WO2021181994A1 - ＳＡＲＳ－ＣｏＶ－２の構造タンパク質に対する抗体のエピトープ、該エピトープに反応する抗体、該抗体を用いてＳＡＲＳ－ＣｏＶ－２を検出する方法、該抗体を含むＳＡＲＳ－ＣｏＶ－２検出キット、該エピトープのポリペプチドを含む抗ＳＡＲＳ－ＣｏＶ－２抗体を検出する方法、該エピトープのポリペプチドを含む抗ＳＡＲＳ－ＣｏＶ－２抗体検出キット、該エピトープのポリペプチドを含むＳＡＲＳ－ＣｏＶ－２用ワクチン及び該抗体を含むＳＡＲＳ－ＣｏＶ－２感染症治療薬 - Google Patents

ＳＡＲＳ－ＣｏＶ－２の構造タンパク質に対する抗体のエピトープ、該エピトープに反応する抗体、該抗体を用いてＳＡＲＳ－ＣｏＶ－２を検出する方法、該抗体を含むＳＡＲＳ－ＣｏＶ－２検出キット、該エピトープのポリペプチドを含む抗ＳＡＲＳ－ＣｏＶ－２抗体を検出する方法、該エピトープのポリペプチドを含む抗ＳＡＲＳ－ＣｏＶ－２抗体検出キット、該エピトープのポリペプチドを含むＳＡＲＳ－ＣｏＶ－２用ワクチン及び該抗体を含むＳＡＲＳ－ＣｏＶ－２感染症治療薬 Download PDF

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WO2021181994A1
WO2021181994A1 PCT/JP2021/005011 JP2021005011W WO2021181994A1 WO 2021181994 A1 WO2021181994 A1 WO 2021181994A1 JP 2021005011 W JP2021005011 W JP 2021005011W WO 2021181994 A1 WO2021181994 A1 WO 2021181994A1
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seq
cov
sars
protein
antibody
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French (fr)
Japanese (ja)
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小笠原　真也
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Denka Co Ltd
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Denka Co Ltd
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Priority to US17/909,816 priority Critical patent/US20230348570A1/en
Priority to CN202180017717.5A priority patent/CN115867573A/zh
Priority to JP2022505853A priority patent/JPWO2021181994A1/ja
Priority to EP21768221.0A priority patent/EP4119575A4/en
Publication of WO2021181994A1 publication Critical patent/WO2021181994A1/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • C07K16/102—Coronaviridae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • C07K16/102—Coronaviridae (F)
    • C07K16/104—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2]
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • 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
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the present invention relates to an epitope of an antibody against a structural protein of SARS-CoV-2, an antibody that reacts with the epitope, a method for detecting SARS-CoV-2 using the antibody, and a SARS-CoV-2 detection kit containing the antibody.
  • the present invention relates to a vaccine and a SARS-CoV-2 infectious disease therapeutic agent containing the antibody.
  • Coronavirus is an enveloped virus with a positive single-stranded RNA genome and a nucleocapsid with spiral symmetry.
  • Beta coronavirus is a coronavirus that infects humans, and among them, MERS coronavirus (MERS-CoV) and SARS coronavirus (SARS-CoV) are known to cause serious respiratory symptoms.
  • MERS coronavirus MERS coronavirus
  • SARS-CoV SARS coronavirus
  • SARS-CoV-2 coronavirus
  • Non-Patent Document 1 Since the outbreak of SARS-CoV-2, there have been reports on the antigenicity and candidate epitope of SARS-CoV-2 (for example, Non-Patent Document 1), but the candidate epitope has been used to develop a vaccine against SARS-CoV-2. An epitope that is oriented and suitable for detecting SARS-CoV-2 and an antibody that recognizes the epitope have not yet been reported.
  • An object of the present invention is to provide an antibody that detects SARS-CoV-2, a method for detecting SARS-CoV-2 using the antibody, and a detection kit containing the antibody. Further, the present invention is an antibody that simultaneously detects human infectious coronavirus that causes serious respiratory symptoms such as MERS-CoV and SARS-CoV in addition to SARS-CoV-2, and SARS-CoV using the antibody. It is an object of the present invention to provide a method for simultaneously detecting -2 and human infectious coronavirus and a detection kit containing the antibody.
  • the viral antigenicity is strong in the region having a specific amino acid sequence in the structural protein constituting SARS-CoV-2, and the specific amino acid sequence is SARS-CoV-2.
  • the present invention has been completed by finding that it is useful as an epitope (polypeptide) recognized by an antibody that detects a virus.
  • the present invention provides a monoclonal antibody that specifically reacts with the structural protein of SARS-CoV-2 or an antigen-binding fragment thereof, and a detection kit for SARS-CoV-2 containing the antibody.
  • the structural protein of SARS-CoV-2 is at least one protein selected from the group consisting of S-protein, N-protein, M-protein and E-protein.
  • the present invention is to contact a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with the structural protein of SARS-CoV-2 with a sample, and detect SARS-CoV-2 by an immunological measurement method.
  • a method for detecting SARS-CoV-2 in a sample including.
  • a sandwich method using at least two types of monoclonal antibodies or antigen-binding fragments thereof for each structural protein is preferable.
