Targeting Claudin18.2 nano antibody and application thereof

Abstract

The invention discloses a Claudin18.2-targeting nano antibody and application thereof, and belongs to the technical field of molecular biology. Aiming at the unique advantages of a nano antibody in the prior art, the invention provides a nano antibody targeting Claudin18.2, the amino acid sequence of the targeting nano antibody is shown as SEQ ID NO.1, and the nano antibody has relatively strong affinity with Claudin18.2 positive cells 293T-Claudin18.2, and can be combined with Claudin18.2 protein expressed on the surfaces of the cells. The targeted Claudin18.2 nano antibody provided by the invention can be prepared through in-vitro engineering bacterium mass expression, can be applied to preparation of protein detection antibodies or therapeutic antibodies, can be used for in-vivo and in-vitro detection of tumor tissues expressed by Claudin18.2, and has important commercial value in clinical disease diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and relates to a nano antibody and an application thereof. Background Art

[0002] Claudins (CLDNs) are a multigene family encoding at least 24 tetraspanins. CLDNs are major components of tight junctions, forming paracellular barriers that control the flow of molecules between cells. CLDNs range in size from 20 to 34 kDa and share a similar overall structure, consisting of a short cytoplasmic N-terminal region, two extracellular loops consisting of four transmembrane domains, and a cytoplasmic C-terminal tail. CLDNs possess a large extracellular loop containing charged amino acids that regulate extracellular ion selectivity for anions and cations. A shorter second extracellular loop participates in dimerization with other CLDNs on the cell membrane through hydrophobic interactions between conserved aromatic residues.

[0003] Claudin18 is the most studied protein among CLDNs. The human Claudin18 gene is approximately 35 kb long and consists of six exons and five introns. Two different exons 1 undergo transcription and differential splicing, resulting in two isoforms, Claudin18.1 and Claudin18.2, each composed of 261 amino acids. These isoforms differ by 21 amino acids in the first 69 amino acids of the N-terminus and by only 8 amino acids in the extracellular domain I. Claudin18.1 is specifically expressed in normal and cancerous lung tissue, while Claudin18.2 is expressed only in differentiated gastric mucosal epithelial cells. Its expression is highly limited in normal healthy tissues, but is elevated in gastric, pancreatic, esophageal, and lung cancer tissues. Studies have shown that Claudin18.2 can restrict and regulate tight junctions between gastric mucosal epithelial cells, thereby affecting the permeability of gastric mucosal epithelial cells and maintaining the barrier function of the gastric wall.

[0004] Currently, the tight junction protein Claudin18.2, as a newly discovered target for tumor treatment, has shown great potential for clinical application. Many pharmaceutical companies around the world have laid out plans for the development of drugs targeting Claudin18.2, among which monoclonal antibody drugs account for the largest proportion, and others include bispecific antibodies, antibody-drug conjugates (ADCs), and chimeric antigen receptor T cell (CAR-T) therapies and other innovative drug forms. Zolbetuximab is a mouse chimeric monoclonal antibody with a human IgG1 constant region that can specifically bind to Claudin18.2 and mediate the specific killing of Claudin18.2-positive cells through antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. Zolbetuximab is the world's first and only approved Claudin18.2 targeted therapeutic drug, mainly used to treat Claudin18.2-positive gastric cancer or gastroesophageal junction cancer.

[0005] Since the first discovery of heavy-chain antibodies in camelids, the application of nanobodies (VHHs) derived from them has rapidly developed in a variety of fields. With a molecular weight of only 12-15 kDa, VHHs are significantly smaller than traditional monoclonal antibodies (approximately 150 kDa), making them the smallest complete antigen-binding fragment. This gives them enhanced tissue penetration and faster tumor uptake, enabling them to more effectively penetrate solid tumors, thereby enhancing therapeutic efficacy. The high solubility and stability of nanobodies enable their large-scale production in bacteria. Furthermore, the longer CDR3s of VHHs enable them to recognize and bind to hidden epitopes that are difficult for traditional antibodies to reach, such as the active sites of enzymes, thereby expanding their potential applications. In recent years, the research and development of nanobody-based drugs has progressed rapidly, demonstrating tremendous potential in areas such as targeted tumor therapy, immunomodulation, diagnostic imaging, and the treatment of infectious diseases. Their unique physicochemical properties and functional advantages have made nanobodies a hot topic in the development of next-generation antibody drugs, laying a solid foundation for their clinical translation and industrial application. Summary of the Invention

[0006] In view of the unique advantages of nanobodies in the prior art, the present invention provides a nanobody targeting Claudin18.2 and its application.

