Bispecific chimeric antigen receptor targeting bcma-cd19 and use thereof

Abstract

The invention provides a bispecific chimeric antigen receptor targeting BCMA-CD19 and application of the bispecific chimeric antigen receptor. The bispecific chimeric antigen receptor comprises an extracellular antigen recognition structural domain; wherein the extracellular antigen recognition structural domain comprises an anti-BCMA extracellular antigen recognition structural domain and an anti-CD19 extracellular antigen recognition structural domain; the anti-CD19 extracellular antigen recognition structural domain comprises CD19VH and CD19VL, and the CD19VH and the CD19VL are selected from one of the first group to the eighth group.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202411958182.3 (22) Application Date 2024.12.27 (71) Applicant Heyuan Kanghua Pharmaceutical Technology (Beijing) Co., Ltd. Address 102204, Beijing Changping District Liucun Town Beiliu Village A6-587 (72) Inventors Li Zhu, Zheng Yaling, Wang Huanyu, Zhang Chao, Liu Lishi, Lin Wang Yongzeng, Lü Lulu (74) Patent Agency Beijing Beixiang Intellectual Property Agency Co., Ltd. 11285 Patent Attorney Zhang Yong, Zhang Guangyu (51) Int.Cl. C07K 19 / 00 (2006.01) C12N 15 / 867 (2006.01) C12N 5 / 10 (2006.01) C12N 15 / 62 (2006.01) A61K 40 / 31(2025.01) A61K 40 / 15(2025.01) A61K 40 / 17(2025.01) A61K 40 / 11(2025.01) A61K 40 / 42(2025.01) A61P 35 / 00(2006.01) A61P 37 / 02(2006.01) (54) Invention Title: Bispecific Chimeric Antigen Receptor Targeting BCMA-CD19 and Its Application (57) Abstract: This invention provides a bispecific chimeric antigen receptor targeting BCMA-CD19 and its application, wherein the bispecific chimeric antigen receptor includes an extracellular antigen recognition domain; wherein: the extracellular antigen recognition domain includes an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain; the anti-CD19 extracellular antigen recognition domain includes CD19VH and CD19VL, which are selected from one of groups 1) to 8). Claims (4 pages), Description (24 pages), Sequence Listing (electronic publication), Figures (10 pages), CN 122302079 A 2026.06.30 CN 1 22 30 20 79 A 1. A bispecific chimeric antigen receptor targeting BCMA-CD19, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain; wherein: the extracellular antigen recognition domain comprises an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain; the anti-CD19 extracellular antigen recognition domain comprises CD19 VH and CD19 VL, selected from one of the following: 1) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:1, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or 2) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:3.The VL sequence includes the amino acid sequence shown in SEQ ID NO:2; or 3) the CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:5; or 4) the CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:6; or 5) the CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:7; or 6) the CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:9; or 7) the CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:11; or 8) the CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:2. 1. The amino acid sequence shown in SEQ ID NO:11. 2. The bispecific chimeric antigen receptor according to claim 1, wherein the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:6; or the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11. 3. The bispecific chimeric antigen receptor according to claim 1 or 2, wherein the BCMA VH sequence comprises the amino acid sequence shown in SEQ ID NO:12, and the BCMA VL sequence comprises the amino acid sequence shown in SEQ ID NO:13. 4. The bispecific chimeric antigen receptor according to claim 1 or 2, wherein the extracellular antigen recognition domain of the bispecific chimeric antigen receptor is an scFv antibody, an sc(Fv)2 antibody, or a [sc(Fv)2]2 antibody. 5. The bispecific chimeric antigen receptor according to claim 4, wherein the scFv antibody comprises any one of the following structures: CD19 VL sequence - first linker sequence - BCMA VL sequence - second linker sequence - BCMA VH sequence - third linker sequence - CD19 VH sequence, BCMA VL sequence - fourth linker sequence - CD19 VL sequence - fifth linker sequence - CD19 VH sequence - sixth linker sequence - BCMAVH sequence, CD19VL sequence - 7th linker sequence - CD19 VH sequence - 8th linker sequence - BCMA VL sequence - 9th linker sequence - BCMA VH sequence, BCMA VL sequence - 10th linker sequence - BCMA VH sequence - 11th linker sequence - CD19 VL sequence - 12th linker sequence - CD19 VH sequence; Optionally, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor includes any one of the following structures: CD19 VL sequence - 1st linker sequence - BCMA VL sequence - 2nd linker sequence - BCMA VH sequence - 3rd linker sequence - CD19 VH sequence and BCMA VL sequence - 4th linker sequence - CD19 VL sequence - 5th linker sequence - CD19 VH sequence - 6th linker sequence - BCMA VH sequence; Claims 1 / 4 page 2 CN 122302079 A Further optionally, 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 independently selected from one or more of the following sequences: SEQ ID NO:14 and SEQ ID NO:15. 6. The bispecific chimeric antigen receptor according to claim 5, wherein the extracellular antigen recognition domain of the bispecific chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28. 7. The bispecific chimeric antigen receptor according to claim 1, wherein 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; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:29; and / or 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 acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:30. 8. The bispecific chimeric antigen receptor according to claim 1, wherein the intracellular domain comprises an intracellular signal transduction region; optionally, the intracellular signal transduction region originates from CD3ζ, CD3γ, CD3δ, CD3ε, CCD5, CD22, CD79a, CD79b, FcRγ.The intracellular signal transduction 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, the intracellular signal transduction region is derived from CD3ζ. Even further optionally, the amino acid sequence of the intracellular signal transduction region is derived from 4-1BB. 9. The bispecific chimeric antigen receptor according to claim 1, wherein the intracellular domain further includes a co-stimulatory signal transduction region; optionally, the co-stimulatory signal transduction region is derived from one, two, 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, the co-stimulatory signal transduction region is derived from 4-1BB. Even further optionally, the amino acid sequence of the co-stimulatory signal transduction region is derived from the amino acid sequence shown in SEQ ID NO:31. 10. The bispecific chimeric antigen receptor according to any one of claims 1-9, further comprising a guide peptide located at the N-terminus of the amino acid sequence of the chimeric antigen receptor; optionally, the guide peptide is derived from CD8α; further optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence shown in SEQ ID NO:33. 11. The bispecific chimeric antigen receptor according to any one of claims 1-9, wherein the bispecific chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. 12. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a bispecific chimeric antigen receptor according to any one of claims 1-11; optionally, the nucleotide sequence encoding the bispecific chimeric antigen receptor comprises a nucleotide sequence encoding CD19 VH and CD19 VL and a nucleotide sequence encoding BCMA VH and BCMA VL, wherein the nucleotide sequences encoding CD19 VH and CD19 VL are selected from a group of: 1) a nucleotide sequence encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:1, as shown in SEQ ID NO:38; and a nucleotide sequence encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:2, as shown in SEQ ID NO:39; and / or 2) a nucleotide sequence encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:3, as shown in SEQ ID NO:40; and a nucleotide sequence encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:2, as shown in SEQ ID NO:39; and / or the claimsPage 2 / 4 3 CN 122302079 A 3) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:4, as shown in SEQ ID NO:41; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:5, as shown in SEQ ID NO:42; and / or 4) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:41; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:6, as shown in SEQ ID NO:43; and / or 5) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:3, as shown in SEQ ID NO:40; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:7, as shown in SEQ ID NO:44; and / or 6) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:8, as shown in SEQ ID NO:45; and nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:41; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:42; and nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:43; and nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:44; and nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:45; and nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:41; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:42; and nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:43; and nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:44; and nucleotide sequences encoding the CD The nucleotide sequences encoding the CD19 VL amino acid sequence shown in SEQ ID NO:9 are as shown in SEQ ID NO:46; and / or 7) the nucleotide sequences encoding the CD19 VH amino acid sequence shown in SEQ ID NO:10 are as shown in SEQ ID NO:47; and the nucleotide sequences encoding the CD19 VL amino acid sequence shown in SEQ ID NO:11 are as shown in SEQ ID NO:48; and / or 8) the nucleotide sequences encoding the CD19 VH amino acid sequence shown in SEQ ID NO:8 are as shown in SEQ ID NO:45; and the nucleotide sequences encoding the CD19 VL amino acid sequence shown in SEQ ID NO:11 are as shown in SEQ ID NO:48; The nucleotide sequences encoding BCMA VH and BCMA VL are: The nucleotide sequences encoding the BCMA VH amino acid sequence shown in SEQ ID NO:12 are as shown in SEQ ID NO:49; and the nucleotide sequences encoding the BCMA VL amino acid sequence shown in SEQ ID NO:13 are as shown in SEQ ID NO:50. 13. A vector comprising the isolated nucleic acid molecule of claim 12; optionally, the vector is an expression vector; further optionally, the vector is a viral vector; and even more optionally, the vector is a lentiviral vector. 14. An engineered immune effector cell comprising a chimeric antigen receptor as described in any one of claims 1-11.15. The engineered immune effector cells according to claim 14, wherein the engineered immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), pluripotent stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, and embryonic stem cells; optionally, the engineered immune effector cells are T lymphocytes; further optionally, the T lymphocytes are derived from autologous T lymphocytes or allogeneic T lymphocytes. 16. A pharmaceutical composition comprising the engineered immune effector cells according to claim 14 or 15 and pharmaceutically acceptable excipients; optionally, the pharmaceutically acceptable excipients include protective agents; optionally, the pharmaceutically acceptable excipients include cell cryopreservation solutions. 17. The pharmaceutical composition according to claim 16, wherein the pharmaceutical composition is a cell suspension or its cryopreserved cells; or the pharmaceutical composition is an intravenous injection. 18. The use of the chimeric antigen receptor of any one of claims 1-11, the isolated nucleic acid of claim 12, the vector of claim 13, or the engineered immune effector cells of claim 14 in the preparation of a medicament for treating a disease or condition associated with BCMA expression. 19. The use according to claim 18, wherein 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. 20. The use according to claim 19, wherein the disease or condition associated with BCMA expression can be an autoimmune disease; optionally, the autoimmune disease can be selected from: systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia. Claims 4 / 4 Page 5 CN 122302079 A Bispecific Chimeric Antigen Receptor Targeting BCMA-CD19 and Its Application Technical Field

[0001] This application relates to the field of biomedicine, specifically to a bispecific chimeric antigen receptor targeting BCMA-CD19 and its application. Background Art

[0002] Cell therapy is an emerging medical technology. Its main principle is to utilize the patient's own immune cells, which are cultured and modified in vitro to give them the ability to specifically kill tumor cells. These modified immune cells are then reinfused into the patient to achieve the purpose of treating tumors. Among them, chimeric antigen receptor T cell (CAR-T) technology is aCAR-T cell therapy is an important cell therapy technology that uses genetic engineering to enable T cells to express a specific antibody, thereby allowing T cells to specifically recognize and kill tumor cells expressing the corresponding antigen. Tumor immunotherapy is a treatment method that utilizes the body's own immune system to fight tumors, with the main goal of enhancing the body's immune system's ability to recognize and kill tumor cells. Among them, CAR-T cell therapy is an important tumor immunotherapy method, which directly introduces T cells with specific killing capabilities into the patient's body to achieve specific killing of tumor cells. Genetic engineering technology is an important part of modern biotechnology, which achieves genetic modification of organisms through operations such as gene cutting, ligation, and transfer. In CAR-T cell therapy, genetic engineering technology is widely used to modify T cells to give them specific anti-tumor capabilities.

[0003] Existing CAR-T cell therapies mainly target single tumor antigens, such as CD19 or BCMA. By designing and constructing CAR-T cells that target specific tumor antigens, specific killing of corresponding tumor cells can be achieved. However, this single-target CAR-T cell therapy has some problems. For example, if the expression level of the antigen on the surface of the tumor cells is low, or if the tumor cells escape antigens, then the CAR-T cells may not be able to effectively kill the tumor cells. 3) Problems of existing technology: Existing single-target CAR-T cell therapy has some problems and limitations. First, due to the heterogeneity and complexity of tumor cells, a single tumor antigen may not meet all treatment needs. Second, tumor cells can evade the attack of CAR-T cells by changing antigen expression or producing antigen escape. In addition, single-target CAR-T cell therapy may also lead to increased drug resistance of tumor cells, thereby affecting the treatment effect. Therefore, how to construct CAR-T cells that simultaneously target multiple tumor antigens to improve the treatment effect is an important challenge facing current cell therapy technology.

