TRPA1 antibody, and preparation method therefor and use thereof

Abstract

Provided are a TRPA1 antibody, and a preparation method therefor and the use thereof. The TRPA1 antibody comprises a light-chain variable region and a heavy-chain variable region, wherein the heavy-chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 56-58, respectively; and / or the light-chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 59, SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The provided TRPA1 antibody has a relatively high affinity for TRPA1, and the screened TRPA1 antibody has an obvious inhibitory effect on a TRPA1 channel activated by AITC, with IC50 lower than 1.54 μM and the minimum being 0.56 μM.

Description

TRPA1 antibody, preparation method and application thereof Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a TRPA1 antibody, a preparation method thereof and an application thereof. Background Art

[0002] Transient receptor potential ankyrin subtype 1 protein (TRPA1) is a Ca-permeable 2+ 、Na + and K + TRPA1 is a non-selective cation channel. It is present in a subset of Aδ-fiber and C-fiber nociceptive sensory neurons and other sensory cells including epithelial cells. In primary sensory neurons, Ca 2+ and Na + It flows into cells through TRPA1, causing membrane depolarization, action potential firing, and neurotransmitter release in peripheral and central neural projections.

[0003] In addition to being activated by cysteine- and lysine-reactive electrophiles and oxidants, TRPA1 can also be indirectly activated by proinflammatory agents through the phospholipase C signaling pathway, in which cytosolic Ca 2+ It is an important regulator of channel gating.

[0004] Multiple lines of evidence implicate TRPA1 in pain perception. TRPA1 is expressed in sensory neurons and colocalizes with pain markers such as TRPV1 and bradykinin receptors. Its expression is increased in animal models of inflammation and neuropathic pain, as well as in DRG neurons following human avulsion injury. TRPA1 agonists induce neurotransmitter release, pain, and inflammation in rodents and humans, while endogenous agonists such as 4-HNE are elevated in human pathological conditions. In several animal models, gene knockout reduces agonist sensitivity, and antagonist treatment reduces pain. TRPA1 is thought to play a role in numerous sensory modalities, including chemical nociception, mechanical nociception, and cold nociception.

[0005] In recent years, opioid addiction and overdose have increased at an alarming rate and have become a serious national crisis in the United States, resulting in numerous deaths daily. The need for new, non-opioid pain medications without addictive potential is greater than ever. Because TRPA1 is involved in both chronic and acute pain, TRPA1 antagonists have the potential to provide the next generation of pain medications that could help alleviate the current opioid crisis.

[0006] Research on the indications of TRPA1 for the treatment of pain, skin diseases and respiratory diseases such as asthma is quite hot. TRPA1 small molecule antagonists mainly include structures such as xanthine, sulfonamide, oxadiazolone, oxadiazole and carboxamide. As of 2019, five small molecules have entered the clinic, but all have been terminated due to poor pharmacokinetic properties. Compared with small molecule drugs, large molecule antibody drugs have obvious advantages. Their limited central nervous system (CNS) permeability (when targeting peripheral treatment), low immunogenicity, high selectivity and favorable half-life make the development of TRPA1 antibodies an attractive alternative to biologics. However, due to the dynamic nature of the TRPA1 conformation and the extremely small extracellular region, screening for functionally active antibodies remains a well-known challenge. Existing commercially available TRPA1 antibodies are mainly polyclonal antibodies used in TRPA1 immunological detection experiments, and their affinity, specificity, functionality and other properties are poor.

[0007] Summary of the Invention

[0008] To address the lack of functionally active antibodies targeting TRPA1 in the prior art, the present invention provides a TRPA1 antibody, method for preparing the same, and its use. The immunogen used in the preparation process is a nanodisc-packaged, purified protein. The antibody is prepared using hybridoma technology. Affinity screening is primarily performed using ELISA and FACS, combined with a FlexStation3 to measure changes in fluorescence intensity caused by calcium ions flowing into cells through ion channels, to screen for antibodies with both affinity and functionality. Through affinity testing, functional testing, and sequence acquisition, several therapeutic antibodies with TRPA1 antagonism were obtained.

[0009] In order to solve the above technical problems, the present invention provides a TRPA1 antibody in a first aspect, comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 56-58, respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 59, 9 and 10, respectively; or,

[0010] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 26-28, respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 29-31, respectively; or,

[0011] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 36-38, respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 39-41, respectively; or,

[0012] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 51-53, respectively; and / or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 54, 35 and 55, respectively.

[0013] In some embodiments of the present invention, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 5-7, respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 8-10, respectively; or,

[0014] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 17-19, respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 8-10, respectively; or,

[0015] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 17-19, respectively; and / or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 46, 9 and 10, respectively.

