Anti-DDR1 antibodies and their applications

Abstract

The present invention discloses an anti-DDR1 antibody and its application, belonging to the field of biomedicine. The technical problem to be solved by the present invention is to provide a new anti-DDR1 antibody, the anti-DDR1 antibody comprises a heavy chain and a light chain; the heavy chain comprises HCDR1, HCDR2, and HCDR3, and the light chain comprises LCDR1, LCDR2, and LCDR3. The antibody provided by the present invention can effectively block the binding of rat tail collagen to DDR1, weakly bind to human DDR2, not bind to mouse DDR1, promote the infiltration of T cells into the tumor, and is used for the treatment of colorectal cancer patients.

Description

Technical Field

[0001] The present invention relates to an anti-DDR1 antibody and an application thereof, and belongs to the technical field of biomedicine. Background Art

[0002] The formation of a tumor growth microenvironment not only depends on some cytokines secreted by the tumor itself, but can also escape immune surveillance and regulation through certain signaling pathways, thereby making the body immune tolerant to tumor cells and leading to rapid tumor progression.

[0003] Immune cells in the tumor microenvironment play an important role in the occurrence and development of tumors, among which the infiltration level of cytotoxic CD8+ T cells in tumors is related to clinical prognosis. In solid tumors, immune exclusion (IE) is characterized by T cells enriched in the tumor stroma and less infiltrated in the tumor epithelium. Existing immunotherapies are less effective for immune-excluded tumors, so regulating the tumor microenvironment and promoting the infiltration of cytotoxic T cells is a potential effective direction for tumor treatment.

[0004] Discoidin domain-containing receptor 1 (DDR1) is a receptor tyrosine kinase with many important physiological functions in cells. Dysregulation of DDR1 has been implicated in a variety of human diseases, including cancer. Studies have reported that DDR1 promotes cancer progression by regulating the interaction between tumor cells and the collagen matrix, and that overexpression of DDR1 promotes tumor growth in vivo. Studies have reported that increased DDR1 expression is associated with tumor progression in multiple cancer types. A negative correlation between DDR1 expression and CD8+ T cells in tumors has been demonstrated, and it has been demonstrated that the interaction of DDR1 with collagen plays an important role in the exclusion of T cells from the tumor microenvironment. DDR1 is activated by binding to fibrillar collagen (types I to III and V) or non-fibrillar collagen (type IV collagen) to its discoidin (DS) domain. Upon collagen binding, DDR1 aggregates on the cell surface and undergoes tyrosine autophosphorylation, triggering a series of downstream signaling pathways.

[0005] Therefore, it is urgent to develop a new drug that can resist DDR1 or bind to DDR1, in order to provide new ideas and methods for the treatment of tumors. Summary of the invention

[0006] In view of the above situation, in order to solve the existing technical problems, the present invention provides an anti-DDR1 antibody and its application, the purpose of which is to block the binding of DDR1 and rat tail collagen by the antibody, so as to provide a new idea and method for the treatment of tumors.

[0007] The first technical solution provided by the present invention is an anti-DDR1 antibody, wherein the anti-DDR1 antibody comprises a heavy chain and a light chain; the heavy chain comprises HCDR1, HCDR2, and HCDR3, and the light chain comprises LCDR1, LCDR2, and LCDR3, and the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are respectively the sequences shown in any one of the groups (a) to (c):

[0008] (a) SEQIDNO.1~SEQIDNO.6;

[0009] (b) SEQIDNO.7~SEQIDNO.12;

[0010] (c) SEQ ID NO.13 ~ SEQ ID NO.18.

[0011] The second technical solution provided by the present invention is a polynucleotide encoding the antibody described in the first technical solution.

[0012] The third technical solution provided by the present invention is an expression vector carrying the polynucleotide described in the second technical solution.

[0013] The fourth technical solution provided by the present invention is a host cell that expresses the antibody described in the first technical solution, or contains the polynucleotide described in the second technical solution, or is transformed with the expression vector described in the third technical solution.

[0014] In certain embodiments, the host cell includes, but is not limited to, bacteria, fungi, mammalian cells, or insect cells.

