TRPM3 antibody, and preparation method therefor and use thereof

Abstract

Provided are a TRPM3 antibody, and a preparation method therefor and a use thereof. The 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 set forth in SEQ ID NOs: 25, 38, and 39, SEQ ID NOs: 5-7, or SEQ ID NOs: 15-17, respectively, and / or the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NOs: 28, 29, and 40, SEQ ID NOs: 8-10, or SEQ ID NOs: 18-20, respectively. The anti-TRPM3 antibody has a relatively high affinity, and the screened antibody has a significant inhibitory effect on a PregS-activated TRPM3 channel.

Description

TRPM3 antibody, preparation method and application thereof Technical Field

[0001] The present invention relates to the field of immunotherapy, and in particular to a TRPM3 antibody, a method for preparing the same, and an application thereof. Background Art

[0002] TRPM3 (TRP (transient receptor potential) intercellular resilin 3) is one of the least studied proteins in the TRP family of ion channels. Heterologously expressed TRPM3 channels are constitutively active, have an outwardly rectifying current-voltage relationship, and are regulated by intracellular Mg. 2+ Despite these fairly common features, which make TRPM3 channels similar to the closely related channels TRPM6 and TRPM7, TRPM3 channels have several unique features that distinguish them from other members of this diverse family. The TRPM3 gene encodes a large number of isoforms, primarily generated by alternative splicing. Only two of (at least) eight sites generating sequence diversity have their functional consequences elucidated: one results in a nonfunctional channel, the other profoundly affects ion selectivity.

[0003] TRPM3 is a homotetrameric channel with a unique ~700 amino acid long TRPM-specific domain at the cytoplasmic N-terminus of the protein. Like TRPV1, TRPM3 is a 2+ TRPM3 is a non-selective cation channel with high permeability and a strong outward rectifying current-voltage relationship. In the nervous system, TRPM3 has been reported to be expressed in the CNS and PNS, including DRG neurons, as well as various non-neuronal tissues.

[0004] TRPM3 is a recently identified pain-sensing channel in the TRP family, alongside TRPV1 and TRPA1. TRPM3 can be activated by heat and chemical ligands, such as the neurosteroid pregnenolone sulfate (PregS) and the synthetic ligand CIM0216. Upon activation, it exhibits increased permeability to calcium ions. Another important finding is that opioids such as morphine can strongly inhibit TRPM3 activity after activating μ receptors, suggesting that TRPM3 may be a peripheral analgesic target for opioid analgesics. Therefore, TRPM3 holds promise as a novel target for the development of analgesic drugs.

[0005] Some TRPM3 antagonists are known, for example, liquiritigenin, a putative TRPM3 blocker, has been described to reduce mechanical hyperalgesia and cold hyperalgesia in a rat pain model (Chen L et al., Scientific Reports, July 2014). There is still a large medical need for drugs for preventing or treating TRPM3-mediated pain, and therefore there is an urgent need to develop drugs with good efficacy, low levels or no side effects (e.g., unlikely to be addictive or toxic like opioids) and good or better pharmacokinetic or kinetic properties, while macromolecular drugs have the advantages of limited central nervous system (CNS) permeability (when targeting peripheral treatment), low immunogenicity, high selectivity and favorable half-life, which can be used to prevent or treat TRPM3-mediated pain (such as inflammatory pain) new, alternative and or better therapeutic drugs.

[0006] Since small molecule drugs have relatively large side effects, while large molecule drugs have advantages such as limited central nervous system (CNS) permeability (when targeting peripheral treatment), low immunogenicity, high selectivity and favorable half-life, there is an urgent need to develop antibody drugs to replace small molecule drugs.

[0007] Summary of the Invention

[0008] To address the above issues, the present invention primarily provides a method for preparing a TRPM3 monoclonal antibody. The immunogen used is a detergent-purified protein, and the antibody is prepared using hybridoma technology. Affinity screening primarily involves ELISA (enzyme-linked immunosorbent assay) and FACS (flow cytometry). The flexstation3 (multi-mode microplate reader) is then used to read changes in fluorescence intensity caused by calcium ions flowing into cells through ion channels, thereby screening for antibodies with both affinity and functionality.