  • the present invention specifically reacts with the structural protein of SARS-CoV-2 and the structural protein of human infectious coronavirus (preferably MERS-CoV and / or SARS-CoV) other than SARS-CoV-2.
  • Detection kit for human infectious coronaviruses other than SARS-CoV-2 and SARS-CoV-2 preferably MERS-CoV and / or SARS-CoV
  • the structural protein of SARS-CoV-2 is at least one protein selected from the group consisting of S-protein, N-protein, M-protein and E-protein, and is SARS-CoV-2.
  • the structural protein of the human infectious coronavirus other than the above is at least one protein selected from the group consisting of S-protein, N-protein, M-protein and E-protein.
  • the present invention also provides an epitope (polypeptide) recognized by an antibody that detects SARS-CoV-2. Furthermore, the present invention relates to a method for detecting an anti-SARS-CoV-2 antibody containing a polypeptide of the epitope, an anti-SARS-CoV-2 antibody detection kit containing the polypeptide of the epitope, and SARS containing the polypeptide of the epitope. -Providing a vaccine for CoV-2 and a therapeutic agent for SARS-CoV-2 infection containing the antibody.
  • an antibody for detecting SARS-CoV-2 it is possible to provide an antibody for detecting SARS-CoV-2, a method for detecting SARS-CoV-2 using the antibody, and a detection kit containing the antibody.
  • the present invention also provides an antibody that simultaneously detects coronavirus that causes serious respiratory symptoms such as MERS-CoV and SARS-CoV in addition to SARS-CoV-2, and a detection kit containing the antibody. be able to.
  • an epitope polypeptide
  • SARS-CoV-2 belongs to the genus Beta coronavirus, which is mainly composed of S-protein (surface protein), N-protein (nucleocapsid protein), M-protein (membrane protein) and E-protein (envelope protein). It is a virus.
  • the monoclonal antibody according to the present embodiment is at least one selected from the group consisting of major structural proteins constituting SARS-CoV-2, that is, S-protein, N-protein, M-protein and E-protein. Reacts specifically with proteins.
  • “specifically” means an antigen antibody at a level at which the antibody can be detected as a protein component other than the protein of SARS-CoV-2 in a liquid system in which a protein and a monoclonal antibody according to the present embodiment are mixed. It means that no reaction occurs, or even if some binding reaction or association reaction occurs, the reaction is clearly weaker than the antigen-antibody reaction of the antibody with the protein of SARS-CoV-2.
  • the SARS-CoV-2 S-protein (surface protein; hereinafter, also simply referred to as "S-protein” in the present specification) is typically composed of 1273 amino acid sequences represented by SEQ ID NO: 1.
  • the S-protein may be a protein consisting of the amino acid sequence of SEQ ID NO: 1 or a protein having 90% or more (preferably 95% or more) sequence identity with SEQ ID NO: 1.
  • the monoclonal antibodies that specifically react with the S-protein of SARS-CoV-2 are S-proteins 66-85 (SEQ ID NO: 5), 87-107 (SEQ ID NO: 6), 105.
  • SEQ ID NO: 7 To 131st (SEQ ID NO: 7), 140 to 157th (SEQ ID NO: 8), 155 to 170th (SEQ ID NO: 9), 522 to 541th (SEQ ID NO: 10), 538 to 562th (SEQ ID NO: 11), 595 Amino acids at positions 611 (SEQ ID NO: 12), 670 to 693 (SEQ ID NO: 13), 761 to 797 (SEQ ID NO: 14), 802 to 818 (SEQ ID NO: 15) or 1235-1273 (SEQ ID NO: 16).
  • the antigenicity score of these amino acid regions was higher than that of the threshold score, and at least 16 amino acid sequences were used. It is a polypeptide consisting of.
  • the monoclonal antibody that specifically reacts with the S-protein of SARS-CoV-2 is preferably the 71st to 83rd, 89th to 103rd, 107th to 127th, and 143rd to 154th S-protein of the S-protein. It binds to the amino acid regions of positions 158 to 167, 524 to 538, 546 to 560, 598 to 608, 672 to 691, 772 to 796, 806 to 816 or 1238 to 1269. These amino acid regions are polypeptides consisting of at least 10 amino acid sequences, with the antigenicity score higher than the threshold score in FIG. 1, which scores the strength of antigenicity for the entire S-protein molecule.
  • the monoclonal antibody that specifically reacts with the S-protein according to the present embodiment is more preferably the 73rd to 79th, 93rd to 99th, 109th to 115th, 145th to 152nd, 160th to 165th of the S-protein. It binds to the amino acid regions of positions 526 to 531, 535 to 558, 599 to 607, 674 to 684, 773 to 795, 808 to 814, or 1257 to 1261. These amino acid regions are polypeptides having the highest antigenicity score compared to the threshold score in FIG. 1, which scores the strength of antigenicity for the entire S-protein molecule, and consists of at least 5 amino acid sequences.
  • N-protein of SARS-CoV-2 (nucleocapsid protein; hereinafter, also simply referred to as "N-protein” in the present specification) consists of 419 amino acid sequences represented by SEQ ID NO: 2.
