Nanometer antibody targeting B cell CD20 and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a nano antibody targeting B cell CD20 and application of the nano antibody. The amino acid sequences of the complementarity determining regions CDR1-CDR3 of the nano antibody are selected from SEQ ID NO.1-9. The nano antibody provided by the invention can be specifically combined with B cells in human blood, so that the B cells generating pathological antibodies are removed. Compared with the traditional antibody, the nano antibody provided by the invention has the advantages of smaller molecular weight, high stability, low cost, easiness in expression and high expression quantity, and is suitable for large-scale production. The nano antibody is combined with the solid-phase carrier, B cells in blood can be cleared in a targeted mode, the number of the B cells in the blood of a patient can be reduced in a short time, the antibody level can be reduced, therefore, the life quality is improved, complications are reduced, and a new treatment choice is provided for the patient who is ineffective or intolerant to traditional treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a nano antibody targeting B cell CD20 and an application thereof. Background Art

[0002] B cells are important lymphocytes in the immune system. Malignant tumors, autoimmune diseases, lymphomas, and leukemia are common B cell-related diseases. The pathogenesis of these diseases is related to the excessive activation of B cells to produce a large number of autoantibodies. Therefore, B lymphocyte targeted therapy targeting B lymphocyte surface molecules has received extensive attention and research in recent years. The B lymphocyte clearance strategy is to use antibody drugs to target specific antigens of B cell subsets, induce B cell apoptosis through antibody-dependent cytotoxicity or complement, or by targeting cytokines required for B cell survival, such as B cell activation factor (-cell activation factor, BAFF), to achieve the purpose of clearing B cells. The main targets of B cell clearance are CD20, CD19, and BAFF, which 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 mainly expressed by B cells, downregulated during the process of initial B cell proliferation and activation to plasma cells, and lost in terminally differentiated plasmablasts and plasma cells. CD20 is specifically expressed in B cells and is not expressed in monocytes and granulocytes, most T cells, and other tissue cells. Drugs targeting CD20 can reduce the level of plasma cells that produce autoimmune antibodies by blocking their differentiation into plasma cells, while retaining the regenerative pool of B cell precursors, naive B cells, and long-lived plasma cells.

[0004] At present, B lymphocyte depletion therapy represented by CD20 monoclonal antibodies is a relatively mature solution, such as Rituximab, Ocrelizumab, and Ofatumumab. In addition to B cell depletion, there are also neutralizing drugs targeting B cell growth factor (BAFF), including GAFF neutralizing antibodies (such as Belimumab). Rituximab is a chimeric anti-CD20 monoclonal antibody (chimeric anti-CD20 mAb) composed of human IgG1 and mouse variable regions. It can specifically bind to the CD20 antigen on the surface of B cells and induce B cell apoptosis (self-destruction) to destroy 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, rheumatoid arthritis, etc. Due to its chimeric structure, Rituximab has better immunogenicity and tolerability than mouse antibodies.

[0005] However, studies have shown that long-term use of Rituximab may lead to temporary suppression of the immune system and increase the risk of infection. Some patients may have allergic reactions to the monoclonal antibody or its components, manifested as rash, difficulty breathing and other symptoms, and lead to 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. Nanobodies are a special type of antibody extracted from camelids (such as camels, alpacas, and llamas) that is only one-tenth the size of conventional antibodies. This allows them to penetrate tissues more easily and reach places that conventional antibodies cannot reach. Nanobodies have extremely high thermal and chemical stability, allowing them to remain active in various harsh environments. Due to the simple structure of nanobodies, they can be easily genetically engineered to produce antibodies with specific functions. In addition, nanobodies can also be mass-produced by microorganisms such as bacteria and yeast, greatly reducing production costs. Nanobodies can recognize multiple antigen epitopes at the same time, which makes them have great potential in the diagnosis and treatment of various diseases. Due to their unique structure and properties, nanobodies show efficient performance in targeted drug delivery, immunodiagnosis, tumor treatment, etc. Nanobodies have been widely used in medicine, biotechnology, agriculture and other fields, including early diagnosis and treatment of diseases, as well as as research tools. The development of nanobodies targeting B cells is of vital importance for the treatment and research of B cell-related diseases.

