Nanobodies targeting b-cell cd20 and uses thereof

Abstract

The application discloses the field of biotechnology, and discloses a kind of nanobody targeting B cell CD20 and application thereof.The amino acid sequence of nanobody complementarity determining region CDR1~CDR3 is selected from SEQ ID NO.1~9.The nanobody provided by the application can specifically bind to B cell in human blood, thereby eliminating B cell producing pathological antibody.The nanobody provided by the application has smaller molecular weight compared with traditional antibody, is stable, low in cost, easy to express and high in expression amount, and is suitable for large-scale production.Nanobody is combined with solid phase carrier in the application, B cell in blood can be targetedly eliminated, the number of B cell in blood of patient is reduced in a short time, antibody level is reduced, thereby improving life quality, reducing complication, and providing new treatment selection for those patients who are ineffective or intolerant to traditional treatment.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a nanobody that targets CD20 on B cells and its applications. Background Technology

[0002] B cells are important lymphocytes in the immune system. Malignant tumors, autoimmune diseases, lymphomas, and leukemia are common B cell-related diseases, and their pathogenesis is related to the overactivation of B cells, leading to the production of large amounts of autoantibodies. Therefore, B-lymphocyte-targeted therapy, which targets B lymphocyte surface molecules, has received widespread attention and research in recent years. B-lymphocyte clearance strategies utilize antibody drugs that target and bind to specific antigens of B cell subsets, inducing B cell apoptosis through antibody-dependent cytotoxicity or complement, or by targeting cytokines necessary for B cell survival, such as B cell activating factor (BAFF), thereby clearing B cells. The main targets for B cell clearance are CD20, CD19, and BAFF, and these drugs are used to treat autoimmune diseases including systemic lupus erythematosus and multiple sclerosis.

[0003] CD20 is a non-glycosylated protein expressed on the surface of B cells. It is primarily expressed by B cells, downregulated during the initial B cell proliferation and activation into plasma cells, and lost in terminally differentiated plasmablasts and plasma cells. CD20 expression is specific to B cells and is not expressed in monocytes, granulocytes, most T cells, or other tissue cells. Drugs targeting CD20 can reduce the level of plasma cells producing autoantibodies by blocking differentiation into plasma cells, while preserving a regenerative pool of B cell precursors, immature B cells, and long-lived plasma cells.

[0004] Currently, B-lymphocyte depletion therapy, represented by CD20 monoclonal antibodies such as Rituximab, Ocrelizumab, and Ofatumumab, is a relatively mature treatment option. Besides B-cell depletion, there are also neutralizing drugs targeting B-cell growth factors (BAFF), including BAFF neutralizing antibodies (such as Belimumab). Rituximab is a chimeric anti-CD20 monoclonal antibody composed of human IgG1 and a murine variable region. It specifically binds to the CD20 antigen on the surface of B cells and induces B-cell apoptosis (self-destruction) to disrupt B-cell function. Rituximab is mainly used to treat certain types of cancer and autoimmune diseases, such as non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and rheumatoid arthritis. Due to its chimeric structure, Rituximab has better immunogenicity and tolerability compared to murine antibodies.

[0005] However, studies have shown that long-term use of Rituximab may lead to temporary suppression of the immune system, increasing the risk of infection. Some patients may experience allergic reactions to the monoclonal antibody or its components, manifesting as symptoms such as rash and difficulty breathing. Furthermore, it can cause a decrease in blood cell counts (such as white blood cells, red blood cells, and platelets), which may affect the patient's health and recovery ability.