  • the monoclonal antibody that specifically reacts with the N-protein according to the present embodiment is the N-protein at positions 1 to 18 (SEQ ID NO: 17), 19 to 49 (SEQ ID NO: 18), and 174 to 207 (SEQ ID NO: 17). 19), 230-252 (SEQ ID NO: 20), 247-267 (SEQ ID NO: 21), 336-350 (SEQ ID NO: 22), 362-379 (SEQ ID NO: 23), 377-395 (SEQ ID NO: 21) 24) or binds to the amino acid region of positions 401-419 (SEQ ID NO: 25).
  • SEQ ID NO: 17 amino acid region of positions 1 to 18
  • 19 to 49 SEQ ID NO: 18
  • 174 to 207 SEQ ID NO: 17
  • 19 230-252
  • SEQ ID NO: 21 247-267
  • 336-350 SEQ ID NO: 22
  • 362-379 SEQ ID NO: 23
  • 377-395 SEQ ID NO: 21
  • these amino acid regions have an antigenicity score higher than that of the threshold score (threshold score), and are amino acids consisting of at least 15 sequences. It is a polypeptide consisting of a sequence.
  • the monoclonal antibody that specifically reacts with the N-protein according to the present embodiment is preferably the 1st to 12th, 20th to 48th, 175th to 206th, 234 to 250th, and 254 to 263th of the N-protein. It binds to the amino acid regions of positions 338 to 348, 366 to 377, 381 to 391 or 403 to 417. These amino acid regions are polypeptides consisting of at least 10 amino acid sequences, with the antigenicity score higher than the threshold score in FIG. 2, which scores the strength of antigenicity for the entire N-protein molecule.
  • the monoclonal antibody that specifically reacts with the N-protein according to the present embodiment is more preferably the 4th to 11th, 27th to 41st, 185th to 196th, 237th to 247th, 256th to 261st of the N-protein. It binds to the amino acid regions of positions 340 to 347, 376 to 375, 384 to 390, or 406 to 415. These amino acid regions are polypeptides having the highest antigenicity score compared to the threshold score in FIG. 2, which scores the strength of antigenicity for the entire N-protein molecule, and consists of at least 6 amino acid sequences.
  • SARS-CoV-2 M-protein membrane protein; hereinafter, also simply referred to as "M-protein” in the present specification
  • M-protein consists of 222 amino acid sequences represented by SEQ ID NO: 3.
  • the monoclonal antibody that specifically reacts with the M-protein according to this embodiment is the M-protein at positions 1 to 24 (SEQ ID NO: 26), 33 to 50 (SEQ ID NO: 27), and 101 to 120 (SEQ ID NO: 26). 28), it binds to the amino acid regions of positions 121 to 139 (SEQ ID NO: 29), 146 to 171 (SEQ ID NO: 30), 169 to 195 (SEQ ID NO: 31) or 193 to 222 (SEQ ID NO: 32).
  • SEQ ID NO: 29 amino acid regions of positions 121 to 139
  • SEQ ID NO: 30 146 to 171
  • 169 to 195 SEQ ID NO: 31
  • 193 to 222 SEQ ID NO: 32.
  • FIG. 3 in which the strength of antigenicity of the entire M-protein molecule was scored, the antigenicity score of these amino acid regions was higher than that of the threshold score, and at least 18 amino acid sequences were used. It is a polypeptide consisting of.
  • the monoclonal antibody that specifically reacts with the M-protein according to the present embodiment is preferably the 1st to 19th, 38th to 45th, 103rd to 118th, 122nd to 138th, 155th to 168th of the M-protein. It binds to the amino acid region of positions 172 to 195 or 196 to 216. These amino acid regions are polypeptides consisting of at least 8 amino acid sequences, with the antigenicity score higher than the threshold score in FIG. 3, which scores the strength of antigenicity for the entire M-protein molecule.
  • the monoclonal antibody that specifically reacts with the M-protein according to the present embodiment is more preferably the 1st to 16th, 40th to 44th, 105th to 117th, 123rd to 136th, 160th to 167th of the M-protein. , 173 to 179th or 205 to 215th amino acid region.
  • These amino acid regions are polypeptides having an amino acid sequence consisting of at least 5 sequences, which has the highest antigenicity score as compared with the threshold score in FIG. 3, which scores the strength of antigenicity for the entire M-protein molecule. ..
  • the SARS-CoV-2 E-protein (envelope protein; hereinafter, also simply referred to as "E-protein” in the present specification) consists of 75 amino acid sequences represented by SEQ ID NO: 4.
  • the monoclonal antibody that specifically reacts with the E-protein binds to the amino acid region of positions 1 to 19 (SEQ ID NO: 33) or 50 to 75 (SEQ ID NO: 34) of the E-protein.
  • SEQ ID NO: 33 amino acid region of positions 1 to 19
  • SEQ ID NO: 34 amino acid region of positions 1 to 19
  • the antigenicity score of these amino acid regions was higher than that of the threshold score, and at least 19 amino acid sequences. It is a polypeptide consisting of.
  • the monoclonal antibody that specifically reacts with the E-protein according to the present embodiment preferably binds to the amino acid region of the 4th to 13th or 51st to 72nd positions of the E-protein.
  • These amino acid regions are polypeptides consisting of at least 10 amino acid sequences, with the antigenicity score higher than the threshold score in FIG. 4, which scores the strength of antigenicity for the entire E-protein molecule.
