BCMA-targeting nanobodies, chimeric antigen receptors and uses thereof

Abstract

We provide a nanobody targeting BCMA and its chimeric antigen receptor. This nanobody contains VHH01 and VHH02, both of which have strong specificity and high affinity for BCMA. The constructed CAR-T cells have high specific killing efficiency against BCMA-positive cell lines, enabling the development of immunotherapeutics targeting BCMA.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to a patent application for invention, filed on December 26, 2022, bearing application number 2022116791873 and entitled "BCMA-targeting nanobodies, chimeric antigen receptors and uses thereof," the contents of which are incorporated by reference in their entirety.

[0002] The present invention belongs to the technical field of biomedicine. Specifically, the present invention relates to a nanobody, a chimeric antigen receptor, and uses thereof that target BCMA. More specifically, the present invention relates to nanobodies VHH01 and VHH02, a chimeric antigen receptor, and uses thereof that target BCMA. [Background technology]

[0003] B cell maturation antigen (BCMA) is an antigen expressed on plasma cells, plasmablasts, and bone marrow plasma cells, but not on B cells or hematopoietic stem cells. BCMA, also known as CD269 or TNFRSF17, belongs to the TNF receptor superfamily and is a 185-amino acid type III transmembrane protein that can bind to B lymphocyte stimulatory factor (BAFF) and proliferation-inducing ligand (APRIL). BCMA expression is associated with many cancers, autoimmune diseases, and infectious diseases. High BCMA expression is associated with several hematological malignancies, including multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, various leukemias, and glioblastoma. BCMA also plays an important role in cell survival, proliferation, metastasis, and drug resistance by mediating downstream signaling pathways. These properties make BCMA an effective target for the treatment of hematological malignancies, particularly multiple myeloma. Currently, novel tumor immunotherapies against BCMA mainly include chimeric antigen receptor T-cell immunotherapy (CAR-T), bispecific antibody (BsAb) drugs, and antibody-drug conjugates (ADC), and recent studies have shown that BCMA-targeting CAR-T cells can effectively eliminate myeloma cells in vivo.

[0004] CAR-T is a novel, highly targeted tumor therapy. Its principle is that T cells modified with chimeric antigen receptors (CARs) can specifically recognize tumor-associated antigens on tumor cell surfaces. The targeting, killing activity, and persistence of effector T cells are all higher than those of conventional immune cells, and they can overcome the local immunosuppressive microenvironment of tumors and break the host's immune tolerance. CARs are the core structure of CAR-Ts, conferring the cells the ability to specifically recognize tumor-associated antigens on tumor cell surfaces. They consist of three functional domains: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain consists of an antigen-binding region and a hinge region, which performs linking functions. The intracellular domain consists of a costimulatory domain and a signaling domain. When the extracellular antigen-binding region binds to a target protein expressed on the surface of a target cell, the costimulatory signal domain and signal transduction domain of the CAR structure are activated, and the CAR-T has both an activating signal and a co-activating signal, enabling it to kill tumor cells while achieving efficient proliferation. The effectiveness of CAR-T therapy depends on properties such as the specificity of the antibody that recognizes the antigen and the high binding affinity of the antibody for the antigen.

[0005] Currently, CAR T therapy typically uses the scFv segment derived from a monoclonal antibody as the antigen-binding region. However, the large molecular weight of scFvs and their tendency to form multimers affect the function of CARs and the therapeutic efficacy of CAR T cells. Nanobodies, the smallest antibody molecules available, have gradually attracted attention since their discovery and have been applied to autoimmune diseases, blood diseases, viral infection-related diseases, and bone diseases, demonstrating significant advantages in anti-infective, anti-inflammatory, and anti-neurodegenerative diseases. The main advantages of nanobodies are as follows: First, their small molecular weight and strong tissue penetration. Nanobodies are only one-tenth the size of conventional antibodies, allowing for strong tissue penetration and direct penetration of the blood-brain barrier, thereby enhancing the therapeutic efficacy against solid tumors. Second, their low toxicity and immunogenicity. Their small molecular weight and small volume result in low immunogenicity and toxicity to the host. Third, their high affinity and stability. Nanobodies have high affinity for antigens, stable nanobody properties, good water solubility, and strong resistance to acids and alkalis. Fourth, various combination methods are available, allowing the preparation of monofunctional, bifunctional, and multifunctional antibodies, enabling simultaneous attack on multiple targets. Fifth, the production cycle is short, production costs are low, and nanobodies are easy to prepare. Once the technology is stable, drug development can be completed in just one to two years. Sixth, the range of applications is wide, allowing them to be used in fields such as disease treatment, medical diagnosis, and immunoaffinity chromatography. To overcome the technical challenges of conventional CAR T therapy and fully utilize the advantages of nanobodies, the present invention provides novel nanobodies VHH01 and VHH02 that target BCMA, as well as CAR-T cells containing these nanobodies. Summary of the Invention

[0006] The present invention overcomes the shortcomings of the prior art and aims to provide a nanobody and chimeric antigen receptor (CAR) targeting BCMA, as well as uses thereof, in the field. The nanobody comprises VHH01 and VHH02, both of which have strong specificity and high affinity for BCMA. CAR-T cells prepared based on the nanobody exhibit high specific killing efficiency against BCMA-positive cell lines, have no adverse effects on cell lines that do not express BCMA, and have broad applicability. Based on this, the following invention has been completed.

[0007] Nanobodies In one aspect, the present invention provides nanobodies that target BCMA.

[0008] Furthermore, the nanobody comprises VHH01, VHH02, The amino acid sequences of CDR1, CDR2, and CDR3 of VHH01 are shown in SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15, respectively.

[0009] Furthermore, the nucleotide sequences of CDR1, CDR2, and CDR3 of said VHH01 are set forth as SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, respectively. Further, the amino acid sequence of said VHH01 is set forth as SEQ ID NO:1. Further, the nucleotide sequence of said VHH01 is set forth as SEQ ID NO:2.

[0010] Furthermore, the nucleotide sequences of CDR1, CDR2, and CDR3 of said VHH02 are set forth in SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16, respectively. Further, the amino acid sequence of said VHH02 is set forth in SEQ ID NO:9. Further, the nucleotide sequence of said VHH02 is set forth in SEQ ID NO:10.

[0011] Nanobodies corresponding to amino acid sequences or nucleotide sequences that have at least 75% identity with the above amino acid sequences or nucleotide sequences, i.e., amino acid sequences or nucleotide sequences obtained by replacing an amino acid or nucleotide at one or more positions of the corresponding amino acid or nucleotide sequence with any other amino acid or nucleotide, are all within the scope of protection of the present invention, provided that they have at least 75% identity with the amino acid sequences or nucleotide sequences described in the present invention.

[0012] In some embodiments, the Nanobodies further comprise functional variants of Nanobody VHH01, functional variants of Nanobody VHH02, in particular, functional variants include, but are not limited to, derivatives that are essentially similar in primary structural sequence but contain, for example, in vitro or in vivo chemical and / or biochemical modifications that are not present in the parent Nanobodies VHH01 or VHH02 of the invention. These modifications include, for example, acetylation, acylation, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, cross-linking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolytic treatment, phosphorylation, and such functional variants are likewise included within the scope of protection of the present invention.

[0013] Alternatively, a functional variant may be a Nanobody comprising an amino acid sequence that comprises one or more amino acid substitutions, insertions, deletions, or a combination thereof, compared to the amino acid sequence of the parent Nanobody VHH01 or VHH02. Furthermore, a functional variant may comprise a truncation of the amino acid sequence at one or both of the amino or carboxyl termini. Compared to the parent Nanobody VHH01 or VHH02, a functional variant of the invention may have the same or a different, higher or lower binding affinity, but still be able to specifically bind to BCMA. For example, compared to the parent Nanobody VHH01 or VHH02, a functional variant of the invention may have improved or decreased binding affinity to BCMA.

[0014] In the present invention, the term "identity" refers to the same amino acid residue between sequences at any particular position in the compared sequences. As used herein, "similarity" refers to the similarity of the type of amino acid residue between sequences at any particular position in the compared sequences. For example, leucine can be substituted with isoleucine or valine. Other amino acids that are typically substituted for each other include, but are not limited to, phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains), lysine, arginine, and histidine (amino acids with basic side chains), aspartic acid and glutamic acid (amino acids with acidic side chains), asparagine and glutamine (amino acids with amide side chains), and cysteine ​​and methionine (amino acids with sulfur-containing side chains). Typically, modification of one or more amino acids in a protein does not affect the function of the protein. It is recognized by those skilled in the art that changes of single amino acids or small percentages of amino acids, or individual additions, deletions, insertions, or substitutions in an amino acid sequence, are conservative modifications, where alterations to a protein result in a protein with similar function, and conservative substitution tables of functionally similar amino acids are provided, as is well known in the art.

[0015] In the present invention, the term "conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of a Nanobody comprising that amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into a Nanobody according to the present invention by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). A conservative substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have already been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0016] In the present invention, the term "BCMA" can be used interchangeably with "CD269" and "TNFRSF17" and generally refers to a B-cell maturation antigen. For example, human BCMA is generally a 184-amino acid protein encoded by a 994-nucleotide primary mRNA transcript (NM_001192.2). The amino acid sequence of human BCMA is represented by UniProtKB accession number Q02223. In the present invention, the term "BCMA" may also include proteins containing mutations, such as proteins containing point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type BCMA. In the present invention, the term "BCMA" may also include portions of the complete BCMA protein, so long as the relevant biological activity is retained.

[0017] Single nanobody-based chimeric antigen receptors In another aspect, the present invention provides a chimeric antigen receptor that targets BCMA based on a single nanobody.

[0018] Further, the chimeric antigen receptor comprises any one of the nanobodies described in the first aspect of the present invention. Further, the chimeric antigen receptor further comprises a transmembrane domain. Further, the chimeric antigen receptor further comprises an intracellular signaling domain. Further, the chimeric antigen receptor further comprises a hinge region. Further, the chimeric antigen receptor further comprises a signal peptide. Further, the chimeric antigen receptor further comprises a costimulatory signal domain. Further, the chimeric antigen receptor further comprises a promoter. Further, the chimeric antigen receptor further comprises a self-cleaving peptide. Further, the chimeric antigen receptor further comprises a detection tag / accessory function element. Further, the chimeric antigen receptor further comprises a tEGFR signal peptide.

[0019] The transmembrane domain includes those of CD8α, CD28, 4-1BB, CD34, CD3ε, PD-1, IgG1, IgG4, OX40, IL-2 receptor, IL-7 receptor, and IL-11 receptor. The intracellular signaling domain includes those of CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, and CD66d. The hinge region includes those of CD8α, CD28, CD34, 4-1BB, OX40, CD3ε, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, and IL-11 receptor. Furthermore, the signal peptides include those of the α and β chains of the T cell receptor, CD3ζ, CD3ε, CD16, CD22, CD33, CD4, CD5, CD8, CD9, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, and GM-CSF. Furthermore, the costimulatory signal domains include those of the 4-1BB (CD137), CD19, CD4, CD27, CD28, ICOS (CD278), CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, and CD226. Furthermore, the promoter includes an EF1α promoter, a CMV promoter, an EFS promoter, a CAG promoter, a CBh promoter, an SFFV promoter, an MSCV promoter, an SV40 promoter, an mPGK promoter, an hPGK promoter, and a UBC promoter. Furthermore, the self-cleaving peptide includes T2A, P2A, E2A, and F2A.Further, the detection tags / auxiliary function elements include tEGFR, tCD34, tCD19, tCD20, tCD22, immune checkpoint inhibitors (CTLA-4, PD-1 / PD-L1, LAG-3, TIM-3, TIGIT, CD226, CD155, CD47, B7-H3, B7-H4), nanobodies, cytokines and their receptors (IL2, IL2 receptor, IL7, IL7 receptor, IL15, IL15 receptor).

[0020] Furthermore, the transmembrane domain is a CD8α transmembrane domain. Furthermore, the intracellular signaling domain is a CD3ζ intracellular signaling domain. Furthermore, the hinge region is a CD8α hinge region. Furthermore, the costimulatory signal domain is a 4-1BB costimulatory signal domain. Furthermore, the promoter is EF1α. Furthermore, the self-cleaving peptide is T2A. Furthermore, the detection tag / auxiliary function element is tEGFR.

[0021] Furthermore, the chimeric antigen receptor comprises EF1α, a signal peptide, any one of the nanobodies described in the first aspect of the present invention, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB costimulatory signal domain, a CD3ζ intracellular signaling domain, T2A, a tEGFR signal peptide, and tEGFR linked in tandem in this order.

[0022] Furthermore, the chimeric antigen receptor is formed by tandemly linking, in this order, EF1α, a signal peptide, VHH01, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB costimulatory signal domain, a CD3ζ intracellular signaling domain, T2A, a tEGFR signal peptide, and tEGFR.

[0023] Furthermore, the chimeric antigen receptor is composed of EF1α, a signal peptide, VHH02, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB costimulatory signal domain, a CD3ζ intracellular signaling domain, T2A, a tEGFR signal peptide, and tEGFR linked in tandem in this order.

