Antibodies against calcitonin gene-related peptide

Abstract

The present invention discloses an antibody against calcitonin gene-related peptide, belonging to the field of biomedicine. The present invention provides an antibody or its antigen-binding fragment, a nucleic acid molecule containing the same, a vector containing the nucleic acid molecule, and a cell containing the nucleic acid molecule or the vector. The present invention also provides products and pharmaceutical compositions related to the aforementioned antibody or its antigen-binding fragment, as well as related applications and methods.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an antibody 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 polypeptide family and plays important roles in regulating metabolism, inflammatory response, vascular tone, and pain in sensory nerve transmission. The main advantages of CGRP receptor antagonists are obvious efficacy, few adverse reactions, and no addiction, so there are no usage taboos for most analgesics.

[0003] Compared with small chemical molecules, antibodies that antagonize CGRP and its receptor have higher specificity, affinity, longer half-life, and lower hepatotoxicity. They can extend the dosing interval, improve patient compliance, and enhance drug efficacy. Therefore, developing 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 its antigen-binding fragment, and the antibody or its antigen-binding fragment comprises:

[0006] Complementary determining regions of the heavy chain variable region:

[0007] 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,

[0008] 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,

[0009] 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,

[0010] Complementary determining regions of the light chain variable region:

[0011] 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 shown in SEQ ID NO:9,

[0012] 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 shown in SEQ ID NO:10.

[0013] VL 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:11;

[0014] The antibody or antigen-binding fragment thereof specifically binds to and / or neutralizes calcitonin gene-related peptide.

[0015] Furthermore, the amino acid sequence of VH CDR1 is as shown in SEQ ID NO:1,

[0016] the amino acid sequence of VH CDR2 is as shown in SEQ ID NO:2,

[0017] the amino acid sequence of VH CDR3 is as shown in SEQ ID NO:3,

[0018] the amino acid sequence of VL CDR1 is as shown in SEQ ID NO:9,

[0019] the amino acid sequence of VL CDR2 is as shown in SEQ ID NO:10,

[0020] the amino acid sequence of VL CDR3 is as shown in SEQ ID NO:11.

[0021] Furthermore, the antibody or antigen-binding fragment thereof further comprises

[0022] Heavy chain variable region framework regions:

[0023] 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,

[0024] 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,

[0025] 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,

[0026] 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,

[0027] Light chain variable region framework region:

[0028] 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 shown in SEQ ID NO:12,

[0029] 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 shown in SEQ ID NO:13,

[0030] 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 shown in SEQ ID NO:14,

[0031] VL 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:15.

[0032] Furthermore, the amino acid sequence of VH FR1 is as shown in SEQ ID NO:4,

[0033] The amino acid sequence of VH FR2 is as shown in SEQ ID NO:5,

[0034] The amino acid sequence of VH FR3 is as shown in SEQ ID NO:6,

[0035] The amino acid sequence of VH FR4 is as shown in SEQ ID NO:7,

[0036] The amino acid sequence of VL FR1 is as shown in SEQ ID NO:12,

[0037] The amino acid sequence of VL FR2 is as shown in SEQ ID NO:13,

[0038] The amino acid sequence of VL FR3 is as shown in SEQ ID NO:14,

[0039] The amino acid sequence of VL FR4 is as shown in SEQ ID NO:15.

[0040] Furthermore, the heavy-chain variable region VH of the antibody or its antigen-binding fragment has an amino acid sequence with 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% identity to the amino acid sequence shown in SEQ ID NO:8,

[0041] and the light-chain variable region VL has an amino acid sequence with 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% identity to the amino acid sequence shown in SEQ ID NO:16.

[0042] Furthermore, the amino acid sequence of the heavy-chain variable region VH is as shown in SEQ ID NO:8,

[0043] and the amino acid sequence of the light-chain variable region VL is as shown in SEQ ID NO:16.

[0044] Furthermore, the antibody or its antigen-binding fragment further comprises a heavy-chain variable region signal peptide, and the amino acid sequence of the heavy-chain variable region signal peptide has 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:17.

[0045] Furthermore, the antibody or its antigen-binding fragment further comprises a light-chain variable region signal peptide, and the amino acid sequence of the light-chain variable region signal peptide has 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:18.

[0046] Furthermore, the amino acid sequence of the heavy-chain variable region signal peptide in the antibody or its antigen-binding fragment is as shown in SEQ ID NO:17.