  • the monoclonal antibody that specifically reacts with the E-protein more preferably binds to the 5th to 10th or 60th to 71st amino acid regions of the E-protein. These amino acid regions are polypeptides having the highest antigenicity score compared to the threshold score in FIG. 4, which scores the strength of antigenicity for the entire E-protein molecule, and consists of at least 6 amino acid sequences.
  • the antigen-binding site can be separated and used. That is, fragments having specific antigen-binding properties (antigen-binding fragments) such as Fab, Fab', F (ab') 2, single-chain antibody (scFv), and VHH produced by a known method are present. It is included in the scope of the invention.
  • the class of the monoclonal antibody according to this embodiment is not limited to IgG, and may be IgY, IgM, Camel Ig or Ig NAR.
  • antibody means “antibody or antigen-binding fragment thereof” unless it is clear from the context.
  • the monoclonal antibody according to the present embodiment uses a known immunological method in SARS-CoV-2 protein or a partial peptide thereof (for example, in the above-mentioned S-protein, N-protein, M-protein or E-protein). It can be obtained by immunizing an immunized animal (polypeptide consisting of a specific amino acid region) and producing a hybridoma using the cells of the immunized animal, or by producing it as a recombinant antibody by a gene recombination technique. ..
  • the length of the peptide used for immunization is not particularly limited, but a peptide of 5 amino acids or more, more preferably 10 amino acids or more, still more preferably 13 amino acids or more can be used as an immunogen.
  • the SARS-CoV-2 protein used as an immunogen can be obtained from a cultured virus solution, but the DNA encoding the SARS-CoV-2 protein is incorporated into a plasmid vector and introduced into a host cell for expression. Can also be obtained by.
  • the SARS-CoV-2 protein as an immunogen or a partial peptide thereof can be expressed as a fusion protein with a protein as exemplified below, and can be used as an immunogen after purification or as unpurified.
  • Glutathione S-transferase GST
  • MBP maltose-binding protein
  • TRX thioredoxin
  • Nus tag, S tag, HSV which are commonly used by those skilled in the art as "protein expression / purification tags" for producing fusion proteins.
  • Tags FLAG tags, polyhistidine tags, Protein tags, Strip-II tags, Myc tags, HA tags, V5 tags, E tags, T7 tags, VSV-G tags, Glu-Glu tags, Avi tags and the like can be used.
  • the fusion protein with these is preferably used as an immunogen after cleaving the protein of SARS-CoV-2 or a partial peptide portion thereof and a tag portion other than the tag portion using a digestive enzyme, separating and purifying the protein.
  • Preparation of monoclonal antibodies from immunized animals can be easily performed by the method of well-known Keller et al. (Kohler et al., Nature, vol. 256, p495-497 (1975)). That is, antibody-producing cells such as splenocytes and lymphocytes are collected from immunized animals and fused with mouse myeloma cells by a conventional method to prepare a hybridoma, and the obtained hybridoma is cloned by a limiting dilution method or the like. , Among the monoclonal antibodies produced by each of the cloned hybridomas, a monoclonal antibody that reacts with the protein of SARS-CoV-2 as an antigen antibody is selected.
  • a known immunoglobulin purification method can be used to purify the monoclonal antibody from ascites or culture supernatant.
  • a fractionation method by salting out using ammonium sulfate or sodium sulfate, a PEG fractionation method, an ethanol fractionation method, a DEAE ion exchange chromatography method, a gel filtration method and the like can be mentioned.
  • It can also be purified by an affinity chromatography method using a carrier to which any of protein A, protein G, and protein L is bound, depending on the immune animal species and the class of the monoclonal antibody.
  • the method for detecting SARS-CoV-2 is at least one selected from the group consisting of SARS-CoV-2 protein, that is, S-protein, N-protein, M-protein and E-protein. It involves contacting a sample with a monoclonal antibody or antigen-binding fragment thereof that specifically reacts with a protein of a species and detecting SARS-CoV-2 by immunoassay.
  • sample used in the detection method according to the present embodiment examples include body fluids such as human or animal blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, tears, ascites or sheep water; mucus; feces; blood vessels.
  • body fluids such as human or animal blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, tears, ascites or sheep water; mucus; feces; blood vessels.
  • organs such as the liver; tissues; cells, or biological samples that may contain the protein of SARS-CoV-2, such as cells or extracts thereof, preferably the oral cavity, the nasal cavity, which are easy to collect.
  • the method for collecting these samples is not particularly limited, and a known method can be adopted. Specifically, a method using a cotton swab can be mentioned.
  • the measurement of SARS-CoV-2 protein according to the present embodiment can be adopted by any immunological measurement method well known to those skilled in the art.
  • the immunological measurement method include a competitive method, an agglutination method, a Western blotting method, an immunostaining method, and a sandwich method.
  • a sandwich method using at least two types of monoclonal antibodies is preferable.
  • the sandwich method itself is well known in the field of immunoassay, and can be performed by, for example, an immunochromatography method, an ELISA method, or the like.
  • the measurement method according to the present embodiment can be carried out by a well-known sandwich method except that a monoclonal antibody that specifically reacts with the above-mentioned SARS-CoV-2 protein is used.