[0015] In certain embodiments, the fungus comprises yeast or mold, and the bacteria comprises Escherichia coli.

[0016] In certain embodiments, the mammalian cells include but are not limited to 293 cells.

[0017] The fifth technical solution provided by the present invention is a pharmaceutical composition, which contains the antibody described in the first technical solution, or the polynucleotide described in the second technical solution, or the expression vector described in the third technical solution, or the host cell described in the fourth technical solution.

[0018] In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or diluent.

[0019] The sixth technical solution provided by the present invention is the use of the antibody described in the first technical solution, or the polynucleotide described in the second technical solution, or the expression vector described in the third technical solution, or the host cell described in the fourth technical solution, or the pharmaceutical composition described in the fifth technical solution in the preparation of drugs for preventing, alleviating, improving and / or inhibiting tumor diseases.

[0020] In certain embodiments, the neoplastic disease is cancer.

[0021] In some embodiments, the application includes at least one of the following effects:

[0022] (1) Blocking rat tail collagen and DDR1 signaling pathway;

[0023] (2) Promote T cell infiltration into the tumor.

[0024] The technical effects of the present invention are as follows:

[0025] The antibody prepared by the present invention can effectively block the rat tail collagen and DDR1 signaling pathway, weakly bind to human DDR2, not bind to mouse DDR1, promote the infiltration of T cells into the tumor, and is used for the treatment of cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the purification map of 1E1, 10C7 and 15A11 hybridoma antibodies in the present invention.

[0027] FIG. 2 is a purification diagram of the recombinant antibody in the present invention.

[0028] FIG. 3 is a curve showing the binding activity of hybridoma antibodies to human DDR1 antigen determined by ELISA method in the present invention.

[0029] FIG. 4 is a curve showing the binding activity of hybridoma antibodies to human DDR2-his measured by ELISA in the present invention.

[0030] FIG5 is a curve showing the binding activity of hybridoma antibodies to mouse DDR1-his measured by ELISA in the present invention.

[0031] FIG. 6 is a curve showing the competitive activity between hybridoma antibodies and ligands measured by ELISA in the present invention.

[0032] FIG. 7 is a curve showing the binding activity of the recombinant antibody to the antigen detected by the ELISA method of the present invention.

[0033] FIG8 is a curve showing the binding activity of the recombinant antibody to human DDR2-his detected by ELISA in the present invention.

[0034] FIG. 9 is a curve showing the binding activity of the recombinant antibody to mouse DDR1-his detected by ELISA in the present invention.

[0035] FIG. 10 is a diagram showing the competitive activity of recombinant antibodies detected by ELISA in the present invention. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0037] The raw materials used in the examples are:

[0038] 1. BALB / C mice were obtained from Shanghai Xipu-Bikai Experimental Animal Co., Ltd.

[0039] 2. SP2 / 0 cells were obtained from Beijing Zhongyuan Heju Biotechnology Co., Ltd.

[0040] 3. IMDM culture medium: purchased from Shanghai Yuanpei Biotechnology Co., Ltd., L610KJ.

[0041] 4. HAT complete medium: 83% IMDMMedium (purchased from Shanghai Yuanpei Biotechnology Co., Ltd., L450KJ) + 15% FBS + 1% P / S + 1% HAT (purchased from Shanghai Yuanpei Biotechnology Co., Ltd., S912JV).

[0042] 5. Goat anti-mouse IgG antibody was purchased from Zhongshan Jinqiao.

[0043] 6. Expi 293F cells were obtained from Thermo Fisher Scientific China Ltd.

[0044] Example 1 Preparation of anti-DDR1 antibodies

[0045] The following steps are involved:

[0046] Step 1: Preparation of protein antigen. Human DDR1 protein (Uniprot No: Q08345) was used as a template. Different tags (hFc, mFc, his) were fused to the extracellular region of human DDR1 protein according to the reference ([1] Leitinger, Birgit. Molecular Analysis of Collagen Binding by the Human Discoidin Domain Receptors, DDR1 and DDR2. [J]. Journal of Biological Chemistry, 2003.). The proteins were named: DDR1-mFc, DDR1-hFc, DDR1-his. The target protein was expressed by Expi293F transient expression system. After 5-7 days of transfection, the target protein was obtained by affinity purification.