[0047] Furthermore, the amino acid sequence of the light-chain variable region signal peptide in the antibody or its antigen-binding fragment is as shown in SEQ ID NO:18.

[0048] Furthermore, the antibody or its antigen-binding fragment further comprises a heavy-chain constant region, and the amino acid sequence of the heavy-chain constant region is as shown in SEQ ID NO:19.

[0049] Furthermore, the antibody or its antigen-binding fragment further comprises a light-chain constant region, and the amino acid sequence of the light-chain constant region is as shown in SEQ ID NO:20.

[0050] Further, the antibody or its antigen-binding fragment further comprises a defucosylated antibody or its antigen-binding fragment.

[0051] 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 two optimally aligned sequences in a comparison window, determine the number of positions where the same nucleic acid bases (such as A, T, C, G, I) or the same amino acid residues (such as 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 percentage of sequence identity.

[0052] Further, the antibody includes diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment, (dsFv)2, dual-specific dsFv, disulfide-stabilized diabody, single-chain antibody molecule, scFv dimer, multispecific antibody, camelized single-domain antibody, nanobody, domain antibody, or bivalent domain antibody.

[0053] The present invention provides a nucleic acid molecule encoding the aforementioned antibody or its antigen-binding fragment.

[0054] Further, the nucleic acid molecule encodes the nucleotide sequences of VH CDR1, VH CDR2, and VH CDR3 in the antibody or its antigen-binding fragment, which have nucleotide sequences with at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequences shown in SEQ ID NO: 21, 22, and 23, respectively.

[0055] The nucleotide sequences encoding VL CDR1, VL CDR2, and VL CDR3 in the antibody or its antigen-binding fragment have nucleotide sequences with at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequences shown in SEQ ID NO: 29, 30, and 31, respectively.

[0056] Further, the nucleotide sequences of VH CDR1, VH CDR2, and VH CDR3 encoded by the nucleic acid molecule in the antibody or its antigen-binding fragment are respectively as shown in SEQ ID NO: 21, 22, and 23.

[0057] The nucleotide sequences encoding VL CDR1, VL CDR2, and VL CDR3 in the encoded antibody or its antigen-binding fragment are shown as SEQ ID NO:29, 30, and 31 respectively.

[0058] Furthermore, the nucleotide sequences encoding VH FR1, VH FR2, VH FR3, and VH FR4 in the encoded 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 nucleotide sequences shown as SEQ ID NO:24, 25, 26, and 27 respectively.

[0059] The nucleotide sequences encoding VL FR1, VL FR2, VL FR3, and VL FR4 in the encoded 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 as SEQ ID NO:32, 33, 34, and 35 respectively.

[0060] Furthermore, the nucleotide sequences encoding VH FR1, VH FR2, VH FR3, and VH FR4 in the encoded antibody or its antigen-binding fragment are shown as SEQ ID NO:24, 25, 26, and 27 respectively.

[0061] The nucleotide sequences encoding VL FR1, VL FR2, VL FR3, and VL FR4 in the encoded antibody or its antigen-binding fragment are shown as SEQ ID NO:32, 33, 34, and 35 respectively.

[0062] Furthermore, the nucleic acid molecule encodes the heavy chain variable region of the antibody or its antigen-binding fragment, 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%, and at least 99% identical to the nucleotide sequence shown as SEQ ID NO:28.

[0063] The nucleic acid molecule encodes the light chain variable region of the antibody or its antigen-binding fragment, 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%, and at least 99% identical to the nucleotide sequence shown as SEQ ID NO:36.

[0064] Furthermore, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody or its antigen-binding fragment is shown as SEQ ID NO:28.

[0065] The nucleotide sequence encoding the light chain variable region of an antibody or an antigen-binding fragment thereof is as shown in SEQ ID NO: 36.

[0066] Furthermore, the nucleic acid molecule encodes the heavy chain variable region signal peptide in an antibody or an antigen-binding fragment thereof, which has a nucleotide sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 37.

[0067] It encodes the light chain variable region signal peptide in an antibody or an antigen-binding fragment thereof, which has a nucleotide sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 38.

[0068] Furthermore, the nucleotide sequence encoding the heavy chain variable region signal peptide in the nucleic acid molecule is as shown in SEQ ID NO: 37.