  • the protein of SARS-CoV-2 to be measured is S-protein
  • the protein of SARS-CoV-2 to be measured is N-protein
  • the 1st to 18th (SEQ ID NO: 17), 19th to 49th (SEQ ID NO: 18), 174 to 207th (SEQ ID NO: 19) , 230-252 (SEQ ID NO: 20), 247-267 (SEQ ID NO: 21), 336-350 (SEQ ID NO: 22), 362-379 (SEQ ID NO: 23), 377-395 (SEQ ID NO: 24).
  • the protein of SARS-CoV-2 to be measured is an E-protein, it can be simultaneously bound to the amino acid regions of positions 1 to 19 (SEQ ID NO: 33) and 50 to 75 (SEQ ID NO: 34). It is preferable to use two types of monoclonal antibodies.
  • the SARS-CoV-2 detection kit is at least one selected from the group consisting of SARS-CoV-2 proteins, that is, S-proteins, N-proteins, M-proteins and E-proteins. Contains a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with the protein of.
  • the SARS-CoV-2 detection kit according to the present embodiment can be used in any form used in the above-mentioned immunological measurement method, but preferably, the SARS-CoV-2 can be used accurately, quickly and easily. It is in the form of an immunochromatographic strip from which proteins can be measured.
  • a preferred embodiment of the SARS-CoV-2 detection kit according to the present embodiment is the 66th to 85th (SEQ ID NO: 5), 87th to 107th (SEQ ID NO: 6), 105th to 131st (SEQ ID NO:) S-proteins of the S-protein.
  • a preferred embodiment of the SARS-CoV-2 detection kit according to the present embodiment is the N-protein at positions 1 to 18 (SEQ ID NO: 17), 19 to 49 (SEQ ID NO: 18), and 174 to 207 (SEQ ID NO: 17). 19), 230-252 (SEQ ID NO: 20), 247-267 (SEQ ID NO: 21), 336-350 (SEQ ID NO: 22), 362-379 (SEQ ID NO: 23), 377-395 (SEQ ID NO: 21) 24) and at least two monoclonal antibodies or antigen-binding fragments thereof capable of simultaneously binding to the amino acid region selected from the group consisting of positions 401 to 419 (SEQ ID NO: 25).
  • a preferred embodiment of the SARS-CoV-2 detection kit according to the present embodiment is the M-protein at positions 1 to 24 (SEQ ID NO: 26), 33 to 50 (SEQ ID NO: 27), and 101 to 120 (SEQ ID NO: 26). 28), Amino acid selected from the group consisting of positions 121 to 139 (SEQ ID NO: 29), 146 to 171 (SEQ ID NO: 30), 169 to 195 (SEQ ID NO: 31) and 193 to 222 (SEQ ID NO: 32). Includes at least two types of monoclonal antibodies according to claim 8 or antigen-binding fragments thereof that can simultaneously bind to a region.
  • a preferred embodiment of the SARS-CoV-2 detection kit according to the present embodiment is to simultaneously bind to the amino acid regions of positions 1 to 19 (SEQ ID NO: 33) and 50 to 75 (SEQ ID NO: 34) of the E-protein. Includes two types of monoclonal antibodies or antigen-binding fragments thereof.
  • the monoclonal antibody according to the present embodiment includes SARS-CoV-2 structural proteins, that is, SARS-CoV-2 S-protein (surface protein), N-protein (nucleocapsid protein), M-protein (membrane protein) and At least one protein selected from the group consisting of E-proteins (envelope proteins), and structural proteins of human infectious coronaviruses other than SARS-CoV-2, that is, human infectious coronas other than SARS-CoV-2. Reacts specifically with at least one protein selected from the group consisting of viral S-protein (surface protein), N-protein (nucleocapsid protein), M-protein (membrane protein) and E-protein (envelope protein). do.
  • the antibody is a protein of SARS-CoV-2 and human infectivity other than SARS-CoV-2 in a liquid system in which a protein and a monoclonal antibody according to the present embodiment are mixed. Even if an antigen-antibody reaction does not occur at a detectable level with a protein component other than the coronavirus protein, or even if some binding reaction or association reaction occurs, the SARS-CoV-2 protein of the antibody and SARS-CoV It means that the reaction is clearly weaker than the antigen-antibody reaction with proteins other than the protein of human infectious coronavirus other than -2.
  • the human infectious coronavirus other than SARS-CoV-2 is not particularly limited as long as it is a coronavirus that causes serious respiratory symptoms in humans, but is preferably MERS-CoV and / or SARS-CoV, more preferably. Is SARS-CoV.
  • the monoclonal antibody according to the present embodiment is the same as the method described in the above [Antibody against SARS-CoV-2, a method for detecting SARS-CoV-2 using the antibody, and a detection kit containing the antibody]. Can be prepared.
  • the method for detecting human infectious coronaviruses other than SARS-CoV-2 and SARS-CoV-2 is a structural protein of SARS-CoV-2, that is, an S-protein of SARS-CoV-2. , N-protein, M-protein and E-protein, at least one protein selected from the group, and structural proteins of human infectious coronavirus other than SARS-CoV-2, that is, other than SARS-CoV-2.
  • a monoclonal antibody that specifically reacts with at least one protein selected from the group consisting of S-protein, N-protein, M-protein and E-protein of human infectious coronavirus or an antigen-binding fragment thereof and a sample thereof. Includes detecting human infectious coronaviruses other than SARS-CoV-2 and SARS-CoV-2 by immunological measures.