[0024] In some embodiments, the nucleotide sequence of EF1α described in the present invention is represented by SEQ ID NO:17, and the amino acid sequences of the signal peptide, CD8α hinge region, CD8α transmembrane domain, 4-1BB costimulatory signal domain, CD3ζ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR are represented by SEQ ID NO:18-SEQ ID NO:25, respectively, and the corresponding nucleotide sequences are represented by SEQ ID NO:26-SEQ ID NO:33, respectively.

[0025] In some embodiments, the EF1α, signal peptide, CD8α hinge region, CD8α transmembrane domain, 4-1BB costimulatory signal domain, CD3ζ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR described in the present invention are not limited to the above sequences, and any amino acid sequence or nucleotide sequence having at least 75% identity with the above amino acid sequence or nucleotide sequence falls within the scope of protection of the present invention.

[0026] In the present invention, the term "chimeric antigen receptor (CAR)" generally refers to a fusion protein comprising an extracellular domain capable of binding to an antigen and at least one intracellular domain. CARs are the core structure of chimeric antigen receptor T cells (CAR-Ts) and may comprise an antigen (e.g., tumor-specific antigen and / or tumor-associated antigen) binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. CARs are engineered receptors, allowing any specific receptor to be transplanted into immune effector cells, particularly T cells. In CARs, VHH fragments or scFv fragments of monoclonal antibodies that specifically recognize tumor antigens can be transplanted into T cells or NK cells. A nucleic acid encoding a CAR can be introduced into T cells, NK cells, or NK T cells, for example, using a retroviral vector. In this way, large numbers of cancer-specific T cells, NK cells, or NK T cells can be generated for adoptive cell transfer. In the present invention, the CAR can be combined with the activating intracellular domain of a T cell receptor based on the antigen (e.g., BCMA) specificity of the antibody. T cells genetically engineered to express a CAR can specifically recognize and eliminate malignant cells that express the target antigen.

[0027] Dual nanobody-based chimeric antigen receptors In another aspect, the present invention provides a dual Nanobody-based BCMA-targeting chimeric antigen receptor, characterized in that said chimeric antigen receptor comprises any one of the Nanobodies according to the first aspect of the invention and any other Nanobody that targets BCMA.

[0028] Furthermore, any one of the other Nanobodies targeting BCMA is Nanobody VHH02. Furthermore, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are set forth in SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15, respectively. Furthermore, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are set forth in SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16, respectively. Furthermore, the amino acid sequence of VHH02 is set forth in SEQ ID NO:9. Furthermore, the nucleotide sequence of VHH02 is set forth in SEQ ID NO:10.

[0029] In some embodiments, the Nanobody VHH02 described in the invention further comprises Nanobodies corresponding to amino acid sequences or nucleotide sequences that have at least 75% identity with the above amino acid sequences or nucleotide sequences, i.e., amino acid sequences or nucleotide sequences that are obtained by replacing an amino acid or nucleotide at any one or more positions on the basis of the corresponding amino acid sequence or nucleotide sequence with any other amino acid or nucleotide, and any amino acid sequence or nucleotide sequence that has at least 75% identity with the amino acid sequences or nucleotide sequences described in the invention falls within the scope of protection of the present invention.

[0030] The chimeric antigen receptor further comprises a transmembrane domain. The chimeric antigen receptor further comprises an intracellular signaling domain. The chimeric antigen receptor further comprises a hinge region. The chimeric antigen receptor further comprises a signal peptide. The chimeric antigen receptor further comprises a costimulatory signal domain. The chimeric antigen receptor further comprises a promoter. The chimeric antigen receptor further comprises a self-cleaving peptide. The chimeric antigen receptor further comprises a detection tag / accessory function element. The chimeric antigen receptor further comprises a tEGFR signal peptide.

[0031] The transmembrane domain includes those of CD8α, CD28, 4-1BB, CD34, CD3ε, PD-1, IgG1, IgG4, OX40, IL-2 receptor, IL-7 receptor, and IL-11 receptor. The intracellular signaling domain includes those of CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, and CD66d. The hinge region includes those of CD8α, CD28, CD34, 4-1BB, OX40, CD3ε, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, and IL-11 receptor. Furthermore, the signal peptides include those of the α and β chains of the T cell receptor, CD3ζ, CD3ε, CD16, CD22, CD33, CD4, CD5, CD8, CD9, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, and GM-CSF. Furthermore, the costimulatory signal domains include those of the 4-1BB (CD137), CD19, CD4, CD27, CD28, ICOS (CD278), CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, and CD226. Furthermore, the promoter includes an EF1α promoter, a CMV promoter, an EFS promoter, a CAG promoter, a CBh promoter, an SFFV promoter, an MSCV promoter, an SV40 promoter, an mPGK promoter, an hPGK promoter, and a UBC promoter. Furthermore, the self-cleaving peptide includes T2A, P2A, E2A, and F2A.Further, the detection tags / auxiliary function elements include tEGFR, tCD34, tCD19, tCD20, tCD22, immune checkpoint inhibitors (CTLA-4, PD-1 / PD-L1, LAG-3, TIM-3, TIGIT, CD226, CD155, CD47, B7-H3, B7-H4), nanobodies, cytokines and their receptors (IL2, IL2 receptor, IL7, IL7 receptor, IL15, IL15 receptor).

[0032] Furthermore, the transmembrane domain is a CD8α transmembrane domain. Furthermore, the intracellular signaling domain is a CD3ζ intracellular signaling domain. Furthermore, the hinge region is a CD8α hinge region. Furthermore, the costimulatory signal domain is a 4-1BB costimulatory signal domain. Furthermore, the promoter is EF1α. Furthermore, the self-cleaving peptide is T2A. Furthermore, the detection tag / auxiliary function element is tEGFR.

[0033] Furthermore, the chimeric antigen receptor comprises EF1α, a signal peptide, any one of the Nanobodies according to the first aspect of the present invention, a linker, any other Nanobody targeting BCMA, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB costimulatory signal domain, a CD3ζ intracellular signaling domain, T2A, a tEGFR signal peptide, and tEGFR linked in tandem in this order.

[0034] Furthermore, the chimeric antigen receptor comprises EF1α, a signal peptide, any one of the other Nanobodies targeting BCMA, a linker, any one of the Nanobodies according to the first aspect of the present invention, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB costimulatory signal domain, a CD3ζ intracellular signaling domain, T2A, a tEGFR signal peptide, and tEGFR linked in tandem in this order.

[0035] Furthermore, the chimeric antigen receptor is formed by tandemly linking, in this order, EF1α, a signal peptide, VHH01, a linker, VHH02, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB costimulatory signal domain, a CD3ζ intracellular signaling domain, T2A, a tEGFR signal peptide, and tEGFR.

[0036] Furthermore, the chimeric antigen receptor is composed of EF1α, a signal peptide, VHH02, a linker, VHH02, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB costimulatory signal domain, a CD3ζ intracellular signaling domain, T2A, a tEGFR signal peptide, and tEGFR linked in tandem in this order.

[0037] Furthermore, the linker is (G4S)5.

[0038] In some embodiments, the linker is a linker commonly used in the art, and may be, but is not limited to, (G4S)5, (GGGGS)n, (GGGS)n, (SSSSG)n, (GSGSA)n, (GGSGG)n, or any other linker, where n can be any integer from 1 to 10.

[0039] In some embodiments, the nucleotide sequence of EF1α described in the present invention is represented by SEQ ID NO:17, and the amino acid sequences of the signal peptide, CD8α hinge region, CD8α transmembrane domain, 4-1BB costimulatory signal domain, CD3ζ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR are represented by SEQ ID NO:18-SEQ ID NO:25, respectively, and the corresponding nucleotide sequences are represented by SEQ ID NO:26-SEQ ID NO:33, respectively.

[0040] In some embodiments, the EF1α, signal peptide, CD8α hinge region, CD8α transmembrane domain, 4-1BB costimulatory signal domain, CD3ζ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR described in the present invention are not limited to the above sequences, and any amino acid sequence or nucleotide sequence having at least 75% identity with the above amino acid sequence or nucleotide sequence falls within the scope of protection of the present invention.

[0041] In some embodiments, the chimeric antigen receptor described herein may further comprise a surrogate marker, such as a cell surface marker (e.g., a truncated cell surface marker) that can be used to confirm that cells have been transduced or engineered to express the receptor. In some embodiments, the surrogate marker comprises all or a portion (e.g., a truncated form) of CD34, NGFR, CD19, or truncated CD19, or epidermal growth factor receptor (e.g., tEGFR). In other embodiments, the cell surface marker comprises a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as superfold GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed, or DsRed2, cyan fluorescent protein (CFP), cyan-green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), and variants thereof, further including species variants, monomeric variants, and codon-optimized and / or enhanced variants of fluorescent proteins. In another embodiment, the surrogate marker comprises an enzyme such as luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT), and further comprises β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or variants thereof.

[0042] nucleic acid molecule In another aspect, the invention provides an isolated nucleic acid molecule.

[0043] Furthermore, the nucleic acid molecule comprises a nucleotide sequence encoding a Nanobody according to the first aspect of the invention, a chimeric antigen receptor according to the second aspect of the invention or a chimeric antigen receptor according to the third aspect of the invention.

[0044] Furthermore, the nucleotide sequence of VHH01 in the Nanobody according to the first aspect of the present invention is set forth in SEQ ID NO:2. Furthermore, the nucleotide sequence of VHH02 in the Nanobody according to the first aspect of the present invention is set forth in SEQ ID NO:10. Furthermore, the nucleotide sequence of EF1α in the chimeric antigen receptor according to the second aspect of the present invention or the chimeric antigen receptor according to the third aspect of the present invention is set forth in SEQ ID NO:17. Furthermore, the nucleotide sequence of the signal peptide in the chimeric antigen receptor according to the second aspect of the present invention or the chimeric antigen receptor according to the third aspect of the present invention is set forth in SEQ ID NO:26. Furthermore, the nucleotide sequence of the CD8α hinge region in the chimeric antigen receptor according to the second aspect of the present invention or the chimeric antigen receptor according to the third aspect of the present invention is set forth in SEQ ID NO:27. Furthermore, the nucleotide sequence of the CD8α transmembrane domain in the chimeric antigen receptor according to the second aspect of the present invention or the chimeric antigen receptor according to the third aspect of the present invention is set forth in SEQ ID NO:28. Furthermore, the nucleotide sequence of the 4-1BB costimulatory signal domain in the chimeric antigen receptor according to the second aspect of the present invention or the chimeric antigen receptor according to the third aspect of the present invention is shown in SEQ ID NO:29. Furthermore, the nucleotide sequence of the CD3ζ intracellular signaling domain in the chimeric antigen receptor according to the second aspect of the present invention or the chimeric antigen receptor according to the third aspect of the present invention is shown in SEQ ID NO:30. Furthermore, the nucleotide sequence of T2A in the chimeric antigen receptor according to the second aspect of the present invention or the chimeric antigen receptor according to the third aspect of the present invention is shown in SEQ ID NO:31. Furthermore, the nucleotide sequence of the tEGFR signal peptide in the chimeric antigen receptor according to the second aspect of the present invention or the chimeric antigen receptor according to the third aspect of the present invention is shown in SEQ ID NO:32. Furthermore, the nucleotide sequence of tEGFR in the chimeric antigen receptor according to the second aspect of the present invention or the chimeric antigen receptor according to the third aspect of the present invention is shown in SEQ ID NO:33.

[0045] As used herein, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0046] Expression vector In another aspect, the present invention provides an expression vector comprising a nucleic acid molecule according to the fourth aspect of the invention.

[0047] Furthermore, the vectors include DNA vectors, RNA vectors, plasmids, and virus-derived vectors, and the virus-derived vectors include lentivirus vectors, retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, and herpesvirus vectors.

[0048] In some embodiments, the present invention is not particularly limited to vectors, and vectors are selected according to the desired function. Non-limiting examples of vectors include plasmid vectors, virus-derived vectors, phage vectors, and other conventional vectors used in genetic engineering, for example. Various plasmids and vectors can be constructed based on methods well known to those skilled in the art.

[0049] In some embodiments, an expression vector according to the invention is capable of directing the replication and expression of a nucleic acid molecule of the invention in a host, thereby ensuring the expression in a selected host of a BCMA-targeting Nanobody or chimeric antigen receptor according to the invention encoded thereby. Expression vectors may be, for example, cloning vectors, binary vectors or integrating vectors. Expression includes transcription of the nucleic acid molecule, for example into translatable mRNA.

[0050] Non-limiting examples of vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), λgt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027 pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1, pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (EdgeBiosystems), pTriEx-Hygro (Novagen), and pCINeo (Promega). Non-limiting examples of plasmid vectors suitable for Pichia pastoris include, for example, plasmids pAO815, pPIC9K, and pPIC3.5K (all Invitrogen). Another vector suitable for expressing proteins in Xenopus embryos, zebrafish embryos, and various mammalian and avian cells is the multipurpose expression vector pCS2+.

