TRPV1 antibody, and preparation method therefor and use thereof

Abstract

Provided are a TRPV1 antibody, and a preparation method therefor and a use thereof. The antibody or a variant thereof comprises 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 as set forth in SEQ ID NOs: 11-13, respectively; and / or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as set forth in SEQ ID NOs: 14-16, respectively; or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as set forth in SEQ ID NOs: 21-23, respectively; and / or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as set forth in SEQ ID NOs: 24-26, respectively; or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as set forth in SEQ ID NOs: 31-33, respectively; and / or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as set forth in SEQ ID NOs: 34-36, respectively. The provided TRPV1 antibody has a relatively high affinity for TRPV1 and has a significant inhibitory effect on TRPV1 channels.

Description

TRPV1 antibody, preparation method and application thereof Technical Field

[0001] The present invention relates to the field of immunotherapy, and in particular to a TRPV1 antibody, a preparation method and application thereof. Background Art

[0002] Transient receptor potential vanilloid subfamily 1 (TRPV1) channel is a member of transient receptor potential (TRP) channel. It is a ligand-gated non-selective cation channel. It causes changes in ion concentration after binding with ligand. When TRPV1 is activated, it can cause Ca 2+ , K + Mg 2+ and Na + Isocation influx, but for Ca 2+ and Mg 2+ TRPV1 has a relatively high selectivity, approximately 5-10 times that of other cations. Upon activation, TRPV1 increases intracellular cation concentrations, causing corresponding physiological and pathological changes. TRPV1 is widely distributed throughout the central nervous system, peripheral nervous system, respiratory system, digestive system, cardiovascular and urinary system, and other systems. Recent studies have revealed that the TRPV1 channel plays a crucial role in the development and progression of pathological pain, making it a promising new target for the treatment of pathological pain.

[0003] TRPV1 channels have always been the main TRP channels for developing new drugs to treat pain, but few analgesics targeting TRPV1 channels have actually been used in clinical practice. Many preclinical and clinical studies have shown that different types of TRPV1 channel antagonists, such as AMG-517 and AZD1386, will increase body temperature, and this adverse reaction is the main reason hindering their clinical development. Due to the relatively large side effects of small molecule drugs, and the advantages of large molecule drugs such as limited central nervous system (CNS) permeability (when targeting peripheral treatment), low immunogenicity, high selectivity and favorable half-life, researchers have a strong interest in the development of large molecules. However, since it is a transmembrane protein, only a small part is exposed in the extracellular region, and screening for functionally active antibodies remains a relatively large challenge.

[0004] Summary of the Invention

[0005] In order to solve the problem of the lack of functional active antibodies targeting TRPV1 in the prior art, the present invention mainly provides a TRPV1 antibody, its preparation method and application. The immunogen used in the preparation process is a protein purified by nanodisc packaging. The antibody is prepared using hybridoma technology. The affinity screening is mainly ELISA and FACS, and then combined with flexstation3 to read the changes in fluorescence intensity caused by calcium ions flowing into the cell through ion channels, and screen out antibodies with both affinity and functionality. The TRPV1 antibody provided by the present invention is used to inhibit the TPRV1 pathway activated by stimulation. The stimulation has the same (or similar) mechanism of activating TRPV1 as capsaicin, and through functional testing, it is found that it specifically inhibits the TRPV1 pathway.

[0006] In order to solve the above technical problems, the first aspect of the present invention provides a TRPV1 antibody, wherein the antibody or variant thereof 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: 11-13, respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 14-16, respectively; or,

[0007] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 21-23, respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 24-26, respectively; or,

[0008] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 31-33, respectively; and / or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 34-36, respectively.

[0009] 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.

[0010] In some preferred embodiments of the present invention, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 10; and / or, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 9; or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 20; and / or, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 19; or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 30; and / or, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 29.

[0011] In some more preferred embodiments of the present invention, the antibody or variant thereof further comprises a heavy chain constant region and a light chain constant region.

[0012] In some further more preferred embodiments of the present invention, the antibody heavy chain constant region is a human or mouse antibody heavy chain constant region; and the antibody light chain constant region is a human or mouse antibody light chain constant region.

