BCMA nano antibody and application thereof

Abstract

The invention belongs to the technical field of nano antibodies, and particularly relates to a BCMA nano antibody and application thereof. The preparation method comprises the following steps: immunizing alpaca by using BCMA recombinant protein, separating peripheral blood lymphocytes, constructing a gene library, and screening an anti-BCMA nano antibody from the library. The nano antibody provided by the invention has unique complementary determining regions 1, 2 and 3, and the antibody has high specific binding capacity to a BCMA antigen. According to the human FC fusion antibody constructed by the nano antibody, the nano antibody has ideal capability of combining multiple myeloma (MM) cells and inducing T cells to kill the MM cells. The compound can be applied independently or in combination with other MM drugs, and is used for preparing MM therapeutic drugs. According to the technical scheme, the technical problems that in the prior art, a nano antibody aiming at BCMA is lacked and the variety of drugs for treating multiple myeloma is limited can be solved, and the nano antibody has ideal application and popularization prospects.

Description

Technical Field

[0001] The present invention belongs to the field of nanoantibody technology, and specifically relates to a BCMA nanoantibody and its application. Background Art

[0002] Multiple myeloma (MM) is a cancer of plasma cells, accounting for approximately 10% of hematologic malignancies. Plasma cells reside in the bone marrow and are an important component of the immune system. MM develops when these cells undergo malignant proliferation within the bone marrow. MM can damage the bones and immune system, with clinical manifestations often including bone pain, osteoporosis, kidney disease, lung inflammation, and hypercalcemia. In recent years, significant progress has been made in targeted therapies for MM, including immunomodulators, proteasome inhibitors, monoclonal antibodies, bispecific antibodies, and chimeric antigen receptor (CAR) cells (CAR-T). Antibodies targeting CD38, SLAMF7, and BCMA, or bispecific antibodies targeting BCMA / CD3, have demonstrated long-lasting efficacy in MM. Currently, MM remains incurable, and most patients eventually relapse after treatment, developing drug resistance and developing refractory multiple myeloma (RR-MM). Achieving effective treatment for RR-MM remains a pressing challenge in MM research.

[0003] B-cell maturation antigen (BCMA), also known as CD269, is a type I transmembrane protein and a member of the tumor necrosis factor receptor (TNFR) superfamily. B-cell activating factor receptors include the TNFR superfamily's BAFF receptor (BAFFR), B-cell maturation antigen (BCMA), and transmembrane activator and calcineurin ligand interactor (TACI). BCMA is selectively expressed on B-lineage cells, including MM cells, and plays a crucial role in various stages of B-cell development. Because it is selectively expressed during plasma cell differentiation, rarely expressed on naive and memory B cells, and lacks expression in other major organs, BCMA is an ideal target for plasma cell cancers.

[0004] BCMA was first discovered in the early 1990s. This 184-amino acid glycoprotein plays a major role in B cell maturation and differentiation into plasma cells. BCMA is composed of three major domains: an extracellular segment (amino acids 1-54), a transmembrane region (amino acids 55-77), and an intracellular segment (amino acids 78-184). In normal human tissues, BCMA protein and mRNA are found almost exclusively on plasma cells and are selectively overexpressed during malignant transformation of plasma cells, promoting tumor cell growth, survival, and the development of drug resistance by activating intracellular signaling cascades involving NFκB, AKT, phosphatidylinositol 3-kinase (PI3K), STAT3, and MAPK. Studies have shown that measuring membrane-bound BCMA can serve not only as a diagnostic and prognostic biomarker for multiple myeloma (MM) but also as a potential predictor of treatment response. Studies have also shown that BCMA expression levels are similar across different stages of multiple myeloma (from untreated to relapsed), suggesting that BCMA may be a therapeutic target throughout the disease course.

[0005] Belantamab Mafodotin is an antibody-drug conjugate (ADC) of a BCMA-targeting antibody and a microtubule inhibitor. In August 2020, the U.S. Food and Drug Administration (FDA) first approved Belantamab Mafodotin for the treatment of relapsed or refractory multiple myeloma in adults. These patients have received at least four prior therapies, including CD38 monoclonal antibodies, proteasome inhibitors, and immunomodulators. Studies have shown that the overall response rate (ORR) of Belantamab Mafodotin 2.5 mg / kg monotherapy was 32%, the median duration of response (DoR) was 11 months, and the median overall survival (OS) was 13.7 months. The main adverse reactions of Belantamab Mafodotin were thrombocytopenia (38%), anemia (27%), infusion-related reactions (21%), and lymphocytopenia (20%). However, the above-mentioned monoclonal antibodies have problems such as limited affinity for antigens, large molecular weight, and limited tissue penetration. In addition, the tumor resistance problem they produce is difficult to overcome, and there is an urgent need to develop new alternative drugs with better performance.

[0006] Nanobodies are artificially designed antibody molecules, also known as single-domain antibodies or VHH antibodies. They originate from herbivorous mammals (such as camels and alpacas). Nanobodies typically have a higher affinity for antigens and are smaller in size, making them more susceptible to binding to difficult epitopes. Furthermore, their small molecular weight, typically around 15 kDa, is one-tenth that of conventional antibodies, enabling them to penetrate tissues with remarkable strength. Because nanobodies naturally lack the Fc segment and their VH (heavy chain) gene sequences are highly homologous to those of humans, nanobodies exhibit very low immunogenicity in humans. The proportion of hydrophilic residues in the framework region (FR) of nanobodies is higher than that of conventional IgG, resulting in improved water solubility and preventing aggregation and adhesion of antibody drugs during transport. Nanobodies have been widely used in medical diagnosis and treatment, biological research, and industry. They can be used to prepare highly sensitive diagnostic reagents, high-affinity separation and purification materials, disease treatment, and targeted drug delivery. However, the existing technology still lacks nanoantibodies targeting B cell maturation antigen (BCMA), and there is an urgent need to research and develop corresponding nanoantibodies to meet the needs of clinical practice in treating relapsed or refractory multiple myeloma. Summary of the Invention

