WO2023068377A1 - ＳＡＲＳ－ＣｏＶ－２のＯＲＦ８タンパク質に結合するモノクローナル抗体及び該抗体を含む炎症抑制組成物 - Google Patents

ＳＡＲＳ－ＣｏＶ－２のＯＲＦ８タンパク質に結合するモノクローナル抗体及び該抗体を含む炎症抑制組成物 Download PDF

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WO2023068377A1
WO2023068377A1 PCT/JP2022/039419 JP2022039419W WO2023068377A1 WO 2023068377 A1 WO2023068377 A1 WO 2023068377A1 JP 2022039419 W JP2022039419 W JP 2022039419W WO 2023068377 A1 WO2023068377 A1 WO 2023068377A1
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amino acid
acid sequence
seq
chain variable
variable region
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French (fr)
Japanese (ja)
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正之 森
泰希 東村
智弘 今村
亜希子 水野
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Ishikawa Prefectural University
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Ishikawa Prefectural University
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    • 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]
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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]
    • 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
    • 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
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present invention relates to a monoclonal antibody that binds to the ORF8 protein of SARS-CoV-2 and an anti-inflammatory composition containing the antibody.
  • SARS-CoV-2 The novel coronavirus infection (COVID-19) caused by SARS-CoV-2 is a major problem worldwide, as it tends to aggravate patients with underlying diseases and obesity.
  • COVID-19 elucidation of the protein function of SARS-CoV-2 is essential.
  • SARS-CoV-2 genome sequencing reveals that SARS-CoV-2 synthesizes at least 16 nonstructural proteins, 4 structural proteins, and at least 6 or 7 accessory proteins in infected cells It has become.
  • the ORF8 protein one of the accessory proteins, has low homology compared to the ORF8 proteins of related viruses and is known to be a characteristic protein in SARS-CoV-2.
  • ORF8 has also been reported to bind to proteins involved in immunity and inflammation. Furthermore, it has been reported that SARS-CoV-2 strains lacking the ORF8 gene region are less likely to develop severe disease. These findings suggest that the ORF8 protein is involved in exacerbation of COVID-19. However, the function of the OFR8 protein has not been elucidated.
  • ORF8 protein To elucidate the function of the ORF8 protein, it is necessary to synthesize a large amount of the ORF8 protein that is homogeneous and maintains its three-dimensional structure (native state). However, in the native state of the ORF8 protein, it has a dimeric structure with three intramolecular disulfide bonds and one intermolecular disulfide bond. Therefore, homogenous ORF8 protein synthesis in E. coli was extremely difficult.
  • ORF8 protein which is the antigen used to obtain polyclonal antibodies or monoclonal antibodies that recognize these ORF8 proteins, is a dimer having three disulfide bonds in the molecule and one disulfide bond in the molecule, which is close to the native state. was not the structure of
  • the present inventors have so far constructed a large-scale protein synthesis system using tobacco cultured cells (tobacco BY-2 cells) as hosts. Therefore, when we tried to synthesize ORF8 protein using this protein synthesis system, we obtained an ORF8 protein with a uniform structure (a dimeric structure with three disulfide bonds in the molecule and one disulfide bond in the molecule). It was confirmed by NMR that it was possible. Furthermore, the present inventors obtained a monoclonal antibody using as an antigen the ORF8 protein, which is a dimeric structure having three disulfide bonds in a molecule close to the native state and one disulfide bond between molecules, and the antibody is It was confirmed to have an anti-inflammatory effect. With the above, the present invention was completed.
  • the present invention is as follows. 1. A monoclonal antibody or an antigen-binding fragment thereof that specifically recognizes a dimer of ORF8 protein having three intramolecular disulfide bonds and one intermolecular disulfide bond of SARS-CoV-2. 2.
  • a heavy chain variable region comprising a polypeptide consisting of an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of a sequence complementary to the described base sequence: 32) A light chain variable region comprising a polypeptide consisting of an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of a sequence complementary to the base sequence of SEQ ID NO:5, and SEQ ID NO:6 A heavy chain variable region comprising a polypeptide consisting of an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of a sequence complementary to the described base sequence: 33) A light chain variable region comprising a polypeptide consisting of an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of a sequence complementary to the base sequence of SEQ ID NO: 7, and a light chain variable region of SEQ ID NO: 8 A heavy chain variable region comprising a polypeptide consisting of an amino acid sequence
  • the isolated monoclonal antibody or antigen-binding fragment thereof which comprises any one of 1) to 10) below; 1) A light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:3 or the amino acid sequence of SEQ ID NO:23, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:4 A heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO: 24: 2) a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:5 or the amino acid sequence of SEQ ID NO:25, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:6; A heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO: 26: 3) a light chain variable region comprising
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 27, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO: 8
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO: 28: 2
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 13 or the amino acid sequence set forth in SEQ ID NO: 33, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 14
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO: 34: and
  • the isolated monoclonal antibody or antigen-binding fragment thereof according to item 2 comprising: 1) A light chain variable region comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 25, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO: 6
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:26. 6.
