Nanobodies targeting cadherin 17 and uses thereof

Abstract

The application discloses a nanobody targeting cadherin 17 and an 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 cadherin 17, wherein the nanobody targeting cadherin 17 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. Western, SPR and flow cytometry detection results show that the nanobody targeting cadherin 17 has higher affinity and specificity relative to traditional antibodies, 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] Cadherin 17 (CDH17), also known as liver intestine-cadherin (LI-cadherin), is an atypical calcium-dependent adhesion molecule. The extracellular segment of CDH17 is composed of seven cadherin repeat sequences, and the intracellular segment is composed of 24 amino acids. CDH17 is highly expressed in the small intestine and colon in normal human cells, and is not expressed or is lowly expressed in other tissues such as the liver, heart and kidney. The function of CDH17 is to maintain the integrity of epithelial tissues as a polypeptide transporter and cell adhesion molecule. Studies have shown that CDH17 is highly expressed in tumors of the digestive system, such as gastric cancer, colorectal cancer, pancreatic cancer and some liver cancer, especially in adenocarcinoma. Moreover, the tumor tissue of metastatic foci still maintains a high level of expression of CDH17 after the primary cancer with high expression of CDH17 molecules metastasizes, which confirms that CDH17 is closely related to the occurrence and development of tumors. TCGA data analysis results show that the expression of CDH17 gene in gastric cancer, colorectal cancer, pancreatic cancer and some liver cancer tissues is higher than that in corresponding normal tissues, and the expression level of CDH17 in normal other organs is very low. In summary, CDH17 has great potential as a new therapeutic target for malignant tumors of the digestive system and metastatic tumors thereof.

[0003] At present, there are not many drugs prepared for CDH17 target molecules, and all of them are in the early clinical or preclinical research stage. Among them, ARB202 and BI-905711, two kinds of double antibody drugs, are in the clinical phase I stage, and CAR-T and CAR-NK drugs are in the early clinical stage. BI905711 is a tetravalent double antibody targeting TRAILR2 and CDH17. TRAILR2 and CDH17 cross-link to induce CDH17-dependent TRAILR2 aggregation, and induce selective apoptosis in tumor cells co-expressing TRAILR2 and CDH17. The current BI905711 for advanced refractory gastrointestinal cancer Ia / Ib phase study (NCT04137289) is recruiting patients. ARB202 is a humanized IgG4 double antibody against CDH17 and CD3 constructed by TriAx technology, which has a unique affinity structure for anti-CDH17 and anti-CD3. It can ensure high specificity and high killing activity, and avoid the risk of systemic immune response caused by excessive activation of T cells. Currently, ARB202 is undergoing phase I clinical study for the treatment of advanced gastrointestinal cancer.

[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, and is currently the smallest antibody known with a molecular weight of 15KD. 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. Nanobodies have high affinity and specificity, 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, can maintain their structure and function under a wide range of environmental conditions, and exhibit high thermal 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 engineering bacteria expression, which has the advantages of easy expression and easy genetic engineering, and has the advantages of 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 cadherin 17 and applications thereof

[0006] One of the purposes of the present application is to provide a nanobody targeting cadherin 17, wherein the amino acid sequence of the nanobody targeting cadherin 17 is shown in SEQ ID NO. 8.

[0007] In a preferred embodiment of the present application, the nanobody targeting cadherin 17 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 cadherin 17 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] The second object of the present application is to provide a nucleic acid comprising a nucleic acid sequence encoding the above-mentioned nanobody targeting cadherin 17 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 nanobody targeting cadherin 17 in the preparation of a protein detection antibody or a therapeutic antibody.

[0015] In a preferred embodiment of the present application, the application refers to the detection of tumor tissues expressing cadherin 17.