[0007] One of the objects of the present invention is to provide a nanobody targeting Claudin18.2, wherein the amino acid sequence of the nanobody targeting Claudin18.2 is shown in SEQ ID NO.1.

[0008] In a preferred embodiment of the present invention, the Nanobody targeting Claudin18.2 comprises complementarity determining regions CDR1, CDR2 and CDR3.

[0009] In a preferred embodiment of the present invention, the amino acid sequence of the CDR1 is shown in SEQ ID NO.2, the amino acid sequence of the CDR2 is shown in SEQ ID NO.3, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.4.

[0010] In a preferred embodiment of the present invention, the nanobody targeting Claudin18.2 further comprises a framework region FR1,

[0011] FR2, FR3 and FR4.

[0012] In a preferred embodiment of the present invention, the amino acid sequence of FR1 is shown as SEQ ID NO.5, the amino acid sequence of FR2 is shown as SEQ ID NO.6, the amino acid sequence of FR3 is shown as SEQ ID NO.7, and the amino acid sequence of FR4 is shown as SEQ ID NO.8.

[0013] A second object of the present invention is to provide a nucleic acid comprising a nucleic acid sequence encoding the above-mentioned nanoantibody targeting Claudin18.2 or its complementary sequence.

[0014] A third object of the present invention is to provide an expression vector containing the above-mentioned nucleic acid.

[0015] A fourth object of the present invention is to provide a host cell containing the above-mentioned expression vector.

[0016] A fifth object of the present invention is to provide the use of the above-mentioned nanoantibody targeting Claudin18.2 in the preparation of protein detection antibodies or therapeutic antibodies.

[0017] In a preferred embodiment of the present invention, the application refers to detecting tumor tissue expressing Claudin18.2.

[0018] Beneficial effects of the present invention: The present invention provides a nanobody targeting Claudin18.2. Effectiveness data demonstrate that the nanobody targeting Claudin18.2 has a strong affinity for the Claudin18.2-positive cell line 293T-Claudin18.2. Immunohistochemical staining also demonstrates that the nanobody targeting Claudin18.2 can bind to Claudin18.2 protein expressed on the cell surface. The nanobody targeting Claudin18.2 can be produced in large quantities through in vitro expression in engineered bacteria and can be used to prepare protein detection antibodies or therapeutic antibodies. It can be used for in vitro and in vivo detection of Claudin18.2-expressing tumor tissues, and has significant commercial value in clinical disease diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Figure 1 shows the results of CLDN18.2 gene cloning and eukaryotic expression vector construction in Example 1; M: DL2000 DNA molecular weight standard;

[0020] Figure 2 This is a diagram showing the identification results of the 293T cell line overexpressing CLDN18.2 in Example 1; M is a protein molecular weight standard;

[0021] Figure 3 This is the result of immunofluorescence identification of CLDN18.2 displayed on the cell membrane surface in Example 1;

[0022] Figure 4 This is a diagram showing the Western identification results of CLDN18.2 cell membrane extraction in Example 1;

[0023] Figure 5 This is a graph showing serum positive conversion after CLDN18.2 alpaca immunization in Example 2;

[0024] Figure 6 Figure 3 shows the capacity detection results of the cDNA library and phage library in Example 3;

[0025] Figure 7 This is a graph showing the recovery rate of the phage panning library based on the membrane protein display system in Example 4;

[0026] Figure 8 This is a diagram of the cell membrane extraction and identification of the CHO-CLDN18.2 cell line constructed in Example 4;

[0027] Figure 9 This is a flow cytometry analysis of the Nanobodies induced to express after three rounds of panning in Example 4;

[0028] Figure 10This is a diagram of the prokaryotic expression and purification of the CLDN18.2-targeting nanobody in Example 5;

[0029] Figure 11 This is a flow cytometry analysis of the binding of the CLDN18.2-targeting nanobody to the CLDN18.2 membrane protein in Example 5;

[0030] Figure 12 Figure 6 shows the results of prokaryotic expression and purification of the streptavidin-fused CLDN18.2 Nanobody in Example 6;

[0031] Figure 13 Figure 6 shows the purification, concentration and identification of the streptavidin-fused CLDN18.2 Nanobody.

