Antibodies targeting adam17 protein and uses

Abstract

This invention belongs to the field of biomedical technology, specifically disclosing antibodies targeting the ADAM17 protein and their applications. This invention proposes ADAM17 as a specific target for biliary tract malignancies, which is highly expressed in biliary tract tumors and lowly expressed in normal tissues. By intervening in this target, tumor cells can be killed while minimizing non-specific attacks on normal cells, thereby improving the efficacy and safety of treatment for patients with biliary tract malignancies. This invention also provides antibodies targeting the ADAM17 protein and their preparation and screening methods. The antibodies specifically recognize the antigen-binding domain of ADAM17 and have a strong affinity for the ADAM17 antigen, making them suitable for treating ADAM17-positive malignancies.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to antibodies targeting the ADAM17 protein and their applications. Background Technology

[0002] Malignant biliary tract tumors (BTC) include intrahepatic cholangiocarcinoma (iCCA), perihepatic cholangiocarcinoma (pCCA), extrahepatic cholangiocarcinoma (eCCA), and gallbladder cancer (GBC), accounting for approximately 3% of digestive system tumors. In recent years, the global incidence and mortality rates of BTC have been on the rise, but treatment strategies remain limited. Surgery is the only radical treatment for BTC patients; however, BTC has a strong invasive and metastatic capacity, and most patients are diagnosed at an advanced stage, having already lost the opportunity for surgical treatment, resulting in a very poor prognosis.

[0003] In patients with advanced unresectable or metastatic biliary tract cancer, chemotherapy remains the cornerstone of treatment. Immunotherapy alone is ineffective, and gemcitabine-cisplatin combined with immunotherapy has become a first-line treatment option. For patients who progress after first-line systemic therapy, next-generation genome sequencing can be used to provide second-line targeted therapies; however, only about 30% of BTC cases have potentially viable genetic alterations. In addition, radiotherapy, ablation therapy, and transarterial interventional therapies such as TACE, SIRT, and HAIC are also used in advanced biliary malignancies. Currently, the treatment of BTC is exploring combined chemotherapy, immunotherapy, and targeted therapies, but overall, the efficacy remains unsatisfactory.

[0004] Currently, there are few specific targets for biliary tract malignancies, and the tumors are highly heterogeneous, making it difficult to cover all tumor cells. Therefore, there is an urgent need to develop new therapeutic targets and related targeted agents to improve the prognosis of BTC patients. Summary of the Invention

[0005] To address the above problems, this invention provides an antibody targeting the ADAM17 protein and its applications.

[0006] A first aspect of the present invention is to provide a specific target for malignant biliary tract tumors, 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) gene encodes a member of the ADAM (de-integrin and de-metalloproteinase domain) family. Members of this family are involved in various cell-cell and cell-matrix biological processes, 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 a crucial 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 critical. We found that ADAM17 is highly expressed in biliary tract tumors and lowly expressed in normal tissues, making it an ideal target for the treatment of biliary tract tumors and other ADAM17-positive tumors.

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

[0009]

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

[0011]

[0012]

[0013] Furthermore, the antibody also includes 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.

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

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

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

[0017] Step 2, preparation of ADAM17 recombinant protein antigen expression: The expression vector constructed in Step 1 was transfected into cells and then purified to obtain ADAM17-FC recombinant protein;

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

[0019] Step 4: Screening for positive clones: Antigen staining is performed on hybridoma cells to identify positive clones; positive clones that bind to ADAM17-FC protein but not to FC control protein are selected by ELISA; finally, three positive clones with high cell positivity rates, 1G9, 2F12, and 6G5, are screened by flow cytometry.

[0020] Further, in step one, 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 the DNA plasmid expressing the human ADAM17 antigen: pcDNA3.1-ADAM17-FC.

[0021] A fourth aspect of the present invention is to provide the use of the antibody targeting the ADAM17 protein described above in the preparation of a medicament for treating malignant tumors of the biliary tract.

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

[0023] 1. This invention proposes ADAM17 as a specific target for biliary tract malignancies. ADAM17 is highly expressed in biliary tract tumors and lowly expressed in normal tissues. By intervening in this target, tumor cells can be killed while non-specific attacks on normal cells can be avoided as much as possible, thereby improving the effectiveness and safety of treatment for patients with biliary tract malignancies.

[0024] 2. This invention provides three antibodies that target the ADAM17 protein. These antibodies 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. Attached Figure Description

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

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

[0028] Figure 3 Results of mouse serum titer detection in Example 2.