[0016] In some embodiments of the present invention, the heavy chain variable region further includes a heavy chain variable region framework region HFWR, and / or the light chain variable region further includes a light chain variable region framework region LFWR, wherein the HFWR is a heavy chain variable region framework region of human or mouse origin, and the LFWR is a light chain variable region framework region of a human or mouse antibody.

[0017] In some preferred embodiments of the present invention, the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 4 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence as shown in SEQ ID NO: 4; and / or, the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 3 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence as shown in SEQ ID NO: 3; or,

[0018] The light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 14, or is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence as shown in SEQ ID NO: 14; and / or the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 13, or is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence as shown in SEQ ID NO: 13; or,

[0019] The light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 14, or is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence as shown in SEQ ID NO: 14; and / or the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 16, or is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence as shown in SEQ ID NO: 16; or

[0020] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14, or is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 14; and / or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 21, or is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 21; or

[0021] The light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 25 or is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as shown in SEQ ID NO: 25; and / or the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 24 or is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as shown in SEQ ID NO: 24; or,

[0022] The light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 35 or is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as shown in SEQ ID NO: 35; and / or the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 34 or is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as shown in SEQ ID NO: 34; or,

[0023] The light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 45 or is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as shown in SEQ ID NO: 45; and / or the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 44 or is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as shown in SEQ ID NO: 44; or,

[0024] The light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:50 or is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as shown in SEQ ID NO:50; and / or the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:49 or is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as shown in SEQ ID NO:49.

[0025] In some more preferred embodiments of the present invention, the TRPA1 antibody further comprises a heavy chain constant region and a light chain constant region.

[0026] In some further more preferred embodiments of the present invention, the TRPA1 antibody heavy chain constant region is a human or mouse antibody heavy chain constant region; the TRPA1 antibody light chain constant region is a human or mouse antibody light chain constant region.

[0027] In some embodiments of the present invention, the TRPA1 antibody is in any of the following antibody forms:

[0028] (a) a complete immunoglobulin molecule;

[0029] (b) an scFv;

[0030] (c) a fusion protein comprising an scFv;

[0031] (d) a Fab fragment;

[0032] (e) a Fab′ fragment;

[0033] (f) a F(ab)2;

[0034] Alternatively, the TRPA1 antibody is a monoclonal antibody or a polyclonal antibody;

[0035] Alternatively, the TRPA1 antibody is a humanized antibody or a bispecific antibody.

[0036] In order to solve the above technical problems, the second aspect of the present invention provides a chimeric antigen receptor, which comprises the TRPA1 antibody as described in the first aspect of the present invention.

[0037] In order to solve the above technical problems, the third aspect of the present invention provides an isolated nucleic acid encoding the TRPA1 antibody as described in the first aspect of the present invention, or the chimeric antigen receptor as described in the second aspect of the present invention.

[0038] In some preferred embodiments of the present invention, the nucleic acid encoding the TRPA1 antibody comprises the polynucleotide sequence shown in SEQ ID NO: 1 and / or as shown in SEQ ID NO: 2; or, comprises the polynucleotide sequence shown in SEQ ID NO: 11 and / or as shown in SEQ ID NO: 12; or, comprises the polynucleotide sequence shown in SEQ ID NO: 15 and / or as shown in SEQ ID NO: 12; or, comprises the polynucleotide sequence shown in SEQ ID NO: 20 and / or as shown in SEQ ID NO: 12; or, comprises the polynucleotide sequence shown in SEQ ID NO: 22 and / or as shown in SEQ ID NO: 23; or, comprises the polynucleotide sequence shown in SEQ ID NO: 32 and / or as shown in SEQ ID NO: 33; or, comprises the polynucleotide sequence shown in SEQ ID NO: 42 and / or as shown in SEQ ID NO: 43; or, comprises the polynucleotide sequence shown in SEQ ID NO: 47 and as shown in SEQ ID NO: 48.

[0039] In order to solve the above technical problems, the fourth aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid as described in the third aspect of the present invention.

[0040] In order to solve the above technical problems, the fifth aspect of the present invention provides a transformant, which contains the recombinant expression vector as described in the fourth aspect in a host cell.

[0041] In order to solve the above technical problems, the sixth aspect of the present invention provides a method for preparing a TRPA1 antibody, which comprises culturing the transformant as described in the fifth aspect of the present application, and obtaining the TRPA1 antibody from the culture.

[0042] In order to solve the above technical problems, the seventh aspect of the present invention provides an antibody-drug conjugate, which comprises a cytotoxic agent and the TRPA1 antibody according to the first aspect of the present invention.