[0013] In some embodiments of the present invention, the antibody is in any of the following antibody forms:

[0014] (a) a complete immunoglobulin molecule;

[0015] (b) an scFv;

[0016] (c) a fusion protein comprising an scFv;

[0017] (d) a Fab fragment;

[0018] (e) a Fab′ fragment;

[0019] (f) a F(ab)2;

[0020] Alternatively, the antibody is a monoclonal antibody or a polyclonal antibody;

[0021] Alternatively, the antibody is a humanized antibody or a bispecific antibody.

[0022] In order to solve the above technical problems, the second aspect of the present invention provides a chimeric antigen receptor, which comprises the antibody as described in the first aspect of the present invention.

[0023] In order to solve the above technical problems, the third aspect of the present invention provides an isolated nucleic acid encoding the 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.

[0024] In some preferred embodiments of the present invention, the nucleic acid encoding the antibody comprises a polynucleotide sequence as shown in SEQ ID NO:7 and / or as shown in SEQ ID NO:8; or, comprises a polynucleotide sequence as shown in SEQ ID NO:17 and / or as shown in SEQ ID NO:18; or, comprises a polynucleotide sequence as shown in SEQ ID NO:27 and / or as shown in SEQ ID NO:28.

[0025] 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.

[0026] 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.

[0027] In order to solve the above technical problems, the sixth aspect of the present invention provides a method for preparing a TRPV1 antibody, which comprises culturing the transformant as described in the fifth aspect of the present application and obtaining the antibody from the culture.

[0028] 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 antibody according to the first aspect of the present invention.

[0029] In order to solve the above technical problems, the eighth aspect of the present invention provides a pharmaceutical composition, which comprises the 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.

[0030] In order to solve the above technical problems, the ninth aspect of the present invention provides the use of the 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 and / or the pharmaceutical composition as described in the eighth aspect of the present invention in the preparation of a medicament for treating pain.

[0031] In some preferred embodiments of the present invention, the pain is pain caused by TRPV1 activation; and the target of the drug is TRPV1.

[0032] In order to solve the above technical problems, the tenth aspect of the present invention provides a kit, which includes the 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 and / or the pharmaceutical composition described in the eighth aspect of the present invention.

[0033] In order to solve the above technical problems, the eleventh aspect of the present invention provides a method for detecting TRPV1, which comprises contacting a sample with the 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.

[0034] In some preferred embodiments of the present invention, the detection is for non-diagnostic purposes.

[0035] In order to solve the above technical problems, the twelfth aspect of the present invention provides a method for diagnosing, treating and / or preventing pain, which comprises administering to a patient in need thereof a therapeutically effective amount of the 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.

[0036] In some preferred embodiments of the present invention, the pain is pain caused by TRPV1 activation; and the target of the drug is TRPV1.

[0037] In order to solve the above technical problems, the thirteenth aspect of the present invention provides the antibody according to the first aspect of the present invention, the chimeric antigen receptor according to the second aspect of the present invention, the isolated nucleic acid according to the third aspect of the present invention, the recombinant expression vector according to the fourth aspect of the present invention, the transformant according to the fifth aspect of the present invention, the antibody-drug conjugate according to the seventh aspect of the present invention, the pharmaceutical composition according to the eighth aspect of the present invention and / or the kit according to the tenth aspect of the present invention, which are used for diagnosing, treating and / or preventing pain.

[0038] In some preferred embodiments of the present invention, the pain is pain caused by TRPV1 activation; and the target of the drug is TRPV1.

[0039] 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.

[0040] The reagents and raw materials used in the present invention are commercially available.

[0041] The positive progress of the present invention is that the anti-TRPV1 antibody provided by the present invention has a relatively high affinity for TRPV1, and its IC 50It is lower than 2.994μM, and can reach as low as 1.914μM. It has a significant inhibitory effect on the TRPV1 channel activated by capsaicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 shows the construction of the TRPV1 sequence on the pEGBacMam vector. The figure shows the cloning of the human TRPV1 sequence on the pEGBacMam vector using seamless cloning technology.

[0043] Figure 2 shows the MSP2N2 sequence constructed on the pET 28a vector.

[0044] Figure 3 shows the results of detergent protein purification of TRPV1. The figure shows the SEC and SDS-PAGE images of the purified human TRPV1 protein in detergent form.

[0045] FIG4 shows the results of TRPV1-Nanodisc protein purification.

[0046] FIG5 shows the titer test results of the 4th and 5th sera from mice immunized with TRPV1-Nanodisc protein.

[0047] FIG6 shows the ELISA test results of three antibodies obtained by screening, wherein A, B and C are the ELISA test results of 29G5, 38A3 and 33D5, respectively.