[0029] Figure 4 Example 2: ELISA results of hybridoma supernatant. A total of 6 96-well plates were used. Figures 4-1 and 4-2 show the 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 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 Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0035] Example 1: Screening Experiment for Targets in Malignant Biliary Tract Tumors

[0036] Experimental Procedure: 1E6 total cells were used for each sample. The cells were surface-stained with ADAM17 PE-conjugated flow cytometry antibody (Catalog #FAB9301P) in 100 μL of FACS buffer for 30 minutes, followed by washing with FACS buffer. ADAM17 expression on the cell surface was quantified using the PE Fluorescence Quantitation Kit (Catalog #340495) according to the manufacturer's recommended procedures. Samples were analyzed using a FACSAria II flow cytometer (BD Biosciences).

[0037] Result: As Figure 1 As shown, ADAM17 is highly expressed in cholangiocarcinoma cell lines HuCCT1, LICCF, QBC939, RBE, ICC772, ICC2935, ICC4715, HCCC-9810 and gallbladder cancer cell lines NOZ, OCUG1, and SGC996, while it is expressed at lower levels in normal cholangiocarcinoma cell line H69 and normal intestinal epithelial cell line FHC. It can serve as a target for the treatment of biliary tract 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] The ADAM17 gene template cDNA (pCR4-TOPO-ADAM17(human), Wuhan Miaoling Biotechnology) was purchased. The corresponding ADAM17 gene sequence is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2. The ADAM17 expression protein sequence ADAM17-FC (sequence shown in SEQ ID NO:3) was designed. Primers were designed to amplify the target fragment using the extracellular segment sequence of the ADAM17 antigen (Arg215-Asn671). An IgGκSP signal peptide sequence was added to the 5' end of the gene, and an FC fusion protein tag was added to the 3' end. The plasmid expressing the human ADAM17 antigen, pcDNA3.1-ADAM17-FC, was constructed into the pcDNA3.1(+) vector. The constructed plasmid was then used for PCR identification and sequencing (Suzhou Genewiz Biotechnology Co., Ltd.). Identification results showed that the PCR amplified target band was consistent with the expected size, and sequencing confirmed correct construction.

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

[0042] The ADAM17-FC recombinant expression plasmid was designed and constructed, and expressed by transient transfection into 293F cells. Antigen purification was performed using protein A purification medium to obtain the ADAM17-FC recombinant protein. The specific procedures are as follows:

[0043] 293F cells were placed in a 5% CO2 constant-temperature shaker and cultured at 37°C and 120 rpm to determine cell density and viability. The cell density at transfection was 4 × 10⁻⁶ cells / year. 6Transfect cells with a cell count / mL and a viability greater than 97%; prepare two 15ml sterile centrifuge tubes. Add 5ml KPM and 100μg sterile plasmid pcDNA3.1-ADAM17-FC to one tube and gently mix. Add 5ml KPM and 500μl TA-293 transfection reagent to the other tube and gently mix (the amount of transfection reagent and plasmid corresponds to a transfection volume of 100ml cells). Transfer all liquid from the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid and gently mix. Incubate at room temperature for 10 minutes to prepare the plasmid-vector complex. Remove the cells from the incubator and add the prepared plasmid-vector complex while shaking. Return the cells to the CO2 incubator for further culture. 24 hours after transfection, add 600μl of the prepared plasmid-vector complex. 293 cell protein expression enhancer (KE-293) and feed were used to increase product expression levels. Cells and supernatant were collected on day 6 post-transfection for subsequent purification. All reagents used for the rapid transfection were purchased from Zhuhai Kairui Biotechnology Co., Ltd. ADAM17-FC protein was purified using Protein A (chromatographic packing material: Saifen Technology, catalog number: MabPurix A65).

[0044] The purified protein was subjected to SDS-PAGE electrophoresis, and the results are as follows: Figure 2 As shown, the ADAM17-FC protein has a distinct band, indicating that the recombinant ADAM17-FC protein was successfully prepared.

[0045] Step 3: Preparation of mouse hybridomas

[0046] Animal immunization: Each animal was immunized intraperitoneally with 100 μg of antigen (ADAM17-FC) per dose. Immunization schedule: A second immunization was administered 15 days later with the same dose mixed with PAP1 adjuvant (purchased from Siger Biotech); a third immunization was administered 21 days later with the same dose mixed with PAP1 adjuvant; blood was collected from the tail 7 days later for serum titer determination using indirect ELISA. Results are as follows... Figure 3 As shown, six mice were immunized with ADAM17 protein. After three immunizations, the serum titer of the mice exceeded 1:128,000, which can be used for subsequent fusion and screening.