[0004] Autoimmune diseases (AIDs) are a class of diseases caused by the immune reaction between pathogenic autoantibodies and self-antigens, resulting in damage to the body's own tissues. Currently, they include more than 80 diseases, affecting about 7.6% to 9.4% of the world's population. The potential advantages of CAR-T therapy in autoimmune diseases such as systemic lupus erythematosus include: 1) the opportunity for immune memory remodeling through deep clearance of B cells in the body. T cells can enter tissues and clear memory B cells in the tissues. 2) long-term remission with a single treatment, avoiding continuous drug administration.

[0005] Autoantibodies and autoreactive B cells are directly related to autoimmune diseases. These pathogenic B cells will recognize self-antigens and produce self-antigen antibodies, leading to the development of autoimmune diseases.B cells are the source of pathogenic antibodies. B cells can differentiate into plasmablasts and long-lived plasma cells. Among them, B cells, memory B cells, and plasmablasts express CD19, while long-lived plasma cells express BCMA but not CD19. Therefore, CAR-T therapy targeting both CD19 and BCMA is expected to achieve better therapeutic effects and a wider range of indications. Specification 1 / 24 pages 6 CN 122302079 A Summary of the Invention

[0006] This application provides a bispecific chimeric antigen receptor targeting BCMA-CD19 and its application. Based on existing CD19 murine antibody sequences, the inventors designed 8 humanized CD19 antibodies to reduce their heterologity. However, during the humanization process, the antibody's functionality (e.g., CAR positivity rate expression, in vitro killing activity, cytokine release, etc.) may change. Therefore, obtaining humanized antibodies with reduced immunogenicity and no loss of functionality is of practical significance. This invention screened two humanized CD19 antibodies with good efficacy and, together with BCMA antibody, constructed multiple bispecific chimeric antigen receptor expression vectors targeting BCMA-CD19, and prepared bispecific CAR-T cells targeting BCMA-CD19. Furthermore, the invention verified at the cellular level that the BCMA-CD19 bispecific CAR-T cells have good tumor-suppressing function.

[0007] This invention provides a bispecific chimeric antigen receptor targeting BCMA-CD19, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain; wherein: the extracellular antigen recognition domain comprises an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain; the anti-CD19 extracellular antigen recognition domain comprises CD19 VH and CD19 VL, selected from one of the following:

[0008] 1) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:1, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or

[0009] 2) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or

[0010] 3) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2. The amino acid sequence shown in NO:5; or

[0011] 4) the CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:6; or

[0012] 5) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:7; or

[0013] 6) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:9; or

[0014] 7) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11; or

[0015] 8) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11.

[0016] In a preferred embodiment of the present invention, the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:6; or

[0017] the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11.

[0018] In a preferred embodiment of the present invention, the BCMA VH sequence comprises the amino acid sequence shown in SEQ ID NO:12, and the BCMA VL sequence comprises the amino acid sequence shown in SEQ ID NO:13.

[0019] In some embodiments, this application further comprises one or more amino acids in the amino acid sequence of any of the above-mentioned bispecific chimeric antigen receptors being substituted, deleted, added, and / or inserted, and having activity equivalent to any of the above-mentioned chimeric antigen receptors. Those skilled in the art will understand that during the humanization process, amino acids in the FR region of the VH and VL sequences can be substituted so that the CDR region of the modified antibody can retain a suitable antigen-binding site. Therefore, this application naturally includes different amino acid sequences obtained by humanizing the FR region of the VH and VL sequences based on the CDR described above in this application. Furthermore, those skilled in the art will also understand that during the humanization process, in order to ensure that the CDR region of the modified antibody retains a suitable antigen-binding site, if necessary, one, two, three, or no more than 10% of the amino acid sequence in the CDR may be substituted, deleted, added, and / or inserted. These contents are also included in this application.

[0020] In a preferred embodiment of the present invention, the extracellular antigen recognition structure of the bispecific chimeric antigen receptor...The domain is scFv antibody, sc(Fv)2 antibody or [sc(Fv)2]2 antibody.

[0021] In a preferred embodiment of the present invention, the scFv antibody comprises any one of the following structures: CD19 VL sequence - first linker sequence - BCMA VL sequence - second linker sequence - BCMA VH sequence - third linker sequence - CD19 VH sequence, BCMA VL sequence - fourth linker sequence - CD19 VL sequence - fifth linker sequence - CD19 VH sequence - sixth linker sequence - BCMA VH sequence, CD19 VL sequence - seventh linker sequence - CD19 VH sequence - eighth linker sequence - BCMA VL sequence - ninth linker sequence - BCMA VH sequence, BCMA VL sequence - tenth linker sequence - BCMA VH sequence - eleventh linker sequence - CD19 VL sequence - twelfth linker sequence - CD19 VH sequence;

[0022] Optionally, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor comprises any one of the following structures: CD19 VL sequence - first linker sequence - BCMA VL sequence - second linker sequence - BCMA VH sequence - third linker sequence - CD19 VH sequence and BCMA VL sequence - fourth linker sequence - CD19 VL sequence - fifth linker sequence - CD19 VH sequence - sixth linker sequence - BCMA VH sequence;

[0023] Further optionally, 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 independently selected from one or more of the following sequences: SEQ ID NO:14 and SEQ ID NO:15.

[0024] In a preferred embodiment of the present invention, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor includes the amino acid sequence shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28.

[0025] In a preferred embodiment of the present invention, 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; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:29; and / or

[0026] the transmembrane region is derived from CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152,One or more of OX40 and Fc70; optionally, the amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:30.

[0027] In a preferred embodiment of the present invention, the intracellular domain comprises an intracellular signal transduction region; optionally, the intracellular signal transduction region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CCD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; further optionally, the intracellular signal transduction region is derived from CD3ζ; even further optionally, the amino acid sequence of the intracellular signal transduction region comprises the amino acid sequence shown in SEQ ID NO:32.

[0028] In a preferred embodiment of the present invention, the intracellular domain further includes a co-stimulatory signal transduction region; optionally, the co-stimulatory signal transduction region is derived from one, two, 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, the co-stimulatory signal transduction region is derived from 4-1BB; even further optionally, the amino acid sequence of the co-stimulatory signal transduction region comprises the amino acid sequence shown in SEQ ID NO:31.

[0029] In a preferred embodiment of the present invention, the above-mentioned bispecific chimeric antigen receptor further includes a guide peptide located at the N-terminus of the chimeric antigen receptor amino acid sequence; optionally, the guide peptide is derived from CD8α; further optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence shown in SEQ ID NO:33. Specification 3 / 24 pages 8 CN 122302079 A

[0030] In a preferred embodiment of the present invention, the bispecific chimeric antigen receptor comprises an amino acid sequence as shown in SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37.

[0031] The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned bispecific chimeric antigen receptor;

[0032] Optionally, the nucleotide sequence encoding the bispecific chimeric antigen receptor comprises a nucleotide sequence encoding CD19 VH and CD19 VL and a nucleotide sequence encoding BCMA VH and BCMA VL, wherein the nucleotide sequence encoding CD19 VH and CD19 VL is selected from one group of the following:

[0033] 1) Encoding CD19 as shown in SEQ ID NO: 1The nucleotide sequence encoding the VH amino acid sequence is shown in SEQ ID NO: 38; and the nucleotide sequence encoding the CD19 VL amino acid sequence is shown in SEQ ID NO: 2, as shown in SEQ ID NO: 39; and / or

[0034] 2) the nucleotide sequence encoding the CD19 VH amino acid sequence is shown in SEQ ID NO: 3, as shown in SEQ ID NO: 40; and the nucleotide sequence encoding the CD19 VL amino acid sequence is shown in SEQ ID NO: 2, as shown in SEQ ID NO: 39; and / or

[0035] 3) the nucleotide sequence encoding the CD19 VH amino acid sequence is shown in SEQ ID NO: 41; and the nucleotide sequence encoding the CD19 VL amino acid sequence is shown in SEQ ID NO: 5, as shown in SEQ ID NO: 42; and / or

[0036] 4 ...42; and the nucleotide sequence encoding the CD19 VH amino acid sequence is shown in SEQ ID NO: 41; and the nucleotide sequence encoding the CD19 VL amino acid sequence is shown in SEQ ID NO: 42; and the nucleotide sequence encoding the CD19 VH amino acid sequence is shown in SEQ ID NO: 42; and the nucleotide sequence encoding the CD19 VL amino acid sequence is shown in SEQ ID NO: 42; and the nucleotide sequence encoding the CD19 VH amino acid sequence is shown in SEQ ID NO: 42; and the nucleotide sequence encoding the CD19 V The nucleotide sequence encoding the CD19 VL amino acid sequence shown in NO:6, as shown in SEQ ID NO:43; and / or

[0037] 5) the nucleotide sequence encoding the CD19 VH amino acid sequence shown in SEQ ID NO:3, as shown in SEQ ID NO:40; and the nucleotide sequence encoding the CD19 VL amino acid sequence shown in SEQ ID NO:7, as shown in SEQ ID NO:44; and / or

[0038] 6) the nucleotide sequence encoding the CD19 VH amino acid sequence shown in SEQ ID NO:8, as shown in SEQ ID NO:45; and the nucleotide sequence encoding the CD19 VL amino acid sequence shown in SEQ ID NO:9, as shown in SEQ ID NO:46; and / or

[0039] 7) the nucleotide sequence encoding the CD19 VH amino acid sequence shown in SEQ ID NO:10, as shown in SEQ ID NO:47; and the nucleotide sequence encoding the CD19 VL amino acid sequence shown in SEQ ID NO:1 ... As shown in NO:48; and / or

[0040] 8) nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:8, as shown in SEQ ID NO:45; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:11, as shown in SEQ ID NO:48;

[0041] The nucleotide sequences encoding BCMA VH and BCMA VL are:

[0042] Encoding as shown in SEQ IDThe nucleotide sequence encoding the BCMA VH amino acid sequence shown in NO:12 is as shown in SEQ ID NO: 49; and the nucleotide sequence encoding the BCMA VL amino acid sequence shown in SEQ ID NO:13 is as shown in SEQ ID NO: 50.

[0043] The present invention also provides a vector comprising the above-isolated nucleic acid molecule;

[0044] Optionally, the vector is an expression vector;

[0045] Further optionally, the vector is a viral vector;

[0046] Even further optionally, the vector is a lentiviral vector. Specification 4 / 24 pages 9 CN 122302079 A

[0047] The present invention also provides an engineered immune effector cell comprising the above-mentioned chimeric antigen receptor, the above-isolated nucleic acid, or the above-mentioned vector.

[0048] In a preferred embodiment of the present invention, the engineered immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), pluripotent stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, and embryonic stem cells;

[0049] Optionally, the engineered immune effector cells are T lymphocytes;

[0050] Further optionally, the T lymphocytes are derived from autologous T lymphocytes or allogeneic T lymphocytes.

[0051] In some embodiments, the surface of the engineered immune effector cells may express or express the chimeric antigen receptor described in this application.

[0052] The present invention also provides a pharmaceutical composition comprising the above-mentioned engineered immune effector cells and pharmaceutically acceptable excipients; pharmaceutically acceptable excipients include one or more of the following: carriers, protectants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives.

[0053] Optionally, pharmaceutically acceptable excipients include protective agents; alternatively, pharmaceutically acceptable excipients include cell cryopreservation solutions.

[0054] In a preferred embodiment of the invention, the pharmaceutical composition is a cell suspension or cryopreserved cells thereof.

[0055] In a preferred embodiment of the invention, the pharmaceutical composition is an intravenous injection.

[0056] The invention also provides the use of the above-described chimeric antigen receptor, the above-described isolated nucleic acid, the above-described carrier, or the above-described engineered immune effector cells in the preparation of a medicament for treating diseases or conditions associated with BCMA expression.

[0057] In a preferred embodiment of the invention, the disease or condition associated with BCMA expression is cancer; alternatively, the cancer is multiple myeloma; further alternatively, the cancer is refractory or relapsed multiple myeloma.

[0058] In a preferred embodiment of the present invention, the disease or condition associated with BCMA expression may be an autoimmune disease; optionally, the autoimmune disease may be selected from the following: systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.

[0059] On the other hand, the present application also provides a method for treating a disease or condition associated with BCMA expression, the method comprising administering an effective dose of the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, the above-mentioned carrier, and / or the above-mentioned engineered immune effector cells to a subject who needs treatment for a disease or condition associated with BCMA expression.

[0060] In some embodiments, the administration may be performed in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, local, or intradermal administration. For example, the administration may be performed by intravenous injection to the subject. In some embodiments, an effective dose of engineered immune effector cells or pharmaceutical composition may be administered to the subject once or in multiple doses over a period of time, such as 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.