[0048] Figure 7 shows the FACS test results of three antibodies obtained by screening, where A, B, and C are the FACS test results of 29G5, 38A3, and 33D5, respectively.

[0049] FIG8 is an SDS-PAGE diagram of three antibodies obtained by purification and screening.

[0050] Figure 9 shows the inhibitory effects of three antibodies obtained by screening, where A, B, C and D are the inhibitory effects of AMG9810, 33D5, 29G5 and 38A3, respectively. DETAILED DESCRIPTION

[0051] 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.

[0052] Example 1 Gene synthesis and plasmid construction

[0053] We sent the wild-type TRPV1 gene, MBP gene, modified clone sequence, and MSP2N2 to Nanjing Qingke Biotechnology Co., Ltd. for synthesis. The MBP+TRPV1 sequence carried EcoR1 and Not1 restriction sites and was ligated and recombined into the pEGBacMam plasmid (Figure 1), and MSP2N2 was ligated and recombined into pET-28a (Figure 2).

[0054] ① The 6×His tag sequence and the MBP sequence, as well as the MBP and the HRV 3C protease cleavage site are connected by a linker.

[0055] MBP amino acid sequence (SEQ ID NO: 1):

[0056] MBP base sequence (SEQ ID NO: 2):

[0057] MSP2N2 amino acid sequence (SEQ ID NO: 3):

[0058] MSP2N2 base sequence (SEQ ID NO: 4):

[0059] Wild-type human TRPV1 (TRPV1-WT) amino acid sequence (SEQ ID NO: 5):

[0060] Wild-type human TRPV1 (TRPV1-WT) base sequence (SEQ ID NO: 6):

[0061] Example 2 Preparation of recombinant baculovirus

[0062] 2.1. Transform the recombinant pEGBacMam plasmid containing the target gene into Escherichia coli through heat shock transformation

[0063] DH10Bac competent cells (Shanghai Weidi) were 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 transferred to 5 mL of LB liquid medium containing three antibiotics (50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline) and cultured at 37°C, 200 rpm for 12-16 hours to extract the recombinant bacmid.

[0064] 2.2. Take a 6-well cell culture plate (Nest), 1×10 6 / 2mL sf9 insect cells, cultured in a constant temperature and humidity incubator at 27℃ for 30min, took 100μL Insect Medium (Sf-900 TM 10 μL of transfection reagent (Cellfectin) was added to 100 μL of Insect Medium (SFM), and 5 μg of recombinant baculovirus plasmid was added to 100 μL of Insect Medium. After mixing, the mixture was incubated at room temperature for 20 minutes. The transfection complex was evenly added dropwise to a 6-well plate and cultured at 27°C for 72 hours. The cells were centrifuged at 6000 rpm at 4°C for 15 minutes. The supernatant was added with 2% FBS and stored in the dark at 4°C. This is the P1 recombinant baculovirus.

[0065] 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 in the dark at 4°C to obtain the P2 generation recombinant baculovirus.

[0066] The separation density is 1×10 6 40 mL of sf9 insect cells (100 μl / mL) were cultured for 24 hours, and then infected with the 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 in the dark at 4°C to obtain the P3 recombinant baculovirus.

[0067] The separation density is 1×10 6200 mL of sf9 insect cells (100 μg / mL) were cultured for 24 hours, and then infected with P3 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 in the dark at 4°C to obtain P4 recombinant baculovirus.

[0068] Example 3 Protein Purification

[0069] 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 the P4 generation recombinant baculovirus to infect mammalian cells at a ratio of 1:10, culture on a shaker for 12-18 h, and add sodium butyrate to a final concentration of 10 mM.

[0070] After 72 hours, 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, pH 7.4, 150 mM NaCl, 1% Protease Inhibitor Cocktail, EDTA-Free, 0.5% LMNG / 0.05% CHS, 1 mM TCEP) and lysed by inversion at 4°C for 3 hours. After membrane dissolution, centrifuge at 40000 rpm for 45 minutes at 4°C. The supernatant was flipped and bound to MBP affinity chromatography filler at 4°C for 2.5 hours. The combined supernatant was passed through a gravity column and eluted with wash buffer (50mM HEPES, pH7.4, 150mM NaCl, 1mM TCEP, 0.01% LMNG / 0.001% CHS). The target protein was eluted with elute buffer (50mM HEPES, pH7.4, 150mM NaCl, 0.01% LMNG / 0.001% CHS, 1mM TCEP, 40mM Maltose). The target protein was concentrated to a volume of 500 μL using a 100KDa ultrafiltration tube and subjected to gel filtration chromatography. The gel column model used was Superrose 6 Increase 10 / 300GL (cytiva) and the buffer was SEC buffer (50mM HEPES, pH 7.4, 150 mM NaCl, 1 mM TCEP, 0.01% LMNG / 0.001% CHS), collect protein samples, test A280 concentration, and perform SDS-PAGE gel electrophoresis to detect the size and purity of the target protein ( Figure 3 ).

