Nanobodies targeting mesothelin and uses thereof

Abstract

The application discloses a nanobody targeting mesothelin and application thereof, and belongs to the technical field of molecular biology. In view of the unique advantages of nanobodies in the prior art, the application provides a nanobody targeting mesothelin, wherein the nanobody targeting mesothelin comprises CDR1 as shown in SEQ ID NO. 1, CDR2 as shown in SEQ ID NO. 2 and CDR3 as shown in SEQ ID NO. 3. The nanobody targeting mesothelin has strong binding force with cells overexpressing mesothelin. It can be seen that the nanobody targeting mesothelin can bind to mesothelin protein expressed on the surface of cells, and can be applied to preparation of a protein detection antibody or a therapeutic antibody, and has important commercial value in clinical disease diagnosis and treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular biology, and relates to a nanobody and application thereof. BACKGROUND

[0002] Mesothelin (MSLN) is a glycosylated phosphatidylinositol-anchored protein, which is usually expressed in small amounts on the surface of mesothelial cells of the pleura, pericardium and peritoneum; the precursor of MSLN is a 71KD glycoprotein, which releases 31KD megakaryocyte potentiating factor (MPF) and 40KD mature MSLN after enzymatic digestion. MSLN is identified as a CA125 receptor mediating cell adhesion, and the interaction of CA125 and MSLN increases the motility and invasion of pancreatic cancer cells. Overexpression of MSLN can activate the Akt / PI3K / NFκB pathway to inhibit cell apoptosis, or promote cell proliferation, migration and metastasis by inducing activation and expression of MMP7 and MMP9. Preclinical and clinical studies have shown that increased tumor burden and low overall survival are associated with increased MSLN expression, and abnormal MSLN expression on tumor cells plays an important role in promoting proliferation and invasion, so MSLN has become one of the popular targets for anti-tumor therapy.

[0003] At present, the immunotherapy strategies targeting MSLN include the use of chimeric monoclonal antibodies (Amatuximab), antibody-drug conjugates (Anetumab ravtansine, BMS-986148 and BAY2287411), immunotoxins (SS1P and LMB-100), cancer vaccines and CAR-T cell immunotherapy. Amatuximab and Anetumab ravtansine have progressed to clinical trial phase II. Amatuximab monoclonal antibody binding to MSLN can trigger antibody-dependent cellular cytotoxicity (ADCC) and inhibit the adhesion of MSLN-expressing tumor cells to MUC16-expressing tumor cells. Anetumab ravtansine (AR) is a human anti-MSLN antibody coupled with a microtubule inhibitor drug DM4, which can destroy the function of microtubules. SS1P immunotoxin is composed of a high-affinity murine-derived antibody variable fragment (Fv) that binds to MSLN MDR and is fused with a truncated form of Pseudomonas exotoxin A (PE). In a mouse model, SS1P combined with gemcitabine has strong anti-tumor activity against MSLN-expressing tumors.

[0004] In 1989, researchers discovered a heavy chain antibody derived from Camelidae, which naturally lacks light chains, with a variable region molecular weight of only 15KD, which is 1 / 10 of the molecular weight of conventional antibodies. Its protein crystal structure is 4nm in length and 2.5nm in diameter, which is the smallest antibody known with a molecular weight. It is called nanobody. Nanobody can more easily penetrate cell membranes and tissue barriers, enter deep tissues and specific cells, and has a longer active binding region than traditional antibodies, with 16-18 amino acids, high affinity and specificity, and can bind to specific epitopes of target molecules to achieve highly accurate recognition and intervention. Nanobody has become an important tool in research and treatment. Nanobody also has good stability, can maintain its structure and function under a wide range of environmental conditions, and has high thermal and chemical stability, allowing it to adapt to various application scenarios and be less susceptible to damage during storage and transportation. In addition, unlike the hybridoma cell preparation method of traditional antibodies, the method for preparing nanobody is the engineering bacteria expression method, which has the advantages of easy expression, easy genetic engineering modification, relatively low production cost and easy large-scale production. SUMMARY

[0005] In view of the unique advantages of nanobody in the prior art, the present application provides a nanobody targeting mesothelin and applications thereof.

[0006] One of the purposes of the present application is to provide a nanobody targeting mesothelin, wherein the amino acid sequence of the nanobody targeting mesothelin is shown in SEQ ID NO. 8.

