TRPA1 antibody as well as preparation method and application thereof

Abstract

The invention discloses a TRPA1 antibody as well as a preparation method and application thereof, the TRPA1 antibody comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of which the amino acid sequences are respectively shown as SEQ ID NO: 36-38; and / or the light chain variable region comprises an LCDR1, an LCDR2 and an LCDR3 of which the amino acid sequences are respectively shown as SEQ ID NO: 39-41. The TRPA1 antibody provided by the invention has relatively high affinity to TRPA1, and the screened TRPA1 antibody has an obvious inhibition effect on a TRPA1 channel activated by AITC, and IC50 of the TRPA1 antibody is 0.66 [mu] M.

Description

[0001] This patent application is a divisional application of the patent application with the application number 2024100844916. The filing date of this patent application is January 19, 2024, and its invention title is "A TRPA1 Antibody, a Method for Preparing the Same, and Applications". Technical Field

[0002] The present invention belongs to the field of biotechnology, and particularly relates to a TRPA1 antibody, a method for preparing the same, and applications. Background Art

[0003] Transient receptor potential ankyrin subtype 1 protein (TRPA1) is a non-selective cation channel permeable to Ca 2+ , Na + and K + . TRPA1 is present in Aδ fiber and C fiber nociceptive sensory neuron subsets and other sensory cells including epithelial cells. In primary sensory neurons, Ca 2+ and Na + flow into the cell through TRPA1, causing membrane depolarization, action potential firing, and neurotransmitter release at peripheral and central nerve projections.

[0004] In addition to being activated by cysteine- and lysine-reactive electrophiles and oxidants, TRPA1 can also be indirectly activated by pro-inflammatory agents through the phospholipase C signaling pathway, where cytoplasmic Ca 2+ is an important regulator of channel gating.

[0005] Multiple lines of evidence suggest that TRPA1 is involved in pain sensation. TRPA1 is expressed in sensory neurons and co-localizes with pain markers such as TRPV1 and bradykinin receptors. Its expression increases in animal models of inflammatory and neuropathic pain and in DRG neurons of human avulsion injuries. TRPA1 agonists cause neurotransmitter release, pain, and inflammation in rodents and humans, and endogenous agonists such as 4-HNE are elevated in human pathological conditions. In several animal models, gene knockout attenuates agonist sensitivity and antagonist treatment alleviates pain. TRPA1 is thought to play a role in many sensory modalities, including chemical nociception, mechanical nociception, and cold nociception.

[0006] In recent years, opioid addiction and overdose have been increasing at an alarming rate. There is a greater need today than ever to develop new non-opioid pain medications with no potential for addiction. Since TRPA1 is involved in 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.

[0007] Research on the indications of TRPA1 for treating pain, skin diseases and respiratory diseases such as asthma is relatively hot. Small molecule antagonists of TRPA1 mainly include structures such as xanthine, sulfonamide, oxadiazolone, oxadiazole and carboxamide. As of 2019, five small molecules have entered clinical trials but have all been terminated due to poor pharmacokinetic properties. Compared with small molecule drugs, macromolecular antibody drugs have obvious advantages. Their limitations in central nervous system (CNS) permeability (when targeting peripheral treatment), low immunogenicity, high selectivity and favorable half-life make the antibody research of TRPA1 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 performance such as affinity, specificity and functionality is poor. Summary of the Invention

[0008] In order to solve the problem of the lack of functionally active antibodies targeting TRPA1 in the prior art, the present invention provides a TRPA1 antibody, a method for preparing the same and an application thereof. The immunogen used in the preparation process is a protein purified by nanodisc packaging. The antibody is prepared by hybridoma technology. The screening of affinity is mainly ELISA and FACS, and then combined with flexstation3 to read the fluorescence intensity change caused by calcium ions flowing into the cells through ion channels, so as to screen out antibodies with both affinity and functionality. Through affinity testing, functional testing, sequence acquisition and other work, several therapeutic antibodies with TRPA1 antagonistic function are obtained.

