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WO2025123292A1 - Trpv1 antibody, and preparation method therefor and use thereof - Google Patents

Trpv1 antibody, and preparation method therefor and use thereof Download PDF

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Publication number
WO2025123292A1
WO2025123292A1 PCT/CN2023/138840 CN2023138840W WO2025123292A1 WO 2025123292 A1 WO2025123292 A1 WO 2025123292A1 CN 2023138840 W CN2023138840 W CN 2023138840W WO 2025123292 A1 WO2025123292 A1 WO 2025123292A1
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Prior art keywords
antibody
chain variable
variable region
seq
trpv1
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French (fr)
Chinese (zh)
Inventor
张进
李健
张雨婷
徐婷婷
周澄杰
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Shenzhen Crystalo Biopharma Technology Co Ltd
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Shenzhen Crystalo Biopharma Technology Co Ltd
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Priority to PCT/CN2023/138840 priority Critical patent/WO2025123292A1/en
Priority to CN202311821240.3A priority patent/CN118005789A/en
Publication of WO2025123292A1 publication Critical patent/WO2025123292A1/en
Pending legal-status Critical Current
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
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    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification

Definitions

  • the present invention relates to the field of immunotherapy, and in particular to a TRPV1 antibody, a preparation method and application thereof.
  • 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. After binding with ligand, it causes changes in ion concentration. When TRPV1 is activated, it can cause the influx of cations such as Ca 2+ , K + , Mg 2+ and Na + , but it has relative selectivity for Ca 2+ and Mg 2+ , which is about 5 to 10 times that of other cations. After TRPV1 is activated, the intracellular cation concentration increases, causing corresponding physiological and pathological changes.
  • TRPV1 Transient receptor potential vanilloid subfamily 1
  • TRPV1 is widely distributed in the central nervous system, peripheral nervous system, respiratory, digestive, cardiovascular and urinary systems. In recent years, studies have found that TRPV1 channels play an important role in the generation and development of pathological pain and are expected to become a new target for the treatment of pathological pain.
  • TRPV1 channel has always been the main TRP channel for developing new drugs to treat pain, but few analgesics targeting TRPV1 channel are actually 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 its clinical development.
  • small molecule drugs have relatively large side effects, and large molecule drugs have the advantages of 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 is still a relatively large challenge.
  • CNS central nervous system
  • the present invention mainly provides a TRPV1 antibody, a preparation method and application thereof.
  • the immunogen used in the preparation process is a protein purified by nanodisc packaging.
  • the preparation of the antibody adopts hybridoma technology.
  • the affinity screening is mainly ELISA and FACS, and the fluorescence intensity changes caused by calcium ions flowing into the cell through ion channels are read by flexstation3 to 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, and the stimulation has the same (or similar) mechanism of activating TRPV1 as capsaicin, and its specific inhibitory effect on the TRPV1 pathway is found through functional testing.
  • the first aspect of the present invention provides a TRPV1 antibody, wherein the antibody or its variant comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are respectively shown in SEQ ID NO: 11-13; and/or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are respectively shown in SEQ ID NO: 14-16; or,
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:21-23, respectively; and/or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:24-26, respectively; or,
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, whose amino acid sequences are respectively shown in SEQ ID NO:31-33; and/or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3, whose amino acid sequences are respectively shown in SEQ ID NO:34-36.
  • the heavy chain variable region also includes a heavy chain variable region framework region HFWR, and/or the light chain variable region also 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.
  • the antibody or variant thereof further comprises a heavy chain constant region and a light chain constant region.
  • 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.
  • the antibody is in any of the following antibody forms:
  • the antibody is a monoclonal antibody or a polyclonal antibody
  • the antibody is a humanized antibody or a bispecific antibody.
  • 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.
  • 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.
  • 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.
  • the fourth aspect of the present invention provides a recombinant expression vector, which It comprises the isolated nucleic acid as described in the third aspect of the present invention.
  • 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.
  • 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.
  • the seventh aspect of the present invention provides an antibody-drug conjugate, which comprises a cytotoxic agent and the antibody as described in the first aspect of the present invention.
  • 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.
  • 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 drug for treating pain.
  • the pain is pain caused by activation of TRPV1; and the target of the drug is TRPV1.
  • the tenth aspect of the present invention provides a kit, which includes 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 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.
  • 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.
  • the detection is for non-diagnostic purposes.
  • the twelfth aspect of the present invention provides a method for diagnosing, treating and/or preventing pain, which method 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.
  • the pain is pain caused by activation of TRPV1; and the target of the drug is TRPV1.
  • the pain is pain caused by activation of TRPV1; and the target of the drug is TRPV1.
  • the reagents and raw materials used in the present invention are commercially available.
  • the positive improvement effect of the present invention is that the anti-TRPV1 antibody provided by the present invention has a relatively high affinity for TRPV1, with an IC 50 lower than 2.994 ⁇ M, and a minimum of 1.914 ⁇ M, and has a significant inhibitory effect on the TRPV1 channel activated by capsaicine.
  • FIG. 1 shows the construction of the TRPV1 sequence on the pEGBacMam vector.
  • the human TRPV1 sequence was cloned into the pEGBacMam vector using the slot cloning technique.
  • Figure 2 shows the MSP2N2 sequence constructed on the pET 28a vector.
  • FIG. 3 shows the results of TRPV1 detergent protein purification.
  • the figure shows the SEC and SDS-PAGE images of the purification of human TRPV1 protein in detergent form.
  • FIG. 4 shows the results of TRPV1-Nanodisc protein purification.
  • FIG. 5 shows the titer test results of the 4th and 5th sera from mice immunized with TRPV1-Nanodisc protein.
  • 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.
  • Figure 7 shows the FACS test results of three antibodies obtained by screening, wherein A, B and C are the FACS test results of 29G5, 38A3 and 33D5 respectively.
  • FIG8 is an SDS-PAGE diagram of three antibodies obtained by purification and screening.
  • sf9 insect cells 200 mL of sf9 insect cells with a density of 1 ⁇ 10 6 /mL were separated and cultured for 24 hours.
  • the P3 generation recombinant baculovirus was used for infection at a ratio of 1:100.
  • the cells were cultured at 27°C and 120 rpm for 96 hours.
  • the cells were centrifuged at 4°C and 6000 rpm for 15 minutes.
  • the supernatant was filtered with a 0.22 ⁇ m filter device, 2% FBS was added, and the cells were stored at 4°C in the dark to obtain the P4 generation recombinant baculovirus.
  • HEK-293S cells were passaged at a concentration of 1.5 ⁇ 10 6 /mL, cultured at 37°C, 8% CO 2 , 60% humidity, and 120 rpm for 24 h, and P4 generation recombinant baculovirus was used to infect mammalian cells at a ratio of 1:10. Sodium butyrate was added for 12-18 h on a shaking platform to a final concentration of 10 mM.
  • 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 the membrane was lysed by flipping at 4°C for 3 hours. After the membrane was dissolved, the mixture was centrifuged at 4°C and 40,000 rpm for 45 minutes. The supernatant was flipped and combined with the MBP affinity chromatography filler at 4°C for 2.5 hours. The combined supernatant was passed through a gravity column.
  • 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
  • wash buffer 50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM TCEP, 0.01% LMNG/0.001% CHS
  • Elute buffer 50 mM HEPES, pH 7.4, 150 mM NaCl, 0.01% LMNG/0.001% CHS, 1 mM TCEP, 40 mM Maltose
  • the target protein was concentrated to a volume of 500 ⁇ L using a 100 KDa ultrafiltration tube and subjected to gel filtration chromatography.
  • the gel column model used was Superrose 6 Increase 10/300 GL (cytiva) and the buffer was SEC buffer.
  • Immunizing animals with antigens is the first and most important step in the preparation of monoclonal antibodies. Whether the animal's immune response to the antigen is good and whether it can produce antibodies with high titers and good specificity directly determines the difficulty of later screening of monoclonal hybridoma cell lines and the effectiveness of the antibodies obtained.
  • the antigen used for immunization was produced by the protein preparation method of TRPV1-nanodisc.
  • the animals for immunization were balb/c, 6-8 weeks old female mice.
  • the amount of TRPV1-nanodics antigen used for the first immunization was 100 ⁇ g protein/mouse, mixed with Freund's complete adjuvant at a volume ratio of 1:1.
  • the insufficient antigen volume was replaced with the corresponding protein purification buffer, emulsified by a shaker, and routinely injected subcutaneously at multiple points on the back to immunize the animals. After that, immunization was performed every 14 days, and the amount of antigen used was half of the initial immunization.
  • the amount of antigen (50 ⁇ g/mouse) was mixed with Freund's incomplete adjuvant at a volume ratio of 1:1.
  • the insufficient amount of antigen was replaced by the corresponding protein purification buffer, and after emulsification, the immunized animals were routinely injected subcutaneously at multiple points on the back. Usually, the blood of the mice was taken one week after the third immunization.
  • the serum titer was 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, and then the color development solution was added for 10 minutes, and the stop solution was added to stop the reaction, and the absorbance value of OD450nm was tested).
  • the serum of the mice was tested every 7 days after each immunization. Generally, when the OD450nm value was positive at a serum dilution of 1:10000, it indicated that the immunization had achieved a good effect (Figure 5), and subsequent experiments could be carried out.
  • Cell fusion is the most important part of 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 the peritoneal antigen with 100 ⁇ g 3 days before fusion. Take spleen cells, wash them twice with serum-free and non-resistant DMEM (20 mL) culture medium, resuspend the cells with 2 mL fusion buffer after centrifugation, count and set aside. The day before fusion, myeloma cells are passaged 1:2. On the day of fusion, SP2/0 is collected, the culture medium is removed by centrifugation, and the cells are washed twice with serum-free and non-resistant DMEM (20 mL).
  • the cells are resuspended with 2 mL fusion buffer, counted and set aside.
  • the electrofusion instrument used is BTX fusion instrument, and the fusion parameters are alternating current 48V, 40s, pulse voltage: 2070V, 30us, PF, 7S.
  • the screening of monoclonal cells is mainly through the detection of hybridoma cell supernatant.
  • the detection methods mainly include ELISA and FACS.
  • ELISA mainly tests the affinity of hybridoma supernatant to TRPV1-nanodisc antigen for immunity. After coating with 2 ⁇ g/mL TRPV1-nanodisc antigen and blocking with 2% BAS, hybridoma supernatant is added for incubation for 1h, goat anti-mouse HRP is added for incubation for 1h, color development solution is added for 10min, and then stop solution is added to terminate the reaction. The absorbance value of OD450nm is tested to test the titer of serum.
  • the ELISA is used for initial screening, and the positive hybridoma supernatant is screened and then flow screening is performed.
  • the flow cytometer used is Beckman Coulter CytoFLEX, and the stable cell line screened is HEK293S-TRPV1.
  • the cells to be tested are suspended cultured and passaged according to the appropriate ratio. 10 ⁇ l of cells are mixed with 10 ⁇ l of trypan blue and added to a hemacytometer for counting. 5E5 cells are added to each well in a 96-well V-type dilution plate.
  • the obtained positive monoclonal cells were expanded and inoculated into 50 mL of serum-free culture medium for suspension culture when the density reached 3 ⁇ 10 5 -5 ⁇ 10 5 /mL.
  • the number of hybridoma cells was counted every day.
  • the culture was generally carried out for 4-5 days.
  • the supernatant was collected by centrifugation, and 200 ⁇ L of Protein A filler was added.
  • the cells were flipped and bound for 2-3 hours.
  • Affinity purification PBS pH 7.3 equilibrium solution, glycine pH 3.0 elution solution
  • the purified antibody was dialyzed with PBS, the buffer was replaced, the concentration was determined, and the purity of the antibody was detected by SDS-PAGE ( Figure 8).
  • the three monoclonal antibodies 29G5, 38A3 and 33D5 were screened, and the hybridoma cells were revived.
  • the density reached 5 ⁇ 10 6 /mL
  • the cells were collected by centrifugation, and the monoclonal cell RNA was extracted according to the FastPure Cell/Tissue Total RNA Isolation Kit of 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 of Novagen.
  • the target band was cut and recovered and the concentration was determined. Then, 2 ⁇ Taq Master Mix was used to add base A at both ends of VH and VL.
  • TaKaRa's pMD TM 18-T Vector Cloning Kit was used to complete the vector construction. After coating, the monoclonal colonies were picked for culture and sent for sequencing. The sequences of VH and VL were obtained after alignment of the sequences (Table 1-3).
  • the antibody function test is mainly to detect calcium flow through an ELISA instrument.
  • the FlexStation3 multi-function plate reader produced by Molecular Devices in the United States can effectively read the fluorescence intensity changes caused by calcium ions flowing into the cell through ion channels.
  • the HEK293T cell line with stable expression of TRPV1 was plated in 96-well plates in advance for adherent culture, grown to a suitable density, and the culture medium was discarded.
  • a membrane-permeable calcium ion concentration indicator (FLIPR Calcium 5Assay kit) prepared with extracellular buffer and antibody solutions of different concentrations (the concentration gradient is adjusted according to needs) were added, and the cells were incubated in an incubator for at least 60 minutes.
  • the positive small molecule was AMG9810, which is a TRPV1 channel-specific inhibitor.
  • the blank control was an extracellular buffer as a solvent, and an extracellular buffer containing a corresponding concentration of cosolvent DMSO (10% DMSO/HBSS or PBS). The whole process was kept away from light. After the incubation was completed, the 96-well plate was placed on the FlexStation3 multi-function plate reader. The sample plate of capsaicine, a specific agonist of TRPV1, was loaded into the sampler of the microplate reader in advance, and the concentration was set to 20 ⁇ M (the final concentration in the well was 5 ⁇ M).
  • the test program was set, and the changes in the fluorescence intensity of each column of cells were immediately monitored after each column of agonists was added. The monitoring lasted for at least two minutes. After all the well plates were tested, the fluorescence increments of all wells before and after the addition of the agonists were calculated according to the quantified fluorescence intensity change curve output by the machine, and the fluorescence increments of the corresponding blank control wells were normalized to obtain the inhibition rate of fluorescence intensity under the action of different concentrations of antibodies.
  • the IC50 values of 38A3, 33D5, AMG9810 and 29G5 were 2.680 ⁇ M, 1.914 ⁇ M, 0.242 ⁇ M and 2.992 ⁇ M, respectively.