[0069] The nucleotide sequence encoding the light chain variable region signal peptide in an antibody or an antigen-binding fragment thereof is as shown in SEQ ID NO: 38.

[0070] Furthermore, the nucleotide sequence encoding the heavy chain constant region in the nucleic acid molecule is as shown in SEQ ID NO: 39;

[0071] The nucleotide sequence encoding the light chain constant region in an antibody or an antigen-binding fragment thereof is as shown in SEQ ID NO: 40.

[0072] Furthermore, the nucleic acid molecule is codon-optimized.

[0073] In some embodiments, the nucleotide sequence in the nucleic acid molecule undergoes substitution with conservative nucleic acids. 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 an amino acid sequence, sequences with the same or related basic structure and sequence. In the substitution of conservative nucleic acids, the degeneracy of the genetic code is applied.

[0074] The present invention provides a vector, which includes the aforementioned nucleic acid molecule.

[0075] Furthermore, the vector further includes elements for controlling expression.

[0076] Furthermore, the vector further includes materials that facilitate the entry of the vector into cells.

[0077] In some embodiments, the most commonly used type of the vector is a plasmid. In other embodiments, the vector is a viral vector, specifically including common retroviruses (such as lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses, poxviruses, baculoviruses, papillomaviruses, etc.

[0078] The present invention provides cells, which comprise the nucleic acid molecules or the vectors described above.

[0079] Furthermore, the cells include prokaryotic cells and eukaryotic cells.

[0080] Furthermore, the prokaryotic cells include Gram-negative or Gram-positive cells.

[0081] Furthermore, the eukaryotic cells include protist cells, animal cells, and fungal cells.

[0082] Furthermore, the animal cells include mammalian cells, avian cells, and insect cells.

[0083] Furthermore, the mammalian cells include 293F cells, 293T cells, CHO cells, AT1080 cells, and A549 cells.

[0084] In some embodiments, the mammalian cells include human and non-human mammalian cells, and the non-human mammalian cells refer to viviparous animal cells other than humans, including murine cells, ovine cells, rabbit cells, equine cells, bovine cells, porcine cells, canine cells, simian cells, etc.

[0085] The present invention provides a product for detecting calcitonin gene-related peptide, which comprises the antibody or its antigen-binding fragment described above.

[0086] Furthermore, the product further includes a reagent for performing an antigen-antibody reaction or a reagent for detecting the reaction.

[0087] Furthermore, the reagent for performing an antigen-antibody reaction includes buffers and salts.

[0088] Furthermore, the product includes a conjugate of an antibody linked to a detectable label.

[0089] Furthermore, the product includes a kit and a test strip.

[0090] The present invention provides a pharmaceutical composition for preventing or treating inflammatory pain, which comprises the antibody or its antigen-binding fragment described above, or the nucleic acid molecule described above, or the vector described above, or the cell described above.

[0091] Furthermore, the inflammatory pain includes migraine and peritoneal visceral inflammatory pain.

[0092] Further, the migraine includes chronic migraine, migraine without aura, tension headache, and cluster headache.

[0093] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

[0094] Further, the pharmaceutical composition further comprises other antibodies.

[0095] The present invention provides any one of the following applications.

[0096] The present invention provides the use of the antibody or its antigen-binding fragment described above, the nucleic acid molecule described above, the vector described above, or the cell described above in the preparation of a product for detecting calcitonin gene-related peptide.

[0097] The present invention provides the use of the antibody or its antigen-binding fragment described above, the nucleic acid molecule described above, the vector described above, or the cell described above in the preparation of a pharmaceutical composition for preventing and / or treating inflammatory pain.

[0098] Further, the inflammatory pain includes migraine and peritoneal visceral inflammatory pain.

[0099] Further, the migraine includes chronic migraine, migraine without aura, tension headache, and cluster headache.

[0100] Further, the pharmaceutical composition is used for inflammatory pain in Balb / c mice.

[0101] The present invention provides any one of the following methods.

[0102] The present invention provides a method for preparing the antibody or its antigen-binding fragment described above, the method comprising: culturing the cell described above and recovering the antibody or its antigen-binding fragment.

[0103] The present invention provides 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 its antigen-binding fragment described above so as to detect the level of calcitonin gene-related peptide in the sample to be tested. Description of the Drawings

[0104] Figure 1 It is a diagram of the SDS-PAGE detection result of the antibody.