[0009] In addition, the present invention provides an application of an anti-TRPM3 antibody for use in inhibiting activation of the TPRM3 pathway by an agonist. The TRPM3 agonist is pregnenolone sulfate (PregS for short), which can specifically activate the mechanism of TRPM3. The antibodies screened by the present invention were found to specifically inhibit the TRPM3 pathway.

[0010] Specifically:

[0011] The first aspect of the present invention provides a TRPM3 antibody, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 25, 38, and 39, respectively; and / or, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 28, 29, and 40, respectively; or,

[0012] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 5-7, 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,

[0013] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 15-17, respectively; and / or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 18-20, respectively.

[0014] In some specific embodiments of the present invention, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 25-27, respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 28-30, respectively; or,

[0015] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 25, 35 and 36, respectively; and / or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 28, 29 and 37, respectively.

[0016] In some specific 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 specific embodiments of the present invention, the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 3 or is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as shown in SEQ ID NO: 3; and / or, the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 4 or is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as shown in SEQ ID NO: 4; or,

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

[0019] The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 23 or is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as shown in SEQ ID NO: 23; and / or the light 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,

[0020] The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:33 or is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as shown in SEQ ID NO:33; and / or the light 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.

[0021] In some specific embodiments of the present invention, the TRPM3 antibody further comprises a heavy chain constant region and a light chain constant region.

[0022] In some specific embodiments of the present invention, the heavy chain constant region of the TRPM3 antibody is a human or mouse antibody heavy chain constant region; and the light chain constant region of the TRPM3 antibody is a human or mouse antibody light chain constant region.

[0023] In some specific embodiments of the present invention, the TRPM3 antibody is in any of the following antibody forms:

[0024] (a) a complete immunoglobulin molecule;

[0025] (b) an scFv;

[0026] (c) a fusion protein comprising an scFv;

[0027] (d) a Fab fragment;

[0028] (e) a Fab′ fragment;

[0029] (f) a F(ab)2;

[0030] Alternatively, the TRPM3 antibody is a monoclonal antibody or a polyclonal antibody;

[0031] Alternatively, the TRPM3 antibody is a humanized antibody or a bispecific antibody.

[0032] The second aspect of the present invention provides a chimeric antigen receptor comprising the TRPM3 antibody according to the first aspect of the present invention.

[0033] The third aspect of the present invention provides an isolated nucleic acid encoding the TRPM3 antibody according to the first aspect of the present invention, or the chimeric antigen receptor according to the second aspect of the present invention.

[0034] In some specific embodiments of the present invention, the nucleic acid encoding the TRPM3 antibody comprises a polynucleotide sequence as shown in SEQ ID NO: 1 and / or as shown in SEQ ID NO: 2; or, comprises a polynucleotide sequence as shown in SEQ ID NO: 11 and / or as shown in SEQ ID NO: 12; or, comprises a polynucleotide sequence as shown in SEQ ID NO: 21 and / or as shown in SEQ ID NO: 22; or, comprises a polynucleotide sequence as shown in SEQ ID NO: 31 and / or as shown in SEQ ID NO: 32.

[0035] The fourth aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described in the third aspect of the present invention.

[0036] In some specific embodiments of the present invention, the recombinant expression vector is a plasmid, cosmid, phage or viral vector.

[0037] In some specific embodiments of the present invention, the viral vector is a baculoviral vector, a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

[0038] The fifth aspect of the present invention provides a transformant, which comprises the recombinant expression vector according to the fourth aspect of the present invention in a host cell.

[0039] In some specific embodiments of the present invention, the host cell is a prokaryotic cell or a eukaryotic cell.

[0040] In some specific embodiments of the present invention, the host cell is selected from yeast cells, insect cells, mammalian cells or other cells suitable for antibody production; the mammalian cells are, for example, HEK293 cells.

[0041] The sixth aspect of the present invention provides a method for preparing a TRPM3 antibody, comprising culturing the transformant according to the fifth aspect of the present invention, and obtaining the TRPM3 antibody from the culture.