[0071] Package the solution using a molar ratio of TRPV1:MSP2N2:soybean of 1:3:200. Calculate the required amount of Soybean Polar Lipid Extract (Avanti). Use a protein loading syringe to aspirate the stock solution (25 mg / mL of soybean stock solution). Pipette the soybean stock solution into the bottom of a clean, dry glass tube. Then, use a nitrogen blower to remove the chloroform residue from the soybean stock solution. A light yellow residue will form at the bottom of the tube. Add SEC buffer and sonicate for 10 minutes. During this time, the solution in the tube will initially become turbid, then gradually clarify and become a light yellow solution. Add TRPV1, MSP2N2, and soybean to the EP tube in the appropriate proportions. Invert at 4°C for 1 hour. Then, add 100 mg / mL Bio-beads SM-2 to remove the detergent from the solution. Invert at 4°C overnight. The Bio-beads SM-2 were removed by gravity filtration, and the filtered protein solution was concentrated to a volume of 500 μL and centrifuged at 13,000 rpm at 4°C for 10 min. Gel filtration chromatography was performed using a Superrose 6 Increase 10 / 300 GL (Cytiva) column in SEC buffer (50 mM HEPES, pH 7.4, 150 mM NaCl). Protein samples were collected, the A280 concentration was measured, and the size and purity of the target protein were determined by SDS-PAGE gel electrophoresis (Figure 4).

[0072] Example 4 Animal Immunization

[0073] 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.

[0074] The antigen used for immunization was produced using the protein preparation method of TRPV1-nanodisc. Balb / c, 6-8 week old female mice were selected for immunization. For the initial immunization, the TRPV1-nanodics antigen dosage was 100 μg protein per mouse, mixed with Freund's complete adjuvant at a volume ratio of 1:1. The insufficient antigen volume was replaced with a purified buffer of the corresponding protein. After emulsification on a shaker, the immunized animals were routinely injected subcutaneously at multiple points on the back. Thereafter, immunization was performed every 14 days, using half the amount of antigen used in the initial immunization (50 μg per mouse), mixed with Freund's incomplete adjuvant at a volume ratio of 1:1. The insufficient antigen volume was replaced with a purified buffer of the corresponding protein. After emulsification, the immunized animals were routinely injected subcutaneously at multiple points on the back. Normally, blood is collected from mice one week after the third immunization. After separating the serum, the serum titer is tested by ELISA (coated with RPV1-nanodics antigen, blocked with 2% BAS, incubated with gradient diluted serum for 1 hour, incubated with goat anti-mouse HRP for 1 hour, developed with colorimetric solution for 10 minutes, then stopped with stop solution, and the absorbance at OD450nm is tested). After that, the serum of 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 5), and subsequent experiments can be carried out.

[0075] Example 5 Cell Fusion

[0076] 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 intraperitoneal antigen 3 days before fusion. Remove spleen cells, wash them twice with serum-free, non-antibody DMEM (20 mL) culture medium, centrifuge and resuspend the cells in 2 mL fusion buffer, count and set aside. The day before fusion, myeloma cells are passaged at a 1:2 ratio. On the day of fusion, collect SP2 / 0 cells, remove the culture medium by centrifugation, wash them twice with serum-free, non-antibody DMEM (20 mL), centrifuge and resuspend the cells in 2 mL fusion buffer, count and set aside. 8E7 splenocytes and 8E7 SP2 / 0 cells were transferred to a new 50mL centrifuge tube. 20mL of fusion buffer was added and the cells were centrifuged twice at a 1:1 ratio (500x g for 5 minutes). The cells were then resuspended in 8mL of fusion buffer and placed in an electroporation cuvette for electrofusion using a BTX fusion instrument. Fusion parameters were alternating current 48V for 40s, pulse voltage 2070V for 30µs, and PF for 7s. The tubes were allowed to stand for 10 minutes. The entire fusion solution was then added to 600mL of culture medium (75% DMEM + 20% FBS + 1% PS + 2% HAT (50×) + 1% Glu + 1% OPI) and incubated at 37°C for 2 hours.