[0061] In some embodiments, the dosage may vary for different indications; the dosage may also vary for patients with different degrees of disease severity. The dosage range can be from 1×10⁵ CAR-positive T cells / kg to 1×10⁷ CAR-positive T cells / kg, for example, 1×10⁵ CAR-positive T cells / kg to 1×10⁶ CAR-positive T cells / kg, 1×10⁶ CAR-positive T cells / kg to 1×10⁷ CAR-positive T cells / kg, 0.5×10⁶ CAR-positive T cells / kg, 0.6×10⁶ CAR-positive T cells / kg, 0.7×10⁶ CAR-positive T cells / kg, 0.8×10⁶ CAR-positive T cells / kg, 0.9×10⁶ CAR-positive T cells / kg, 1.0×10⁶ CAR-positive T cells / kg, 1.1×10⁶ CAR-positive T cells / kg, 1.2 ... 1.3 × 10⁶ CAR-positive T cells / kg, 1.4 × 10⁶ CAR-positive T cells / kg, 1.5 × 10⁶ CAR-positive T cells / kg, 1.6 × 10⁶ CAR-positive T cells / kg, 1.7 × 10⁶ CAR-positive T cells / kg, 1.8 × 10⁶ CAR-positive T cells / kg, 1.9 × 10⁶ CAR-positive T cells / kg, 2.0 × 10⁶ CAR-positive T cells / kg.

[0062] In some embodiments, the subject may include humans and non-human animals. For example, the subject may include, but is not limited to, mice, rats, cats, dogs, horses, pigs, cattle, sheep, rabbits, or monkeys.

[0063] Compared with the prior art, the bispecific chimeric antigen receptor targeting BCMA-CD19 of the present invention has the following beneficial effects:

[0064] The dual-target humanized CD19 / BCMACAR-T of the present invention has the same pharmacological efficacy as single-target CD19 CAR-T and single-target BCMACAR-T, and the dual-target humanized CD19 / BCMACAR-T has lower immunogenicity than single-target murine CD19 CAR-T, thereby having better clinical efficacy and safety. Brief Description of the Drawings

[0065] Figure 1: Positive rate of CD19-CAR cells at different time points.

[0066] Figure 2: Statistical graph of CD19-CAR MFI at different time points.

[0067] Figure 3: In vitro killing activity against positive target cells Nalm6.

[0068] Figure 4: Detection of cytokine release during Nalm6 cell killing.

[0069] Figure 5: CAR-T cell expansion fold during multiple rounds of Nalm6 stimulation.

[0070] Figure 6: CD19-CAR positivity rate and BCMA-CAR positivity rate in different groups on Day 9.

[0071] Figure 7: Scoring results of CD19 humanized sequences using three biological computer evaluation tools (T20 score, Hscore, QASis).

[0072] Figure 8: Immunogenicity risk prediction results of CD19 humanized sequences using two immunogenicity prediction tools (AlphaMHC v2, CD4 T cell Immunogenicity prediction tool).

[0073] Figure 9: In vitro killing activity against Nalm6 cells.

[0074] Figure 10: In vitro killing activity against K562-CD19 cells.

[0075] Figure 11: In vitro killing activity against MM.1S cells.

[0076] Figure 12: In vitro killing activity against target cells K562-BCMA.

[0077] Figure 13: Detection of IL-2 cytokine release during killing of target cells Nalm6 and MM.1S.

[0078] Figure 14: Detection of IFN-γ cytokine release during killing of target cells Nalm6 and MM.1S.

[0079] Figure 15: CAR-T cell expansion fold during multiple rounds of stimulation of target cells Nalm6.

[0080] Figure 16: CAR-T cell expansion fold during multiple rounds of stimulation of target cells RPMI-8226.

[0081] Figure 17: Imaging fluorescence statistics of different groups of mice in the Nalm6 tumor-bearing animal model.

[0082] Figure 18: Imaging fluorescence statistics of different groups of mice in the MM.1S tumor-bearing animal model.

[0083] In Figures 1-2, each bar, from left to right, represents CNCT19, Q11, Q14, Q23, Q33, Q54, GS32, GS21, and GS22. In Figures 3-4, each bar, from left to right, represents UTD (untransduced CAR T cells), CNCT19, Q11, Q14, Q33, Q54, GS32, GS21, and GS22. In Figure 5, each bar, from left to right, represents CNCT19, Q11, Q14, Q33, Q54, GS32, GS21, and GS22. In Figures 9-14, each bar, from left to right, represents UTD (untransduced CAR T cells), CNCT19, Q33, GS21, O2G, O2G-Q33, Q33-O2G, O2G-GS21, and GS21-O2G. In Figures 15-16, each column from left to right represents CNCT19, Q33, 02G, and 02G-Q33 respectively. Specification 6 / 24 pages 11 CN 122302079 A Detailed Description

[0084] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0085] The following further describes the invention: In this invention, unless otherwise stated, the scientific and technical terms used herein have meanings commonly understood by those skilled in the art. Furthermore, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology-related terms and laboratory procedures used herein are all widely used terms and routine procedures in the corresponding fields. Meanwhile, to better understand the invention, definitions and explanations of relevant terms are provided below.

[0086] In this application, the term "Chimeric Antigen Receptor" (CAR) is a core component of CAR cell therapy drugs, which may include an extracellular antigen recognition domain (e.g., a portion that binds to tumor-associated antigens (TAAs)), 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 means to conquer tumors. CAR-T cells utilize genetic modification to enable T cells to express CAR proteins. These CAR proteins are capable of recognizing intact proteins on the membrane surface without relying on antigen presentation, thereby activating and functionally affecting T cells.

[0087] In this application, the term "extracellular antigen recognition domain" refers to an antigen recognition domain.Domain (ARD). CAR cell therapy products (such as CAR-T cells) can specifically recognize and / or bind to target antigens expressed by tumor cells by relying on extracellular antigen recognition domains. To date, antigen recognition domains are derived from the single-chain variable fragment (scFv) of antibodies, or from receptor-ligand interactions, TCR mimics, and variable lymphocyte receptors (VLRs). The most common source to date is the scFv segment of antibodies. The scFv includes the antibody heavy chain variable region and the light chain variable region, linked by a peptide chain, such as the 18-amino acid linker sequence GSTGSGSGKPGSGEGSTKG. Common CDR (Canonical Receiver Registry) rules for antibodies include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well-known to those skilled in the art. When applying websites that execute these rules, simply inputting the VH and VL sequences and selecting the corresponding rule will yield CDR sequences according to different rules. Those skilled in the art should understand that the scope of protection of this application covers combinations of CDR sequences obtained by analysis using different rules.

[0088] In this application, the term "hinge region" refers to the connecting segment that acts between the extracellular antigen recognition domain and the transmembrane domain. This region allows CAR to recognize antigens by giving the antigen recognition domain a certain range of activity. 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 some residues that participate in CAR dimerization, which helps to enhance antigen sensitivity.

[0089] In this application, "transmembrane region" refers to the transmembrane domain that connects the intracellular and extracellular components of the CAR structure. Different transmembrane domains can affect the expression and stability of CAR to a certain extent, but they do not directly participate in signal transduction. However, they can enhance downstream signal transduction through interaction. The transmembrane region may originate from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70.

[0090] In this application, the term "intracellular domain" includes intracellular signal transduction regions and may also include co-stimulatory signal transduction regions.

[0091] In this application, the term "intracellular signal transduction region" refers to the activation of at least one normal effector function of immune effector cells responsible for expressing CAR. The intracellular signal transduction region may originate from CD3ζ, CD3γ, CD3δ, CD3ε, CCD5, CD22, CD79a, CD79b, and FcRγ.One or more of FcRβ, CD66d, DAP10, DAP12, and Syk.

[0092] In this application, the term "co-stimulatory signal transduction region" is used because, in addition to the stimulation of antigen-specific signals (as described in the specification 7 / 24 page 12 CN 122302079 A), many immune effector cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate the effector functions of cells. In some embodiments, the CAR may further include one or more co-stimulatory signal transduction regions, wherein the co-stimulatory signal transduction regions may be derived from one, two, 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.

[0093] In this application, the term "scFv" has the conventional meaning in the art, referring to a single-chain variable fragment (scFv), which is an antibody formed by linking the heavy chain variable fragment and the light chain variable fragment through a short peptide (linker).

[0094] In this application, terms such as "Sc(Fv)2" and "[Sc(Fv)2]2" without specific explanation also have the conventional meaning in the art.

[0095] In this application, the term "isolated" generally refers to something obtained artificially from its natural state. If a "isolated" substance or component appears in nature, it may be because its natural environment has changed, or the substance has been isolated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide may exist naturally in the body of a living animal, and a high-purity identical polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" does not exclude substances obtained artificially from their natural state and then processed artificially or synthesized, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0096] In this application, the term "guide peptide" refers to a short peptide preceding the extracellular antigen recognition domain (such as the scFv sequence), whose function is to guide the export of recombinant proteins synthesized intracellularly to the extracellular space. Commonly used guide peptides include human CD8α signal peptide or human GM-CSF receptor α signal peptide.

[0097] In this application, one of the key factors determining the efficacy of CAR-immunotherapy is the selection of tumor target antigens. In this application, the term "BCMA" refers to B cell maturation antigen, a member of the tumor necrosis factor receptor superfamily. Human BCMA is expressed almost exclusively in plasma cells and multiple myeloma cells. BCMA can be used for immune responses against multiple myeloma.Suitable tumor antigen targets for therapeutic agents. However, due to the heterogeneity of specific antigens on the surface of multiple myeloma cells, the selection of antigen targets is not necessarily singular. By selecting appropriate targets, the anti-tumor activity of CAR-T cells can be optimized. The "CD19" molecule is currently the main target for the treatment of B-lymphocyte-derived hematologic malignancies and is also a hot topic in CAR-T cell therapy research. Most B-cell-derived malignant tumor cells express CD19 molecules on their surface. Multiple myeloma, as a B-cell lineage tumor, generally does not express CD19 molecules, therefore CD19 is usually not used as a target for the treatment of multiple myeloma. However, some literature studies suggest that some trace amounts of drug-resistant and relapsed multiple myeloma clones also have a CD19+ phenotype. At the same time, compared with preparing CAR-immune cells targeting different targets separately and using them together, dual-target CAR-immune cells have the following advantages: 1. Fewer immune cells are required, preparation is convenient and cost-effective; 2. From the perspective of drug administration, the safety and operability of using one product are much higher than using two products.

[0098] In this application, the term "humanized antibody" also refers to a humanized antibody. Methods of humanization are known (e.g., WO96 / 02576). The purpose of humanization is to reduce heterology while substantially preserving the affinity and specificity of the parent antibody. For example, in the case where the CDR is derived from a mouse antibody, primer 25 (the corresponding primer can be obtained by referring to the method described in WO98 / 13388) can be synthesized and used to link the CDR of the mouse antibody to the frame region (FR) of the human antibody.

[0099] In this application, the term "linking 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 together. The linking sequence may consist of different amino acid residues (e.g., glycine and serine) so that adjacent protein domains can move freely relative to each other. Longer linking sequences can be used when it is desirable to ensure that two adjacent domains do not interfere with each other spatially. Specification 8 / 24 pages 13 CN 122302079 A

[0100] In this application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotide, deoxyribonucleotide, or ribonucleotide of any length, which may be isolated from its natural environment or artificially synthesized analogs.

[0101] In this application, during CAR gene transduction / transfection and target gene expression, the gene transduction / transfection methods mainly include viral and non-viral methods. For example: through γ-retroviral vectors, lentiviral vectors, adenovirus-associated viral vectors, plasmid DNA-dependent vectors, transposon-dependent gene transfer, and mRNA-mediated gene transduction.

[0102] The term "vector" generally refers to a vector into which a polynucleotide encoding a protein can be inserted to enable protein expression.A vector is a nucleic acid delivery vehicle. Vectors can transform, transduce, or transfect host cells, enabling the expression of their carried genetic material elements within the host cells. Examples of vectors include: plasmids; phage particles; Cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain replication initiation sites. Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances. The term "transposon" refers to a discontinuous DNA segment with the ability to migrate between chromosomal loci and carry genetic information, such as the Sleeping Beauty SB system and the PB system derived from lepidopteran insects. In some embodiments, electroporation can also be used to transduce mRNA into T cells.

[0103] In this application, the term "immune effector cell" generally refers to a cell that participates in an immune response, such as promoting an immune effector response. Immune effector cells may be selected from one or more of the following groups: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), pluripotent stem cells, T lymphocytes differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, and embryonic stem cells.

[0104] In this application, the term "pharmaceutical composition" generally refers to a pharmaceutical composition suitable for administration to a patient, which may contain the immune effector cells described in this application and may also contain one or more pharmaceutically acceptable excipients, such as one or more of the following: carriers, protectants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives. In some embodiments, pharmaceutically acceptable excipients include cryoprotectants, such as cell cryopreservation solutions. In some embodiments, the pharmaceutical composition of this application is a cell suspension or its cryopreserved cells.