[0009] In order to solve the above technical problems, in the first aspect of the present invention, there is provided a TRPA1 antibody, the TRPA1 antibody comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences respectively shown in SEQ ID NO: 56-58; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences respectively shown in SEQ ID NO: 59, SEQ ID NO: 9 and SEQ ID NO: 10; or,

[0010] the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences respectively shown in SEQ ID NO: 26-28; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences respectively shown in SEQ ID NO: 29-31; or,

[0011] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 36 - 38 respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 39 - 41 respectively; or,

[0012] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 51 - 53 respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 54, SEQ ID NO: 35 and SEQ ID NO: 55 respectively.

[0013] In some embodiments of the present invention, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 5 - 7 respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 8 - 10 respectively; or,

[0014] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 17 - 19 respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 8 - 10 respectively; or,

[0015] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 17 - 19 respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 46, SEQ ID NO: 9 and SEQ ID NO: 10 respectively.

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

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

[0018] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% 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 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 13; or,

[0019] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% 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 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence 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 has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% 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 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 21; or,

[0021] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% 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 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 24; or,

[0022] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% 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 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 34; or,

[0023] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% 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 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 44; or,

[0024] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 50 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% 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 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence 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 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.

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

[0028] (a) A complete immunoglobulin molecule;

[0029] (b) A scFv;

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

[0031] (d) A Fab fragment;

[0032] (e) A Fab′ fragment;

[0033] (f) An 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] To solve the above technical problems, a second aspect of the present invention provides a chimeric antigen receptor comprising the TRPA1 antibody as described in the first aspect of the present invention.

[0037] To solve the above technical problems, a 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 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 sequence shown in SEQ ID NO: 47 and SEQ ID NO: 48.

[0039] To solve the above technical problems, the fourth aspect of the present invention provides a recombinant expression vector, which comprises the isolated nucleic acid as described in the third aspect of the present invention.

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

[0041] 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] 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 as described in the first aspect of the present invention.

[0043] 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] 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 caused by TRPA1 activation; the target of the medicament is TRPA1.

[0046] To solve the above technical problems, the tenth aspect of the present invention provides a kit, 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, 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] 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 a TRPA1 antibody as described in the first aspect of the present invention, a chimeric antigen receptor as described in the second aspect of the present invention, an antibody-drug conjugate as described in the seventh aspect of the present invention, a pharmaceutical composition as described in the eighth aspect of the present invention, and / or a kit as 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] To solve the above technical problems, the twelfth aspect of the present invention provides a method for treating and / or preventing pain, the method comprising administering a therapeutically effective amount of a TRPA1 antibody as described in the first aspect of the present invention, a chimeric antigen receptor as described in the second aspect of the present invention, an isolated nucleic acid as described in the third aspect of the present invention, a recombinant expression vector as described in the fourth aspect of the present invention, a transformant as described in the fifth aspect of the present invention, an antibody-drug conjugate as described in the seventh aspect of the present invention, a pharmaceutical composition as described in the eighth aspect of the present invention, and / or a kit as described in the tenth aspect of the present invention to a patient in need thereof.

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

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

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

[0053] On the basis of conforming to common general knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

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

[0055] The positive and progressive effects of the present invention are as follows: the anti-TRPA1 antibody provided by the present invention has a relatively high affinity for TRPA1, and the screened antibody has an obvious inhibitory effect on the AITC-activated TRPA1 channel, with an IC50 lower than 1.54 μM, and the lowest can reach 0.56 μM. Brief Description of the Drawings

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

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

[0058] Figure 3 It is the result of the purification of the TRPA1-Nanodisc protein.

[0059] Figure 4 It is the result of the detection of the serum titers of the 4th and 5th immunizations of the mice immunized with the TRPA1-Nanodisc protein.

[0060] Figures 5a - 5h It is the result of the ELISA detection of 8 antibodies obtained by screening.

[0061] Figures 6a - 6h It is the result of the FACS detection of 8 antibodies obtained by screening.

[0062] Figure 7 It is the SDS-PAGE diagram of the purified 8 antibodies obtained by screening.

[0063] Figures 8a - 8i It is the inhibitory effect of the positive small molecule A967079 and 8 antibodies screened on the TRPA1 channel current. Detailed Implementation Modes

[0064] The present invention will be further illustrated below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product instructions.