[0007] The present invention aims to provide a BCMA nanobody to solve the technical problems in the prior art of the lack of nanoantibodies targeting B cell maturation antigen (BCMA) and the limited types of drugs for treating multiple myeloma.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions: The amino acid sequence of the complementary determining region 1 of the nanobody against B cell maturation antigen is: (1-1): any one of the amino acid sequences of FSFTEGEIGEFR, FTFRDHEEGQFR, FSFTTESMLNFR, RSESAQSLSEFR, FSESLESLSFFR, NILSARTRGWYR, FSFSASMSRFR, FSFSFFAGRSWFR, and FSFRSASMGRFR; Or (1-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences in (1-1), and having the same function as any of the amino acid sequences in (1-1); or (1-3): an amino acid sequence having greater than 80% identity with any of the amino acid sequences in (1-1); The amino acid sequence of its complementarity determining region 2 is: (2-1): any one of the amino acid sequences of GETNYQGSVK, GTTNEALSVK, GDVSNYADQVK, GDESNVQGSQK, GGVEKYARQVK, TGSRSQADSVK, GGNVSNYADSVK, GGEVSNYRGSVK, and GGNVSNYADSVK; Or (2-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences in (2-1), and having the same function as any of the amino acid sequences in (2-1); or (2-3): an amino acid sequence that is more than 80% identical to any of the amino acid sequences in (2-1); The amino acid sequence of its complementarity determining region 3 is: (3-1): any one of the amino acid sequences LSAFETSRLRWGQ, LSAQCTTRLYENQ, CQGGISQRLERDQ, CQGRLSGREVRGQ, CTGERSGRLVRGQ, YFSFSYEFDWGQ, QGALNGRLARGQ, QGFENGFLERGQ, and QGELQGVLAEGQ; Or (3-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences in (3-1), and having the same function as any of the amino acid sequences in (3-1); Or (3-3): an amino acid sequence having 80% or more identity with any of the amino acid sequences in (3-1).

[0009] Furthermore, the amino acid sequence of the framework region 1 of the nanobody against B cell maturation antigen is: (4-1): any one of the amino acid sequences DVQLQESGGGLVQPGGSLRLTCVASG, DVQLQESGGGLVQPGGSLRLSCAASG, and DVQLQESGGGLVQAGGSLRLSCTVSG; Or (4-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences in (4-1), and having the same function as any of the amino acid sequences in (4-1); or (4-3): an amino acid sequence having greater than 80% identity with any of the amino acid sequences in (4-1); The amino acid sequence of its framework region 2 is: (5-1): any one of the amino acid sequences QAPGKEREGVSCIGRFG, HFPGKGIEWVSGISAG, QGPGKQRELVARLTP, and HFPGKGLEWVSGISA; Or (5-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences in (5-1), and having the same function as any of the amino acid sequences in (5-1); or (5-3): an amino acid sequence that is more than 80% identical to any of the amino acid sequences in (5-1); The amino acid sequence of its framework region 3 is: (6-1): Any one of the amino acid sequences of GRFAISRDNAKNTVYLQMNSLKPEDTAVYYCAAQYSLRA, GRFFVSRDNAKSTLYLQMNNLTPEDTALYF, GRFTISRDNAKNMVYLQMNSLKPEDTAVYYCNAAG, and GRFTVSRDNAKSTLYLQMNSLTPEDTALYFC; Or (6-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences in (6-1), and having the same function as any of the amino acid sequences in (6-1); or (6-3): an amino acid sequence having greater than 80% identity with any of the amino acid sequences in (6-1); The amino acid sequence of its framework region 4 is: (7-1):GTQVTVSS; Or (7-2): an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids of GTQVTVSS, and having the same function as GTQVTVSS; Or (7-3): an amino acid sequence that is more than 80% identical to GTQVTVSS.

[0010] Furthermore, the amino acid sequence of the nanobody against B cell maturation antigen is: (8-1): any one of the amino acid sequences of SEQ ID NO.1 to SEQ ID NO.9; Or (8-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences in (8-1), and having the same function as any of the amino acid sequences in (8-1); or (8-3): an amino acid sequence having 80% or greater identity with any of the amino acid sequences in (8-1).

[0011] Furthermore, the amino acid sequence of the nanobody against B cell maturation antigen is: (9-1): any one of the amino acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.9; Or (9-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences in (9-1), and having the same function as any of the amino acid sequences in (9-1); or (9-3): an amino acid sequence having 80% or more identity with any one of the amino acid sequences in (9-1).

[0012] Furthermore, the amino acid sequence of the nanobody against B cell maturation antigen is: (10-1): SEQ ID NO. 3; Or (10-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO.3, and having the same function as SEQ ID NO.3; Or (10-3): an amino acid sequence having 80% or greater identity with SEQ ID NO. 3.

[0013] The present technical solution also provides a dimer or multimer or humanized nanobody or fusion-expressed nanobody or conjugate formed based on nanobodies against B cell maturation antigens.

[0014] Furthermore, the conjugate is a nanobody containing a chemical label or a biological label, an anti-B cell maturation antigen antibody dimer, an anti-B cell maturation antigen antibody multimer, a humanized nanobody or a fusion-expressed nanobody.

[0015] The chemical label of the present invention is an isotope, immunotoxin, and / or chemical drug; the biological label is biotin, avidin, or an enzyme label. The enzyme label is preferably horseradish peroxidase (HRP) or alkaline phosphatase. The immunotoxin is preferably a mycotoxin, diphtheria toxin, Pseudomonas aeruginosa exotoxin, ricin, abrin, mistletoe lectin, modeccin, PAP, herbivorin, gelonin, or luffa toxin. The conjugated drug includes, but is not limited to, paclitaxel, cisplatin, gemcitabine, and the like.

[0016] Furthermore, anti-B cell maturation antigen antibody dimers or anti-B cell maturation antigen antibody multimers or humanized nanobodies or fusion-expressed nanobodies or conjugates are used to couple with solid or semisolid media.

[0017] The solid medium or non-solid medium is selected from colloidal gold, polystyrene plates or magnetic beads.

[0018] The present technical solution also provides a gene encoding an anti-B cell mature antigen antibody dimer or an anti-B cell mature antigen antibody multimer or a humanized nanobody or a fusion-expressed nanobody.

[0019] This technical solution also provides a gene encoding a nanobody against B cell maturation antigen.

[0020] The present technical solution also provides a protein expression vector integrated with a gene encoding an anti-B cell mature antigen antibody dimer or an anti-B cell mature antigen antibody multimer or a humanized nanobody or a fusion-expressed nanobody.

