage of CAR+ cells.

[0139] Before antigen stimulation, the CAR positive rates of the four bispecific CAR-T cells (Tan CD19-BCMA, Tan BCMA-CD19, Loop CD19-BCMA, Loop BCMA-CD19), BCMA CAR-T cells and CD19 CAR-T cells obtained in Example 1 were all adjusted using UTD to a level consistent with the rate of the group of CAR-T cells with the lowest CAR positive rate. The CAR positive rates of Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells and BCMA CAR-T cells are based on the detection data of BCMA antigen, and the CAR positive rate of CD19 CAR-T cells is based on the detection data of CD19 antigen. In the multiple antigen stimulation experiment, each group of CAR-T cells was co-cultured with positive target cells at an effector-target ratio of 1:2 in 24-well plates with 2ml of X-VIVO15 medium per well, with three wells repeated for each cell group. The positive target cells used were MM.1S and NALM6, respectively, which were equivalent to BCMA and CD19 as antigens for multiple stimulations to test the effect of multiple stimulations with different antigens on the sustained proliferation of BCMA-CD19 bispecific CAR-T cells. MM.1S cells did not express the CD19 antigen, so they were not used to detect CD19 CAR-T cells. Similarly, NALM6 cells did not express the BCMA antigen, so they were not used to detect BCMA CAR-T cells.

[0140] After co-culture of CAR-T cells with positive target cells for 3 days, 500 μL of cells were taken out and CAR staining was performed using fluorescently labeled CD3 antibody (manufacturer: BioLegend, catalog number: 300312) and BCMA antigen and CD19 antigen (same as in Example 2) (Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells, BCMA CAR-T cells were stained with BCMA antigen, and CD19 CAR-T cells were stained with CD19 antigen). Detection and analysis were performed by flow cytometry to show the percentage and number of CAR positive cells in CD3 positive cells. The number of CAR positive cells in CD3 positive cells can also be calculated based on the volume multiple conversion (CD3 is a marker that distinguishes whether they are T cells or not). Then, according to the calculation results, a certain amount of CAR-T cells were taken out from each group and added to the corresponding positive target cells at an effector-target ratio of 1:2 for new stimulation, which was repeated for 3 to 4 stimulations.

[0141] After multiple antigen stimulation experiments, the results of CD3+ cell proliferation (i.e., T cell proliferation) stimulated with MM.1S and NALM6 cells, respectively, are shown in Figure 5A and Figure 5B, and the results of CAR+ cell proliferation stimulated with MM.1S and NALM6 cells, respectively, are shown in Figure 5C and Figure 5D. As shown in Figure 5A and Figure 5C, after multiple antigen stimulation experiments, the number of CD3+ cells (T cell proliferation) and the number of CAR+ cells in Tan BCMA-CD19 cells stimulated with MM.1S cells were significantly lower than those in other CAR-T cell groups. As shown in Figure 5B and Figure 5D, after multiple antigen stimulation experiments, the number of CD3+ cells and the number of CAR+ cells in Tan CD19-BCMA cells stimulated with NALM6 cells were significantly lower than those in other CAR-T cell groups. Comparing Figure 5A with Figure 5B and Figure 5C with Figure 5D, it can be seen that the proliferation ability of CAR-T cells stimulated with MM.1S cells was stronger than that of CAR-T cells stimulated with NALM6 cells. The above results show that when the scFv far from the cell membrane of Tan-structured BCMA-CD19 bispecific CAR-T cells was stimulated with antigen, the sustained proliferation ability of CAR-T cells was relatively poor, but the sustained proliferation ability of Loop-structured CAR-T cells was generally stronger than that of Tan-structured CAR-T cells, and when the cells were stimulated with two antigens, BCMA and CD19, they had sustained proliferation ability, and the proliferation ability of CAR-T cells stimulated with BCMA antigen was stronger.

[0142] In conclusion, the in vitro pharmacodynamics studies showed that Tan CD19-BCMA, Tan BCMA-CD19, Loop CD19-BCMA and Loop BCMA-CD19 structures had superior performance in terms of expression on the surface of T cells, cytokine release and in vitro cell killing. In terms of sustained proliferation of CAR-T, the Loop CD19-BCMA and Loop BCMA-CD19 structures were significantly superior and performed better than the Tan CD19-BCMA and Tan BCMA-CD19 structures. In addition, the Loop CD19-BCMA and Loop BCMA-CD19 structures performed similarly to BCMA CAR-T and CD19 CAR-T in various experiments and tests, and had full functionality for dual targeting.