[0042] The seventh aspect of the present invention provides an antibody-drug conjugate, which comprises a cytotoxic agent and the TRPM3 antibody according to the first aspect of the present invention.

[0043] The eighth aspect of the present invention provides a pharmaceutical composition, comprising the TRPM3 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] The ninth aspect of the present invention provides a TRPM3 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 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 for use in the preparation of a medicament for treating pain.

[0045] In some specific embodiments of the present invention, the pain is pain caused by TRPM3 activation; and the target of the drug is TRPM3.

[0046] The tenth aspect of the present invention provides a kit, comprising the TRPM3 antibody according to the first aspect of the present invention, the chimeric antigen receptor according to the second aspect of the present invention, the 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, or the pharmaceutical composition according to the eighth aspect of the present invention.

[0047] The eleventh aspect of the present invention provides a method for detecting TRPM3, comprising contacting a sample with the TRPM3 antibody according to any one of the first aspect of the present invention, the chimeric antigen receptor according to the second 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.

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

[0049] The twelfth aspect of the present invention provides a method for diagnosing, treating and / or preventing pain, comprising administering to a subject in need thereof a therapeutically effective amount of the TRPM3 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 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 specific embodiments of the present invention, the pain is pain caused by TRPM3 activation; and the drug target is TRPM3.

[0051] The thirteenth aspect of the present invention provides the TRPM3 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 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 diagnosing, treating and / or preventing pain.

[0052] In some specific embodiments of the present invention, the pain is pain caused by TRPM3 activation; and the target of the drug is TRPM3.

[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 immunogen used in the present invention is a detergent-purified protein, and a special cloning method is used to make the protein have better activity, stability and functionality. The anti-TRPM3 antibodies screened by the hybridoma method have relatively high affinity, and the screened antibodies have a significant inhibitory effect on the TRPM3 channel activated by PregS. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 shows the results of TRPM3 detergent protein purification.

[0057] FIG2 shows the titer test results of the 4th and 5th immune sera of mice immunized with TRPM3 detergent protein.

[0058] Figure 3 shows the ELISA test results of four antibodies obtained by screening; among them, Figure 3a is the ELISA test result of the 15B1 antibody, Figure 3b is the ELISA test result of the 19C4 antibody, Figure 3c is the ELISA test result of the 27F10 antibody, and Figure 3d is the ELISA test result of the 17F9 antibody.

[0059] Figure 4 shows the FACS test results of four antibodies obtained by screening; among them, Figure 4a is the FACS test result of 17F9 antibody, Figure 4b is the FACS test result of 27F10 antibody, Figure 4c is the FACS test result of 19C4 antibody, and Figure 4d is the FACS test result of 15B1 antibody.

[0060] FIG5 is an SDS-PAGE diagram of four antibodies obtained by purification and screening.

[0061] Figure 6 shows the inhibitory effects of four antibodies obtained by screening; among them, Figure 6a shows the inhibitory effect of small molecule 193, Figure 6b shows the inhibitory effect of 15B1 antibody, Figure 6c shows the inhibitory effect of 17F9 antibody, Figure 6d shows the inhibitory effect of 19C4 antibody, and Figure 6e shows the inhibitory effect of 27F10 antibody. DETAILED DESCRIPTION

[0062] 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.

[0063] Example 1: Extraction of bacmids

[0064] By heat shock transformation, the recombinant pEGBacMam plasmid containing the nucleotide sequence of the TRPM3 truncate linked to the MBP protein 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 halogenated indole-β-galactoside (Thermofish), and 40 μg / mL isopropyl-β-D-thiogalactoside (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 at 200 rpm for 12-16 hours to extract the recombinant bacmid.

[0065] Example 2: Baculovirus Preparation

[0066] Take a 6-well cell culture plate (Nest), 1×10 6 / 2mL sf9 insect cells were cultured in a constant temperature and humidity incubator at 27℃ for 30min, and 100μL insect culture medium (Sf-900 TM 10 μL of transfection reagent (Cellfectin) was added to 100 μL of insect culture medium. 5 μg of recombinant baculovirus plasmid was added to another 100 μL of insect culture 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 culture was centrifuged at 6000 rpm at 4°C for 15 minutes. The supernatant was added with 2% fetal bovine serum and stored at 4°C in the dark to obtain the P1 recombinant baculovirus.