[0016] The present application has the following advantages:

[0017] The present application provides a nanobody targeting cadherin 17, and western results show that the nanobody targeting cadherin 17 provided by the present application can specifically bind to cadherin 17 recombinant protein, SPR results show that the binding constant KD of the above-mentioned nanobody and recombinant CDH17-EC protein is 7.15nM, and flow cytometry results show that the above-mentioned nanobody can specifically bind to membrane protein expressing cadherin 17.

[0018] The nanobody targeting cadherin 17 provided by the present application has higher affinity and specificity than traditional antibodies, can specifically bind to CDH17 expressed on the membrane surface, can be prepared by large-scale expression of in vitro engineering bacteria, can be applied to the preparation of protein detection antibodies or therapeutic antibodies, can be used for in vivo and in vitro detection of tumor tissues expressing cadherin 17, and has important commercial value in clinical disease diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is the construction result diagram of the eukaryotic expression vector pCAGGS-SP-CDH17-EC-6His in Example 1; M is DL15000 DNA marker;

[0020] Figure 2Figure for identification of CDH17-EC recombinant protein expression in Example 1; M is protein molecular weight standard;

[0021] Figure 3 Figure for identification of Ni-CDH17-EC recombinant protein in Example 1; 2+ Figure for identification of Ni-CDH17-EC recombinant protein in Example 1; Figure for identification of Ni-CDH17-EC recombinant protein in Example 1;

[0022] Figure 4 Figure for determination of llama serum antibody titer in Example 2;

[0023] Figure 5 Figure for detection of cDNA library and phage library capacity in Example 3;

[0024] Figure 6 Figure for construction results of CDH17 membrane protein 293T overexpression cell line in Example 4; M is protein molecular weight standard;

[0025] Figure 7 Figure for flow cytometry identification results of overexpression cell line in Example 4;

[0026] Figure 8 Figure for determination of phage panning library recovery rate in Example 4;

[0027] Figure 9 Figure for indirect ELISA identification results in Example 5;

[0028] Figure 10 Figure for nanobody amino acid phylogenetic tree analysis in Example 5;

[0029] Figure 11 Figure for prokaryotic expression identification of nanobody in Example 5; M1 is protein molecular weight standard;

[0030] Figure 12 Figure for Ni-CDH17-EC recombinant protein in Example 5; 2+ Figure for Ni-CDH17-EC recombinant protein in Example 5;

[0031] Figure 13 Figure for western identification of nanobody in Example 5; M is protein molecular weight standard;

[0032] Figure 14Figure for identification of binding affinity of nanobody to CDH17-EC recombinant protein in Example 5;

[0033] Figure 15 Figure for identification of truncated expression of CDH17-EC recombinant protein in Example 6; M is a protein molecular weight standard;

[0034] Figure 16 Figure for identification of fusion expression of VHH and Fc tag protein in Example 6; M is a protein molecular weight standard;

[0035] Figure 17 Figure for identification of nanobody binding region targeting CDH17 in Example 6; M is a protein molecular weight standard;

[0036] Figure 18 Figure for identification of nanobody targeting CDH17 as protein detection antibody in Example 7; M is a protein molecular weight standard;

[0037] Figure 19 Figure for identification of nanobody targeting CDH17 as flow detection antibody in Example 7. DETAILED DESCRIPTION

[0038] Those skilled in the art can modify the process parameters according to the content herein. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application described herein to realize and apply the present application technology without departing from the content and scope of the present application.

[0039] In order to make the purpose, technical scheme and advantages of the present application more clear and explicit, the present application will be 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.

[0040] Cadherin 17: Cadherin 17, CDH17;

[0041] The western used in this embodiment includes the following steps:

[0042] S1: Collect protein samples for SDS-PAGE polyacrylamide gel electrophoresis, and perform constant voltage electrophoresis under the condition of 120V;

[0043] S2: After electrophoresis in S1, transfer the protein sample from the gel to a nitrocellulose membrane and incubate at 230°C in an ice box.

