Nanobodies targeting nectin-4 and uses thereof

Abstract

The application discloses a nano antibody targeting Nectin-4 and application thereof, and belongs to the technical field of molecular biology. In view of the unique advantages of the nano antibody in the prior art, the application provides the nano antibody targeting Nectin-4, wherein the nano antibody targeting Nectin-4 comprises CDR1 shown as SEQ ID NO. 1, CDR2 shown as SEQ ID NO. 2 and CDR3 shown as SEQ ID NO. 3. The flow cytometry and western detection results show that the nano antibody targeting Nectin-4 provided by the application can be specifically combined with Nectin-4 protein expressed on the cell surface; the nano antibody can be applied to preparation of a protein detection antibody or a therapeutic antibody, and can be used for in-vivo and in-vitro detection of tumor tissues with Nectin-4 expression, and has important commercial value in clinical disease diagnosis.

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] Nectin-4, also known as PVRL4, belongs to the Nectin family and is a kind of cell adhesion protein and I-type transmembrane glycoprotein, which plays an important role in cell adhesion, signal transduction and cell-cell interaction. Nectin-4 has a high content in normal embryonic and fetal tissues, and a low expression in adult healthy tissues. The molecular weight of Nectin-4 is 55kDa, the extracellular domain is three lg-like domains, a single transmembrane helix structure, and an intracellular domain containing an Afadin binding motif, wherein Afadin is an F-actin binding protein that can recruit a variety of proteins required for intercellular adhesion. Studies have found that Nectin-4 promotes the proliferation, differentiation, migration and invasion of tumor cells by activating the PI3K / Akt pathway, and is highly expressed in a variety of tumor cells. Solid tumors with high expression of Nectin-4 include bladder cancer, pancreatic cancer, triple-negative breast cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, ovarian cancer and many other cancers, and are believed to play an important role in the occurrence and metastasis of cancer.

[0003] At present, there are not many drugs prepared for Nectin-4 target molecules, and all of them are in the early clinical or preclinical development stage. Among them, Enfortumab Vedotin and 9MW-2821 two kinds of double antibody drugs have faster development progress. The structure of Enfortumab Vedotin is composed of three parts: antibody, linker and cytotoxic drug part. The antibody part is a specific antibody against human Nectin-4. The antibody part is connected to the drug through a linker that can be hydrolyzed by protease. The linker has a certain stability, so it will not separate too early in the blood. The drug part is a kind of microtubule toxin, MMAE, which can be released into the cell interior to play a role after the antibody binds to Nectin-4. When Enfortumab Vedotin binds to Nectin-4, it is internalized into the cell and cleaved by protease in the lysosome, releasing cytotoxin MMAE, which binds to microtubules, inhibits the division process of cells, and induces tumor cell apoptosis.

[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 15 KD, which is 1 / 10 of the molecular weight of conventional antibodies. Its protein crystal structure is 4 nm in length and 2.5 nm in diameter, and it is currently the smallest antibody known with a molecular weight of 15 KD. It is called a nanobody. Nanobodies can more easily penetrate cell membranes and tissue barriers, and enter deep tissues and specific cells. Compared with traditional antibodies, the active binding region of nanobodies is longer, with as many as 16-18 amino acids, which can bind to specific epitopes of target molecules, achieve highly accurate recognition and intervention, and make nanobodies an important tool in research and treatment. Nanobodies also have good stability and can maintain their structure and function under a wide range of environmental conditions, showing high thermal stability and chemical stability, allowing them 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 nanobodies is an engineering bacteria expression method, which has the advantages of easy expression, easy genetic engineering, relatively low production cost and easy large-scale production. SUMMARY

[0005] In view of the unique advantages of nanobodies in the prior art, the present application provides a nanobody targeting Nectin-4 and applications thereof.

[0006] One of the purposes of the present application is to provide a nanobody targeting Nectin-4, wherein the amino acid sequence of the nanobody targeting Nectin-4 is shown in SEQ ID NO. 8.

[0007] In a preferred embodiment of the present application, the nanobody targeting Nectin-4 comprises complementarity determining regions CDR1, CDR2 and CDR3.