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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

一种TRPV1抗体、其制备方法及应用TRPV1 antibody, preparation method and application thereof 技术领域Technical Field

本发明涉及免疫治疗领域，具体涉及一种TRPV1抗体、其制备方法及应用。The present invention relates to the field of immunotherapy, and in particular to a TRPV1 antibody, a preparation method and application thereof.

背景技术Background Art

瞬时感受器电位香草酸亚型1(transient receptor potential vanilloid subfamily 1TRPV1)通道是瞬时感受器电位(transient receptor potential,TRP)通道的一员，是一种配体门控非选择性阳离子通道，与配体结合后引起离子浓度变化，TRPV1被激活时，能引起Ca2+、K+、Mg2+和Na+等阳离子内流，但对Ca2+和Mg2+有相对选择特异性，为其他阳离子的5～10倍左右，TRPV1激活后使胞内阳离子浓度升高，引起相应的生理和病理变化。中枢神经、外周神经、呼吸、消化、心脑血管和泌尿等系统皆可见TRPV1的广泛分布。近年来研究发现，TRPV1通道在病理性疼痛的产生和发展中扮演着重要的角色，有望成为治疗病理性疼痛的新靶点。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. After binding with ligand, it causes changes in ion concentration. When TRPV1 is activated, it can cause the influx of cations such as Ca 2+ , K + , Mg 2+ and Na + , but it has relative selectivity for Ca 2+ and Mg 2+ , which is about 5 to 10 times that of other cations. After TRPV1 is activated, the intracellular cation concentration increases, causing corresponding physiological and pathological changes. TRPV1 is widely distributed in the central nervous system, peripheral nervous system, respiratory, digestive, cardiovascular and urinary systems. In recent years, studies have found that TRPV1 channels play an important role in the generation and development of pathological pain and are expected to become a new target for the treatment of pathological pain.

TRPV1通道一直是开发治疗疼痛的新药物的主要TRP通道，但鲜有靶向TRPV1通道的镇痛药真正应用于临床。很多临床前和临床研究表明，不同类别的TRPV1通道拮抗剂如AMG-517、AZD1386，均会使体温升高，而这种不良反应是阻碍其临床发展的最主要原因。由于小分子药物的副作用比较大，而大分子药物具有其限制中枢神经系统(CNS)渗透性(当靶向外周治疗时)、低免疫原性、高选择性和有利的半衰期的优势，使得研究人员对大分子的研发产生浓厚的兴趣，然而，由于其是一种跨膜蛋白，只有很少的部分暴露在胞外区域，筛选到功能活性抗体仍然是比较大的挑战。TRPV1 channel has always been the main TRP channel for developing new drugs to treat pain, but few analgesics targeting TRPV1 channel are actually 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 its clinical development. Since small molecule drugs have relatively large side effects, and large molecule drugs have the advantages of 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 is still a relatively large challenge.