[0067] 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 at adherence, centrifuged at 6000 rpm at 4°C for 15 minutes, the supernatant was added with 2% fetal bovine serum, and stored at 4°C in the dark to obtain the P2 generation recombinant baculovirus.

[0068] The separation density is 1×10 6 40 mL of sf9 insect cells (100 μg / 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% fetal bovine serum was added, and the cells were stored in the dark at 4°C to obtain the P3 recombinant baculovirus.

[0069] The separation density is 1×10 6 200 mL of sf9 insect cells (100 mL / 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% fetal bovine serum was added, and the cells were stored in the dark at 4°C to obtain P4 recombinant baculovirus.

[0070] Example 3: Protein purification test

[0071] 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.

[0072] 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 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 150 mM NaCl, 1% protease inhibitor cocktail, EDTA-free, 0.5% dodecylmaltose neopentyl glycol / 0.05% cholesterol succinate, 1 mM tris(2-carboxyethyl)phosphine) 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 the MBP affinity chromatography medium at 4°C for 2.5 hours. The combined supernatant was passed through a gravity column and the impurities were eluted with a washing buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 150 mM NaCl, 0.02% dodecylmaltose neopentyl glycol / 0.002% cholesterol succinate monoester, 1 mM tris(2-carboxyethyl)phosphine). The target protein was eluted with an ultrafiltration tube of 100 kDa and concentrated to a volume of 500 μL. The target protein was then subjected to gel filtration chromatography using a Superrose 6 Increase 10 / 300 GL (cytiva) gel column model and SEC buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 150 mM NaCl, 0.02% dodecyl maltose neopentyl glycol / 0.002% cholesterol succinate monoester). Protein samples were collected, the A280 concentration was tested, and the size and purity of the target protein were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The test results are shown in Figure 1.

[0073] Animal Immunization

[0074] 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.

[0075] The antigen used for immunization was produced using a protein preparation method using a TRPM3 detergent. Balb / c, 6-8 week old female mice were selected for immunization. The initial TRPM3 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 solution of the corresponding protein. After emulsification on a shaker, the immunized animals were routinely injected subcutaneously at multiple points on the back. Immunizations were then 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 solution 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 the serum is separated, the titer of the serum is tested by ELISA (coated with TRPM3 antigen, blocked with 2% bovine serum albumin, incubated with graded diluted serum for 1 hour, incubated with goat anti-mouse horseradish peroxidase for 1 hour, developed with colorimetric solution for 10 minutes, stopped with stop solution, and the absorbance 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 2), and subsequent experiments can be carried out.

[0076] Example 4: Cell Fusion

[0077] 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-containing DMEM (20 mL) 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 SP 2 / 0, remove the medium by centrifugation, wash them twice with serum-free, non-antibody-containing DMEM (20 mL), centrifuge and resuspend the cells in 2 mL fusion buffer, count and set aside. 8E7 splenocytes and 8E7 SP 2 / 0 cells were transferred to a new 50 mL centrifuge tube. 20 mL of fusion buffer was added and the cells were centrifuged twice at a 1:1 ratio (500 x g for 5 minutes). The cells were resuspended in 8 mL of fusion buffer and placed in an electroporation cuvette for electrofusion using a BTX fusion instrument. Fusion parameters were alternating current 48 V for 40 seconds, pulse voltage 2070 V for 30 μs, and PF for 7 seconds. The tubes were allowed to stand for 10 minutes. The entire fusion solution was then added to 600 mL of culture medium (75% DMEM + 20% fetal bovine serum + 1% penicillin-streptomycin + 2% HAT (50×) + 1% glucose + 1% OPI) and incubated at 37°C for 2 hours.

[0078] 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.