[0007] In a preferred embodiment of the present application, the nanobody targeting mesothelin comprises complementarity determining regions CDR1, CDR2 and CDR3.

[0008] In a preferred embodiment of the present application, the amino acid sequence of CDR1 is shown in SEQ ID NO. 1, the amino acid sequence of CDR2 is shown in SEQ ID NO. 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 3.

[0009] In a preferred embodiment of the present application, the nanobody targeting mesothelin further comprises framework regions FR1, FR2, FR3 and FR4.

[0010] In a preferred embodiment of the present application, the amino acid sequence of FR1 is shown in SEQ ID NO. 4, the amino acid sequence of FR2 is shown in SEQ ID NO. 5, the amino acid sequence of FR3 is shown in SEQ ID NO. 6, and the amino acid sequence of FR4 is shown in SEQ ID NO. 7.

[0011] The second object of the present application is to provide a nucleic acid comprising a nucleic acid sequence encoding the above-mentioned mesothelin-targeting nanobody or a complementary sequence thereof.

[0012] The third object of the present application is to provide an expression vector containing the above-mentioned nucleic acid.

[0013] The fourth object of the present application is to provide a host cell containing the above-mentioned expression vector.

[0014] The fifth object of the present application is to provide the use of the above-mentioned mesothelin-targeting nanobody in the preparation of a protein detection antibody or a therapeutic antibody.

[0015] In a preferred embodiment of the present application, the use refers to the detection of tumor tissues expressing mesothelin.

[0016] The present application has the following advantages:

[0017] The present application provides a mesothelin-targeting nanobody. The flow cytometry detection results show that the mesothelin-targeting nanobody of the present application can specifically bind to 293T-MSLN cells expressing MSLN protein on the cell surface, and the above-mentioned nanobody can be prepared by large-scale expression of an in vitro engineering bacterium, can be applied to the preparation of a protein detection antibody or a therapeutic antibody, and can be used for in vivo and in vitro detection of tumor tissues expressing MSLN, and has important commercial value in clinical disease diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure 1 is a diagram of the construction results of the eukaryotic expression vector pCAGGS-SP-MSLN-6His in Example 1; M1 is a DL15000 DNA marker, and M2 is a DL2000 DNA marker;

[0019] Figure 2 Figure 2 is a diagram of the identification of MSLN recombinant protein expression in Example 1; M is a protein molecular weight standard; MSLN-EC is MSLN recombinant protein;

[0020] Figure 3 Figure 3 is a diagram of the Ni 2+ affinity purification identification of MSLN recombinant protein in Example 1; M is a protein molecular weight standard; lane 1 is supernatant of expressed protein, lane 2 is effluent of column loading, lane 3 is sample washing liquid, lanes 4-6 are eluent of pH=3.5, lanes 7-9 are eluent of pH=3.0, lanes 10-12 are eluent of pH=2.8, lanes 13-15 are eluent of pH=2.6, and lanes 16-18 are eluent of pH=2.5;

[0021] Figure 4 This is a graph showing the serum antibody titer determination in Example 2;

[0022] Figure 5 This is a graph showing the capacity detection of the cDNA library and phage library in Example 3;

[0023] Figure 6 This is a diagram showing the construction results of the MSLN membrane protein overexpression cell line in Example 4; M represents the protein molecular weight standard.

[0024] Figure 7 This is a graph showing the flow cytometry results from Example 4.

[0025] Figure 8 This is a graph showing the recovery rate of the phage library in Example 4.

[0026] Figure 9 This is a graph showing the indirect ELISA identification results of the nanobody in Example 5;

[0027] Figure 10 Ni is the prokaryotic expression protein of the nanobody in Example 5. 2+ Affinity purification identification diagram; M is the molecular weight standard of the protein; lane 1 is the supernatant of the expressed protein, lane 2 is the elution solution, lanes 3-4 are the sample washing solution, lanes 5-7 are 50mM imidazole elution solution, lanes 8-10 are 100mM imidazole elution solution, lanes 11-12 are 200mM imidazole elution solution, lanes 13-14 are 300mM imidazole elution solution, lanes 15-16 are 400mM imidazole elution solution, and lanes 17-18 are 500mM imidazole elution solution;

[0028] Figure 11 This is a Western blot image of the purified 3A12 nanobody from Example 5; lane 1 is the purified 3A12 nanobody; lane 2 is the positive control.