[0021] This technical solution also provides a protein expression vector integrated with the gene of the nano-antibody against B cell maturation antigen.

[0022] This technical solution also provides a host cell or cell line expressing nanobodies against B cell maturation antigens.

[0023] The present technical solution also provides a host cell or cell line expressing an anti-B cell mature antigen antibody dimer or an anti-B cell mature antigen antibody multimer or a humanized nanobody or a fusion-expressing nanobody.

[0024] This technical solution also provides the use of nanobodies against B cell maturation antigens in the preparation of diagnostic reagents and / or kits for B cell maturation antigen-related diseases.

[0025] This technical solution also provides the use of nanobodies against B cell maturation antigens in the preparation of drugs for preventing and / or treating B cell maturation antigen-related diseases.

[0026] This technical solution also provides the use of nanobodies against B cell maturation antigens in the preparation of drugs for preventing and / or treating diseases related to multiple myeloma.

[0027] The present technical solution also provides the use of anti-B cell maturation antigen antibody dimers or anti-B cell maturation antigen antibody multimers or humanized nanobodies or fusion-expressed nanobodies or conjugates in the preparation of B cell maturation antigen-related disease diagnostic reagents and / or kits.

[0028] The present technical solution also provides the use of anti-B cell maturation antigen antibody dimers or anti-B cell maturation antigen antibody multimers or humanized nanobodies or fusion-expressed nanobodies or conjugates in the preparation of drugs for preventing and / or treating B cell maturation antigen-related diseases.

[0029] The present technical solution also provides the use of anti-B cell mature antigen antibody dimers or anti-B cell mature antigen antibody multimers or humanized nanobodies or fusion-expressed nanobodies or conjugates in the preparation of drugs for preventing and / or treating multiple myeloma-related diseases.

[0030] The principle of this technical solution is: The present invention relates to a novel BCMA (B cell maturation antigen) nanobody and its applications, belonging to the field of nanoantibody technology. BCMA is a type I transmembrane protein and a member of the TNFR superfamily. It is specifically and highly expressed in multiple myeloma (MM) cells and is involved in promoting tumor cell growth, survival, and drug resistance. However, existing treatments, such as monoclonal antibodies, suffer from limited affinity, large molecular weight, and insufficient tissue penetration, limiting their clinical efficacy.

[0031] The present invention constructs a heavy chain antibody gene library targeting BCMA by immunizing animals with a recombinant protein fused to the extracellular domain of BCMA and isolating peripheral blood lymphocytes after four immunizations. Using an enzyme-linked immunosorbent assay (ELISA), the recombinant BCMA protein is immobilized on an enzyme-labeled plate, and phage display technology is used to screen for nanobodies with high BCMA binding capacity from the gene library. Furthermore, these nanobodies are expressed and purified using an E. coli expression system, ensuring the high purity and activity of the final product. The purified BCMA nanobodies have strong binding affinity to BCMA. The IgG-fused anti-BCMA nanobodies designed in the present invention can effectively induce T cells to kill MM cells.

[0032] The beneficial effects of this technical solution are: (1) High affinity and selectivity: The BCMA nanobody in this invention is designed to increase the binding affinity for BCMA, thereby enhancing the recognition and targeting ability of multiple myeloma cells. Compared with traditional monoclonal antibodies, nanobodies are more effectively able to enter the tumor microenvironment and bind tightly to the target antigen due to their small size. The small molecular weight of nanobodies makes it easier for them to penetrate tissue barriers and reach the tumor site, providing higher local concentrations and thus improving the therapeutic effect.

[0033] (2) Low immunogenicity: Since nanoantibodies naturally lack the FC segment, the immune response they cause in the human body is extremely low, reducing the risk of immune rejection or allergic reactions caused by long-term use.

[0034] (3) Multifunctional application potential: In addition to direct therapeutic applications, the present invention also covers the application scenarios of coupling BCMA nanobodies with other drugs or markers, such as preparing diagnostic kits or developing new treatments. In addition, its clinical application performance can be further optimized through humanization or fusion expression with other antibody fragments.

[0035] (4) Easy to produce and prepare on a large scale: The production process based on the Escherichia coli expression system simplifies the manufacturing process and reduces costs, while ensuring the quality and consistency of the product, making it possible for large-scale clinical applications in the future.

[0036] In summary, the BCMA nanobody proposed in the present invention not only solves the limitations of existing treatment methods, but also opens up new avenues for the treatment of multiple myeloma and other related diseases, and demonstrates broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the electrophoresis diagram of the pComb3XSS vector recovery in Example 3.

[0038] Figure 2 These are the experimental results for detecting the antibody-mediated tumor killing effect of Example 9. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below with reference to the following examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods and can be performed according to the recombinant techniques described (see Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); the materials and reagents used are all commercially available.

[0040] Example 1: Expression and purification of BCMA protein (1) Using conventional methods, the 54 amino acids of the BCMA extracellular domain known in the prior art were fused to the human IgG1 Fc region via a "GGGGSGGGGSGGGGS" linker peptide and then constructed into a pcAGGS vector to obtain a recombinant vector, pcAGGS-BCMA-IGG vector. After sequencing confirmed successful cloning, the recombinant bacteria were expanded and cultured.

[0041] (2) Extract a large amount of plasmid from the recombinant vector pcAGGS-BCMA-IGG. The specific steps are as follows: Transfer 120 mL of overnight bacterial culture to a suitable centrifuge bottle. Centrifuge at 4500-6000 g for 10 minutes at 4°C to pellet the cells. Discard the supernatant. Perform large-scale plasmid extraction according to the kit instructions. Dissolve the precipitate with an appropriate amount of DNA dissolution buffer (200-500 μL) (shaking in a 37°C water bath may aid dissolution).

[0042] (3) Protein purification Grow 293 cells to the logarithmic phase, digest with 2.5% trypsin, and split into 10 cm culture dishes. After 24 hours, replace the medium with DMEM serum-free medium. Mix 2 μg of plasmid and 5 μl of PEI at a ratio of 2 μg and add to the cell supernatant. 6 hours after transfection, replace the medium with DMEM 10% FBS medium. After 3 days, collect the cell supernatant, centrifuge at 12,000 rpm at 4°C for 10 minutes, and filter through a 0.22 μm filter membrane. Collect the supernatant. Prepare the following solution: Solution A: 20 mM Na3PO4, pH 7.0 (pH adjusted with phosphoric acid); Solution B: 0.1 M glycine, pH 3.0 (adjust pH with hydrochloric acid); Solution C: 1 M tris, pH 9.0.