[0079] Example 5: Monoclonal cell screening

[0080] 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 TRPM3 detergent antigen used for immunity. After coating with 2 μg / mL TRPM3 detergent antigen and blocking with 2% bovine serum albumin, 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 OD 450nm is measured to test the titer of the 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-TRPM3. The cells to be tested are suspension cultured and passaged at an appropriate ratio. 10 μL of cells are mixed with 10 μL 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 or directly use a microtiter plate for loading. Detection is performed using Beckman CytoFLEX, and the MFI value is output using Cytoexpert. The data is analyzed by comparing the difference with the positive and negative controls. If a positive result is obtained, two rounds of subcloning are performed by limiting dilution to finally screen for positive monoclonal cells (Figures 3a-d, 4a-d).

[0081] Example 6: Monoclonal Antibody Preparation

[0082] 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 hybridoma cell mass 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 sodium dodecyl sulfate polyacrylamide gel electrophoresis (Figure 5).

[0083] Example 7: Monoclonal Antibody Functional Testing

[0084] The antibody function test is mainly carried out by detecting calcium flow with a 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. HEK293T cells transfected with human TRPM3 protein for about 24 hours were plated in 96-well plates for adherent culture in advance. After growing to an appropriate density, the culture medium was discarded. At the same time, 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. The cells were incubated in an incubator for at least 60 minutes. The positive small molecule was 193, which is a TRPM3 channel-specific inhibitor synthesized by us. 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 incubation, place the 96-well plate on the FlexStation3 multi-function plate reader. Load the TRPM3 specific agonist pregnenolone sulfate (PregS) sample plate into the sampler of the microplate reader in advance, set the concentration to 60μM (the final concentration in the well is 15μM), set the test program, and immediately monitor the changes in the fluorescence intensity of each column of cells after adding the agonist for at least two minutes. After all well plates are tested, calculate the fluorescence increase of all wells before and after the addition of the agonist 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 antibody concentrations. 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 , in order to judge the inhibitory effect of the antibody on TRPM3, the results are shown in Figure 6a-e.

[0085] Example 8: Antibody sequence acquisition

[0086] The four monoclonal antibodies obtained were screened and the hybridoma cells were recovered and the density reached 5×10 6 , the cells were collected by centrifugation, and the monoclonal cell RNA was extracted 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 pMDTM18-T Vector Cloning Kit was used to complete the vector construction and coating. After the monoclonal colonies were cultured and sent for sequencing, the sequences of VH and VL were obtained after alignment of the sequenced sequences (Tables 1, 2, 3, 4).

[0087] The CDR sequences of the heavy chain variable regions and light chain variable regions of the above-mentioned antibodies 19C4 and 15B1 are very similar. The present invention summarizes their CDR sequences as follows:

[0088] SEQ ID NO: 38: IDPX1NGX2T, wherein X1 is V or A; X2 is N or K.

[0089] SEQ ID NO:39: X3RSX4X5X6X7, wherein X3 is S or A; X4 is N or T; X5 is S or Y; X6 is V or A; and X7 is empty or Y.

[0090] SEQ ID NO:40: QQX8SSYPFT, wherein X8 is R or K.

[0091] 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 TRPM3 antibody, characterized in that The TRPM3 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 25, 38 and 39, respectively; and / or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 28, 29 and 40, respectively; or, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 5-7, 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, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 15-17, respectively; and / or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 18-20, respectively.

2. The TRPM3 antibody according to claim 1, wherein The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 25-27, respectively; and / or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 28-30, respectively; or, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 25, 35 and 36, respectively; and / or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 28, 29 and 37, respectively.

3. The TRPM3 antibody according to claim 1 or 2, wherein 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 origin antibody; Preferably, the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 3 or has at least 80% identity with the amino acid sequence as shown in SEQ ID NO: 3; and / or, the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 4 or has at least 80% identity with the amino acid sequence as shown in SEQ ID NO: 4; or, The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13 or is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 13; and / or the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14 or is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 14; or, The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23 or is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 23; and / or the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24 or is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 24; or, The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33 or the amino acid sequence shown in SEQ ID NO:

33. and / or the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 34 or has at least 80% identity with the amino acid sequence as shown in SEQ ID NO: 34; More preferably, the TRPM3 antibody further comprises a heavy chain constant region and a light chain constant region; Further preferably, the TRPM3 antibody heavy chain constant region is a human or mouse antibody heavy chain constant region; and the TRPM3 antibody light chain constant region is a human or mouse antibody light chain constant region.