[0006] Nanobodies are a special type of antibody extracted from camelids (such as camels, alpacas, and vicuñas), and are only one-tenth the size of conventional antibodies. This allows them to penetrate tissues more easily and reach places inaccessible to conventional antibodies. Nanobodies possess extremely high thermal and chemical stability, enabling them to maintain activity under various harsh environments. Due to their simple structure, nanobodies can be easily genetically engineered to produce antibodies with specific functions. Furthermore, nanobodies can be mass-produced using microorganisms such as bacteria and yeast, significantly reducing production costs. Nanobodies can simultaneously recognize multiple antigenic epitopes, making them highly promising for the diagnosis and treatment of various diseases. Due to their unique structure and properties, nanobodies exhibit high efficiency in targeted drug delivery, immunodiagnostics, and tumor therapy. Nanobodies have been widely applied in medicine, biotechnology, agriculture, and other fields, including early diagnosis and treatment of diseases, as well as as research tools. Developing nanobodies that target B cells is of crucial significance for the treatment and research of B-cell-related diseases.

[0007] Blood purification is a crucial medical technology that treats various diseases and poisoning conditions by removing harmful substances from the blood. By using specific adsorbents, such as immunoadsorbents, specific pathogenic factors, such as autoantibodies or inflammatory mediators, can be targeted for adsorption. Immunoadsorption is a technique that utilizes specific antigens, antibodies, or ligands with unique physicochemical affinities to bind to an adsorption medium, forming a selective or specific adsorption column. When used in conjunction with drug therapy, immunoadsorption can significantly improve patient responsiveness to drugs, thereby enhancing drug efficacy, reducing side effects, shortening treatment time, and lowering the risk of disease recurrence. By combining antibodies targeting B cells with a solid-phase carrier, B cells in human blood can be specifically bound, thereby eliminating B cells that produce pathological antibodies. This method offers high adsorption efficiency, stable adsorption performance, targeted removal of B cells, low non-specific adsorption, good specificity, good safety in clinical treatment, and high economic benefits. It may become a new approach for the treatment of B-cell-related diseases. Summary of the Invention

[0008] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a nanobody that targets CD20 on B cells and its application.

[0009] The technical solution adopted in this invention is:

[0010] In a first aspect, the present invention provides a nanobody targeting CD20 on B cells, comprising a framework region and a complementarity-determining region, wherein the amino acid sequences of the complementarity-determining regions CDR1 to CDR3 of the nanobody are selected from any of the following combinations:

[0011] Combination 1, the amino acid sequences of CDR1 to CDR3 are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively;

[0012] Combination 2, the amino acid sequences of CDR1 to CDR3 are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively;

[0013] Combination 3, the amino acid sequences of CDR1 to CDR3 are shown in SEQ ID NO.7 to SEQ ID NO.9, respectively.

[0014] In some instances, it is a chimeric antibody, a humanized antibody, or a nanobody fused with an Fc fragment.

[0015] In some instances, the amino acid sequence of the nanobody is selected from any combination of the following:

[0016] CA05:

[0017] QVQLVESGGGPVQSGGSLRLSCAGSRNIYS PYSMGDYGLS WFRQAPGEEREGVA LIDRDDSTFYADSV KG RFTISQDDAKTNLFLQMNSLKPEDSAMYYCAA AAKPKEHGLFDS WGQGTQVTVSS;

[0018] CB01:

[0019] QVQLQESGGGPVQAGDSLRLSCAASGNIYS GNIYSRHSMG WFRQVPGKEREGVA INRDDSS RFTLSQDDAKTNLFLQMNSLKPTDTAYYCAA GWMATLAWSDWF RGRGTQVTVSS;

[0020] CD09:

[0021] QVQLQESGGGPVQSGGSLRLSCAAS RNIYSRPYMA WFRQAPGKEREGVAT SSTFGSTVYADSVKGRFTISRDDAKTNLFLQMNSLKPEDSAMYYC AAGFDYLSPSDYGY WGQGTQVTVSS.

[0022] In the above sequence, the underlined part is the CDR area, and the unmarked part is the FR area.

[0023] Some examples of nanobodies include chimeric antibodies, humanized antibodies, and nanobodies fused with Fc fragments.