[0051] Typically, vectors may contain one or more origins of replication (ori) and genetic systems for cloning or expression, one or more markers for selection in the host (e.g., antibiotic resistance), and one or more expression cassettes. Furthermore, the coding sequence contained in the vector can be linked to transcriptional regulatory elements and / or other amino acid coding sequences using established methods. Such regulatory sequences are well known to those skilled in the art and include, but are not limited to, regulatory sequences ensuring transcription initiation, internal ribosome entry sites (IRES), and optional regulatory elements ensuring transcription termination and transcript stability. Non-limiting examples of such regulatory elements ensuring transcription initiation include a promoter, translation initiation codon, enhancer, insulator, and / or regulatory element ensuring transcription termination contained downstream of the nucleic acid molecule of the present invention. Further examples include Kozak sequences, intervening sequences flanking donor and acceptor sites for RNA splicing, nucleotide sequences encoding secretion signals, or signal sequences capable of directing the expressed protein to an intracellular compartment or the medium, depending on the expression system used. The vector may also contain another expressible polynucleotide encoding one or more protein chaperones to facilitate correct folding of the protein.

[0052] As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or may be present in an in vitro expression system. Expression vectors include all expression vectors known in the art, such as cosmids, plasmids (e.g., naked plasmids or plasmids contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) into which a recombinant polynucleotide has been incorporated.

[0053] Engineered host cells In another aspect, the present invention provides engineered host cells.

[0054] Furthermore, the engineered host cell comprises the expression vector according to the fifth aspect of the present invention. Furthermore, the host cell is a eukaryotic or prokaryotic cell. Furthermore, the host cell is an immune cell. Furthermore, the immune cell includes a T cell, a B cell, a NK cell, an iNKT cell, a CTL cell, a dendritic cell, a medullary cell, a monocyte, a macrophage, a gdT cell, any immune cell derived from iPS, or any combination thereof. Furthermore, the immune cell is a T cell.

[0055] In the present invention, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus oryzae, insect cells such as Drosophila S2 cells or Sf9 cells, fibroblasts, and animal cells such as CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells. In a specific embodiment of the present invention, the host cell is preferably an immune cell, including, but not limited to, T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, medullary cells, monocytes, macrophages, or any combination thereof, preferably T cells. Furthermore, the "host cell" according to the present invention may include a single cell or a cell population; i.e., the "engineered host cell" according to the present invention includes both an engineered single host cell and an engineered host cell population.

[0056] Immunoconjugates or detection reagents In another aspect, the invention provides reagents for detecting immunoconjugates or BCMA proteins.

[0057] Furthermore, said immunoconjugates comprise a Nanobody according to the first aspect of the invention and conjugates that bind thereto.

[0058] Additionally, the conjugate may comprise a detectable marker, a radionuclide, a cytokine, a therapeutic agent, a cytotoxin, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral capsid protein or VLP, or a combination thereof.

[0059] Furthermore, the detectable marker includes a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computed tomography) imaging agent. 131 I, 32 P, 89 Sr, 90 Y, 223 Ra, 125 I, 103Pd. Furthermore, the cytokines include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN-γ, TNF-β, TNF-α, G-CSF, and M-CSF. Furthermore, the therapeutic agents include alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, chromatin function inhibitors, antiangiogenic agents, antiestrogens, antiandrogens, and immunomodulators. Furthermore, the alkylating agents include bischloroethylmethylamine, chlorambucil, melphalan, pipobroman, prednimustine, estracyte, cyclophosphamide, altretamine, ifosfamide, fotemustine, thiotepa, carmustine, streptozotocin, improsulfan, dacarbazine, cisplatin, oxaliplatin, and carboplatin. Furthermore, the antimetabolites include methotrexate, 5-fluorouracil, floxuridine, 5-fluorodeoxyuracil, capecitabine, cytarabine, fludarabine, 6-mercaptopurine, 2-chlorodeoxyadenosine, 5-azacytidine, 2,2-difluorodeoxycytidine, cladribine, deoxycoformycin, and pentostatin. Furthermore, the antitumor antibiotics include daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin C, valrubicin, mitoxantrone hydrochloride, bleomycin, dactinomycin, mithramycin, and procarbazine. Furthermore, the mitotic inhibitors include docetaxel, vinblastine, paclitaxel, vincristine, vindesine, and vinorelbine. Furthermore, the chromatin function inhibitors include irinotecan, etoposide, topotecan, etoposide phosphate, and teniposide. Furthermore, the antiangiogenic agents include prinomastat, tanomastat, ilomastat, razoxane, marimastat, batimastat, CGS-27023A, halofuginone, COL-3, neovastat, BMS-275291, and thalidomide. Furthermore, the antiestrogens include toremifene, raloxifene, tamoxifen, anastrozole, letrozole, droloxifene, iodoxifene, and exemestane.Further, the antiandrogen includes nilutamide, bicalutamide, spironolactone, flutamide, finasteride, cyproterone acetate, and cimetidine. Further, the immunomodulator includes interleukin, tumor necrosis factor, interferon, lentinan, sizofiran, roquinimex, pidotimod, methoxypolyethylene glycol succinamide adenosine deaminase, and thymosin preparations. Further, the cytotoxin includes MMAE, DM1, ozogamicin, Dxd, SN-38, MMAF, PBD, DM2, amnitin, DM4, PNU-159682, IR700, PE-38, PE24, PE-T20, PE-T20-KDEL, PE4E, and PE40.

[0060] Furthermore, the reagent for detecting said BCMA protein comprises a Nanobody or said immunoconjugate according to the first aspect of the invention.

[0061] Additionally, the reagent further comprises a diagnostic agent that binds to the Nanobody or immunoconjugate.

[0062] Furthermore, the diagnostic agent includes a radionuclide, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, and a photosensitive diagnostic agent. 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 86 Y, 90 Y, 89 Zr, 120 I, 123 I, 124 I, 125 I, 131 I, 13 N, 15 O. 186 Re, 188 Re, 51 Mn, 55 Co, 72The chemiluminescent agent includes luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt, and oxalate. The bioluminescent agent includes fluorescein, luciferase, and aequorin. The paramagnetic ion includes chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), and erbium(III). The enzyme includes horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase. Furthermore, the photosensitive diagnostic agent includes silicon dihydroxyl phthalocyanine, methylene blue, protoporphyrin, hematoporphyrin, and photofrin.

[0063] Drug composition or kit In another aspect, the present invention provides a pharmaceutical composition or kit.

[0064] Furthermore, the pharmaceutical composition comprises a Nanobody according to the first aspect of the invention, an engineered host cell according to the sixth aspect of the invention and / or an immunoconjugate according to the seventh aspect of the invention.

[0065] Furthermore, the pharmaceutical composition further comprises one or more pharmaceutically or physiologically acceptable vectors and / or auxiliary materials, and is used for treating and / or preventing BCMA-positive associated diseases.

[0066] Furthermore, the BCMA-positive associated diseases include multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastric cancer, liver cancer, kidney cancer, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal gland tumor, bladder tumor, non-small cell lung cancer, glioma, endometrial cancer, fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, and pancreatic cancer.

[0067] Furthermore, the kit comprises a Nanobody according to the first aspect of the invention, an immunoconjugate or a reagent according to the seventh aspect of the invention.

[0068] Furthermore, the kit is used to detect BCMA protein.

[0069] In some embodiments, the kit further comprises a container, an instruction manual, a buffer, etc., and in other embodiments, the kit further comprises a dissolution medium for dissolving the test sample, general-purpose reagents and buffers necessary for detection, such as various buffers, a detection marker, a detection substrate, etc. The detection kit may be an in vitro diagnostic device.

[0070] In some embodiments, the pharmaceutical composition according to the invention may further comprise other therapeutic agents that can be used to assist in the treatment and / or prevention of BCMA-positive associated diseases, and the Nanobody according to the first aspect of the invention, the engineered host cell according to the sixth aspect of the invention, the immunoconjugate according to the seventh aspect of the invention and / or the other therapeutic agents in said pharmaceutical composition may be administered simultaneously, separately or sequentially.

[0071] In some embodiments, the pharmaceutically or physiologically acceptable vector and / or adjuncts may comprise a sterile injectable fluid (e.g., an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such a sterile injectable fluid is selected from water for injection (WFI), sterile water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a surfactant solution (e.g., 0.01% polysorbate 20), a pH buffer (e.g., phosphate buffer), Ringer's solution, and any combination thereof.

[0072] In some embodiments, the pharmaceutical composition contains a "therapeutically effective amount" or a "prophylactically effective amount" of a Nanobody according to the first aspect of the invention, an engineered host cell according to the sixth aspect of the invention, and / or an immunoconjugate according to the seventh aspect of the invention, where a "prophylactically effective amount" refers to an amount sufficient to prevent, inhibit, or delay the onset of disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient suffering from the disease. The therapeutically effective amount may vary depending on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition, such as age, weight, and sex, the method of administration of the drug, and other concurrently used treatments.

[0073] In some embodiments, the dosage and frequency (single or multiple doses) of administering the pharmaceutical composition to a subject can vary depending on various factors, such as whether the mammal is suffering from another disease and the route of administration, the subject's age, sex, health status, weight, body mass index, and diet, the nature and extent of symptoms of the disease being treated (e.g., cancer symptoms and the severity of such symptoms), the type of concomitant therapy, complications of the disease being treated, or other health-related problems. Other treatments or agents can be used in combination with the pharmaceutical compositions and treatment methods described in the present invention. Adjustment and manipulation of established dosages (e.g., frequency and duration) are fully within the capabilities of one of ordinary skill in the art.

[0074] In some embodiments, the pharmaceutical composition may be in any dosage form selected from tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. It may be administered in single or multiple doses. The pharmaceutical composition may be administered in the form of a liquid formulation, powder, aerosol, capsule, vaginal tablet, capsule, or suppository. Routes of administration may include, but are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary, and rectal administration. For oral administration, the active ingredient in the pharmaceutical composition may be formulated with a coating to protect it from degradation in the stomach. The active ingredient may also be administered by any device capable of delivering it to target cells. In specific embodiments, the pharmaceutical composition provided by the present invention may be formulated into various dosage forms according to actual needs. Clinicians can determine and administer the optimal dose for each patient based on factors such as the type, age, weight, and general condition of the patient, as well as the method of administration. The method of administration may be, for example, injection or any other suitable method of administration known to those skilled in the art.

[0075] In some embodiments, the drug compositions, nanobodies, engineered host cells, immunoconjugates, etc. provided herein can be used in combination with another therapy, treatment, or agent, including, but not limited to, chemotherapy, radiation therapy, surgery, transplantation, adoptive cell therapy, antibodies, cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitors, antihormones, kinase inhibitors, antiangiogenic agents, cardioprotective agents, immunostimulators, immunosuppressants, immune checkpoint inhibitors, antibiotics, angiogenesis inhibitors, metabolic modulators, or other therapeutic agents, or any combination thereof. In some embodiments, the other agent is a protein, peptide, nucleic acid, small molecule drug, cell, toxin, lipid, carbohydrate, or a combination thereof, or any other type of therapeutic agent, such as radiation. In some embodiments, the other therapy, agent, or treatment comprises surgery, chemotherapy, radiation therapy, transplantation, administration of cells expressing a recombinant receptor (e.g., a chimeric antigen receptor), kinase inhibitors, immune checkpoint inhibitors, mTOR pathway inhibitors, immunosuppressants, immunomodulators, antibodies, immunoablative agents, antibodies and / or antigen-binding fragments thereof, antibody conjugates, other antibody therapies, cytotoxins, steroids, cytokines, peptide vaccines, hormone therapy, antimetabolites, metabolic modulators, drugs that inhibit the calcium-dependent phosphatase calcineurin or the p70S6 kinase FK506 or p70S6 kinase, alkylating agents, anthracyclines, vinca alkaloids, proteasome inhibitors, GITR agonists, protein tyrosine phosphatase inhibitors, protein kinase inhibitors, oncolytic viruses, and / or other types of immunotherapy. In some embodiments, the other agent or treatment is bone marrow transplant, T-cell ablation therapy using a chemotherapeutic agent (e.g., fludarabine), external beam radiation therapy (XRT), cyclophosphamide, and / or antibody therapy.

[0076] Methods of production, preparation, detection and diagnosis In other aspects, the invention provides any one of the following methods:

[0077] (1) A method for producing a nanobody according to the first aspect of the invention. (a) culturing an engineered host cell according to the sixth aspect of the invention under conditions suitable for the production of a Nanobody to obtain a culture comprising said Nanobody; (b) isolating or recovering said Nanobodies from the culture obtained in step (a); (c) purifying the Nanobody obtained in step (b) to obtain a Nanobody according to the first aspect of the invention.

[0078] (2) A method for preparing an engineered host cell according to the sixth aspect of the invention, comprising the step of introducing into a host cell a nucleic acid molecule according to the fourth aspect of the invention or an expression vector according to the fifth aspect of the invention. Furthermore, the introduction method includes lipofection, microinjection, electroporation, DNA vectors, RNA vectors, retroviral vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenovirus vectors, and adeno-associated virus vectors. Furthermore, the host cells are eukaryotic cells or prokaryotic cells. Furthermore, the host cells are immune cells. Furthermore, the immune cells include T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, medullary cells, monocytes, macrophages, gdT cells, any immune cell derived from iPS cells, or any combination thereof. Furthermore, the immune cells are T cells.