[0006] Blood purification is a vital medical technology that treats a variety of 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 adsorbed in a targeted manner. Immunoadsorption is a technology that uses specific antigens, antibodies, or ligands with unique physicochemical affinity to bind to an adsorption medium to form a selective or specific adsorption column. When used in combination with drug therapy, immunoadsorption can significantly improve the patient's responsiveness to drugs, thereby enhancing drug effects, reducing side effects, shortening treatment time, and reducing the risk of disease recurrence. By combining antibodies targeting B cells with solid phase carriers, B cells in human blood can be specifically bound to eliminate B cells that produce pathological antibodies. High adsorption efficiency, stable adsorption performance, targeted elimination of B cells, low nonspecific adsorption, good specificity, good safety performance in clinical treatment, and high economic benefits. It may become a new way to treat B cell-related diseases. Summary of the invention

[0007] The object of the present invention is to overcome at least one deficiency of the prior art and to provide a nanobody targeting B cell CD20 and its application.

[0008] The technical solution adopted by the present invention is: In a first aspect, the present invention provides a nanobody targeting B cell CD20, comprising a framework region and a complementarity determining region, wherein the amino acid sequence of the complementarity determining region CDR1 to CDR3 of the nanobody is selected from any one of the following combinations: Combination 1, wherein the amino acid sequences of CDR1 to CDR3 are shown as SEQ ID NO.1 to SEQ ID NO.3, respectively; Combination 2, wherein the amino acid sequences of CDR1 to CDR3 are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively; Combination 3, the amino acid sequences of CDR1 to CDR3 are shown in SEQ ID NO.7 to SEQ ID NO.9, respectively.

[0009] In some examples, it is a chimeric antibody, a humanized antibody, a Nanobody fused to an Fc fragment.

[0010] In some examples, the Nanobody amino acid sequence is selected from any one of the following combinations: CA05: QVQLVESGGGPVQSGGSLRLSCAGSRNIYS PYSMGDYGLS WFRQAPGEEREGVA LIDRDDSTFYADSV KG RFTISQDDAKTNLFLQMNSLKPEDSAMYYCAA AAKPKEHGLFDS WGQGTQVTVSS; CB01: QVQLQESGGGPVQAGDSLRLSCAASGNIYS GNIYSRHSMG WFRQVPGKEREGVA INRDDSS RFTLSQDDAKTNLFLQMNSLKPTDTAYYCAA GWMATLAWSDWF RGRGTQVTVSS; CD09: QVQLQESGGGPVQSGGSLRLSCAAS RNIYSRPYMA WFRQAPGKEREGVAT SSTFGST VYADSVKGRFTISRDDAKTNLFLQMNSLKPEDSAMYYC AAGFDYLSPSDYGY WGQGTQVTVSS.

[0011] In the above sequences, the underlined parts are CDR regions, and the unmarked parts are FR regions.

[0012] In some examples of Nanobodies, they are chimeric antibodies, humanized antibodies, Nanobodies fused to an Fc fragment.

[0013] By introducing the antibody Fc region, the dimerization of nanobodies can be achieved. The introduction of the Fc region can also utilize the affinity of Fc to simplify the purification operation of the antibody. The introduction of Fc is also conducive to extending the half-life of the antibody and improving the stability of the antibody in vivo. There are no special requirements for the antibody Fc region, and the Fc region of a human antibody is preferred. The antibody Fc region includes the Fc segment of human IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgM, IgE or its variants and modifications.

[0014] In a second aspect, the present invention provides a bivalent or multivalent Nanobody comprising at least one Nanobody as described in the first aspect of the present invention.