[0065] Example 1 Preparation of Recombinant Baculovirus

[0066] 1.1 Obtaining the Recombinant Baculovirus Plasmid

[0067] Through heat shock transformation, the recombinant pEGBacMam plasmid containing the target gene (synthesized by Nanjing Qingke Biotechnology Co., Ltd., carrying EcoR1 and Not1 restriction enzyme sites, ligated and recombined into the pEGBacMam plasmid, the recombinant pEGBacMam plasmid containing TRPA1 and MBP is as Figure 1 shown, and the recombinant pET 28a plasmid containing MSP2N2 is as Figure 2It was introduced into Escherichia coli DH10Bac competent cells (Shanghai Weidi), and cultured at 37 °C for 48 - 72 hours on 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 colonies were selected and transferred to 5 mL of LB liquid medium containing three antibiotics (50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline), and cultured at 37 °C under the condition of 200 rpm for 12 - 16 hours, and the recombinant baculovirus plasmid was extracted.

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

[0069]

[0070] TRPA1 base sequence (SEQ ID NO: 61):

[0071]

[0072] MSP2N2 amino acid sequence (SEQ ID NO: 62):

[0073] STFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQGTPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQE

[0074] MSP2N2 base sequence (SEQ ID NO: 63):

[0075] TCTACCTTCAGTAAACTTCGCGAACAACTGGGCCCCGTGACGCAGGAATTCTGGGACAACCTGGAAAAAGAAACCGAGGGACTGCGTCAGGAAATGTCCAAAGATTTAGAAGAGGTGAAGGCCAAGGTTCAGCCATATCTCGATGACTTTCAGAAAAAATGGCAGGAAGAGATGGAATTATATCGTCAAAAGGTGGAACCGCTGCGTGCGGAACTGCAAGAGGGGGCACGCCAAAAACTCCATGAGCTCCAAGAGAAGCTCAGCCCATTAGGCGAAGAAATGCGCGATCGCGCCCGTGCACATGTTGATGCACTCCGGACTCATTTGGCGCCGTATTCGGATGAACTTCGCCAGCGTTTGGCCGCACGTCTCGAGGCGCTGAAAGAAAACGGGGGTGCCCGCTTGGCTGAGTACCACGCGAAAGCGACAGAACACCTGAGCACCTTGAGCGAAAAAGCGAAACCGGCGCTGGAAGATCTACGCCAGGGCTTATTGCCTGTTCTTGAGAGCTTTAAAGTCAGTTTTCTGTCAGCTCTGGAAGAATATACTAAAAAGCTGAATACCCAGGGTACCCCCGTGACGCAGGAATTCTGGGACAACCTGGAAAAAGAAACCGAGGGACTGCGTCAGGAAATGTCCAAAGATTTAGAAGAGGTGAAGGCCAAGGTTCAGCCATATCTCGATGACTTTCAGAAAAAATGGCAGGAAGAGATGGAATTATATCGTCAAAAGGTGGAACCGCTGCGTGCGGAACTGCAAGAG

[0076] 1.2 Preparation of recombinant baculovirus

[0077] Take a 6-well cell culture plate (Nest), 1×10 per well 6 / 2 mL Gibco™ sf9 insect cells were adherently cultured in a constant temperature and humidity incubator at 27 °C for 30 min. 100 μL of Insect Medium (Sf-900™ III SFM) was added to 10 μL of transfection reagent (Cellfectin™ II), and 100 μL of Insect Medium was added to 5 μg of recombinant baculovirus plasmid. After mixing, it was incubated at room temperature for 20 minutes. The transfection complex was evenly dropped into a 6-well plate and further cultured at 27 °C for 72 hours. It was centrifuged at 6000 rpm at 4 °C for 15 minutes. The supernatant was taken and 2% FBS was added, and it was stored at 4 °C in the dark to obtain the P1 generation recombinant baculovirus.

[0078] Take the P1 generation recombinant baculovirus and infect 2 mL of sf9 insect cells with a cell density of 5×10 5 / mL at 27 °C under adherent culture for 72 hours. It was centrifuged at 6000 rpm at 4 °C for 15 minutes. The supernatant was taken and 2% FBS was added, and it was stored at 4 °C in the dark to obtain the P2 generation recombinant baculovirus.