发明内容Summary of the invention

为了解决现有技术中缺少靶向TRPV1的功能活性抗体的问题，本发明主要提供一种TRPV1抗体、其制备方法及应用，制备过程中所使用的免疫原为nanodisc包装纯化的蛋白，抗体的制备采用杂交瘤技术，亲和力的筛选主要是ELISA和FACS，再结合flexstation3读取细胞通过离子通道流入胞内的钙离子引起的荧光强度变化，筛选出既有亲和力又有功能性的抗体。本发明提供的TRPV1抗体，用于抑制通过刺激激活的TPRV1通路，所述刺激具有与辣椒素相同(或类似)的激活TRPV1的机制，且通过功能的测试发现其具体抑制TRPV1通路的作用。In order to solve the problem of lack of functional active antibodies targeting TRPV1 in the prior art, the present invention mainly provides a TRPV1 antibody, a preparation method and application thereof. The immunogen used in the preparation process is a protein purified by nanodisc packaging. The preparation of the antibody adopts hybridoma technology. The affinity screening is mainly ELISA and FACS, and the fluorescence intensity changes caused by calcium ions flowing into the cell through ion channels are read by flexstation3 to 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, and the stimulation has the same (or similar) mechanism of activating TRPV1 as capsaicin, and its specific inhibitory effect on the TRPV1 pathway is found through functional testing.

为了解决上述技术问题，本发明第一方面提供了一种TRPV1抗体，所述抗体或其变体包括轻链可变区和重链可变区，其中，所述重链可变区包含氨基酸序列分别如SEQ ID NO:11-13所示的HCDR1、HCDR2和HCDR3；和/或，所述轻链可变区包含氨基酸序列分别如SEQ ID NO:14-16所示的LCDR1、LCDR2和LCDR3；或，In order to solve the above technical problems, the first aspect of the present invention provides a TRPV1 antibody, wherein the antibody or its variant comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are respectively shown in SEQ ID NO: 11-13; and/or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are respectively shown in SEQ ID NO: 14-16; or,

所述重链可变区包含氨基酸序列分别如SEQ ID NO:21-23所示的HCDR1、HCDR2和HCDR3；和/或，所述轻链可变区包含氨基酸序列分别如SEQ ID NO:24-26所示的LCDR1、LCDR2和LCDR3；或，The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:21-23, respectively; and/or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:24-26, respectively; or,

所述重链可变区包含氨基酸序列分别如SEQ ID NO:31-33所示的HCDR1、HCDR2和HCDR3；和/或，所述轻链可变区包含氨基酸序列分别如SEQ ID NO:34-36所示的LCDR1、LCDR2和LCDR3。The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, whose amino acid sequences are respectively shown in SEQ ID NO:31-33; and/or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3, whose amino acid sequences are respectively shown in SEQ ID NO:34-36.

在本发明的一些实施方案中，所述重链可变区还包括重链可变区框架区HFWR，和/或，所述轻链可变区还包括轻链可变区框架区LFWR，其中，所述HFWR为人源或小鼠源的重链可变区框架区，所述LFWR为人源或小鼠源抗体的轻链可变区框架区。In some embodiments of the present invention, the heavy chain variable region also includes a heavy chain variable region framework region HFWR, and/or the light chain variable region also 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.

在本发明的一些优选实施方案中，所述轻链可变区包含氨基酸序列如SEQ ID NO:10所示的序列；和/或，所述重链可变区包含氨基酸序列如SEQ ID NO:9所示的序列；或，所述轻链可变区包含氨基酸序列如SEQ ID NO:20 所示的序列；和/或，所述重链可变区包含氨基酸序列如SEQ ID NO:19所示的序列；或，所述轻链可变区包含氨基酸序列如SEQ ID NO:30所示的序列；和/或，所述重链可变区包含氨基酸序列如SEQ ID NO:29所示的序列。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. The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 19; or the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 30; and/or the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 29.

在本发明的一些更优选实施方案中，所述的抗体或其变体还包括重链恒定区和轻链恒定区。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.

在本发明的一些进一步更优选实施方案中，所述的抗体重链恒定区为人源或小鼠源抗体重链恒定区；所述的抗体轻链恒定区为人源或小鼠源抗体轻链恒定区。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.

在本发明的一些实施方案中，所述的抗体为以下任何一种抗体形式：In some embodiments of the present invention, the antibody is in any of the following antibody forms:

(a)一种完整的免疫球蛋白分子；(a) a complete immunoglobulin molecule;

(b)一种scFv；(b) a scFv;

(c)一种包含scFv的融合蛋白；(c) a fusion protein comprising scFv;

(d)一种Fab片段；(d) a Fab fragment;

(e)一种Fab′片段；(e) a Fab′ fragment;

(f)一种F(ab)2；(f) a F(ab) 2 ;

或者，所述的抗体为单克隆抗体或者多克隆抗体；Alternatively, the antibody is a monoclonal antibody or a polyclonal antibody;

或者，所述的抗体为人源化抗体或者双特异性抗体。Alternatively, the antibody is a humanized antibody or a bispecific antibody.

为了解决上述技术问题，本发明第二方面提供了一种嵌合抗原受体，其包含如本发明第一方面所述的抗体。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.

为了解决上述技术问题，本发明第三方面提供了一种分离的核酸，其编码如本发明第一方面所述的抗体、或如本发明第二方面所述的嵌合抗原受体。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.

在本发明的一些优选实施方案中，编码所述抗体的核酸包含如SEQ ID NO:7所示和/或如SEQ ID NO:8所示的多核苷酸序列；或，包含如SEQ ID NO:17所示和/或如SEQ ID NO:18所示的多核苷酸序列；或，包含如SEQ ID NO:27所示和/或如SEQ ID NO:28所示的多核苷酸序列。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.

为了解决上述技术问题，本发明第四方面提供了一种重组表达载体，其 包含如本发明第三方面所述的分离的核酸。In order to solve the above technical problems, the fourth aspect of the present invention provides a recombinant expression vector, which It comprises the isolated nucleic acid as described in the third aspect of the present invention.

为了解决上述技术问题，本发明第五方面提供了一种转化体，其在宿主细胞中包含如第四方面所述的重组表达载体。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.

为了解决上述技术问题，本发明第六方面提供了一种TRPV1抗体的制备方法，其包括培养如本申请第五方面所述的转化体，从培养物中获得所述抗体。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.

为了解决上述技术问题，本发明第七方面提供了一种抗体药物偶联物，其包含细胞毒性剂，以及如本发明第一方面所述的抗体。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 as described in the first aspect of the present invention.

为了解决上述技术问题，本发明第八方面提供了一种药物组合物，其包含如本发明第一方面所述的抗体、如本发明第二方面所述的嵌合抗原受体或如本发明第七方面所述的抗体药物偶联物，以及药学上可接受的载体。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.

为了解决上述技术问题，本发明第九方面提供了如本发明第一方面所述的抗体、如本发明第二方面所述的嵌合抗原受体、如本发明第三方面所述的分离的核酸、如本发明第四方面所述的重组表达载体、如本发明第五方面所述的转化体、如本发明第七方面所述的抗体药物偶联物和/或如本发明第八方面所述的药物组合物在制备治疗疼痛的药物中的应用。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 drug for treating pain.

在本发明的一些优选实施方案中，所述疼痛为TRPV1激活导致的疼痛；所述药物的靶点为TRPV1。In some preferred embodiments of the present invention, the pain is pain caused by activation of TRPV1; and the target of the drug is TRPV1.

为了解决上述技术问题，本发明第十方面提供了一种试剂盒，其包括如本发明第一方面所述的抗体、如本发明第二方面所述的嵌合抗原受体、如本发明第七方面所述的抗体药物偶联物和/或如本发明第八方面所述的药物组合物。In order to solve the above technical problems, the tenth aspect of the present invention provides a kit, which includes 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 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.

为了解决上述技术问题，本发明第十一方面提供了一种检测TRPV1的方法，其包括使用本发明第一方面所述的抗体、如本发明第二方面所述的嵌合抗原受体、如本发明第七方面所述的抗体药物偶联物、如本发明第八方面所述的药物组合物和/或如本发明第十方面所述的试剂盒与样品接触。 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.