[0024] By introducing the Fc region of an antibody, the dimerization of nanobodies can be achieved. The introduction of the Fc region also simplifies antibody purification procedures by utilizing the affinity of Fc. Furthermore, the introduction of Fc helps to prolong the antibody's half-life and improve its stability in vivo. There are no special requirements for the antibody Fc region, but it is preferably the Fc region of a human antibody. The antibody Fc region includes the Fc fragment of human IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgM, or IgE, or its variants or modified forms.

[0025] In a second aspect, the present invention provides a bivalent or multivalent nanobody, comprising at least one of the nanobodies described in the first aspect of the present invention.

[0026] Bivalent or multivalent nanobodies are formed by linking single nanobodies together through a linker element. The linker element is preferably a flexible peptide, including but not limited to (G4S)n, where n is an integer of 1 or higher, preferably an integer from 1 to 4. The nanobodies in a bivalent or multivalent nanobody may be the same or different.

[0027] Thirdly, the application of the nanobodies described in the first and second aspects of this invention includes, but is not limited to:

[0028] Preparation of B-cell immunosorbents;

[0029] Preparation of B-cell detection reagents;

[0030] Preparation of CD20-targeting immunoconjugates;

[0031] Prepare antibody drugs for the treatment of B-cell-related diseases.

[0032] In some instances, the immunoconjugate consists of a nanobody and a conjugate attached to the nanobody, such as an ADC drug composed of an antibody and a small molecule.

[0033] In some instances, the conjugate is a drug.

[0034] In some instances, the B-cell-related diseases are selected from tumors and autoimmune diseases.

[0035] In some instances, tumors include, but are not limited to, multiple myeloma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and glioblastoma; autoimmune diseases include, but are not limited to, lupus erythematosus, myasthenia gravis, rheumatoid arthritis, idiopathic inflammatory myositis, systemic sclerosis, and primary Sjögren's syndrome.

[0036] Fourthly, the present invention provides an immunoadsorbent comprising a solid support on which nanobodies as described in the first and second aspects of the present invention are coupled.

[0037] In some instances, the solid support is selected from at least one of chitosan, agarose, cellulose, dextran, resin, and cellulose.

[0038] Fifthly, the present invention provides a blood purification device, comprising the immunosorbent described in the fourth aspect of the present invention.

[0039] In a sixth aspect, the present invention provides a gene encoding a nanobody as described in the first or second aspect of the present invention.

[0040] In a seventh aspect, the present invention provides an expression vector that expresses the nanobody described in the first or second aspect of the present invention, or contains the gene described in the sixth aspect of the present invention.

[0041] The beneficial effects of this invention are:

[0042] The nanobodies provided by this invention can specifically bind to B cells in human blood, thereby eliminating B cells that produce pathological antibodies. Compared with traditional antibodies, the nanobodies provided by this invention have a smaller molecular weight, higher stability, lower cost, are easier to express, and can achieve high expression levels, making them suitable for large-scale production.

[0043] This invention combines nanobodies with a solid-phase carrier, which may target and eliminate B cells in the blood, reduce the number of B cells in the patient's blood in a short period of time, lower antibody levels, thereby improving quality of life, reducing complications, and providing a new treatment option for patients who are unresponsive to or intolerant of traditional treatments. Attached Figure Description

[0044] Figure 1 The image shows the electrophoresis pattern of the purified nanobody. The molecular weights of the marker bands from top to bottom are 180kD, 130kD, 100kD, 70kD, 55kD, 40kD, 35kD, 25kD, 15kD, and 10kD. Detailed Implementation

[0045] The following disclosure provides many different implementations or examples for different ways of implementing the present invention.

[0046] As used herein, the term "antigen-binding fragment" refers to one or more portions of an antibody structure that specifically bind to a target antigen, including but not limited to VHH fragments and fragments containing CDRs. "Specific binding" as used herein refers to a non-covalent interaction between a nanobody or its antigen-binding fragment and the antigen. In this invention, the term "affinity" refers to the strength of the interaction between a macromolecule, particularly a nanobody, and its corresponding antigen. This binding ability reflects the interaction characteristics between the nanobody and a specific antigen. To accurately assess the affinity of the nanobodies in this invention, various in vitro experimental methods can be used for measurement, including surface plasmon resonance (SPR) technology and ELISA assays.