[0043] In order to solve the above technical problems, the eighth aspect of the present invention provides a pharmaceutical composition, which comprises the TRPA1 antibody as described in the first aspect of the present invention, the chimeric antigen receptor as described in the second aspect of the present invention or the antibody-drug conjugate as described in the seventh aspect of the present invention, and a pharmaceutically acceptable carrier.

[0044] In order to solve the above technical problems, the ninth aspect of the present invention provides the use of the TRPA1 antibody as described in the first aspect of the present invention, the chimeric antigen receptor as described in the second aspect of the present invention, the isolated nucleic acid as described in the third aspect of the present invention, the recombinant expression vector as described in the fourth aspect of the present invention, the transformant as described in the fifth aspect of the present invention, the antibody-drug conjugate as described in the seventh aspect of the present invention, or the pharmaceutical composition as described in the eighth aspect of the present invention in the preparation of a medicament for treating pain.

[0045] In some preferred embodiments of the present invention, the pain is pain caused by TRPA1 activation; and the target of the drug is TRPA1.

[0046] In order to solve the above technical problems, the tenth aspect of the present invention provides a kit, which includes the TRPA1 antibody as described in the first aspect of the present invention, the chimeric antigen receptor as described in the second aspect of the present invention, the antibody-drug conjugate as described in the seventh aspect of the present invention and / or the pharmaceutical composition as described in the eighth aspect of the present invention.

[0047] In order to solve the above technical problems, the eleventh aspect of the present invention provides a method for detecting TRPA1, which comprises contacting a sample with the TRPA1 antibody described in the first aspect of the present invention, the chimeric antigen receptor described in the second aspect of the present invention, the antibody-drug conjugate described in the seventh aspect of the present invention, the pharmaceutical composition described in the eighth aspect of the present invention and / or the kit described in the tenth aspect of the present invention.

[0048] In some preferred embodiments of the present invention, the detection is for non-diagnostic purposes.

[0049] In order to solve the above technical problems, the twelfth aspect of the present invention provides a method for treating and / or preventing pain, which comprises administering to a patient in need thereof a therapeutically effective amount of the TRPA1 antibody as described in the first aspect of the present invention, the chimeric antigen receptor as described in the second aspect of the present invention, the isolated nucleic acid as described in the third aspect of the present invention, the recombinant expression vector as described in the fourth aspect of the present invention, the transformant as described in the fifth aspect of the present invention, the antibody-drug conjugate as described in the seventh aspect of the present invention, the pharmaceutical composition as described in the eighth aspect of the present invention and / or the kit as described in the tenth aspect of the present invention.

[0050] In some preferred embodiments of the present invention, the pain is pain caused by TRPA1 activation; and the target of the drug is TRPA1.

[0051] In order to solve the above technical problems, the thirteenth aspect of the present invention provides the TRPA1 antibody as described in the first aspect of the present invention, the chimeric antigen receptor as described in the second aspect of the present invention, the isolated nucleic acid as described in the third aspect of the present invention, the recombinant expression vector as described in the fourth aspect of the present invention, the transformant as described in the fifth aspect of the present invention, the antibody-drug conjugate as described in the seventh aspect of the present invention, the pharmaceutical composition as described in the eighth aspect of the present invention and / or the kit as described in the tenth aspect of the present invention, which are used for treating and / or preventing pain.

[0052] In some preferred embodiments of the present invention, the pain is pain caused by TRPA1 activation; and the target of the drug is TRPA1.

[0053] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0054] The reagents and raw materials used in the present invention are commercially available.

[0055] The positive progress of the present invention is that the anti-TRPA1 antibody provided by the present invention has a relatively high affinity for TRPA1, and the screened antibody has a significant inhibitory effect on the TRPA1 channel activated by AITC, and its IC 50 Below 1.54μM, the lowest can reach 0.56μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 is a map of the pEGBacMam recombinant plasmid containing TRPA1 and MBP.

[0057] Figure 2 is a map of the pET 28a recombinant plasmid containing MSP2N2.

[0058] Figure 3 shows the results of TRPA1-Nanodisc protein purification.

[0059] FIG4 shows the titer test results of the 4th and 5th sera from mice immunized with TRPA1-Nanodisc protein.

[0060] Figures 5a-5h are the ELISA test results of the 8 antibodies obtained by screening.

[0061] Figures 6a-6h are the FACS detection results of the 8 antibodies obtained by screening.

[0062] FIG7 is an SDS-PAGE image of 8 antibodies obtained after purification.