[0047] Preparation of mouse myeloma cell suspension: Two weeks in advance, mouse myeloma cells SP2 / 0 were resuscitated and cultured and passaged in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. On the day of fusion, the status of SP2 / 0 cells was observed. Microscopic examination showed that the confluence of SP2 / 0 cells was 70%-80%, indicating that the cells were in the logarithmic growth phase. SP2 / 0 cells were collected by centrifugation at 300g for 5 minutes, washed once with 20ml PBS, and centrifuged at 1000rpm for 5 minutes. The supernatant was discarded. The cells were resuspended in an appropriate amount of 1640 medium to adjust the SP2 / 0 cell concentration to 1×10⁻⁶ cells / mL. 7 Quantity / ml, ready for use.

[0048] Preparation of mouse spleen lymphocyte suspension: On days 3-4 after booster immunization, spleen cells from immunized mice were fused with SP2 / 0. Mice were euthanized by cervical retraction, the peritoneum was aseptically cut open, the spleen was removed, and the surrounding connective tissue was peeled off. The mouse spleen cells were ground and filtered through a 70μm sieve into a 50ml centrifuge tube pre-filled with 15ml of 1640 medium to prepare a single-cell suspension; washed at 1500rpm for 5min with 1640 medium, and the supernatant was discarded; erythrocytes were lysed with 4ml of erythrocyte lysis buffer (Sigma) for about 2min; an equal volume of 1640 medium was added, centrifuged at 1500rpm for 5min, and the upper erythrocyte layer was discarded; the cells were resuspended in 20ml of PBS, washed twice, and centrifuged at 1500rpm for 5min. After the second wash, resuspend the cells in 15 ml of PBS. If any clumps of connective tissue remain, sieve all the cells and transfer them to a new 50 ml centrifuge tube. Count the spleen lymphocytes, resuspend them in an appropriate amount of 1640 medium, and adjust the spleen cell count to 1 × 10⁻⁶ cells / mL. 7 1 x 10^6 cells / ml. 8 Spleen cells were mixed with SP2 / 0 cells at a 1:1 ratio, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in BTX electrofusion buffer and mixed thoroughly. Electrofusion was performed according to the BTX electrofusion instrument manual. The fused cells were incubated at room temperature for 30 minutes, then added to 1640 selective medium containing 1×HAT and 10% fetal bovine serum. The mixture was then placed in a T175 flask and cultured at 37°C with 5% CO2 for 5 days. Finally, the fused hybridoma cell clones were cultured in medium containing 1×HT.

[0049] Step 4: Screening for positive clones

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

[0051] Indirect ELISA: The ELISA plate was coated with ADAM17-FC antigen, and a plate coated with control FC protein was also prepared for detection. The plate was incubated overnight at 4°C, blocked with irrelevant protein (BSA) at 37°C for 2 hours, and then the supernatant of hybridoma cell culture obtained from the above experiments was added. The plate was incubated at 37°C for 1 hour, washed 5-6 times with 0.05% Tween 20-PBS, and then incubated with secondary antibody (e.g., HRP-GAM) at 37°C for another 2 hours. After washing, the plate was developed with the substrate. Once the colorimetric level reached a certain point, stop solution (3M H2SO4) was added to terminate the reaction. The OD492 was read on a microplate reader. Results are as follows: Figure 4As shown, there are a total of 6 96-well hybridoma plates. The supernatant of each hybridoma plate was tested to see if it binds to the ADAM17-FC protein and to see if it binds to the FC control protein. Clones that bind to the ADAM17-FC protein but not to the FC control protein were selected and marked as positive wells for subsequent flow cytometry detection.

[0052] Flow cytometry: ADAM17-positive tumor cell lines were selected as experimental cells. The supernatant of ELISA-positive hybridoma samples was subjected to flow cytometry staining to confirm the cell positivity rate of different hybridoma clones. 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 for subsequent hybridoma sequencing based on the positive rate results.

[0053] Step 5: Antibody variable region sequencing

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

[0055] Example 3: Antibody Efficacy Verification Experiment

[0056] I. Antibody Affinity Detection

[0057] The binding kinetics of three clones (1G9, 2F12, and 6G5) to ADAM17 were determined using Octet-Red biolayer interferometry (BLI). First, EDC and S-NHS were mixed in a 1:1 ratio and used to activate the AR2G biosensor. Then, the recombinant ADAM17-Fc protein was diluted to 30 μg / mL with sodium acetate buffer (pH 5.0), and the antigen was loaded onto the sensor until saturation, followed by blocking with 1M ethanolamine (pH 8.5). The antigen-loaded AR2G probe was immersed in the antibody-diluted wells for 300 s. The ADAM17-Fc antigen-antibody complex was then dissociated in PBST (PBS + 0.2% Tween) buffer. Affinity was calculated using Data Analysis software version 10.0 with a 1:1 binding model. The KON (binding rate constant), KOFF (dissociation rate constant), and KD (equilibrium dissociation constant) values ​​of the three antibody clones 1G9, 2F12, and 6G5 were determined by means of the average binding curves with R² values ​​greater than 95% confidence level.