[0105] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to mice, rats, cats, dogs, rabbits, horses, pigs, cattle, sheep, or monkeys.

[0106] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements.

[0107] In this application, the term "about" generally refers to a range of fluctuations acceptable to those skilled in the art above or below a specified value, such as variations within ±0.5% to 10%, for example, 0.5%, 1%, or more above or below a specified value.The percentage varies within the range of 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%.

[0108] On the other hand, this application also provides the chimeric antigen receptor, the isolated nucleic acid molecule, the vector, and / or the engineered immune effector cells, which can be used to treat diseases or conditions associated with BCMA expression.

[0109] In some embodiments, the diseases or conditions associated with BCMA expression may include non-solid tumors, optionally, the non-solid tumors being hematologic malignancies.

[0110] In some embodiments, the diseases or conditions associated with BCMA expression may include multiple myeloma. Specification 9 / 24 pages 14 CN 122302079 A

[0111] In some embodiments, the multiple myeloma is relapsed or refractory multiple myeloma.

[0112] Not intended to be limited by any theory, the embodiments described below are merely for illustrating the chimeric antigen receptor, engineered immune effector cells, preparation methods, and uses of this application, and are not intended to limit the scope of the invention. The embodiments do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in numerous publications, including Sambrook, J., Fritsch, E.F., and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0113] The murine antibody and humanized antibody scFv of the present invention

[0114] >HI19a(CNCT19)scFv

[0115] DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSG TDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKRGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKIS CKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCA RKTISSVVDFYFDYWGQGTTLTVSS(SEQ ID NO:51)

[0116] >Q11 scFv

[0117] DIQLTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSG TDFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSC KASGYAFSSYWMNWVRQAPGQGLEWMGQIYPGDGDTNYNGKFKGRVTMTRDTSTSTAYMELSSLRSEDTAVYFCAR KTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:16)

[0118] Q11 scFv contains Q11 VH and Q11 VL, wherein the amino acid sequence of Q11 VH is as follows:

[0119] QVQLVQSGAEVKKPGASVKVSCKASGYAFSSYWMNWVRQAPGQGLEW MGQIYPGDGDTNYNGKFKGR VTMTRDTSTSTAYMELSSLRSEDTAVYFCARK TISSVVDFYFDYWGQGTTVTVSS (SEQ ID NO: 1)

[0120] The nucleotide sequence encoding Q11 VH is as follows:

[0121] CAAGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCTCCGGTGAAGGTGAGCTGCAA AGCCTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAAGCCCCCGGCCAAGGCCTGGAGTGGATG GGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAGAGTCACCATGACAAGAGACA CAAGCACAAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTTCTGCGCTAGAAA GACAATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC(SEQ ID NO:38)

[0122] The amino acid sequence of Q11 VL is as follows:

[0123] DIQLTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGS GTDFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKLE IK (SEQ ID NO: 2)

[0124] The nucleotide sequence encoding Q11 VL is as follows:

[0125] GACATTCAGCTGACACAGAGCCCTAGCAGCCTGAGCGCCTCCGTGGGCGACAGAGTGACCATCACATG CAAGGCCTCCCAAAACGTGGGCACCAACGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCCCCTGATC TACAGCGCCACCTACAGAAACAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGA CCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTTCTGTCAGCAGTACAACAGATACCCCTACACATTTGG CGGGGGCACAAAGCTGGAGATTAAG (SEQ ID NO:39)

[0126] >Q14 scFv Specification 10 / 24 pages 15 CN 122302079 A

[0127] DIQLTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSG TDFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSC KASGYAFSSYWMNWVRQAPGQGLEWMGQIYPGDGDTNYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYFCAR KTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:17)

[0128] Q14 scFv contains Q14 VH and Q14 VL, wherein the amino acid sequence of Q14 VH is as follows:

[0129] QVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEW MGQIYPGDGDTNYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYFCARKT ISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:3)

[0130] The nucleotide sequence encoding Q14 VH is shown below:

[0131] CAAGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCTCCTCCGTGAAGGTGAGCTGCAA AGCCTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAAGCCCCCGGCCAAGGCCTGGAGTGGATG GGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAGAGTCACCATCACAGCAGACA AAAGCACAAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTTCTGCGCTAGAAA GACAATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC(SEQ ID NO: 40)

[0132] The amino acid sequence of Q14 VL is shown below:

[0133] DIQLTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSA TYRNSGVPSRFSGSGS GTDFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKLE IK(SEQ ID NO: 2)

[0134] The nucleotide sequence encoding Q14 VL is shown below (SEQ ID NO: 39):

[0135] GACATTCAGCTGACACAGAGCCCTAGCAGCCTGAGCGCCTCCGTGGGCGACAGAGTGACCATCACATG CAAGGCCTCCCAAAACGTGGGCACCAACGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCCCCTGATC TACAGCGCCACCTACAGAAACAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGA CCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTTCTGTCAGCAGTACAACAGATACCCCTACACATTTGG CGGGGGCACAAAGCTGGAGATTAAG(SEQ ID NO: 39)

[0136] >Q23 scFv

[0137] DIQLTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKSPKPLIYSATYRNSGVPSRFSGSGSG TDFTLTISSLQPKDFATYFCQQYNRYPYTSGGGTKLEIKGGGGSGGGGSGGGSEVQLVQSGAEVKKPGESLKISC KASGYAFSSYWMNWVRQMPGKGLEWMGQIYPGDGDTNYNGKFKGQVTLSADKSISTAYLQWSSLKASDTAMYFCAR KTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:18)

[0138] Q23 scFv contains Q23 VH and Q23 VL, wherein the amino acid sequence of Q23 VH is as follows:

[0139] EVQLVQSGAEVKKPGESLKISCKASGYAFSSYWMNWVRQMPGKGLEWM GQIYPGDGDTNYNGKFKGQ VTLSADKSISTAYLQWSSLKASDTAMYFCARKTI SSVVDFYFDYWGQGTTVTVSS (SEQ ID NO: 4)

[0140] The nucleotide sequence encoding Q23 VH is as follows:

[0141] GAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGCAA AGCTTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAGATGCCCGGCAAGGGCCTGGAGTGGATG GGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCCAAGTGACCCTGAGCGCCGACA AGAGCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCCGACACCGCCATGTACTTCTGCGCTAGAAA GACCATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC(SEQ ID NO:41)

[0142] The amino acid sequence of Q23 VL is as follows: Specification 11 / 24 page 16 CN 122302079 A

[0143] DIQLTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKSPKPLIYSATYRNSGVPSRFSGSGS GTDFTLTISSLQPKDFATYFCQQYNRYPYTSGGGTKLE IK (SEQ ID NO: 5)

[0144] The nucleotide sequence encoding Q23 VL is as follows:

[0145] GACATTCAGCTGACACAGAGCCCTAGCAGCCTGAGCGCCTCCGTGGGCGACAGAGTGACCATCACCTG CAAGGCCTCCCAAAACGTGGGCACCAACGTGGCCTGGTATCAGCAGAAGCCCGGCAAGAGCCCCAAGCCCCTGATC TACAGCGCCACCTACAGAAACAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGA CCATCAGCTCCCTGCAGCCCAAGGACTTCGCCACCTACTTCTGTCAGCAGTACAACAGATACCCCTACACATCCGG GGGCGGCACCAAGCTGGAAATCAAG (SEQ ID NO:42)

[0146] >Q33 scFv

[0147] EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRPLIYSATYRNSGIPARFSGSGSG TEFTLTISSLQSEDFAVYFCQQYNRYPYTfGGGTKLEIKGGGGSGGGGSGGGSEVQLvQSGAEvkkPGeSlKISC KASGYAFSSYWMNWVrQmPGkGLEWmGQIYPGDGDTNYNGKFKGQvTLsADKSiSTAYlQwSsLkasDtAmYFCAR KTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:19)

[0148] Q33 scFv contains Q33 VH and Q33 VL, wherein the amino acid sequence of Q33 VH is as follows:

[0149] EVQLvQSGAEvkkPGeSlKISCKASGYAFSSYWMNWVrQmPGkGLEWmGQI YPGDGDTNYNGKFKGQ vTLsADKSiSTAYlQwSsLkasDtAmYFCARKTISSVVDF YFDYWGQGTTVTVSS(SEQ ID NO:4)

[0150] The nucleotide sequence encoding Q33 VH is shown below:

[0151] GAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGCAA AGCTTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAGATGCCCGGCAAGGGCCTGGAGTGGATG GGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCCAAGTGACCCTGAGCGCCGACA AGAGCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCCGACACCGCCATGTACTTCTGCGCTAGAAA GACCATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC(SEQ ID NO: 41)

[0152] The amino acid sequence of Q33 VL is as follows:

[0153] EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRPLIYS ATYRNSGIPARFSGSGS GTEFTLTISSLQSEDFAVYFCQQYNRYPYTFGGGTKLE IK(SEQ ID NO: 6)

[0154] The nucleotide sequence encoding Q33 VL is as follows:

[0155] GAAATCGTGATGACCCAGTCCCCTGCTACACTGAGCGTGTCCCCAGGCGAGCGGGCCACACTGTCTTG CAAGGCCTCCCAAAACGTGGGCACCAACGTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGGCCCCTGATC TACAGCGCCACCTACAGAAACAGCGGCATCCCTGCCAGATTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGA CCATCAGCAGCCTGCAGTCCGAGGACTTCGCCGTCTACTTCTGTCAGCAGTACAACAGATACCCCTACACATTCGG CGGGGGGACCAAGCTGGAGATCAAA(SEQ ID NO: 43)

[0156] >Q54 scFv

[0157] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLvQSGAEvkkPGSSVKvSC KASGYAFSSYWMNWVrQaPGQGLEWmGQIYPGDGDTNYNGKFKGrvTiTADKStSTAYMeLSsLrSEDtAVYFCAR KTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:20)

[0158] Q54 scFv contains Q54 VH and Q54 VL, wherein the amino acid sequence of Q54 VH is as follows:

[0159] QVQLvQSGAEvkkPGSSVKvSCKASGYAFSSYWMNWVrQaPGQGLEWmGQ IYPGDGDTNYNGKFKGr Instruction manual 12 / 24 pages 17 CN 122302079 A vTiTADKStSTAYMeLSsLrSEDtAVYFCARKTISSVVDF YFDYWGQGTTVTVSS (SEQ ID NO:3)

[0160] The nucleotide sequence encoding Q54 VH is as follows:

[0161] CAAGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCTCCTCCGTGAAGGTGAGCTGCAA AGCCTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAAGCCCCCGGCCAAGGCCTGGAGTGGATG GGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAGAGTCACCATCACAGCAGACA AAAGCACAAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTTCTGCGCTAGAAA GACAATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC (SEQ ID NO: 40) The amino acid sequence of Q54 VL is as follows: DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGS GTEFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKVE IK (SEQ ID NO:7)

[0164] The nucleotide sequence encoding Q54 VL is as follows:

[0165] GACATCCAACTGACCCAGAGCCCCTCCTTCCTGAGCGCCTCTGTCGGCGATAGAGTGACAATCACCTG TAAGGCCAGCCAGAACGTGGGCACCAAGTGTGGCCTGGTACCAGCAGAAACCAGGCAAGGCTCCTAAGCCTCTGATC TACTCCGCTACATATCGGAACAGCGGCGTGCCTTCGAGATTTTCTGGCAGCGGCCTGGAACCGAGTTCACCCTGA CCATCTCCTCTCTGCAGCCTGAGGACTTCGCCACCTACTTCTGCCAGCAGTACAACAGATACCCCTACACCTTCGG AGGCGGCACCAAGGTGGAGATTAAG (SEQ ID NO:44)

[0166] >GS32 scFv

[0167] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSG TEFTLTISSLQPEDFADYFCQQYNRYPYTFGGGTKVEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVS CKASGYAFSSYWMNWVRQAPGQGLEWIGQIYPGDGDTNYNGKFKGRATITADKSTSTAYMELSSLRSEDTAVYFCA RKTISSVVDFYFDYWGQGTLVTVSS(SEQ ID NO:21)

[0168] GS32 scFv contains GS32 VH and GS32 VL, wherein the amino acid sequence of GS32 VH is as follows:

[0169] QVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWI GQIYPGDGDTNYNGKFKGR ATITADKSTSTAYMELSSLRSEDTAVYFCARKTIS SVVDFYFDYWGQGTLVTVSS(SEQ ID NO:8)

[0170] The nucleotide sequence encoding GS32 VH is shown below:

