Antibodies directed against calcitonin gene related peptides

Abstract

The invention discloses an antibody aiming at a calcitonin gene related peptide, belongs to the field of biological medicines, and provides an antibody or an antigen binding fragment thereof, a nucleic acid molecule containing the antibody, a carrier containing the nucleic acid molecule, and a cell containing the nucleic acid molecule or the carrier. The invention also provides products and pharmaceutical compositions related to the antibodies or the antigen binding fragments thereof, and related applications and methods.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to antibodies against calcitonin gene-related peptide. Background Art

[0002] Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide widely expressed in the central and peripheral nervous systems. It belongs to the calcitonin peptide family and plays an important role in regulating metabolism, inflammatory response, vascular tension and pain in sensory nerve transmission. The main advantages of CGRP receptor antagonists are obvious efficacy, few adverse reactions and no addiction, so they do not have the contraindications of most analgesics.

[0003] Compared with chemical small molecules, antibodies that antagonize CGRP and its receptors have higher specificity, affinity, longer half-life, and less hepatotoxicity, which can extend the medication interval, improve patient compliance, and enhance drug efficacy. Therefore, the development of antibodies against CGRP has good application prospects. Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0005] The present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises: Heavy chain variable region complementarity determining region: VH CDR1, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO: 1, VH CDR2, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:2, VH CDR3, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:3, Light chain variable region complementarity determining region: VL CDR1, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence set forth in SEQ ID NO:9, VL CDR2, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 10, VL CDR3, which comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 11; The antibody or antigen-binding fragment thereof specifically binds to the CGRP and / or neutralizes the CGRP.

[0006] Furthermore, the amino acid sequence of VH CDR1 is shown in SEQ ID NO: 1, The amino acid sequence of VH CDR2 is shown in SEQ ID NO: 2, The amino acid sequence of VH CDR3 is shown in SEQ ID NO: 3, The amino acid sequence of VL CDR1 is shown in SEQ ID NO:9, The amino acid sequence of VL CDR2 is shown in SEQ ID NO: 10, The amino acid sequence of VL CDR3 is shown in SEQ ID NO:11.

[0007] Furthermore, the antibody or antigen-binding fragment thereof further comprises: Heavy chain variable region framework region: VH FR1, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:4, VH FR2, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:5, VH FR3, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:6, VH FR4, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:7, Light chain variable region framework region: VL FR1, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 12, VL FR2, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 13, VL FR3, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 14, VL FR4, which comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence shown in SEQ ID NO:15.

[0008] Furthermore, the amino acid sequence of VH FR1 is shown in SEQ ID NO: 4, The amino acid sequence of VH FR2 is shown in SEQ ID NO:5, The amino acid sequence of VH FR3 is shown in SEQ ID NO:6, The amino acid sequence of VH FR4 is shown in SEQ ID NO:7, The amino acid sequence of VL FR1 is shown in SEQ ID NO: 12, The amino acid sequence of VL FR2 is shown in SEQ ID NO: 13, The amino acid sequence of VL FR3 is shown in SEQ ID NO: 14, The amino acid sequence of VL FR4 is shown in SEQ ID NO:15.

[0009] Furthermore, the heavy chain variable region VH of the antibody or antigen-binding fragment thereof has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 8, The light chain variable region VL has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO:16.

[0010] Furthermore, the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO: 8, The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO:16.

[0011] Furthermore, the antibody or antigen-binding fragment thereof also includes a heavy chain variable region signal peptide, the amino acid sequence of the heavy chain variable region signal peptide having an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO:17.

[0012] Furthermore, the antibody or antigen-binding fragment thereof also includes a light chain variable region signal peptide, the amino acid sequence of the light chain variable region signal peptide having an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO:18.

[0013] Furthermore, the amino acid sequence of the heavy chain variable region signal peptide in the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 17.

[0014] Furthermore, the amino acid sequence of the light chain variable region signal peptide in the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:18.