[0015] A bivalent or multivalent nanobody is formed by connecting a single nanobody 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 more, preferably, n is an integer of 1 to 4. The nanobodies in a bivalent or multivalent nanobody may be the same or different.

[0016] In a third aspect, the use of the Nanobodies described in the first aspect and the second aspect of the present invention, the use includes but is not limited to: preparing B cell immunoadsorbent; preparing B cell detection reagents; preparing CD20-targeted immunoconjugates; Preparation of antibody drugs for treating B cell related diseases.

[0017] In some examples, the immunoconjugate is composed of a nanobody and a conjugate connected to the nanobody, such as an ADC drug composed of an antibody and a small molecule.

[0018] In some examples, the conjugate is a drug.

[0019] In some examples, the B cell-related disease is selected from a tumor and an autoimmune disease.

[0020] In some examples, tumors include but are not limited to multiple myeloma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, glioblastoma; autoimmune diseases include but are not limited to lupus erythematosus, myasthenia gravis, rheumatoid arthritis, idiopathic inflammatory myositis, systemic sclerosis, primary Sjögren's syndrome.

[0021] In a fourth aspect, the present invention provides an immunosorbent comprising a solid phase carrier, to which the nanobodies described in the first aspect of the present invention and the second aspect of the present invention are coupled.

[0022] In some examples, the solid phase carrier is selected from at least one of chitosan, agarose, cellulose, dextran, resin, and cellulose.

[0023] In a fifth aspect, the present invention provides a blood purification device comprising the immunosorbent described in the fourth aspect of the present invention.

[0024] In a sixth aspect, the present invention provides a gene encoding the Nanobody according to the first aspect of the invention or the second aspect of the invention.

[0025] In a seventh aspect, the present invention provides an expression vector that expresses the Nanobody described in the first aspect of the present invention or the second aspect of the present invention, or contains the gene described in the sixth aspect of the present invention.

[0026] The beneficial effects of the present invention are: The nanobody provided by the present invention can specifically bind to B cells in human blood, thereby eliminating B cells that produce pathological antibodies. Compared with traditional antibodies, the nanobody provided by the present invention has a smaller molecular weight, strong stability, low cost, easy expression and high expression amount, and is suitable for large-scale production.

[0027] The present invention combines nano-antibodies with solid phase carriers, which may target and eliminate B cells in the blood, reduce the number of B cells in the patient's blood in a shorter period of time, and reduce antibody levels, thereby improving the quality of life and reducing complications, providing a new treatment option for patients who are ineffective or intolerant to traditional treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the electrophoresis diagram of the purified nanobody, in which the molecular weights of the marker bands are 180kD, 130kD, 100kD, 70kD, 55kD, 40kD, 35kD, 25kD, 15kD, and 10kD from top to bottom. DETAILED DESCRIPTION

[0029] The following disclosure provides many different embodiments or examples for implementing different solutions of the present invention.

[0030] The term "antigen binding fragment" used herein refers to one or more parts of an antibody structure that specifically binds to a target antigen, including but not limited to VHH, fragments containing CDRs, etc. "Specific binding" mentioned herein refers to a non-covalent interaction between a nanobody or its antigen binding fragment and an antigen. In the present invention, the term "affinity" refers to the intensity of the interaction between a macromolecule, especially a nanobody, and its corresponding antigen. This binding ability reflects the interaction characteristics between a nanobody and a specific antigen. To accurately evaluate the affinity of the nanobody in the present invention, a variety of in vitro experimental methods can be used for measurement, including surface plasmon resonance technology and ELISA determination.