[0171] CAAGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGTAA AGCTTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAAGCCCCCGGCCAAGGCCTGGAGTGGATC GGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAGAGCCACCATCACCGCCGACA AGAGCACAAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTTCTGCGCTAGAAA GACCATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCCTGGTGACCGTGAGCAGC(SEQ ID NO:45)

[0172] The amino acid sequence of GS32 VL is as follows:

[0173] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSA TYRNSGVPSRFSGSGS GTEFTLTISSLQPEDFADYFCQQYNRYPYTFGGGTKVE IKR(SEQ ID NO:9)

[0174] The nucleotide sequence encoding GS32 VL is as follows:

[0175] GACATTCAGCTGACACAGAGCCCTAGCTTCCTGAGCGCCTCCGTGGGCGACAGAGTGACCATCACCTG CAAGGCTTCCCAAAACGTGGGCACCAACGTGGCCTGGTATCAGCAGAAACCCGGCAAGGCCCCCAAGCCCCTGATC TACAGCGCCACCTACAGAAACAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGA Description Page 13 / 24 18 CN 122302079 A CAATCAGCAGCCTGCAGCCCGAGGACTTCGCCGACTACTTCTGTCAGCAGTACAACAGATACCCCTACACCTTCGG CGGGGGCACCAAGGTGGAGATCAAGCGG(SEQ ID NO:46)

[0176] >GS21 scFv

[0177] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQYNRYPYTSGGGTKVEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVS CKASGYAFSSYWMNWVRQAPGQGLEWMGQIYPGDGDTNYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCA RKTISSVVDFYFDYWGQGTLVTVSS (SEQ ID NO: 22)

[0178] GS21 scFv contains GS21 VH and GS21 VL, where the amino acid sequence of GS21 VH is as follows:

[0179] QVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEW MGQIYPGDGDTNYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARK TISSVVDFYFDYWGQGTLVTVSS (SEQ ID NO: 10)

[0180] The nucleotide sequence encoding GS21 VH is as follows:

[0181] CAAGTGCAGCTGGTCCAGAGCGGCGCCGAGGTGAAGAAACCTGGCTCTAGCGTGAAAGTGTCCTGCAA GGCCTCTGGCTACGCCTTTTCCAGCTATTGGATGAACTGGGTGCGGCAGGCTCCTGGCCAGGGCCTGGAATGGATG GGCCAGATCTACCCGGAGATGGCGATACCAACTACAATGGCAAGTTCAAGGGCAGAGTGACCATCACCGCTGACA AGTCTACAAGCACAGCCTACATGGAGCTGTCCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGTGCCAGAAA GACCATCTCCTCTGTGGTGGACTTCTACTTCGACTACTGGGGACAGGGCACGCTGGTGACCGTGTCCTCT(SEQ ID NO:47)

[0182] The amino acid sequence of GS21 VL is as follows:

[0183] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSA TYRNSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQYNRYPYTSGGGTKVE IKR (SEQ ID NO: 11)

[0184] The nucleotide sequence encoding GS21 VL is as follows:

[0185] GACATCCAACTGACCCAGAGCCCCTCCTTCCTGAGCGCCTCTGTCGGCGATAGAGTGACAATCACCTG TAAGGCCAGCCAGAACGTGGGCACCAAGTGTGGCCTGGTACCAGCAGAAACCAGGCAAGGCTCCTAAGCCTCTGATC TACTCCGCTACATATCGGAACAGCGGCGTGCCTTCGAGATTTTCTGGCAGCGGCCTGGAACCGAGTTCACCCTGA CCATCTCCTCTCTGCAGCCTGAGGACTTCGCCACCTACTTCTGCCAGCAGTACAACAGATACCCCTACACCTCCGG AGGCGGCACCAAGGTGGAAATCAAGCGG (SEQ ID NO:48)

[0186] >GS22 scFv

[0187] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQYNRYPYTSGGGTKVEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVS CKASGYAFSSYWMNWVRQAPGQGLEWIGQIYPGDGDTNYNGKFKGRATITADKSTSTAYMELSSLRSEDTAVYFCA RKTISSVVDFYFDYWGQGTLVTVSS(SEQ ID NO:23)

[0188] GS22 scFv contains GS22 VH and GS22 VL, wherein the amino acid sequence of GS22 VH is as follows:

[0189] QVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWI GQIYPGDGDTNYNGKFKGR ATITADKSTSTAYMELSSLRSEDTAVYFCARKTIS SVVDFYFDYWGQGTLVTVSS(SEQ ID NO:8)

[0190] The nucleotide sequence encoding GS22 VH is shown below:

[0191] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGATCTAGCGTGAAAGTGTCCTGCAA GGCCTCCGGCTACGCCTTCAGCTCCTATTGGATGAACTGGGTGCGGCAGGCTCCTGGACAAGGCCTGGAATGGATC GGCCAGATCTACCCTGGCGATGGCGATACCAACTACAATGGCAAGTTCAAGGGCAGAGCCACCATCACCGCCGACA AGTCCACATCTACCGCCTACATGGAGCTGAGCAGCCTGAGATCCGAGGACACCGCTGTGTACTTTTGTGCCAGAAA Description 14 / 24 Page 19 CN 122302079 A GACCATCAGCTCCGTGGTGGACTTCTACTTCGACTACTGGGGCCAGGGCACCCTGGTCACAGTGTCTTCT(SEQ ID NO: 45)

[0192] The amino acid sequence of GS22 VL is shown below:

[0193] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSA TYRNSGVPSRFSGSGS GTEFTLTISSLQPEDFATYFCQQYNRYPYTSGGGTKVE IKR(SEQ ID NO: 11)

[0194] The nucleotide sequence encoding GS22 VL is shown below:

[0195] GACATCCAACTGACCCAGAGCCCCTCCTTCCTGAGCGCCTCTGTCGGCGATAGAGTGACAATCACCTG TAAGGCCAGCCAGAACGTGGGCACCAATGTGGCCTGGTACCAGCAGAAACCAGGCAAGGCTCCTAAGCCTCTGATC TACTCCGCTACATATCGGAACAGCGGCGTGCCTTCGAGATTTTCTGGCAGCGGCTCTGGAACCGAGTTCACCCTGA CCATCTCCTCTCTGCAGCCTGAGGACTTCGCCACCTACTTCTGCCAGCAGTACAACAGATACCCCTACACCTCCGG AGGCGGCACCAAGGTGGAAATCAAGCGG(SEQ ID NO: 48)

[0196] >02G scFv

[0197] EIVMTQSPSTLSASVGDRVIINCQSSPSVYNNYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGS GAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLR LSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFC ARDLDYVIDLWGPGTLVTVSS(SEQ ID NO: 24)

[0198] 02G scFv comprises 02G VH and 02G VL, wherein the amino acid sequence of 02G VH is as follows:

[0199] EVQLVESGGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIG VISSSGSTYYASWAKGRF TISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVI DLWGPGTLVTVSS(SEQ ID NO: 12)

[0200] The nucleotide sequence encoding 02G VH is as follows:

[0201] GAAGTGCAGCTGGTGGAGTCCGGCGGTGGACTGGTGCAACCGGGAGGCTCACTCAGATTGTCATGCAC CGCCTCTGGCTTTAGTCTCTCCACCTATCATATGACTTGGGTGAGGCAGGCACCCGGCAAGGGCCTGGAATGGATC GGCGTGATCTCTTCCAGCGGTAGCACCTATTACGCCTCTTGGGCGAAGGGCAGGTTTACCATCAGCCGCGACAACA GCAAGAATACCGTTTACCTGCAGATGAATAGCCTGAGGGCCGAAGACACGGCGGTCTATTTCTGTGCACGGGACCT TGACTACGTTATTGACCTGTGGGGCCCTGGGACCCTCGTAACTGTGAGCAGC(SEQ ID NO: 49)

[0202] The amino acid sequence of 02G VL is as follows:

[0203] EIVMTQSPSTLSASVGDRVIINCQSSPSVYNNYLSWYQQKPGKAPKLLIYE TSTLASGVPSRFSGSG SGAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGTKLTVL(SEQ ID NO: 13)

[0204] The nucleotide sequence encoding 02G VL is shown below:

[0205] GAGATCGTGATGACCCAGTCCCCAAGTACACTGAGCGCCTCCGTGGGCGACCGCGTGATCATAAACTG TCAAAGCTCACCCTCTGTTTACAACAATTACCTGTCTTGGTATCAACAGAAGCCCGGTAAGGCCCCCAAACTGCTC ATTTACGAGACATCCACCCTGGCATCCGGGGTGCCAAGCCGCTTCTCCGGGAGTGGGTCTGGCGCCGAGTTCACCC TGACCATATCTTCCCTGCAGCCCGACGACTTCGCAACGTACTATTGCGCCGGAACCTATGTAAGTGGGGATAGACG CGCCTTCGGGCAGGGCACGAAGTTGACCGTGCTG(SEQ ID NO: 50)

[0206] Dual-target scFv

[0207] 02G‑Q33 scFv

[0208] EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRPLIYSATYRNSGIPARFSGSGSG TEFTLTISSLQSEDFAVYFCQQYNRYPYTFGGGTKLEIKGGGGSEIVMTQSPSTLSASVGDRVIINCQSSPSVYNN YLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGTKLT DESCRIPTION 15 / 24 Page 20 CN 122302079 A VLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYY ASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVIDLWGPGTLVTVSSGGGGSEVQLVQSGAEVKKP GESLKISCKASGYAFSSYWMNWVRQMPGKGLEWMGQIYPGDGDTNYNGKFKGQVTLSADKSISTAYLQWSSLKASD TAMYFCARKTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO: 25)

[0209] 02G‑Q33scFv contains 02G VH, 02G VL and Q33 VH, Q33 VL

[0210] The structure of 02G-Q33 scFv is as follows:

[0211] Q33 VL-connection sequence (GGGGS, SEQ ID NO:14)-02G VL-connection sequence (GGGGSGGGGSGGGGS, SEQ ID NO:15)-02G VH-connection sequence (GGGGS, SEQ ID NO:14)-Q33 VH

[0212] Q33-02G scFv

[0213] EIVMTQSPSTLSASVGDRVIINCQSSPSVYNNYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGS GAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGTKLTVLGGGGSEIVMTQSPATLSVSPGERATLSCKASQNV GTNVAWYQQKPGQAPRPLIYSATYRNSGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYNRYPYTFGGGTKLE IKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKISCKASGYAFSSYWMNWVRQMPGKGLEWMGQIYPGDGDTN YNGKFKGQVTLSADKSISTAYLQWSSLKASDTAMYFCARKTISSVVDFYFDYWGQGTTVTVSSGGGGSEVQLVESG GGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNS LRAEDTAVYFCARDLDYVIDLWGPGTLVTVSS(SEQ ID NO:26)

[0214] Q33-02G scFv contains Q33 VH, Q33 VL and 02G VH, 02G VL

[0215] The structure of Q33-02G scFv is as follows:

[0216] 02G VL-connection sequence (GGGGS, SEQ ID NO:14)-Q33 VL-connection sequence (GGGGSGGGGSGGGGS, SEQ ID NO:15)-Q33 VH-connection sequence (GGGGS, SEQ ID NO:14)-02G VH

[0217] 02G-GS21 scFv

[0218] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQYNRYPYTSGGGTKVEIKRGGGGSEIVMTQSPSTLSASVGDRVIINCQSSPSVYN NYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGTKL TVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTY YASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVIDLWGPGTLVTVSSGGGGSQVQLVQSGAEVKK PGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWMGQIYPGDGDTNYNGKFKGRVTITADKSTSTAYMELSSLRSE DTAVYYCARKTISSVVDFYFDYWGQGTLVTVSS(SEQ ID NO: 27)

[0219] 02G‑GS21 scFv comprises 02G VH, 02G VL, GS21 VH and GS21 VL

[0220] The structure of 02G‑GS21 scFv is:

[0221] GS21 VL-linker sequence (GGGGS, SEQ ID NO: 14)-02G VL-linker sequence (GGGGSGGGGSGGGGS, SEQ ID NO: 15)-02G VH-linker sequence (GGGGS, SEQ ID NO: 14)-GS21 VH

[0222] GS21‑02G scFv

[0223] EIVMTQSPSTLSASVGDRVIINCQSSPSVYNNYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGS GAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGTKLTVLGGGGSDIQLTQSPSFLSASVGDRVTITCKASQNV GTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQYNRYPYTSGGGTKVE IKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWMGQIYPGDGDTNYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARKTISSVVDFYFDYWGQGTLVTVSSGGGGSEVQLVES GGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYYASWAKGRFTISRDNSKNTVYLQMN Specification 16 / 24 pages 21 CN 122302079 A SLRAEDTAVYFCARDLDYVIDLWGPGTLVTVSS(SEQ ID NO:28)