[0079] (3) A method for detecting BCMA protein, comprising the step of contacting a test sample with a Nanobody according to the first aspect of the invention and / or an immunoconjugate or reagent according to the seventh aspect of the invention, and detecting the presence of an antibody-antigen complex.

[0080] (4) A method for diagnosing whether a subject is suffering from a BCMA-positive associated disease, comprising the steps of contacting a test sample derived from the subject with a Nanobody according to the first aspect of the invention and / or an immunoconjugate or reagent according to the seventh aspect of the invention, and detecting the formation of a complex between said Nanobody, immunoconjugate and / or reagent and BCMA, or detecting the amount of said complex, wherein the formation of said complex indicates the presence of BCMA or cells expressing BCMA and that said subject is suffering from a BCMA-positive associated disease.

[0081] In some embodiments, the method for diagnosing whether a subject has a BCMA-positive associated disease may further comprise comparing the amount of BCMA in a sample from the subject with a reference value. The reference value may be the level of BCMA in a sample from a subject known not to have a BCMA-related disease (e.g., a healthy control) (also referred to as a "negative reference value"). For example, if the amount of BCMA in the sample from the subject is elevated relative to the negative reference value, this indicates that the subject has a BCMA-related disease.

[0082] As used herein, the term "test sample" refers to a sample (e.g., a fluid, cell, or tissue) isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary test samples include, for example, blood, serum and serous fluid, plasma, lymphatic fluid, urine, saliva, bursal fluid, lachrymal fluid, excrement, sputum, mucosal secretions of secretory tissues or secretory organs, vaginal secretions, ascites, pleural, pericardial, peritoneal, abdominal and other body cavity fluids, fluids collected from bronchial lavage, synovial fluid, and other biological fluids; liquid solutions that have come into contact with a subject or biological source, such as culture media (including conditioned media), lavage fluids, tissue biopsies, fine needle aspirates, surgically removed tissues, organ cultures, or cell cultures.

[0083] As used herein, the term "subject" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals), such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. In some embodiments, the "subject" is preferably a human.

[0084] (5) A method for treating a BCMA-positive associated disease, comprising administering to a subject in need thereof an effective amount of a nanobody according to the first aspect of the invention, an engineered host cell according to the sixth aspect of the invention, an immunoconjugate according to the seventh aspect of the invention and / or a pharmaceutical composition according to the eighth aspect of the invention.

[0085] In some embodiments, the administration modes include parenteral, pulmonary, and intranasal administration, with intralesional administration being used when localized treatment is required. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, such as injection, e.g., intravenous or subcutaneous injection, depending on whether the administration is intended to be short-term or long-term. Various administration time regimens are contemplated herein, including, but not limited to, multiple administrations at single or multiple time points, push administration, and pulse infusion.

[0086] Use in the preparation of therapeutic and diagnostic reagents, and in diagnostics and therapeutics In other aspects, the present invention provides any one of the following uses:

[0087] (1) Use of a nanobody according to the first aspect of the invention in the detection of BCMA protein and / or in the diagnosis of a BCMA-positive associated disease.

[0088] (2) Use of a nanobody according to the first aspect of the invention in the preparation of a reagent or kit for detecting BCMA protein and / or diagnosing a BCMA-positive associated disease.

[0089] (3) Use of the reagent according to the seventh aspect of the present invention or the kit according to the eighth aspect of the present invention in detecting BCMA protein and / or diagnosing a BCMA-positive associated disease.

[0090] (4) Use of a nanobody according to the first aspect of the invention, a chimeric antigen receptor according to the second aspect of the invention, a chimeric antigen receptor according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention or an expression vector according to the fifth aspect of the invention in the preparation of an engineered host cell for treating and / or preventing a BCMA-positive associated disease.

[0091] (5) Use of a Nanobody according to the first aspect of the invention, a chimeric antigen receptor according to the second aspect of the invention, a chimeric antigen receptor according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, an expression vector according to the fifth aspect of the invention or an engineered host cell according to the sixth aspect of the invention in the preparation of an immunoconjugate for the treatment and / or prevention of a BCMA-positive associated disease.

[0092] (6) Use of a nanobody according to the first aspect of the invention, a chimeric antigen receptor according to the second aspect of the invention, a chimeric antigen receptor according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, an expression vector according to the fifth aspect of the invention, an engineered host cell according to the sixth aspect of the invention or an immunoconjugate according to the seventh aspect of the invention in the preparation of a medicament for treating and / or preventing a BCMA-positive associated disease.

[0093] (7) Use of a nanobody according to the first aspect of the invention, a chimeric antigen receptor according to the second aspect of the invention, a chimeric antigen receptor according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, an expression vector according to the fifth aspect of the invention, an engineered host cell according to the sixth aspect of the invention, an immunoconjugate according to the seventh aspect of the invention or a pharmaceutical composition according to the eighth aspect of the invention in the preparation of a biological preparation for the treatment and / or prevention of a BCMA-positive associated disease.

[0094] (8) Use of a nanobody according to the first aspect of the invention, a chimeric antigen receptor according to the second aspect of the invention, a chimeric antigen receptor according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, an expression vector according to the fifth aspect of the invention, an engineered host cell according to the sixth aspect of the invention, an immunoconjugate according to the seventh aspect of the invention or a pharmaceutical composition according to the eighth aspect of the invention in the treatment and / or prevention of a BCMA-positive associated disease.

[0095] Furthermore, the BCMA-positive associated diseases include multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastric cancer, liver cancer, kidney cancer, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal gland tumor, bladder tumor, non-small cell lung cancer, glioma, endometrial cancer, fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, and pancreatic cancer.

[0096] In the present invention, the term "BCMA-positive associated disease" refers to any disease or condition associated with BCMA expression, including, but not limited to, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastric cancer, liver cancer, kidney cancer, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer, glioma, endometrial cancer, fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, and pancreatic cancer. Any disease or condition associated with BCMA expression falls within the scope of protection of the present invention.

[0097] In the present invention, the term "treatment" refers to the complete or partial improvement or alleviation of a disease, condition, symptom, side effect, outcome, or associated phenotype. Desired therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological outcome of a disease, prevention of cancer metastasis, reduction of the rate of disease progression, amelioration or alleviation of disease symptoms, and alleviation or improvement of prognosis. The term does not imply complete cure of a disease, complete elimination of all symptoms, or effectiveness against all symptoms and outcomes.

[0098] In the present invention, the term "delaying disease progression" refers to slowing, inhibiting, slowing, halting, stabilizing, inhibiting, and / or slowing the progression of a disease (e.g., cancer). Depending on the disease being treated and / or the medical history of the subject, this delay may vary in duration. A sufficient or significant delay may actually include preventing an individual from developing the disease. As an example, the progression of late-stage cancer, such as cancer metastasis, may be slowed.

[0099] In the present invention, the term "prevention" includes providing a prophylactic effect against the occurrence or recurrence of a disease in a subject in an individual who is at high risk of the disease but has not yet been diagnosed with the disease. In some embodiments, the provided molecules and compositions are used to delay the onset of the disease or slow the progression of the disease.

[0100] In the present invention, the term "inhibition" of a function or activity refers to a decrease in the function or activity when compared to a condition in which all other aspects are the same except for the condition or parameter of interest, or when compared to another condition. For example, an antibody, composition, or cell that inhibits tumor growth reduces the rate of tumor growth compared to when the antibody, composition, or cell is not present.

[0101] The present invention has the following advantages and beneficial effects over the prior art:

[0102] (1) The present invention provides BCMA-targeting nanobodies VHH01 and VHH02. The amino acid sequences of CDR1, CDR2, and CDR3 of nanobody VHH01 are set forth in SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of nanobody VHH02 are set forth in SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15, respectively. These nanobodies all have high specificity and affinity for BCMA, and can specifically bind to BCMA-positive cell lines with high affinity. They also have advantages such as high stability, simple structure, and amenable engineering, providing a new approach to the development of effective BCMA-targeting immunotherapeutics.

[0103] (2) The present invention provides a chimeric antigen receptor comprising nanobody VHH01 and / or VHH02, and further provides BCMA-targeting CAR-T cells prepared based on the chimeric antigen receptor. The CAR-T cells can specifically recognize BCMA-positive tumor cell lines, have high specific killing efficiency against BCMA-positive cell lines, and have no adverse effects on cell lines that do not express BCMA, thereby avoiding off-target therapeutic toxicity and safety issues. This provides a potential treatment for BCMA-associated diseases in this field and has significant potential for application and clinical value. [Brief explanation of the drawings]

[0104] [Figure 1] FIG. 1 is a schematic diagram of the alpaca immunization and antibody screening flow. [Figure 2] Statistical diagram of the nanobody library enrichment and screening results. [Figure 3] Statistical graph of the screening results of monoclonal anti-BCMA nanobodies. [Figure 4] FIG. 1 shows the results of flow cytometry detection of overexpressing cell line K562-BCMA. [Figure 5]Schematic diagram of the structure of a single VHH CAR, in which panel A is a schematic diagram of the construction of a single VHH CAR structure, and panel B is a schematic diagram of the display of a single VHH CAR structure on the cell surface. [Figure 6] Representative results of measuring the transduction rate of single VHH CAR-T cells. [Figure 7] Figure 14 shows the statistical results of MFI values ​​on the surface of single VHH CAR-T cells. [Figure 8] Figure 1 shows the growth curve of single VHH CAR-T cells. [Figure 9] This is a graph showing the statistical results of the CD4 / CD8 ratio during the culture process of single VHH CAR-T. [Figure 10] Figure 1 shows the results of a killing experiment of single VHH CAR-T cells against K562-BCMA. [Figure 11] 1 shows a schematic diagram of the structure of a dual VHH CAR, in which panel A shows a schematic diagram of the construction of the dual VHH CAR structure, and panel B shows a schematic diagram of the display of the dual VHH CAR structure on the cell surface. [Figure 12] This is a graph showing the statistical results of MFI values ​​on the surface of K562-dNMC003-A and K562-dNMC003-B cells. [Figure 13] Representative results of transduction rate measurement of dNMC003-A & dNMC003-B CAR-T cells. [Figure 14] Growth curve of dNMC003-A & dNMC003-B CAR-T cells. [Figure 15] This is a graph showing the statistical results of survival rates during the culture process of dNMC003-A & dNMC003-B CAR-T cells. [Figure 16] This is a graph showing the statistical results of the CD4 / CD8 ratio during the culture process of dNMC003-A & dNMC003-B CAR-T. [Figure 17]These are graphs showing the in vitro functional verification results of dNMC003-A & dNMC003-B CAR-T, of which Figure A is a graph showing the killing results of dNMC003-A & dNMC003-B, NMC003-01 & nmC003-02 against cell lines K562-BCMA and K562, and Figure B is a graph showing the killing results of dNMC003-A & dNMC003-B against cell lines RPMI 8226 and U87-MG. [Figure 18] Figure 1 shows the results of IFN-γ detection after co-incubation of dNMC003-A & dNMC003-B CAR-T cells with K562-BCMA, RPMI8226, and U87-MG cells. [Figure 19] Figure 1 shows the affinity detection results (ELISA method) for Nb003-01, Nb003-02, dNb003-A, and dNb003-B, where Figure A shows the affinity detection results for Nb003-01 with human BCMA antigen, Figure B shows the affinity detection results for Nb003-02 with human BCMA antigen, Figure C shows the affinity detection results for dNb003-A with human BCMA antigen, and Figure D shows the affinity detection results for dNb003-B with human BCMA antigen. [Figure 20] These are graphs showing the results of affinity detection (SPR method) for Nb003-01, Nb003-02, and dNb003-B, where Figure A is a graph showing the results of affinity detection between Nb003-01 and human BCMA antigen, Figure B is a graph showing the results of affinity detection between Nb003-02 and human BCMA antigen, and Figure C is a graph showing the results of affinity detection between dNb003-B and human BCMA antigen. [Figure 21] This is a diagram showing the results of verification of the specificity of Nb003-01, Nb003-02, dNb003-A, and dNb003-B. DETAILED DESCRIPTION OF THE INVENTION

[0105] The present invention will be further described below in conjunction with specific examples, which are merely for the purpose of illustrating the present invention and should not be construed as limiting the present invention. It will be understood by those skilled in the art that various changes, modifications, substitutions, and alterations can be made to these examples without departing from the principles and spirit of the present invention, and that the scope of the present invention is limited by the claims and their equivalents. Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and unless otherwise specified, the reagents, materials, etc. used in the following examples are commercially available.

[0106] Example 1 Screening of anti-BCMA nanobodies 1. Animal immunization and titer measurement (1) Antigen preparation RNA was extracted from RPMI 8226 cells using an RNA extraction kit. SuperScript TM Following the instructions for II Reverse Transcriptase, reverse transcription was performed using random primers to obtain cDNA. Using the cDNA as a template, PCR was performed to obtain the gene sequence for the extracellular domain of the BCMA antigen. The gene sequence for the BCMA extracellular domain was ligated into a protein expression vector, expressed, and purified using a Ni column to obtain purified BCMA protein.