[0031] In the present invention, the term "variable" used refers to the differentiated characteristics of the variable region in the antibody in terms of sequence, which contributes to the precise binding and recognition of different antibodies to their specific antigens. The variability of antibodies is mainly concentrated in the three complementary determining regions (CDRs) of the heavy chain variable region, also known as hypervariable regions. In the natural heavy chain variable region, there are four framework regions (FR regions), which are relatively conservative and usually present a β-folded configuration. The FR region is connected by three CDRs that form a connecting loop, and these CDRs locally form a β-folded structure. The CDRs in each chain are closely adjacent through the FR region and together with the CDRs of the other chain constitute the antigen binding site of the antibody. Although the constant region is not directly involved in the binding process of the antibody to the antigen, they exhibit different effector functions, such as participating in mediating antibody-dependent cellular toxicity.

[0032] In immunology, an "epitope" refers to an area on the surface of an antigen molecule that can be recognized and bound by a single antibody molecule. Typically, an antigen may have multiple different epitopes, which allows it to react with a variety of different antibodies. Epitopes are mainly divided into two categories: linear epitopes and conformational epitopes. Linear epitopes are composed of continuous amino acid sequences in antigen molecules, while conformational epitopes are formed by discontinuous amino acid residues in antigen molecules that are close to each other through spatial structures. Both types of epitopes are key sites for antibody recognition and binding.

[0033] "Biepitopic 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.

[0034] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse / camelid antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse / camelid antibody.

[0035] The term "humanized antibody" refers to an antibody produced by transplanting the CDR sequence of a camel-derived VHH into a human antibody variable region framework, i.e., different types of human germline antibody framework sequences. The heterologous reaction induced by chimeric antibodies due to carrying a large amount of camel-derived protein components can be overcome. Such framework sequences can be obtained from public DNA databases or public references including germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be obtained in 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. In order to avoid a decrease in immunogenicity and a decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies after affinity maturation of CDRs by phage display.

[0036] The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen.

[0037] The term "antibody framework" as used herein refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loop (CDR) of the variable domain. In essence, it is a variable domain without CDRs.

[0038] The technical solution of the present invention is further illustrated below in combination with examples and experimental data.

[0039] Example 1: Preparation of protein with CD20 as antigen The following antigens were used to prepare anti-CD20 antibodies. A polypeptide of amino acid residues 265-290 at the C-terminus of the amino acid sequence of human CD20 (Uniprot accession number P11836-1, with a specific amino acid sequence of MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP, as shown in SEQ ID NO. 13) was used as an antigen.

[0040] The preparation method of CD20 antigen includes: preparing a nucleic acid sequence molecule of an antigen containing amino acid residues 265-290 of human CD20 with a human His tag at its C-terminus, cloning it into a plasmid pcDNA3.1, transforming the cloned vector into HEK293T cells for expression, and purifying it using a cobalt ion column to obtain the CD20 antigen.

[0041] Example 2: Alpaca Immunization with CD20 Antigen During the immunization process, about 0.5 mg of antigen is used each time, and the total volume does not exceed 1.5 mL. The injection site is near the alpaca's cervical lymph nodes, divided into the left and right sides for injection, 2 injection points on each side, and about 0.4 mL of emulsified antigen is injected at each point. Immunization is performed every two weeks, and at least 5 times are required. Blood samples need to be collected before each immunization for immune effect evaluation. 5-7 days after the 5th immunization, a large amount of blood is collected to separate lymphocytes.

[0042] The isolation of lymphocytes is as follows: blood is collected from the jugular vein of the alpaca, and a 50 mL blood sample is collected using an anticoagulant (EDTA). The collected blood is mixed and diluted with an equal volume of phosphate buffered saline (PBS) or serum-free medium, and then the lymphocyte separation medium is added to the centrifuge tube, and the diluted blood is slowly added to the upper layer of the separation medium for density gradient centrifugation, usually at 400g to 600g at room temperature for 20 to 30 minutes. After centrifugation, the lymphocyte layer is carefully aspirated from the formed layer, transferred to a new centrifuge tube, and the cells are washed with a large amount of PBS or serum-free medium, followed by centrifugation at 200g to 300g for 10 minutes to precipitate the cells and discard the supernatant, and repeat the wash 2 times. Finally, the lymphocytes are resuspended in the culture medium for counting and survival assessment by trypan blue staining.