[0105] Figure 2 It is a diagram of the SEC-HPLC detection result of the antibody.

[0106] Figure 3It is a graph showing the results of antibody binding experiments. Among them, in A, ELISA test starts from 4 μg / ml and is diluted 5-fold, and in B, the initial concentration of the antibody is increased, starting from 100 μg / ml and diluted 2-fold.

[0107] Figure 4 It is a graph showing the results of the writhing experiment of treating mice with peritoneal visceral inflammatory pain with antibodies. Detailed implementation manners

[0108] Unless otherwise specified, all technical and scientific terms used in the present invention generally have the same meanings as those commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0109] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the specific implementation manners described herein are presented by way of example and are not intended to limit the present invention. Without departing from the scope of the present invention, the main features of the present invention can be used in various implementation manners.

[0110] Example 1 Preparation and purification method of ZRQ1Ab antibody

[0111] 1. Experimental method

[0112] Expression method: Expressed by CHO-S cells.

[0113] The heavy chain and light chain genes of the antibody are synthesized, and the specific sequences are shown in Table 1. They are 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 is detected by ELISA. Select an appropriate time to harvest the antibody. Then, it is purified and concentrated by a protein purification instrument. Finally, the purity of the antibody is verified by SDS and HPLC.

[0114] Among them, transfection: Transfection is carried out when the CHO-S cells are cultured to an appropriate confluence and the viability is above 95%. High-quality plasmid DNA, with an OD260 / OD280 ratio between 1.8 - 2.0 and a concentration ranging from several hundred ng / μl to several μg / μl. Under sterile conditions, an appropriate amount of plasmid DNA is diluted into serum-free medium and gently mixed. An appropriate amount of transfection reagent is also diluted into serum-free medium and gently mixed. The diluted plasmid DNA solution and transfection reagent solution are slowly mixed and gently inverted and mixed, and left standing at room temperature for a certain time (usually 1 - 5 minutes) to form a plasmid-transfection reagent complex. Add it to the CHO-S cell flask and slowly add it while shaking. Place the cell flask back on the cell shaker and set it at 37°C, 125 r, 8% CO2, and continue culturing.

[0115] ELISA detection of antibody expression level: Select an ELISA plate with good adsorption performance, low blank value, and high bottom transparency of the wells. Dilute the CGRP protein to an appropriate concentration (such as 2 μg / ml) using a coating buffer (such as phosphate buffer with pH 7.2 - 7.4), add a certain volume (such as 0.1 ml) to each well, and incubate overnight at 4°C or for 2 hours at 37°C. After coating, block the non-specific binding sites with a blocking solution (such as 5% BSA) to reduce background noise, add a certain volume (such as 0.20 ml) to each well, and block at 37°C for 1 - 2 hours. Add the diluted antibody to be detected, a certain volume (such as 0.1 ml) to each well, and incubate at 37°C for 1 hour. Wash 3 times with a washing buffer (such as PBST), 1 minute each time, to remove unbound samples. Add the enzyme-labeled secondary antibody, a certain volume (such as 0.1 ml) to each well, and incubate at 37°C for 30 - 60 minutes. Wash again 3 times with the washing buffer, 1 minute each time, to remove unbound enzyme-labeled antibody. Add the enzyme substrate (such as TMB), a certain volume (such as 0.1 ml) to each well, and incubate at 37°C in the dark for 1 - 15 minutes until an obvious color gradient change appears. Add the stop solution (such as 2M sulfuric acid), a certain volume (such as 0.1 ml) to each well, to terminate the enzyme reaction. Measure the absorbance (OD value) of each well at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader, and calculate the concentration of the antibody in the sample according to the standard curve.