[0107] (2) Animal immunity Alpacas were immunized using the self-purified BCMA-His protein of the present invention, and the specific immunization flow for alpacas is shown in Figure 1. Immunizations were performed once a week for a total of six consecutive immunizations. Before the sixth immunization, 5 mL of blood was collected for preliminary titer determination, and 100 mL of peripheral blood was collected seven days after the final immunization for antibody library construction and screening.

[0108] 2. Nanobody Library Construction, Enrichment, and Screening (1) Construction of nanobody library Seven days after the final immunization, 100 mL of peripheral blood was collected from the alpaca, peripheral blood mononuclear cells were separated by Ficoll density gradient centrifugation, RNA was extracted, and cDNA was prepared using a reverse transcription kit.

[0109] Using cDNA as a template and immunoglobulin heavy chain region-specific primers CALL001 and CALL002, all variable regions of immunoglobulin heavy chains (VHs and VHHs) were amplified from the cDNA. Of these, approximately 700 bp fragments represented the heavy chain-only antibody library, while approximately 1000 bp fragments corresponded to the heavy chains of conventional antibodies. The amplification products were analyzed using a 1% (wt / Vol) agarose gel. Approximately 700 bp of the product was recovered by gel recovery.

[0110] Using the gel-collected product as a template, the VHH sequences were specifically amplified using the degenerate primers VHH-BACK and VHH-FOR. The amplified products were analyzed on a 1% (wt / vol) agarose gel. They were then ligated into the pMES4 phage display vector, and the ligated products were electrotransformed into electrocompetent cells TG1. The resulting bacterial library was a BCMA single-domain heavy chain antibody phage display library. After library construction was completed, 25 clones were randomly selected and subjected to colony PCR using primers MP57 and GIII to detect the insertion efficiency of the library. The PCR products were then subjected to Sanger sequencing. The nanobody library construction flow is shown in Figure 1, and the primer sequences used are listed in Table 1.

[0111] [Table 1]

[0112] (2) Antibody library enrichment and screening The TG1 E. coli nanobody library was transferred to 2-YT liquid medium and grown at 37°C and 200 rpm until an OD of 0.5 was reached. The cells were then infected with helper phage VCSM13. After gentle mixing, the cells were incubated at 37°C for 30 minutes. The bacterial culture was centrifuged to remove traces of glucose. The precipitate was resuspended in 2-YT medium containing both ampicillin and kanamycin resistance and cultured overnight at 37°C with shaking at 200 rpm to amplify nanobody-displaying phages. The overnight culture was transferred to a 50 mL centrifuge tube, centrifuged, and the supernatant was removed. The phage was precipitated by adding a 20% (wt / vol) PEG6000 / 2.5 M NaCl solution. The supernatant was discarded and the precipitate was resuspended in 1 mL of PBS. After centrifugation, the supernatant was transferred to a new centrifuge tube, glycerol was added to a final concentration of 20%, and the mixture was stored at -80°C. To measure the titer of the phage nanobody library, the phages were diluted 10-fold, and different dilutions were used to infect logarithmically growing TG1 bacteria. The bacteria were then cultured at 37°C overnight, and the titer of the phage nanobody library was estimated from the number of plaques the next day.

[0113] Nanobody panning was performed using the ELISA method. Recombinant BCMA-His protein was coated onto ELISA plates and incubated overnight at 4°C. The ELISA plates were washed three times with 250 μL of PBST, 200 μL of blocking solution was added, and the plates were incubated at room temperature for 2 hours. The corresponding phage was added to each well and incubated at room temperature for 2 hours. The ELISA plates were washed 15 times with 250 μL of PBST. 100 μL of trypsin was added to each well and incubated at room temperature at 700 rpm for 0.5 hours. Phages were eluted using AEBSF, and titer measurements and phage infection and propagation were performed on the eluted phage. Panning was stopped when the number of eluted phages reached a positive:negative ratio of ≥ 100. The antibody library enrichment and screening flow chart is shown in Figure 1.

[0114] 3. Identification of monoclonal antibodies A single clone was selected from the TG1 E. coli library obtained after two rounds of screening, expanded, and infected with helper phage VCSM13 to prepare monoclonal phage. The monoclonal phage was added to an ELISA plate coated with BCMA protein and blocked with 2% nonfat dry milk, and incubated at room temperature for 2 hours. After washing the ELISA plate with PBST, HA-HRP antibody was added and incubated at room temperature for 1 hour. After washing the ELISA plate with PBST, 100 μL of TMB single-component color development solution was added, and after 30 minutes of incubation at room temperature, 100 μL of stop solution was added. The absorbance at 450 nm was measured using an ELISA reader. A ratio of OD450 values ​​of the sample well to the blank control greater than 2 was considered a positive clone. The monoclonal identification process is shown in Figure 1. Positive clones were subjected to bacterial liquid PCR and Sanger sequencing. Sequence comparison of the Sanger-sequenced monoclonals was performed using the software DNAMAN. Specific clones were screened for sequence identity.

[0115] 4. Experimental results The results of nanobody library construction showed that a BCMA nanobody library was successfully constructed, with a library capacity of 3E8 and an insertion rate close to 95%.

[0116] The results of antibody library panning are shown in Figure 2. After two rounds of enrichment and screening, the final number of phage was 262.1 times the positive:negative ratio, meeting the monoclonal screening criteria. Therefore, after two rounds of panning, the panning was stopped and the next monoclonal was screened and identified.

[0117] After sequence identification, two clones with unique sequences and high OD450 values ​​by ELISA were finally screened and named Nb003-01 (VHH01) and Nb003-02 (VHH02), respectively. The OD450 results are shown in Figure 3. Here, the amino acid sequences of CDR1, CDR2, and CDR3 of nanobody VHH01 are shown as SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, respectively, and the corresponding nucleotide sequences are shown as SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, respectively. The amino acid sequence of nanobody VHH01 is shown as SEQ ID NO:1, and the nucleotide sequence is shown as SEQ ID NO:2. The amino acid sequences of CDR1, CDR2 and CDR3 of Nanobody VHH02 are shown in SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:15, respectively, and the corresponding nucleotide sequences are shown in SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16, respectively; the amino acid sequence of Nanobody VHH02 is shown in SEQ ID NO:9, and the nucleotide sequence is shown in SEQ ID NO:10.

[0118] Example 2: Construction of K562-BCMA stable transfected cell line 1. Experimental method Using the cDNA obtained in Example 1 as a template, PCR was performed to obtain the full-length BCMA sequence, which was then inserted into the lentiviral vector pLVX-Puro by double digestion and ligation, resulting in a recombinant plasmid designated pLVX-BCMA-Puro. This recombinant plasmid utilizes the CMV promoter and contains a puromycin resistance gene.

[0119] The target plasmid pLVX-BCMA-Puro was packaged with a helper plasmid for lentiviral synthesis. Prior to CAR-T cell preparation, the target plasmid was co-transfected with three helper plasmids (pMD2.G, pRSV-REV, and pMDLg / RRE) into 293FT cells under the influence of PEI-Pro. Six hours after packaging, the medium was replaced. Lentivirus was harvested 48 hours after packaging. The harvested lentivirus stock was concentrated by ultracentrifugation. The lentiviral particles were resuspended in DMEM high-glucose medium and aliquoted for use.

[0120] 2. Experimental results The results are shown in Figure 4, which demonstrates that the constructed K562-BCMA stable transfected cell line highly expresses BCMA. This means that the present invention has successfully constructed a K562-BCMA stable transfected cell line.

[0121] Example 3 Preparation of single VHH CAR-T cells and in vitro functional validation 1. Construction of single VHH CAR constructs A single VHH CAR construct was constructed using sequence-specific clones. First, the VHH sequence of the positive clone was amplified using primers NCAR-F1 and NCAR-R1, using the successfully sequenced plasmid as a template. After the first PCR, a second PCR was performed using primers NCAR-F2 and NCAR-R2 as a template. The second PCR product was ligated into the vector Senl-S88BZ by homologous recombination, and the vector was digested with NotI. This resulted in the successful construction of a CAR construct containing a single VHH targeting BCMA. The structural diagram is shown in Figure 5, and the primer sequences are shown in Table 2.

[0122] [Table 2]

[0123] A total of two single VHH CAR constructs were constructed, designated NMC003-01 and NMC003-02, respectively. The structures are shown in Figure 5. EF1α is the promoter of elongation factor 1α, Leader is the coding sequence for the signal peptide, VHH is the coding sequence for the anti-BCMA nanobody, CD8αH+TM is the CD8α hinge region and transmembrane domain, 4-1BB and CD3ζ intracellular signaling region are intracellular costimulatory domains, and the tEGFR extracellular domain is expressed via T2A peptide ligation, allowing for easy detection of CAR expression after lentiviral transduction. NMC003-01 is a tandem linkage of EF1α, signal peptide, VHH01, CD8α hinge region, CD8α transmembrane domain, 4-1BB costimulatory signal domain, CD3ζ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR, in this order, and NMC003-02 is a tandem linkage of EF1α, signal peptide, VHH02, CD8α hinge region, CD8α transmembrane domain, 4-1BB costimulatory signal domain, CD3ζ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR, in this order. Here, the nucleotide sequence of EF1α is represented by SEQ ID NO:17, and the amino acid sequences of the signal peptide, CD8α hinge region, CD8α transmembrane domain, 4-1BB costimulatory signal domain, CD3ζ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR are represented by SEQ ID NO:18-SEQ ID NO:25, respectively, and the corresponding nucleotide sequences are represented by SEQ ID NO:26-SEQ ID NO:33, respectively.

[0124] 2. Lentivirus packaging Before preparing CAR-T cells, lentivirus was first packaged. That is, the target plasmid was co-transfected with three helper plasmids (pMD2.G, pRSV-REV, pMDLg / RRE) into 293FT cells under the influence of PEI-Pro. Six hours after packaging, the medium was changed. 48 hours after packaging, lentivirus was harvested. The harvested lentivirus stock was concentrated by ultracentrifugation. The lentivirus particles were resuspended in DMEM high glucose medium and aliquoted for use.

[0125] 3. Preparation of Single VHH CAR-T Cells After lentiviral packaging was completed, CAR-T cells were prepared. Peripheral blood mononuclear cells (PBMCs) were collected from patients or healthy donors and selected for αβ T cells using CD3 magnetic beads. The selected αβ T cells were cultured in TexMACS GMP medium (MACS). Lentiviral transduction was performed two days later, and CAR-T cells were harvested after 12–14 days of culture to obtain BCMA-targeting VHH CAR-T cells (designated NMC003-01 and NMC003-02, respectively). Flow cytometry was performed to measure the percentage of CAR+ cells during the culture process. tEGFR expression was detected using an anti-EGFR antibody, and nanobody expression on the cell surface was detected using BCMA-His protein as the primary antibody and an anti-His tag antibody as the secondary antibody. The MFI values ​​of the CAR-T cell surface VHHs detected by the BCMA-His antigen were calculated. The number of cells was counted on days 6, 9, and 12 of cell culture, and cell proliferation was statistically analyzed. Samples were taken on days 6 and 12 of culture, and flow cytometry was performed to statistically analyze the CD4 / CD8 ratio of CAR-T cells.

[0126] 4. In vitro functional validation of single VHH CAR-T cells To verify the in vitro biological activity of the anti-BCMA VHH CAR-T cells prepared in this example, an in vitro killing experiment was performed during the culture process. First, target cells were collected. The overexpressing cell line K562-BCMA (K562 is a human chronic myeloid leukemia cell) described in Example 2 was collected, centrifuged at 2000 rpm for 5 minutes, resuspended in DPBS, and counted. 1 × 10 5 The cells were added to a 96-well plate at a concentration of 1000 cells / well. Then, effector cells were added to target cells at different effector cell to target cell ratios (E:T = 0.3:1, 1:1, 3:1). After mixing, the cells were incubated for 4 hours, and the cell killing rate was detected by flow cytometry.

[0127] 5. Experimental results CAR-T cells were prepared by transducing T cells with the single VHH construct. Representative flow cytometry results after 6 days of culture are shown in Figure 6. In the figure, CAR(Erb) indicates the expression of tEGFR on the cell surface, and CAR(BCMA) indicates the expression of nanobodies on the cell surface. The results demonstrate that both CAR-T cells, NMC003-01 and NMC003-02, specifically express CAR(Erb) and CAR(BCMA). Furthermore, the MFI of nanobodies on the CAR-T cell surface was statistically analyzed (Figure 7). The results demonstrate that the MFI of nanobodies on both NMC003-01 and NMC003-02 cells was significantly improved compared to blank T cells. These results demonstrate the normal expression of tEGFR and nanobodies in the single VHH CAR construct.