在本发明的一些优选实施方案中，所述检测为非诊断目的。In some preferred embodiments of the present invention, the detection is for non-diagnostic purposes.

为了解决上述技术问题，本发明第十二方面提供了一种诊断、治疗和/或预防疼痛的方法，所述方法包括向有需要的患者施用治疗有效量的如本发明第一方面所述的抗体、如本发明第二方面所述的嵌合抗原受体、如本发明第三方面所述的分离的核酸、如本发明第四方面所述的重组表达载体、如本发明第五方面所述的转化体、如本发明第七方面所述的抗体药物偶联物、如本发明第八方面所述的药物组合物和/或如本发明第十方面所述的试剂盒。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 method 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.

在本发明的一些优选实施方案中，所述疼痛为TRPV1激活导致的疼痛；所述药物的靶点为TRPV1。In some preferred embodiments of the present invention, the pain is pain caused by activation of TRPV1; and the target of the drug is TRPV1.

为了解决上述技术问题，本发明第十三方面提供了本发明第一方面所述的抗体、如本发明第二方面所述的嵌合抗原受体、如本发明第三方面所述的分离的核酸、如本发明第四方面所述的重组表达载体、如本发明第五方面所述的转化体、如本发明第七方面所述的抗体药物偶联物、如本发明第八方面所述的药物组合物和/或如本发明第十方面所述的试剂盒，其用于诊断、治疗和/或预防疼痛。In order to solve the above technical problems, the thirteenth aspect of the present invention provides 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, which are used for diagnosing, treating and/or preventing pain.

在本发明的一些优选实施方案中，所述疼痛为TRPV1激活导致的疼痛；所述药物的靶点为TRPV1。In some preferred embodiments of the present invention, the pain is pain caused by activation of TRPV1; and the target of the drug is TRPV1.

在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

本发明所用试剂和原料均市售可得。The reagents and raw materials used in the present invention are commercially available.

本发明的积极进步效果在于：本发明提供的抗TRPV1的抗体对TRPV1具有比较高的亲和力，其IC50低于2.994μM，最低可达1.914μM，对Capsaicine(辣椒素)激活的TRPV1通道具有明显的抑制作用。The positive improvement effect of the present invention is that the anti-TRPV1 antibody provided by the present invention has a relatively high affinity for TRPV1, with an IC 50 lower than 2.994 μM, and a minimum of 1.914 μM, and has a significant inhibitory effect on the TRPV1 channel activated by capsaicine.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1为TRPV1序列构建在pEGBacMam载体上，图中展示的是通过无 缝克隆技术将人源TRPV1序列克隆在pEGBacMam载体上。Figure 1 shows the construction of the TRPV1 sequence on the pEGBacMam vector. The human TRPV1 sequence was cloned into the pEGBacMam vector using the slot cloning technique.

图2为MSP2N2序列构建在pET 28a载体上。Figure 2 shows the MSP2N2 sequence constructed on the pET 28a vector.

图3为TRPV1去垢剂蛋白纯化结果。图中展示的是去垢剂形式的人源TRPV1蛋白纯化的SEC图与SDS-PAGE图。Figure 3 shows the results of TRPV1 detergent protein purification. The figure shows the SEC and SDS-PAGE images of the purification of human TRPV1 protein in detergent form.

图4为TRPV1-Nanodisc蛋白纯化结果。FIG. 4 shows the results of TRPV1-Nanodisc protein purification.

图5为TRPV1-Nanodisc蛋白免疫小鼠第4、5免血清效价检测结果。FIG. 5 shows the titer test results of the 4th and 5th sera from mice immunized with TRPV1-Nanodisc protein.

图6为筛选获得三个抗体ELISA检测结果，其中，A、B和C分别为29G5、38A3和33D5的ELISA检测结果。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.

图7为筛选获得三个抗体FACS检测结果。其中，A、B和C分别为29G5、38A3和33D5的FACS检测结果。Figure 7 shows the FACS test results of three antibodies obtained by screening, wherein A, B and C are the FACS test results of 29G5, 38A3 and 33D5 respectively.

图8为纯化筛选获得三个抗体SDS-PAGE图。FIG8 is an SDS-PAGE diagram of three antibodies obtained by purification and screening.

图9为筛选获得三个抗体抑制作用，A、B、C和D分别为AMG9810、33D5、29G5和38A3的抑制作用。FIG9 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.

具体实施方式DETAILED DESCRIPTION

下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

实施例1基因合成及质粒构建Example 1 Gene synthesis and plasmid construction

我们将野生型TRPV1的基因、MBP基因、改造克隆序列、MSP2N2送由南京擎科生物技术有限公司进行合成，其中MBP+TRPV1序列携带EcoR1和Not1酶切位点，连接重组至pEGBacMam质粒(图1)，MSP2N2连接重组至pET-28a(图2)。We sent the wild-type TRPV1 gene, MBP gene, modified clone sequence, and MSP2N2 to Nanjing Qingke Biotechnology Co., Ltd. for synthesis, in which 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).

①6×His标签序列与MBP序列之间，以及MBP与HRV 3C蛋白酶切割位点间通过linker连接。①The 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.

MBP氨基酸序列(SEQ ID NO:1)：
MBP amino acid sequence (SEQ ID NO: 1):

MBP碱基序列(SEQ ID NO:2)：

MBP base sequence (SEQ ID NO: 2):

MSP2N2氨基酸序列(SEQ ID NO:3)：
MSP2N2 amino acid sequence (SEQ ID NO: 3):

MSP2N2碱基序列(SEQ ID NO:4)：

MSP2N2 base sequence (SEQ ID NO:4):

野生型人源TRPV1(TRPV1-WT)氨基酸序列(SEQ ID NO:5)：

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

野生型人源TRPV1(TRPV1-WT)碱基序列(SEQ ID NO:6)：


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


实施例2重组杆状病毒的制备Example 2 Preparation of recombinant baculovirus

2.1、通过热激转化，将含有目的基因的重组pEGBacMam质粒导入到大肠杆菌2.1. Introduce the recombinant pEGBacMam plasmid containing the target gene into Escherichia coli through heat shock transformation

DH10Bac感受态细胞(上海唯地)中，在含有50μg/mL卡那霉素(aladdin)、7μg/mL庆大霉素(aladdin)、10μg/mL四环霉素(aladdin)、100μg/mL Bluo-gal(Thermofish)、40μg/mLIPTG(aladdin)的LB固体培养基中，于37℃培养48-72小时。挑选均匀的白斑至5mL含有三种抗生素(50μg/mL卡那霉素、7μg/mL庆大霉素、10μg/mL四环霉素)的LB液体培养基中，37℃，200rpm条件下培养12-16小时，抽提重组杆状病毒质粒。DH10Bac competent cells (Shanghai Weidi) were cultured at 37°C for 48-72 hours in LB solid medium containing 50μg/mL kanamycin (aladdin), 7μg/mL gentamicin (aladdin), 10μg/mL tetracycline (aladdin), 100μg/mL Bluo-gal (Thermofish), and 40μg/mL IPTG (aladdin). Uniform white spots were selected and added to 5mL LB liquid medium containing three antibiotics (50μg/mL kanamycin, 7μg/mL gentamicin, and 10μg/mL tetracycline), and cultured at 37°C and 200rpm for 12-16 hours to extract the recombinant bacillivirus plasmid.

2.2、取细胞培养6孔板(Nest),每孔1×106/2mL sf9昆虫细胞，于27℃恒温恒湿培养箱中贴壁培养30min，取100μL Insect Medium(Sf-900TM III SFM)加入10μL转染试剂(Cellfectin)，另100μL Insect Medium加入5μg重组杆状病毒质粒，混合后室温下孵育20分钟，将转染复合物均匀滴加到6孔板中，于27℃条件下继续培养72小时，于4℃6000rpm离心15分钟，取上清加入2％的FBS，避光4度保存即为P1代重组杆状病毒。2.2. Take a 6-well cell culture plate (Nest), with 1×10 6 /2mL sf9 insect cells in each well, and culture them in a constant temperature and humidity incubator at 27°C for 30 minutes. Take 100μL Insect Medium (Sf-900 TM III SFM) and add 10μL transfection reagent (Cellfectin). Add 5μg recombinant baculovirus plasmid to another 100μL Insect Medium. Incubate at room temperature for 20 minutes after mixing. Evenly add the transfection complex to the 6-well plate, continue to culture at 27°C for 72 hours, centrifuge at 4°C 6000rpm for 15 minutes, take the supernatant and add 2% FBS, and store it at 4 degrees in the dark to obtain the P1 generation recombinant baculovirus.