[0047] In this invention, the term "variable" refers to the sequence-specific differences in the variable regions of an antibody, which enable different antibodies to precisely bind to and recognize specific antigens. Antibody variability is primarily concentrated in the three complementarity-determining regions (CDRs) of the heavy chain variable region, also known as hypervariable regions. In the native heavy chain variable region, there are four frame regions (FRs), which are relatively conserved and typically exhibit a β-sheet configuration. The FRs are linked by three CDRs forming a linker loop, which locally form a β-sheet structure. The CDRs in each chain are closely adjacent through the FRs and together with the CDRs of the other chain, constitute the antibody's antigen-binding site. While constant regions are not directly involved in the antibody-antigen binding process, they exhibit different effector functions, such as mediating antibody-dependent cytotoxicity.

[0048] In immunology, an "epitaph" is a region located on the surface of an antigen molecule that can be recognized and bound by a single antibody molecule. Typically, an antigen may possess multiple different epitopes, enabling it to react with a variety of different antibodies. Epitopes are mainly classified into two types: linear epitopes and conformational epitopes. Linear epitopes consist of a continuous amino acid sequence in the antigen molecule, while conformational epitopes are formed by discontinuous amino acid residues in the antigen molecule being spatially adjacent to each other. Both types of epitopes are key sites for antibody recognition and binding.

[0049] "Dual epitope specificity" refers to the ability of an antibody or immune receptor to specifically recognize and bind to two or more different epitopes on the same target protein, or multiple epitopes on different target proteins.

[0050] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine / camel-derived antibody with the constant region of a human antibody. It can reduce the immune response induced by murine / camel-derived antibodies.

[0051] The term "humanized antibody" refers to an antibody generated by transplanting the CDR sequence of a camel-derived VHH antibody into the variable region framework of a human antibody, i.e., a human germline antibody framework sequence of different types. This overcomes the heterologous response induced by chimeric antibodies carrying a large amount of camel-derived protein components. Such framework sequences can be obtained from public DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be obtained from the "VBase" human germline sequence database (Internet www.mrccpe.com.ac.uk / vbase), and in Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity along with a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse or reversion mutations to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies further matured by phage display with affinity for the CDR.

[0052] The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments of the antibody that maintain its ability to specifically bind to antigens.

[0053] As used in this article, the term "antibody framework" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0054] The technical solution of the present invention will be further illustrated below with examples and experimental data.

[0055] Example 1: Preparation of protein with CD20 as antigen

[0056] Anti-CD20 antibodies were prepared using the following antigen. The peptide derived from the C-terminal amino acid residues 265-290 of the human CD20 (Uniprot accession number P11836-1, specific amino acid sequence: MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP, as shown in SEQ ID NO.13) was used as the antigen.

[0057] The method for preparing CD20 antigen includes: preparing a nucleic acid sequence molecule containing the human His tag to the C-terminus of human CD20 amino acid residues 265-290, cloning it into plasmid pcDNA3.1, transforming the cloned vector into HEK293T cells for expression, and purifying it using a cobalt ion column to obtain CD20 antigen.

[0058] Example 2: Alpaca Immunization with CD20 Antigen

[0059] During immunization, approximately 0.5 mg of antigen is used each time, with a total volume not exceeding 1.5 mL. The injection site is near the lymph nodes in the alpaca's neck, administered on both sides, with two injection points on each side, and approximately 0.4 mL of emulsified antigen injected at each point. Immunization is performed every two weeks, for a minimum of five doses. Blood samples are collected before each immunization for assessing the immunization effect. Five to seven days after the fifth immunization, a large volume of blood is collected to isolate lymphocytes.