[0116] SDS-PAGE: Mix the antibody sample with the loading buffer, which contains SDS (sodium dodecyl sulfate) and a reducing agent (such as β-mercaptoethanol) to disrupt the spatial structure and disulfide bonds of the protein, causing the protein to depolymerize into polypeptide chains. The mixing ratio of the sample and the loading buffer is usually 4:1 or 5:1. Heat the mixed sample in boiling water for 5 - 10 minutes to completely denature the protein. Prepare a polyacrylamide gel, usually including two layers: a stacking gel (5% concentration) and a separating gel (select the concentration according to the protein molecular weight, such as 10%, 12%, etc.). After the gel is prepared, place it in the electrophoresis tank and add the electrophoresis buffer. Add the treated sample and the protein molecular weight standard (Marker) into the sample wells of the gel respectively. Connect the power supply and start electrophoresis. During electrophoresis, protein molecules are separated in gels with different concentrations according to their molecular weights and charges. The electrophoresis conditions are usually: voltage 80 - 100 V in the stacking gel stage, voltage 120 - 150 V in the separating gel stage, and the electrophoresis time depends on the migration of the bromophenol blue indicator. After electrophoresis, take out the gel and stain it with Coomassie Brilliant Blue staining solution, usually staining for 1 - 2 hours. After staining, decolorize with the decolorizing solution until the background is clear and the protein bands are obvious. Observe the protein bands on the gel. If there is only one clear band in the antibody sample and the molecular weight is consistent with the expected value, it indicates that the antibody has a high purity.

[0117] HPLC: Prepare the antibody sample to be tested, ensuring that it is dissolved in an appropriate solvent and at an appropriate concentration. Select an appropriate chromatographic column and mobile phase according to the properties of the antibody. Commonly used chromatographic columns include reverse-phase columns, ion-exchange columns, etc. Equilibrate the HPLC system with the same buffer used for sample dilution until the baseline is stable. Load the prepared sample into the chromatographic system by means of an autosampler or manually. Pass through the chromatographic column at a constant flow rate, and the antibody will be eluted successively according to its size and properties. Record the elution curve through a detector (such as a UV detector). Analyze the elution profile to identify protein peaks and possible impurity peaks. Perform quantitative analysis by integrating the peak areas to calculate the purity of the antibody.

[0118] Table 1

[0119]

[0120]

[0121]

[0122]

[0123] 2. Experimental Results

[0124] After culturing the antibody (using the Human IgG1-Kappa tag) with CHO-S cells, its expression level was 283.60 mg / L. Subsequently, molecular analysis was used to obtain a predicted MW of 49 / 23 kDa under reducing conditions and a predicted MW of 144 kDa under non-reducing conditions for the antibody.

[0125] Subsequently, SDS-PAGE was used to detect that the purity of the antibody under reducing conditions was greater than 95%, and the results were as Figure 1 shown. SEC-HPLC was used for antibody purity detection, and the result was an antibody purity of 98.463%, and the results were as Figure 2 shown. The endotoxin level was detected to be 0 - 1 EU / mg.

[0126] The binding result of the antibody ZRQ1Ab was as Figure 3 shown, and its IC50 value was 15.45 μg / ml. ZR8Ab is Galcanezuma, a marketed antibody of Eli Lilly and Company, used as a positive control.

[0127] Example 2 Animal Writhing Experiment to Determine the Therapeutic Function of ZRQ1Ab Antibody

[0128] 1. Experimental Method

[0129] The acetic acid-induced abdominal constriction (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, thereby causing central and peripheral sensitization and hyperalgesia. In mice, the nociceptive cascade after acetic acid administration results in repeated abdominal contractions or writhing.

[0130] Male and female Balb / c mice, each weighing 20 ± 2 g (n = 4 per group), and ICR mice weighing 40 g ± 2 g (n = 4 per group) were used. The injection material was acetic acid. One day before the experiment, antibodies (5 mg / Kg) were subcutaneously injected according to the usage method of the marketed drug, and the control group was injected with normal saline. The next day, the mice were acclimated in a plexiglass cage for 15 minutes and then intraperitoneally injected with 100 μl / 10 g of a solution containing 0.6% acetic acid (v / v).

[0131] Typical writhing responses within 30 minutes after acetic acid injection were recorded, which were characterized by longitudinal extension of the trunk, concave extension of the back, and extension of the hind limbs. Each writhing was counted as one when the normal posture was restored.

[0132] 2. Experimental results

[0133] The results of the writhing experiment are as Figure 4 shown. The results showed that compared with the control group, the use of ZRQ1Ab antibody in the present invention could significantly reduce the writhing intensity of Balb / c mice. The specific results are shown in Table 2.

[0134] Table 2

[0135]

[0136] Table 3 shows the effects of different antibodies on the writhing intensity in ICR mice. The use of ZRQ1Ab antibody in the present invention did not play a role in reducing the writhing intensity in ICR mice. The specific results are as Figure 4 shown.