[0128] The cell proliferation rate and CD4 / CD8 ratio were statistically analyzed during the single VHH CAR-T culture. Figure 8 shows the proliferation curves of single VHH CAR-T. From the start of culture to harvest on day 12, the proliferation rates of NMC003-01, NMC003-02, and blank T cells were 26.8, 20.6, and 29.2, respectively. The proliferation rates of NMC003-01 and NMC003-02 correspond to those of blank T cells. The CD4 / CD8 ratios during the cell culture process are shown in Figure 9. The CD4 / CD8 ratios varied somewhat during the culture process. At the final harvest, the CD4 / CD8 ratios of NMC003-01 and NMC003-02 were 2.75 and 2.56, respectively, and the CD4 / CD8 ratio of blank T cells was 2.07. These results demonstrated that single VHH CAR-T cells proliferated normally and that the CD4 / CD8 ratio tended to approach normal levels during the proliferation process.

[0129] The in vitro biological activity of single VHH CAR-T cells was verified by killing experiments against K562-BCMA. Figure 10 shows the in vitro killing results of single VHH CAR-Ts against K562-BCMA. At killing ratios of 0.3:1, 1:1, and 3:1, both NMC003-01 and NMC003-02 demonstrated specific killing of K562-BCMA. The killing rate gradually increased with increasing effector cell to target cell ratio. The killing rates of NMC003-01 and NMC003-02 against K562-BCMA were significantly higher than those of blank T cells. These results demonstrate that single VHH CAR-T cells specifically killed cell lines positively expressing BCMA.

[0130] Example 4 Preparation and functional verification of dual VHH CAR-T (dNMC003-A, dNMC003-B) cells 1. Construction of dNMC003-A and dNMC003-B structures The nanobodies Nb003-01 and Nb003-02 screened in this study were used to construct a target plasmid with a dual VHH CAR structure, the structural diagram of which is shown in Figure 11. The dNMC003-A antigen-binding region contains two VHH02s, and the dNMC003-B antigen-binding region contains VHH01 and VHH02. First, the VHH02 sequence was amplified by PCR. After the first PCR was completed, a second PCR was performed using the first PCR product as a template. The primer sequences are shown in Table 3. Next, the second PCR product was ligated into vectors NMC003-01 and NMC003-02 by homologous recombination, and the vectors were single-cleaved with Not I. The recombinant plasmid ligated into vector NMC003-02 was named dNMC003-A, and the recombinant plasmid ligated into vector NMC003-01 was named dNMC003-B. The structural diagrams are shown in Figure 11.

[0131] EF1α is the promoter of elongation factor 1α, Leader is the coding sequence for the signal peptide, VHH01 and VHH02 are the coding sequences for the anti-BCMA nanobody, CD8αH+TM is the CD8α hinge region and transmembrane domain, 4-1BB and CD3ζ intracellular signaling region are intracellular costimulatory domains, and the expression of the tEGFR extracellular domain via T2A peptide ligation allows for easy detection of CAR expression after lentiviral transduction. The dNMC003-A comprises EF1α, a signal peptide, VHH02, a linker ((G4S)5), VHH02, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB costimulatory signal domain, a CD3ζ intracellular signaling domain, T2A, a tEGFR signal peptide, and tEGFR, linked in tandem in this order. The dNMC003-B comprises EF1α, a signal peptide, VHH01, a linker ((G4S)5), VHH02, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB costimulatory signal domain, a CD3ζ intracellular signaling domain, T2A, a tEGFR signal peptide, and tEGFR, linked in tandem in this order. Here, the sequence information of EF1α, signal peptide, CD8α hinge region, CD8α transmembrane domain, 4-1BB costimulatory signal domain, CD3ζ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR is shown in Example 3.

[0132] [Table 3]

[0133] 2. Preparation of K562-dNMC003-A and K562-dNMC003-B Cells Lentivirus packaging was performed according to the preparation flow in Example 3. After lentivirus packaging was completed, the lentivirus was transduced into the K562 cell line. K562 cells transduced with the dNMC003-A and dNMC003-B series lentiviruses were cultured for 3 days, after which flow cytometry detection was performed to detect the expression of nanobodies on the cell surface using BCMA-His protein as the primary antibody and an anti-His tag antibody as the secondary antibody. The MFI of VHHs on the K562 surface was calculated.

[0134] 3. Preparation of dNMC003-A and dNMC003-B CAR-T cells dNMC003-A and dNMC003-B CAR-T cells were cultured according to the preparation flow of Example 3, and NMC003-01 and NMC003-02 CAR-T cells were simultaneously cultured. Flow cytometry was performed during the culture process to measure the percentage of CAR+ cells, detect tEGFR expression using an anti-EGFR antibody, and detect nanobody expression on the cell surface using BCMA-His protein as the primary antibody and an anti-His tag antibody as the secondary antibody. Cell numbers were counted on days 6, 9, and 13 of cell culture, and cell proliferation and viability were analyzed. Samples were then analyzed by flow cytometry to determine the CD4 / CD8 ratio of CAR-T cells.

[0135] 4. In vitro functional validation of dNMC003-A and dNMC003-B CAR-T cells To verify the in vitro biological activity of the dNMC003-A, dNMC003-B, NMC003-01, and NMC003-02 CAR-T cells prepared in this example, in vitro killing experiments were performed during the culture process. First, target cells were collected. K562 (human chronic myeloid leukemia cells) and the BCMA-overexpressing cell line K562-BCMA were collected, centrifuged at 2000 rpm for 5 min, resuspended in DPBS, and counted. 1 × 10 5The cells were added to a 96-well plate at a concentration of 1000p / well. The corresponding effector cells (dNMC003-A, dNMC003-B, NMC003-01, and NMC003-02 CAR-T cells) were then added to the target cells at an effector cell:target cell (E:T) ratio of 3:1. The mixture was incubated for 4 hours, and the cell killing rate was detected by flow cytometry.

[0136] At the same time, the in vitro killing effects of dNMC003-A and dNMC003-B CAR-T cells against human multiple myeloma peripheral blood B lymphocytes RPMI 8226 (purchased from Kitano Biosciences) and BCMA-negative expressing human glioma cell line U87-MG (purchased from Kitano Biosciences) were measured according to the above methods.

[0137] Also, take target cells and increase the cell density to 2 x 10 6 The concentration was adjusted to 100 μL / mL and added to a 96-well plate at a well density of 2 × 10 5 Appropriate amounts of effector cells dNMC003-A and dNMC003-B CAR-T cells were added to the target cells in each well at an effector cell:target cell (E:T) ratio of 3:1. After uniform mixing, the cells were co-incubated for 18 hours, and the supernatant was collected to detect IFN-γ secretion.

[0138] 5. Experimental results After transduction of the dual VHH construct into K562 cells, the MFI statistical results of BCMA antigen staining of K562-dNMC003-A and K562-dNMC003-B cells are shown in Figure 12. The results show that the expression intensity of VHH on the surface of K562-dNMC003-A and K562-dNMC003-B was higher than that of blank T cells, indicating that the nanobodies in the dual VHH CAR construct were successfully expressed in K562 cells.

[0139] Representative flow cytometry results after culturing CAR-T cells for 6 days are shown in Figure 13, where CAR(Erb) indicates the expression of tEGFR on the cell surface and CAR(BCMA) indicates the expression of nanobody on the cell surface. The results show that both the two types of dual VHH CAR-T cells, dNMC003-A and dNMC003-B, specifically expressed CAR(Erb) and CAR(BCMA).

[0140] During the CAR-T cell culture process in this example, the cell proliferation fold, CAR-T cell viability, and CD4 / CD8 ratio were statistically analyzed. Figure 14 shows the proliferation curves of NMC003-01, NMC003-02, dNMC003-A, and dNMC003-B CAR-Ts. From the figure, it can be seen that on day 13 of proliferation, the proliferation fold of NMC003-01 was 56.9-fold, that of NMC003-02 was 60.6-fold, that of dNMC003-A was 63.6-fold, that of dNMC003-B was 59.1-fold, and that of blank T cells was 68.5-fold. Figure 15 shows the statistical results of cell viability during the culture of NMC006-04, NMC006-06, and dNMC006-A CAR-T cells. The figure demonstrates that CAR-T cells maintained high cell viability throughout the entire expansion process. Figure 16 shows the CD4 / CD8 ratios during the cell culture process. The CD4 / CD8 ratios decreased during the culture. At the final harvest, the CD4 / CD8 ratios of NMC003-01, NMC003-02, dNMC003-A, and dNMC003-B were 0.24, 0.31, 0.26, and 0.23, respectively, which was comparable to the level of blank T cells (0.36). These results demonstrate that dual VHH CAR-T cells expanded normally, had high viability during the expansion process, and tended to have CD4 / CD8 ratios approaching normal levels compared to blank T cells.

[0141] The in vitro biological activity of dual VHH CAR-T cells was verified by killing experiments against K562, K562-BCMA, RPMI 8226, and U87-MG. Figure 17A shows the in vitro killing results of dual VHH CAR-T against K562 and K562-BCMA. The results indicate that all four CAR-Ts exhibited specific killing of K562-BCMA, and that the killing rates of dual VHH CAR-Ts were higher than those of single VHH CAR-Ts at an effector cell to target cell ratio of 3:1. Figure 17B shows the in vitro killing results of dual VHH CAR-T against RPMI 8226 and U87-MG. The results indicate that dual VHH CAR-Ts exhibited specific killing of the BCMA-positive cell line RPMI 8226, but no significant killing of the BCMA-negative cell line U87-MG.

[0142] Furthermore, the INF-γ concentration in the culture supernatant after co-incubation of dual VHH CAR-T cells with K562-BCMA and RPMI 8226 was much higher than that in the culture medium after incubation with U87-MG (see Figure 18). These results demonstrate that all dual VHH CAR-T cells exhibited high killing activity against BCMA-positive expressing cell lines, but not against BCMA-negative expressing cell lines, indicating that the killing of BCMA-positive cell lines by the two dual VHH CAR-T cells was specific.

[0143] Example 5 Identification of Antibody Characteristics 1. Expression of recombinant antibodies To further confirm the properties of the nanobodies Nb003-01 and Nb003-02 and the double nanobodies dNb003-A and dNb003-B screened in Example 1, the nanobodies were expressed and purified in vitro in this Example. First, the nanobodies Nb003-01, Nb003-02, dNb003-A, and dNb003-B were inserted between the BamH I and NHE I enzyme cleavage sites of the pET-28a-Sumo-Nb-Fc plasmid (stored in our laboratory) by molecular cloning. The nanobody sequence contained a SUMO tag at its N-terminus (SUMO-tagged proteins are small ubiquitin-like modified proteins. Research has shown that SUMO can be used as a fusion tag and molecular chaperone for recombinant protein expression, further improving the expression level of fusion proteins, as well as resisting protease hydrolysis, promoting correct folding of target proteins, and improving the solubility of recombinant proteins). The C-terminus of the nanobody sequence contained a human IgG1 Fc tag for protein purification. After accurate sequencing, the plasmid was extracted and then transformed into E. coli strain BL21 for protein expression under IPTG induction. After expression was complete, the cells were harvested and sonicated to obtain crude protein. High-purity nanobodies were obtained by purification using Protein A affinity chromatography. The purified nanobodies were named Nb003-01-Fc, Nb003-02-Fc, dNb003-A-Fc, and dNb003-B-Fc, respectively.

[0144] 2. Affinity detection (1) Detection of antibody affinity by ELISA In this example, the affinity of nanobodies Nb003-01-Fc, Nb003-02-Fc, dNb003-A-Fc, and dNb003-B-Fc for human BCMA was measured using an ELISA assay. BCMA-His protein prepared in Example 1 was coated overnight, and the expressed and purified nanobodies at different dilutions were used as primary antibodies, followed by anti-human IgG1 Fc-HRP antibody as secondary antibodies to detect the affinity of each antibody to its corresponding antigen.

[0145] (2) Detection of antibody affinity by SPR method The expressed and purified nanobodies Nb003-01-Fc, Nb003-02-Fc, and dNb003-B-Fc were used for SPR measurements. The affinity of nanobodies Nb003-01-Fc, Nb003-02-Fc, and dNb003-B-Fc for human BCMA was measured by SPR using a Biacore 8K analysis system.

[0146] The affinity of each nanobody to BCMA-His was detected as follows: First, BCMA-His was immobilized on an NTA chip using the capture method, and serially diluted Nb003-01-Fc, Nb003-02-Fc, and dNb003-B-Fc were injected as the flow phase at a flow rate of 30 μL / min. Dissociation was then performed, and the results were analyzed using the Biacore T200 Evaluation Software Kinetic 1:1 Binding mode.

[0147] 3. Specificity detection method The specificity of Nb003-01, Nb003-02, dNb003-A, and dNb003-B for BCMA was detected by flow cytometry.The specificity of Nb003-01-Fc, Nb003-02-Fc, dNb003-A-Fc, and dNb003-B-Fc for BCMA-positive cell lines was detected by flow cytometry. The purified biotinylated nanobodies Nb003-01-Fc, Nb003-02-Fc, dNb003-A-Fc, and dNb003-B-Fc were used as primary antibodies, respectively, and SA-PE antibody was used as secondary antibody. The binding status of each nanobody to the human BCMA-positive cell line K562-BCMA, RPMI 8226, and the BCMA-negative cell line K562 was detected by flow cytometry, thereby determining the specificity of each nanobody for the human BCMA antigen.