[0047] Step 2, mouse immunization, the purified DDR1-hFc antigen is emulsified with complete Freund's adjuvant, and 6-8 week old BALB / C mice are immunized by subcutaneous injection or intraperitoneal injection, the immunization dose is 50 μg / mouse, and the second immunization is performed two weeks later, the antigen is emulsified with incomplete Freund's adjuvant and injected subcutaneously, the immunization dose is 50 μg / mouse; after two immunizations, tail blood is collected and the serum titer is determined by gradient dilution by ELISA; according to the serum titer results, it is determined whether to boost the immunization, and the mouse with the highest antibody titer is selected for subsequent cell fusion, and B cells that can secrete specific target protein antibodies are produced in the mouse body.

[0048] Step 2 consists of the following two parts:

[0049] Part I: Antigen emulsification: according to the concentration of DDR1-hFc antigen protein, take out 200 μg, dilute it to 500 μl volume with PBS (PBS is the abbreviation of phosphate buffered saline), and draw it into a new 2 mL syringe, marked as the first syringe A; take another new 2 mL syringe, draw 500 μl of incomplete Freund's adjuvant, marked as the second syringe B; the first syringe A and the second syringe B draw the liquid in the needle back into the syringe cavity, and discard the needle; screw the double female Luer tip into the first syringe A, exhaust the air in the first syringe A, the second syringe B and the Luer tip; quickly connect the second syringe B to the Luer tip of the first syringe A and tighten it; use another third syringe to add a drop of incomplete Freund's adjuvant to the bottom of the piston rod of the first syringe A and the second syringe B; push the liquid in the first syringe A and the second syringe B back and forth for 30-60 minutes. After completion, there is no need to disassemble it and place it at 4℃ for use;

[0050] Part II: Subcutaneous injection method: Take out a 1mL syringe, draw the required injection to the corresponding scale and remove the bubbles, cover it with a needle cap and place it on the right side of the operating area; secure the mouse, pick up the prepared syringe with one hand, and let the needle cap fall naturally to the table; pierce the skin at the injection site at an angle of less than 30° with the needle bevel facing upward; once the needle pierces the skin and feels a sense of empty space, immediately adjust the needle angle to about 0° and continue to insert the needle for about 1-2cm; slowly push the injection, and after the push is completed, the syringe rotates 180° so that the bevel of the needle faces downward, and then withdraws from the skin; put the syringe back on the operating table and the mouse back into the breeding ground.

[0051] In step 2, if the serum titer result is ≥243000, booster immunization is performed; if the serum titer result is <243000, booster immunization is not performed. The booster immunization in step 2 is performed by intraperitoneal injection or tail vein injection. The booster immunization still uses the antigen used for conventional immunization in order to increase the serum titer of mice.

[0052] Step 3, feeder cell preparation: prepare complete culture medium as needed; kill healthy BALB / c mice by cervical dislocation and disinfect by immersion in 75% alcohol; place the mice in supine position, cut the mouse skin on the abdomen under a sterile environment to expose the peritoneum; draw about 5 mL of IMDM culture medium with a syringe, pinch the peritoneum with tweezers, pierce the needle horizontally into the pinched peritoneum, and inject the liquid in the syringe; rotate the needle so that the needle bevel faces downward, at this time the needle cannot be pulled out and the peritoneum is slightly lifted, and the back of the mouse abdomen is massaged with fingers to allow the culture medium to fully wash the abdominal cavity; aspirate the fluid in the mouse peritoneal cavity, centrifuge at 1200 rpm for 3 minutes, discard the supernatant, obtain the feeder cells and resuspend them in the above-configured culture medium.

[0053] Step 4, preparation of spleen cells: kill the immune mice by cervical dislocation, collect serum, and obtain fresh spleen cells; disinfect the dead mice by soaking in 75% alcohol, cut the skin and mucosa below the left rib of the mouse under a sterile environment to expose the spleen; gently clamp the tissue around the spleen with tweezers, lift the spleen indirectly, remove the tissues connecting the spleen to the body one by one with scissors, and finally gently clamp the spleen and remove it; disinfect the spleen with alcohol and rinse the spleen with IMDM medium, grind the spleen on the cell filter with a syringe core, and rinse the cells several times with 10~20 mL IMDM medium in the middle; centrifuge at 1200 rpm for 3 minutes, discard the supernatant, resuspend with 20~30 mL IMDM medium, and count the live spleen cells for use. If the spleen cells do not need to be fused immediately, they can be frozen according to the SP2 / 0 cell freezing operation.