[0063] Figures 8a-8i show the inhibitory effects of the positive small molecule A967079 and the eight screened antibodies on TRPA1 channel currents. DETAILED DESCRIPTION

[0064] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0065] Example 1 Preparation of recombinant baculovirus

[0066] 1.1 Obtaining recombinant baculovirus plasmid

[0067] By heat shock transformation, the recombinant pEGBacMam plasmid containing the target gene (synthesized by Nanjing Qingke Biotechnology Co., Ltd., carrying EcoR1 and Not1 restriction sites, ligated and recombined into the pEGBacMam plasmid; the pEGBacMam recombinant plasmid containing TRPA1 and MBP is shown in Figure 1 , and the pET 28a recombinant plasmid containing MSP2N2 is shown in Figure 2 ) was introduced into Escherichia coli DH10Bac competent cells (Shanghai Weidi) and cultured at 37°C for 48-72 hours in LB solid medium containing 50 μg / mL kanamycin (aladdin), 7 μg / mL gentamicin (aladdin), 10 μg / mL tetracycline (aladdin), 100 μg / mL Bluo gal (Thermofish), and 40 μg / mL IPTG (aladdin). Uniform white spots were selected and added to 5 mL of LB liquid culture medium containing three antibiotics (50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline). The culture was carried out at 37°C and 200 rpm for 12-16 hours to extract the recombinant bacmid.

[0068] TRPA1 amino acid sequence (SEQ ID NO: 60):

[0069] TRPA1 base sequence (SEQ ID NO: 61):

[0070] MSP2N2 amino acid sequence (SEQ ID NO: 62):

[0071] MSP2N2 base sequence (SEQ ID NO: 63):

[0072] 1.2 Preparation of recombinant baculovirus

[0073] Take a 6-well cell culture plate (Nest), 1×10 6 / 2mL Gibco TM sf9 insect cells were cultured in a constant temperature and humidity incubator at 27°C for 30 min, and 100 μL Insect Medium (Sf-900 TM III SFM) and add 10 μL transfection reagent (Cellfectin TM II), add 5 μg of recombinant baculovirus plasmid to another 100 μL of Insect Medium, mix, and incubate at room temperature for 20 minutes. The transfection complex is evenly added dropwise to a 6-well plate and cultured at 27°C for 72 hours. Centrifuge at 6000 rpm at 4°C for 15 minutes. The supernatant is added with 2% FBS and stored at 4°C in the dark to obtain the P1 recombinant baculovirus.

[0074] The P1 recombinant baculovirus was taken at a ratio of 1:100 to infect 2 mL of cells with a density of 5×10 5 / mL sf9 insect cells were cultured at 27°C for 72 hours, centrifuged at 6000 rpm at 4°C for 15 minutes, the supernatant was added with 2% FBS, and stored at 4°C in the dark to obtain the P2 recombinant baculovirus.

[0075] The separation density is 1×10 6 / mL of sf9 insect cells, culture for 24 hours, and then infect with P2 recombinant baculovirus at a ratio of 1:100. The cells were cultured at 27°C and 120 rpm for 96 hours. The cells were centrifuged at 6000 rpm at 4°C for 15 minutes. The supernatant was filtered through a 0.22 μm filter, 2% FBS was added, and the cells were stored at 4°C in the dark to obtain P3 recombinant baculovirus.

[0076] The separation density is 1×10 6200 mL of sf9 insect cells (100 mL / mL) were cultured for 24 hours, and then infected with the P3 generation recombinant baculovirus at a ratio of 1:100. The cells were cultured at 27°C and 120 rpm for 96 hours. The cells were centrifuged at 6000 rpm at 4°C for 15 minutes. The supernatant was filtered through a 0.22 μm filter, 2% FBS was added, and the cells were stored at 4°C in the dark to obtain the P4 generation recombinant baculovirus.

[0077] Example 2 Protein Purification

[0078] HEK-293S cells were passaged at a concentration of 1.5 × 10 6 / mL, culture under 37°C, 8% CO2, 60% humidity, 120 rpm conditions for 24 h, take P4 generation recombinant baculovirus to infect HEK-293S cells at a ratio of 1:10, culture on a shaker for 12-18 h, and then add sodium butyrate to a final concentration of 10 mM.

