Antibody targeting ADAM17 protein and application

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses an antibody targeting ADAM17 protein and application of the antibody. The invention provides the ADAM17 specific target spot aiming at the malignant tumor of the biliary tract, the ADAM17 is highly expressed in the tumor of the biliary tract and is relatively low in expression in normal tissues, and by intervening the target spot, tumor cells can be killed, and non-specific attack to normal cells can be avoided as much as possible, so that the effectiveness and the safety of treating a patient with the malignant tumor of the biliary tract are improved. The invention also provides an antibody targeting ADAM17 protein and a preparation and screening method thereof, and the antibody can specifically recognize an antigen binding domain of ADAM17, has strong affinity with an ADAM17 antigen, and can be used for treating ADAM17 positive malignant tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to antibodies targeting ADAM17 protein and applications thereof. Background Art

[0002] Biliary tract cancer (BTC) includes intrahepatic cholangiocarcinoma (iCCA), perihilar cholangiocarcinoma (pCCA), extrahepatic cholangiocarcinoma (eCCA) and gallbladder cancer (GBC), accounting for about 3% of digestive system tumors. In recent years, the incidence and mortality of BTC have been increasing worldwide, but treatment strategies are still limited. Surgery is the only way to radically treat BTC patients. However, BTC has strong invasion and metastasis capabilities. Most patients are in the advanced stage at the time of initial diagnosis and have lost the opportunity for surgical treatment, with a very poor prognosis.

[0003] In patients with advanced unresectable or metastatic biliary tract cancer, chemotherapy remains the basis for the treatment of advanced BTC. Immunotherapy alone is ineffective, and gemcitabine-cisplatin combined immunotherapy has become a first-line treatment option. For patients who progress after first-line systemic treatment, second-generation genome sequencing can be used to provide second-line targeted drug treatment, but only about 30% of BTC have potentially viable genetic changes. In addition, radiotherapy, ablation therapy, and transarterial interventional therapy such as TACE, SIRT, and HAIC are also used in advanced biliary malignancies. At present, the treatment of BTC is exploring a combined treatment model of chemotherapy, immunotherapy, and targeted therapy, but overall its efficacy is still not ideal.

[0004] Currently, there are few specific target options for biliary malignancies, and the intratumor heterogeneity is high, making it difficult to cover all tumor cells. Therefore, there is an urgent need to develop new therapeutic targets and related targeted preparations to improve the prognosis of BTC patients. Summary of the invention

[0005] In order to solve the above problems, the present invention provides antibodies targeting ADAM17 protein and applications thereof.

[0006] The first aspect of the present invention is to provide a specific target for biliary malignancies, the target being ADAM17, the nucleotide sequence of which is shown in SEQ ID NO:1, and the amino acid sequence of which is shown in SEQ ID NO:2.

[0007] The ADAM17 (ADAM Metallopeptidase Domain 17, ADAM17) gene encodes a member of the ADAM (disintegrin and metalloproteinase domain) family. Members of this family are involved in a variety of biological processes of cell-cell and cell-matrix interactions, including fertilization, muscle development, and neurogenesis. The protease encoded by this gene plays a role in the shedding of the extracellular domain of tumor necrosis factor-α. This protease is also involved in the processing of many other substrates, including cell adhesion proteins, cytokine and growth factor receptors, and epidermal growth factor (EGF) receptor ligands, and plays an important role in the activation of the Notch signaling pathway. Due to the high heterogeneity and complex tumor microenvironment of solid tumors, the selection of therapeutic targets is crucial. We found that ADAM17 is highly expressed in biliary tumors and less expressed in normal tissues, making it an ideal target for the treatment of biliary tumors and other ADAM17-positive tumors.

[0008] The second aspect of the present invention is to provide the sequence of an antibody targeting ADAM17 protein. The antibody targeting ADAM17 protein comprises a heavy chain variable region VH and a light chain variable region VL, and the heavy chain variable region and the light chain variable region both comprise three complementary determining regions CDR1, CDR2 and CDR3, and the antibody is selected from any one of the three antibodies 1G9, 2F12, and 6G5, and the nucleotide sequences of the variable regions and complementary determining regions of the three antibodies 1G9, 2F12, and 6G5 are shown in the following table:

[0009]

[0010] Furthermore, the amino acid sequences of the variable regions and complementary determining regions of the three antibodies 1G9, 2F12, and 6G5 are shown in the following table:

[0011]

[0012]

[0013] Furthermore, the antibody further comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region is selected from IgG1, IgG2, IgG3, or IgG4, and the light chain constant region is a κ or λ chain.