[0043] Wash the protein A column with water before equilibration with Solution A. Mix the supernatant with Solution A at a 1:1 ratio, shake well, and pass it over the protein A column. After the supernatant is removed, remove the supernatant and elute with Solution A, followed by Solution B. Immediately after the peak elutes, add 10% Solution C. Replace the column with PBS using an ultrafiltration tube. Measure the protein concentration and freeze (contains BCMA-FC protein).

[0044] Example 2: Animal immunization and serum titer detection The purified BCMA-FC recombinant protein was diluted to 1 mL with PBS (200 μg BCMA-FC protein), mixed with an equal volume of adjuvant and emulsified evenly. Alpacas were immunized once every two weeks for a total of 4 immunizations, with Freund's complete adjuvant used for the first immunization and incomplete adjuvant used for the remaining immunizations.

[0045] BCMA protein was diluted to 1.5 μg / mL in 0.05 M carbonate coating buffer, and 150 μL was added to each well at 4°C overnight. The next day, the coating buffer was discarded and the plates were washed four times, each for 4 minutes. 300 μL of 2.5% BSA blocking buffer was added to each well and incubated at 37°C for 1-2 hours. After washing, the plates were dried and set aside. 100 μL of serum to be tested, diluted in sample diluent, was added to each well and incubated at room temperature for 2 hours. The plates were washed five times, each for 3 minutes. After patting dry, HRP-conjugated secondary antibody was added and incubated at room temperature for 1 hour. The plates were washed five times, each for 3 minutes. 100 μL of TMB substrate was added to each well and incubated at room temperature for 15-30 minutes until uniform color was developed. The reaction was terminated by adding 50 μL to each well. The results are shown in Table 1.

[0046] Table 1: ELISA test results of serum titer of immune animals

[0047] Example 3: Construction of Nanobody Library (1) Construction of vector Isolate the peripheral blood lymphocytes of the immunized alpaca, and take an appropriate amount of lymphocytes for RNA extraction. Reverse transcription is performed using the extracted RNA as a template. Using cDNA as a template, the total RNA obtained is reverse transcribed using the Takara reverse transcription kit. Divide the total RNA sample extracted each time into two parts, one using the OligodT Primer in the kit as a primer, and the other using the Random 6 in the kit as a primer. According to the instructions of the reverse transcription kit, the total RNA obtained in the previous step is used as a template for reverse transcription into cDNA, and stored in 2 centrifuge tubes respectively as subsequent PCR templates. Two rounds of PCR amplification are performed with single domain specific primers. 1.2% agarose gel identification. In the first round, antibody gene fragments of about 600bp are recovered, and single domain antibody gene fragments less than 400bp are recovered. The fragments and vectors are digested overnight with restriction endonucleases, and the pComb3XSS vector recovers fragments of about 4000bp ( Figure 1 The recovered fragment was ligated to the vector using T4 ligase overnight at 16°C. TG1 competent electroporation was then performed.

[0048] (2) Bacterial library construction (2.1) Perform electroporation reactions using at least 20 competent cells as described above.

[0049] (2.2) After 1 h of resuscitation at 37°C, 100 μL of the culture was diluted 10-fold and plated, and cultured at 37°C overnight.

[0050] (2.3) Collect all the remaining bacterial suspension and evenly spread it onto five 245 mm square culture plates (2×YT containing 100 μg / mL Amp), and culture at 37°C overnight.

[0051] (2.4) Calculate the number of transformed colonies obtained from all reactions based on the dilution factor and the number of single colonies. This is the library capacity. The calculation formula is: C = E × P × 20. Where C is the library capacity, E is the ligation efficiency, and P is the positive clone rate.

[0052] (2.5) At the same time, 48 single clones were randomly selected from the gradient dilution plate for colony PCR to verify the clone positive rate of the bacterial library.

[0053] (2.6) Scrape the colonies from the overnight culture on a 245 mm square plate using 2×YT liquid medium, place them in a 50 mL centrifuge tube, measure the OD600 value, and add glycerol to a final concentration of 20% and store at -80°C.

[0054] (3) Phage library preparation (3.1) Calculate the volume of bacterial library to be added to 100 mL of 2×YT liquid medium based on the OD600 of the bacterial library. Based on the calculated result, inoculate the bacterial library into 100 mL of 2×YT liquid medium (containing 100 μg / mL Amp) and incubate at 37°C, 250 rpm until the OD600 reaches 0.5-0.55. Calculate the volume of the inoculated bacterial culture according to the following formula:

[0055] Wherein, V is the volume of the transferred bacterial solution (unit: μL), and OD600 is the total OD600 of the constructed bacterial library.

[0056] (3.2) Add helper phage at a ratio of 1:20 (number of bacteria: number of phages) based on the helper phage titer. Incubate at 37°C, 250 rpm for 30 min. Calculate the helper phage volume using the following formula:

[0057] Where V is the volume of helper phage added (unit: mL), T helper-phage is the titer of the helper phage used, and OD600 is the OD600 value of the bacterial solution.

[0058] (3.3) Add Kanamycin to a final concentration of 50 μg / mL and incubate overnight at 30°C and 250 rpm.

[0059] (3.4) Centrifuge the overnight culture at 4000 rpm for 20 min at 4°C, and transfer the supernatant to a new 50 mL centrifuge tube.

[0060] (3.5) Add 1 / 4 of pre-cooled 20% PEG / 2.5M NaCl, mix well, and incubate on ice for 30 min.

[0061] (3.6) Centrifuge at 4000 rpm for 20 min at 4°C, discard the supernatant, and invert to dry for 2 min.

[0062] (3.7) Add 1 mL of PBS and resuspend in a new centrifuge tube. Centrifuge at 12,000 rpm for 20 min at 4°C.

[0063] (3.8) After centrifugation, transfer the supernatant to a new centrifuge tube. Add 1 / 4 volume of pre-cooled 20% PEG / 2.5M NaCl, mix well, and incubate on ice for 10 min.