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

[0009] In a preferred embodiment of the present application, the nanobody targeting Nectin-4 further comprises framework regions FR1, FR2, FR3 and FR4.

[0010] In a preferred embodiment of the present application, 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.

[0011] A second object of the present application is to provide a nucleic acid comprising a nucleic acid sequence encoding the above-mentioned Nectin-4-targeting nanobody or a complementary sequence thereof.

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

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

[0014] A fifth object of the present application is to provide the use of the above-mentioned Nectin-4-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 detecting tumor tissues expressing Nectin-4.

[0016] Advantages of the present application:

[0017] The present application provides a Nectin-4-targeting nanobody. Flow cytometry detection results show that the Nectin-4-targeting nanobody provided by the present application has strong affinity with 293T-Nectin-4 cells which are Nectin-4 positive cells; and the Nectin-4-targeting nanobody can specifically bind to SW780 cells which express Nectin-4 protein on the surface of natural cells. The above-mentioned nanobody can be prepared by large-scale expression of in vitro engineering bacteria, and 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 Nectin-4, and has important commercial value in clinical disease diagnosis and treatment. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a construction result diagram of the eukaryotic expression vector pCAGGS-SP-Nectin-4-EC-6His in Example 1; M1 is DL2000 DNA maker, and M2 is DL15000 DNA maker;

[0019] Figure 2 It is a Nectin-4 recombinant protein expression identification diagram in Example 1; M is a protein molecular weight standard, and Nectin-4-EC is a Nectin-4 recombinant protein.

[0020] Figure 3 The recombinant Nectin-4 protein Ni in Example 1 2+ Affinity purification identification diagram; M represents the molecular weight standard of the protein; lane 1 is the supernatant of the expressed protein, lane 2 is the elution solution, lane 3 is the sample washing solution, lanes 4-5 are the elution solution at pH=3.5, lanes 6-8 are the elution solution at pH=3.0, lanes 9-11 are the elution solution at pH=2.8, lanes 12-14 are the elution solution at pH=2.6, and lanes 15-18 are the elution solution at 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 Nectin-4 membrane protein overexpression cell line in Example 4; M represents the protein molecular weight standard.

[0024] Figure 7 This is a flow cytometry result of the immune serum in Example 4;

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

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

[0027] Figure 10 This is the phylogenetic tree analysis diagram of amino acids in Example 5;

[0028] Figure 11 Ni is the prokaryotic expression protein of the nanobody in Example 5. 2+ Affinity purification identification diagram; M is the protein molecular weight standard, lane 1 is the bacterial supernatant, lanes 2-3 are the washing buffer, and lanes 4-17 are the elution buffer;

[0029] Figure 12 This is a Western blot image of the purified nanobody in Example 5; M represents the protein molecular weight standard.

[0030] Figure 13 This is an identification diagram of the Nectin-4-targeting nanobody used in Example 6 as an antibody for flow cytometry detection. 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 preferred embodiments, and those skilled in the art 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] The western used in this embodiment includes the following steps:

[0034] S1: Collect protein samples for SDS-PAGE polyacrylamide gel electrophoresis, and perform constant voltage electrophoresis at 120V;

[0035] 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 2h in an ice box;

[0036] 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;

[0037] 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

[0038] and incubated at room temperature for 2h;

[0039] 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

[0040] and incubated at room temperature for 1h;

[0041] 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.

[0042] Example 1: Preparation of Nectin-4-EC recombinant protein

[0043] (1) The Nectin-4 extracellular segment target gene Nectin-4-EC was amplified using pMD18-T-Nectin-4 plasmid as a template;

[0044] (2) The eukaryotic expression vector pCAGGS-SP-Nectin-4-EC-6His was constructed by amplifying the vector pCAGGS-SP and the target gene obtained in (1) through overlap extension PCR technology, and the PCR verification of the obtained eukaryotic expression vector was performed, the results are shown in Figure 1 . The PCR product was sequenced, and after success, the plasmid was extracted in large quantities. The PCR verification and sequencing results proved that the eukaryotic expression vector was successfully constructed, and the eukaryotic expression vector pCAGGS-SP-Nectin-4-EC-6His was obtained.