[0148] 4. Experimental results The results of antibody affinity detection by ELISA are shown in Figure 19. The results showed that the EC50 value of Nb003-01-Fc and human BCMA was 2.048nm, the EC50 value of Nb003-02-Fc and human BCMA was 0.6518nm, the EC50 value of dNb003-A-Fc and human BCMA was 0.02227nm, and the EC50 value of dNb003-B-Fc and human BCMA was 0.3458nm.

[0149] The results of antibody affinity detection by SPR are shown in Table 4 and Figure 20. The affinity constant of Nb003-01 with human BCMA was 6.20 × 10 -8 The affinity constant of Nb003-02 for human BCMA is 1.79 × 10 -9 The affinity constant of dNb003-B for human BCMA is 1.94 × 10 -9 It was.

[0150] The above results show that the two anti-BCMA nanobodies Nb003-01 and Nb003-02 obtained by screening and identification in the present invention have high affinity for human BCMA, and the dNb003-B double nanobody has high affinity for human BCMA, which tends to be higher than that of single nanobodies.

[0151] Flow cytometry results showed that Nb003-01-Fc, Nb003-02-Fc, dNb003-A-Fc, and dNb003-B-Fc did not specifically bind to K562, but all specifically bound to the BCMA-positive cell lines K562-BCMA and RPMI 8226 (see Figure 21), demonstrating that Nb003-01-Fc, Nb003-02-Fc, dNb003-A-Fc, and dNb003-B-Fc are BCMA-specific antibodies.

[0152] [Table 4]

[0153] The above description of the examples is only for understanding the method and gist of the present invention, and those skilled in the art can make various improvements and modifications to the present invention, and these improvements and modifications are also intended to fall within the scope of protection of the claims of the present invention.

Claims

1. In nanobodies targeting BCMA, Includes VHH01 or VHH02, The amino acid sequences of CDR1, CDR2, and CDR3 of VHH01 are shown as SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown by SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively. The above nanobody.

2. i) The nucleotide sequences of CDR1, CDR2, and CDR3 of VHH01 are shown as SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, respectively, for example, ia) The amino acid sequence of VHH01 is shown as SEQ ID NO: 1, or, for example, ib) The amino acid sequence of VHH01 is shown as SEQ ID NO: 1, and the nucleotide sequence of VHH01 is shown as SEQ ID NO: 2, or ii) The nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, for example, ii) The amino acid sequence of VHH02 is shown in SEQ ID NO: 9, or, for example, iib) The amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is shown in SEQ ID NO:

10. The nanobody according to claim 1.

3. A chimeric antigen receptor based on a single nanobody that targets BCMA, wherein the chimeric antigen receptor comprises the nanobody described in claim 1 or 2, for example, i) further comprising a transmembrane domain, for example, the transmembrane domain comprising the transmembrane domain of a molecule of CD8α, CD28, 4-1BB, CD34, CD3ε, PD-1, IgG1, IgG4, OX40, IL-2 receptor, IL-7 receptor, or IL-11 receptor, for example, the transmembrane domain being a CD8α transmembrane domain, or for example, ii) Further comprising a transmembrane domain, further comprising an intracellular signaling domain, for example, the intracellular signaling domain comprising an intracellular signaling domain of the molecules CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, or CD66d, for example, the intracellular signaling domain being the CD3ζ intracellular signaling domain, or for example, iii) further comprising a transmembrane domain, further comprising an intracellular signaling domain, and further comprising a hinge region, for example, the hinge region comprising a hinge region of a molecule of CD8α, CD28, CD34, 4-1BB, OX40, CD3ε, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, or IL-11 receptor, for example, the hinge region being a CD8α hinge region, or for example, iv) further comprising a transmembrane domain, further comprising an intracellular signaling domain, and further comprising a signal peptide, for example, the signal peptide comprising the α and β chains of the T cell receptor, CD3ζ, CD3ε, CD16, CD22, CD33, CD4, CD5, CD8, CD9, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, or a GM-CSF molecule, or for example, v) Further comprising a transmembrane domain, further comprising an intracellular signaling domain, further comprising a signal peptide, and further comprising a co-stimulatory signaling domain, for example, the co-stimulatory signaling domain comprising a co-stimulatory signaling domain of a molecule of 4-1BB (CD137), CD19, CD4, CD27, CD28, ICOS (CD278), CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, or CD226, for example, the co-stimulatory signaling domain is the 4-1BB co-stimulatory signaling domain, or, for example, vi) further comprising a transmembrane domain, further comprising an intracellular signaling domain, further comprising a signal peptide, further comprising a co-stimulatory signaling domain, and further comprising a promoter, for example, the promoter comprising an EF1α promoter, a CMV promoter, an EFS promoter, a CAG promoter, a CBh promoter, a SFFV promoter, an MSCV promoter, an SV40 promoter, an mPGK promoter, an hPGK promoter, or a UBC promoter, for example, the promoter being EF1α, or for example, vii) further comprising a transmembrane domain, further comprising an intracellular signaling domain, further comprising a signal peptide, further comprising a co-stimulatory signaling domain, further comprising a promoter, further comprising a self-cleaving peptide, for example, the self-cleaving peptide comprising T2A, P2A, E2A, or F2A, for example, the self-cleaving peptide being T2A, or for example, viiii) further comprising a transmembrane domain, further comprising an intracellular signaling domain, further comprising a signal peptide, further comprising a co-stimulatory signaling domain, further comprising a promoter, further comprising a self-cleaving peptide, and further comprising a detection tag / auxiliary functional element, for example, the detection tag / auxiliary functional element comprising tEGFR, tCD34, tCD19, tCD20, tCD22, an immune checkpoint inhibitor (CTLA-4, PD-1 / PD-L1, LAG-3, TIM-3, TIGIT, CD226, CD155, CD47, B7-H3, or B7-H4), a nanobody, a cytokine or its receptor (IL2, IL2 receptor, IL7, IL7 receptor, IL15, or IL15 receptor), for example, the detection tag / auxiliary functional element is tEGFR, and optionally the chimeric antigen receptor is EF1α, a signal peptide, and the nanobody according to claim 1 or 2 The chimeric antigen receptor is obtained by sequentially linking a nobody, CD8α hinge region, CD8α transmembrane domain, 4-1BB costimulatory signaling domain, CD3ζ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR in series, for example, a) the chimeric antigen receptor is obtained by sequentially linking EF1α, signal peptide, VHH01, CD8α hinge region, CD8α transmembrane domain, 4-1BB costimulatory signaling domain, CD3ζ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR in series, or for example, b) the chimeric antigen receptor is obtained by sequentially linking EF1α, signal peptide, VHH02, CD8α hinge region, CD8α transmembrane domain, 4-1BB costimulatory signaling domain, CD3ζ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR in series, or for example, ix) further comprising a transmembrane domain, further comprising an intracellular signaling domain, further comprising a signal peptide, further comprising a co-stimulatory signaling domain, further comprising a promoter, further comprising a self-cleaving peptide, further comprising a detection tag / auxiliary functional element, further comprising a tEGFR signal peptide, The above-mentioned chimeric antigen receptor.

4. A chimeric antigen receptor based on a dual nanobody targeting BCMA, wherein the chimeric antigen receptor comprises one of the nanobodies described in claim 1 or 2 and one other nanobody targeting BCMA, for example, i) Any other nanobody targeting the BCMA is VHH02, or, for example, ii) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are indicated by SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, or, for example, iii) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are indicated by SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, and the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are indicated by SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, or, for example, iv) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are indicated by SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are indicated by SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is indicated by SEQ ID NO: 9, or, for example, v) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are indicated by SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are indicated by SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is indicated by SEQ ID NO: 9, the nucleotide sequence of VHH02 is indicated by SEQ ID NO: 10, or, for example, vi) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, the nucleotide sequence of VHH02 is shown in SEQ ID NO: 10, and the chimeric antigen receptor further comprises a transmembrane domain, or, for example, vii) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, the nucleotide sequence of VHH02 is shown in SEQ ID NO: 10, and the chimeric antigen receptor further comprises a transmembrane domain, further comprises an intracellular signaling domain, or, for example, viiii) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, the nucleotide sequence of VHH02 is shown in SEQ ID NO: 10, and the chimeric antigen receptor further comprises a transmembrane domain, further comprises an intracellular signaling domain, further comprises a hinge region, or, for example, ix) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, the nucleotide sequence of VHH02 is shown in SEQ ID NO: 10, and the chimeric antigen receptor further comprises a transmembrane domain, further comprises an intracellular signaling domain, further comprises a hinge region, further comprises a signal peptide, or, for example, x) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, the nucleotide sequence of VHH02 is shown in SEQ ID NO: 10, and the chimeric antigen receptor further comprises a transmembrane domain, further comprises an intracellular signaling domain, further comprises a hinge region, further comprises a signal peptide, further comprises a co-stimulatory signaling domain, or, for example, xi) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, the nucleotide sequence of VHH02 is shown in SEQ ID NO: 10, and the chimeric antigen receptor further comprises a transmembrane domain, further comprises an intracellular signaling domain, further comprises a hinge region, further comprises a signal peptide, further comprises a co-stimulatory signaling domain, further comprises a promoter, or, for example, xi) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated by NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, a signal peptide, a co-stimulatory signaling domain, a promoter, a self-cleaving peptide, or, for example, xiiii) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated by NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, a signal peptide, a co-stimulatory signaling domain, a promoter, a self-cleaving peptide, a detection tag / auxiliary functional element, or, for example, xiv) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated by NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, a signal peptide, a co-stimulatory signaling domain, a promoter, a self-cleaving peptide, a detection tag / auxiliary functional element, a tEGFR signal peptide, or, for example, xv) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated as NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, which comprises the transmembrane domain of a molecule of CD8α, CD28, 4-1BB, CD34, CD3ε, PD-1, IgG1, IgG4, OX40, IL-2 receptor, IL-7 receptor, or IL-11 receptor, for example, xva) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, the nucleotide sequence of VHH02 is shown in SEQ ID NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, the transmembrane domain is a CD8α transmembrane domain, or, for example, xvi) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated as NO: 10, the chimeric antigen receptor further comprises a transmembrane domain and an intracellular signaling domain, for example, the intracellular signaling domain comprises the intracellular signaling domains of the molecules CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, or CD66d, for example, xvia) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, the nucleotide sequence of VHH02 is shown in SEQ ID NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, further comprises an intracellular signaling domain, the intracellular signaling domain is a CD3ζ intracellular signaling domain, or, for example, xvii) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated as NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, and a hinge region, for example, the hinge region comprising the hinge region of a molecule of CD8α, CD28, CD34, 4-1BB, OX40, CD3ε, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, or IL-11 receptor, for example, xvia) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, the nucleotide sequence of VHH02 is shown in SEQ ID NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, further comprises an intracellular signaling domain, further comprises a hinge region, the hinge region is a CD8α hinge region, or, for example, xviiii) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated by NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, and a signal peptide, for example, the signal peptide comprises the signal peptides of the α and β chains of the T cell receptor, CD3ζ, CD3ε, CD16, CD22, CD33, CD4, CD5, CD8, CD9, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, or GM-CSF molecules, or, for example, xix) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated by NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, a signal peptide, and a co-stimulatory signaling domain, for example, the co-stimulatory signaling domain comprises the co-stimulatory signaling domain of the molecules 4-1BB (CD137), CD19, CD4, CD27, CD28, ICOS (CD278), CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, or CD226, for example, xixa) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated as NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, a signal peptide, and a costimulatory signaling domain, wherein the costimulatory signaling domain is a 4-1BB (CD137) costimulatory signaling domain, or, for example, xx) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated by NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, a signal peptide, a co-stimulatory signaling domain, and a promoter, for example, the promoter comprising an EF1α promoter, a CMV promoter, an EFS promoter, a CAG promoter, a CBh promoter, a SFFV promoter, an MSCV promoter, an SV40 promoter, an mPGK promoter, an hPGK promoter, or a UBC promoter, for example, xxa) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated as NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, a signal peptide, a costimulatory signaling domain, and a promoter which is EF1α, or, for example, xxi) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated by NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, a signal peptide, a co-stimulatory signaling domain, a promoter, and a self-cleaving peptide, for example, the self-cleaving peptide comprising T2A, P2A, E2A, or F2A, for example, xxia) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated as NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, a signal peptide, a co-stimulatory signaling domain, a promoter, and a self-cleaving peptide, wherein the self-cleaving peptide is T2A, or, for example, xxii) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated as NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, a signal peptide, a co-stimulatory signaling domain, a promoter, a self-cleaving peptide, and a detection tag / auxiliary functional element, for example, the detection tag / auxiliary functional element comprises tEGFR, tCD34, tCD19, tCD20, tCD22, an immune checkpoint inhibitor (CTLA-4, PD-1 / PD-L1, LAG-3, TIM-3, TIGIT, CD226, CD155, CD47, B7-H3, or B7-H4) nanobody, a cytokine or its receptor (IL2, IL2 receptor, IL7, IL7 receptor, IL15, or IL15 receptor), for example, xxiiia) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated as NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, a signal peptide, a co-stimulatory signaling domain, a promoter, a self-cleaving peptide, and a detection tag / auxiliary functional element, wherein the detection tag / auxiliary functional element is tEGFR, or, for example, xxiii) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated as NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, a signal peptide, a co-stimulatory signaling domain, a promoter, a self-cleaving peptide, and a detection tag / auxiliary functional element, wherein the detection tag / auxiliary functional element is tEGFR, and the chimeric antigen receptor comprises EF1α, a signal peptide, any one of the nanobodies described in claim 1, a linker, any one other nanobodies targeting BCMA, and CD The chimeric antigen receptor is obtained by sequentially linking the 8α hinge region, CD8α transmembrane domain, 4-1BB costimulatory signaling domain, CD3ζ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR in series, or, for example, the chimeric antigen receptor is obtained by sequentially linking EF1α, signal peptide, VHH01, linker, VHH02, CD8α hinge region, CD8α transmembrane domain, 4-1BB costimulatory signaling domain, CD3ζ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR in series, or, for example, xxiv) Any other nanobody targeting the BCMA is VHH02, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively, the amino acid sequence of VHH02 is shown in SEQ ID NO: 9, and the nucleotide sequence of VHH02 is SEQ ID Indicated as NO: 10, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, a signal peptide, a co-stimulatory signaling domain, a promoter, a self-cleaving peptide, and a detection tag / auxiliary functional element, wherein the detection tag / auxiliary functional element is tEGFR, and the chimeric antigen receptor comprises EF1α, a signal peptide, any one of the nanobodies described in claim 1 or 2, a linker, any one other nanobodies targeting BCMA, and CD8α-hinge The chimeric antigen receptor is obtained by sequentially linking the following in series: the EF1α, signal peptide, VHH02, linker, VHH02, CD8α hinge region, CD8α transmembrane domain, 4-1BB costimulatory signaling domain, CD3ζ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR. For example, the chimeric antigen receptor is obtained by sequentially linking the following in series: EF1α, signal peptide, VHH02, linker, VHH02, CD8α hinge region, CD8α transmembrane domain, 4-1BB costimulatory signaling domain, CD3ζ intracellular signaling domain, T2A, tEGFR signal peptide, and tEGFR. The linker may be (G4S). ５ That is, The above-mentioned chimeric antigen receptor.