[0224] GS21-02G scFv includes GS21 VH, GS21 VL and 02G VH, 02G VL

[0225] The structure of GS21-02G scFv is:

[0226] 02G VL-connection sequence (GGGGS, SEQ ID NO:14)-GS21 VL linker sequence (GGGGSGGGGSGGGGS, SEQ ID NO:15)-GS21 VH--linker sequence (GGGGS, SEQ ID NO:14)-02G VH

[0227] Amino acid sequence of CD8 hinge region:

[0228] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:29)

[0229] Amino acid sequence of CD8 transmembrane region:

[0230] IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:30)

[0231] Amino acid sequence of 4-1BB intracellular signal transduction region:

[0232] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR (SEQ ID NO:31)

[0233] Amino acid sequence of CD3ζ co-stimulatory signal transduction region:

[0234] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:32)

[0235] Amino acid sequence of CD8α guide peptide:

[0236] MALPVTALLL PLALLLHAARP (SEQ ID NO:33)

[0237] The amino acid sequence of the first CD19 / BCMA-CAR (containing CD8α guide chain, O2G-Q33 scFv, CD8 hinge region, CD8 transmembrane region, 4-1BB intracellular domain and CD3ζ in sequence) is as follows:

[0238] MALPVTALLLPLALLLHAARPEIVMTQSPATLSVSPGERATLSCKASQNV

[0239] GTNVAWYQQKPGQAPRPLIYSATYRNSGIPARFSGSGSGTEFTLTISSLQSEDF

[0240] AVYFCQQYNRYPYTFGGGTKLEIKGGGGSEIVMTQSPSTLSASVGDRVIINCQS

[0241] SPSVYNNYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQ

[0242] PDDFATYYCAGTYVSGDRRAFGQGTKLTVLGGGGSGGGGSGGGGSEVQLVES

[0243] GGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYYA

[0244] SWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVIDLWGPGTLV

[0245] TVSSGGGGSEVQLVQSGAEVKKPGESLKISCKASGYAFSSYWMNWVRQMPGK

[0246] GLEWMGQIYPGDGDTNYNGKFKGQVTLSADKSISTAYLQWSSLKASDTAMYF

[0247] CARKTISSVVDFYFDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACR

[0248] PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFK

[0249] QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYN

[0250] ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE

[0251] IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 34)

[0252] The amino acid sequence of the second CD19 / BCMA-CAR (which sequentially comprises a CD8α leader chain, Q33-02G scFv, a CD8 hinge region, a CD8 transmembrane domain, the intracellular domain of 4-1BB and CD3ζ) is as follows:

[0253] MALPVTALLLPLALLLHAARPEIVMTQSPSTLSASVGDRVIINCQSSPSVYN

[0254] NYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFA

[0255] TYYCAGTYVSGDRRAFGQGTKLTVLGGGGSEIVMTQSPATLSVSPGERATLSC

[0256] KASQNVGTNVAWYQQKPGQAPRPLIYSATYRNSGIPARFSGSGSGTEFTLTISS

[0257] LQSEDFAVYFCQQYNRYPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVQS Description Page 17 of 24 22 CN 122302079 A

[0258] GAEVKKPGESLKISCKASGYAFSSYWMNWVRQMPGKGLEWMGQIYPGDGDT

[0259] NYNGKFKGQVTLSADKSISTAYLQWSSLKASDTAMYFCARKTISSVVDFYFDY

[0260] WGQGTTVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSTYHMTW

[0261] VRQAPGKGLEWIGVISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAED

[0262] TAVYFCARDLDYVIDLWGPGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEAC

[0263] RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF

[0264] KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY

[0265] NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS

[0266] EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 35)

[0267] The amino acid sequence of the third CD19 / BCMA-CAR (sequentially comprising a CD8α leader chain, 02G-GS21 scFv, a CD8 hinge region, a CD8 transmembrane domain, the intracellular domain of 4-1BB and CD3ζ) is as follows:

[0268] MALPVTALLLPLALLLHAARPDIQLTQSPSFLSASVGDRVTITCKASQNVG

[0269] TNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSGTEFTLTISSLQPEDFA

[0270] TYFCQQYNRYPYTSGGGTKVEIKRGGGGSEIVMTQSPSTLSASVGDRVIINCQS

[0271] SPSVYNNYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQ

[0272] PDDFATYYCAGTYVSGDRRAFGQGTKLTVLGGGGSGGGGSGGGGSEVQLVES

[0273] GGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYYA

[0274] SWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVIDLWGPGTLV

[0275] TVSSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPG

[0276] QGLEWMGQIYPGDGDTNYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVY

[0277] YCARKTISSVVDFYFDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEAC

[0278] RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF

[0279] KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY

[0280] NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS

[0281] EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:36)

[0282] The amino acid sequence of the fourth CD19 / BCMA-CAR (comprising CD8α leader chain, GS21-02G scFv, CD8 hinge region, CD8 transmembrane domain, intracellular domain of 4-1BB and CD3ζ in this order) is as follows:

[0283] MALPVTALLLPLALLLHAARPEIVMTQSPSTLSASVGDRVIINCQSSPSVYN

[0284] NYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGTKLTVLGGGGSDIQLTQSPSFLSSVGDRVTITC KASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSGTEFTLTISS QSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWMGQIYPGDG DTNYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARKTISSVVDFYFD

[0291] WVRQAPGKGLEWIGVISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAE

[0292] DTAVYFCARDLDYVIDLWGPGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEA

[0293] CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI

[0294] FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQL Instructions 18 / 24 Page 23 CN 122302079 A

[0295] YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY

[0296] SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:37)

[0297] Example 1: Preparation of humanized CD19 CAR-T cells

[0298] I. Humanization screening of anti-CD19 murine antibodies

[0299] 1. Construction of humanized anti-CD19-CAR and lentiviral packaging

[0300] The murine antibody HI19a (named CNCT19 in this patent, the amino acid sequence of its scFv is as shown in SEQ ID NO:51) was humanized. The humanized antibody sequence numbers are Q11 (the amino acid sequence of its scFv is as shown in SEQ ID NO:16, the amino acid sequence of VH is as shown in SEQ ID NO:1, and the amino acid sequence of VL is as shown in SEQ ID NO:1).(Shown NO:2), Q14 (its scFv amino acid sequence is shown in SEQ ID NO:17, VH amino acid sequence is shown in SEQ ID NO:3, VL amino acid sequence is shown in SEQ ID NO:2), Q23 (its scFv amino acid sequence is shown in SEQ ID NO:18, VH amino acid sequence is shown in SEQ ID NO:4, VL amino acid sequence is shown in SEQ ID NO:5), Q33 (its scFv amino acid sequence is shown in SEQ ID NO:19, VH amino acid sequence is shown in SEQ ID NO:4, VL amino acid sequence is shown in SEQ ID NO:6), Q54 (its scFv amino acid sequence is shown in SEQ ID NO:20, VH amino acid sequence is shown in SEQ ID NO:3, VL amino acid sequence is shown in SEQ ID NO:7), GS32 (its scFv amino acid sequence is shown in SEQ ID NO:21, VH amino acid sequence is shown in SEQ ID NO:8, VL amino acid sequence is shown in SEQ ID NO:9), GS21 (its scFv amino acid sequence is shown in SEQ ID NO:21). NO:22, the amino acid sequence of VH is shown in SEQ ID NO:10, the amino acid sequence of VL is shown in SEQ ID NO:11), GS22 (the amino acid sequence of its scFv is shown in SEQ ID NO:23, the amino acid sequence of VH is shown in SEQ ID NO:8, the amino acid sequence of VL is shown in SEQ ID NO:11). Different anti-CD19 humanized scFvs were combined with CD8α guide chain signal peptide (as shown in SEQ ID NO:33), CD8 hinge region (as shown in SEQ ID NO:29), CD8 transmembrane region (as shown in SEQ ID NO:30), 4-1BB intracellular domain (as shown in SEQ ID NO:31) and CD3ζ (as shown in SEQ ID NO:23). (As shown in NO:32) CAR genes were constructed together, and different anti-CD19 CAR genes were constructed into different lentiviral master plasmids (manufacturer: SBI, catalog number: CD500-CD800, as described in Example 1 of WO2021 / 121227 for routine resistance modification) to obtain CAR expression vectors. The master plasmids and three packaging plasmids (pMD2.G (purchased from Biovector, product number Biovector012259), pMDLg / pRRE (purchased from Biovector, product number Biovector012251), and pRSV-Rev (purchased from Biovector, product number Biovector012253)) were co-transfected into 293T cells. Lentiviral viruses containing different anti-CD19-CARs were collected for infecting T cells.

[0301] 2. Preparation of different humanized anti-CD19 CAR-T cells

[0302] The T cell culture medium was Optimizer basal medium supplemented with OpTmizer amplification additive, ISR and GlutaMAX (commercially available), and cytokines IL-7 (10 ng / mL) and IL-15 (5 ng / mL) were added.

[0303] PBMC cells were resuscitated on day -1;

[0304] On day 0, T cells in PBMCs were sorted with CD4 magnetic beads and CD8 magnetic beads, and T cells were activated with Thermo CD3 / CD28 activation magnetic beads.

[0305] On day 1, T cells were infected with different humanized anti-CD19-CAR lentiviruses in groups CNCT19, Q11, Q14, Q23, Q33, Q54, GS32, GS21, and GS22.

[0306] T cell culture medium was added every 2-3 days according to the culture status. Samples were taken and counted to detect cell viability and CD19-CAR positivity. After culturing for 9 days, CAR-T cells (humanized Q11 CAR-T cells, Q14 CAR-T cells, Q23 CAR-T cells, Q33 CAR-T cells, Q54 CAR-T cells, GS32 CAR-T cells, GS21 CAR-T cells, GS22 CAR-T cells and one type of mouse-derived CNCT19 CAR-T cells) were harvested and subjected to in vitro functional testing.

[0307] After humanizing the CNCT19 sequence, Q11, Q14, Q23, Q33, Q54, GS32, GS21, and GS22 were selected for a series of experimental functional verifications. The experimental results are shown in Figures 1-5.

[0308] 1. Detection of CAR-T positive ratio in CAR-T cells

[0309] Eight types of humanized CAR-T cells and one type of mouse-derived CNCT19 CAR-T cells obtained in Example 1 were mixed with CD19 antigen-conjugated PE fluorescein and incubated at room temperature in the dark for 15 minutes. The supernatant was removed by centrifugation, and the CAR molecule positive ratio of various cells was detected by flow cytometry at different days after transduction (Day 3, Day 6 and Day 9). The detection results are shown in Figure 1 and Figure 2: except for the poor CAR positive rate expression of Q23 structure (Q23 CAR-T cells), the expression of other structures was normal; except for the low CAR expression MFI (mean fluorescence intensity) of Q23 structure (Q23 CAR-T cells), the expression of other structures was normal.

[0310] 2. In vitro killing of CAR-T cells (short-term)

[0311] 1. Target cell plating

[0312] 1.1 Target cell preparation:

[0313] 1.1.1 Target cells are in good logarithmic growth phase with a cell viability of over 85%.

[0314] 1.1.2 Count the target cells according to the standard cell counting procedure. Take 2×10⁶ target cells (Nalm6, K562-CD19, MM.1S, K562-BCMA) into a 15ml centrifuge tube, centrifuge at 300g for 8min, remove the supernatant, and resuspend the target cells in TO medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) until the cell density is 2×10⁵ cells / ml.

[0315] 1.1.3 Carefully transfer the diluted target cells into a sterile sample loading trough, and add the target cells to a black-bottomed transparent 96-well plate using a multi-channel pipette, 50μL / well.

[0316] 1.2 Effector cell plating

[0317] 1.2.1 Effector cell preparation: Calculate the cell dosage according to the effector-target ratio of 9:1, 3:1, and 1:1, which are 9×10⁴ cells, 3×10⁴ cells, and 1×10⁴ cells, respectively. Count the effector cells, take the required number of CAR-T cell samples into a 15ml centrifuge tube, centrifuge at 300g for 8min, and discard the supernatant.

[0318] 1.2.2 Resuspend the CAR-T cells in TO medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax), and adjust the effector cell density according to the effector-to-target ratio.

[0319] 1.2.3 Add effector cells to the well plates at effector-to-target ratios of 9:1, 3:1, and 1:1, 50μL / well.

[0320] 1.3 Target cell control group setup

[0321] Add 50μL of target cells and 50μL of TO medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) to the wells of the target cell control group, so that the total culture volume of the target cell control group is consistent with the total culture volume of the co-culture experimental group.