取P1代重组杆状病毒按照1:100的比例感染2mL细胞密度为 5×105/mLsf9昆虫细胞，27℃的条件下贴壁进行培养72小时，于4℃6000rpm离心15分钟，取上清加入2％的FBS，避光4度保存即为P2代重组杆状病毒。Take the P1 recombinant baculovirus and infect 2 mL of cells at a ratio of 1:100. 5×10 5 /mL sf9 insect cells were cultured at 27°C for 72 hours, centrifuged at 4°C and 6000 rpm for 15 minutes, the supernatant was added with 2% FBS, and stored at 4°C in the dark to obtain the P2 generation recombinant baculovirus.

分出密度为1×106/mL的40mL sf9昆虫细胞，培养24小时后，取P2代重组杆状病毒按照1:100的比例感染，27℃，120rpm的条件下进行培养96小时，于4℃6000rpm离心15分钟，上清液用0.22μm过滤装置过滤后，加入2％的FBS，避光4度保存即为P3代重组杆状病毒。40 mL of sf9 insect cells with a density of 1×10 6 /mL were separated and cultured for 24 hours. The P2 generation recombinant baculovirus was used for infection at a ratio of 1:100. The cells were cultured at 27°C and 120 rpm for 96 hours. The cells were centrifuged at 4°C and 6000 rpm for 15 minutes. The supernatant was filtered with a 0.22 μm filter device, 2% FBS was added, and the cells were stored at 4°C in the dark to obtain the P3 generation recombinant baculovirus.

分出密度为1×106/mL的200mL sf9昆虫细胞，培养24小时后，取P3代重组杆状病毒按照1:100的比例感染，27℃，120rpm的条件下进行培96小时，于4℃6000rpm离心15分钟，上清液用0.22μm过滤装置过滤后，加入2％的FBS，避光4度保存即为P4代重组杆状病毒。200 mL of sf9 insect cells with a density of 1×10 6 /mL were separated and cultured for 24 hours. The P3 generation recombinant baculovirus was used for infection at a ratio of 1:100. The cells were cultured at 27°C and 120 rpm for 96 hours. The cells were centrifuged at 4°C and 6000 rpm for 15 minutes. The supernatant was filtered with a 0.22 μm filter device, 2% FBS was added, and the cells were stored at 4°C in the dark to obtain the P4 generation recombinant baculovirus.

实施例3蛋白纯化Example 3 Protein Purification

将HEK-293S细胞传代，浓度为1.5×106/mL，37℃，8％CO2，60％湿度，120rpm条件下培养24h，取P4代重组杆状病毒按照1:10的比例感染哺乳动物细胞，摇床培养12-18h加入丁酸钠，终浓度为10mM。HEK-293S cells were passaged at a concentration of 1.5×10 6 /mL, cultured at 37°C, 8% CO 2 , 60% humidity, and 120 rpm for 24 h, and P4 generation recombinant baculovirus was used to infect mammalian cells at a ratio of 1:10. Sodium butyrate was added for 12-18 h on a shaking platform to a final concentration of 10 mM.

72小时后，4℃，6000rpm离心15分钟，收集细胞，用Lysis buffer(50mM HEPES,pH7.4,150mM NaCl,1％Protease Inhibitor Cocktail,EDTA-Free,0.5％LMNG/0.05％CHS,1mM TCEP)重悬细胞沉淀，4℃下翻转溶膜3小时。溶膜结束后，于4℃，40000rpm条件下离心45分钟，上清液与MBP亲和层析填料4℃翻转结合2.5小时，结合上清液过重力柱，用Wash buffer(50mM HEPES,pH7.4，150mM NaCl，1mM TCEP，0.01％LMNG/0.001％CHS)洗脱杂蛋白，Elute buffer(50mM HEPES,pH7.4,150mM NaCl,0.01％LMNG/0.001％CHS，1mM TCEP,40mM Maltose)洗脱目的蛋白100KDa的超滤管对目的蛋白进行浓缩，浓缩至体积为500μL，进行凝胶过滤层析，所用凝胶柱型号为Superrose 6Increase 10/300GL(cytiva)，缓冲液为SEC buffer (50mM HEPES,pH7.4,150mM NaCl，1mM TCEP,0.01％LMNG/0.001％CHS)，收集蛋白样品，测试A280浓度，进行SDS-PAGE凝胶电泳检测目的蛋白大小与纯度(图3)。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 the membrane was lysed by flipping at 4°C for 3 hours. After the membrane was dissolved, the mixture was centrifuged at 4°C and 40,000 rpm for 45 minutes. The supernatant was flipped and combined with the MBP affinity chromatography filler at 4°C for 2.5 hours. The combined supernatant was passed through a gravity column. Wash buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM TCEP, 0.01% LMNG/0.001% CHS) was used to elute the impurities. Elute buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.01% LMNG/0.001% CHS, 1 mM TCEP, 40 mM Maltose) was used to elute the target protein. The target protein was concentrated to a volume of 500 μL using a 100 KDa ultrafiltration tube and subjected to gel filtration chromatography. The gel column model used was Superrose 6 Increase 10/300 GL (cytiva) and the buffer was SEC buffer. (50 mM 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).

按照TRPV1:MSP2N2:soybean＝1:3:200物质的量比例进行包装。计算需要的Soybean Polar Lipid Extract Polar(Avanti)，蛋白上样针吸取母液(soybean母液为25mg/mL)，将soybean母液打到一干净干燥玻璃管的底部，然后用氮吹仪将玻璃管中soybean母液的氯仿吹干，会在玻璃管底部形成一点淡黄色残留物。再加入SEC buffer，超声10min，期间玻璃管中液体会先出现白色混浊再逐渐澄清后变成淡黄色的溶液，按比例将TRPV1，MSP2N2，soybean加入EP管，4℃翻转1h。随后加入100mg/mL Bio-beads SM-2用于除去溶液中的detergent，4℃翻转过夜。使用重力柱过滤除去Bio-beads SM-2，将滤过的蛋白液浓缩至体积为500μL，13000rpm，4℃离心10min，进行凝胶过滤层析，所用凝胶柱型号为Superrose 6Increase 10/300GL(cytiva)，缓冲液为SEC buffer(50mM HEPES,pH7.4,150mM NaCl)，收集蛋白样品，测试A280浓度，进行SDS-PAGE凝胶电泳检测目的蛋白大小与纯度(图4)。Package according to the molar ratio of TRPV1:MSP2N2:soybean=1:3:200. Calculate the required Soybean Polar Lipid Extract Polar (Avanti), draw up the mother liquor with the protein loading needle (soybean mother liquor is 25mg/mL), and beat the soybean mother liquor to the bottom of a clean and dry glass tube. Then use a nitrogen blower to blow dry the chloroform of the soybean mother liquor in the glass tube, which will form a little light yellow residue at the bottom of the glass tube. Then add SEC buffer and sonicate for 10 minutes. During this period, the liquid in the glass tube will first appear white and turbid, then gradually clarify and become a light yellow solution. Add TRPV1, MSP2N2, and soybean to the EP tube in proportion and flip at 4℃ for 1h. Then add 100mg/mL Bio-beads SM-2 to remove the detergent in the solution, and flip overnight at 4℃. Use gravity column filtration to remove Bio-beads SM-2, concentrate the filtered protein solution to a volume of 500 μL, centrifuge at 13000 rpm, 4°C for 10 min, and perform gel filtration chromatography. The gel column model used is Superrose 6 Increase 10/300 GL (cytiva), and the buffer is SEC buffer (50 mM HEPES, pH 7.4, 150 mM NaCl). Collect protein samples, test A280 concentration, and perform SDS-PAGE gel electrophoresis to detect the size and purity of the target protein (Figure 4).

实施例4动物免疫Example 4 Animal Immunization

用抗原对动物(通常是小鼠)进行免疫是单克隆抗体制备过程的第一步，也是至关重要的一步。动物对抗原免疫应答反应是否良好，能否产生滴度高，特异性好的抗体直接决定了后期筛选单克隆杂交瘤细胞株的难度和所获抗体的有效性。Immunizing animals (usually mice) with antigens is the first and most important step in the preparation of monoclonal antibodies. Whether the animal's immune response to the antigen is good and whether it can produce antibodies with high titers and good specificity directly determines the difficulty of later screening of monoclonal hybridoma cell lines and the effectiveness of the antibodies obtained.