  • the sample used in the method according to the present embodiment, the protein of SARS-CoV-2, and the protein of human infectious coronavirus other than SARS-CoV-2 were measured by the above [antibody against SARS-CoV-2, said antibody. It is the same as the sample and measurement method described in the method for detecting SARS-CoV-2 and the detection kit containing the antibody].
  • the detection kit for human infectious coronaviruses other than SARS-CoV-2 and SARS-CoV-2 is a structural protein of SARS-CoV-2, that is, an S-protein of SARS-CoV-2.
  • At least one protein selected from the group consisting of N-protein, M-protein and E-protein and structural protein of human infectious coronavirus other than SARS-CoV-2, that is, human other than SARS-CoV-2. Includes a monoclonal antibody or antigen-binding fragment thereof that specifically reacts with at least one protein selected from the group consisting of S-protein, N-protein, M-protein and E-protein of infectious coronavirus.
  • polypeptide recognized by antibody that detects SARS-CoV-2
  • the polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 5 to 34 according to the present embodiment can be used as an epitope recognized by an antibody that detects SARS-CoV-2 present in a sample. Further, the polypeptide according to the present embodiment can be used as an epitope recognized by an antibody that detects human infectious coronavirus other than SARS-CoV-2 in addition to SARS-CoV-2 present in the sample. ..
  • the polypeptide according to this embodiment is an anti-SARS-CoV-produced by an antibody against SARS-CoV-2 present in a sample (preferably in blood) (that is, an in vivo defense mechanism against SARS-CoV-2). It can be used as a hapten to detect (2 antibody titers). Therefore, as one embodiment of the present invention, one or more polypeptides having the amino acid sequences of SEQ ID NOs: 5 to 34 are brought into contact with a sample (preferably blood), and anti-SARS- by immunological measurement method. A method for detecting an anti-SARS-CoV-2 antibody in a sample, including detecting a CoV-2 antibody, is provided. Further, as an embodiment of the present invention, an anti-SARS-CoV-2 antibody detection kit containing one or more polypeptides having the amino acid sequences of SEQ ID NOs: 5 to 34 is provided.
  • the polypeptide according to this embodiment is an antibody against SARS-CoV-2 present in a sample (preferably in blood) and an antibody against human infectious coronavirus other than SARS-CoV-2 (that is, SARS-CoV-). It can be used as a hapten to detect (anti-human infectious coronavirus antibody titer) produced by an in vivo defense mechanism against human infectious coronavirus other than 2. Therefore, as one embodiment of the present invention, one or more polypeptides having the amino acid sequences of SEQ ID NOs: 5 to 34 are brought into contact with a sample (preferably blood), and anti-SARS- by an immunological measurement method.
  • anti-human infectious coronavirus antibodies other than CoV-2 and anti-SARS-CoV-2 antibodies.
  • a method for detecting an anti-human infectious coronavirus antibody is provided.
  • other than the anti-SARS-CoV-2 antibody and the anti-SARS-CoV-2 antibody which contain one or more polypeptides having the amino acid sequences of SEQ ID NOs: 5 to 34.
  • An anti-human infectious coronavirus antibody detection kit is provided.
  • the polypeptide according to this embodiment can be used as a basic skeleton of a vaccine for the purpose of preventing infection with SARS-CoV-2 (for example, a peptide vaccine, a cocktail peptide vaccine, a fusion peptide, or a fragment domain containing the peptide).
  • SARS-CoV-2 for example, a peptide vaccine, a cocktail peptide vaccine, a fusion peptide, or a fragment domain containing the peptide.
  • the polypeptide according to the present embodiment has the possibility of being used as a basic skeleton of a vaccine for the purpose of preventing infection with human infectious coronavirus other than SARS-CoV-2 in addition to SARS-CoV-2. ..
  • the polypeptide according to this embodiment is the basic skeleton of a vaccine (for example, a peptide vaccine, a cocktail peptide vaccine, a fusion peptide, or a fragment domain containing the peptide) for the purpose of preventing the aggravation of SARS-CoV-2 infection.
  • a vaccine for example, a peptide vaccine, a cocktail peptide vaccine, a fusion peptide, or a fragment domain containing the peptide
  • the polypeptide according to the present embodiment is the basic framework of a vaccine aimed at preventing the aggravation of human infectious coronavirus infections other than SARS-CoV-2 in addition to SARS-CoV-2 infections. Has the possibility of being used as.
  • a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with the polypeptide according to the present embodiment has potential as an antibody drug (neutralizing antibody) for the treatment of SARS-CoV-2 infection. ..
  • the monoclonal antibody or the antigen-binding fragment thereof that specifically reacts with the polypeptide according to the present embodiment is used for human infectious coronavirus infections other than SARS-CoV-2 in addition to SARS-CoV-2 infections. It has the potential as an antibody drug (neutralizing antibody) for the treatment of.
  • the human infectious coronavirus other than SARS-CoV-2 in each of the above-described embodiments is not particularly limited as long as it is a coronavirus that causes serious respiratory symptoms in humans, but is preferably MERS-CoV and / or SARS. -CoV, more preferably SARS-CoV.