[0060] Lymphocyte isolation was performed as follows: Blood was collected from the jugular vein of an alpaca, using 50 mL of EDTA-containing medium. The collected blood was diluted with an equal volume of phosphate-buffered saline (PBS) or serum-free medium. Lymphocyte separation medium was then added to a centrifuge tube, and the diluted blood was slowly added to the top layer of the separation medium for density gradient centrifugation, typically at room temperature, at 400-600g for 20-30 minutes. After centrifugation, the lymphocyte layer was carefully aspirated from the formed layers and transferred to a new centrifuge tube. The cells were washed with copious amounts of PBS or serum-free medium, followed by centrifugation at 200-300g for 10 minutes to precipitate the cells and discard the supernatant. This washing process was repeated twice. Finally, the lymphocytes were resuspended in medium for counting and viability assessment using trypan blue staining.

[0061] The next steps are RNA extraction and cDNA synthesis: Lymphocytes are mixed thoroughly with chloroform and centrifuged. The supernatant is then transferred to a new centrifuge tube. Isopropanol is added to precipitate the RNA, followed by washing the precipitate with 75% ethanol. Finally, the precipitate is dissolved in RNase-free water. Following the instructions of the reverse transcription kit, the extracted RNA is reverse transcribed into cDNA.

[0062] The antibody fragment amplification steps are as follows: First, using cDNA as a template, the VHH antibody fragment is amplified by PCR using specific primers. The target band is separated by agarose gel electrophoresis. Then, using the DNA recovered after the first PCR amplification as a template, PCR amplification is performed again to increase the content of specific antibody fragments. Finally, the amplified antibody fragment is cloned into a phage plasmid.

[0063] Example 3: Construction and screening of CD20 nanobody phage library

[0064] The process of constructing a phage library mainly includes three key steps: First, two restriction enzyme sites, SacI and SpeI, are selected. Diverse antibody gene sequences obtained through PCR amplification are ligated into the phage vector pComb3XSS to form a recombinant vector, which is then transformed into *E. coli* TG1 competent cells. Cell transformation is performed using electroporation, followed by culture in resuscitation medium. 10 ml of *E. coli* TG1 cells in logarithmic growth phase are transferred to a new sterile 50 ml centrifuge tube, and...

[0065] 4×10 10After gently mixing the PFU M13KO7 helper phage, incubate at 37°C for 30 minutes to allow the phage to infect *E. coli*. Centrifuge at 2,800g at room temperature for 10 minutes, carefully removing the supernatant to remove glucose from the culture medium. Resuspend the cells in 50ml of 2×TY medium (containing 100μg / ml ampicillin and 25μg / ml kanamycin) in a 250ml Erlenmeyer flask and incubate overnight at 30°C and 200rpm. Centrifuge again, add 20% PEG300 / 2.5M NaCl solution to the supernatant to precipitate the phage particles and obtain the phage library.

[0066] Coat each well with recombinant human CD20 antigen at 100 µL / well (i.e., 5 µg / well) and incubate overnight at 4°C. Discard the coating solution and wash the plate three times with ELISA wash buffer (PBST: 0.05% Tween-20 PBS). Block the plate with 5% skim milk powder and wash three times. Then add 100 µL of phage library solution to each well and incubate at 37°C for 2 hours. Wash the plate three times. Add 100 µL of Glycine-HCl buffer (pH=2.2) to each well and gently shake for 10 minutes at room temperature. Aspirate the eluent and quickly add 1M Tris-HCl buffer (pH=9.6) to the eluent to adjust the pH to 7.0-8.0, completing the first round of screening. Collect all eluted phages and add them to 10 mL of TG1 bacterial culture in the logarithmic growth phase. Incubate at 37°C for 30 min, then incubate at 220 rpm for 30 min–1 h. After adding helper phages and culturing, precipitate the phage particles as before. Repeat the above steps for the second and third rounds of screening.

[0067] Ninety-six single clones were picked from the titration plates of the elution library after the third round of screening and cultured separately. 1 μg / mL CD20 antigen was coated onto each well of a 96-well microplate, phage stock solution was added, followed by 1:5000 M13-HRP secondary antibody. TMB was used for color development, and the OD450 nm value was read using a microplate reader. The three with the highest OD values ​​were selected, as shown in Table 1.