[0064] (3.9) Centrifuge at 12,000 rpm for 10 min at 4°C, discard the supernatant, resuspend in 1 mL of PBS, and centrifuge at 12,000 rpm for 2 min. Transfer the supernatant to a new centrifuge tube and store at -80°C. This is the purified phage library.

[0065] (3.10) Take 10 μL of the prepared phage library and dilute it in a 1.5 mL centrifuge tube in a 10-fold gradient, for a total of 12 gradients. That is, take 10 μL of the phage library and dilute it to 100 μL, then take 10 μL and dilute it to 100 μL, and so on, for a total of 12 dilution gradients to 10-12. Vortex to mix.

[0066] (3.11) Add 90 μL of TG1 (OD600 approximately 0.5) bacterial solution to each dilution centrifuge tube, mix well, and incubate at 37°C for 30 min.

[0067] (3.12) Take 5 μL of the solution from each dilution centrifuge tube and add it dropwise to 2×YT solid medium (Amp). Incubate at 37°C overnight. Count the number of colonies on the plate at the dilution at which single colonies can be clearly distinguished. Calculate the phage library titer according to the following formula:

[0068] Where, is the titer of phage library (unit), D is the dilution factor, and N is the number of single colonies on the plate with the corresponding dilution factor.

[0069] The results showed that the titer of the phage library reached 4.8×10 13 pfu / mL.

[0070] Example 4: Immunoscreening of antibodies (1) First round of screening Thaw BCMA on ice; coat the screening antigen in the immunotube (50µg / tube, coating solution is PBS, pH 9.6, 2mL / tube), rotate slowly at 4°C overnight, and coat BSA in parallel as a control; discard the liquid in the overnight coated immunotube, add 1.9mL PBS buffer and wash the immunotube three times at room temperature, rotating for 5 minutes each time; add 1.9mL blocking solution (3% BSA) solution and rotate and block at room temperature for 2 hours; discard the liquid in the blocked immunotube, and add 1.5mL PBS buffer and wash the immunotube three times at room temperature, rotating for 5 minutes each time; discard the washing solution in the immunotube, add 1.9mL PBS, calculate according to the following formula and add 20μL of the prepared phage library as the first round of screening input phage library, and incubate with rotation at room temperature for 1 hour.

[0071] The volume of phage added was calculated by the following formula:

[0072] Where V is the volume of phage added (unit: μl), T library is the phage titer.

[0073] Discard the liquid in the immunotube and add 1.9 mL of PBST (1×PBS plus 0.1% Tween20, the same below) buffer to wash the immunotube 25 times at room temperature, rotating for 5-10 minutes each time; discard the liquid in the immunotube to remove as much residual liquid as possible, add 1 mL of 0.25 mg / mL Trypsin solution, and elute by rotating at room temperature for 30 minutes; add 10 μL of 10% AEBSF to terminate the elution, and transfer the solution in the immunotube to a new 1.5 mL centrifuge tube, which is the phage eluate for the first round of screening.

[0074] (2) First round of phage eluate titer detection The preserved TG1 strain was streaked with single colonies on 2×YT solid medium and cultured overnight at 37°C. A single colony was picked from the single colony plate and transferred to 5 mL of 2×YT medium and cultured overnight at 37°C. 500 μL of the overnight culture was transferred to 5 mL of 2×YT liquid medium and cultured at 37°C, 250 rpm for about 40 min-60 min.

[0075] Take 10 μL of the first round of phage elution and dilute it 10-fold in a 1.5 mL centrifuge tube, for a total of 12 dilutions. That is, take 10 μL of the first round of phage elution and dilute it to 100 μL, then take 10 μL and dilute it to 100 μL, and so on, for a total of 12 dilutions to 10-12, and shake to mix. Add 90 μL of TG1 bacterial solution to each dilution centrifuge tube, mix well and incubate at 37°C for 30 minutes. Take 5 μL from each dilution centrifuge tube and add it dropwise to 2×YT solid culture medium (Amp), invert and culture at 37°C overnight. Count the number of single colonies on the dilution plate that can clearly distinguish single colonies, and calculate the number of phages per milliliter of phage solution, that is, the phage library titer, according to the following formula:

[0076] Where T is the phage titer (unit: pfu / mL), D is the dilution factor, and N is the number of single colonies at the corresponding dilution factor.

[0077] (3) Amplification of the first round of phage eluate The stored TG1 strain was streaked with single colonies on 2×YT solid medium and cultured at 37°C overnight (stored at 4°C for one week). A single colony was picked from the single colony plate and transferred to 5 mL of 2×YT medium and cultured at 37°C overnight.

[0078] 500 μL of bacterial solution was transferred to 5 mL of 2×YT liquid medium and cultured at 37°C and 250 rpm for about 60 min until the OD600 value was 0.5; 500 μL of phage eluate obtained after the first round of screening was added to the bacterial solution; culture was continued at 37°C and 250 rpm for 30 min; the entire bacterial solution was evenly spread on a 245 mm square culture medium plate containing 2% agarose containing 100 μg / mL Amp and cultured at 37°C overnight; 5 mL of 2×YT liquid medium (containing 100 μg / mL Amp) was added to the surface of the culture plate after overnight culture, and the colonies on the square plate were gently scraped off with a spreading rod and the bacterial solution was collected into a 15 mL centrifuge tube, which is the amplified bacterial sub-library. At the same time, the OD600 value of the bacterial solution was measured using a spectrophotometer, and glycerol was added to a final concentration of 20%, which is the first round bacterial library. Calculate the amount of bacterial solution from the eluate bacterial library according to the following formula and transfer it to 100 mL of 2×YT liquid culture medium (containing 100 μg / mL Amp) to make the initial OD600 of 0.1.

[0079] (4) First round of phage purification (4.1) Transfer the overnight culture to a new 50 mL centrifuge tube and centrifuge at 4000 rpm at 4°C for 10 min.

[0080] (4.2) Transfer the supernatant after centrifugation to a new 50 mL centrifuge tube, add 1 / 4 volume of 4°C pre-cooled 20% PEG / 2.5 M NaCl, mix thoroughly, and place on ice for 30 min.

[0081] (4.3) Centrifuge at 4000 rpm, 4°C for 20 min, discard the supernatant, and invert on paper for 2 min.

[0082] (4.4) Add 1 mL of PBS to resuspend the pellet. Transfer the resuspension to a new 1.5 mL centrifuge tube and centrifuge at 13,000 rpm and 4°C for 20 min.