[0032] Figure 14 Figure 2 shows the immunohistochemical identification of the binding of the streptavidin-fused CLDN18.2 nanobody to gastric cancer specimens in Example 6. DETAILED DESCRIPTION

[0033] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained from commercial channels by those skilled in the art.

[0035] In the following examples, Claudin18.2 is referred to as CLDN18.2.

[0036] The Western blotting used in this example includes the following steps:

[0037] S1: Collect protein samples for SDS-PAGE polyacrylamide gel electrophoresis at a constant voltage of 120 V;

[0038] S2: After the electrophoresis in S1, transfer the protein samples in the gel to a nitrocellulose membrane. Place the membrane in an ice box and transfer at a constant current of 230 mA for 2 h.

[0039] S3: After the transfer is completed, the membrane in S2 is placed in blocking solution (5% skim milk powder) and blocked at room temperature for 2 hours;

[0040] S4: After blocking in S3, the membrane was washed three times with 1×TBST solution, each time for 10 min, and then a

[0041] Antibody solution, incubate at room temperature for 2 h;

[0042] S5: After the primary antibody incubation in S4, the membrane was washed three times with 1×TBST solution, each time for 10 min, and then

[0043] Add the secondary antibody solution and incubate at room temperature for 1 hour;

[0044] S6: After the incubation with the secondary antibody in S5, the membrane was washed three times with 1×TBST solution for 10 minutes each time. Then, the developing solution was prepared for development. The membrane was then developed, photographed, and stored in a gel imager.

[0045] Example 1: Preparation of CLDN18.2 Immunogen

[0046] (1) The extracellular segment of CLDN18.2 target gene was amplified using the pUC19-CLDN18.2 plasmid as a template;

[0047] (2) The lentiviral plasmid pLVSIN-CLDN18.2-HA was constructed by overlap extension PCR technique, and the recombinant plasmid obtained above was verified by PCR. The results are as follows: Figure 1 As shown; and sequence the positive plasmids identified, and after successful sequencing and alignment, extract a large amount of plasmids for standby use;

[0048] (3) The recombinant plasmid pLVSIN-CLDN18.2-HA obtained in (2) was transiently transfected into 293T mammalian cells together with the packaging plasmids Gag-pol and VS VG for virus packaging. The packaged lentivirus was then used to infect 293T cells to construct the CLDN18.2-overexpressing cell line 293T-hCLDN18.2-HA. The cell membrane protein was then extracted using a kit. After the protein concentration was determined, it was stored at -80°C for future use.

[0049] In this example, Western blotting was performed on the membrane proteins extracted from the CLDN18.2 overexpressing cell line obtained above. The results are as follows: Figure 2 As shown, the target band appeared at the 23 kDa position, indicating that the CLDN18.2 overexpression cell line was successfully constructed.

[0050] In this example, the expression location of CLDN18.2 was further detected. 293T-vector and 293T-CLDN18.2 cell lines were plated in six-well plates. After 24 hours, the culture medium was aspirated and the cells were fixed with methanol for 10 minutes. The cells were then incubated with primary antibody (CLDN18.2 monoclonal antibody) at room temperature for 2 hours, washed three times with PBS for 5 minutes each time, and then incubated with secondary antibody (Alexa Fluor 500). 594-anti-mouse fluorescent antibody) at room temperature for 1 h, washed with PBS three times, 5 min each time, stained with DAPI nuclei for 30 s, and then photographed using an inverted fluorescence microscope. The results are shown in Figure 2. Figure 3 As shown, CLDN18.2 is expressed on the cell membrane surface.

[0051] This example further performed Western identification on the extracted membrane proteins, using 293T-vector as a control. The results are as follows: Figure 4 As shown, the extracted 293T-CLDN18.2 membrane protein can detect specific bands, proving that the membrane protein was successfully extracted and can be used for subsequent immunization.

[0052] Example 2: Animal Immunization Process

[0053] (1) 0.5 mg of the CLDN18.2 immunogen (CLDN18.2 membrane protein) obtained in Example 1 was mixed and emulsified with 1 mL of Freund's adjuvant to obtain a CLDN18.2 immunogen mixture, which was stored at 4°C until use;

[0054] (2) Select alpacas and record their ear numbers. Inject subcutaneously on the left and right sides of the alpaca's buttocks, with two injections on each side. Each injection point is 0.4 mL of the CLDN18.2 immunogen mixture obtained in (1). Observe for 30 minutes after the immunization to confirm that the alpaca is in good condition and has no discomfort symptoms. Vaccinate once every two weeks, for a total of four immunizations. Before each immunization, draw blood from the alpaca's neck vein, taking 10 mL of blood each time, separate the serum, and store it at -80°C for later use. On the 5th day after the last immunization, draw 100 mL of peripheral blood from the alpaca's neck to obtain the immunized alpaca's peripheral anticoagulant blood sample, i.e., the immune blood sample.