[0079] Separate 40 mL of sf9 insect cells with a density of 1×10 6 / mL. After culturing for 24 hours, take the P2 generation recombinant baculovirus and infect it at a ratio of 1:100. Culture it at 27 °C and 120 rpm for 96 hours. It was centrifuged at 6000 rpm at 4 °C for 15 minutes. The supernatant was filtered through a 0.22 μm filter device and 2% FBS was added, and it was stored at 4 °C in the dark to obtain the P3 generation recombinant baculovirus.

[0080] Separate 200 mL of sf9 insect cells with a density of 1×10 6 / mL. After culturing for 24 hours, take the P3 generation recombinant baculovirus and infect it at a ratio of 1:100. Culture it at 27 °C and 120 rpm for 96 hours. It was centrifuged at 6000 rpm at 4 °C for 15 minutes. The supernatant was filtered through a 0.22 μm filter device and 2% FBS was added, and it was stored at 4 °C in the dark to obtain the P4 generation recombinant baculovirus.

[0081] Example 2 Protein Purification

[0082] Subculture HEK-293S cells at a concentration of 1.5×10 6 / mL at 37 °C, 8% CO2, 60% humidity, and 120 rpm for 24 h. Take the P4 generation recombinant baculovirus and infect HEK-293S cells at a ratio of 1:10. Add sodium butyrate to the shaker culture for 12 - 18 h, with a final concentration of 10 mM.

[0083] After culturing for 72 hours, centrifuge at 4°C and 6000 rpm for 15 minutes to collect the cells. Resuspend the cell pellet with 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 (Maclean, T819166)), and invert the tube to lyse the membrane at 4°C for 3 hours. After the lysis is completed, centrifuge at 4°C and 40000 rpm for 45 minutes. The supernatant is combined with MBP affinity chromatography resin (NEB, E8021L) and incubated with inversion at 4°C for 2.5 hours. The combined supernatant is passed through a gravity column, and the impurity proteins are eluted with Wash buffer (25 mM HEPES pH 7.4, 150 mM NaCl, 1 mM TCEP, 0.01% LMNG / 0.001% CHS). The target protein of 100 kDa is eluted with Elute buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 0.01% LMNG / 0.001% CHS, 1 mM TCEP, 40 mM Maltose (Adamas, 73054C)), and the target protein is concentrated using an ultrafiltration tube (Millopore, UFC910096). Package according to the molar ratio of TRPA1: MSP2N2: soybean lipid = 1: 3: 200. Calculate the required Soybean Polar Lipid Extract Polar (Avanti). Use a protein loading pipette to aspirate the stock solution (the soybean stock solution is 25 mg / mL), and pipette the soybean stock solution to the bottom of a clean and dry glass tube. Then use a nitrogen evaporator to dry the chloroform in the soybean stock solution in the glass tube, and a little pale yellow residue will form at the bottom of the glass tube. Add SEC buffer and sonicate for 10 min. During this period, the liquid in the glass tube will first become white and turbid, then gradually clear, and finally become a pale yellow solution. Add TRPA1, MSP2N2, and soybean to the EP tube according to the ratio and incubate on ice for 1 h. Subsequently, add 100 mg / mL Bio - beads SM - 2 (Bio - Beads™ SM - 2 Resin, Bio - Rad) to remove the detergent in the solution, and invert the tube at 4°C overnight.Remove Bio-beads SM-2 by gravity column filtration, and concentrate the filtered protein solution to a volume of 500 μL; centrifuge at 13,000 rpm for 10 min at 4°C, perform gel filtration chromatography, the gel column model used is Superrose 6 Increase 10 / 300 GL (cytiva), the buffer is SEC buffer (25 mM HEPES, pH 7.4, 150 mM NaCl), collect the protein sample, measure the A280 concentration, and perform SDS-PAGE gel electrophoresis to detect the size and purity of the target protein. The SDS-PAGE gel electrophoresis pattern is as follows. Figure 3 shown.

[0084] Example 3 Animal Immunization

[0085] Immunizing animals (usually mice) with an antigen is the first and crucial step in the preparation of monoclonal antibodies. Whether the animal has a good immune response to the antigen and can produce antibodies with high titer and good specificity directly determines the difficulty of screening monoclonal hybridoma cell lines in the later stage and the effectiveness of the obtained antibodies.