[0054] Step 5, cell fusion. Take the immunized mice, kill them and take spleen cells. According to the principle of SP2 / 0 cells: spleen cells = 1:5 (cell number ratio), the immunized mouse cell fusion is performed according to the standard operating procedures for cell fusion. The specific fusion steps are as follows: aspirate an appropriate amount of SP2 / 0 cell suspension and add it to the spleen cell suspension. After slightly mixing, centrifuge at 1200 rpm for 3 minutes, discard the supernatant, and knock to break up the cell precipitate for later use; after breaking up the cell precipitate, place the centrifuge tube in a 37°C water bath, and slowly add 37°C preheated polyethylene glycol PEG to the bottom of the tube (add 1 mL PEG in about 60 seconds). While adding, shake the tube body with a small amplitude to ensure that PEG and cells are fully in contact and the water bath is sufficient; after adding PEG, let the tube stand in a 37°C water bath for about 1 minute, and then add IMDM culture medium along the wall of the tube in the following order: add 1 mL culture medium in the first minute, add 2 mL culture medium in the second minute, add 5 mL culture medium in the third minute, and then add culture medium to 20-30 mL; after standing at 37°C for 5 minutes, centrifuge at 800 rpm for 3 minutes, discard the supernatant for later use; resuspend the fused cells in HAT complete medium containing feeder cells, mix well, add 250 μl / well to a 96-well cell plate, and culture at 37°C.

[0055] Step 6, fusion screening, the fused cells were tested by ELISA, and the wells that had strong binding to human DDR1-his, strong competition with rat tail collagen ligand, and no or weak binding to human DDR2 and rat DDR1 were selected for subsequent subcloning;

[0056] Step seven, subcloning. In order to finally obtain a stable clone, subclone cells were detected, positive cells were cloned, and cells were further purified. The specific steps are as follows: 100 μg DDR1-his was aspirated, diluted into 100 mL CBS, and added to the ELISA plate at 50 μL / well, coated at 37°C for 2 hours, washed and blocked, and dried, 50 μL of cell clone culture supernatant was added to each well, incubated at 37°C for 1 hour, the supernatant was discarded, and the ELISA plate was washed 5 times with phosphate buffer containing 0.05% Tween 20, 50 μL of 1:4000 diluted horseradish peroxidase-labeled goat anti-mouse IgG antibody was added to each well, incubated at 37°C for 30 minutes, the secondary antibody was discarded, and the ELISA plate was washed 3 times with phosphate buffer containing 0.05% Tween 20, 50 μL TMB color development solution (Tiangen) was added to each well for color development for 5 minutes, 50 μL 2MH2SO4 was added to each well to terminate the reaction, and the OD was read with an enzyme reader. 450 value. Mark the positive cell line number. Perform limiting dilution on the cells in the positive wells, measure the ELISA value 7-10 days after each limiting dilution, select the monoclonal wells with higher positive values ​​for limiting dilution until the ELISA result of the whole 96-well plate is positive; select the monoclonal lines with high positive values ​​and name them as cell lines 1E1, 10C7 and 15A11.

[0057] Step 8: Hybridoma antibody expression: The monoclonal cell line was expanded and cultured in a T75 culture flask. When the confluence rate in the flask was greater than 50%, it could be used for antibody expression. The cells in the flask were resuspended by blowing with a pipette. The suspension was counted and 6×10 6 Collect the cell suspension in a 15 mL centrifuge tube.