  • Example 1 Measurement of antibody titer
  • 3 BALB / c mice were intraperitoneally administered at a dose of 100 ⁇ g / 200 ⁇ L / head.
  • a sigma adjuvant system was used as an adjuvant. Blood was collected over time during the immunization period from the tail vein. The antibody titer was confirmed by the peptide conjugate solid-phase ELISA (Enzyme-Linked Immuno Sorbent Assay) method using BSA according to the following procedure. 1) A total of 24 BSA peptide conjugate solutions diluted with PBS to 2 ⁇ g / mL were added to 96-well immunoplates at 100 ⁇ L / well increments, and solid-phased overnight at 4 ° C.
  • Example 2 Reactivity of antibody to SARS-CoV-2 inactivated antigen
  • EPI_ISL_406034 the inactivating antigen of SARS-CoV-2
  • ELISA Enzyme-Linked Immuno Sorbent Assay
  • Example 2 As shown in FIG. 6, it was confirmed that all of the 24 types of antibodies obtained in Example 1 reacted with the SARS-CoV-2 inactivating antigen.
  • Example 3 Analysis of the epitope of the antibody present in the antiserum when the SARS-CoV-2 inactivated antigen is used as an immunogen
  • BALB / c mice were immunized using the SARS-CoV-2 inactivated antigen used in Example 2 as an immunogen in the same procedure as in Example 1.
  • the epitope of the antibody present in the obtained antiserum was analyzed by the following procedure.
  • the sequence of the N-protein of SARS-CoV-2 (SEQ ID NO: 2) is slid from the N-terminal to the C-terminal side by one amino acid, and is a polypeptide consisting of 15 consecutive amino acids, and each polypeptide chain SARS-CoV-2 N-protein peptide set (BioTides TM Biotinylated Peptides: manufactured by JPT Peptide Technologies GmbH) in which the N-terminal is labeled with biotin and one residue of glycine is added to the C-terminal of each polypeptide chain.
  • BioTides TM Biotinylated Peptides: manufactured by JPT Peptide Technologies GmbH in which the N-terminal is labeled with biotin and one residue of glycine is added to the C-terminal of each polypeptide chain.
  • BioTides TM Biotinylated Peptides described in 1) above was dissolved in DMF (N, N-dimethylformamide) to prepare BioTides TM Biotinylated Peptides-immobilized LumAvidin Microspheres in LumAvidin Microspheres (manufactured by Liminex) via a biotin-avidin reaction. .. Details of this preparation were in accordance with Luminex's xMAP (registered trademark) Cookbook (Chapter 4.2.1).
  • Example 3 As shown in FIG. 7, it was confirmed that the antiserum obtained in Example 3 had a predominant antibody titer against N-protein epitope sequences other than SEQ ID NOs: 21 and 22.
  • Example 4 Analysis of the epitope of an antibody present in antiserum when recombinant N-protein is used as an immunogen
  • BALB / c mice were immunized using recombinant N-protein (full-length; SEQ ID NO: 2) as an immunogen in the same procedure as in Example 1.
  • SEQ ID NO: 2 full-length; SEQ ID NO: 2
  • the epitope of the antibody present in the obtained antiserum was analyzed by the same procedure as in Example 3. The result is shown in FIG.
  • Example 4 As shown in FIG. 8, it was confirmed that the antiserum obtained in Example 4 had a predominant antibody titer against the entire epitope sequence of the N-protein of SEQ ID NOs: 17 to 25.
  • Example 5 Reactivity of antibody in antiserum to epitope sequence of N-protein
  • the antisera obtained in Examples 3 and 4 the reactivity of the antibody in the antiserum to the epitope sequence of the N-protein of SEQ ID NOs: 17 to 25 was confirmed by the following procedure. 1) Biotin labeling is applied to the N-terminal of each polypeptide of SEQ ID NO: 17 to 25, the polypeptide is dissolved in DMF (N, N-dimethylformamide), and LumAvidin Microspheres (manufactured by Liminex) is subjected to a biotin-avidin reaction. Biotinylated epitope-peptides-immobilized LumAvidin Microspheres were prepared.