[0015] Furthermore, the antibody or antigen-binding fragment thereof further includes a heavy chain constant region, and the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:19.

[0016] Furthermore, the antibody or antigen-binding fragment thereof further includes a light chain constant region, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:20.

[0017] Furthermore, the antibody or antigen-binding fragment thereof also includes a defucosylated antibody or antigen-binding fragment thereof.

[0018] In the present invention, the "identity" is synonymous with "sequence identity" and refers to the degree of identity between two sequences. The specific calculation rules are as follows: compare the two best aligned sequences in the window, determine the number of positions where the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) appear in the two sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiply the result by 100 to obtain the sequence identity percentage.

[0019] Further, the antibody includes a diabody, Fab, Fab', F(ab')2, Fd, an Fv fragment, a disulfide-stabilized Fv fragment, (dsFv)2, a dual-specific dsFv, a disulfide-stabilized diabody, a single-chain antibody molecule, a scFv dimer, a multi-specific antibody, a camelized single-domain antibody, a nanobody, a domain antibody or a bivalent domain antibody.

[0020] The present invention provides a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0021] Further, the nucleic acid molecule encodes the nucleotide sequences of VH CDR1, VH CDR2, and VHCDR3 in the antibody or antigen-binding fragment thereof, which have nucleotide sequences that are at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the nucleotide sequences shown in SEQ ID NOs: 21, 22, and 23, respectively. The nucleotide sequences encoding VL CDR1, VL CDR2, and VL CDR3 in the antibody or antigen-binding fragment thereof have nucleotide sequences that are at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the nucleotide sequences shown in SEQ ID NOs: 29, 30, and 31, respectively.

[0022] Furthermore, the nucleotide sequences of VH CDR1, VH CDR2, and VHCDR3 in the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 21, 22, and 23, respectively. The nucleotide sequences encoding VL CDR1, VL CDR2, and VL CDR3 in the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 29, 30, and 31, respectively.

[0023] Further, the nucleic acid molecule encodes the nucleotide sequences of VH FR1, VH FR2, VH FR3, and VH FR4 in the antibody or antigen-binding fragment thereof, which have nucleotide sequences that are at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the nucleotide sequences shown in SEQ ID NOs: 24, 25, 26, and 27, respectively. The nucleotide sequences encoding VL FR1, VL FR2, VL FR3, and VL FR4 in the antibody or its antigen-binding fragment have nucleotide sequences that are at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the amino acid sequences shown in SEQ ID NOs: 32, 33, 34, and 35, respectively.

[0024] Furthermore, the nucleotide sequences of VH FR1, VH FR2, VH FR3, and VH FR4 in the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 24, 25, 26, and 27, respectively. The nucleotide sequences encoding VL FR1, VL FR2, VL FR3, and VL FR4 in the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 32, 33, 34, and 35, respectively.

[0025] Further, the nucleic acid molecule encodes a heavy chain variable region of an antibody or antigen-binding fragment thereof, which has a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence shown in SEQ ID NO: 28, A light chain variable region encoding an antibody or antigen-binding fragment thereof having a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence shown in SEQ ID NO:36.

[0026] Furthermore, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 28, The nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:36.

[0027] Further, the nucleic acid molecule encodes a heavy chain variable region signal peptide in an antibody or antigen-binding fragment thereof, which has a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence shown in SEQ ID NO: 37, A nucleotide sequence encoding a light chain variable region signal peptide in an antibody or an antigen-binding fragment thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence shown in SEQ ID NO:38.

[0028] Furthermore, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region signal peptide in the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 37, The nucleotide sequence encoding the light chain variable region signal peptide in the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:38.

[0029] Furthermore, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain constant region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 39; The nucleotide sequence encoding the light chain constant region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:40.

[0030] Furthermore, the nucleic acid molecule is codon optimized.