[0043] Next is the RNA extraction and cDNA synthesis step: the lymphocytes are mixed with chloroform, centrifuged, and the supernatant is transferred to a new centrifuge tube. Isopropanol is added to precipitate the RNA, and then the precipitate is washed with 75% ethanol and finally dissolved in RNase-free water. According to the operating instructions of the reverse transcription kit, the extracted RNA is reverse transcribed into cDNA.

[0044] The steps of antibody fragment amplification are as follows: First, cDNA is used as a template and specific primers are used to perform PCR amplification on the VHH antibody fragment. The target band is separated by agarose gel electrophoresis. Then, the DNA recovered after the first PCR amplification is used as a template, and PCR amplification is performed again to increase the content of the specific antibody fragment. Finally, the amplified antibody fragment is cloned into a phage plasmid.

[0045] Example 3: Construction and screening of CD20 nanobody phage library The process of constructing a phage library mainly includes three key steps: First, select two restriction sites, SacI and SpeI, and connect the diversified antibody gene sequences obtained by PCR amplification with the phagemid vector pComb3XSS to form a recombinant vector and transform it into E. coli TG1 competent cells. Use electroporation technology to transform the cells, and then culture them in recovery medium. Transfer 10ml of E. coli TG1 cultured to the logarithmic phase to a new sterile 50ml centrifuge tube and add 4×10 10pfu of M13KO7 helper phage, gently mix, and incubate at 37℃ water bath for 30 minutes to allow phage to infect E. coli. Centrifuge at 2,800g, room temperature for 10 minutes, and carefully remove the supernatant to remove the glucose in the culture medium. Then resuspend the bacteria in 50ml 2×TY culture medium (containing 100μg / ml ampicillin and 25μg / ml kanamycin) in a 250ml conical flask, and culture overnight at 30℃, 200rpm. Centrifuge, add 20% PEG300 / 2.5MNaCl solution to the supernatant, and precipitate the phagemid to obtain the phage library.

[0046] Coat the ELISA plate with recombinant human CD20 antigen at 100µL / well (i.e. 5µg / well) overnight at 4°C, discard the coating solution, wash the plate three times with ELISA washing solution (PBST: PBS with 0.05% Tween-20); block with 5% skimmed milk powder, wash the plate three times, then add 100µL of phage library solution to each well, incubate at 37°C for 2h, and wash the plate three times; add 100µL of Glycine-HCl buffer (pH=2.2) to each well, shake gently at room temperature for 10min, aspirate the eluate, and then quickly add 1M Tris-HCl buffer (pH=9.6) to the eluate to adjust the pH to 7.0-8.0 to complete the first round of screening. Pipette all eluted phages and add them to 10 mL of TG1 bacterial solution grown to the logarithmic phase. After standing at 37°C for 30 minutes, culture at 220 rpm for 30 minutes to 1 hour. After adding auxiliary phages, precipitate phagemids as above. Repeat the above steps for the second and third rounds of screening.

[0047] 96 monoclones were picked from the elution library titration plate after the third round of screening and cultured separately. 1 μg / mL CD20 antigen was coated in a 96-well ELISA plate, phage stock solution was added, 1:5000 M13-HRP secondary antibody was added, TMB was used for color development, and the OD450 nm value was read using an ELISA reader. The three with the highest OD values ​​were selected, as shown in Table 1.

[0048] Table 1. ELISA results of anti-human CD20 nanoantibody alpaca immune titer

[0049] As can be seen from Table 1, different nanobodies have different affinities for the antigen CD20 protein, and CA05 has a higher affinity.

[0050] The above three nanobodies were sequenced, and the amino acid sequences of different nanobodies were obtained as shown in Table 2.

[0051] Table 2. Amino acid sequences of different CD20-targeting nanobodies

[0052] 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.