[0055] (3) First add 3 mL of cell separation solution to a 15 mL centrifuge tube, then slowly add 3 mL of the immune blood sample dilution solution obtained in (2), pre-cool the centrifuge, centrifuge at 400g for 30 minutes, observe the blood separation in the centrifuge tube, use a 200 μl pipette to draw the cotton-like upper immune cells in the middle into a new 15 mL centrifuge tube, and store the upper plasma in a new centrifuge tube and store at -80°C; add 10 mL of PBS buffer at room temperature to the above centrifuge tube, centrifuge at 400g for 20 minutes, discard the supernatant, and then continue to add 5 mL of PBS buffer at room temperature, centrifuge at 400g for 20 minutes; use a hemocytometer to count the number of cells, centrifuge and discard the supernatant, use Trizol (Sigma) to dissolve and separate the lymphocytes according to the cell number and the instructions, and store at -80°C.

[0056] In this example, positive conversion test was performed on serum from immunized alpacas with CLDN18.2. The results are as follows: Figure 5 As shown, pET22b empty vector bacteria were induced as a control, and the recombinant plasmid pET22b-CLDN 18.2-4ECL1 bacteria containing four CLDN18.2 extracellular ECL1s were induced as positive. Alpaca immune serum was used as the primary antibody, and HRP-labeled goat anti-alpaca antibody was used as the secondary antibody. The Western test results showed that four ECL1 tandemly expressed target bands were detected at the 25kDa band, indicating that the serum was successfully converted to positive after antigen immunization.

[0057] Example 3: Preparation of Nanobody Library Targeting CLDN18.2

[0058] Total RNA was extracted from the lymphocytes obtained in Example 2 using an RNA extraction kit (Invitrogen), and a cDNA library was generated using a reverse transcription kit (Invitrogen). Nested PCR was performed using specific primers for the alpaca heavy chain antibody and the heavy chain antibody variable region to amplify the VHH gene fragment; the primer sequences were the upstream primer FR1-RSCF as shown in SEQ ID NO.9 and the downstream primer VHH-RSCB as shown in SEQ ID NO.10; the above-mentioned VHH gene fragment was ligated with the phage expression vector pCombo by enzyme digestion to construct a recombinant phage vector, and the above-mentioned recombinant phage vector was transformed into Escherichia coli TG1 competent cells by electroporation for amplification. With the help of helper phage, VHH was displayed on the surface of the phage to form a VHH phage library, that is, a nanobody library targeting CLDN18.2.

[0059] In this example, the capacity of the cDNA library and phage library obtained above was tested, and the results were as follows: Figure 6As shown, the cDNA library capacity is 7.8×10 7 pfu / mL, the phage library capacity is 4.5×10 13 cfu / mL.

[0060] Example 4: Selection and identification of nanobodies targeting CLDN18.2

[0061] 1. Selection of Nanobodies Based on Phage Display Technology

[0062] (1) Negative panning

[0063] S1: Prepare negative elutriation cells 293T-vector cell line, calculate the cell number, and use 3.5×10 cells each time. 8 cells;

[0064] S2: Wash the cells in S1 twice with PBS, resuspending them in 30 mL of PBS each time, centrifuging at 4°C and 500 g for 5 min, discarding the supernatant, and resuspending them in 2 mL of PBS for the final time before placing them in cryovials.

[0065] S3: The resuspended cells obtained in S2 were centrifuged at 4°C and 500g for 5 minutes, the supernatant was discarded, and 500 μL of phage-milk / PBS was added for resuspending and incubated at room temperature for 30 minutes; then centrifuged at 13,000 rpm for 2 minutes, and the phage supernatant, i.e., the phage eluate, was collected.

[0066] (2) Positive panning

[0067] S1: Prepare positive elutriation cells 293T-CLDN18.2-HA cell line, calculate the cell number, and use 5×10 cells each time. 8 cells;

[0068] S2: Wash the cells in S1 twice with PBS, resuspending them in 30 mL of PBS each time, centrifuging at 4°C and 500 g for 5 min, discarding the supernatant, and resuspending them in 2 mL of PBS for the final time before placing them in cryovials.