[0058] The collected cell suspension was centrifuged at 1200 rpm for 5 minutes, the supernatant in the tube was discarded, and 5 mL of expression medium was added to the tube to resuspend. The resuspended cell suspension in each tube was added to a 125 mL cell shake flask, and 25 mL of expression medium was added to the flask; the corresponding cell line name and batch number were marked on the 125 mL cell shake flask, and it was placed in a shaker at 135 rpm and cultured at 37°C; after 4-5 days, the shake flask was removed, and the cell suspension in the shake flask was collected in a 50 mL centrifuge tube, and the cell suspension was transferred to a clean new centrifuge tube after centrifugation at 3000 rpm for 10 min. The corresponding cell line name and batch number were marked on the centrifuge tube, and it was transferred to the downstream for antibody purification.

[0059] Step 9, mouse antibody purification, hybridoma cells are expanded, cell supernatant is collected, and mouse antibody purification is performed according to the protein affinity purification standard operating procedures. As shown in Figure 1, the experimental results show that the purity of the purified mouse antibody is greater than 90%.

[0060] Step 10: Cultivate hybridoma cells to obtain a small amount of purified high-purity RNA, reverse transcribe it into cDNA as a template, and use specific PCR primers to amplify the target fragment in vitro; connect the positive PCR product to the T vector for plasmid recombination and transformation, and select positive clones for culture and sequencing through blue-white spot screening and PCR verification. Analyze the sequencing results and determine the correct sequence to complete the antibody sequencing.

[0061] Step 11: By molecular biological methods, the variable regions of the heavy and light chains of the antibodies obtained by sequencing were cloned into a modified mammalian cell expression vector (PTT5, General Biotechnology (Anhui) Co., Ltd.) for efficient expression to obtain anti-DDR1 recombinant antibodies. The recombinant antibodies were expressed in Expi 293F cells, and the cell supernatant was collected and purified according to the standard protein purification procedures, such as Figure 2 As shown, the purity of the purified humanized antibody is greater than 90%.

[0062] 1E1 Antibody Light Chain:

[0063] DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYGTPWTFGGGTKLEIK (SEQ ID NO. 19).

[0064] 1E1 Antibody Heavy Chain:

[0065] EVKLVESGGGLVQPGGSLKLSCIASGFFTFSSYTMSWVRQTPEKRLEYVAYITNGDGRTYYPDIVKGRFTISRDDNAKNTLYLQMNSLKSDDTAMYYCASPNAMDSWGQGTLVTVSS (SEQ ID NO. 20).

[0066] 10C7 antibody light chain:

[0067] DILMTQSPSSLSASLGDTITITCHASQNINIWLTWYQQKPGNIPKLLIYKASDLHTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFGGGTKLEIK (SEQ ID NO. 21).

[0068] 10C7 antibody heavy chain:

[0069] QVQLKQSGAELVRPGTSVKISCKASGYTFTNSWLGWVKQRPGHGLEWLGDIYPGGGYTNYNEKFKGKATLTADTSSSTAYMQLISLTSEDSAVYFCARSGNYGKAFLAYWGQGTLVTVSA (SEQ ID NO. 22).

[0070] 15A11 Antibody Light Chain:

[0071] DILMTQSPSSLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPLTFGAGTKLELK (SEQ ID NO. 23).

[0072] 15A11 antibody heavy chain:

[0073] QVQLKQSGAELVRPGTSVKISCKASGYTFTNSWLGWVKQRPGHGLEWLGDIYPGGGYTNYNEKFKGKATLTADTSSSTAYMQLISLTSEDSAVYFCARSGNYGKAFLAYWGQGTLVTVSA (SEQ ID NO. 24).

[0074] Wherein, the DDR1 antibody includes the first antibody (1E1) or the second antibody (10C7) or the third antibody (15A11), the first antibody includes three first heavy chain CDR regions and three first light chain CDR regions; the three first heavy chain CDR regions include: HCDR1 is the amino acid sequence shown in SEQIDNO.1, HCDR2 is the amino acid sequence shown in SEQIDNO.2, and HCDR3 is the amino acid sequence shown in SEQIDNO.3; the three first light chain CDR regions include: LCDR1 is the amino acid sequence shown in SEQIDNO.4, LCDR2 is the amino acid sequence shown in SEQID NO.5, and LCDR3 is the amino acid sequence shown in SEQIDNO.6. The specific sequence information is as follows: SEQIDNO.1: SYTMS. SEQIDNO.2: YITNGDGRTYYPDIVKG. SEQIDNO.3: PNAMDS. SEQIDNO.4: KASQDVSTAVA. SEQIDNO.5: WASTRHT. SEQIDNO.6: QQHYGTPWT.