[0014] Furthermore, the antibody is a monoclonal antibody, Fab, Fab'-SH, (Fab')2, Fv, single-chain Fv (scFv), diabody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody or a fusion protein comprising the antigen binding portion of an antibody.

[0015] The third aspect of the present invention is to provide a method for preparing and screening the above-mentioned 1G9, 2F12 and 6G5 antibodies targeting ADAM17 protein, comprising the following steps:

[0016] Step 1: Synthesis of ADAM17 antigen gene and construction of expression vector;

[0017] Step 2: Expression and preparation of ADAM17 recombinant protein antigen: transfect cells with the expression vector constructed in step 1, and then purify to obtain ADAM17-FC recombinant protein;

[0018] Step 3: Preparation of mouse hybridoma: electrofusion of mouse myeloma cells SP2 / 0 and spleen lymphocytes to obtain hybridoma cells;

[0019] Step 4: Screening of positive clones: antigen staining of hybridoma cells was performed to determine positive clones; positive clones that bound to ADAM17-FC protein but not to FC control protein were selected by ELISA; finally, three positive clones with higher cell positivity rates, 1G9, 2F12, and 6G5, were screened by flow cytometry.

[0020] Furthermore, in the step 1, the expression vector construction method is as follows: design the ADAM17 expression protein sequence ADAM17-FC as shown in SEQ ID NO: 3, select the extracellular segment sequence Arg215-Asn671 of the ADAM17 antigen to design primers to amplify the target fragment, add the IgGκSP signal peptide sequence to the 5' end of the gene, add the FC fusion protein tag to the 3' end of the gene, construct it into the pcDNA3.1(+) vector, and obtain a DNA plasmid expressing the human ADAM17 antigen: pcDNA3.1-ADAM17-FC.

[0021] The fourth aspect of the present invention is to provide the use of the above-mentioned antibody targeting ADAM17 protein in the preparation of a drug for treating biliary malignancies.

[0022] The present invention has the following beneficial effects:

[0023] 1. The present invention proposes ADAM17, a specific target for biliary malignancies. ADAM17 is highly expressed in biliary tumors and less expressed in normal tissues. By intervening in this target, it can kill tumor cells and avoid nonspecific attacks on normal cells as much as possible, thereby improving the effectiveness and safety of treatment for patients with biliary malignancies.

[0024] 2. The present invention provides three antibodies targeting ADAM17 protein, which can specifically recognize the antigen binding domain of ADAM17 and can be used to treat ADAM17-positive malignant tumors.

[0025] 3. The present invention also provides a method for preparing and screening antibodies targeting ADAM17 protein, which can obtain antibodies with strong affinity and good specificity for ADAM17 antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : The expression results of ADAM17 in different cell lines in Example 1.

[0027] Figure 2 : The result of 4-20% SDS-PAGE reduction electrophoresis of ADAM17-FC protein in Example 2. In the figure, the left band is the ADAM17-FC recombinant protein, and the right band is the marker.

[0028] Figure 3 : Mouse serum titer detection results in Example 2.

[0029] Figure 4 : ELISA test results of hybridoma supernatants in Example 2. There are 6 96-well plates in total, and Figures 4-1 and 4-2 show the test results of three 96-well plates respectively.

[0030] Figure 5 : Flow cytometry results of hybridoma supernatant in Example 2.

[0031] Figure 6 : Variable region sequence information of the three antibodies 1G9, 2F12, and 6G5 in Example 2.

[0032] Figure 7 : Affinity test results of the three antibodies 1G9, 2F12 and 6G5 in Example 3.

[0033] Figure 8 : Specificity detection results of the three antibodies 1G9, 2F12 and 6G5 in Example 3. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0035] Example 1: Biliary tract malignancy target screening experiment

[0036] Experimental procedures: 1E6 total cells were used for each sample, and surface staining was performed using ADAM17 PE-conjugated flow antibody (Catalog #FAB9301P) in a total volume of 100 μL FACS buffer for 30 minutes, and then the cells were washed with FACS buffer. Cell surface ADAM17 expression was quantified using the PE Fluorescence Quantitation Kit (Catalog #340495) and following the manufacturer's recommended procedures. Samples were analyzed using a FACSAria II flow cytometer (BD Biosciences).

[0037] Results: Figure 1 As shown, it can be concluded that ADAM17 is highly expressed in cholangiocarcinoma cell lines HuCCT1, LICCF, QBC939, RBE, ICC772, ICC2935, ICC4715, HCCC-9810 and gallbladder carcinoma cell lines NOZ, OCUG1, and SGC996, while is less expressed in the normal bile duct cell line H69 and the normal intestinal epithelial cell line FHC, and can be used as a target for the treatment of biliary tumors and other ADAM17-positive tumors.