[0069] S3: The resuspended cells obtained in S2 were centrifuged at 4°C and 500g for 5 minutes, the supernatant was discarded, and then 500μL of phage eluate collected by negative panning obtained in (1) was added, resuspended, and incubated at room temperature for 30 minutes; washed five times with PBS, centrifuged at 500g for 2 minutes at room temperature, and PBS was removed; 150μL Elution Buffer (glycine solution, pH 2.2) was added, and reacted at room temperature for 10 minutes; 10μL 2M Tris Base solution (glycine solution, pH 9.0) was added for neutralization; and then centrifuged at 13000rpm for 2 minutes to collect the phage eluate;

[0070] S4: Take 10 μL of the phage eluate obtained in S3 and dilute it into 5 gradients (10 1-5 Add 90 μL OD 600 TG1 bacterial solution with a concentration of 0.5-0.6 was incubated in a 37°C water bath for 15 minutes. Five gradient bacterial solutions were then spread onto 2×YT culture plates containing ampicillin resistance, inverted and cultured overnight at 37°C. The number of single colonies on the culture plates was counted to calculate the titer.

[0071] S5: Take 10 mL of the remaining phage eluate from S3 and add it to the OD 600 The mixture was added to a TG1 bacterial solution with a concentration of 0.5-0.6, and allowed to stand at room temperature for 30 minutes; 0.1% Amp resistance was added, and the culture was continued at 37°C and 250rpm for 30 minutes; then 100μL of helper phage was added, and the mixture was allowed to stand at room temperature for 30 minutes, and centrifuged at 8000rpm for 10 minutes. The precipitate after the centrifugation was transferred to a 100mL 2×YT culture plate containing 0.1% ampicillin and kanamycin resistance, and cultured overnight at 37°C and 220rpm. The phage was concentrated the next day and set aside.

[0072] (3) The VHH phage library obtained in Example 3 was incubated with the 293T-vector / 293T-h-CLDN18.2-HA cell line obtained in Part 1 of this Example. After three rounds of negative and positive panning, VHH phage that specifically binds to the CLDN18.2 membrane antigen and is highly enriched was obtained, i.e., the positive phage screening library.

[0073] In this example, the phage recovery amount of the obtained positive phage screening library was tested, and the results were as follows: Figure 7 As shown in Figure 2, the amount of phage recovered increased with each round, and the final amount of phage recovered reached 8×10 8 pfu.

[0074] 2. Construction of a CHO cell line overexpressing CLDN18.2

[0075] The lentiviral solution packaged in Example 1 was used to infect CHO cell lines to construct CHO-vector and CHO-CLDN18.2-HA cell lines. Membrane proteins were then extracted from the two cell lines. CHO-vector membrane protein was used as a control. Western blotting was performed using a CLDN18.2-specific antibody (Proteintech) as the primary antibody and an HRP-labeled goat anti-mouse secondary antibody (Thermo) as the secondary antibody to identify the CHO-CLDN18.2 membrane protein. The results are shown in Figure 2. Figure 8 As shown, the target band of 25 kDa appeared, indicating that the cell line was successfully constructed and CLDN18.2 was expressed on the surface of the CHO cell membrane.

[0076] In this Example 4, the constructed CHO-CLDN18.2 cell line was used to identify the phages induced to express nanobodies after the above three rounds of panning by flow cytometry. The primary antibody was phage No. 4 and the isotype control phage, and the secondary antibody was APC-anti-HA1.1 flow cytometry antibody (Biolegend). The results are shown in FIG. Figure 9 As shown, compared with the negative control group and the isotype control group, the No. 4 phage obtained by screening was deflected by 97.6%. The results showed that the No. 4 phage obtained by screening can specifically bind to CLDN18.2 on the membrane surface. The phage was then infected with TG1 for sequence determination.