[0322] 2. Detection of lucif value

[0323] 2.1 Cell co-culture: Place the 96-well plate with the sample added in a carbon dioxide incubator and co-culture for 4 hours.

[0324] 2.2 Addition of detection reagent: Melt steadyglo luciferase (commercially available) at 4°C or room temperature in the dark. Dilute the steadyglo required for the experiment with PBS 3 times and mix well. Add 50 μL to each well and place on a 96-well plate shaker. Shake at 100 rpm for 15 min.

[0325] 2.3 Detection on the instrument: Turn on the multi-mode microplate reader and computer software. Place the shaken 96-well plate into the multi-mode microplate reader and detect the lucif fluorescence intensity. Remove the 96-well plate and turn off the instrument and computer.

[0326] 3. Calculation of Cell Killing Activity

[0327] After obtaining the luc values ​​of all wells, the killing activity was calculated according to the following formula

[0328] Cell killing activity = (average luc value of individual target cell group - luc value of co-culture well) / average luc value of individual target cell group × 100%

[0329] The detection results are shown in Figure 3: There was no significant difference in the killing activity of CAR-T cells against target cells Nalm6 in all groups.

[0330] 3. Cytokine detection instructions 20 / 24 pages 25 CN 122302079 A

[0331] 1. CAR-T cells and target cells were mixed at an effector-target ratio of 1:1

[0332] 2. Incubated in carbon dioxide medium for 24 hours

[0333] 3. After incubation, centrifuged at 500g for 10 minutes and collected the supernatant

[0334] 4. The concentration of cytokines in the supernatant was detected using the Human Th1 Panel (5-Plex) with Filter Plate V02 kit.

[0335] The detection results are shown in Figures 4A and 4B: The IL-2 release of CAR-T cells from humanized structures Q11, Q14, Q33, Q54, GS32, GS21 and other groups was higher than that of mouse CNCT19. The IFN-γ release of humanized CAR-T cells from the Q11, Q33, GS32, and GS21 groups was higher than that of the mouse-derived CNCT19. This experimental result indicates that the humanization modification of the mouse antibody in this application improved the antibody affinity, thereby making the humanized CAR-T cells of this application higher than those of the mouse-derived CNCT19 in terms of IL-2 release and IFN-γ release.

[0336] 4. Multiple rounds of stimulation

[0337] 1. CAR-T cells and target cells were mixed at an effector-to-target ratio of 1:1 and a cell number of 0.5×106:0.5×106

[0338] 2. Every 2 days, the number of CAR-T cells was detected, and 0.5×106 CAR-T cells were taken and 0.5×106 target cells were added

[0339] 3. After 4 rounds of target cell stimulation, the expansion fold of CAR-T cells was calculated.

[0340] The detection results are shown in Figure 5: After four rounds of target cell stimulation, the CAR-T amplification fold of humanized structures Q11, Q33, and GS21 was consistent with that of the mouse-derived structure CNCT19.

[0341] In addition, the humanization scores of the VH and VL chains of CD19 were scored using three biological computer tools: T20 score, HS score, and QASis. The scores of the three tools were consistent, with Q11, Q14, Q23, Q33, Q54, GS32, GS21, and GS22 showing higher humanization scores compared to the mouse-derived CNCT19 (see Figure 6). The immunogenicity of the humanized antibodies was predicted using two immunogenicity prediction tools, Alpha MHC v2 and CD4 T cell Immunogenicity prediction tool. The prediction results were basically consistent, showing that Q11, Q14, Q23, Q33, Q54, and GS21 had the lowest immunogenicity risk (see Figure 7).

[0342] The immunogenicity of antibody drugs may trigger an anti-drug immune response in the body, thereby inducing the production of anti-drug antibodies (ADA). The immunogenicity of antibodies is mainly evaluated by the method of anti-drug antibody (ADA) analysis. The immunogenicity of CAR-T causes an anti-CAR immune response, which in turn destroys and eliminates CAR-T cells, and is one of the factors for CAR-T treatment failure. The scFv used in CAR-T contains mouse-derived sequences, which is an important factor in generating anti-CAR immune responses. Humanizing mouse-derived scFvs to avoid anti-CAR immune responses related to such mouse-derived scFvs is an important strategy, but whether the antibody functional reduction or loss can be avoided during the modification process is the key to the success or failure of this strategy.

[0343] Based on the in vitro functional data of different humanized CD19 CAR-Ts, combined with the antibody humanization degree score and antibody immunogenicity prediction data, the Q33 and GS21 sequences have better CAR positive rate expression, higher in vitro killing activity and cytokine release, and lower immunogenicity. We selected the Q33 and GS21 sequences to construct a dual-target CAR-T for further validation.

[0344] Example 2, Preparation and Screening Verification of BCMA-CD19 Bispecific CAR-T Cells

[0345] 1. Construction of Humanized Anti-CD19 / BCMA-CAR and Lentiviral Packaging

[0346] Humanized anti-CD19 sequences (Q33, GS21) and humanized anti-BCMA sequences (O2G, whose scFv amino acid sequence is shown in SEQ ID NO:24, VH amino acid sequence is shown in SEQ ID NO:12, VL amino acid sequence is shown in SEQ ID NO:13) were used to construct different scFvs. The dual-target scFv antibody sequences are 02G-Q33 (whose scFv amino acid sequence is shown in SEQ ID NO:25), Q33-02G (whose scFv amino acid sequence is shown in SEQ ID NO:26), 02G-GS21 (whose scFv amino acid sequence is shown in SEQ ID NO:27), and GS21-02G (whose scFv amino acid sequence is shown in SEQ ID NO:28). The four dual-target scFvs are combined with CD8. (Instructions page 21 / 24, 26 CN 122302079 A) The α-guide chain is a signal peptide (as shown in SEQ ID NO:33), a CD8 hinge region (as shown in SEQ ID NO:29), a CD8 transmembrane region (as shown in SEQ ID NO:30), an intracellular domain of 41BB (as shown in SEQ ID NO:31), and CD3ζ (as shown in SEQ ID NO:32).(Show) The CD19 / BCMA-CAR gene was constructed together, and different anti-CD19 / BCMA-CAR genes were constructed into different lentiviral main plasmids. The main plasmids were mixed with three packaging plasmids (pMD2.G (purchased from Biovector, product number Biovector012259), pMDLg / pRRE (purchased from Biovector, product number Biovector012251), and pRSV-Rev (purchased from Biovector, product number Biovector012253)) and co-transfected into 293T cells. Lentiviral viruses containing different anti-CD19 / BCMA-CAR were collected for infecting T cells.

[0347] 2. Preparation of different anti-CD19 / BCMA-CAR T cells

[0348] The T cell culture medium was Optimizer basal medium with OpTmizer amplification additive and ISR (commercially available), and a certain concentration of cytokines IL-7 (10 ng / mL) and IL-15 (5 ng / mL) were added.

[0349] PBMC cell resuscitation was performed on day -1;

[0350] T cells in PBMCs were sorted using CD4 and CD8 magnetic beads on day 0, and T cells were activated using Thermo CD3 / CD28 activation magnetic beads.

[0351] On day 1, T cells were infected with the corresponding anti-CD19 / BCMA-CAR lentivirus in the O2G, O2G-Q33, Q33-O2G, O2G-GS21, and GS21-O2G groups.

[0352] T cell culture medium was added every 2-3 days according to the culture status. After culturing to 9, CAR-T cells (02G CAR-T cells, 02G-Q33 CAR-T cells, Q33-02G CAR-T cells, 02G-GS21 CAR-T cells and GS21-02G CAR-T cells, respectively) were harvested. The positive rates of CD19-CAR and BCMA-CAR were detected and in vitro functional tests were performed. The experimental results are shown in Figures 8-18.

[0353] 1. Detection of CAR-T positive proportion in CAR-T cells

[0354] CNCT19 CAR-T cells, Q33 CAR-T cells, GS21 CAR-T cells obtained in Example 1 and 02G CAR-T cells, 02G-Q33 CAR-T cells, Q33-02G CAR-T cells, 02G-GS21 CAR-T cells and GS21-02G CAR-T cells or UTD (representing untransduced T cell group) obtained in Example 2 were mixed with CD19 antigen-coupled PE fluorescein or BCMA antigen-coupled FITC fluorescein and incubated at room temperature in the dark for 15 minutes; the supernatant was removed by centrifugation and various CAR-T cells were analyzed by flow cytometry.The CAR molecule positivity rate of cells was detected, and the results are shown in Figure 8: Among the four dual-target structures, the CAR positivity rate of the O2G-Q33 structure was the best.

[0355] 2. In vitro killing of CAR-T cells (short time)

[0356] 1. Target cell plate preparation

[0357] 1.1 Target cell preparation:

[0358] 1.1.1 The target cells are in good logarithmic growth phase with a cell viability of more than 85%.

[0359] 1.1.2 Target cells are counted according to standard cell counting procedures. 2×10⁶ cells of target cells Nalm6, K562-CD19, MM.1S, and K562-BCMA are placed in a 15ml centrifuge tube, centrifuged at 300g for 8min, the supernatant is removed, and the target cells are resuspended in TO medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) until the cell density is 2×10⁵ cells / ml.

[0360] 1.1.3 Carefully transfer the diluted target cells into a sterile sample loading tank, and use a multi-channel pipette to add the target cells into a black, transparent, flat-bottomed 96-well plate, 50 μL / well.

[0361] 1.2 Effector cell plating

[0362] 1.2.1 Effector cell preparation: Calculate the cell quantities according to the effector-target ratios of 9:1, 3:1, and 1:1, respectively, to be 9 × 10⁴ cells, 3 × 10⁴ cells, and 1 × 10⁴ cells. Count the effector cells, take the required number of CAR-T cell samples into a 15 ml centrifuge tube, centrifuge at 300 g for 8 min, and discard the supernatant. Instructions 22 / 24 pages 27 CN 122302079 A

[0363] 1.2.2 Resuspend the CAR-T cells in TO medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax), and adjust the effector cell density according to the effector-target ratio.

[0364] 1.2.3 Add effector cells to the well plates at effector-to-target ratios of 9:1, 3:1, and 1:1, 50 μL / well.

[0365] 1.3 Setting up the target cell control group

[0366] Add 50 μL of target cells and 50 μL of TO medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) to the wells of the target cell control group, so that the total culture volume of the target cell control group is consistent with the total culture volume of the co-culture experimental group.

[0367] 2. Detection of luc value

[0368] 2.1 Cell co-culture: Place the 96-well plate with the added samples in a carbon dioxide incubator and co-culture for 4 hours.

[0369] 2.2 Adding the detection reagent: Melt steadyglo luciferase (commercially available) at 4°C or room temperature in the dark. Dilute the required amount of steadyglo with PBS 3 times and mix well. Add 50 μL to each well and place on a 96-well plate shaker. Shake at 100 rpm for 15 min.

[0370] 2.3 Detection on the instrument: Turn on the multi-functional microplate reader and computer software, put the shaken 96-well plate into the multi-functional microplate reader, and detect the luc fluorescence intensity. Take out the 96-well plate and turn off the instrument and computer.

[0371] 3. Calculation of cell killing activity

[0372] After obtaining the luc values ​​of all wells, calculate the killing activity according to the following formula

[0373] Cell killing activity = (average luc value of individual target cell group - luc value in co-culture well) / average luc value of individual target cell group × 100%

[0374] The detection results are shown in Figure 9: On the killing of CD19+ target cells Nalm6, the killing activities of the four dual-target CD19 / BCMA CAR-T (02G-Q33, Q33-02G, 02G-GS21, GS21-02G) are consistent with those of the single-target CD19 CAR-T (CNCT19, Q33, GS21).

[0375] The detection results are shown in Figure 10: In killing CD19+ target cells K562-CD19, the killing activities of the four dual-target CD19 / BCMA CAR-T (02G-Q33, Q33-02G, 02G-GS21, GS21-02G) were consistent with those of the single-target CD19 CAR-T (CNCT19, Q33, GS21).

[0376] The detection results are shown in Figure 11: In killing BCMA+ target cells MM.1S, the killing activities of the four dual-target CD19 / BCMA CAR-T (02G-Q33, Q33-02G, 02G-GS21, GS21-02G) were higher than those of the single-target BCMA CAR-T (02G).

[0377] The detection results are shown in Figure 12: In killing BCMA+ target cells K562-BCMA, the killing activity of dual-target CD19 / BCMACAR-T (Q33-O2G, GS21-O2G) was higher than that of single-target BCMA CAR-T (O2G). In killing BCMA+ target cells K562-BCMA, the killing activity of dual-target CD19 / BCMA CAR-T (O2G-Q33, O2G-GS21) was consistent with that of single-target BCMACAR-T (O2G).