[0044] Constant current membrane transfer at mA conditions for 1-2 hours;

[0045] S3: After the S2 transfer membrane is completed, place the membrane in the sealing solution (5% skim milk powder) and seal it at room temperature for 2 hours;

[0046] S4: Wash the membrane after blocking in S3 three times with 1×TBST solution for 5 min each time, then add primary antibody.

[0047] Incubate the solution at room temperature for 2 hours;

[0048] S5: Wash the membrane after primary antibody incubation in S4 three times with 1×TBST solution for 5 min each time, then add...

[0049] Secondary antibody solution, incubate at room temperature for 1 hour;

[0050] S6: The membrane after incubation with the secondary antibody in S5 was washed three times with 1×TBST solution for 5 minutes each time. Then, a developing solution was prepared for development, followed by development, photography, and storage in a gel imaging system.

[0051] Example 1: Preparation of CDH17-EC recombinant protein

[0052] (1) The extracellular segment of CDH17 target gene CDH17-EC was amplified using pMD18-T-CDH17 plasmid as a template.

[0053] (2) The eukaryotic expression vector pCAGGS-SP-CDH17-EC-6his was constructed using overlap extension PCR technology and the target gene obtained in (1). The eukaryotic expression vector obtained above was verified by PCR, and the results are as follows: Figure 1 As shown in the figure; the PCR product was sequenced, and a large number of plasmids were extracted after successful sequencing. PCR verification and sequencing results both proved that the eukaryotic expression vector was successfully constructed, and the eukaryotic expression vector pCAGGS-SP-CDH17-EC-6His was obtained.

[0054] (3) The recombinant protein was expressed by transfecting the HEK293 mammalian suspension expression system with the plasmid obtained in (2). Then, the target protein was purified by using the Ni Sepharose excel affinity chromatography packing material from Cytiva and the AKATA instrument. After the protein concentration was determined, the CDH17-EC recombinant protein was obtained and stored at -80℃ for later use.

[0055] The western detection of the obtained CDH17-EC recombinant protein was carried out, and the primary antibody used in the western detection was an anti-His tag monoclonal antibody (Proteintech), and the secondary antibody was an HRP-labeled goat anti-mouse antibody (Thermo); the results are shown in Figure 2 It can be seen that the CDH17-EC recombinant protein is successfully expressed.

[0056] The Ni 2+ affinity purification of the obtained CDH17-EC recombinant protein was identified, and the results are shown in Figure 3 It can be seen that the CDH17-EC recombinant protein is successfully purified.

[0057] Example 2: Animal immunization process

[0058] (1) 0.5 mg of the CDH17-EC recombinant protein obtained in Example 1 was mixed and emulsified with 2 mL of Freund's adjuvant to obtain a CDH17-EC recombinant protein mixture, which was stored at 4°C for standby;

[0059] (2) One alpaca was selected and the ear number was recorded, and the alpaca was injected subcutaneously on the hips with the CDH17-EC recombinant protein mixture obtained in (1) on both left and right sides, with 2 points on each side, and 0.4 mL of the CDH17-EC recombinant protein mixture was injected at each point. After immunization, the alpaca was observed for 30 min to confirm that the alpaca was in good condition and had no discomfort. Immunization was performed every 2 weeks, a total of 4 times. Before each immunization, 10 mL of blood was taken from the alpaca's jugular vein, and the serum was separated and stored at -80°C for standby. On the 5th day after the last immunization, 100 mL of peripheral blood was collected from the alpaca's jugular vein to obtain an immune alpaca peripheral anticoagulant blood sample, i.e., an immune blood sample;

[0060] (3) In a 15 mL centrifuge tube, 3 mL of cell separation solution was first added, then 3 mL of the immune blood sample obtained in (2) was slowly added, and the centrifuge was pre-cooled at 400 g for 30 min. The blood separation in the centrifuge tube was observed, and 200 μl of pipette was used to suck the middle cotton-like immune cells into a new 15 ml centrifuge tube, and the upper layer of blood plasma was saved into a new centrifuge tube and stored at -80°C. 10 mL of PBS buffer at room temperature was added to the above-mentioned centrifuge tube, and centrifuged at 400 g for 20 min. The supernatant was discarded, and then 5 mL of PBS buffer at room temperature was added, and centrifuged at 400 g for 20 min. The cell number was calculated using a hemocytometer, the supernatant was discarded, and the lymphocytes were separated and dissolved using Trizol (Sigma) according to the cell number and the instructions, and stored at -80°C.