[0143] Array Description SEQ ID NO:1: BCMA VH CDR1 SEQ ID NO:2: BCMA VH CDR2 SEQ ID NO:3: BCMA VH CDR3 SEQ ID NO: 4: BCMA VL CDR1 SEQ ID NO: 5: BCMA VL CDR2 (ETS, Glu Thr Ser) SEQ ID NO: 6: BCMA VL CDR3 SEQ ID NO:7: CD19 VH CDR1 SEQ ID NO: 8: CD19 VH CDR2 SEQ ID NO: 9: CD19 VH CDR3 SEQ ID NO: 10: CD19 VL CDR1 SEQ ID NO: 11: CD19 VL CDR2 (SAT, Ser Ala Thr) SEQ ID NO: 12: CD19 VL CDR3 SEQ ID NO: 13: BCMA VH sequence (humanized) SEQ ID NO: 14: BCMA VL sequence (humanized) SEQ ID NO: 15: BCMA VH sequence (from rabbit) SEQ ID NO: 16: BCMA VL sequence (from rabbit) SEQ ID NO: 17: CD19 VH sequence SEQ ID NO: 18: CD19 VL sequence SEQ ID NO: 19: Amino acid sequence of scFv in the Loop CD19-BCMA structure SEQ ID NO: 20: Amino acid sequence of scFv in Loop BCMA-CD19 structure SEQ ID NO: 21: Amino acid sequence of the hinge region SEQ ID NO: 22: Amino acid sequence of the transmembrane domain SEQ ID NO: 23: Amino acid sequence of the intracellular signal transduction domain SEQ ID NO:24: Amino acid sequence of the costimulatory signaling domain SEQ ID NO: 25: Amino acid sequence of guide peptide SEQ ID NO: 26: Amino acid sequence of Tan CD19-BCMA structure SEQ ID NO: 27: Amino acid sequence of Tan BCMA-CD19 structure SEQ ID NO: 28: Amino acid sequence of the Loop CD19-BCMA structure SEQ ID NO: 29: Amino acid sequence of Loop BCMA-CD19 structure SEQ ID NO: 30: Amino acid sequence of the CD19-2A-BCMA structure SEQ ID NO: 31: Amino acid sequence of the BCMA-2A-CD19 structure SEQ ID NO: 32: Amino acid sequence of the CD19 CAR structure SEQ ID NO: 33: Amino acid sequence of the BCMA CAR structure SEQ ID NO: 34: Linker sequence SEQ ID NO: 35: Linker sequence SEQ ID NO: 36: Linker sequence SEQ ID NO: 37: Nucleotide sequence encoding the BCMA VH amino acid sequence represented by SEQ ID NO: 13 SEQ ID NO: 38: Nucleotide sequence encoding the BCMA VL amino acid sequence represented by SEQ ID NO: 14 SEQ ID NO: 39: Nucleotide sequence encoding the CD19 VH amino acid sequence represented by SEQ ID NO: 17 SEQ ID NO: 40: Nucleotide sequence encoding the CD19 VL amino acid sequence represented by SEQ ID NO: 18

Claims

1. It comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain; the extracellular antigen recognition domain comprises an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain; the anti-BCMA extracellular antigen recognition domain comprises a BCMA VH and a BCMA VL; the amino acid sequences of the BCMA VH complementarity determining regions CDR1, CDR2 and CDR3 comprise the amino acid sequences represented by SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; The amino acid sequences of the BCMA VL complementarity determining regions CDR1, CDR2 and CDR3 are represented by SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; BCMA-A bispecific chimeric antigen receptor targeting CD19.