[0075] The second antibody includes three second heavy chain CDR regions and three second light chain CDR regions; the three second heavy chain CDR regions include: HCDR4 is the amino acid sequence shown in SEQ ID NO.7, HCDR5 is the amino acid sequence shown in SEQ ID NO.8, and HCDR6 is the amino acid sequence shown in SEQ ID NO.9; the three second light chain CDR regions include: LCDR4 is the amino acid sequence shown in SEQ ID NO.10, LCDR5 is the amino acid sequence shown in SEQ ID NO.11, and LCDR6 is the amino acid sequence shown in SEQ ID NO.12. The specific sequence information is as follows: SEQ ID NO.7: NSWLG. SEQ ID NO.8: DIYPGGGYTNYNEKFKG. SEQ ID NO.9: SGNYGKAFLAY. SEQ ID NO.10: HASQNINIWLT. SEQ ID NO.11: KASDLHT. SEQ ID NO.12: QQGQSYPLT.

[0076] The third antibody includes three third heavy chain CDR regions and three third light chain CDR regions; the three third heavy chain CDR regions include: HCDR7 is the amino acid sequence shown in SEQ ID NO.13, HCDR8 is the amino acid sequence shown in SEQ ID NO.14, and HCDR9 is the amino acid sequence shown in SEQ ID NO.15; the three third light chain CDR regions include: LCDR7 is the amino acid sequence shown in SEQ ID NO.16, LCDR8 is the amino acid sequence shown in SEQ ID NO.17, and LCDR9 is the amino acid sequence shown in SEQ ID NO.18. The specific sequence information is as follows: SEQ ID NO.13: TYYLH. SEQ ID NO.14: WIFPGSDKTNYNENFKG. SEQ ID NO.15: NYGYLYYSLDY. SEQ ID NO.16: RASQEISGYLS. SEQ ID NO.17: AASTLDS. SEQ ID NO.18: LQYASYPLT.

[0077] Example 2 Antibody Performance Test

[0078] 1. ELISA method to detect the binding activity of mouse antibodies to human DDR1-hFc

[0079] The test antigen human DDR1-hFc was coated on an ELISA plate (0.5 µg / mL, 50 µL / well), and the mouse antibody prepared in Example 1 was added, and the OD value was measured to detect the binding activity of the mouse antibody to the target antigen. As shown in Figure 3, the three hybridoma antibodies 1E1, 10C7 and 15A11 bound strongly to the antigen, with EC50 of 0.3868, 0.7703 and 0.3696 nM, respectively.

[0080] 2. ELISA method to detect the binding activity of mouse antibodies to human DDR2-hFc

[0081] The antigen human DDR2-hFc to be tested (sequence from Uniprot, NO. Q16832, protein made by Meisai Bio) was coated on an ELISA plate (0.5 µg / mL, 50 µL / well), and the antibody prepared in Example 1 was added, and the OD value was measured to detect the binding activity of the mouse antibody to the target antigen. As shown in Figure 4, the three hybridoma antibodies 1E1 and 15A11 did not bind to the antigen human DDR2--hFc, and 10C7 had a weak binding trend at high concentrations.

[0082] 3. ELISA to detect the binding activity of mouse antibodies to mouse DDR1-hFc

[0083] The test antigen mouse DDR1-hFc (sequence from Uniprot, NO.Q03146, protein made by Meisai Biotechnology) was coated on the ELISA plate (0.5 μg / mL, 50 μL / well), and the mouse antibody prepared in Example 1 was added to measure the OD value to detect the binding activity of the mouse antibody to the target antigen. Figure 5 As shown, the three hybridoma antibodies 1E1, 10C7 and 15A11 have a tendency to bind to the mouse DDR1-hFc antigen at high concentrations, among which 1E1 has the strongest binding.