[0061] The antibody titer of the unimmunized serum and the immunized serum was detected in this embodiment, and the results are shown in Figure 4 As shown, the antibody titer of the immunized serum was 5 times that of the unimmunized serum at a serum dilution concentration of 1 / 1000, and the antibody titer of the immunized serum was stably higher than that of the unimmunized serum at a serum dilution concentration in the range of 1 / 1000-1 / 2048000.

[0062] Example 3: Preparation of a nanobody library targeting CDH17

[0063] 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). Nest-PCR was performed using specific primers for llama heavy chain antibodies and heavy chain antibody variable regions, and VHH gene fragments were amplified. 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 fragments were ligated to the phage expression vector pCombo by enzyme digestion to construct a recombinant phage vector, and the recombinant phage vector was transformed into E. coli TG1 competent cells by electroporation to amplify. With the help of helper phage, VHH was displayed on the surface of the phage to form a VHH phage library, i.e., a nanobody library targeting CDH17.

[0064] The capacity of the cDNA library and the phage library obtained above was detected in this embodiment, and the results are shown in Figure 5 As shown, the capacity of the cDNA library was 9.17 x 10 7 cfu / ml, and the capacity of the phage library was 7.1 x 10 13 cfu / ml.

[0065] Example 4: Panning of nanobodies targeting CDH17

[0066] 1. Construction of CDH17 overexpression cell line

[0067] The lentiviral plasmid pLVSIN-CDH17, 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 CDH17 overexpression cell line 293T-h-CDH17, and the CDH17 overexpression cell line 293T-h-CDH17 was obtained.

[0068] The western detection of the obtained CDH17 overexpression cell line 293T-h-CDH17 was carried out, and the primary antibody used in the western detection was anti-CDH17 monoclonal antibody (Proteintech), and the secondary antibody was HRP-labeled goat anti-mouse antibody (Thermo); the results are shown in Figure 6 It can be seen that the CDH17 overexpression cell line 293T-h-CDH17 was successfully constructed.

[0069] The immune serum obtained in the (2) part of Example 2 was verified by flow cytometry, and the results are shown in Figure 7 Compared with the control group and the non-immune serum group, the serum after immunization was deflected by 81.7% at a dilution of 1:2000, indicating that the serum obtained by immunization can specifically bind to the membrane surface CDH17, and the 293T-h-CDH17 cell line can be used for subsequent screening.

[0070] 2. Screening of nanobodies based on phage display technology

[0071] (1) Negative washing

[0072] S1: Prepare the negative washing cell 293T cell line, calculate the cell number, and use 3.5x10 8 cells each time;

[0073] S2: Wash the cells in S1 with PBS twice, each time using 30ml PBS to resuspend, centrifuge at 4℃, 500g for 5min, discard the supernatant, and resuspend with 2ml PBS before placing in a cryotube;

[0074] S3: Centrifuge the resuspended cells obtained in S2 at 4℃, 500g for 5min, discard the supernatant, then add 500μl phage-milk / PBS to resuspend, incubate at room temperature for 30min; then centrifuge at 13000rpm for 2min, collect the phage supernatant, i.e. phage eluate.