[0038] Example 2: Preparation and screening of antibodies targeting ADAM17

[0039] Step 1: Synthesis of ADAM17 antigen gene and construction of expression vector

[0040] ADAM17 gene template cDNA (pCR4-TOPO-ADAM17 (human), Wuhan Miaoling Biotechnology) was purchased, and the corresponding ADAM17 gene sequence was shown in SEQ ID NO: 1, and the amino acid sequence was shown in SEQ ID NO: 2; ADAM17 expression protein sequence ADAM17-FC (sequence shown in SEQ ID NO: 3) was designed, and the extracellular segment sequence of ADAM17 antigen (Arg215-Asn671) was selected to design primers to amplify the target fragment, and the IgGκSP signal peptide sequence was added to the 5' end of the gene, and the FC fusion protein tag was added to the 3' end of the gene. It was constructed into the pcDNA3.1 (+) vector to obtain a DNA plasmid expressing human ADAM17 antigen: pcDNA3.1-ADAM17-FC. The constructed plasmid was used for PCR identification and sequencing (Suzhou Jinweizhi Biotechnology Co., Ltd.), and the identification results showed that the PCR amplification target band was consistent with the expected size, and sequencing indicated that the construction was correct.

[0041] Step 2: Expression and preparation of ADAM17 recombinant protein antigen

[0042] The ADAM17-FC recombinant expression plasmid was designed and constructed, and the ADAM17-FC recombinant protein was obtained by transient transfection of 293F cells and antigen purification was completed using protein A purification medium. The specific operations are as follows:

[0043] 293F cells were placed in a 5% CO2 constant temperature shaker at 37°C and 120 rpm to determine their cell density and survival rate. The cell density at the time of transfection was 4×10 6 / ml, with a survival rate greater than 97% of cells transfected; prepare two 15ml sterile centrifuge tubes, add 5ml KPM and 100μg sterile plasmid pcDNA3.1-ADAM17-FC to one of them, and gently blow to mix; take another tube and add 5ml KPM and 500μl TA-293 transfection reagent, and gently blow to mix (the transfection volume corresponding to the amount of transfection reagent and plasmid used is 100ml cells; transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid, and gently blow to mix; let it stand at room temperature for 10 minutes to prepare the plasmid-vector complex; remove the cells from the constant temperature shaker, add the prepared plasmid-vector complex while shaking, and return to the CO2 constant temperature shaker for culture. 600μl can be added 24 hours after transfection 293 cell protein expression enhancer (KE-293) and feed were used to increase product expression; cells and supernatant were collected 6 days after transfection for subsequent purification. The above transient transfection reagents were purchased from Zhuhai Kairui Biotechnology Co., Ltd. ADAM17-FC protein was purified using Protein A (chromatographic filler: Saifen Technology, catalog number: MabPurix A65).

[0044] The purified protein was subjected to SDS-PAGE electrophoresis. Figure 2 As shown, ADAM17-FC protein had obvious bands, and ADAM17-FC recombinant protein was successfully prepared.

[0045] Step 3. Preparation of mouse hybridoma

[0046] Animal immunization: 100 μg antigen (ADAM17-FC) per animal per time, intraperitoneal immunization. Immunization procedure: After 15 days, the same dose was mixed with PAP1 adjuvant (purchased from Sige Biotech) for the second immunization; after 21 days, the same dose was mixed with PAP1 adjuvant for the third immunization; after 7 days, blood was collected from the tail and the serum titer was measured by indirect ELISA method. The results are as follows Figure 3 As shown, 6 mice were immunized with ADAM17 protein. After three immunizations, the titer of mouse immune serum exceeded 1:128,000 and could be used for subsequent fusion and screening.

[0047] Prepare mouse myeloma cell suspension: resuscitate mouse myeloma SP2 / 0 cells two weeks in advance, culture and passage in 1640 medium containing 10% fetal bovine serum at 37°C, 5% CO2. On the day of fusion, observe the state of SP2 / 0 cells. Microscopic examination of SP2 / 0 cell confluence is 70%-80%, which indicates that the cells are in the logarithmic growth phase. Collect SP2 / 0 cells by centrifugation at 300g for 5 minutes, wash once with 20ml PBS, 1000rpm, 5min, discard the supernatant; resuspend with an appropriate amount of 1640 medium, and adjust the SP2 / 0 cell concentration to 1×10 7 pcs / ml, set aside.