[0031] In some embodiments, the nucleotide sequence in the nucleic acid molecule is replaced by a conservative nucleic acid. Conservative nucleic acids refer to those nucleic acids that encode the same amino acid sequence, or in the case where the nucleic acid molecule does not encode the amino acid sequence, the basic structure and sequence are the same or related sequences. In the replacement of conservative nucleic acids, the degeneracy of the genetic code is applied.

[0032] The present invention provides a vector, which comprises the nucleic acid molecule described above.

[0033] Furthermore, the vector also includes elements for controlling expression.

[0034] Furthermore, the vector also includes materials that facilitate the vector to enter cells.

[0035] In some embodiments, the most commonly used type of vector is a plasmid. In other embodiments, the vector is a viral vector, specifically including common retroviruses (such as lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses, poxviruses, baculoviruses, papillomaviruses, etc.

[0036] The present invention provides a cell, which comprises the aforementioned nucleic acid molecule or the aforementioned vector.

[0037] Furthermore, the cells include prokaryotic cells and eukaryotic cells.

[0038] Furthermore, the prokaryotic cells include Gram-negative or Gram-positive cells.

[0039] Furthermore, the eukaryotic cells include protist cells, animal cells, and fungal cells.

[0040] Furthermore, the animal cells include mammalian cells, bird cells, and insect cells.

[0041] Furthermore, the mammalian cells include 293F cells, 293T cells, CHO cells, AT1080 cells, and A549 cells.

[0042] In some embodiments, the mammalian cells include human and non-human mammalian cells, and the non-human mammalian cells refer to cells of fetal animals other than humans, including mouse cells, sheep cells, rabbit cells, horse cells, cow cells, pig cells, dog cells, monkey cells, etc.

[0043] The present invention provides a product for detecting calcitonin gene-related peptide, which comprises the above-mentioned antibody or antigen-binding fragment thereof.

[0044] Further, the product also includes reagents for performing an antigen-antibody reaction or reagents for detecting a reaction.

[0045] Furthermore, the reagents used to perform the antigen-antibody reaction include a buffer and a salt.

[0046] Further, the product includes a conjugate of an antibody linked to a detectable label.

[0047] Furthermore, the product includes a test kit and a test strip.

[0048] The present invention provides a pharmaceutical composition for preventing or treating inflammatory pain, wherein the pharmaceutical composition comprises the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned nucleic acid molecule, or the above-mentioned vector, or the above-mentioned cell.

[0049] Furthermore, the inflammatory pain includes migraine and peritoneal visceral inflammatory pain.

[0050] Furthermore, the migraine includes chronic migraine, migraine without aura, tension headache, and cluster pain.

[0051] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable adjuvant.

[0052] Furthermore, the pharmaceutical composition also includes other antibodies.

[0053] The present invention provides any of the following applications.

[0054] The present invention provides the use of the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned nucleic acid molecule, the above-mentioned vector, or the above-mentioned cell in the preparation of a product for detecting calcitonin gene-related peptide.

[0055] The present invention provides use of the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned nucleic acid molecule, the above-mentioned vector, or the above-mentioned cell in the preparation of a pharmaceutical composition for preventing and / or treating inflammatory pain.

[0056] Furthermore, the inflammatory pain includes migraine and peritoneal visceral inflammatory pain.

[0057] Furthermore, the migraine includes chronic migraine, migraine without aura, tension headache, and cluster pain.

[0058] Furthermore, the pharmaceutical composition is used for inflammatory pain in Balb / c mice.

[0059] The present invention provides any of the following methods.

[0060] The present invention provides a method for preparing the above-mentioned antibody or antigen-binding fragment thereof, the method comprising: culturing the above-mentioned cell and recovering the antibody or antigen-binding fragment thereof.

[0061] The present invention provides a method for detecting CGRP in a sample for non-diagnostic purposes, the method comprising: contacting the sample to be tested with the above-mentioned antibody or antigen-binding fragment thereof, thereby detecting the level of CGRP in the sample to be tested. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is the result of antibody SDS-PAGE detection.

[0063] Figure 2 This is a graph showing the antibody SEC-HPLC test results.