[0045] (3) The recombinant protein was expressed by transfecting the HEK293 mammalian suspension expression system with the plasmid obtained in (2), and then purified by Ni Sepharose excel affinity chromatography filler of Cytiva company, and the target protein was purified by AKATA instrument. After determination of the protein concentration, the Nectin-4-EC recombinant protein was obtained and stored at -80℃ for standby.

[0046] The Nectin-4 recombinant protein obtained above was subjected to western detection, and the primary antibody used in the western detection was His / Nectin-4 monoclonal antibody (Proteintech), and the secondary antibody was HRP labeled goat anti-mouse antibody (Thermo); the results are shown in Figure 2 . It can be seen that the Nectin-4-EC recombinant protein is successfully expressed.

[0047] The Nectin-4 recombinant protein obtained above was subjected to western detection, and the primary antibody used in the western detection was His / Nectin-4 monoclonal antibody (Proteintech), and the secondary antibody was HRP labeled goat anti-mouse antibody (Thermo); the results are shown in 2+ . It can be seen that the Nectin-4-EC recombinant protein is successfully expressed. Figure 3

[0048] Example 2: Animal immunization process

[0049] (1) 0.5 mg of Nectin-4-EC recombinant protein obtained in Example 1 was mixed and emulsified with 0.5 mL Freund's adjuvant to obtain a Nectin-4-EC recombinant protein mixture, which was stored at 4℃ for standby;

[0050] ​(2) Select one alpaca and record the ear number, inject subcutaneously in the left and right sides of the alpaca's hips, 2 points per side, 0.4 mL of the Nectin-4-EC recombinant protein mixture obtained in (1) per point, observe the alpaca for 30 min after immunization, confirm that the alpaca is in good condition and has no discomfort, immunize once every 2 weeks, a total of 4 times; and before each immunization, take 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, take 100 mL of peripheral blood from the alpaca's jugular vein, obtain the peripheral anticoagulated blood sample of the immunized alpaca, i.e., the immune blood sample;

[0051] (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), dilute by a factor of 2, pre-cool the centrifuge, centrifuge at 400 g for 30 min, observe the blood separation in the centrifuge tube, use a 200 μl pipette to transfer the middle cotton-like upper layer of immune cells to a new 15 mL centrifuge tube, and store the upper layer of blood plasma in a new centrifuge tube, store at -80°C; in the above centrifuge tube, add 10 mL of PBS buffer at room temperature, 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 hemocytometer to count 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, store at -80°C.

[0052] This example detects the antibody titer of non-immune serum and immune serum, and the results are shown in Figure 4 The antibody titer of immune serum is 5 times that of non-immune serum at a serum dilution concentration of 1 / 16000, and the antibody titer of immune serum is stably higher than that of non-immune serum at a serum dilution concentration in the range of 1 / 500-1 / 256000.

[0053] Example 3: Preparation of a nanobody library targeting Nectin-4

[0054] 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 the 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 Nectin-4.

[0055] The cDNA library and phage library capacity obtained above were detected in this example, and the results are shown in Table 1. Figure 5 As shown in Table 1, the cDNA library capacity was 2.88 x 10 6 pfu / ml, and the phage library capacity was 3.8 x 10 13 cfu / ml.

[0056] Example 4: Panning of nanobodies targeting Nectin-4

[0057] 1. Construction of Nectin-4 overexpression cell line

[0058] The lentiviral plasmid pLVSIN-Nectin-4, 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 a Nectin-4 overexpression cell line 293T-Nectin-4.

[0059] The Nectin-4 overexpression cell line 293T-Nectin-4 obtained above was detected by western blot in this example, and the primary antibody used in the western blot was a Nectin-4 monoclonal antibody (Proteintech), and the secondary antibody was a HRP-labeled goat anti-mouse antibody (Thermo); and the results are shown in Figure 2. Figure 6 As shown in Figure 2, the Nectin-4 overexpression cell line 293T-Nectin-4 was successfully constructed.

[0060] 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 post-immune serum was deflected by 74.6% at a dilution of 1:2000, 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 Nectin-4, and the 293T-Nectin-4 cell line can be used for subsequent panning.