[0075] (2) Positive washing

[0076] S1: Prepare the positive washing cell 293T-h-CDH17 cell line, calculate the cell number, and use 5x10 8 cells each time;

[0077] S2: Wash the cells in S1 with PBS twice, each time using 30ml PBS to resuspend, centrifuge at 4℃, 500g for 5min, discard the supernatant, and resuspend with 2ml PBS before placing in a cryotube;

[0078] S3: Resuspend the cells obtained in S2, centrifuge at 500g for 5 min at 4°C, discard the supernatant, then add 500 μl of the phage eluate obtained in (1) to the negative panning collection, resuspend, and incubate at room temperature for 30 min; wash five times with PBS, centrifuge at 500g for 2 min at room temperature, and remove the PBS; add 150 μl of Elution Buffer (glycine solution, pH 2.2), and react at room temperature for 10 min; neutralize by adding 10 μl of 2M Tris Base solution (glycine solution, pH 9.0); then centrifuge at 13000 rpm for 2 min, and collect the phage eluate;

[0079] S4: Take 10 μl of the phage eluate obtained in S3, dilute by 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 in a 37°C water bath for 15 min; then spread the 5 gradients of bacterial solution onto 2xYT culture plates containing ampicillin resistance, and incubate at 37°C overnight, count the number of single colonies on the culture plates, and calculate the titer;

[0080] S5: Take 10 mL of the remaining phage eluate obtained 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 and 250 rpm for 30 min; then add 100 μl of helper phage, incubate at room temperature for 30 min, centrifuge at 8000 rpm for 10 min, transfer the precipitate after centrifugation to 100 mL of 2xYT culture plates containing 0.1% ampicillin and kanamycin resistance, and incubate at 37°C and 220 rpm overnight; the next day, concentrate the phage, and reserve for use.

[0081] (3) Incubate the VHH phage library obtained in Example 3 with the 293T-h-CDH17 cell line obtained in Part 1 of this example, and obtain a VHH phage library that specifically binds to CDH17 membrane antigen and is highly enriched, i.e., a positive phage screening library, by 3 rounds of negative panning and positive panning.

[0082] This example detects the phage recovery amount of the obtained positive phage screening library, and the results are shown in Figure 8 Table 1. It can be seen that the phage recovery amount increases round by round, and the final phage recovery amount reaches 10 9 pfu.

[0083] Example 5: Identification of nanobodies targeting CDH17

[0084] 1. Identification by indirect ELISA

[0085] The phage eluate obtained from the third round of negative panning and positive panning in Example 4 was coated on a plate, 95 bacterial clones were randomly picked and incubated at 37°C overnight. 10 μl of each of the bacterial solutions was transferred to 2xYT medium (1 mL / well) in a 96-deep well plate, and cultured to the logarithmic phase. The remaining bacterial solution was added to 50% sterile glycerol, mixed well, and stored at -80°C. The final concentration of IPTG was 0.2 mM, and the expression was induced at 37°C and 220 rpm for 12 h. The bacteria were centrifuged at 4000 rpm and 4°C for 15 min, and then frozen at -20°C for 30 min. After the temperature was restored to room temperature, the bacteria were resuspended in PBS (1 mL / well), and shaken at 300 rpm and 4°C for 30 min. The supernatant, i.e., the crude nanobody extract, was obtained by centrifugation at 4000 rpm and 4°C for 10 min.

[0086] The recombinant protein CDH17-EC obtained in Example 1 was diluted with the coating solution, and added to the well plate at a standard of 400 ng / well and 100 μl / well, and incubated at 4°C overnight. On the second day, the plate was washed with PBST for 3 times, each for 2 min. 5% skim milk was added to the ELISA plate for blocking for 2 h, 300 μl per well. The plate was washed with PBST for 3 times, each for 2 min. The crude nanobody extract obtained above was added to the ELISA plate as the primary antibody, 100 μl / well, and incubated at 37°C for 2 h. The plate was washed with PBST for 3 times, each for 2 min. Anti-HA-HRP antibody was added as the secondary antibody, 100 μl / well, and incubated at 37°C for 1 h. The plate was washed with PBST for 3 times, each for 2 min. TMB color developing solution was added, 100 μl / well, and reacted at 37°C. 2M sulfuric acid was added to stop the reaction, 50 μl / well, and the OD 450 nm value was read.