[0048] Preparation of mouse spleen lymphocyte suspension: 3-4 days after booster immunization, spleen cells of immune mice were taken and fused with SP2 / 0. The mice were killed by pulling the neck, the peritoneum was cut aseptically, the spleen was removed and the surrounding connective tissue was stripped off. After grinding the mouse spleen cells, 70μm mesh was filtered into a 50ml centrifuge tube pre-filled with 15ml 1640 culture medium to prepare a single cell suspension; 1500rpm, 5min, 1640 culture medium was washed, and the supernatant was discarded; 4ml red blood cell lysis buffer (Sigma) was added to lyse the red blood cells for about 2min; an equal volume of 1640 culture medium was added, 1500rpm, 5min centrifuged, and the upper red blood cell layer was discarded; resuspended with 20ml PBS, washed twice, and centrifuged at 1500rpm, 5min. After the second wash, resuspend the cells with 15 ml PBS. If there are clumping connective tissue residues, all the cells can be sieved and transferred to a new 50 ml centrifuge tube. Count the spleen lymphocytes and resuspend them with an appropriate amount of 1640 medium to adjust the spleen cells to 1×10 7 / ml. Take 1×10 8 Splenocytes were mixed with SP2 / 0 cells at a ratio of 1:1, centrifuged at 1500rpm for 5 minutes, and the supernatant was discarded. Resuspended and mixed with BTX electrofusion buffer, and electrofusion was performed according to the instructions of the BTX electrofusion instrument. The fused cells were allowed to stand at room temperature for 30 minutes, and 1640 selection medium containing 1×HAT and 10% fetal bovine serum was added to them, and placed in a T175 bottle for culture at 37°C and 5% CO2 for 5 days. Then, the culture medium containing 1×HT was used to replace the culture medium and culture the fused hybridoma cell clones.

[0049] Step 4: Positive clone screening

[0050] Antigen staining: Hybridoma cells were stained with antigens, and antigen-positive monoclones were sorted into 96-well cell culture plates. After 7 days of culture, the supernatant was taken for ELISA and flow staining to identify positive clones.

[0051] Indirect ELISA: Coat the ELISA plate with ADAM17-FC antigen, and set up an ELISA plate coated with control FC protein for detection, incubate at 4℃ overnight, block with irrelevant protein (BSA) at 37℃ for 2h, add the culture supernatant of hybridoma cells that passed the above experiment, incubate at 37℃ for 1h, wash 5-6 times with 0.05% Tween 20-PBS, add secondary antibody (such as HRP-GAM), incubate at 37℃ for 2h, wash as above, develop color with substrate, add stop solution (3M H2SO4) after color development to a certain extent, stop the reaction, read OD492 on the ELISA instrument, the result is as follows Figure 4As shown, there are a total of 6 96-well plates of hybridomas. The hybridoma supernatant of each well plate is tested for binding to ADAM17-FC protein and FC control protein. The clones that bind to ADAM17-FC protein but not to FC control protein are selected and marked as positive wells for subsequent flow cytometry detection.

[0052] Flow cytometry: ADAM17-positive tumor cell lines were selected as experimental cells, and the supernatant of ELISA-positive hybridoma samples was stained by flow cytometry to confirm the cell positivity rate of different hybridoma clones. The results are as follows: Figure 5 As shown, a total of 7 flow cytometry-positive clones were screened, and three clones, 1G9, 2F12, and 6G5, were selected according to the positive rate results for subsequent hybridoma sequencing.

[0053] Step 5: Antibody variable region sequencing

[0054] Hybridoma cells were sent to Suzhou Baida Biotechnology Co., Ltd. for antibody sequencing. The hybridoma antibody sequencing experimental process includes RNA extraction by Trizol method, sample reverse transcription to obtain cDNA, amplification and acquisition of heavy chain and light chain variable region sequences, sequencing, comparison of sequencing results to the IMGT database, and extraction of CDR1 / 2 / 3 information. The variable region sequence information of 1G9, 2F12, and 6G5 is as follows: Figure 6 shown.

[0055] Example 3: Antibody effect verification experiment

[0056] 1. Antibody affinity testing

[0057] Octet-Red was used to perform biolayer interferometry (BLI) to determine the binding kinetics of the three clones 1G9, 2F12, and 6G5 to ADAM17. First, EDC and S-NHS were mixed in a 1:1 reaction and used to activate the AR2G biosensor. The recombinant ADAM17-Fc protein was then diluted to 30ug / mL with sodium acetate buffer at pH 5.0, and the antigen was loaded onto the sensor until saturated and blocked with 1M ethanolamine at pH 8.5. The AR2G probe loaded with antigen was immersed in the antibody-diluted well for 300s. The ADAM17-Fc antigen-antibody complex was then dissociated in PBST (PBS + 0.2% Tween) buffer. Data Analysis software version 10.0 was used to fit the 1:1 binding model and calculate the affinity. The kon (association rate constant), koff (dissociation rate constant) and KD (equilibrium dissociation constant) values ​​of the three antibody clones 1G9, 2F12 and 6G5 were determined by averaging the binding curves at R2 values ​​greater than 95% confidence level.