[0086] For the antigen used in immunization, we use the protein produced by the protein preparation method of TRPA1-nanodisc in Examples 1 and 2. The immunized animals are balb / c female mice at 6-8 weeks old. The initial immunization dose of TRPA1-nanodics antigen is 100 μg protein / mouse, which is mixed with Freund's complete adjuvant at a volume ratio of 1:1. The insufficient part of the antigen volume is replaced by the purification buffer of the corresponding protein. After emulsification with a shaker, the animals are immunized by multiple subcutaneous injections at multiple points on the back routinely. After that, immunization is carried out every 14 days. The antigen dose is half of the initial immunization dose (50 μg / mouse), which is mixed with Freund's incomplete adjuvant at a volume ratio of 1:1. The insufficient part of the antigen volume is replaced by the purification buffer of the corresponding protein. After emulsification, the animals are immunized by multiple subcutaneous injections at multiple points on the back routinely. Usually, one week after the third immunization, the blood of the mice is taken. After separating the serum, the titer of the serum is tested by ELISA (coating with TRPA1-nanodics antigen, incubating with the serum diluted in gradient (starting from 1:100, diluted 10 times by 3-fold) after blocking with 2% BAS for 1 h, incubating with goat anti-mouse HRP (SA00001-1, proteintech) for 1 h, adding the chromogenic solution to develop color for 10 min, and then adding the stop solution to terminate the reaction, and measuring the absorbance value at OD450nm). After that, the serum of the mice is tested every 7 days after each boost immunization. Generally, when the OD450nm value is positive at a serum dilution of 1:10,000, it indicates that the immunization has achieved a good effect ( Figure 4 ) and subsequent experiments can be carried out.

[0087] Example 4 Cell Fusion

[0088] Cell fusion is the most important step in the hybridoma method. The efficiency of fusion directly affects whether specific antibodies can be screened. Select mice with antibody titers meeting the fusion requirements, boost them with 100 μg of peritoneal antigen 3 days before fusion, take spleen cells, wash them twice with serum-free and antibiotic-free DMEM (20 mL) medium, resuspend the cells in 2 mL of fusion buffer after centrifugation, and count for standby. Passage the myeloma cells at a ratio of 1:2 one day before fusion. On the day of fusion, collect SP2 / 0 mouse myeloma cells (Cell Bank of the Chinese Academy of Sciences, TCM18), centrifuge to remove the medium, wash them twice with serum-free and antibiotic-free DMEM (20 mL), resuspend the cells in 2 mL of fusion buffer after centrifugation, count, and take for standby. Take 8×10 7 cells each of spleen cells and SP2 / 0 into a new 50 mL centrifuge tube, add 20 mL of fusion buffer, centrifuge together at a ratio of 1:1 (centrifuge at 500×g for 5 minutes) twice, then resuspend in 8 mL of fusion buffer and add to the electroporation cuvette for electrofusion. The electrofusion instrument used is the BTX ECM2001 fusion instrument, and the fusion parameters are alternating current 48 V, 40 s, pulse voltage: 2070 V, 30 μs, PF, 7 S. Let it stand for 10 min, take all the fusion liquid and add it to the pre-prepared 600 mL medium, where the main components of the medium are (75% DMEM + 20% FBS + 1% PS + 2% HAT (50×) + 1% Gln + 1% OPI), and incubate at 37°C for 2 h. After mixing, plate on 42 plates, 150 μL per well. On the fourth day after fusion, replace the medium completely with the medium supplemented with HT, and take the supernatant for testing on the 7th day.