[0061] 2. Panning of nanobodies based on phage display technology

[0062] (1) Negative panning

[0063] S1: Prepare the negative panning cell line 293T, calculate the number of cells, use 3.5 x 10 8 cells each time;

[0064] S2: Wash the cells in S1 twice with PBS, each time use 30 ml PBS to resuspend, centrifuge at 4°C, 500g for 5 min, discard the supernatant, and resuspend with 2 ml PBS before placing in a cryotube;

[0065] S3: Centrifuge the resuspended cells obtained in S2 at 4°C, 500g for 5 min, discard the supernatant, then add 500 μl of phage-milk / PBS resuspension, incubate at room temperature for 30 min; then centrifuge at 13000 rpm for 2 min, collect the phage supernatant, i.e. phage eluate.

[0066] (2) Positive panning

[0067] S1: Prepare the positive panning cell line 293T-Nectin-4, calculate the number of cells, use 5 x 10 8 cells each time;

[0068] S2: Wash the cells in S1 twice with PBS, each time use 30 ml PBS to resuspend, centrifuge at 4°C, 500g for 5 min, discard the supernatant, and resuspend with 2 ml PBS before placing in a cryotube;

[0069] S3: Centrifuge the resuspended cells obtained in S2 at 4°C, 500g for 5 min, discard the supernatant, then add 500 μl of phage-milk / PBS resuspension, incubate at room temperature for 30 min; use PBS to wash five times, centrifuge at 500g at room temperature for 2 min, remove the PBS; add 150 μl of Elution Buffer (glycine solution, pH 2.2), react at room temperature for 10 min; neutralize with 10 μl of 2M Tris Base solution (glycine solution, pH 9.0); then centrifuge at 13000 rpm for 2 min, collect the phage eluate;

[0070] 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 on 2xYT culture plates containing ampicillin resistance, and incubate at 37 °C overnight, count the number of single colonies on the culture plates to calculate the titer.

[0071] 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, 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, transfer the above-mentioned centrifuged precipitate to 100 mL 2xYT culture plate containing 0.1% ampicillin and kanamycin resistance, and incubate at 37 °C, 220 rpm overnight; the next day, concentrate the phage for standby.

[0072] (3) Incubate the VHH phage library obtained in Example 3 with the 293T-Nectin-4 cell line obtained in Part 1 of this example, and obtain VHH phage that specifically binds to Nectin-4 membrane antigen and is highly enriched, i.e., the positive phage screening library, through 3 rounds of negative and positive panning.

[0073] This example detects the phage recovery amount of the obtained positive phage screening library, and the results are shown in Figure 8 Table 1, which shows that the phage recovery amount increases round by round, and the final phage recovery amount reaches 10 9 pfu.

[0074] Example 5: Identification of nanobodies targeting Nectin-4

[0075] 1. Indirect ELISA identification

[0076] Take the phage eluate obtained by the third round of negative and positive panning in Example 4 to coat the plate, randomly pick 56 bacterial clones, and incubate at 37°C overnight; take 10 μl of each of the above bacterial solutions, transfer to 2xYT medium (1 mL / well) in a 96-deep well plate, and incubate to the logarithmic phase, then add the remaining bacterial solution to 50% sterile glycerol, shake well, and store at -80°C; add IPTG at a final concentration of 0.2 mM, and induce expression at 37°C, 220 rpm for 12 h; centrifuge at 4000 rpm, 4°C for 15 min, then freeze the bacterial cells at -20°C for 30 min; after returning to room temperature, resuspend the bacterial cells in PBS, 1 mL / well, and shake at 300 rpm, 4°C for 30 min; centrifuge at 4000 rpm, 4°C for 10 min, and obtain the supernatant, which is the crude nanobody extract.