[0083] (4.5) Transfer the supernatant after centrifugation to a new 1.5 mL centrifuge tube, add 1 / 4 volume of pre-chilled 20% PEG / 2.5 M NaCl solution, mix well, and place on ice for 10 min.

[0084] (4.6) Centrifuge at 13,000 rpm and 4°C for 10 min, discard the supernatant, and resuspend the pellet in 1 mL of PBS.

[0085] (4.7) Centrifuge at 13,000 rpm and 4°C for 2 min. Transfer the supernatant to a new 1.5 mL centrifuge tube. This is the first round of screening of the phage sub-library. Store at -80°C for long-term storage or at -20°C for short-term storage (1-2 weeks).

[0086] The second and third rounds of screening were carried out according to the above method.

[0087] (5) Monoclonal ELISA detection The TG1 strain was streaked with single colonies on 2×YT solid medium and cultured overnight at 37°C. A single colony was picked from the plate and transferred to 5 mL of 2×YT medium and cultured overnight at 37°C. 500 μL of the overnight culture solution was transferred to 5 mL of 2×YT liquid medium and cultured at 37°C at 250 rpm for 40 min-60 min. 10 μL of the phage eluate after the third round of screening was diluted 10-fold in a centrifuge tube for a total of 12 dilutions and oscillated to mix. 90 μL of a bacterial solution with an OD600 value of 0.5-0.55 was added and mixed evenly. The solution was cultured at 37°C at 250 rpm for 30 min. The bacterial solution was evenly spread on a solid medium plate containing 100 μg / mL Amp and cultured overnight at 37°C. Single clones were randomly picked from the plate and placed in a sterile 96-well cell culture plate (P1-P3), and 200 μL of 2×YT was added to each well. Culture medium (containing 100 μg / mL Amp) at 37°C overnight; transfer 2 μL of the overnight culture to 200 μL of 2×YT liquid medium (containing 100 μg / mL Amp) per well of a new 96-well cell culture plate and incubate at 37°C for 3-5 hours. Store the overnight culture at 4°C before transfer. Add helper phage M13K07 to each well to achieve a bacterial count of 1:20 using the following formula. The volume of helper phage added is calculated using the following formula:

[0088] Where V is the volume of helper phage added (unit: mL), T helper-phage is the helper phage titer used.

[0089] The plates were incubated at 37°C for 30 min, and kanamycin was added to a final concentration of 50 μg / mL. The plates were then incubated at 30°C overnight. The 96-well plates were centrifuged at 4000 rpm for 10 min at 4°C and stored at 4°C until use. BCMA-FC was coated onto the microplate (1 ng / μL, 100 μL / well of CBS, pH 9.6) and BSA was coated in parallel as a control overnight at 4°C. The overnight coated microplate contents were discarded, 200 μL of PBS buffer was added to each well, and the plates were washed three times at room temperature for 10 min each. 200 μL of blocking buffer (3% BSA) was added to each well and blocked for 1 h at room temperature. The blocking buffer was discarded, and 200 μL of PBST (1× PBS with 0.1% Tween 20, the same below) buffer was added to each well. The plates were washed three times at room temperature for 10 min each. 120 μL of 3% After adding BSA, add 80µl of supernatant and incubate at room temperature for 2 hours; discard the liquid in the ELISA plate, add 200µl PBST buffer to each well and wash three times, 10 minutes each time; add M13 Bacteriophage Antibody (HRP) mouse monoclonal antibody, diluted 1:30,000 in blocking buffer, 100µL / well, and incubate at room temperature for 1 hour; discard the liquid in the ELISA plate, add 200µl PBST buffer to each well and wash three times, 10 minutes each time; add 100µl TMB single-component colorimetric solution to each well, develop in the dark for 3 minutes, add 100µl of stop buffer to each well, and read the OD450nm value using a microplate reader.

[0090] (6) Secondary verification of positive clones by ELISA To exclude false positive results, a second ELISA validation was performed using the same method as above, but this time the coating antigen included an FC control.

[0091] Example 4: Sequencing of positive clones Positive clones were selected based on ELISA results. A 5µl aliquot of the positive clone culture from the monoclonal ELISA plate was inoculated into 1mL of 2×YT medium (containing 100µg / mL Amp) and cultured at 37°C, 250 rpm, until the OD600 reached 0.8-1.0 (approximately 6-8 hours). A 0.5mL aliquot was sequenced, and the remaining aliquot was supplemented with 30% glycerol to a final concentration and stored at -20°C. Sequences were aligned using MegAlign software, and the antibody sequences were translated into amino acid sequences.

[0092] After screening, a total of 25 positive clones were obtained and sequenced. Fifteen of these clones were normal (sequencing completed normally and the sequences were typical Nanobody sequences, labeled BS01 to BS09). The Nanobody sequences were derived from the sequencing results based on the preceding and following sequences. The 15 sequences were translated into amino acids, sorted, and subjected to multiple sequence alignment, resulting in nine distinct Nanobody sequences (labeled BS01 to BS09). Each Nanobody has the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, meaning the variable region is composed of alternating complementarity-determining regions (CDRs) and framework regions (FRs). The amino acid sequences and composition of the nine Nanobodies are shown in Table 2.

[0093] Table 2: Amino acid sequences and compositions of 9 nanobodies

[0094] Example 6: Nanobody Expression The genes corresponding to the nine sequences described above were amplified by PCR using conventional methods. The PCR system consisted of 1 μL of template, 1 μL of upstream primer, 1 μL of downstream primer, 25 μL of 2× PCR Master Mix, and 22 μL of DNase / RNase-free water. The upstream and downstream primers were designed according to conventional methods and included restriction sites to facilitate subsequent construction of the recombinant vector.

[0095] After PCR is complete, the amplified bands are identified and separated by agarose gel electrophoresis. The fragments are then recovered and digested with enzymes, translating into the region between the NdeI and XhoI restriction sites of pet20a. Once the recombinant vector has been sequenced correctly, the plasmid is transformed into a BL21 Rocetta expression-competent cell. After identifying antibody strains expressing in the supernatant through small-scale expression, the expression system can be scaled up for large-scale production of nanobodies. The specific steps are as follows: (1) Select the antibody strain expressing in the supernatant and resuscitate its BL21 glycerol bacteria into 5 mL LB liquid medium, then transfer it into 500 mL LB liquid medium and culture it at 37°C with shaking until the OD600 of the bacterial solution is about 0.5.