免疫所用的抗原我们采用TRPV1-nanodisc的蛋白制备方法所生产的蛋白。免疫的动物选取balb/c，6-8周雌鼠，初次免疫TRPV1-nanodics抗原用量为100μg蛋白/只，与弗氏完全佐剂按体积比1:1混合，抗原体积不足部分用对应蛋白的纯化用buffer代替，经过震荡仪乳化，常规背部多点皮下注射免疫动物。之后每间隔14天进行一次免疫，抗原用量为初次免疫一半的 抗原量(50μg/只)，与弗氏不完全佐剂按体积比1:1混合，抗原体积不足部分用对应蛋白的纯化用buffer代替，经过乳化，常规背部多点皮下注射免疫动物。通常情况下，完成第三次免疫之后间隔一周会取小鼠的血，分离血清后，通过ELISA的方法(包被RPV1-nanodics抗原，2％BAS封闭后加梯度稀释的血清孵育1h，加羊抗鼠的HRP孵育1h，加显色液显色10min后，加终止液终止反应，测试OD450nm的吸光度值)来测试血清的效价，之后每加免一次都间隔7天测试小鼠的血清，一般血清稀释度1:10000时OD450nm值为阳性时说明免疫已经达到较好的效果(图5)，可以进行后续的实验。The antigen used for immunization was produced by the protein preparation method of TRPV1-nanodisc. The animals for immunization were balb/c, 6-8 weeks old female mice. The amount of TRPV1-nanodics antigen used for the first immunization was 100 μg protein/mouse, mixed with Freund's complete adjuvant at a volume ratio of 1:1. The insufficient antigen volume was replaced with the corresponding protein purification buffer, emulsified by a shaker, and routinely injected subcutaneously at multiple points on the back to immunize the animals. After that, immunization was performed every 14 days, and the amount of antigen used was half of the initial immunization. The amount of antigen (50 μg/mouse) was mixed with Freund's incomplete adjuvant at a volume ratio of 1:1. The insufficient amount of antigen was replaced by the corresponding protein purification buffer, and after emulsification, the immunized animals were routinely injected subcutaneously at multiple points on the back. Usually, the blood of the mice was taken one week after the third immunization. After the serum was separated, the serum titer was 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, and then the color development solution was added for 10 minutes, and the stop solution was added to stop the reaction, and the absorbance value of OD450nm was tested). After that, the serum of the mice was tested every 7 days after each immunization. Generally, when the OD450nm value was positive at a serum dilution of 1:10000, it indicated that the immunization had achieved a good effect (Figure 5), and subsequent experiments could be carried out.

实施例5细胞融合Example 5 Cell Fusion

细胞融合是杂交瘤方法的最重要的环节，融合效率的高低直接影响能否筛选到特异性的抗体，取效价达到融合需求的小鼠，在融合前3天进行腹腔抗原100μg的加强，取脾细胞，用无血清、无抗性的DMEM(20mL)培养基清洗两遍，离心后用2mL融合缓冲液重悬细胞，计数备用。融合前一天骨髓瘤细胞1:2传代，融合当天收集SP2/0，离心去除培养基，用无血清、无抗性的DMEM(20mL)清洗两遍，离心后用2mL融合缓冲液重悬细胞，计数，取备用。将脾细胞和SP2/0各取8E7个细胞至一新的50mL出离心管中，加入20mL融合缓冲液，以1:1的比例一起离心(500x g下离心5分钟)2次，之后用8mL融合缓冲液重悬后加入电转杯中进行电融合，电融合仪器使用的是BTX融合仪，融合参数为交替电流48V，40s，脉冲电压：2070V，30us，PF，7S。静置10min，取全部融合液加入到已经配制好的600mL培养基中，其中培养基中的主要成分为(75％DMEM+20％FBS+1％PS+2％HAT(50×)+1％Glu+1％OPI)，37℃静置培养2h。Cell fusion is the most important part of 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 the peritoneal antigen with 100 μg 3 days before fusion. Take spleen cells, wash them twice with serum-free and non-resistant DMEM (20 mL) culture medium, resuspend the cells with 2 mL fusion buffer after centrifugation, count and set aside. The day before fusion, myeloma cells are passaged 1:2. On the day of fusion, SP2/0 is collected, the culture medium is removed by centrifugation, and the cells are washed twice with serum-free and non-resistant DMEM (20 mL). After centrifugation, the cells are resuspended with 2 mL fusion buffer, counted and set aside. Take 8E7 cells of spleen cells and SP2/0 cells respectively and put them into a new 50mL centrifuge tube, add 20mL fusion buffer, centrifuge them together at a ratio of 1:1 (centrifuge at 500x g for 5 minutes) twice, then resuspend them with 8mL fusion buffer and put them into the electroporation cup for electrofusion. The electrofusion instrument used is BTX fusion instrument, and the fusion parameters are alternating current 48V, 40s, pulse voltage: 2070V, 30us, PF, 7S. Let it stand for 10min, take all the fusion solution and add it to the prepared 600mL culture medium, in which the main components of the culture medium are (75% DMEM + 20% FBS + 1% PS + 2% HAT (50×) + 1% Glu + 1% OPI), and let it stand at 37℃ for 2h.

混匀后铺42板，每孔150μL。融合第四天用添加HT的培养基全换液，第7天取上清测试。 After mixing, spread 42 plates with 150 μL per well. On the fourth day of fusion, replace the medium with HT-added medium and take the supernatant for testing on the seventh day.

实施例6单克隆细胞筛选Example 6 Monoclonal cell screening

单克隆细胞的筛选主要通过检测杂交瘤细胞的上清，检测使用的方法主要包括ELISA和FACS，ELISA主要是测试杂交瘤上清与免疫用TRPV1-nanodisc抗原的亲和力，包被2μg/mL的TRPV1-nanodisc抗原，2％BAS封闭后加杂交瘤上清孵育1h，加羊抗鼠的HRP孵育1h，加显色液显色10min后，加终止液终止反应，测试OD450nm的吸光度值)来测试血清的效价通过ELISA初筛，筛选阳性的杂交瘤上清再进行流式筛选，所使用的流式细胞仪为beckman coulter CytoFLEX，筛选的稳定细胞系为HEK293S-TRPV1，使用的，待测细胞为悬浮培养，按照适合的比例进行传代，取10μl细胞与10μl台盼蓝混合，加入血球计数板，计数，每孔加入5E5个细胞于96孔V型稀释板中。1500rpm离心5分钟，拍板去除培养基，加入ELISA阳性的杂交瘤上清4℃孵育1小时，使用PBS清洗3次后，加入1:200稀释的二抗(jackson，Allophycocyanin-AffiniPure F(ab')2Fragment Donkey Anti-Mouse lgG(H+L))，4℃孵育30分钟后PBS清洗3次，最后每孔加入150μl PBS重悬细胞，转移至1.5mL EP管中，或者直接使用微孔板上样，Beckman CytoFLEX检测，使用Cytoexpert将MFI值输出，通过与阳性对照和阴性对照的差异，来分析数据，得到阳性结果，通过有限稀释的方法进行两轮的亚克隆，最终筛选获得阳性的单克隆细胞(图6，图7)。The screening of monoclonal cells is mainly through the detection of hybridoma cell supernatant. The detection methods mainly include ELISA and FACS. ELISA mainly tests the affinity of hybridoma supernatant to TRPV1-nanodisc antigen for immunity. After coating with 2μg/mL TRPV1-nanodisc antigen and blocking with 2% BAS, hybridoma supernatant is added for incubation for 1h, goat anti-mouse HRP is added for incubation for 1h, color development solution is added for 10min, and then stop solution is added to terminate the reaction. The absorbance value of OD450nm is tested to test the titer of serum. The ELISA is used for initial screening, and the positive hybridoma supernatant is screened and then flow screening is performed. The flow cytometer used is Beckman Coulter CytoFLEX, and the stable cell line screened is HEK293S-TRPV1. The cells to be tested are suspended cultured and passaged according to the appropriate ratio. 10μl of cells are mixed with 10μl of trypan blue and added to a hemacytometer for counting. 5E5 cells are added to each well in a 96-well V-type dilution plate. Centrifuge at 1500 rpm for 5 minutes, tap the plate to remove the culture medium, add ELISA-positive hybridoma supernatant and incubate at 4°C for 1 hour, wash three times with PBS, add 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 wash three times with PBS. Finally, add 150 μl PBS to each well to resuspend the cells, transfer to a 1.5 mL EP tube, or directly use a microtiter plate for loading, Beckman CytoFLEX for detection, use Cytoexpert to output the MFI value, analyze the data by the difference with the positive control and negative control, and obtain a positive result. Perform two rounds of subcloning by the limiting dilution method, and finally screen to obtain positive monoclonal cells (Figures 6 and 7).