[0077] Dilute the Nectin-4-EC recombinant protein obtained in Example 1 with the coating solution, and add to the well plate at a standard of 400 ng / well and 100 μL / well, and incubate at 4°C overnight; on the second day, wash 3 times with PBST, each for 2 min; then add 5% skim milk to the ELISA plate to block for 2 h, 300 μl per well; wash the plate 3 times with PBST, each for 2 min; add the crude nanobody extract obtained above as the primary antibody to the ELISA plate, 100 μl / well, and incubate at 37°C for 2 h; wash the plate 3 times with PBST, each for 2 min; add Anti-HA-HRP antibody as the secondary antibody, 100 μl / well, and incubate at 37°C for 1 h; wash the plate 3 times with PBST, each for 2 min; add TMB color developing solution, 100 μl / well, and react at 37°C, then add 2 M sulfuric acid to stop the reaction, 50 μl / well, and read the OD 450 nm value.

[0078] This example detects the crude nanobody extract induced by indirect ELISA, and the identification results are shown in Figure 9 , OD 450 >1.0 is determined as a positive colony.

[0079] 2. Nanobody sequence determination and amino acid phylogenetic tree analysis

[0080] After re-shaking and activating the positive clone colonies identified in Part 1 of this example, send them to Jilin Kumuyi Biotechnology Co., Ltd. for sequencing, align the amino acid sequences of the screened nanobodies, and perform phylogenetic tree analysis, and the results are shown in Figure 10 , select the VHH with the highest frequency of occurrence in the phylogenetic relationship branch for prokaryotic expression identification.

[0081] 3. Prokaryotic expression, purification, specificity, and affinity identification of nanobodies

[0082] The positive sequence determined by the second part of the present embodiment sequencing was cloned, and a prokaryotic expression vector PET22b-Nectin-4-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 after centrifugation, and the bacterial pellet, i.e., the nanobody targeting Nectin-4, was obtained.

[0083] The nanobody obtained in the third part of the present embodiment was subjected to protein purification using the AKATA protein purification system through a Ni Sepharose excel affinity chromatography column, and the purified product was identified by SDS-PAGE. The results, as shown in FIG. 3, indicated that the nanobody targeting Nectin-4 was successfully obtained after purification and elution. Figure 11 The purified nanobody was identified by western blotting. The primary antibody was an anti-His tag monoclonal antibody (Proteintech), and the secondary antibody was an HRP-labeled goat anti-mouse antibody (Thermo). The results, as shown in FIG. 4, indicated that the purified protein obtained in the present embodiment was a recombinant nanobody. Figure 12

[0084] Example 6: Application of the Nanobody Targeting Nectin-4

[0085] In the present embodiment, the cell line SW780 naturally expressing Nectin-4 membrane protein was used as the research object. The cultured cell line was counted, and 1 x 10 6 The cells were washed with 500 μl of PBS solution at 500 g for 5 min, and the supernatant was discarded. Then, 2 μg of the purified nanobody targeting CDH17 in Example 5 was added as the primary antibody, and the mixture was incubated at 4°C for 45 min. Then, 500 μl of PBS solution was added, and the mixture was centrifuged at 500 g for 5 min. The supernatant was discarded, and the washing was repeated twice. Then, APC-anti-His flow cytometry antibody (Biolegend) was added as the secondary antibody, and the mixture was incubated at 4°C for 45 min. Then, 500 μl of PBS solution was added, and the mixture was centrifuged at 500 g for 5 min. The supernatant was discarded, and the washing was repeated twice. The mixture was resuspended with 1 ml of PBS solution, and finally filtered with a 200-mesh filter cloth. ​

[0086] The filtered nanobodies were subjected to flow detection, and the results are shown in Figure 13 Compared with the control group, the positive cell deflection was 80%, indicating that the Nectin-4 targeted nanobodies provided by the application can specifically bind to the Nectin-4 protein on the cell membrane surface, and thus can be used as a detection and therapeutic antibody.

[0087] 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 Nectin-4, characterized in that, The amino acid sequence of the Nectin-4-targeting nanobody is shown as SEQ ID NO. 8; The Nectin-4-targeting nanobody 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 Nectin-4-targeting nanobody 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 Nectin-4-targeting nanobody of claim 1.

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

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

5. Use of the Nectin-4-targeting nanobody of claim 1 in the preparation of a reagent for detecting Nectin-4.

6. Use according to claim 5, characterized in that, The use refers to detecting tumor tissues expressing Nectin-4.

Images