[0096] (2) Add an appropriate amount of IPTG to the bacterial solution to make the final concentration of IPTG 0.5 mM, and culture at 15°C with shaking for 24 h.

[0097] (3) After 24 h, collect the bacterial precipitate by centrifugation, wash the precipitate twice with 1 / 10 volume (50 mL) of PBS, and then resuspend the precipitate with 1 / 10 volume (50 mL) of deionized water.

[0098] (4) Use an ultrasonic device to break and lyse the bacteria. The ultrasonic conditions are 4 seconds for 4 seconds and 6 seconds for 1 hour. The total time is 1 hour. Keep the sample incubated on ice during the ultrasonic process.

[0099] (5) Centrifuge the sample (12000×g, 30 min) and collect the supernatant into a new centrifuge tube. The supernatant is then subjected to affinity chromatography on Ni-NTA resin.

[0100] (6) The purified antibody was subjected to SDS PAGE electrophoresis to determine its purity.

[0101] (7) Concentrate the collected eluate using a concentrator tube and replace it with PBS multiple times to remove impurities such as imidazole.

[0102] (8) The concentration of nanoantibodies was determined using the Bradford protein quantification kit.

[0103] Example 7: In vitro binding ability detection of nanobodies To detect the binding ability of the expressed nanobody to the target protein, an indirect enzyme-linked immunosorbent assay was used to verify the affinity of the expressed antibody to BCMA, and the ELISA method was used for detection: BCMA-FC and FC protein were diluted with coating solution (PBS) to a concentration of 1 μg / mL, added to a 96-well ELISA plate, 50 μL / well, and incubated at 4°C overnight; the next day, the plate was washed once with PBST and patted dry, and blocking solution (PBS containing 3% BSA) was added at 100 μL / well, and incubated at 37°C for 1 hour; the plate was washed once with PBST and patted dry; another 96-well dilution plate was used, and the antibody (BS01-09) was diluted in equal proportions, and then transferred to a 96-well ELISA plate at 50 μL / well for 3 hours. Incubate at 7°C for 45 minutes; wash the plate three times with PBST and pat dry; dilute the tag antibody in blocking buffer and add 50 μL / well to a 96-well microtiter plate. Incubate at 37°C for 30 minutes; wash the plate three times with PBST and pat dry; add 50 μL / well of TMB one-component colorimetric solution and incubate at room temperature or 37°C for several minutes. Once color develops to the appropriate depth (usually 5-10 minutes), add 50 μL / well of stop solution and gently tap the plate to mix. Read the absorbance at 450 nm using a microplate reader. The results are shown in Table 3, which shows the absorbance at 450 nm of BS01-BS09 at the same concentration against BCMA-FC or FC, demonstrating the affinity of BS01-BS09 for BCMA.

[0104] Table 3: In vitro binding ability test results of nanobodies (absorbance value at 450nm, repeated twice)

[0105] ELISA results showed that the ELISA signals for BS03 and BCMA-FC were 2.7056 and 2.6051, respectively, with average values higher than those of other nanobodies in the same group. This suggests that BS03 has a higher affinity for BCMA-FC and a lower affinity for FC than other nanobodies.

[0106] Example 7: Fusion expression of nanobodies and IgG BS03 was linked to human IgG1 to create a synthetic sequence (BS03+IgG FC, SEQ ID NO. 10). To improve antibody assembly efficiency, a hole-in-knob structure was employed, and T366S, L368A, and Y407V mutations were introduced into one of the FC chains. After large-scale plasmid extraction, the recombinant construct was transiently transfected into HEK293 cells using PEI for expression. After 48 hours, the supernatant was purified by Protein A affinity chromatography. The purified antibody was concentrated using ultrafiltration, and the buffer was replaced with PBS.

[0107] SEQ ID NO.10 is specifically (the underline is the connecting peptide, and the wavy line is the human IgG1 FC region): DVQLQESGGGLVQPGGSLRLSCAASGFSFTTESMLNFRHFPGKGTEWVSGISAGGDVSNYADQVKGRFFVSRDNAKSTLYLQMNNLTPEDTALYFCQGGISQRLERDQGTQVTVSS GGGGSGGGGSGGGGSDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK .

[0108] Example 9: Detection of Antibody-Mediated Tumor Killing Effect The ability of the BS03 antibody (BS03 + IgG FC, SEQ ID NO. 10; BS01 + IgG FC, constructed using the same method, was used as a comparison) to kill the BCMA-H929 cell line was tested. BCMA-H929 target cells and PBMC effector cells were prepared in RPMI-1640 medium containing 1% FBS. 5 ml of medium was added to a 15 ml centrifuge tube. Frozen PBMCs (including lymphocytes and monocytes) were thawed in a 37°C water bath and then transferred to 5 ml of medium. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, resuspend the tube, count the cells, and adjust the effector cell density to 1.25 × 106 / ml and placed in a 37°C, 5% CO2 incubator until ready for use. Pre-thaw target BCMA-H929 cells were cultured and centrifuged at 1500 rpm for 5 minutes when cell viability exceeded 70%. Discard the supernatant, wash the cells twice with culture medium, resuspend the cells, and use a cell counter to adjust the target cell density to 1.88 × 10 5 / ml and incubate at 37°C, 5% CO2 until ready. Dilute the antibody to a 0.7 μM concentration in the first well, then dilute four-fold in succession. Transfer 60 μl of each to each well of a 96-well plate for incubation with BCMA-H929 target cells and PBMC effector cells. Simultaneously, set up spontaneous wells for BCMA-H929 and PBMC, spontaneous wells for target cells, and wells for maximum target cell LDH release. Transfer 60 μl of each diluted antibody to each well of the 96-well plate, mix thoroughly, and incubate overnight at 37°C, 5% CO2 for 24 hours. 30 minutes before the end of co-culture, add 20 μl of 10x lysis buffer to the well for maximum target cell LDH release, and incubate the cells at 37°C, 5% CO2 for 30 minutes. Centrifuge the co-cultured 96-well plate at 1500 rpm for 2 minutes, transfer 10 μL of the supernatant to a new microplate, add 10 μL of CytoTox96 reagent to each well, centrifuge at 1000 rpm for 1 minute, and incubate at room temperature in the dark for 30 minutes. Add 10 μL of stop solution to each well. Record the absorbance at 490 nm using a microplate reader and analyze the data using Prism. For detailed experimental results, see [ 10 μL of CytoTox96 reagent ](https: / / www.cytotox.com / products / cytotox96 / ). Figure 2 Experimental data show that BS03 antibody has a stronger tumor-killing effect than other antibodies (such as SBS01).