[0029] Figure 12 This is an affinity identification diagram of the purified nanobody in Example 5.

[0030] Figure 13 This is an identification diagram of the mesothelin-targeting nanobody 3A12 as a flow cytometry antibody in Example 6. Detailed Implementation

[0031] Those skilled in the art can improve the process parameters according to the content herein. In particular, it is pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present application, to realize and apply the present application technology.

[0032] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below in combination with specific embodiments and the drawings of the specification. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art.

[0033] Mesothelin: Mesothelin, MSLN;

[0034] The western used in this embodiment includes the following steps:

[0035] S1: Collect protein samples for SDS-PAGE polyacrylamide gel electrophoresis, and perform constant voltage electrophoresis at 120V;

[0036] S2: Transfer the protein sample in the gel after electrophoresis in S1 to a nitrocellulose membrane, and transfer the membrane at a constant current of 230mA for 1h in an ice box;

[0037] S3: After the transfer in S2 is completed, the membrane is placed in blocking solution (5% skim milk powder) for room temperature blocking for 2h;

[0038] S4: The membrane after blocking in S3 is washed with 1xTBST solution for 3 times, 5min each time, and then a primary antibody solution is added

[0039] and incubated at room temperature for 2h;

[0040] S5: The membrane after incubation of the primary antibody in S4 is washed with 1xTBST solution for 3 times, 5min each time, and then a secondary antibody solution is added

[0041] and incubated at room temperature for 1h;

[0042] S6: The membrane after incubation of the secondary antibody in S5 is washed with 1xTBST solution for 3 times, 5min each time, and then a developing solution is prepared for development, and then developed, photographed and saved in a gel imager.

[0043] Example 1: Preparation of MSLN-EC recombinant protein

[0044] (1) The MSLN extracellular segment target gene MSLN-EC was amplified using the pMD18-T-MSLN plasmid as a template;

[0045] (2) The eukaryotic expression vector pCAGGS-SP-MSLN-EC-6his was constructed by overlap extension PCR technology and the target gene obtained in (1), and PCR verification was performed on the eukaryotic expression vector obtained above, the results are shown in Figure 1 ; and the PCR product was sequenced, and after success, the plasmid was extracted in large quantities, and the PCR verification and sequencing results proved that the eukaryotic expression vector was successfully constructed, and the eukaryotic expression vector pCAGGS-SP-MSLN-EC-6His was obtained.

[0046] (3) The plasmid obtained in (2) was used to transfect the HEK293 mammalian suspension expression system to express the recombinant protein, and then the Ni Sepharose excel affinity chromatography filler of Cytiva Company was used to purify the target protein, and after protein concentration determination, the MSLN-EC recombinant protein was obtained, which was stored at -80°C for standby.

[0047] The MSLN recombinant protein obtained above was subjected to western detection, and the primary antibody used in the western detection was His / MSLN monoclonal antibody (Proteintech), and the secondary antibody was HRP-labeled goat anti-mouse antibody (Thermo); the results are shown in Figure 2 , and it can be seen that the MSLN-EC recombinant protein is successfully expressed.

[0048] The MSLN-EC recombinant protein obtained above was subjected to Ni 2+ affinity purification, and the results are shown in Figure 3 , and the recombinant target protein was obtained at 40KD by PH elution, and it can be seen that the MSLN recombinant protein is successfully purified.

[0049] Example 2: Animal immunization process

[0050] (1) 0.5 mg of the MSLN-EC recombinant protein obtained in Example 1 was mixed and emulsified with 0.5 mL of Freund's adjuvant to obtain a MSLN-EC recombinant protein mixture, which was stored at 4°C for standby;

[0051] (2) Select one alpaca and record the ear number, inject subcutaneously in the left and right sides of the alpaca's hips, inject 0.4 mL of the MSLN-EC recombinant protein mixture obtained in (1) at 2 points per side, observe the alpaca for 30 min after the immunization injection, confirm that the alpaca is in good condition and has no discomfort symptoms, immunize and inject once every 2 weeks, a total of 4 times; and before each immunization injection, collect 10 mL of blood from the alpaca's jugular vein, separate the serum, and store it at -80°C for later use; on the 5th day after the last immunization injection, extract 100 mL of peripheral blood from the alpaca's jugular vein, and obtain the peripheral anticoagulated blood sample of the immunized alpaca, i.e., the immune blood sample;