[0089] Example 5 Screening of Monoclonal Cells

[0090] The screening of monoclonal cells is mainly through detecting the supernatant of hybridoma cells. The methods used for detection mainly include ELISA and FACS. ELISA is mainly used to test the affinity between the hybridoma supernatant and the immunized TRPA1-nanodisc antigen. Coat 2 μg / mL of TRPA1-nanodisc antigen, block with 2% BAS, add the hybridoma supernatant and incubate for 1 h, add goat anti-mouse HRP and incubate for 1 h, add the chromogenic solution to develop color for 10 min, then add the stop solution to terminate the reaction, and test the absorbance value at OD450nm) to test the serum titer through ELISA primary screening. Screen the positive hybridoma supernatants by flow cytometry. The flow cytometer used is the beckmancoulter CytoFLEX, and the cell line used for screening is HEK293S-TRPA1. The cells to be tested are cultured in suspension, passaged at an appropriate ratio, take 10 µl of cells and mix with 10 µl of trypan blue, add to the hemocytometer, count, and add 5×10 5Cells were placed in a 96-well V-bottom dilution plate. Centrifuge at 1500 rpm for 5 minutes, tap the plate to remove the medium, add the hybridoma supernatant positive for ELISA, incubate at 4°C for 1 hour, wash 3 times with PBS, add the secondary antibody diluted 1:200 (jackson, Allophycocyanin-AffiniPure F(ab')2 Fragment Donkey Anti-Mouse lgG (H+L)), incubate at 4°C for 30 minutes, wash 3 times with PBS, finally resuspend the cells in 150 µl of PBS per well, transfer to a 1.5 mL EP tube, or directly load the sample on the microplate, detect with Beckman CytoFLEX, output the MFI value using Cytoexpert, analyze the data by comparing with the positive and negative controls to obtain positive results, and perform two rounds of subcloning by the method of limiting dilution, and finally screen to obtain positive monoclonal cells, as Figures 5a - 5h and Figures 6a - 6h shown.

[0091] Example 6 Preparation of Monoclonal Antibody

[0092] The obtained positive monoclonal cells were expanded in culture. When the density reached 3×10 5 ~5×10 5 / mL, they were inoculated into 50 mL of serum-free medium for suspension culture. The cell count of the hybridoma was counted every day. Generally, the culture was carried out for 4-5 days. The supernatant was collected by centrifugation, 200 μL of Protein A packing material was added, and it was inverted and combined for 2-3 h for affinity purification (PBS pH 7.3 equilibration buffer, glycine pH 3.0 elution buffer). The purified antibody was dialyzed with PBS to replace the buffer, the concentration was measured, and the purity of the antibody was detected by SDS-PAGE. The SDS-PAGE results are as Figure 7 shown.

[0093] Example 7 Function Test of Monoclonal Antibody

[0094] The test of antibody function is mainly carried out by detecting calcium flux with a microplate reader. The FlexStation3 multifunctional microplate reader produced by Molecular Devices in the United States can effectively read the change in fluorescence intensity caused by calcium ions flowing into cells through ion channels. The HEK293T cell line stably expressing TRPA1 was seeded onto a 96-well plate in advance for adherent culture. When it grew 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 at different concentrations (the concentration gradient was adjusted according to requirements) were added simultaneously. The cells were incubated in an incubator for at least 60 minutes. The positive small molecule was A967079, a specific inhibitor of the TRPA1 channel. The blank control was the extracellular buffer as the solvent, and the extracellular buffer containing the corresponding concentration of co-solvent DMSO (10% DMSO / HBSS or PBS). The whole process was placed in the dark. After incubation, the 96-well plate was placed on the FlexStation3 multifunctional microplate reader (Molecular Devices). The sample plate of the TRPA1 agonist AITC (propyl isothiocyanate) 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. Immediately after adding each column of the agonist, the change in intracellular fluorescence intensity of this column of cells was monitored for at least two minutes. After all the well plates were tested, according to the quantitative fluorescence intensity change curve output by the machine, the fluorescence increment before and after adding the agonist in all wells was calculated, and it was normalized with the fluorescence increment of the corresponding blank control well to obtain the inhibition rate under the action of antibodies at different concentrations. 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 50 of the antibody inhibiting the channel was calculated to judge the inhibitory effect of the antibody on TRPA1. The results are shown in Figures 8a - 8i . The IC 50 values of 33E8, 20D5, 38B3, 20E6, 4A11, 39C6, 33E6, and 36F6 were 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.