[0109] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. A nanobody against a B cell maturation antigen, characterized in that: The amino acid sequence of its complementarity determining region 1 is: (1-1): any one of the amino acid sequences of FSFTEGEIGEFR, FTFRDHEEGQFR, FSFTTESMLNFR, RSESAQSLSEFR, FSESLESLSFFR, NILSARTRGWYR, FSFSASMSRFR, FSFSFFAGRSWFR, and FSFRSASMGRFR; Or (1-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences in (1-1), and having the same function as any of the amino acid sequences in (1-1); or (1-3): an amino acid sequence having greater than 80% identity with any of the amino acid sequences in (1-1); The amino acid sequence of its complementarity determining region 2 is: (2-1): any one of the amino acid sequences of GETNYQGSVK, GTTNEALSVK, GDVSNYADQVK, GDESNVQGSQK, GGVEKYARQVK, TGSRSQADSVK, GGNVSNYADSVK, GGEVSNYRGSVK, and GGNVSNYADSVK; Or (2-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences in (2-1), and having the same function as any of the amino acid sequences in (2-1); or (2-3): an amino acid sequence that is more than 80% identical to any of the amino acid sequences in (2-1); The amino acid sequence of its complementarity determining region 3 is: (3-1): any one of the amino acid sequences LSAFETSRLRWGQ, LSAQCTTRLYENQ, CQGGISQRLERDQ, CQGRLSGREVRGQ, CTGERSGRLVRGQ, YFSFSYEFDWGQ, QGALNGRLARGQ, QGFENGFLERGQ, and QGELQGVLAEGQ; Or (3-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences in (3-1), and having the same function as any of the amino acid sequences in (3-1); Or (3-3): an amino acid sequence having 80% or more identity with any of the amino acid sequences in (3-1).

2. The nanobody against B cell maturation antigen according to claim 1, characterized in that: The amino acid sequence of its framework region 1 is: (4-1): any one of the amino acid sequences DVQLQESGGGLVQPGGSLRLTCVASG, DVQLQESGGGLVQPGGSLRLSCAASG, and DVQLQESGGGLVQAGGSLRLSCTVSG; Or (4-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences in (4-1), and having the same function as any of the amino acid sequences in (4-1); or (4-3): an amino acid sequence having greater than 80% identity with any of the amino acid sequences in (4-1); The amino acid sequence of its framework region 2 is: (5-1): any one of the amino acid sequences QAPGKEREGVSCIGRFG, HFPGKGIEWVSGISAG, QGPGKQRELVARLTP, and HFPGKGLEWVSGISA; Or (5-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences in (5-1), and having the same function as any of the amino acid sequences in (5-1); or (5-3): an amino acid sequence that is more than 80% identical to any of the amino acid sequences in (5-1); The amino acid sequence of its framework region 3 is: （6-1）： Any one of the amino acid sequences of GRFAISRDNAKNTVYLQMNSLKPEDTAVYYCAAQYSLRA, GRFFVSRDNAKSTLYLQMNNLTPEDTALYF, GRFTISRDNAKNMVYLQMNSLKPEDTAVYYCNAAG, and GRFTVSRDNAKSTLYLQMNSLTPEDTALYFC; Or (6-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences in (6-1), and having the same function as any of the amino acid sequences in (6-1); or (6-3): an amino acid sequence having greater than 80% identity with any of the amino acid sequences in (6-1); The amino acid sequence of its framework region 4 is: (7-1):GTQVTVSS; Or (7-2): an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids of GTQVTVSS, and having the same function as GTQVTVSS; Or (7-3): an amino acid sequence that is more than 80% identical to GTQVTVSS.

3. The nanobody against B cell maturation antigen according to claim 2, characterized in that: Its amino acid sequence is: (8-1): any one of the amino acid sequences of SEQ ID NO.1 to SEQ ID NO.9; Or (8-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences in (8-1), and having the same function as any of the amino acid sequences in (8-1); or (8-3): an amino acid sequence having 80% or more identity with any of the amino acid sequences in (8-1).

4. The nanobody against B cell maturation antigen according to claim 3, characterized in that: Its amino acid sequence is: (9-1): any one of the amino acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.9; Or (9-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any of the amino acid sequences in (9-1), and having the same function as any of the amino acid sequences in (9-1); or (9-3): an amino acid sequence having 80% or more identity with any one of the amino acid sequences in (9-1).

5. The nanobody against B cell maturation antigen according to claim 4, characterized in that: Its amino acid sequence is: (10-1): SEQ ID NO. 3; Or (10-2): an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO. 3, and having the same function as SEQ ID NO. 3; Or (10-3): an amino acid sequence having 80% or greater identity with SEQ ID NO.

3.

6. The dimer or multimer formed by the Nanobody against B cell maturation antigen or the humanized Nanobody or the fusion-expressed Nanobody or the conjugate according to any one of claims 1 to 5, characterized in that: The conjugate is a nanobody, dimer, multimer, humanized nanobody or fusion-expressed nanobody comprising a chemical label or a biological label; the dimer or multimer or humanized nanobody or fusion-expressed nanobody or conjugate is used to couple with a solid medium or a semi-solid medium.

7. A gene encoding the dimer or multimer or humanized Nanobody or fusion-expressed Nanobody according to claim 6.

8. A gene encoding the nanobody against B cell maturation antigen according to any one of claims 1 to 5.

9. A host cell or cell line expressing the Nanobody against B cell maturation antigen according to any one of claims 1 to 5.

10. Use of the anti-B cell maturation antigen Nanobody according to any one of claims 1 to 5 in the preparation of a diagnostic reagent and / or kit for a B cell maturation antigen-related disease; or in the preparation of a medicament for preventing and / or treating a B cell maturation antigen-related disease; or in the preparation of a medicament for preventing and / or treating a disease related to multiple myeloma.

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