[0052] (3) In a 15 mL centrifuge tube, first add 3 mL of cell separation solution, then slowly add 3 mL of the immune blood sample obtained in (2), a dilution solution, pre-cool the centrifuge, centrifuge at 400 g for 30 min, observe the blood separation in the centrifuge tube, use a 200 ul pipette to extract the middle cotton-like upper immune cells into a new 15 mL centrifuge tube, and store the upper blood plasma into a new centrifuge tube, and store it at -80°C; add 10 mL of PBS buffer at room temperature to the above centrifuge tube, centrifuge at 400 g for 20 min, discard the supernatant, then continue to add 5 mL of PBS buffer at room temperature, centrifuge at 400 g for 20 min; use a blood cell counting plate to calculate the number of cells, discard the supernatant, and use Trizol (Sigma) to dissolve and separate the lymphocytes according to the number of cells and the instructions, and store them at -80°C.

[0053] This example detects the antibody titer of the non-immune serum and the immune serum, and the results are shown in Figure 4 The antibody titer of the immune serum is 5 times that of the non-immune serum at a serum dilution concentration of 1 / 16000, and the antibody titer of the immune serum is stably higher than that of the non-immune serum at a serum dilution concentration in the range of 1 / 500-1 / 128000.

[0054] Example 3: Preparation of a nanobody library targeting MSLN

[0055] The 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), and a VHH gene fragment was amplified by nest PCR using specific primers for llama heavy chain antibodies and heavy chain antibody variable regions; the primer sequences were an upstream primer FR1-RSCF as shown in SEQ ID NO. 9 and a downstream primer VHH-RSCB as shown in SEQ ID NO. 10; the above VHH gene fragment was ligated to a phage expression vector pCombo by enzyme digestion to construct a recombinant phage vector, and the above recombinant phage vector was transformed into E. coli TG1 competent cells by electroporation for amplification, and the VHH was displayed on the surface of the phage with the help of helper phage to form a VHH phage library, i.e., a nanobody library targeting MSLN.

[0056] The cDNA library and the phage library obtained above were detected for capacity, and the results are shown in Table 1. Figure 5 As shown in Table 1, the cDNA library capacity was 2.02 x 10 8 cfu / ml, and the phage library capacity was 1.54 x 10 13 cfu / ml.

[0057] Example 4: Panning of nanobodies targeting MSLN

[0058] 1. Construction of MSLN overexpression cell line

[0059] The lentiviral plasmid pLVSIN-h-MSLN, the packaging plasmid Gag-pol and VSV-G were co-transfected into 293T cells for virus packaging, and the above packaged virus was used to infect 293T cells to construct an MSLN overexpression cell line 293T-MSLN, and the MSLN overexpression cell line 293T-MSLN was obtained;

[0060] The MSLN overexpression cell line 293T-MSLN obtained above was detected by western blot, and the primary antibody used in the western blot was a β-tubulin / MSLN monoclonal antibody (Proteintech), and the secondary antibody was an HRP-labeled goat anti-mouse antibody; and the results are shown in Figure 2. Figure 6 As shown in Figure 2, the MSLN overexpression cell line 293T-MSLN was successfully constructed.

[0061] The immune serum obtained in Example 2 (2) was verified by flow cytometry in this example, and the results are shown in Figure 3. Figure 7As shown, the serum after immunization deflected 93.4% at 1:2000 dilution, compared with the control group and the non-immune serum group, indicating that the nanobody library obtained by immunization can specifically bind to the membrane surface MSLN, and the 293T-MSLN cell line can be used for subsequent panning.

[0062] 2. Panning of nanobodies based on phage display technology

[0063] (1) Negative panning

[0064] S1: Prepare the negative panning cell line 293T, calculate the number of cells, use 3.5x10 8 cells each time;

[0065] S2: Wash the cells in S1 with PBS twice, each time use 30ml PBS to resuspend, centrifuge at 4°C, 500g for 5min, discard the supernatant, and resuspend with 2ml PBS after the last time, then place it in a cryotube;

[0066] S3: Centrifuge the resuspended cells obtained in S2 at 4°C, 500g for 5min, discard the supernatant, then add 500μl of phage-milk / PBS resuspension, incubate at room temperature for 30min; then centrifuge at 13000rpm for 2min, collect the phage supernatant, i.e. phage eluate.