5. In an isolated nucleic acid molecule, the nucleic acid molecule comprises a nucleotide sequence encoding a nanobody according to claim 1 or 2 or a chimeric antigen receptor according to claim 3 or 4, optionally a) The nucleotide sequence of VHH01 in the nanobody according to claim 1 or 2 is indicated by SEQ ID NO: 2, or b) The nucleotide sequence of VHH02 in the nanobody according to claim 1 or 2 is indicated by SEQ ID NO: 10, or c) The nucleotide sequence of EF1α in the chimeric antigen receptor according to claim 3 or 4 is indicated by SEQ ID NO: 17, or d) The nucleotide sequence of the signal peptide in the chimeric antigen receptor according to claim 3 or 4 is indicated by SEQ ID NO: 26, or e) The nucleotide sequence of the CD8α hinge region in the chimeric antigen receptor according to claim 3 or 4 is shown as SEQ ID NO: 27, or f) The nucleotide sequence of the CD8α transmembrane domain in the chimeric antigen receptor according to claim 3 or 4 is indicated by SEQ ID NO: 28, or g) The nucleotide sequence of the 4-1BB costimulatory signaling domain in the chimeric antigen receptor according to claim 3 or 4 is indicated by SEQ ID NO: 29, or h) The nucleotide sequence of the CD3ζ intracellular signaling domain in the chimeric antigen receptor according to claim 3 or 4 is indicated by SEQ ID NO: 30, or i) The nucleotide sequence of T2A in the chimeric antigen receptor according to claim 3 or 4 is indicated by SEQ ID NO: 31, or j) The nucleotide sequence of the tEGFR signal peptide in the chimeric antigen receptor according to claim 3 or 4 is indicated by SEQ ID NO: 32, or k) The nucleotide sequence of the tEGFR in the chimeric antigen receptor according to claim 3 or 4 is indicated by SEQ ID NO:

33. The nucleic acid molecule mentioned above.

6. An expression vector comprising the nucleic acid molecule described in claim 5, Depending on the circumstances, the vector may include a DNA vector, an RNA vector, a plasmid, or a virus-derived vector, and depending on the circumstances, the virus-derived vector may include a lentiviral vector, a retroviral vector, an adenovirus vector, an adeno-associated virus vector, a poxvirus vector, or a herpesvirus vector. The expression vector described above.

7. An engineered modified host cell comprising the expression vector described in claim 6, Depending on the case, the host cell may be a eukaryotic or prokaryotic cell, for example, the host cell may be an immune cell, for example, the immune cell may include T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, medullary cells, mononuclear cells, macrophages, or any combination thereof, for example, the immune cell may be a T cell. The above-mentioned host cells.

8. An immunoconjugate comprising a nanobody according to claim 1 or 2 and a conjugate bound thereto, wherein the conjugate optionally comprises a detectable marker, a radionuclide, a cytokine, a therapeutic agent, a cytotoxin, an enzyme, gold nanoparticles / nanorods, nanomagnetic particles, a viral capsid protein or VLP, or a combination thereof, for example, a) The detectable marker includes a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or, for example, b) The radioactive nuclide is, １３１ I, ３２ P, ８９ Sr, ９０ Y, ２２３ Ra, １２５ I, １０３ Including Pd, or for example, c) The cytokines include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN-γ, TNF-β, TNF-α, G-CSF, or M-CSF, or, for example, d) The therapeutic agent comprises an alkylating agent, an antimetabolite, an antitumor antibiotic, a mitotic inhibitor, a chromatin function inhibitor, an anti-angiogenic agent, an anti-estrogen, an anti-androgen, or an immunomodulator, for example, d1) The alkylating agent includes bischloroethylmethylamine, chlorambucil, melphalan, pipobromane, prednimustine, estracyte, cyclophosphamide, altretamine, ifosfamide, fotemustine, thiotepa, carmustine, streptozotocin, improsulfan, dacarbazine, cisplatin, oxaliplatin, or carboplatin, or, for example, d2) The antimetabolite includes methotrexate, 5-fluorouracil, phloxuridine, 5-fluorodeoxyuracil, capecitabine, cytarabine, fludarabine, 6-mercaptopurine, 2-chlorodeoxyadenosine, 5-azacitidine, 2,2-difluorodeoxycytidine, cladribine, deoxycoformycin, or pentostatin, or, for example, d3) The antitumor antibiotic includes daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin C, barurubicin, mitoxantrone hydrochloride, bleomycin, dactinomycin, mithramycin, or procarbazine, or, for example, d4) The mitotic inhibitor comprises docetaxel, vinblastine, paclitaxel, vincristine, vindesine, or vinorelbine, or, for example, d5) The chromatin function inhibitor comprises irinotecan, etoposide, topotecan, etoposide phosphate, or teniposide, for example, d6) The anti-angiogenic agent includes prinomast, tanomast, ilomast, razoxane, marimast, batimast, CGS-27023A, halofdinone, COL-3, neovastat, BMS-275291, or thalidomide, or, for example, d7) The anti-estrogen agent includes toremifene, raloxifene, tamoxifen, anastrozole, letrozole, droloxifene, iodoxifene, or exemestane, or, for example, d8) The antiandrogen agent comprises nilutamide, bicalutamide, spironolactone, flutamide, finasteride, cyproterone acetate, or cimetidine, or, for example, d9) The immunomodulatory agent comprises interleukin, tumor necrosis factor, interferon, lentinan, schizophyllan, lokinimex, pyrotimod, methoxypolyethylene glycol succinamide adenosine deaminase, or thymosine preparations, or, for example, e) The cytotoxin comprises MMAE, DM1, Ozogamicin, Dxd, SN-38, MMAF, PBD, DM2, Amanitin, DM4, PNU-159682, IR700, PE-38, PE24, PE-T20, PE-T20-KDEL, PE4E, or PE40. The above immunoconjugate.

9. A reagent for detecting BCMA proteins comprising a nanobody according to claim 1 or 2 or an immunoconjugate according to claim 8, further comprising, for example, a diagnostic agent that binds to the nanobody or immunoconjugate, optionally comprising a radionuclide, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, or a photosensitive diagnostic agent, for example, a) The radionuclide is １８ F, ５２ Fe, ６２ Cu, ６４ Cu, ６７ Cu, ８６ Y, ９０ Y, ８９ Zr, １２０ I, １２３ I, １２４ I, １２５ I, １３１ I, １３ N, １５ O, １８６ Re, １８８ Re, ５１ Mn, ５５ Co, or ７２ As, or, for example, b) The chemiluminescent agent comprises luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt, or oxalate, or, for example, c) The bioluminescent agent comprises fluorescein, luciferase, or aequorin, or, for example, d) The paramagnetic ions include chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), or holmium(III), erbium(III), or, for example, e) The enzyme includes horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase, or, for example, f) The photosensitive diagnostic agent comprises silicon dihydroxylphthalocyanine, methylene blue, protoporphyrin, hematoporphyrin, or photofrin. The above reagents.

10. A drug composition comprising a nanobody according to claim 1 or 2, a chimeric antigen receptor according to claim 3 or 4, a nucleic acid molecule according to claim 5, an expression vector according to claim 6, an engineered modified host cell according to claim 7, and / or an immunoconjugate according to claim 8, the drug composition optionally further comprising one or more pharmaceutically or physiologically acceptable vectors and / or auxiliary materials.

11. A kit comprising a nanobody according to claim 1 or 2, a chimeric antigen receptor according to claim 3 or 4, a nucleic acid molecule according to claim 5, an expression vector according to claim 6, an engineered modified host cell according to claim 7, an immunoconjugate according to claim 8, and / or a reagent according to claim 9, the kit optionally used to detect a BCMA protein.

12. A method for generating nanobodies according to claim 1 or 2, (1) A step of culturing the engineered host cells described in claim 7 under conditions suitable for the generation of nanobodies to obtain a culture containing nanobodies, (2) A step of isolating or recovering nanobodies from the culture obtained in step (1), (3) A step of purifying the nanobody obtained in step (2) to obtain the nanobody described in claim 1 or 2, The above method, including.

13. A method for preparing an engineered host cell according to claim 7, comprising the step of introducing a nucleic acid molecule according to claim 5 or an expression vector according to claim 6 into a host cell, Depending on the case, the methods of introduction include lipofection, microinjection, electroporation, DNA vectors, RNA vectors, retroviral vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenovirus vectors, and adeno-associated virus vectors. In some cases, the host cell is a eukaryotic or prokaryotic cell, for example, an immune cell, and in some cases, the immune cell includes T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, medullary cells, mononuclear cells, macrophages, or any combination thereof, and in some cases, the immune cell is a T cell. The above method.

14. A method for providing an indicator to diagnose whether or not a subject has a BCMA-positive related disease, The procedure includes the step of contacting a test sample derived from a subject with a nanobody according to claim 1 or 2, a chimeric antigen receptor according to claim 3 or 4, a nucleic acid molecule according to claim 5, an expression vector according to claim 6, an engineered modified host cell according to claim 7, an immunoconjugate according to claim 8, and / or a reagent according to claim 9, and detecting the formation of a complex between the nanobody, immunoconjugate, and / or reagent and BCMA, or detecting the amount of said complex. The above method indicates that the formation of a complex is due to the presence of BCMA or cells expressing BCMA, and that the subject suffers from a BCMA-positive related disease.

15. In methods for treating BCMA-positive related diseases, A composition for use in the treatment, prevention, and / or diagnosis of BCMA-positive related diseases, comprising a nanobody according to claim 1 or 2, a chimeric antigen receptor according to claim 3 or 4, a nucleic acid molecule according to claim 5, an expression vector according to claim 6, an engineered modified host cell according to claim 7, an immunoconjugate according to claim 8, and / or a reagent according to claim 9, wherein the BCMA-positive related disease is multiple myeloma, acute myeloid leukemia The above composition comprises a disease, chronic myeloid leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, gastric cancer, liver cancer, kidney cancer, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer, glioma, endometrial cancer, fibrosarcoma, myxoid sarcoma, liposarcoma, mesothelioma, malignant lymphoma, or pancreatic cancer.

16. The use of the nanobody according to claim 1 or 2, the chimeric antigen receptor according to claim 3 or 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the engineered modified host cell according to claim 7, the immunoconjugate according to claim 8, the reagent according to claim 9, and / or the drug composition according to claim 10 in the preparation of a drug for treating, preventing, and / or diagnosing BCMA-positive related disease, wherein the BCMA-positive related disease is a multiple bone disease The above uses include myeloma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, diffuse large B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, gastric cancer, liver cancer, kidney cancer, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer, glioma, endometrial cancer, fibrosarcoma, myxoid sarcoma, liposarcoma, mesothelioma, malignant lymphoma, or pancreatic cancer.

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