[0137] Table 3

[0138]

[0139] The description of the above embodiments is only for understanding the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises: Complementary determining regions of the heavy-chain variable region: The amino acid sequence of VH CDR1 is as shown in SEQ ID NO:1, The amino acid sequence of VH CDR2 is as shown in SEQ ID NO:2, The amino acid sequence of VH CDR3 is as shown in SEQ ID NO:3, Complementary determining regions of the light-chain variable region: The amino acid sequence of VL CDR1 is as shown in SEQ ID NO:9, The amino acid sequence of VL CDR2 is as shown in SEQ ID NO:10, The amino acid sequence of VL CDR3 is as shown in SEQ ID NO:11; The antibody or the antigen-binding fragment thereof specifically binds to calcitonin gene-related peptide and / or neutralizes calcitonin gene-related peptide.

2. The antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody or the antigen-binding fragment thereof further comprises Framework regions of the heavy-chain variable region: The amino acid sequence of VH FR1 is as shown in SEQ ID NO:4, The amino acid sequence of VH FR2 is as shown in SEQ ID NO:5, The amino acid sequence of VH FR3 is as shown in SEQ ID NO:6, The amino acid sequence of VH FR4 is as shown in SEQ ID NO:7, Framework regions of the light-chain variable region: The amino acid sequence of VL FR1 is as shown in SEQ ID NO:12, The amino acid sequence of VL FR2 is as shown in SEQ ID NO:13, The amino acid sequence of VL FR3 is as shown in SEQ ID NO:14, The amino acid sequence of VL FR4 is as shown in SEQ ID NO:

15.

3. The antibody or the antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy-chain variable region VH of the antibody or the antigen-binding fragment thereof is as shown in SEQ ID NO:8, and the amino acid sequence of the light-chain variable region VL is as shown in SEQ ID NO:

16.

4. The antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody or the antigen-binding fragment thereof further comprises a heavy-chain variable region signal peptide, and the amino acid sequence of the heavy-chain variable region signal peptide is as shown in SEQ ID NO:

17.

5. The antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody or the antigen-binding fragment thereof further comprises a light-chain variable region signal peptide, and the amino acid sequence of the light-chain variable region signal peptide is as shown in SEQ ID NO:

18.

6. The antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody or the antigen-binding fragment thereof further comprises a heavy-chain constant region, and the amino acid sequence of the heavy-chain constant region is as shown in SEQ ID NO:

19.

7. The antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody or the antigen-binding fragment thereof further comprises a light-chain constant region, and the amino acid sequence of the light-chain constant region is as shown in SEQ ID NO:

20.

8. The antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody or the antigen-binding fragment thereof further comprises a defucosylated antibody or an antigen-binding fragment thereof.

9. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-8.

10. The nucleic acid molecule according to claim 9, wherein the nucleotide sequences encoding VH CDR1, VH CDR2, and VH 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%, or at least 99% identical to the nucleotide sequences shown in SEQ ID NO: 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%, or at least 99% identical to the nucleotide sequences shown in SEQ ID NO: 29, 30, and 31, respectively.

11. The nucleic acid molecule according to claim 9, wherein the nucleotide sequences encoding VH CDR1, VH CDR2, and VH CDR3 in the antibody or antigen-binding fragment thereof are respectively as shown in SEQ ID NO: 21, 22, and 23. The nucleotide sequences encoding VL CDR1, VL CDR2, and VL CDR3 in the antibody or antigen-binding fragment thereof are respectively as shown in SEQ ID NO: 29, 30, and 31.

12. The nucleic acid molecule according to claim 9, wherein the nucleotide sequences encoding VH FR1, VH FR2, VH FR3, and VH FR4 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%, or at least 99% identical to the nucleotide sequences shown in SEQ ID NO: 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 have nucleotide sequences that are at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequences shown in SEQ ID NO: 32, 33, 34, and 35, respectively.

13. The nucleic acid molecule according to claim 9, wherein the nucleotide sequences encoding VH FR1, VH FR2, VH FR3, and VH FR4 in the antibody or antigen-binding fragment thereof are respectively as shown in SEQ ID NO: 24, 25, 26, and 27. The nucleotide sequences encoding VL FR1, VL FR2, VL FR3, and VL FR4 in the antibody or antigen-binding fragment thereof are respectively as shown in SEQ ID NO: 32, 33, 34, and 35.