[0095] Example 8 Obtaining Antibody Sequences

[0096] For several monoclonal antibodies obtained by screening, the hybridoma cells were revived. When the density reached 5×10 6 / mL, centrifuge to collect cells, extract the RNA of monoclonal cells according to the FastPure Cell / Tissue Total RNA Isolation Kit of Novoprotein. After that, using the extracted RNA as a template, amplify the variable regions of the VH and VL chains of the monoclonal antibody with the HiScript-TS 5' / 3' RACE Kit of Novoprotein. After gel electrophoresis, cut and recover the target band and measure its concentration. Then, add base A to both ends of VH and VL using 2×Taq MasterMix. Finally, complete the vector construction with the pMDTM18-T Vector Cloning Kit of TaKaRa, coat, pick monoclonal colonies for culture, send for sequencing, and obtain the sequences of VH and VL after aligning the sequenced sequences (as shown in Table 1-8).

[0097] Table 1 V sequence information of 33E8 antibody

[0098]

[0099] Table 2 V sequence information of 20D5 antibody

[0100]

[0101] Table 3 V sequence information of 38B3 antibody

[0102]

[0103] Table 4 V sequence information of 20E6 antibody

[0104]

[0105] Table 5 V sequence information of 4A11 antibody

[0106]

[0107] Table 6 V sequence information of 39C6 antibody

[0108]

[0109] Table 7 V sequence information of 33E6 antibody

[0110]

[0111] Table 8 V sequence information of 36F6 antibody

[0112]

[0113] 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, and the CDR sequences thereof are summarized by the present invention as follows:

[0114] SEQ ID NO: 56: GYX1FTDYW, where X1 is T or K.

[0115] SEQ ID NO: 57: IDSSDSYX2, where X2 is S or T.

[0116] SEQ ID NO: 58: X3RGDNSGYAI, where X3 is A or V.

[0117] SEQ ID NO: 59: QTIVHX4TGNTY, where X4 is S or T.

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: 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.

2. The TRPA1 antibody according to claim 1, 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: 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; More preferably, the TRPA1 antibody further includes a heavy chain constant region and a light chain constant region; Further preferably, the heavy chain constant region of the TRPA1 antibody is a human - derived or mouse - derived heavy chain constant region; the light chain constant region of the TRPA1 antibody is a human - derived or mouse - derived light chain constant region.

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

4. A chimeric antigen receptor, which comprises the TRPA1 antibody according to any one of claims 1 - 3.

5. An isolated nucleic acid, which encodes the TRPA1 antibody according to any one of claims 1 - 3, or the chimeric antigen receptor according to claim 4; Preferably, the nucleic acid comprises the polynucleotide sequence shown in SEQ ID NO: 32 and / or as shown in SEQ ID NO:

33.

6. A recombinant expression vector, which comprises the isolated nucleic acid according to claim 5.

7. A transformant, which comprises the recombinant expression vector according to claim 6 in a host cell.

8. A method for preparing a TRPA1 antibody, which includes culturing the transformant according to claim 7 and obtaining the TRPA1 antibody from the culture.

9. An antibody - drug conjugate, which comprises a cytotoxic agent and the TRPA1 antibody according to any one of claims 1 - 3.

10. A pharmaceutical composition comprising a TRPA1 antibody as described in any one of claims 1-3, a chimeric antigen receptor as described in claim 4, or an antibody-drug conjugate as described in claim 9, and a pharmaceutically acceptable carrier.

11. Use of a TRPA1 antibody as described in any one of claims 1-3, a chimeric antigen receptor as described in claim 4, a separated nucleic acid as described in claim 5, a recombinant expression vector as described in claim 6, a transformant as described in claim 7, an antibody-drug conjugate as described in claim 9, or a pharmaceutical composition as described in claim 10 in the preparation of a medicament for treating pain; Preferably, the pain is caused by TRPA1 activation; the target of the medicament is TRPA1.

12. A kit comprising a TRPA1 antibody as described in any one of claims 1-3, a chimeric antigen receptor as described in claim 4, an antibody-drug conjugate as described in claim 9, and / or a pharmaceutical composition as described in claim 10.

13. A method for detecting TRPA1, comprising contacting a sample with a TRPA1 antibody as described in any one of claims 1-3, a chimeric antigen receptor as described in claim 4, an antibody-drug conjugate as described in claim 9, a pharmaceutical composition as described in claim 10, and / or a kit as described in claim 12; Preferably, the detection is for non-diagnostic purposes.

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