[0079] After 72 hours of culture, the cells were collected by centrifugation at 6000 rpm for 15 minutes at 4°C, and the cell pellet was resuspended in Lysis buffer (50 mM HEPES (Sigma-Aldrich, V900477), pH 7.4, 150 mM NaCl (Adamas, 82999B), 1% Protease Inhibitor Cocktail, EDTA-Free, 0.5% LMNG / 0.05% CHS (Anatrace, NG310-CH210), 1 mM TCEP (McLean, T819166)) and lysed by inversion at 4°C for 3 hours. After membrane dissolution, the membrane was centrifuged at 40,000 rpm for 45 minutes at 4°C. The supernatant was tumbled and bound to MBP affinity chromatography medium (NEB, E8021L) at 4°C for 2.5 hours. The combined supernatant was passed through a gravity column and the impurities were eluted with Wash buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM TCEP, 0.01% LMNG / 0.001% CHS) and washed with Elute buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 0.01% LMNG / 0.001% CHS, 1 mM TCEP, 40 mM The 100 kDa target protein was eluted with Maltose (Adamas, 73054C) and concentrated using ultrafiltration tubes (Millopore, UFC910096). The protein was packaged at a molar ratio of TRPA1:MSP2N2:soybean lipids of 1:3:200. The required amount of Soybean Polar Lipid Extract (Avanti) was calculated and the mother liquor (25 mg / mL soybean liquor) was aspirated using a protein loading needle. The soybean liquor was pipetted to the bottom of a clean, dry glass tube. The chloroform in the soybean liquor was then blown away using a nitrogen blower, resulting in a light yellow residue at the bottom of the tube. Then add SEC buffer and sonicate for 10 minutes. During this period, the liquid in the glass tube will first become white and turbid, then gradually become clear and finally turn into a light yellow solution. Add TRPA1, MSP2N2 and soybean into the EP tube in proportion and incubate on ice for 1 hour. Then add 100 mg / mL Bio-beads SM-2 (Bio-Beads TMSM-2 Resin (Bio-Rad) was used to remove detergent from the solution, and the mixture was tumbled overnight at 4°C. The Bio-beads SM-2 were removed using gravity filtration, and the filtered protein solution was concentrated to a volume of 500 μL. The solution was centrifuged at 13,000 rpm at 4°C for 10 min, and then subjected to gel filtration chromatography using Superrose 6 Increase 10 / 300 GL (Cytiva) columns in SEC buffer (25 mM HEPES, pH 7.4, 150 mM NaCl). Protein samples were collected, A280 concentrations were measured, and SDS-PAGE gel electrophoresis was performed to determine the size and purity of the target protein. The SDS-PAGE gel electrophoresis is shown in Figure 3.

[0080] Example 3 Animal Immunization

[0081] Immunizing an animal (usually a mouse) with an antigen is the first and most crucial step in the production of monoclonal antibodies. The effectiveness of the animal's immune response to the antigen and its ability to produce high-titer, specific antibodies directly determines the difficulty of subsequent screening of monoclonal hybridoma cell lines and the effectiveness of the resulting antibodies.

[0082] The antigen used for immunization was the protein produced by the protein preparation method of TRPA1-nanodisc in Examples 1 and 2. The immunized animals were balb / c, 6-8 week old female mice. The amount of TRPA1-nanodics antigen used for the initial immunization was 100 μg protein / mouse, mixed with Freund's complete adjuvant at a volume ratio of 1:1. The insufficient amount of antigen was replaced with the purified buffer of the corresponding protein. After emulsification by a shaker, the immunized animals were routinely injected subcutaneously at multiple points on the back. Thereafter, immunization was performed every 14 days, with the amount of antigen being half of the amount of the initial immunization (50 μg / mouse), mixed with Freund's incomplete adjuvant at a volume ratio of 1:1. The insufficient amount of antigen was replaced with the purified buffer of the corresponding protein. After emulsification, the immunized animals were routinely injected subcutaneously at multiple points on the back. Normally, one week after the third immunization, the blood of the mice is collected. After separating the serum, the titer of the serum is tested by ELISA (coated with TRPA1-nanodics antigen, blocked with 2% BAS, and then incubated with gradient (1:100 starting, 3-fold dilution 10 times) diluted serum for 1 hour, incubated with goat anti-mouse HRP (SA00001-1, proteintech) for 1 hour, added with color development solution for 10 minutes, and then added with stop solution to terminate the reaction, and the absorbance value at OD450nm is tested). After that, the serum of the mice is tested every 7 days after each immunization. Generally, when the OD450nm value is positive at a serum dilution of 1:10000, it indicates that the immunization has achieved a good effect (Figure 4), and subsequent experiments can be carried out.