[0058] The results are as follows Figure 7 As shown, the equilibrium dissociation constants K of the three clones 1G9, 2F12, and 6G5 are D They are 0.16nM, 0.36nM, 0.06nM, respectively, and K D The values ​​are all very small, proving that the three antibodies have a strong affinity for the ADAM17 antigen.

[0059] 2. Antibody Specificity Detection

[0060] The three antigens ADAM8, ADAM9, and ADAM10 were diluted separately and coated in a 96-well plate at 4°C overnight. After washing the plate three times, the blocking solution was added and blocked at room temperature for 1 hour, and the plate was washed again for 3 times. The antibody was diluted to 6 concentration gradients with a starting concentration of 100nM, 100ul / well was added to the well plate, and the plate was washed 3 times with washing solution. The enzyme-linked antibody was diluted to the working concentration, mixed and added to the well plate at 100ul / well, and incubated at room temperature for 1 hour. The substrate ABTS was then added to detect the enzyme activity, and the signal was read at 405nm using a microplate spectrophotometer. The data was fitted to the curve using GraphPad Prism version 8.0. The results are shown in Figure 8 As shown, only the 2F12 antibody had a weaker binding to ADAM10 at a higher concentration, proving that the three antibodies 1G9, 2F12, and 6G5 had good specificity.

[0061] This specific implementation is only an explanation of the present invention, not a limitation of the present invention. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. An antibody targeting ADAM17 protein, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region and the light chain variable region both comprise three complementary determining regions, CDR1, CDR2 and CDR3, characterized in that: The antibody is selected from any one of the three antibodies 1G9, 2F12, and 6G5. The nucleotide sequences of the variable regions and complementary determining regions of the three antibodies 1G9, 2F12, and 6G5 are shown in the following table:

2. The antibody targeting ADAM17 protein according to claim 1, characterized in that The amino acid sequences of the variable regions and complementary determining regions of the three antibodies 1G9, 2F12, and 6G5 are shown in the following table:

3. The antibody targeting ADAM17 protein according to claim 1 or 2, characterized in that: The antibody further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is selected from IgG1, IgG2, IgG3, or IgG4, and the light chain constant region is a κ or λ chain.

4. The antibody targeting ADAM17 protein according to claim 1 or 2, characterized in that: The antibody is a monoclonal antibody, Fab, Fab'-SH, (Fab')2, Fv, single-chain Fv (scFv), diabody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody or a fusion protein comprising an antigen binding portion of an antibody.

5. The antibody targeting ADAM17 protein according to claim 1 or 2, characterized in that: The preparation and screening method of the antibody is as follows: Step 1: Synthesis of ADAM17 antigen gene and construction of expression vector; Step 2: Expression and preparation of ADAM17 recombinant protein antigen: transfect cells with the expression vector constructed in step 1, and then purify to obtain ADAM17-FC recombinant protein; Step 3: Preparation of mouse hybridoma: electrofusion of mouse myeloma cells SP2 / 0 and spleen lymphocytes to obtain hybridoma cells; Step 4: Screening of positive clones: antigen staining of hybridoma cells was performed to determine positive clones; positive clones that bound to ADAM17-FC protein but not to FC control protein were selected by ELISA; finally, three positive clones with higher cell positivity rates, 1G9, 2F12, and 6G5, were screened by flow cytometry.

6. The antibody targeting ADAM17 protein according to claim 5, characterized in that In the step 1, the expression vector construction method is as follows: design the ADAM17 expression protein sequence ADAM17-FC as shown in SEQ ID NO: 3, select the extracellular segment sequence Arg215-Asn671 of the ADAM17 antigen to design primers to amplify the target fragment, add the IgGκSP signal peptide sequence at the 5' end of the gene, add the FC fusion protein tag at the 3' end of the gene, construct it into the pcDNA3.1(+) vector, and obtain a DNA plasmid expressing the human ADAM17 antigen: pcDNA3.1-ADAM17-FC.

7. Use of the antibody targeting ADAM17 protein according to any one of claims 1 to 4 in the preparation of a drug for treating malignant tumors of the biliary tract, characterized in that: The nucleotide sequence of the ADAM17 protein is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.

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