实施例7单克隆抗体制备Example 7 Monoclonal Antibody Preparation

获得的阳性单克隆细胞扩培，待密度达到3×105～5×105/mL接入的50mL无血清的培养基中悬浮培养，每天计数杂交瘤的细胞量，一般培养4-5天，离心收集上清，加200μL的ProteinA的填料，翻转结合2-3h，亲和纯化(PBS pH7.3平衡液，甘氨酸pH3.0洗脱液)，纯化完成的抗体PBS透析，置换缓冲液，测定浓度并通过SDS-PAGE检测抗体的纯度(图8)。 The obtained positive monoclonal cells were expanded and inoculated into 50 mL of serum-free culture medium for suspension culture when the density reached 3×10 5 -5×10 5 /mL. The number of hybridoma cells was counted every day. The culture was generally carried out for 4-5 days. The supernatant was collected by centrifugation, and 200 μL of Protein A filler was added. The cells were flipped and bound for 2-3 hours. Affinity purification (PBS pH 7.3 equilibrium solution, glycine pH 3.0 elution solution) was performed. The purified antibody was dialyzed with PBS, the buffer was replaced, the concentration was determined, and the purity of the antibody was detected by SDS-PAGE (Figure 8).

实施例8抗体序列获取Example 8 Antibody Sequence Acquisition

筛选获得的三个单克隆抗体29G5、38A3和33D5，复苏杂交瘤细胞，待密度达到5×106/mL，离心收集细胞，根据诺唯赞的FastPure Cell/Tissue Total RNA Isolation Kit完成单克隆细胞RNA的提取，之后已提取的RNA为模板，用诺唯赞的HiScript-TS 5'/3'RACE Kit完成单抗VH、VL链可变区的扩增，凝胶电泳后目的条带切胶回收后测定浓度，接着用2×Taq Master Mix完成VH和VL两端碱基A的添加，最后用TaKaRa的pMDTM18-T Vector Cloning Kit完成载体构建，涂布完成，之后挑单克隆菌落培养，送测序，测序完成的序列比对后获得VH、VL的序列(表1-3)。The three monoclonal antibodies 29G5, 38A3 and 33D5 were screened, and the hybridoma cells were revived. When the density reached 5×10 6 /mL, the cells were collected by centrifugation, and the monoclonal cell RNA was extracted according to the FastPure Cell/Tissue Total RNA Isolation Kit of 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 of Novagen. After gel electrophoresis, the target band was cut and recovered and the concentration was determined. Then, 2×Taq Master Mix was used to add base A at both ends of VH and VL. Finally, TaKaRa's pMD TM 18-T Vector Cloning Kit was used to complete the vector construction. After coating, the monoclonal colonies were picked for culture and sent for sequencing. The sequences of VH and VL were obtained after alignment of the sequences (Table 1-3).

表1 29G5抗体V序列信息

Table 1 29G5 antibody V sequence information

表2 38A3抗体V序列信息

Table 2 38A3 antibody V sequence information

表3 33D5抗体V序列信息
Table 3 33D5 antibody V sequence information

实施例9单克隆抗体功能测试Example 9 Monoclonal Antibody Function Test

抗体功能的测试主要是通过酶标仪检测钙流的方法，使用美国Molecular Devices生产的FlexStation3多功能读板机可以有效地读取细胞通过离子通道流入胞内的钙离子引起的荧光强度变化。将TRPV1稳定表达的HEK293T细胞系提前铺至96孔板贴壁培养，生长到合适密度，弃去培养液，同时加入用细胞外缓冲液配制的可透膜的钙离子浓度指示剂(FLIPR Calcium 5Assay kit)和不同浓度的抗体溶液(浓度梯度根据需求调整)，在培养箱中对细胞进行孵育至少60分钟，阳性小分子为AMG9810，是TRPV1通道特异性的抑制剂，空白对照为作为溶剂的细胞外缓冲液，以及包含相应浓度的助溶剂DMSO的细胞外缓冲液(10％DMSO/HBSS或者PBS)。全程避光放置，孵育完成后将96孔板置于FlexStation3多功能读板机，提前将TRPV1的特异性激动剂Capsaicine(辣椒素)样品板装载到酶标仪的加样器中，设置浓度为20μM(孔中终浓度为5μM)，设定好测试程序，每加一列激动剂立即开始监测这一列细胞内荧光强度的变化，至少监测两分钟，所有孔板测试完成之后，根据机器输出的量化的荧光强度变化曲线计算所有孔在加激动剂前后的荧光增值，并和相应的空白对照孔的荧光增值做均一化处理，得出不同浓度抗体作用下荧光强度的抑制率，在graphpad中通过曲线拟合(Y＝100/(1+10^((LogIC50-X)*HillSlope)))得出浓度对数对抑制率的抑制曲线，计算出抗体抑制通道的半抑制浓度IC50(图9)，以此来判断抗体对TRPV1的抑制效果。38A3、33D5、AMG9810和29G5的IC50分别为2.680μM、1.914μM、0.242μM和2.992μM。 The antibody function test is mainly to detect calcium flow through an ELISA instrument. The FlexStation3 multi-function plate reader produced by Molecular Devices in the United States can effectively read the fluorescence intensity changes caused by calcium ions flowing into the cell through ion channels. The HEK293T cell line with stable expression of TRPV1 was plated in 96-well plates in advance for adherent culture, grown to a suitable density, and the culture medium was discarded. At the same time, a membrane-permeable calcium ion concentration indicator (FLIPR Calcium 5Assay kit) prepared with extracellular buffer and antibody solutions of different concentrations (the concentration gradient is adjusted according to needs) were added, and the cells were incubated in an incubator for at least 60 minutes. The positive small molecule was AMG9810, which is a TRPV1 channel-specific inhibitor. The blank control was an extracellular buffer as a solvent, and an extracellular buffer containing a corresponding concentration of cosolvent DMSO (10% DMSO/HBSS or PBS). The whole process was kept away from light. After the incubation was completed, the 96-well plate was placed on the FlexStation3 multi-function plate reader. The sample plate of capsaicine, a specific agonist of TRPV1, was loaded into the sampler of the microplate reader in advance, and the concentration was set to 20 μM (the final concentration in the well was 5 μM). The test program was set, and the changes in the fluorescence intensity of each column of cells were immediately monitored after each column of agonists was added. The monitoring lasted for at least two minutes. After all the well plates were tested, the fluorescence increments of all wells before and after the addition of the agonists were calculated according to the quantified fluorescence intensity change curve output by the machine, and the fluorescence increments of the corresponding blank control wells were normalized to obtain the inhibition rate of fluorescence intensity under the action of different concentrations of antibodies. The inhibition curve of the logarithm of concentration against the inhibition rate was obtained by curve fitting (Y=100/(1+10^((LogIC50-X)*HillSlope))) in Graphpad, and the half-inhibitory concentration IC50 of the antibody inhibition channel was calculated (Figure 9), so as to judge the inhibitory effect of the antibody on TRPV1. The IC50 values of 38A3, 33D5, AMG9810 and 29G5 were 2.680 μM, 1.914 μM, 0.242 μM and 2.992 μM, respectively.