[0064] Figure 3 These are the results of the antibody binding experiment, where A is the ELISA test with a 5-fold dilution starting from 4ug / ml, and B is the increase in the initial antibody concentration, with a 2-fold dilution starting from 100ug / ml.

[0065] Figure 4 This is a diagram showing the results of a writhing experiment in mice with peritoneal visceral inflammatory pain treated with antibodies. DETAILED DESCRIPTION

[0066] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0067] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0068] Example 1 ZRQ1Ab antibody preparation and purification method 1. Experimental methods Expression method: CHO-S cell expression.

[0069] The heavy chain and light chain genes of the antibody were synthesized, and the specific sequences were shown in Table 1. They were optimized to adapt to the expression of CHO-S cells, transfected into CHO-S cells, cultured for 8-10 days, and the expression level of the antibody was detected by ELISA. The antibody was harvested at an appropriate time. Then, it was purified and concentrated by a protein purifier. Finally, the purity of the antibody was verified by SDS and HPLC.

[0070] Among them, transfection: Wait until CHO-S cells are cultured to a suitable confluence and the viability is above 95% for transfection. High-quality plasmid DNA, OD260 / OD280 ratio between 1.8-2.0, concentrations ranging from a few hundred nanograms / microliters to a few micrograms / microliters. Under sterile conditions, dilute an appropriate amount of plasmid DNA into serum-free culture medium and mix gently. Also dilute an appropriate amount of transfection reagent into serum-free culture medium and mix gently. Slowly mix the diluted plasmid DNA solution and transfection reagent solution, gently invert to mix, and let stand at room temperature for a certain time (usually 1-5 minutes) to form a plasmid-transfection reagent complex. Add to the CHO-S cell shake flask and add slowly while shaking. Put the cell shake flask back on the cell shaker and set it to 37°C, 125r, 8% CO2, and continue to culture.

[0071] ELISA detection of antibody expression level: Select an ELISA plate with good adsorption performance, low blank value, and high transparency of the bottom of the well. Use coating buffer (such as pH7.2-7.4 phosphate buffer) to dilute the CGRP protein to an appropriate concentration (such as 2μg / ml), add a certain volume (such as 0.1ml) to each well, and coat at 4℃ overnight or 37℃ for 2 hours. After coating, block nonspecific binding sites with blocking solution (such as 5% BSA) to reduce background noise, add a certain volume (such as 0.20ml) to each well, and block at 37℃ for 1-2 hours. Add the diluted antibody to be tested, a certain volume (such as 0.1ml) to each well, and incubate at 37℃ for 1 hour. Wash 3 times with washing buffer (such as PBST) for 1 minute each time to remove unbound samples. Add enzyme-labeled secondary antibody, a certain volume (such as 0.1ml) to each well, and incubate at 37℃ for 30-60 minutes. Wash again with washing buffer 3 times for 1 minute each time to remove unbound enzyme-labeled antibodies. Add enzyme substrate (such as TMB) to each well at a certain volume (such as 0.1 ml), incubate at 37°C in the dark for 1-15 minutes until a clear color gradient change appears. Add stop solution (such as 2M sulfuric acid) to each well at a certain volume (such as 0.1 ml) to terminate the enzyme reaction. Use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm, and calculate the concentration of the antibody in the sample based on the standard curve.