2. the anti-CD19 extracellular antigen recognition domain comprises CD19 VH and CD19 VL; the amino acid sequences of the CD19 VH complementarity determining regions CDR1, CDR2 and CDR3 comprise the amino acid sequences represented by SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; 2. The bispecific chimeric antigen receptor of claim 1, wherein the amino acid sequences of the CD19 VL complementarity determining regions CDR1, CDR2 and CDR3 comprise the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

3. the BCMA VH sequence comprises the amino acid sequence represented by SEQ ID NO: 13 and the BCMA VL sequence comprises the amino acid sequence represented by SEQ ID NO: 14; or 3. The bispecific chimeric antigen receptor of claim 1 or 2, wherein the BCMA VH sequence comprises the amino acid sequence represented by SEQ ID NO: 15 and the BCMA VL sequence comprises the amino acid sequence represented by SEQ ID NO:

16.

4. The bispecific chimeric antigen receptor of claim 2, wherein the CD19 VH sequence comprises the amino acid sequence represented by SEQ ID NO: 17 and the CD19 VL sequence comprises the amino acid sequence represented by SEQ ID NO:

18.

5. the extracellular antigen recognition domain of the bispecific chimeric antigen receptor comprises any one structure selected from the group consisting of CD19 VL sequence-first linker sequence-CD19 VH sequence-second linker sequence-BCMA VL sequence-third linker sequence-BCMA VH sequence, BCMA VL sequence-fourth linker sequence-BCMA VH sequence-fifth linker sequence-CD19 VL sequence-sixth linker sequence-CD19 VH sequence, BCMA VL sequence-seventh linker sequence-CD19 VL sequence-eighth linker sequence-CD19 VH sequence-ninth linker sequence-BCMA VH sequence, and CD19 VL sequence-tenth linker sequence-BCMA VL sequence-eleventh linker sequence-BCMA VH sequence-twelfth linker sequence-CD19 VH sequence; Optionally, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor comprises any one structure selected from the group consisting of BCMA VL sequence-7th linker sequence-CD19 VL sequence-8th linker sequence-CD19 VH sequence-9th linker sequence-BCMA VH sequence and CD19 VL sequence-10th linker sequence-BCMA VL sequence-11th linker sequence-BCMA VH sequence-12th linker sequence-CD19 VH sequence; 2. The bispecific chimeric antigen receptor of claim 1, further optionally, wherein the first linker sequence, the second linker sequence, the third linker sequence, the fourth linker sequence, the fifth linker sequence, the sixth linker sequence, the seventh linker sequence, the eighth linker sequence, the ninth linker sequence, the tenth linker sequence, the eleventh linker sequence and the twelfth linker sequence are each independently selected from one or more of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO:

36.

6. The bispecific chimeric antigen receptor of claim 5, wherein the extracellular antigen recognition domain of the bispecific chimeric antigen receptor comprises the amino acid sequence represented by SEQ ID NO: 19 or SEQ ID NO:

20.

7. the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84 and CD8α, optionally the amino acids of the hinge region are derived from CD8α, and further optionally the amino acid sequence of the hinge region comprises the amino acid sequence set forth in SEQ ID NO:21; 2. The bispecific chimeric antigen receptor of claim 1, wherein the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40 and Fc70, optionally wherein the amino acid sequence of the transmembrane region is derived from CD8α, and further optionally wherein the amino acid sequence of the transmembrane region comprises the amino acid sequence set forth in SEQ ID NO:

22.

8. 2. The bispecific chimeric antigen receptor of claim 1, wherein the intracellular domain comprises an intracellular signaling region, optionally wherein the intracellular signaling region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12 and Syk, further optionally wherein the intracellular signaling region is derived from CD3ζ, and further optionally wherein the amino acid sequence of the intracellular signaling region comprises the amino acid sequence represented by SEQ ID NO:

23.

9. 2. The bispecific chimeric antigen receptor of claim 1, wherein the intracellular domain further comprises a costimulatory signaling region, optionally wherein the costimulatory signaling region is derived from one or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3 and MyD88, further optionally wherein the costimulatory signaling region is derived from CD28 or 4-1BB, and further optionally wherein the amino acid sequence of the costimulatory signaling region comprises the amino acid sequence represented by SEQ ID NO:

24.

10. 10. The bispecific chimeric antigen receptor of claim 1, further comprising a guide peptide located at the N-terminus of the amino acid sequence of the chimeric antigen receptor, optionally wherein the guide peptide is derived from CD8α, and further optionally wherein the amino acid sequence of the guide peptide comprises the amino acid sequence represented by SEQ ID NO:

25.