[0378] 3. Cytokine detection

[0379] 1. CAR-T cells and target cells were mixed at an effector-target ratio of 1:1.

[0380] 2. Incubated in carbon dioxide medium for 24 hours.

[0381] 3. After incubation, the mixture was centrifuged at 500g for 10 minutes, and the supernatant was collected.

[0382] 4. The cytokine concentration in the supernatant was detected using the Human Th1 Panel (5-Plex) with Filter Plate V02 kit.

[0383] The detection results are shown in Figures 13A and 13B: In killing CD19+ target cells Nalm6, O2G-Q33 was found in four dual-target structures.The IL-2 release level was the highest. In killing BCMA+ target cells MM.1S, the IL-2 release level of the four dual-target CD19 / BCMACAR-T (02G-Q33, Q33-02G, 02G-GS21, GS21-02G) was lower than that of the single-target BCMACAR-T (02G).

[0384] The detection results are shown in Figures 14A and 14B: In killing CD19+ target cells Nalm6, the IFN-γ release level of the four dual-target CD19 / BCMACAR-T (02G-Q33, Q33-02G, 02G-GS21, GS21-02G) was higher than that of the single-target BCMACAR-T (02G). In killing BCMA+ target cells MM.1S, the IFN-γ release level of the four dual-target CD19 / BCMACAR-T (02G-Q33, Q33-02G, 02G-GS21, GS21-02G) was lower than that of the single-target BCMACAR-T (02G).

[0385] 4. Multiple rounds of stimulation

[0386] 1. CAR-T cells were mixed with CD19+ target cells Nalm6 or BCMA+ target cells RPMI-8226 at an effector-target ratio of 1:1 and a cell number of 0.5×106:0.5×106

[0387] 2. Every 2 days, the number of CAR-T cells was detected, and 0.5×106 of CAR-T cells were taken and 0.5×106 of target cells were added

[0388] 3. After 4 rounds of target cell stimulation, the expansion fold of CAR-T cells was calculated.

[0389] The detection results are shown in Figure 15: After four rounds of target cell stimulation, the CAR-T amplification fold of the dual-target structure O2G-Q33 was higher than that of the single-target CNCT19 and Q33.

[0390] The detection results are shown in Figure 16: After four rounds of target cell stimulation, the CAR-T amplification fold of the dual-target structure O2G-Q33 was consistent with that of the single-target structure O2G.

[0391] 4. Animal experiments and in vivo efficacy experiments

[0392] 1. 5×10⁵ CD19+ target cells Nalm.6-luc or 2×10⁶ BCMA+ target cells MM.1S-luc were injected into the tail vein of NSG mice.

[0393] 2. Three days after tumor bearing, 3×10⁶ CAR-T cells were injected into the tail vein for detection.

[0394] 3. Fluorescence imaging was performed on mice at time points (Day 0, Day 3, Day 7, Day 10, Day 14, Day 21, Day 28), and the fluorescence intensity was recorded.

[0395] The detection results are shown in Figure 17: O2G-Q33 dual-target CD19 / BCMACAR-T has the same efficacy as Q33 single-target CD19 CAR-T.

[0396] The detection results are shown in Figure 18: the efficacy of 02G-Q33 dual-target CD19 / BCMACAR-T is the same as that of 02G single-target BCMACAR-T.

[0397] In the validation of CAR-T cells constructed with four dual-target structures (02G-Q33, Q33-02G, 02G-GS21, GS21-02G), based on the expression, in vitro killing activity, and cytokine release data of CD19-CAR and BCMA-CAR positive rates, the 02G-Q33 group showed the best expression and function.

[0398] Based on the multi-round stimulation amplification data and the in vivo efficacy data of animal experiments, the dual-target CD19 / BCMA CAR-T (02G-Q33) has the same efficacy as single-target CD19 CAR-T and single-target BCMACAR-T. Instruction manual, page 24 / 24; 29 CN 122302079 A; Figure 1; Figure 2; Figure 3; Instruction manual drawing 1 / 10 page; 30 CN 122302079 A; Figure 4A; Figure 4B; Instruction manual drawing 2 / 10 page; 31 CN 122302079 A; Figure 5; Instruction manual drawing 3 / 10 page; 32 CN 122302079 A; Figure 6; Instruction manual drawing 4 / 10 page; 33 CN 122302079 A; Figure 7; Figure 8; Instruction manual drawing 5 / 10 page; 34 CN 122302079 A; Figure 9; Figure 10; Figure 11; Instruction manual drawing 6 / 10 page; 35 CN 122302079 A; Figure 12; Figure 13A; Instruction manual drawing 7 / 10 page; 36 CN 122302079 A; Figure 13B; Figure 14A; Instruction manual drawing 8 / 10 page; 37 CN 122302079 A; Figure 14B Figure 15 Appendix to the Specification, Page 9 / 10, 38 CN 122302079 A Figure 16 Figure 17 Figure 18 Appendix to the Specification, Page 10 / 10, 39 CN 122302079 A Abstract The present invention provides a bispecific chimeric antigen receptor targeting BCMA-CD19 and the use thereof. The bispecific chimeric antigen receptor comprises an extracellular antigen recognition domain; wherein the extracellular antigenrecognition domain comprises an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain; and the anti-CDl9 extracellular antigen recognition domain comprises CD19 VH and CD19 VL, which are selected from one of groups 1) to 8). 1:1 3:1 9:1 -20 0 20 40 60 80 100 E:T C y to to x ic it y ( % ) UTD CNCT19 Q33 GS21 02G 02G-Q33 Q33-02G 02G-GS21 GS21-02G Target MM.1S(CD19-BCMA+) Figure 11

Claims

1. A bispecific chimeric antigen receptor targeting BCMA-CD19, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain; wherein: The extracellular antigen recognition domain includes an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain; the anti-CD19 extracellular antigen recognition domain includes CD19 VH and CD19VL, which are selected from one of the following: 1) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:1, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or 2) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or 3) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:5; or 4) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:6; or 5) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:7; or 6) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:9; or 7) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11; or 8) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:

11.

2. The bispecific chimeric antigen receptor of claim 1, wherein, The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:6; or The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:

11.

3. The bispecific chimeric antigen receptor of claim 1 or 2, wherein, The BCMA VH sequence comprises the amino acid sequence shown in SEQ ID NO:12, and the BCMA VL sequence comprises the amino acid sequence shown in SEQ ID NO:

13.

4. The bispecific chimeric antigen receptor of claim 1 or 2, wherein, The extracellular antigen recognition domain of the bispecific chimeric antigen receptor is scFv antibody, sc(Fv)2 antibody or [sc(Fv)2]2 antibody.

5. The bispecific chimeric antigen receptor of claim 4, wherein, The scFv antibody comprises any one of the following structures: CD19 VL sequence - 1st linker sequence - BCMA VL sequence - 2nd linker sequence - BCMA VH sequence - 3rd linker sequence - CD19 VH sequence, BCMA VL sequence - 4th linker sequence - CD19 VL sequence - 5th linker sequence - CD19 VH sequence - 6th linker sequence - BCMA VH sequence, CD19 VL sequence - 7th linker sequence - CD19 VH sequence - 8th linker sequence - 9th linker sequence - BCMA VH sequence, BCMA VL sequence - 10th linker sequence - BCMA VH sequence - 11th linker sequence - CD19 VL sequence - 12th linker sequence - CD19 VH sequence; Optionally, the extracellular antigen recognition domain of the bispecific chimeric antigen receptor includes any one of the following structures: CD19 VL sequence-first linker sequence-BCMA VL sequence-second linker sequence-BCMA VH sequence-third linker sequence-CD19 VH sequence and BCMA VL sequence-fourth linker sequence-CD19 VL sequence-fifth linker sequence-CD19 VH sequence-sixth linker sequence-BCMA VH sequence; Further optionally, the first linking sequence, the second linking sequence, the third linking sequence, the fourth linking sequence, the fifth linking sequence, the sixth linking sequence, the seventh linking sequence, the eighth linking sequence, the ninth linking sequence, the tenth linking sequence, the eleventh linking sequence, and the twelfth linking sequence are independently selected from one or more of the following sequences: SEQ ID NO:14 and SEQ ID NO:

15.

6. The bispecific chimeric antigen receptor of claim 5, wherein, The extracellular antigen recognition domain of the bispecific chimeric antigen receptor includes the amino acid sequence shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:

28.

7. The bispecific chimeric antigen receptor according to claim 1, wherein, 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; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:29; and / or 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 acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:

30.

8. The bispecific chimeric antigen receptor according to claim 1, wherein, The intracellular domain includes an intracellular signal transduction region; optionally, the intracellular signal transduction region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CCD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; further optionally, the intracellular signal transduction region is derived from CD3ζ; even further optionally, the amino acid sequence of the intracellular signal transduction region comprises the amino acid sequence shown in SEQ ID NO:

32.

9. The bispecific chimeric antigen receptor according to claim 1, wherein, The intracellular domain further includes a co-stimulatory signal transduction region; optionally, the co-stimulatory signal transduction region is derived from one, two, 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, the co-stimulatory signal transduction region is derived from 4-1BB; even further optionally, the amino acid sequence of the co-stimulatory signal transduction region comprises the amino acid sequence shown in SEQ ID NO:

31.

10. The bispecific chimeric antigen receptor according to any one of claims 1-9, further comprising a guide peptide located at the N-terminus of the amino acid sequence of the chimeric antigen receptor; optionally, the guide peptide is derived from CD8α; further optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence shown in SEQ ID NO:

33.

11. The bispecific chimeric antigen receptor according to any one of claims 1-9, wherein, The bispecific chimeric antigen receptor includes the amino acid sequence shown in SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:

37.

12. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a bispecific chimeric antigen receptor as described in any one of claims 1-11; Optionally, the nucleotide sequence encoding the bispecific chimeric antigen receptor comprises nucleotide sequences encoding CD19 VH and CD19 VL and nucleotide sequences encoding BCMA VH and BCMA VL, wherein the nucleotide sequences encoding CD19 VH and CD19 VL are selected from one group of the following: 1) A nucleotide sequence encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:1, as shown in SEQ ID NO:38; and a nucleotide sequence encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:2, as shown in SEQ ID NO:39; and / or 2) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:3, as shown in SEQ ID NO:40; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:2, as shown in SEQ ID NO:39; and / or 3) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:4, as shown in SEQ ID NO:41; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:5, as shown in SEQ ID NO:42; and / or 4) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:4, as shown in SEQ ID NO:41; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:6, as shown in SEQ ID NO:43; and / or 5) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:3, as shown in SEQ ID NO:40; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:7, as shown in SEQ ID NO:44; and / or 6) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:8, as shown in SEQ ID NO:45; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:9, as shown in SEQ ID NO:46; and / or 7) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:10, as shown in SEQ ID NO:47; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:11, as shown in SEQ ID NO:48; and / or 8) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:8, as shown in SEQ ID NO:45; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:11, as shown in SEQ ID NO:48; The nucleotide sequences encoding BCMA VH and BCMA VL are as follows: Nucleotide sequences encoding the BCMA VH amino acid sequence as shown in SEQ ID NO:12, as shown in SEQ ID NO:49; and nucleotide sequences encoding the BCMA VL amino acid sequence as shown in SEQ ID NO:13, as shown in SEQ ID NO:

50.

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

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

15. The engineered immune effector cells according to claim 14, wherein, The engineered immune effector cells are selected from one or more of the following: 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, and embryonic stem cells. Optionally, the engineered immune effector cells are T lymphocytes; Further, optionally, the T lymphocytes are derived from autologous T lymphocytes or allogeneic T lymphocytes.

16. A pharmaceutical composition comprising engineered immune effector cells as described in claim 14 or 15 and pharmaceutically acceptable excipients; optionally, the pharmaceutically acceptable excipients comprise protectants; optionally, the pharmaceutically acceptable excipients comprise cell cryopreservation solutions.

17. The pharmaceutical composition according to claim 16, wherein, The pharmaceutical composition is a cell suspension or frozen cells thereof; or the pharmaceutical composition is an intravenous injection.

18. The use of the chimeric antigen receptor of any one of claims 1-11, the isolated nucleic acid of claim 12, the vector of claim 13, or the engineered immune effector cell of claim 14 in the preparation of a medicament for treating diseases or conditions associated with BCMA expression.

19. The use according to claim 18, wherein, 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.

20. The use according to claim 19, wherein, The disease or condition associated with BCMA expression may be an autoimmune disease; optionally, the autoimmune disease may be selected from the following: systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.

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