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

[0059] II. Antibody Specificity Detection

[0060] ADAM8, ADAM9, and ADAM10 antigens were diluted and coated in 96-well plates overnight at 4°C. After washing three times, blocking buffer was added and the plates were blocked at room temperature for 1 hour, followed by three more washes. Antibodies were serially diluted to six concentrations starting at 100 nM, and 100 μL / well was added to each well. The plates were washed three times with washing buffer. The enzyme-linked antibody was diluted to the working concentration, mixed well, and 100 μL / well was added to each well. The plates were incubated at room temperature for 1 hour. The substrate ABTS was then added to detect enzyme activity, and the signal was read at 405 nm using a microplate spectrophotometer. GraphPad Prism version 8.0 was used to fit the data to curves. The results are as follows: Figure 8 As shown, only the 2F12 antibody exhibits a weaker binding to ADAM10 at higher concentrations, demonstrating that the 1G9, 2F12, and 6G5 antibodies possess excellent specificity.

[0061] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims of the present invention, will be protected by patent law.

Claims

1. An antibody targeting the ADAM17 protein, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein both the heavy chain variable region and the light chain variable region comprise three complementarity-determining regions (CDR1, CDR2, and CDR3), characterized in that, The antibody is a 6G5 antibody, and the nucleotide sequences of the variable region and complementarity-determining region of the 6G5 antibody are as follows: The nucleotide sequence of VH is shown in SEQ ID NO: 18, the nucleotide sequence of VH CDR1 is shown in SEQ ID NO: 19, the nucleotide sequence of VH CDR2 is shown in SEQ ID NO: 20, and the nucleotide sequence of VH CDR3 is shown in SEQ ID NO:

21. The nucleotide sequence of VL is shown in SEQ ID NO:22, the nucleotide sequence of VL CDR1 is shown in SEQ ID NO:23, the nucleotide sequence of VL CDR2 is CTCACATCC, and the nucleotide sequence of VL CDR3 is shown in SEQ ID NO:

24.

2. The antibody targeting ADAM17 protein according to claim 1, characterized in that, The amino acid sequences of the variable region and complementarity-determining region of the 6G5 antibody are as follows: The amino acid sequence of VH is shown in SEQ ID NO: 39, the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 40, the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 41, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO:

42. The amino acid sequence of VL is shown in SEQ ID NO:43, the amino acid sequence of VL CDR1 is shown in SEQ ID NO:44, the amino acid sequence of VLCDR2 is LTS, and the amino acid sequence of VL CDR3 is shown in SEQ ID NO:

45.

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), multispecific antibody, chimeric antibody, or humanized antibody.

5. The antibody targeting the ADAM17 protein according to claim 1 or 2, characterized in that, The antibody preparation and screening method is as follows: Step 1: Synthesis of ADAM17 antigen gene and construction of expression vector; Step 2, preparation of ADAM17 recombinant protein antigen expression: The expression vector constructed in Step 1 was transfected into cells and then purified to obtain ADAM17-FC recombinant protein; Step 3: Preparation of mouse hybridomas: Mouse myeloma cells SP2 / 0 and spleen lymphocytes were electrofused to obtain hybridoma cells; Step 4: Screening for positive clones: Antigen staining is performed on hybridoma cells to identify positive clones; then, positive clones that bind to ADAM17-FC protein but not to FC control protein are selected by ELISA; finally, positive clones with high cell positivity rates are screened by flow cytometry.

6. The antibody targeting ADAM17 protein according to claim 5, characterized in that, In step one, the expression vector is constructed as follows: the ADAM17 expression protein sequence ADAM17-FC as shown in SEQ ID NO: 3 is designed, the extracellular segment sequence Arg215-Asn671 of the ADAM17 antigen is selected for primer design to amplify the target fragment, the IgGκSP signal peptide sequence is added to the 5' end of the gene, the FC fusion protein tag is added to the 3' end of the gene, and the DNA plasmid expressing human ADAM17 antigen is obtained: pcDNA3.1-ADAM17-FC.

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