[0072] SDS-PAGE: Mix the antibody sample with a loading buffer containing SDS (sodium dodecyl sulfate) and a reducing agent (such as β-mercaptoethanol) to destroy the spatial structure and disulfide bonds of the protein and depolymerize the protein into polypeptide chains. The mixing ratio of sample and loading buffer is usually 4:1 or 5:1. The mixed sample is heated in boiling water for 5-10 minutes to completely denature the protein. Prepare polyacrylamide gel, which usually includes two layers: stacking gel (5% concentration) and separation gel (concentration selected according to the molecular weight of the protein, such as 10%, 12%, etc.). After the gel is prepared, put it in the electrophoresis tank and add electrophoresis buffer. Add the treated sample and protein molecular weight standard (Marker) to the sample wells of the gel respectively. Turn on the power and start electrophoresis. During the electrophoresis process, protein molecules are separated in gels of different concentrations according to their molecular weight and charge. The electrophoresis conditions are usually: the voltage of the stacking gel stage is 80-100V, the voltage of the separation gel stage is 120-150V, and the electrophoresis time is determined according to the migration of the bromophenol blue indicator. After electrophoresis, remove the gel and stain it with Coomassie Brilliant Blue staining solution, usually for 1-2 hours. After staining, destain with destaining solution until the background is clear and the protein bands are obvious. Observe the protein bands on the gel. If the antibody sample has only one clear band and the molecular weight is consistent with expectations, it means that the antibody purity is high.

[0073] HPLC: Prepare the antibody sample to be tested and ensure that it is dissolved in the appropriate solvent and reaches the appropriate concentration. Choose the appropriate chromatographic column and mobile phase according to the properties of the antibody. Commonly used chromatographic columns include reversed phase columns, ion exchange columns, etc. Use the same buffer as the sample dilution to balance the HPLC system until the baseline is stable. Load the prepared sample into the chromatographic system by an automatic sampler or manually. Passing through the chromatographic column at a constant flow rate, the antibodies will be eluted in sequence according to their size and properties. Record the elution curve by a detector (such as a UV detector). Analyze the elution profile to identify protein peaks and possible impurity peaks. Perform quantitative analysis by peak area integration to calculate the purity of the antibody.

[0074] Table 1

[0075] 2. Experimental results After culturing the antibody in CHO-S cells (using Human IgG1-Kappa tag), its expression level was 283.60 mg / L. Molecular analysis was then used to obtain the predicted MW of the antibody under reducing conditions of 49 / 23 kDa and the predicted MW under non-reducing conditions of 144 kDa.

[0076] Then SDS-PAGE was used to detect the purity of the antibody under reducing conditions, which was greater than 95%. Figure 1 The antibody purity was tested by SEC-HPLC, and the result was 98.463%. Figure 2 The endotoxin level detected was 0-1EU / mg.

[0077] The binding results of antibody ZRQ1Ab are as follows Figure 3 As shown, its IC50 value is 15.45 μg / ml. ZR8Ab is Galcanezuma, an antibody marketed by Eli Lilly and Company, used as a positive control.

[0078] Example 2 Animal writhing experiment to determine the therapeutic function of ZRQ1Ab antibody 1. Experimental methods The acetic acid-induced abdominal contraction (writhing) test is a model of peritoneal visceral inflammatory pain. Acetic acid activates visceral and somatic nociceptors in the peritoneum, leading to inflammation and tissue damage in the abdominal wall muscles and visceral organs, causing central and peripheral sensitization and hyperalgesia. In mice, the nociceptive cascade after acetic acid administration results in repetitive abdominal contractions or writhing.

[0079] Balb / c mice (20±2g, male and female) and ICR mice (40g±2g, female and male) were used for injection of acetic acid. The day before the experiment, the antibody (5mg / Kg) was injected subcutaneously according to the method of marketed drugs. The control group was injected with saline. The next day, the mice were acclimated in plexiglass cages for 15 minutes and intraperitoneally injected with 100μl / 10g of 0.6% acetic acid solution (v / v).

[0080] Typical writhing reactions were recorded within 30 minutes after the injection of acetic acid, characterized by longitudinal extension of the trunk, concave extension of the back and extension of the hind limbs, and one writhing reaction was completed when the normal posture was restored.

[0081] 2. Experimental results The results of the twisting test are as follows Figure 4 As shown, the results show that compared with the control group, the use of ZRQ1Ab antibody in the present invention can significantly reduce the writhing strength of Balb / c mice. The specific results are shown in Table 2.

[0082] Table 2

[0083] Table 3 shows the effects of different antibodies on the torsion strength in ICR mice. The ZRQ1Ab antibody used in the present invention did not play a role in reducing the torsion strength in ICR mice. The specific results are as follows Figure 4 shown.