14. The nucleic acid molecule according to claim 9, wherein the nucleic acid molecule encodes a heavy chain variable region of an antibody or an antigen-binding fragment thereof, and 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. It encodes a light chain variable region of an antibody or an antigen-binding fragment thereof, and 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:

36.

15. The nucleic acid molecule according to claim 9, wherein the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody or an 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 an antigen-binding fragment thereof is as shown in SEQ ID NO:

36.

16. The nucleic acid molecule according to claim 9, wherein the nucleic acid molecule encodes a signal peptide of the heavy chain variable region in the antibody or an antigen-binding fragment thereof, and 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. It encodes a signal peptide of the light chain variable region in the antibody or an antigen-binding fragment thereof, and 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:

38.

17. The nucleic acid molecule according to claim 9, wherein the nucleotide sequence of the nucleic acid molecule encoding the signal peptide of the heavy chain variable region in the antibody or an antigen-binding fragment thereof is as shown in SEQ ID NO:

37. The nucleotide sequence of the nucleic acid molecule encoding the signal peptide of the light chain variable region in the antibody or an antigen-binding fragment thereof is as shown in SEQ ID NO:

38.

18. The nucleic acid molecule according to claim 9, wherein the nucleotide sequence of the nucleic acid molecule encoding the heavy chain constant region in the antibody or an antigen-binding fragment thereof is as shown in SEQ ID NO: 39; the nucleotide sequence of the nucleic acid molecule encoding the light chain constant region in the antibody or an antigen-binding fragment thereof is as shown in SEQ ID NO:

40.

19. A vector, wherein the vector comprises the nucleic acid molecule according to any one of claims 9-18.

20. The vector according to claim 19, wherein the vector further comprises an element for controlling expression.

21. The vector according to claim 19, wherein the vector further comprises a material that facilitates the entry of the vector into cells.

22. A cell, wherein the cell comprises the nucleic acid molecule according to any one of claims 9-18 or the vector according to any one of claims 19-21.

23. The cell according to claim 22, wherein the cell comprises a prokaryotic cell or a eukaryotic cell.

24. The cell according to claim 23, wherein the prokaryotic cell comprises a Gram-negative or Gram-positive cell.

25. The cell according to claim 23, wherein the eukaryotic cell comprises a protist cell, an animal cell, or a fungal cell.

26. The cell according to claim 25, wherein the animal cell comprises a mammalian cell, a bird cell, or an insect cell.

27. The cell according to claim 26, wherein the mammalian cell comprises a 293F cell, a 293T cell, a CHO cell, an AT1080 cell, or an A549 cell.

28. A product for detecting calcitonin gene-related peptide, the product comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-8.

29. The product according to claim 28, wherein the product further comprises a reagent for performing an antigen-antibody reaction or a reagent for detecting the reaction.

30. The product according to claim 29, wherein the reagent for performing an antigen-antibody reaction comprises a buffer and a salt.

31. The product according to claim 28, wherein the product comprises a conjugate of an antibody linked to a detectable label.

32. A pharmaceutical composition for preventing or treating peritoneal visceral inflammatory pain, the pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-8, or the nucleic acid molecule according to any one of claims 9-18, or the vector according to any one of claims 19-21, or the cell according to any one of claims 22-27.

33. The pharmaceutical composition according to claim 32, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

34. The pharmaceutical composition according to claim 32, wherein the pharmaceutical composition further comprises other antibodies.

35. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-8, the nucleic acid molecule according to any one of claims 9-18, the vector according to any one of claims 19-21, or the cell according to any one of claims 22-27 in the preparation of a product for detecting calcitonin gene-related peptide.

36. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-8, the nucleic acid molecule according to any one of claims 9-18, the vector according to any one of claims 19-21, or the cell according to any one of claims 22-27 in the preparation of a pharmaceutical composition for preventing and / or treating peritoneal visceral inflammatory pain.

37. A method for preparing an antibody or an antigen-binding fragment thereof according to any one of claims 1-8, the method comprising: Cultivate the cell according to any one of claims 22-27 and recover the antibody or antigen-binding fragment thereof.

38. A method for detecting calcitonin gene-related peptide in a test sample for non-diagnostic purposes, the method comprising: Contact the antibody or antigen-binding fragment thereof according to any one of claims 1-8 with a sample to be tested, so as to detect the level of calcitonin gene-related peptide in the sample to be tested.

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