[0053] Table 3. Amino acid sequences of Nanobody CDRs

[0054] Example 4: Detection of the expression level of nanobodies The two restriction sites NheI and XhoI were selected to transfer the nucleotide sequence encoding the above-mentioned nanoantibody into the plasmid PET28a, and expressed in Escherichia coli BL21 (DE) 3. 10 μL of the expression strain glycerol bacteria was added to 50 mL LB liquid culture medium, cultured at 37°C and 200 rpm for 4 hours, and then IPTG was added to a final concentration of 0.5 mM. Expression was induced by shaking culture at 37°C and 180 rpm. The bacteria were collected 6 hours after induction, and the supernatants of the three strains were obtained after ultrasonic disruption. The supernatants were purified with nickel ion chelating fillers, balanced with PBS solution, loaded, and then eluted with 500 mM imidazole to collect the eluate to obtain the purified antibodies. The identification was performed by electrophoresis on a 12% separation gel, and the electrophoresis pattern is shown as follows Figure 1 The protein concentration was determined using a BCA kit, and the results are shown in Table 4.

[0055] Table 4. Detection results of expression levels of different nanobodies

[0056] From the expression level, CA05 has the highest expression level. Combining the expression level and antibody activity, CA05 and CB01 have better effects. Figure 1 It can be seen that the molecular weights of the three antibodies are all around 14 kD, which is consistent with expectations.

[0057] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A nanobody targeting B cell CD20, comprising a framework region and a complementarity determining region, characterized in that: The amino acid sequence of the complementary determining regions CDR1 to CDR3 of the Nanobody is selected from any one of the following combinations: Combination 1, wherein the amino acid sequences of CDR1 to CDR3 are shown as SEQ ID NO.1 to SEQ ID NO.3, respectively; Combination 2, wherein the amino acid sequences of CDR1 to CDR3 are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively; Combination 3, the amino acid sequences of CDR1 to CDR3 are shown in SEQ ID NO.7 to SEQ ID NO.9, respectively.

2. The Nanobody according to claim 1, characterized in that The amino acid sequence of the Nanobody is selected from one of the following sequences: CA05: QVQLVESGGGPVQSGGSLRLSCAGSRNIYSPYSMGDYGLSWFRQAPGEEREGVALIDRDDSTFYADSVKGRFTISQDDAKTNLFLQMNSLKPEDSAMYYCAAAAKPKEHGLFDSWGQGTQVTVSS; CB01: QVQLQESGGGPVQAGDSLRLSCAASGNIYSGNIYSRHSMGWFRQVPGKEREGVAINRDDSSRFTLSQDDAKTNLFLQMNSLKPTDTAYYCAAGWMATLAWSDWFRGRGTQVTVSS; CD09: QVQLQESGGGPVQSGGSLRLSCAASRNIYSRPYMAWFRQAPGKEREGVATSSTFGSTVYADSVKGRFTISRDDAKTNLFLQMNSLKPEDSAMYYCAAGFDYLSPSDYGYWGQGTQVTVSS.

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

4. A di- or multivalent nanobody, characterized in that: Comprising at least one Nanobody according to any one of claims 1 to 3.

5. The use of the nanobody according to any one of claims 1 to 4, characterized in that: The applications include: preparing B cell immunoadsorbent; preparing B cell detection reagents; preparing CD20-targeted immunoconjugates; Preparation of antibody drugs for treating B cell related diseases.

6. The use according to claim 5, characterized in that: The conjugate is a drug, and the B cell-related disease is selected from tumors and autoimmune diseases.

7. An immunosorbent, comprising a solid phase carrier, characterized in that: The solid phase carrier is coupled with the nanobody according to any one of claims 1 to 4, and preferably, the solid phase carrier is selected from at least one of chitosan, agarose, cellulose, dextran, resin, and cellulose.

8. A blood purification device, characterized in that: Comprising the immunoadsorbent according to claim 7.

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

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

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