[0084] Table 3

[0085] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. An antibody or an antigen-binding fragment thereof, comprising: Heavy chain variable region complementarity determining region: VH CDR1, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO: 1, VH CDR2, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:2, VH CDR3, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:3, Light chain variable region complementarity determining region: VL CDR1, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence set forth in SEQ ID NO:9, VL CDR2, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 10, VL CDR3, which comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 11; The antibody or antigen-binding fragment thereof specifically binds to the CGRP and / or neutralizes the CGRP.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of VH CDR1 is as shown in SEQ ID NO: 1, The amino acid sequence of VH CDR2 is shown in SEQ ID NO: 2, The amino acid sequence of VH CDR3 is shown in SEQ ID NO: 3, The amino acid sequence of VL CDR1 is shown in SEQ ID NO:9, The amino acid sequence of VL CDR2 is shown in SEQ ID NO: 10, The amino acid sequence of VL CDR3 is shown in SEQ ID NO: 11; Preferably, the antibody or antigen-binding fragment thereof further comprises, Heavy chain variable region framework region: VH FR1, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:4, VH FR2, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:5, VH FR3, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:6, VH FR4, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:7, Light chain variable region framework region: VL FR1, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 12, VL FR2, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 13, VL FR3, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 14, VL FR4 comprising an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 15; Preferably, the amino acid sequence of VH FR1 is as shown in SEQ ID NO: 4, The amino acid sequence of VH FR2 is shown in SEQ ID NO:5, The amino acid sequence of VH FR3 is shown in SEQ ID NO:6, The amino acid sequence of VH FR4 is shown in SEQ ID NO:7, The amino acid sequence of VL FR1 is shown in SEQ ID NO: 12, The amino acid sequence of VL FR2 is shown in SEQ ID NO: 13, The amino acid sequence of VL FR3 is shown in SEQ ID NO: 14, The amino acid sequence of VL FR4 is shown in SEQ ID NO:

15.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region VH of the antibody or antigen-binding fragment thereof has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 8, The light chain variable region VL has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence shown in SEQ ID NO: 16; Preferably, the amino acid sequence of the heavy chain variable region VH is as shown in SEQ ID NO: 8, The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO: 16; Preferably, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region signal peptide, the amino acid sequence of the heavy chain variable region signal peptide having an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence shown in SEQ ID NO: 17; Preferably, the antibody or antigen-binding fragment thereof further comprises a light chain variable region signal peptide, the amino acid sequence of the light chain variable region signal peptide having an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence shown in SEQ ID NO: 18; Preferably, the amino acid sequence of the heavy chain variable region signal peptide in the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 17; Preferably, the amino acid sequence of the light chain variable region signal peptide in the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 18; Preferably, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region, and the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 19; Preferably, the antibody or antigen-binding fragment thereof further comprises a light chain constant region, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 20; Preferably, the antibody or antigen-binding fragment thereof further comprises a defucosylated antibody or antigen-binding fragment thereof.

4. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; Preferably, the nucleic acid molecule encodes the nucleotide sequences of VH CDR1, VH CDR2, and VH CDR3 in the antibody or antigen-binding fragment thereof, which have nucleotide sequences that are at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the nucleotide sequences shown in SEQ ID NOs: 21, 22, and 23, respectively. Nucleotide sequences encoding VL CDR1, VL CDR2, and VL CDR3 in an antibody or antigen-binding fragment thereof, which have nucleotide sequences that are at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the nucleotide sequences shown in SEQ ID NOs: 29, 30, and 31, respectively; Preferably, the nucleotide sequences of VH CDR1, VH CDR2 and VH CDR3 in the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 21, 22 and 23, respectively. The nucleotide sequences encoding VL CDR1, VL CDR2, and VL CDR3 in the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 29, 30, and 31, respectively; Preferably, the nucleic acid molecule encodes the nucleotide sequences of VH FR1, VH FR2, VH FR3, and VHFR4 in the antibody or antigen-binding fragment thereof, which have nucleotide sequences that are at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the nucleotide sequences shown in SEQ ID NOs: 24, 25, 26, and 27, respectively. A nucleotide sequence encoding VL FR1, VL FR2, VL FR3, and VL FR4 in an antibody or antigen-binding fragment thereof, which has a nucleotide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the amino acid sequence shown in SEQ ID NOs: 32, 33, 34, and 35, respectively; Preferably, the nucleotide sequences of VH FR1, VH FR2, VH FR3, and VHFR4 in the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 24, 25, 26, and 27, respectively. The nucleotide sequences encoding VL FR1, VL FR2, VL FR3, and VL FR4 in the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 32, 33, 34, and 35, respectively; Preferably, the nucleic acid molecule encodes a heavy chain variable region of an antibody or antigen-binding fragment thereof, which has a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleotide sequence shown in SEQ ID NO: 28, a nucleotide sequence encoding a light chain variable region of an antibody or antigen-binding fragment thereof that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleotide sequence set forth in SEQ ID NO:36; Preferably, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 28, The nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:36; Preferably, the nucleic acid molecule encodes a heavy chain variable region signal peptide in an antibody or antigen-binding fragment thereof, which has a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleotide sequence shown in SEQ ID NO: 37, A nucleotide sequence encoding a light chain variable region signal peptide in an antibody or antigen-binding fragment thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 38; Preferably, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region signal peptide in the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 37, The nucleotide sequence encoding the light chain variable region signal peptide in the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 38; Preferably, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain constant region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 39; The nucleotide sequence encoding the light chain constant region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:

40.

5. A vector comprising the nucleic acid molecule of claim 4; Preferably, the vector further comprises an element for controlling expression; Preferably, the vector further comprises a material that facilitates the vector to enter cells.

6. A cell comprising the nucleic acid molecule of claim 4 or the vector of claim 5; Preferably, the cells include prokaryotic cells and eukaryotic cells; Preferably, the prokaryotic cells include Gram-negative or Gram-positive cells; Preferably, the eukaryotic cells include protist cells, animal cells, and fungal cells; Preferably, the animal cells include mammalian cells, avian cells, and insect cells; Preferably, the mammalian cells include 293F cells, 293T cells, CHO cells, AT1080 cells, and A549 cells.

7. A product for detecting calcitonin gene-related peptide, the product comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; Preferably, the product further comprises reagents for performing an antigen-antibody reaction or reagents for detecting a reaction; Preferably, the reagents for performing the antigen-antibody reaction include a buffer, a salt; Preferably, the product comprises a conjugate of an antibody linked to a detectable label.

8. A pharmaceutical composition for preventing or treating inflammatory pain, the pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the nucleic acid molecule according to claim 4, or the vector according to claim 5, or the cell according to claim 6; Preferably, the inflammatory pain includes migraine, peritoneal visceral inflammatory pain; Preferably, the migraine includes chronic migraine, migraine without aura, tension headache, cluster pain; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant; Preferably, the pharmaceutical composition further comprises other antibodies.

9. Any of the following applications: 1) Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, or the cell according to claim 6 in the preparation of a product for detecting calcitonin gene-related peptide; 2) Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, or the cell according to claim 6 in the preparation of a pharmaceutical composition for preventing and / or treating inflammatory pain; Preferably, the inflammatory pain includes migraine, peritoneal visceral inflammatory pain; Preferably, the migraine includes chronic migraine, migraine without aura, tension headache, and cluster pain.

10. Any of the following methods: 1) A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the method comprising: Cultivating the cell according to claim 6, and recovering the antibody or antigen-binding fragment thereof; 2) A method for detecting calcitonin gene-related peptide in a sample for non-diagnostic purposes, the method comprising: contacting the sample to be tested with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, thereby detecting the level of calcitonin gene-related peptide in the sample to be tested.

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