[0077] 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.

[0078] Example 6 Monoclonal Cell Screening

[0079] Monoclonal cell screening 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 TRPV1-nanodisc antigen. After coating with 2μg / mL TRPV1-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. After adding color development solution for 10 minutes, the reaction is terminated by adding stop solution, and the absorbance value at OD450nm is measured 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 stable cell line screened is HEK293S-TRPV1. The cells to be tested are suspension cultured and passaged at an appropriate ratio. 10μl of cells are mixed with 10μl of trypan blue and counted in a hemocytometer. 5E5 cells are added to each well of a 96-well V-type dilution plate. Centrifuge at 1500 rpm for 5 minutes, remove the culture medium by tapping, add the supernatant of ELISA-positive hybridomas, incubate at 4°C for 1 hour, wash three times with PBS, add a 1:200 diluted secondary antibody (Jackson, Allophycocyanin-AffiniPure F(ab')2Fragment Donkey Anti-Mouse IgG(H+L)), incubate at 4°C for 30 minutes, and then wash three times with PBS. Finally, add 150 μl of PBS to each well to resuspend the cells and transfer them to a 1.5 mL EP tube. Alternatively, directly use a microtiter plate for loading and detect using Beckman CytoFLEX. Use Cytoexpert to output the MFI value and analyze the data by comparing it with the positive and negative controls. If a positive result is obtained, perform two rounds of subcloning by limiting dilution to finally obtain positive monoclonal cells (Figures 6 and 7).

[0080] Example 7 Monoclonal Antibody Preparation

[0081] 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 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 detected by SDS-PAGE (Figure 8).

[0082] Example 8 Antibody Sequence Acquisition

[0083] The three monoclonal antibodies 29G5, 38A3 and 33D5 were screened and the hybridoma cells were revived and 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 HiScript-TS 5' / 3' RACE Kit from Novagen. 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 pMD TM The 18-T Vector Cloning Kit was used to construct the vector and complete the plating. Monoclonal colonies were then cultured and sent for sequencing. After alignment of the sequenced fragments, the VH and VL sequences were obtained (Tables 1-3).

[0084] Table 1 29G5 antibody V sequence information

[0085] Table 2 38A3 antibody V sequence information

[0086] Table 3 33D5 antibody V sequence information

[0087] Example 9 Monoclonal Antibody Functional Test

[0088] 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 TRPV1 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 AMG9810, a TRPV1 channel-specific inhibitor. The blank control was the extracellular buffer as a solvent and the extracellular buffer containing the corresponding concentration of co-solvent DMSO (10% DMSO / HBSS or PBS). Place in the dark during the whole process. After the incubation is completed, place the 96-well plate on the FlexStation3 multi-function plate reader, load the TRPV1 specific agonist Capsaicine (capsaicin) sample plate into the sampler of the microplate reader in advance, set the concentration to 20μM (the final concentration in the well is 5μM), set the test program, and immediately start monitoring the changes in the fluorescence intensity of each column of cells after adding a column of agonists for at least two minutes. After all well plates are tested, calculate the fluorescence increase of all wells before and after the addition of agonists based on the quantified fluorescence intensity change curve output by the machine, and normalize it with the fluorescence increase of the corresponding blank control wells to obtain the inhibition rate of fluorescence intensity under the action of different concentrations of antibodies. In GraphPad, curve fitting (Y=100 / (1+10^((LogIC50-X)*HillSlope))) is used to obtain the inhibition curve of the logarithm of concentration against the inhibition rate, and calculate the half-inhibitory concentration IC of the antibody inhibition channel. 50 (Figure 9) to determine the inhibitory effect of the antibody on TRPV1. IC 50 They are 2.680 μM, 1.914 μM, 0.242 μM and 2.992 μM respectively.

Claims

1. A TRPV1 antibody, characterized in that, The antibody or its variant 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 as shown in SEQ ID NOs: 11-13 respectively; and / or, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 14-16 respectively; or, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 21-23 respectively; and / or, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 24-26 respectively; or, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 31-33 respectively; and / or, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 34-36 respectively.