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PCT/JP2021/005011 2020-03-10 2021-02-10 ＳＡＲＳ－ＣｏＶ－２の構造タンパク質に対する抗体のエピトープ、該エピトープに反応する抗体、該抗体を用いてＳＡＲＳ－ＣｏＶ－２を検出する方法、該抗体を含むＳＡＲＳ－ＣｏＶ－２検出キット、該エピトープのポリペプチドを含む抗ＳＡＲＳ－ＣｏＶ－２抗体を検出する方法、該エピトープのポリペプチドを含む抗ＳＡＲＳ－ＣｏＶ－２抗体検出キット、該エピトープのポリペプチドを含むＳＡＲＳ－ＣｏＶ－２用ワクチン及び該抗体を含むＳＡＲＳ－ＣｏＶ－２感染症治療薬 Ceased WO2021181994A1 (ja)

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US17/909,816 US20230348570A1 (en) 2020-03-10 2021-02-10 Epitope of antibody against structural protein of sars-cov-2, antibody reacting with epitope, method for detecting sars-cov-2 using antibody, detection kit for sars-cov-2 containing antibody, method for detecting anti-sars-cov-2 antibody containing polypeptide of epitope, detection kit for anti-sars-cov-2 antibody containing polypeptide of epitope, vaccine for sars-cov-2 containing polypeptide of epitope, and therapeutic agent for sars-cov-2 infection containing antibody
CN202180017717.5A CN115867573A (zh) 2020-03-10 2021-02-10 针对SARS-CoV-2的结构蛋白的抗体的表位、与该表位反应的抗体、该表位的多肽、包含它们的检测方法、检测试剂盒、疫苗及治疗药
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EP21768221.0A EP4119575A4 (en) 2020-03-10 2021-02-10 Epitope of antibody against structural protein of sars-cov-2, antibody reacting with epitope, method for detecting sars-cov-2 using antibody, detection kit for sars-cov-2 containing antibody, method for detecting anti-sars-cov-2 antibody containing polypeptide of epitope, detection kit for anti-sars-cov-2 antibody containing polypeptide of epitope, vaccine for sars-cov-2 containing polypeptide of epitope, and therapeutic agent for sars-cov-2 infection containing antibody
JP2024109831A JP7801399B2 (ja) 2020-03-10 2024-07-08 ＳＡＲＳ－ＣｏＶ－２の構造タンパク質に対する抗体のエピトープ、該エピトープに反応する抗体、該抗体を用いてＳＡＲＳ－ＣｏＶ－２を検出する方法、該抗体を含むＳＡＲＳ－ＣｏＶ－２検出キット、該エピトープのポリペプチドを含む抗ＳＡＲＳ－ＣｏＶ－２抗体を検出する方法、該エピトープのポリペプチドを含む抗ＳＡＲＳ－ＣｏＶ－２抗体検出キット、該エピトープのポリペプチドを含むＳＡＲＳ－ＣｏＶ－２用ワクチン及び該抗体を含むＳＡＲＳ－ＣｏＶ－２感染症治療薬

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WO2022265065A1 (ja) * 2021-06-16 2022-12-22 積水メディカル株式会社 ＳＡＲＳ－ＣｏＶ－２の免疫測定方法及び免疫測定キット、並びにモノクローナル抗体又はその抗体断片
JP7105970B1 (ja) 2021-06-16 2022-07-25 積水メディカル株式会社 ＳＡＲＳ－ＣｏＶ－２の免疫測定方法及び免疫測定キット
JP2022191725A (ja) * 2021-06-16 2022-12-28 積水メディカル株式会社 ＳＡＲＳ－ＣｏＶ－２の免疫測定方法及び免疫測定キット
WO2022265066A1 (ja) * 2021-06-16 2022-12-22 積水メディカル株式会社 ＳＡＲＳ－ＣｏＶ－２の免疫測定方法及び免疫測定キット
JPWO2023068343A1 (https=) * 2021-10-21 2023-04-27
WO2023068282A1 (ja) * 2021-10-21 2023-04-27 美幸 徳田 飲食品、食品添加物、サプリメント、化粧品、抗体、医薬、およびその製造方法
WO2023068343A1 (ja) * 2021-10-21 2023-04-27 美幸 徳田 飲食品、食品添加物、サプリメント、化粧品、抗体、医薬、およびその製造方法
CN113943349A (zh) * 2021-10-22 2022-01-18 华瑞同康生物技术（深圳）有限公司 靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组及应用
WO2023066396A1 (zh) * 2021-10-22 2023-04-27 华瑞同康生物技术（深圳）有限公司 靶向识别抗新冠病毒中和抗体N-IgY-pAbs的核心氨基酸序列组及应用
WO2023121264A1 (ko) * 2021-12-20 2023-06-29 아이진 주식회사 변이 sars-cov-2 백신 조성물 및 이의 용도
WO2023167317A1 (ja) * 2022-03-04 2023-09-07 公立大学法人横浜市立大学 変異株を含む、体液中のＳＡＲＳ－ＣｏＶ－２抗原に対する抗ＳＡＲＳ－ＣｏＶ－２抗体、該抗体を用いてＳＡＲＳ－ＣｏＶ－２を検出する方法、および該抗体を含むキット
CN114560929B (zh) * 2022-03-21 2023-07-04 深圳国家感染性疾病临床医学研究中心 针对冠状病毒np蛋白的单克隆抗体及其应用
CN114560929A (zh) * 2022-03-21 2022-05-31 深圳国家感染性疾病临床医学研究中心 针对冠状病毒np蛋白的单克隆抗体及其应用
CN114790239A (zh) * 2022-03-29 2022-07-26 苏州东抗生物科技有限公司 一种抗冠状病毒n蛋白的抗体及其应用
KR20250135899A (ko) 2023-02-08 2025-09-15 덴카 주식회사 SARS-CoV-2의 검출 방법 및 이를 위한 키트
KR20250138260A (ko) 2023-02-08 2025-09-19 덴카 주식회사 항sars-cov-2 모노클로날 항체와 이를 이용한 sars-cov-2의 면역 측정 방법 및 면역 측정 기구
KR20250140089A (ko) 2023-02-08 2025-09-24 덴카 주식회사 SARS-CoV-2의 검출 방법 및 이를 위한 키트
KR20250140090A (ko) 2023-02-08 2025-09-24 덴카 주식회사 SARS-CoV-2의 검출 방법 및 이를 위한 키트

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