[0087] The crude nanobody extract induced was detected by indirect ELISA in this example, and the identification results are shown in Figure 9 , OD 450 >1.0 was determined as a positive colony.

[0088] 2. Nanobody sequence determination and phylogenetic tree analysis

[0089] The positive clone colonies obtained in Part 1 of this example were reactivated by shaking, and sent to Jilin Kumuyi Biotechnology Co., Ltd. for sequencing. The amino acid sequences of the screened nanobodies were aligned and phylogenetic tree analysis was performed, and the results are shown in Figure 10 , and the VHH with the highest frequency of occurrence in the phylogenetic relationship branch was selected for prokaryotic expression identification.

[0090] 3. Prokaryotic expression, purification, specificity and affinity identification of nanobodies

[0091] The positive sequence determined by sequencing in part 2 of this example was cloned, and a prokaryotic expression vector PET22b-CDH17-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 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 expression was induced using 0.2 mM IPTG at 16°C for 12 h. The supernatant was discarded after centrifugation, and the bacterial pellet containing the CDH17-targeted nanobody protein was obtained.

[0092] The nanobodies obtained in part 3 of this example were identified by SDS-PAGE, and the results are shown in FIG. 3. The target band appeared at 15 KD. Figure 11

[0093] The nanobodies obtained in part 3 of this example were then subjected to protein purification using the AKATA protein purification system through a Ni Sepharose excel affinity chromatography column. The purified nanobodies were identified by SDS-PAGE, and the results are shown in FIG. 4. This example successfully obtained the purified nanobody protein targeting CDH17 after elution. Figure 12

[0094] The purified nanobody protein was identified by Western blotting in this example. The primary antibody used in the Western blotting was an anti-His tag monoclonal antibody (Proteintech), and the secondary antibody was an HRP-labeled goat anti-mouse antibody (Thermo). The results are shown in FIG. 5, indicating that the purified protein obtained in this example was a recombinant nanobody. Figure 13

[0095] The purified recombinant nanobody protein was subjected to SPR test by Biacore 8k instrument in this example to verify the binding force of the purified nanobody protein to the recombinant protein CDH17-EC, and the KD constant was calculated and analyzed. The results are shown in FIG. 6. It can be seen that the KD value of the nanobody protein after panning and purification to the recombinant protein CDH17-EC is 7.15 nM, which has higher affinity than traditional antibodies. Figure 14

[0096] Example 6: Verification of the binding domain of the CDH17-targeted nanobody ​​​​

[0097] (1) Truncation expression of CDH17 recombinant protein

[0098] According to the EC1-EC7 domains of the CDH17 extracellular region, gene fragments with deletion of EC1, EC1-2, EC1-3, EC1-4 and EC1-5 domains were cloned, respectively, and the above cloned gene fragments were constructed into the eukaryotic expression vector pCAGGS-SP-C-6his. After successful identification of the recombinant plasmid, it was transiently transfected into HEK293 suspension cells for secretory expression and purification. The identification results are shown in Figure 15 , indicating that the CDH17 recombinant protein was successfully expressed.

[0099] (2) Fusion expression of nanobodies targeting CDH17 and Fc tag

[0100] The 5 strains of nanobodies purified in Example 5 were constructed into the eukaryotic expression vector pFuse-hIgG1-Fc vector by gene cloning technology. After successful identification of the recombinant plasmid, it was transiently transfected into HEK293 suspension cells for secretory expression and purification. The identification results are shown in Figure 16 , indicating that the recombinant protein VHH-Fc was successfully expressed.