2. The 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 or murine heavy chain variable region framework region, and the LFWR is a human or murine antibody light chain variable region framework region; Preferably, the light chain variable region comprises a sequence with an amino acid sequence as shown in SEQ ID NO: 10; and / or, the heavy chain variable region comprises a sequence with an amino acid sequence as shown in SEQ ID NO: 9; or, the light chain variable region comprises a sequence with an amino acid sequence as shown in SEQ ID NO: 20; and / or, the heavy chain variable region comprises a sequence with an amino acid sequence as shown in SEQ ID NO: 19; or, the light chain variable region comprises a sequence with an amino acid sequence as shown in SEQ ID NO: 30; and / or, the heavy chain variable region comprises a sequence with an amino acid sequence as shown in SEQ ID NO: 29; More preferably, the antibody or its variant further includes a heavy chain constant region and a light chain constant region; Further preferably, the heavy chain constant region of the antibody is a human or murine heavy chain constant region; the light chain constant region of the antibody is a human or murine antibody light chain constant region.

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

4. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises the antibody as described in any one of claims 1-3.

5. An isolated nucleic acid, characterized in that, The nucleic acid encodes the antibody as described in any one of claims 1-3, or the chimeric antigen receptor as described in claim 4; Preferably, the nucleic acid encoding the antibody comprises the polynucleotide sequence shown in SEQ ID NO:7 and / or SEQ ID NO:8; or, comprises the polynucleotide sequence shown in SEQ ID NO:17 and / or SEQ ID NO:18; or, comprises the polynucleotide sequence shown in SEQ ID NO:27 and / or SEQ ID NO:

28.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid as claimed in claim 5.

7. A transformant, characterized in that, The transformant comprises the recombinant expression vector as claimed in claim 6 in a host cell.

8. A method for preparing a TRPV1 antibody, characterized in that, The preparation method comprises culturing the transformant as claimed in claim 7 and obtaining the antibody from the culture.

9. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate comprises a cytotoxic agent and the antibody as claimed in any one of claims 1-3.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody as claimed in any one of claims 1-3, the chimeric antigen receptor as claimed in claim 4 or the antibody-drug conjugate as claimed in claim 9, and a pharmaceutically acceptable carrier.

11. Use of the antibody according to any one of claims 1-3, the chimeric antigen receptor according to claim 4, the isolated nucleic acid according to claim 5, the recombinant expression vector according to claim 6, the transformant according to claim 7, the antibody-drug conjugate according to claim 9 or the pharmaceutical composition according to claim 10 in the preparation of a drug for treating pain; Preferably, the pain is caused by TRPV1 activation; the target of the drug is TRPV1.

12. A kit, characterized in that, The kit comprises the antibody as claimed in any one of claims 1-3, the chimeric antigen receptor as claimed in claim 4, the antibody-drug conjugate as claimed in claim 9 and / or the pharmaceutical composition as claimed in claim 10.

13. A method for detecting TRPV1, characterized in that, The method comprises contacting a sample with the antibody as claimed in any one of claims 1-3, the chimeric antigen receptor as claimed in claim 4, the antibody-drug conjugate as claimed in claim 9, the pharmaceutical composition as claimed in claim 10 and / or the kit as claimed in claim 12; Preferably, the detection is for non-diagnostic purposes.

14. A method for diagnosing, treating and / or preventing pain, characterized in that, The method comprises administering to a patient in need a therapeutically effective amount of the antibody as claimed in any one of claims 1-3, the chimeric antigen receptor as claimed in claim 4, the isolated nucleic acid as claimed in claim 5, the recombinant expression vector as claimed in claim 6, the transformant as claimed in claim 7, the antibody-drug conjugate as claimed in claim 9, the pharmaceutical composition as claimed in claim 10 and / or the kit as claimed in claim 12; Preferably, the pain is caused by TRPV1 activation; the target of the drug is TRPV1.

15. The antibody according to any one of claims 1-3, the chimeric antigen receptor according to claim 4, the isolated nucleic acid according to claim 5, the recombinant expression vector according to claim 6, the transformant according to claim 7, the antibody-drug conjugate according to claim 9, the pharmaceutical composition according to claim 10 and / or the kit according to claim 12, which are used for diagnosing, treating and / or preventing pain; Preferably, the pain is caused by TRPV1 activation; the target of the drug is TRPV1.

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