[0077] Example 5: Prokaryotic expression, purification and identification of nanobodies targeting CLDN18.2

[0078] The positive sequence determined by sequencing in Part 2 of this Example 4 was cloned, and the prokaryotic expression vector PET22b-CLDN18.2-VHH-6his was constructed by overlap extension technology. The positive plasmid was transformed into Rosetta (DE3) competent cells for induced expression. The bacteria were picked and placed in 6 mL of LB liquid medium containing ampicillin resistance, and cultured at 37°C and 220 rpm / min for 12-15 hours to obtain a culture solution; the above 2 mL of culture solution was activated and placed in LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37°C and 220 rpm / min for 3-4 hours; when the OD value of the culture solution was 0.4, the above culture solution was induced to express with 0.2 mM IPTG at 16°C for 12 hours, centrifuged, and the supernatant, i.e., the nanobody solution containing CLDN18.2, was collected.

[0079] The nanoantibody obtained in Example 4 was purified by Ni Sepharose excel affinity chromatography using the AKATA protein purification system and then purified and identified by SDS-PAGE. The results are shown in FIG. Figure 10As shown, it was shown that the purified nanobody protein targeting CLDN18.2 was successfully obtained in this example.

[0080] The purified nanobody protein was further identified by flow cytometry using 293T-vector and 293T-CLDN18.2 cell lines as research objects. The purified nanobody was used as the primary antibody and the APC-anti-His flow cytometry antibody (Biolegend) was used as the secondary antibody. The results are shown in Figure 2. Figure 11 As shown, the 293T-h-CLDN18.2-HA cell line incubated with the nanobody group deflected 99.8% relative to the control group, indicating that the purified nanobody can specifically bind to the CLDN18.2 protein on the membrane surface.

[0081] Example 6: Application of Nanobodies Targeting CLDN18.2

[0082] In this Example 6, VHH was ligated with a prokaryotic expression vector pET22b-SA with a streptavidin (SA) tag to construct a prokaryotic expression vector expressing a VHH-SA fusion protein. The vector was transformed into a Rosetta (DE3) competent cell and induced for expression. The vector was purified by nickel column affinity chromatography. The results are shown in FIG. Figure 12 As shown, the VHH-SA fusion protein was purified and then subjected to gradient dialysis renaturation and ultrafiltration concentration. Figure 13 As shown, SDS-PAGE analysis showed that the purity of the purified VHH-SA protein reached more than 90%.

[0083] In Example 6, VHH-SA was used as the primary antibody and biotinylated horseradish peroxidase was used as the secondary antibody to detect the binding ability of VHH to CLDN18.2 in natural gastric tissue. Figure 14 As shown, immunohistochemistry (IHC) results showed that the VHH could specifically recognize CLDN18.2 protein in gastric tissue with good specificity compared with the control antibody. The Nanobodies targeting CLDN18.2 provided by the present invention can specifically bind to CLDN18.2 protein on the cell membrane surface and can therefore be used as detection and therapeutic antibodies.

[0084] Any matters not described in detail in this specification are well known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, these are not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A nanobody targeting Claudin18.2 and its application, characterized in that: The amino acid sequence of the nanobody targeting Claudin18.2 is shown in SEQ ID NO.

1.

2. The nanobody targeting Claudin18.2 according to claim 1, characterized in that The nanobody targeting Claudin18.2 includes complementary determining regions CDR1, CDR2 and CDR3.

3. The nanobody targeting Claudin18.2 according to claim 2, characterized in that The amino acid sequence of the CDR1 is shown in SEQ ID NO.2, the amino acid sequence of the CDR2 is shown in SEQ ID NO.3, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.

4.

4. The nanobody targeting Claudin18.2 according to claim 1, characterized in that The nanobody targeting Claudin18.2 further comprises framework regions FR1, FR2, FR3 and FR4.

5. The nanobody targeting Claudin18.2 according to claim 4, characterized in that The amino acid sequence of the FR1 is shown in SEQ ID NO.5, the amino acid sequence of the FR2 is shown in SEQ ID NO.6, the amino acid sequence of the FR3 is shown in SEQ ID NO.7, and the amino acid sequence of the FR4 is shown in SEQ ID NO.

8.

6. A nucleic acid, characterized in that The nucleic acid comprises a nucleic acid sequence encoding the Nanobody targeting Claudin18.2 according to any one of claims 1 to 5 or its complementary sequence.

7. An expression vector, characterized in that The expression vector contains the nucleic acid according to claim 6.

8. A host cell, characterized in that The host cell contains the expression vector according to claim 7.

9. Use of the nanobody targeting Claudin18.2 according to any one of claims 1 to 5 in the preparation of a protein detection antibody reagent or a therapeutic antibody.

10. The use according to claim 9, characterized in that The application refers to detecting tumor tissue expressing Claudin18.2.

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