4. The TRPM3 antibody according to any one of claims 1 to 3, wherein The TRPM3 antibody is in any of the following antibody forms: (a) a complete immunoglobulin molecule; (b) an scFv; (c) a fusion protein comprising an scFv; (d) a Fab fragment; (e) a Fab′ fragment; (f) a F(ab)2; Alternatively, the TRPM3 antibody is a monoclonal antibody or a polyclonal antibody; Alternatively, the TRPM3 antibody is a humanized antibody or a bispecific antibody.

5. A chimeric antigen receptor, characterized in that It comprises the TRPM3 antibody according to any one of claims 1 to 4.

6. An isolated nucleic acid encoding the TRPM3 antibody of any one of claims 1 to 4, or the chimeric antigen receptor of claim 5; Preferably, the nucleic acid encoding the antibody comprises a polynucleotide sequence as shown in SEQ ID NO: 1 and / or as shown in SEQ ID NO: 2; or, comprises a polynucleotide sequence as shown in SEQ ID NO: 11 and / or as shown in SEQ ID NO: 12; or, comprises a polynucleotide sequence as shown in SEQ ID NO: 21 and / or as shown in SEQ ID NO: 22; or, comprises a polynucleotide sequence as shown in SEQ ID NO: 31 and / or as shown in SEQ ID NO:

32.

7. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid according to claim 6; Preferably, the recombinant expression vector is a plasmid, cosmid, phage or viral vector; More preferably, the viral vector is a baculoviral vector, a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

8. A transformant, characterized in that The transformant comprises the recombinant expression vector according to claim 7 in a host cell; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; More preferably, the host cell is selected from yeast cells, insect cells, mammalian cells or other cells suitable for antibody production; the mammalian cells are, for example, HEK293 cells.

9. A method for preparing a TRPM3 antibody, characterized in that: The method comprises culturing the transformant according to claim 8 and obtaining the antibody from the culture.

10. An antibody-drug conjugate, characterized in that: The antibody-drug conjugate comprises a cytotoxic agent and the TRPM3 antibody according to any one of claims 1 to 4.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the TRPM3 antibody according to any one of claims 1 to 4, the chimeric antigen receptor according to claim 5, or the antibody-drug conjugate according to claim 10, and a pharmaceutically acceptable carrier.

12. Use of the TRPM3 antibody according to any one of claims 1 to 4, the chimeric antigen receptor according to claim 5, the nucleic acid according to claim 6, the recombinant expression vector according to claim 7, the transformant according to claim 8, the antibody-drug conjugate according to claim 10, or the pharmaceutical composition according to claim 11 in the preparation of a medicament for treating pain; Preferably, the pain is pain caused by TRPM3 activation; and the target of the drug is TRPM3.

13. A kit, characterized in that The kit comprises the TRPM3 antibody according to any one of claims 1 to 4, the chimeric antigen receptor according to claim 5, the antibody-drug conjugate according to claim 10, or the pharmaceutical composition according to claim 11.

14. A method for detecting TRPM3, comprising contacting a sample with the TRPM3 antibody according to any one of claims 1 to 4, the chimeric antigen receptor according to claim 5, the antibody-drug conjugate according to claim 10, the pharmaceutical composition according to claim 11, and / or the kit according to claim 13; Preferably, the detection is for non-disease diagnosis purposes.

15. A method for diagnosing, treating and / or preventing pain, comprising administering to a subject in need thereof a therapeutically effective amount of the TRPM3 antibody according to any one of claims 1 to 4, the chimeric antigen receptor according to claim 5, the nucleic acid according to claim 6, the recombinant expression vector according to claim 7, the transformant according to claim 8, the antibody-drug conjugate according to claim 10, the pharmaceutical composition according to claim 11 and / or the kit according to claim 13; Preferably, the pain is pain caused by TRPM3 activation; and the drug target is TRPM3.

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