[0083] Example 4 Cell Fusion

[0084] Cell fusion is the most important step in the hybridoma method. The fusion efficiency directly affects whether specific antibodies can be screened. Take mice with titers that meet the fusion requirements, and boost them with 100 μg of peritoneal antigen 3 days before fusion. Take spleen cells, wash them twice with serum-free and resistance-free DMEM (20 mL) culture medium, resuspend the cells with 2 mL fusion buffer after centrifugation, count and set aside. The day before fusion, myeloma cells are passaged at a ratio of 1:2. On the day of fusion, SP2 / 0 mouse myeloma cells (TCM18, Chinese Academy of Sciences Cell Bank) are collected, the culture medium is removed by centrifugation, and the cells are washed twice with serum-free and resistance-free DMEM (20 mL). After centrifugation, the cells are resuspended with 2 mL fusion buffer, counted and set aside. Take 8×10 spleen cells and SP2 / 0 cells respectively. 7 Transfer cells to a new 50mL centrifuge tube, add 20mL of fusion buffer, and centrifuge twice at a 1:1 ratio (500×g for 5 minutes). Resuspend in 8mL of fusion buffer and place in an electroporator for electrofusion using a BTX ECM2001 fusion instrument. Fusion parameters are alternating current 48V for 40s, pulse voltage 2070V for 30μs, and PF for 7s. Let stand for 10 minutes, then add the entire fusion solution to 600mL of prepared culture medium (75% DMEM + 20% FBS + 1% PS + 2% HAT (50×) + 1% Glu + 1% OPI) and incubate at 37°C for 2 hours.

[0085] After mixing, plate 42 wells with 150 μL per well. On the fourth day of fusion, replace the medium with HT-supplemented medium. On the seventh day, collect the supernatant for testing.

[0086] Example 5 Monoclonal Cell Screening

[0087] The screening of monoclonal cells is mainly carried out by detecting the supernatant of hybridoma cells. The detection methods mainly include ELISA and FACS. ELISA mainly tests the affinity of hybridoma supernatant to the immune TRPA1-nanodisc antigen. After coating 2μg / mL TRPA1-nanodisc antigen and blocking with 2% BAS, hybridoma supernatant is added and incubated for 1 hour, goat anti-mouse HRP is added and incubated for 1 hour, color development solution is added for 10 minutes, and then stop solution is added to terminate the reaction. The absorbance value at OD450nm is tested to test the titer of serum. After initial screening by ELISA, positive hybridoma supernatants are screened and then flow cytometry screening is performed. The flow cytometer used is Beckman Coulter CytoFLEX. The cell line used for screening is HEK293S-TRPA1. The cells to be tested are suspended cultured and passaged according to the appropriate ratio. 10μl of cells are mixed with 10μl of trypan blue and added to a hemocytometer for counting. 5×10 5 Cells were plated in a 96-well V-shaped dilution plate. Centrifugation was performed at 1500 rpm for 5 minutes, and the plate was flicked to remove the culture medium. The supernatant of an ELISA-positive hybridoma was added and incubated at 4°C for 1 hour. After washing three times with PBS, a 1:200 dilution of secondary antibody (Jackson, Allophycocyanin-AffiniPure F(ab')2Fragment Donkey Anti-Mouse IgG (H+L)) was added. The cells were incubated at 4°C for 30 minutes and washed three times with PBS. Finally, 150 μl of PBS was added to each well to resuspend the cells and transfer them to a 1.5 mL EP tube or directly plate the cells. The cells were detected using a Beckman CytoFLEX assay and the MFI values ​​were output using Cytoexpert. The data were analyzed by comparing the difference with the positive and negative controls. Positive results were obtained by two rounds of subcloning using the limiting dilution method, and positive monoclonal cells were finally screened, as shown in Figures 5a-5h and 6a-6h.

[0088] Example 6 Monoclonal Antibody Preparation

[0089] The positive monoclonal cells were expanded and cultured until the density reached 3×10 5 ~5×10 5 / mL was inoculated into 50mL of serum-free culture medium for suspension culture. The cell mass of the hybridoma was counted every day. The culture was generally carried out for 4-5 days. The supernatant was collected by centrifugation and 200μL of Protein A filler was added. The mixture was flipped and bound for 2-3h. Affinity purification was performed (PBS pH 7.3 equilibrium solution, glycine pH 3.0 elution solution). The purified antibody was dialyzed against PBS to replace the buffer, the concentration was determined, and the purity of the antibody was tested by SDS-PAGE. The SDS-PAGE results are shown in Figure 7.