[0067] (2) Positive panning

[0068] S1: Prepare the positive panning cell line 293T-MSLN, calculate the number of cells, use 5x10 8 cells each time;

[0069] S2: Wash the cells in S1 with PBS twice, each time use 30ml PBS to resuspend, centrifuge at 4°C, 500g for 5min, discard the supernatant, and resuspend with 2ml PBS after the last time, then place it in a cryotube;

[0070] S3: Centrifuge the resuspended cells obtained in S2 at 4°C, 500g for 5min, discard the supernatant, then add 500μl of phage-milk / PBS resuspension, incubate at room temperature for 30min; use PBS to wash five times, centrifuge at 500g at room temperature for 2min, remove PBS; add 150μl Elution Buffer (glycine solution, pH 2.2), react at room temperature for 10min; neutralize with 10μl 2M Tris Base solution (glycine solution, pH 9.0); then centrifuge at 13000rpm for 2min, collect the phage eluate;

[0071] S4: Take 10 μl of the phage eluate obtained in S3, dilute 5 gradients (10 1-5 fold dilution), add 90 μl of TG1 bacterial solution with OD 600 0.5-0.6 to each EP tube, and incubate at 37°C for 15 min; then spread the 5 gradients of bacterial solution onto 2xYT culture plates containing ampicillin, and incubate at 37°C overnight, count the number of single colonies on the culture plates to calculate the titer.

[0072] S5: Take the remaining 10 mL of phage eluate in S3, add to TG1 bacterial solution with OD 600 0.5-0.55, and incubate at room temperature for 30 min; add 0.1% Amp resistance, and continue to incubate at 37°C, 250 rpm for 30 min; add 100 μl of helper phage, incubate at room temperature for 30 min, centrifuge at 8000 rpm for 10 min, and transfer the precipitate after centrifugation to 100 mL 2xYT culture plates containing 0.1% ampicillin and kanamycin, and incubate at 37°C, 220 rpm overnight; the next day, concentrate the phage, and reserve for use.

[0073] (3) Incubate the VHH phage library obtained in Example 3 with the 293T-MSLN cell line obtained in Part 1 of this example, and obtain VHH phage that specifically binds to MSLN membrane antigen and is highly enriched, i.e. the positive phage screening library, by 3 rounds of negative and positive panning.

[0074] This example detects the phage recovery amount of the obtained positive phage screening library, and the results are shown in Table 1. Figure 8 As shown in Table 1, the phage recovery amount increases round by round, and the final phage recovery amount reaches 2x10 7 pfu.

[0075] Example 5: Identification of nanobodies targeting MSLN

[0076] 1. Indirect ELISA identification

[0077] Take the phage eluent obtained from the third round of negative and positive washes in Example 4, spread it on a plate, randomly select 95 bacterial clones, and culture them statically overnight at 37°C. Take 10 μL of each of the above bacterial solutions and transfer them to 2xYT medium in a 96-well plate (1 mL / well). Culture until the logarithmic phase. Add 50% sterile glycerol to the remaining bacterial solution, shake well, and store at -80°C. Add IPTG to a final concentration of 0.2 mM and induce expression for 12 h at 37°C and 220 rpm. Centrifuge at 4000 rpm and 4°C for 15 min, then place the bacterial cells at -20°C and freeze for 30 min. After returning to room temperature, resuspend the bacterial cells in PBS (1 mL / well) and shake at 300 rpm and 4°C for 30 min. Centrifuge at 4000 rpm and 4°C for 10 min to obtain the supernatant, which is the crude nanobody extract.

[0078] The MSLN-EC recombinant protein obtained in Example 1 was diluted with coating buffer and added to wells at 400 ng / well and 100 μl / well, respectively, and incubated overnight at 4°C. On the second day, the plates were washed three times with PBST for 2 min each time. 300 μl of 5% skim milk was added to each well to block the ELISA plate for 2 h. The plates were then washed three times with PBST for 2 min each time. 100 μl of the crude nanobody extract obtained above was added to each well as the primary antibody and incubated at 37°C for 2 h. The plates were then washed three times with PBST for 2 min each time. 100 μl of Anti-HA-HRP antibody (abmart) was added as the secondary antibody and incubated at 37°C for 1 h. The plates were washed three times with PBST for 2 min each time. 100 μl of TMB chromogenic solution was added to each well and the reaction was carried out at 37°C. 50 μl of 2M sulfuric acid was added to terminate the reaction, and the OD was read. 450 nm value.