Claims (15)

一种TRPV1抗体，其特征在于，所述抗体或其变体包括轻链可变区和重链可变区，其中，所述重链可变区包含氨基酸序列分别如SEQ ID NO:11-13所示的HCDR1、HCDR2和HCDR3；和/或，所述轻链可变区包含氨基酸序列分别如SEQ ID NO:14-16所示的LCDR1、LCDR2和LCDR3；或，A TRPV1 antibody, characterized in that the antibody or its variant comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are respectively shown in SEQ ID NO:11-13; and/or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are respectively shown in SEQ ID NO:14-16; or, 所述重链可变区包含氨基酸序列分别如SEQ ID NO:21-23所示的HCDR1、HCDR2和HCDR3；和/或，所述轻链可变区包含氨基酸序列分别如SEQ ID NO:24-26所示的LCDR1、LCDR2和LCDR3；或，The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:21-23, respectively; and/or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:24-26, respectively; or, 所述重链可变区包含氨基酸序列分别如SEQ ID NO:31-33所示的HCDR1、HCDR2和HCDR3；和/或，所述轻链可变区包含氨基酸序列分别如SEQ ID NO:34-36所示的LCDR1、LCDR2和LCDR3。The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, whose amino acid sequences are respectively shown in SEQ ID NO:31-33; and/or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3, whose amino acid sequences are respectively shown in SEQ ID NO:34-36. 如权利要求1所述的抗体，其特征在于，所述重链可变区还包括重链可变区框架区HFWR，和/或，所述轻链可变区还包括轻链可变区框架区LFWR，其中，所述HFWR为人源或小鼠源的重链可变区框架区，所述LFWR为人源或小鼠源抗体的轻链可变区框架区；The antibody according to claim 1, characterized in that 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 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; 优选地，所述轻链可变区包含氨基酸序列如SEQ ID NO:10所示的序列；和/或，所述重链可变区包含氨基酸序列如SEQ ID NO:9所示的序列；或，所述轻链可变区包含氨基酸序列如SEQ ID NO:20所示的序列；和/或，所述重链可变区包含氨基酸序列如SEQ ID NO:19所示的序列；或，所述轻链可变区包含氨基酸序列如SEQ ID NO:30所示的序列；和/或，所述重链可变区包含氨基酸序列如SEQ ID NO:29所示的序列；Preferably, 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; 更优选地，所述的抗体或其变体还包括重链恒定区和轻链恒定区；More preferably, the antibody or variant thereof further comprises a heavy chain constant region and a light chain constant region; 进一步优选地，所述的抗体重链恒定区为人源或小鼠源抗体重链恒定区；所述的抗体轻链恒定区为人源或小鼠源抗体轻链恒定区。Further preferably, 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. 如权利要求1或2所述的抗体，其特征在于，所述的抗体为以下任何 一种抗体形式：The antibody according to claim 1 or 2, characterized in that the antibody is any of the following A form of antibody: (a)一种完整的免疫球蛋白分子；(a) a complete immunoglobulin molecule; (b)一种scFv；(b) a scFv; (c)一种包含scFv的融合蛋白；(c) a fusion protein comprising scFv; (d)一种Fab片段；(d) a Fab fragment; (e)一种Fab′片段；(e) a Fab′ fragment; (f)一种F(ab)2；(f) a F(ab) 2 ; 或者，所述的抗体为单克隆抗体或者多克隆抗体；Alternatively, the antibody is a monoclonal antibody or a polyclonal antibody; 或者，所述的抗体为人源化抗体或者双特异性抗体。Alternatively, the antibody is a humanized antibody or a bispecific antibody. 一种嵌合抗原受体，其特征在于，所述嵌合抗原受体包含如权利要求1-3中任一项所述的抗体。A chimeric antigen receptor, characterized in that the chimeric antigen receptor comprises the antibody according to any one of claims 1 to 3. 一种分离的核酸，其特征在于，所述核酸编码如权利要求1-3中任一项所述的抗体、或如权利要求4所述的嵌合抗原受体；An isolated nucleic acid, characterized in that the nucleic acid encodes the antibody according to any one of claims 1 to 3, or the chimeric antigen receptor according to claim 4; 优选地，编码所述抗体的核酸包含如SEQ ID NO:7所示和/或如SEQ ID NO:8所示的多核苷酸序列；或，包含如SEQ ID NO:17所示和/或如SEQ ID NO:18所示的多核苷酸序列；或，包含如SEQ ID NO:27所示和/或如SEQ ID NO:28所示的多核苷酸序列。Preferably, 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. 一种重组表达载体，其特征在于，所述重组表达载体包含如权利要求5所述的分离的核酸。A recombinant expression vector, characterized in that the recombinant expression vector comprises the isolated nucleic acid according to claim 5. 一种转化体，其特征在于，所述转化体在宿主细胞中包含如权利要求6所述的重组表达载体。A transformant, characterized in that the transformant contains the recombinant expression vector according to claim 6 in a host cell. 一种TRPV1抗体的制备方法，其特征在于，所述制备方法包括培养如权利要求7所述的转化体，从培养物中获得所述抗体。A method for preparing a TRPV1 antibody, characterized in that the preparation method comprises culturing the transformant according to claim 7, and obtaining the antibody from the culture. 一种抗体药物偶联物，其特征在于，所述抗体药物偶联物包含细胞毒性剂，以及如权利要求1-3任一项所述的抗体。An antibody-drug conjugate, characterized in that the antibody-drug conjugate comprises a cytotoxic agent and the antibody according to any one of claims 1 to 3. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求1- 3任一项所述的抗体、如权利要求4所述的嵌合抗原受体或如权利要求9所述的抗体药物偶联物，以及药学上可接受的载体。A pharmaceutical composition, characterized in that the pharmaceutical composition comprises as claimed in claim 1- 3, the chimeric antigen receptor according to claim 4, or the antibody-drug conjugate according to claim 9, and a pharmaceutically acceptable carrier. 如权利要求1-3任一项所述的抗体、如权利要求4所述的嵌合抗原受体、如权利要求5所述的分离的核酸、如权利要求6所述的重组表达载体、如权利要求7所述的转化体、如权利要求9所述的抗体药物偶联物或如权利要求10所述的药物组合物在制备治疗疼痛的药物中的应用；Use of the antibody according to any one of claims 1 to 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; 优选地，所述疼痛为TRPV1激活导致的疼痛；所述药物的靶点为TRPV1。Preferably, the pain is pain caused by TRPV1 activation; and the target of the drug is TRPV1. 一种试剂盒，其特征在于，所述试剂盒包括如权利要求1-3任一项所述的抗体、如权利要求4所述的嵌合抗原受体、如权利要求9所述的抗体药物偶联物和/或如权利要求10所述的药物组合物。A kit, characterized in that the kit comprises the antibody according to any one of claims 1 to 3, the chimeric antigen receptor according to claim 4, the antibody-drug conjugate according to claim 9 and/or the pharmaceutical composition according to claim 10. 一种检测TRPV1的方法，其特征在于，所述方法包括使用如权利要求1-3中任一项所述的抗体、如权利要求4所述的嵌合抗原受体、如权利要求9所述的抗体药物偶联物、如权利要求10所述的药物组合物和/或如权利要求12所述的试剂盒与样品接触；A method for detecting TRPV1, characterized in that the method comprises contacting a sample with the antibody according to any one of claims 1 to 3, the chimeric antigen receptor according to claim 4, the antibody-drug conjugate according to claim 9, the pharmaceutical composition according to claim 10 and/or the kit according to claim 12; 优选地，所述检测为非诊断目的。Preferably, the detection is for non-diagnostic purposes. 一种诊断、治疗和/或预防疼痛的方法，其特征在于，所述方法包括向有需要的患者施用治疗有效量的如权利要求1-3任一项所述的抗体、如权利要求4所述的嵌合抗原受体、如权利要求5所述的分离的核酸、如权利要求6所述的重组表达载体、如权利要求7所述的转化体、如权利要求9所述的抗体药物偶联物、如权利要求10所述的药物组合物和/或如权利要求12所述的试剂盒；A method for diagnosing, treating and/or preventing pain, characterized in that the method comprises administering to a patient in need thereof a therapeutically effective amount of the antibody according to any one of claims 1 to 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; 优选地，所述疼痛为TRPV1激活导致的疼痛；所述药物的靶点为TRPV1。Preferably, the pain is pain caused by TRPV1 activation; and the target of the drug is TRPV1. 如权利要求1-3任一项所述的抗体、如权利要求4所述的嵌合抗原受体、如权利要求5所述的分离的核酸、如权利要求6所述的重组表达载体、如权利要求7所述的转化体、如权利要求9所述的抗体药物偶联物、如权利要求10所述的药物组合物和/或如权利要求12所述的试剂盒，其用于诊断、 治疗和/或预防疼痛；The antibody according to any one of claims 1 to 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, for use in diagnosis, Treat and/or prevent pain; 优选地，所述疼痛为TRPV1激活导致的疼痛；所述药物的靶点为TRPV1。 Preferably, the pain is pain caused by TRPV1 activation; and the target of the drug is TRPV1.
PCT/CN2023/138840 2023-12-14 2023-12-14 Trpv1 antibody, and preparation method therefor and use thereof Pending WO2025123292A1 (en)

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