11. The bispecific chimeric antigen receptor according to any one of claims 1 to 10, comprising the amino acid sequence represented by SEQ ID NO:28 or SEQ ID NO:

29.

12. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific chimeric antigen receptor of any one of claims 1 to 11, Optionally, the nucleotide sequence encoding the bispecific chimeric antigen receptor comprises 1) a nucleotide sequence encoding the BCMA VH amino acid sequence represented by SEQ ID NO: 13, represented by SEQ ID NO: 37, and a nucleotide sequence encoding the BCMA VL amino acid sequence represented by SEQ ID NO: 14, represented by SEQ ID NO: 38, and / or 2) An isolated nucleic acid molecule comprising a nucleotide sequence encoding the CD19 VH amino acid sequence represented by SEQ ID NO: 17, represented by SEQ ID NO: 39, and a nucleotide sequence encoding the CD19 VL amino acid sequence represented by SEQ ID NO: 18, represented by SEQ ID NO:

40.

13. A vector comprising the isolated nucleic acid molecule of claim 12, Optionally, the vector is an expression vector, Further optionally, said vector is a viral vector; Further optionally, said vector is a lentiviral vector.

14. 14. An engineered immune effector cell comprising the chimeric antigen receptor of any one of claims 1 to 11, the isolated nucleic acid molecule of claim 12, or the vector of claim 13.

15. the engineered immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells; Optionally, said engineered immune effector cells are T lymphocytes; 15. The engineered immune effector cell of claim 14, further optionally, wherein the source of the T lymphocytes is an autologous or allogeneic T lymphocyte.

16. 16. A pharmaceutical composition comprising the engineered immune effector cells of claim 14 or 15 and a pharma- ceutically acceptable adjuvant, Optionally, the pharma- ceutically acceptable adjuvant comprises a protectant; Optionally, the pharma- ceutical composition, wherein said pharma- ceutical acceptable adjuvant comprises a cell cryopreservation solution.

17. The pharmaceutical composition of claim 16, for intravenous injection.

18. 16. Use of a chimeric antigen receptor according to any one of claims 1 to 11, an isolated nucleic acid molecule according to claim 12, a vector according to claim 13, or an engineered immune effector cell according to claim 14 or 15, in the preparation of a medicament for use in the treatment of a disease or condition associated with expression of BCMA.

19. 19. The use of claim 18, wherein the disease or condition associated with expression of BCMA is cancer, optionally wherein the cancer is multiple myeloma, and further optionally wherein the cancer is refractory or relapsed multiple myeloma.

20. 19. The use of claim 18, wherein the disease or condition associated with expression of BCMA is an autoimmune disease, and optionally the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.

21. A method for treating a disease or condition associated with BCMA expression, comprising administering to a subject in need of treatment thereof an effective amount of an engineered immune effector cell of claim 14 or 15, or a pharmaceutical composition of claim 16 or 17.

22. 22. The method of claim 21, wherein the disease or condition associated with expression of BCMA is cancer, optionally wherein the cancer is multiple myeloma, and further optionally wherein the cancer is refractory or relapsed multiple myeloma.

23. 22. The method of claim 21, wherein the disease or condition associated with expression of BCMA is an autoimmune disease, and optionally, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.

24. The administration method is intravenous injection, Optionally, said method of administration is administering to a subject an effective amount of the engineered immune effector cells of claim 14 or 15 or the pharmaceutical composition of claim 16 or 17 in a single injection; Further optionally, an effective amount of the engineered immune effector cells of claim 14 or 15 or the pharmaceutical composition of claim 16 or 17 is 1×10 ５ ~1×10 ７ The method according to any one of claims 21 to 23, wherein the amount of cells is per kg.

25. 18. The engineered immune effector cell of claim 14 or 15 or the pharmaceutical composition of claim 16 or 17 for use in the treatment of a disease or condition associated with expression of BCMA.

26. 26. The engineered immune effector cell or pharmaceutical composition of claim 25, wherein the disease or condition associated with expression of BCMA is cancer, optionally wherein the cancer is multiple myeloma, further optionally wherein the cancer is refractory or relapsed multiple myeloma.

27. 26. The engineered immune effector cell or pharmaceutical composition of claim 25, wherein the disease or condition associated with expression of BCMA is an autoimmune disease, and optionally the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.

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