[0079] In this embodiment, the crude extract of the induced nanobody was detected by indirect ELISA, and the identification results are as follows: Figure 9 As shown, OD 450 >1.5 was identified as a positive colony.

[0080] 2. Prokaryotic expression, purification, specificity and affinity identification of nanobodies

[0081] The positive sequence determined by sequencing in part 2 of this example was cloned, and a prokaryotic expression vector PET22b-MSLN-VHH-6his was constructed by overlap extension technology. The positive plasmid was transformed into Rosetta (DE3) competent cells for induction expression. The bacteria were picked and placed in 6 mL LB liquid medium containing ampicillin resistance, and cultured at 37°C and 220 rpm / min for 12-15 h to obtain the culture broth. The 2 mL culture broth 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 h. When the OD value of the broth was 0.4, the broth was induced to express at 16°C using 0.2 mM IPTG for 12 h. The supernatant was discarded by centrifugation, and the bacterial pellet, i.e., the MSLN-targeted nanobody, was obtained.

[0082] The nanobody obtained in part 3 of this example was subjected to protein purification by Ni Sepharose excel affinity chromatography column using an AKTA protein purification system. The purified nanobody was identified by SDS-PAGE, and the results are shown in FIG. 3. The purified nanobody was detected by western, and the results are shown in FIG. 4. The results show that the MSLN-targeted nanobody was successfully obtained after purification and elution. Figure 10 Figure 11 The results show that the MSLN-targeted nanobody was successfully obtained after purification and elution. Figure 12 The results show that the MSLN-targeted nanobody 3A12 obtained by purification has the best affinity.

[0083] Example 6: Application of MSLN-targeted nanobody

[0084] In this example, the cell line 293T-MSLN expressing MSLN membrane protein was used as the research object. The cultured cell line was counted, and 1 × 10 6 The cells were washed with 500 μl PBS solution at 500 g for 5 min, and the supernatant was discarded. 2 μg of the purified MSLN-targeted nanobody in Example 5 was added as the primary antibody, and incubated at 4°C for 45 min. Then, 500 μl PBS solution was added, and centrifuged at 500 g for 5 min. The supernatant was discarded, and the washing was repeated twice. APC-anti-His flow antibody (Biolegend) was added as the secondary antibody, and incubated at 4°C for 45 min. Then, 500 μl PBS solution was added, and centrifuged at 500 g for 5 min. The supernatant was discarded, and the washing was repeated twice. The sample was resuspended with 1 mL PBS solution, and filtered with a 200-mesh filter cloth.

[0085] The filtered nanobody was subjected to flow detection, and the results are shown in FIG. 6. Figure 13 ​As shown, the positive cells deflect 99.5% compared with the control group, indicating that the MSLN-targeted nanobody 3A12 provided by the application can specifically bind to the MSLN protein on the surface of the cell membrane, and thus can be used as a detection and therapeutic antibody.

[0086] The content not described in detail in the specification of the present application is the technology known to those skilled in the art. Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A nanobody targeting mesothelin, characterized in that, The amino acid sequence of the nanobody targeting mesothelin is shown as SEQ ID NO. 8; The nanobody targeting mesothelin comprises complementarity determining regions CDR1, CDR2 and CDR3; The amino acid sequence of the CDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 3; The nanobody targeting mesothelin further comprises framework regions FR1, FR2, FR3 and FR4; The amino acid sequence of the FR1 is shown as SEQ ID NO. 4, the amino acid sequence of the FR2 is shown as SEQ ID NO. 5, the amino acid sequence of the FR3 is shown as SEQ ID NO. 6, and the amino acid sequence of the FR4 is shown as SEQ ID NO.

7.

2. A nucleic acid, characterized in that, The nucleic acid encodes the nanobody targeting mesothelin according to claim 1.

3. An expression vector, characterized by, The expression vector contains the nucleic acid according to claim 2.

4. A host cell, characterized in that, The host cell contains the expression vector according to claim 3.

5. Use of the nanobody targeting mesothelin according to claim 1 in the preparation of a reagent for detecting mesothelin.

6. Use according to claim 5, characterized in that, The use refers to detecting tumor tissues expressing mesothelin.

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