[0101] (3) Identification of nanobodies targeting CDH17 and CDH17 binding domain

[0102] The CDH17 recombinant proteins with deletion of different domains successfully expressed in (1) were subjected to SDS-PAGE electrophoresis, and then transferred to a nitrocellulose membrane (NC membrane). After blocking with 5% skim milk for 2 h, the membrane was washed with TBST solution for 3 times, 10 min each time. The primary antibody of recombinant protein VHH-Fc was incubated at room temperature for 2 h, and the membrane was washed with TBST solution for 3 times, 10 min each time. The secondary antibody of HRP-mouse anti-human Fc antibody from invitrogen company was incubated at room temperature for 1 h, and the membrane was washed with TBST solution for 3 times, 10 min each time. ECL luminescent liquid was added for imaging detection, and the results are shown in Figure 17 , indicating that the domain of the nanobodies targeting CDH17 binding to CDH17 is the EC1 region of the CDH17 extracellular region.

[0103] Example 7: Application of nanobodies targeting CDH17

[0104] (1) Application of nanobodies targeting CDH17 in protein detection antibodies

[0105] The present embodiment takes 293T or 293T-h-CDH17 cell line as a positive control, and carries out the culture of AGS-control / AGS-CDH17 and HGC-control / HGC-CDH17 cell lines in vitro. After the cells grow in the cell culture bottle, the cells are washed with PBS solution, and then cell lysis solution (RIPA+PMSF+ nuclease) is added, and the cells are lysed in ice bath for 30 min, and centrifuged at 12000 rpm for 15 min, and the cell supernatant is collected, and the protein concentration of the obtained cell supernatant is determined by using the BCA concentration determination kit of Biyun Tian Company; 50 μg of each sample is loaded, and the commercial CDH17 monoclonal antibody (Proteintech) is used as a control detection antibody, and the purified nanobody targeting CDH17 obtained in Example 5 is used as a primary antibody, and the HRP-labeled sheep anti-mouse antibody (Thermo) is used as a secondary antibody, and western detection is carried out, and the results are shown in Figure 18 The nanobody targeting CDH17 provided by the present application can specifically bind to the CDH17 protein after cell lysis, and therefore can be used as a protein detection antibody.

[0106] (2) Application of the nanobody targeting CDH17 in flow cytometry detection antibody

[0107] In the present embodiment, the cell line AGS-CDH17 expressing CDH17 membrane protein is taken as the research object, and the cultured cell line is counted, and 1x10 6 cells are taken, 500 μl of PBS solution is added for washing, and centrifuged at 500 g for 5 min, and the supernatant is discarded; 2 μg of the purified nanobody targeting CDH17 in Example 5 is added as a primary antibody, and incubated at 4°C for 45 min; then 500 μl of PBS solution is added, and centrifuged at 500 g for 5 min, and the supernatant is discarded, and the washing is repeated twice; then the APC-anti-His flow cytometry antibody (Biolegend) is added as a secondary antibody, and incubated at 4°C for 45 min, and then 500 μl of PBS solution is added, and centrifuged at 500 g for 5 min, and the supernatant is discarded, and the washing is repeated twice, and resuspended with 1 ml of PBS solution, and finally filtered with a 200-mesh filter cloth.

[0108] The filtered cells after staining are subjected to flow cytometry detection, and the results are shown in Figure 19 Compared with the control group, the positive cell deflection is 73.4%, which indicates that the nanobody targeting CDH17 provided by the present application can specifically bind to the CDH17 protein on the cell membrane surface, and therefore can be used as a detection and therapeutic antibody.

[0109] The content not described in detail in the specification of the present application is the technology known to the person 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 cadherin 17, characterized in that, The amino acid sequence of the nanobody targeting cadherin 17 is shown as SEQ ID NO. 8; the nanobody targeting cadherin 17 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 cadherin 17 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 comprises a nucleic acid sequence encoding the nanobody targeting cadherin 17 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 cadherin 17 according to claim 1 in the preparation of a reagent for detecting cadherin 17.

6. Use according to claim 5, characterized in that, The use refers to detecting tumor tissues expressing cadherin 17.

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