[0090] Example 7 Monoclonal Antibody Functional Test

[0091] The antibody function test is mainly carried out by detecting calcium flow with an enzyme-linked microplate reader. The FlexStation3 multi-function plate reader produced by Molecular Devices in the United States can effectively read the changes in fluorescence intensity caused by calcium ions flowing into the cell through ion channels. The HEK293T cell line stably expressing TRPA1 was pre-plated into 96-well plates for adherent culture and grown to an appropriate density. The culture medium was discarded, and a membrane-permeable calcium ion concentration indicator (FLIPR Calcium 5 Assay kit) prepared with extracellular buffer and antibody solutions of different concentrations (the concentration gradient was adjusted according to needs) were added at the same time. The cells were incubated in an incubator for at least 60 minutes. The positive small molecule was A967079, a TRPA1 channel-specific inhibitor. The blank control was the extracellular buffer as a solvent, and the extracellular buffer containing the corresponding concentration of the co-solvent DMSO (10% DMSO / HBSS or PBS). The whole process was kept away from light. After the incubation was completed, the 96-well plate was placed on the FlexStation3 multi-function plate reader (Molecular Devices). The TRPA1 agonist AITC (propyl isothiocyanate) sample plate was loaded into the sampler of the microplate reader in advance and the concentration was set to 400 μM (the final concentration in the well was 100 μM). The test program was set. After each column of agonists was added, the changes in the fluorescence intensity of the cells in this column were immediately monitored for at least two minutes. After all the well plates were tested, the fluorescence increase of all wells before and after the addition of the agonist was calculated based on the quantified fluorescence intensity change curve output by the machine, and the fluorescence increase was normalized with the corresponding blank control well to obtain the inhibition rate under different concentrations of the antibody. Two replicate wells were set for each antibody concentration, and then the inhibition curve of the logarithm of the concentration against the inhibition rate was obtained by curve fitting (Y=100 / (1+10^((LogIC50-X)*HillSlope))) in GraphPad, and the half-inhibitory concentration IC of the antibody inhibition channel was calculated. 50 , in order to determine the inhibitory effect of the antibody on TRPA1, the results are shown in Figures 8a-8i. The IC 50 They are 0.65 μM, 1.54 μM, 0.70 μM, 0.86 μM, 0.56 μM, 0.66 μM, 1.18 μM and 0.56 μM respectively.

[0092] Example 8 Antibody Sequence Acquisition

[0093] Several monoclonal antibodies were screened and hybridoma cells were recovered. When the density reached 5×10 6 / mL, centrifuged to collect cells, and extracted monoclonal cell RNA using the FastPure Cell / Tissue Total RNA Isolation Kit from Novagen. The extracted RNA was then used as a template to amplify the variable regions of the monoclonal antibody VH and VL chains using the Novagen HiScript-TS 5' / 3' RACE Kit. After gel electrophoresis, the target bands were excised and recovered, and the concentration was determined. 2× Taq Master Mix was then used to add base A at both ends of VH and VL. Finally, TaKaRa's pMDTM18-T Vector Cloning Kit was used to complete vector construction and plating. After plating, monoclonal colonies were picked for culture and sent for sequencing. After alignment of the sequenced sequences, the VH and VL sequences were obtained (as shown in Tables 1-8).

[0094] Table 1 33E8 antibody V sequence information

[0095] Table 2 20D5 antibody V sequence information

[0096] Table 3 38B3 antibody V sequence information

[0097] Table 4 20E6 antibody V sequence information

[0098] Table 5 4A11 antibody V sequence information

[0099] Table 6 39C6 antibody V sequence information

[0100] Table 7 33E6 antibody V sequence information

[0101] Table 8 36F6 antibody V sequence information

[0102] Among them, the CDR sequences of the heavy chain variable regions and light chain variable regions of the above-mentioned antibodies 33E8, 20D5, 38B3, 20E6 and 33E6 are very similar. The present invention summarizes their CDR sequences as follows:

[0103] SEQ ID NO:56: GYX1FTDYW, wherein X1 is T or K.

[0104] SEQ ID NO:57:IDSSDSYX2, wherein X2 is S or T.

[0105] SEQ ID NO:58: X3RGDNSGYAI, wherein X3 is A or V.

[0106] SEQ ID NO:59: QTIVHX4TGNTY, wherein X4 is S or T.

[0107] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A TRPA1 antibody, characterized in that, It includes a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO:56 - 58 respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NO:59, SEQ ID NO:9 and SEQ ID NO:10 respectively; or, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO:26 - 28 respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NO:29 - 31 respectively; or, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO:36 - 38 respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NO:39 - 41 respectively; or, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO:51 - 53 respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NO:54, SEQ ID NO:35 and SEQ ID NO:55 respectively.

2. A TRPA1 antibody, characterized in that, It includes a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO:5 - 7 respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NO:8 - 10 respectively; or, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO:17 - 19 respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NO:8 - 10 respectively; or, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO:17 - 19 respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NO:46, SEQ ID NO:9 and SEQ ID NO:10 respectively.