[0084] IV. ELISA method to detect the competitive activity of mouse antibodies and rat tail collagen

[0085] The antigen to be tested, rat tail collagen (Sigma, C7661), was coated on an ELISA plate (20 µg / mL, 50 μL / well), and the antibody prepared in Example 1 and the antigen human DDR1-hFc were added. The OD value was measured to detect the competitive activity of the mouse antibody and the antigen human DDR1-hFc (2.0 µg / mL). Figure 6 As shown, the OD values ​​of the three antibodies have a significant difference trend, proving that the antibodies have strong competitive activity.

[0086] 5. ELISA to detect the binding activity of recombinant antibodies to human DDR1-mFc

[0087] The test antigen human DDR1-mFc was coated on an ELISA plate (0.5 µg / mL, 50 µL / well), and the antibody prepared in Example 1 was added, and the OD value was measured to detect the binding activity of the mouse antibody to the target antigen. As shown in Figure 7, the three chimeric antibodies 1E1-H / L, 10C7-H / L and 15A11-H / L bound strongly to the antigen, with EC50 of 0.02391, 0.035483 and 0.05982 nM, respectively.

[0088] VI. ELISA to detect the binding activity of recombinant antibodies to human DDR2-mFc

[0089] The antigen human DDR2-mFc to be tested was coated on an ELISA plate (0.5 µg / mL, 50 µL / well), and the antibody prepared in Example 1 was added, and the OD value was measured to detect the binding activity of the mouse antibody to the target antigen. As shown in Figure 8, 1E1-H / L and 10C7-H / L had weak binding to the antigen at high concentrations, and 15A11-H / L had the strongest binding activity to human DDR2-mFc.

[0090] VII. ELISA to detect the binding activity of recombinant antibodies to mouse DDR1-mFc

[0091] The test antigen mouse DDR2-mFc was coated on an ELISA plate (0.5 µg / mL, 50 µL / well), and the antibody prepared in Example 1 was added, and the OD value was measured to detect the binding activity of the mouse antibody to the target antigen. As shown in Figure 9, the three recombinant antibodies 1E1-H / L, 10C7-H / L and 15A11-H / L did not bind to the antigen.

[0092] 8. ELISA method to detect the competitive activity between recombinant antibodies and rat tail collagen-mFc

[0093] The test plate was coated with 2.0 µg / mL human DDR1-his, and a concentration gradient was set. Isotype-HYHEL (antiHELantibody, produced by CHO-K1) was used as an isotype control (published in Li Y, Li H, Smith-Gill SJ, et al. Three-Dimensional Structures of the Free and Antigen-BoundFab from Monoclonal Antilysozyme Antibody HyHEL-63 [J]. Biochemistry, 2000, 39(21):6296-6309. DOI:10.1021 / bi000054l.). The OD value was measured to detect the competitive activity of 1E1-H / L, 10C7-H / L and 15A11-H / L recombinant antibodies with 3.0 µg / mL rat tail collagen-mFc ligand. As shown in Figure 10, the OD values ​​of the three antibodies 1E1-H / L, 10C7-H / L and 15A11-H / L have obvious trends, and the upper and lower platforms of the curves are clear, indicating strong competitive activity.

[0094] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An anti-DDR1 antibody, characterized in that The anti-DDR1 antibody comprises a heavy chain and a light chain; the heavy chain comprises HCDR1, HCDR2, and HCDR3, and the light chain comprises LCDR1, LCDR2, and LCDR3, and the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as shown in any one of the groups (a) to (b): (a) SEQIDNO.1~SEQIDNO.6; (b) SEQ ID NO.7 ~ SEQ ID NO.

12.

2. A polynucleotide encoding the antibody according to claim 1.

3. An expression vector carrying the polynucleotide of claim 2.

4. A host cell expressing the antibody of claim 1, or containing the polynucleotide of claim 2, or transformed with the expression vector of claim 3.

5. The host cell according to claim 4, characterized in that The host cell is a bacterium, a fungus, a mammalian cell or an insect cell; the mammalian cell is a 293 cell.

6. The host cell according to claim 5, characterized in that The fungi include yeast or mold, and the bacteria include Escherichia coli.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the antibody according to claim 1, or the polynucleotide according to claim 2, or the expression vector according to claim 3, or the host cell according to any one of claims 4 to 6.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or diluent.

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