[0068] Table 1. Results of ELISA for anti-human CD20 nanobody immunogenicity in alpacas

[0069]

[0070] As shown in Table 1, different nanobodies have different affinities for the CD20 antigen protein, with CA05 exhibiting a higher affinity.

[0071] The three nanobodies were sequenced, and the amino acid sequences of the different nanobodies are shown in Table 2.

[0072] Table 2. Amino acid sequences of different CD20-targeting nanobodies

[0073]

[0074] The amino acid sequence of the heavy chain variable region was analyzed from the nucleotide sequence encoding the selected antibody, and the complementarity-determining region (CDR) was determined according to the Kabat definition. The amino acid sequences of the CDRs of different CD20-targeting nanobodies are shown in Table 3.

[0075] Table 3. Amino acid sequence of nanobody CDR

[0076]

[0077] Example 4: Detection of nanobody expression levels

[0078] The nucleotide sequence encoding the above-mentioned nanobody was transformed into plasmid PET28a by selecting two restriction enzyme sites, NheI and XhoI. Expression was performed using *E. coli* BL21(DE)3. 10 μL of the expression strain's glycerol was added to 50 mL of LB broth and cultured at 37°C and 200 rpm for 4 h. IPTG was then added to a final concentration of 0.5 mM, and expression was induced by shaking at 37°C and 180 rpm. Six h after induction, bacterial cells were collected, and the supernatant from three bacterial strains was obtained after sonication. The supernatant was purified using nickel ion chelating packing material, equilibrated with PBS, loaded, and eluted with 500 mM imidazole. The eluent was collected to obtain the purified antibody. Identification was performed by electrophoresis on a 12% separating gel. The electrophoresis pattern is shown below. Figure 1 As shown in Table 4, protein concentration was determined using a BCA kit.

[0079] Table 4. Detection results of expression levels of different nanobodies

[0080]

[0081] From the expression levels, CA05 showed the highest expression level. Considering both expression levels and antibody activity, CA05 and CB01 were more effective. Figure 1 It can be seen that the molecular weight of the three antibodies is around 14 kD, which is consistent with expectations.

[0082] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A nanobody targeting B cell CD20, comprising a framework region and complementarity determining regions, characterized in that, The amino acid sequences of the complementarity determining regions CDR1-CDR3 of the nanobody are shown in SEQ ID NO. 1-3, respectively.

2. The Nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody is QVQLVESGGGPVQSGGSLRLSCAGSRNIYSPYSMGDYGLSWFRQAPGEEREGVALIDRDDSTFYADSVKGRFTISQDDAKTNLFLQMNSLKPEDSAMYYCAAAAKPKEHGLFDSWGQGTQVTVSS.

3. The Nanobody according to claim 1, characterized in that, It is a nanobody that is a chimeric antibody, a humanized antibody, or a fusion Fc fragment.

4. A bivalent or multivalent nanobody, characterized in that, It comprises at least one nanobody according to any one of claims 1-3.

5. Use of a Nanobody according to any one of claims 1 to 4, characterized in that, The application comprises: Preparation of a B cell immunoadsorbent. Preparation of a B cell detection reagent.

6. An immunoadsorbent comprising a solid support, characterized in that, The solid phase carrier is coupled with the nanobody according to any one of claims 1-4.

7. The immunoadsorbent of claim 6, wherein the polymer is a polyethylene glycol. The solid phase carrier is selected from at least one of chitosan, agarose, cellulose, dextran, and resin.

8. A blood purification apparatus, characterized by comprising: It comprises the immunoadsorbent according to claim 6 or 7.

9. A gene encoding the nanobody according to any one of claims 1-4.

10. An expression vector, characterized in that, It expresses the nanobody according to any one of claims 1-4, or contains the gene according to claim 9.

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