3. The TRPA1 antibody according to claim 1 or 2, characterized in that, The heavy chain variable region further includes a heavy chain variable region framework region HFWR, and / or, the light chain variable region further includes a light chain variable region framework region LFWR, wherein the HFWR is a human - derived or mouse - derived heavy chain variable region framework region, and the LFWR is a human - derived or mouse - derived light chain variable region framework region of an antibody; Preferably, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4 or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 4; and / or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3 shown or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 3; or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14 or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 14; and / or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13 or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 13; or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14 or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 14; and / or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16 or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 16; or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14 or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 14; and / or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 21 or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 21; or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25 or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 25; and / or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24 or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 24; or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35 or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 35; and / or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34 or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 34; or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45 or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 45; and / or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 44 or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 44; or, The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 50 or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 50; and / or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 49 or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 49; More preferably, the TRPA1 antibody further comprises a heavy chain constant region and a light chain constant region; Even more preferably, the heavy chain constant region of the TRPA1 antibody is a human or murine heavy chain constant region; the light chain constant region of the TRPA1 antibody is a human or murine light chain constant region.

4. The TRPA1 antibody according to any one of claims 1-3, characterized in that The TRPA1 antibody is any one of the following antibody forms: (a) A complete immunoglobulin molecule; (b) A scFv; (c) A fusion protein comprising a scFv; (d) A Fab fragment; (e) A Fab′ fragment; (f) A F(ab)2; Alternatively, the TRPA1 antibody is a monoclonal antibody or a polyclonal antibody; Alternatively, the TRPA1 antibody is a humanized antibody or a bispecific antibody.

5. A chimeric antigen receptor comprising the TRPA1 antibody according to any one of claims 1-4.

6. An isolated nucleic acid encoding the TRPA1 antibody according to any one of claims 1-4, or the chimeric antigen receptor according to claim 5; Preferably, the nucleic acid comprises the polynucleotide sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2; or, comprises the polynucleotide sequence shown in SEQ ID NO: 11 and / or SEQ ID NO: 12; or, comprises the polynucleotide sequence shown in SEQ ID NO: 15 and / or SEQ ID NO: 12; or, comprises the polynucleotide sequence shown in SEQ ID NO: 20 and / or SEQ ID NO: 12; or, comprises the polynucleotide sequence shown in SEQ ID NO: 22 and / or SEQ ID NO: 23; or, comprises the polynucleotide sequence shown in SEQ ID NO: 32 and / or SEQ ID NO: 33; or, comprises the polynucleotide sequence shown in SEQ ID NO: 42 and / or SEQ ID NO: 43; or, comprises the polynucleotide sequences shown in SEQ ID NO: 47 and SEQ ID NO:

48.

7. A recombinant expression vector comprising the isolated nucleic acid according to claim 6.

8. A transformant comprising the recombinant expression vector according to claim 7 in a host cell.

9. A method for preparing a TRPA1 antibody, which comprises culturing the transformant according to claim 8 and obtaining the TRPA1 antibody from the culture.

10. An antibody-drug conjugate comprising a cytotoxic agent and the TRPA1 antibody according to any one of claims 1-4.

11. A pharmaceutical composition comprising a TRPA1 antibody as described in any one of claims 1-4, a chimeric antigen receptor as described in claim 5, or an antibody-drug conjugate as described in claim 10, and a pharmaceutically acceptable carrier.

12. Use of a TRPA1 antibody as described in any one of claims 1-4, a chimeric antigen receptor as described in claim 5, an isolated nucleic acid as described in claim 6, a recombinant expression vector as described in claim 7, a transformant as described in claim 8, an antibody-drug conjugate as described in claim 10, or a pharmaceutical composition as described in claim 11 in the preparation of a medicament for treating pain; Preferably, the pain is caused by TRPA1 activation; the target of the medicament is TRPA1.

13. A kit comprising a TRPA1 antibody as described in any one of claims 1-4, a chimeric antigen receptor as described in claim 5, an antibody-drug conjugate as described in claim 10, and / or a pharmaceutical composition as described in claim 11.

14. A method for detecting TRPA1, comprising contacting a sample with a TRPA1 antibody as described in any one of claims 1-4, a chimeric antigen receptor as described in claim 5, an antibody-drug conjugate as described in claim 10, a pharmaceutical composition as described in claim 11, and / or a kit as described in claim 13; Preferably, the detection is for non-diagnostic purposes.

15. A method for treating and / or preventing pain, the method comprising administering to a patient in need a therapeutically effective amount of a TRPA1 antibody as described in any one of claims 1-4, a chimeric antigen receptor as described in claim 5, an isolated nucleic acid as described in claim 6, a recombinant expression vector as described in claim 7, a transformant as described in claim 8, an antibody-drug conjugate as described in claim 10, a pharmaceutical composition as described in claim 11, and / or a kit as described in claim 13; Preferably, the pain is caused by TRPA1 activation; the target of the medicament is TRPA1.

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