  • the monoclonal antibody or antigen-binding fragment thereof according to the preceding item 1, wherein the monoclonal antibody or antigen-binding fragment thereof is any one of the following 1) to 47): 1) A light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:3 or the amino acid sequence of SEQ ID NO:23, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:4 A heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO: 24: 2) a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 9 or the amino acid sequence set forth in SEQ ID NO: 29, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 10; A heavy chain variable region comprising a polypeptide consisting of the amino
  • Fab, Fab', F(ab') 2 7.
  • a host cell comprising the nucleic acid according to 9 above.
  • An anti-inflammatory composition comprising the monoclonal antibody or antigen-binding fragment thereof according to 4 above as an active ingredient.
  • a therapeutic or prophylactic agent for coronavirus comprising the anti-inflammatory composition according to 11 above. 13. 13.
  • the present invention includes the following. 2. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to the ORF8 protein of SARS-CoV-2, comprising any one of the following 1) to 8); 1) a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 17 or the amino acid sequence set forth in SEQ ID NO: 37, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 18; A heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:38: 2) a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 13 or the amino acid sequence set forth in SEQ ID NO:
  • An isolated monoclonal antibody or antigen-binding fragment thereof that binds to ORF8 protein of SARS-CoV-2 comprising any one of 1) to 6) below; 1) A light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 29, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO: 10
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:30: 2) a light chain variable region in which one or more amino acids have been deleted, inserted, substituted or added as compared with the antibody having the light chain variable region and the heavy chain variable region described in 1) above, and A heavy chain variable region in which one or more amino acids have been deleted, inserted, substituted or added: 3) a light chain variable region with an amino acid sequence identity of 95% or more, and an amino acid sequence with an amino acid sequence
  • An isolated monoclonal antibody or antigen-binding fragment thereof that binds to ORF8 protein of SARS-CoV-2 comprising any one of 1) to 6) below; 1) A light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 169 or the amino acid sequence of SEQ ID NO: 319, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO: 170
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:320: 2) a light chain variable region in which one or more amino acids have been deleted, inserted, substituted or added as compared with the antibody having the light chain variable region and the heavy chain variable region described in 1) above, and A heavy chain variable region in which one or more amino acids have been deleted, inserted, substituted or added: 3) a light chain variable region with an amino acid sequence identity of 95% or more, and an amino acid sequence with
  • An isolated monoclonal antibody or antigen-binding fragment thereof that binds to the ORF8 protein of SARS-CoV-2 comprising any one of the following 1) to 13); 1) A light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 29, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO: 10
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:30: 2) a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 115 or the amino acid sequence set forth in SEQ ID NO: 265, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 116;
  • a heavy chain variable region comprising a polypeptide consisting of the
  • An isolated monoclonal antibody or antigen-binding fragment thereof that binds to ORF8 protein of SARS-CoV-2 comprising any one of 1) to 90) below; 1) A light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:3 or the amino acid sequence of SEQ ID NO:23, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:4
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:24.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:5 or the amino acid sequence of SEQ ID NO:25, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:6;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:26.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:7 or the amino acid sequence set forth in SEQ ID NO:27, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:8;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:28.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 9 or the amino acid sequence set forth in SEQ ID NO: 29, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 10;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:30.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 11 or the amino acid sequence set forth in SEQ ID NO: 31, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 12;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:32.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 13 or the amino acid sequence set forth in SEQ ID NO: 33, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 14
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:34.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 15 or the amino acid sequence set forth in SEQ ID NO: 35, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 16
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:36.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 17 or the amino acid sequence set forth in SEQ ID NO: 37, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 18;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:38.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 19 or the amino acid sequence set forth in SEQ ID NO: 39, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 20;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:40.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:21 or the amino acid sequence set forth in SEQ ID NO:41, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:22
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:42.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:55 or the amino acid sequence set forth in SEQ ID NO:205, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:56
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:206.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:57 or the amino acid sequence set forth in SEQ ID NO:207, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:58;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:208.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:59 or the amino acid sequence set forth in SEQ ID NO:209, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:60;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:210.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:61 or the amino acid sequence set forth in SEQ ID NO:211, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:62
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:212.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 63 or the amino acid sequence set forth in SEQ ID NO: 213, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 64;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:214.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:65 or the amino acid sequence set forth in SEQ ID NO:215, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:66;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:216.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:67 or the amino acid sequence of SEQ ID NO:217, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:68
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:218.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:69 or the amino acid sequence set forth in SEQ ID NO:219, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:70;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:220.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 71 or the amino acid sequence set forth in SEQ ID NO: 221, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 72
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:222.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:73 or the amino acid sequence set forth in SEQ ID NO:223, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:74;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:224.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:75 or the amino acid sequence set forth in SEQ ID NO:225, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:76
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:226.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:77 or the amino acid sequence set forth in SEQ ID NO:227, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:78;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:228.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:79 or the amino acid sequence set forth in SEQ ID NO:229, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:80;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:230.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:81 or the amino acid sequence set forth in SEQ ID NO:231, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:82;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:232.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:83 or the amino acid sequence set forth in SEQ ID NO:233, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:84;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:234.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:85 or the amino acid sequence set forth in SEQ ID NO:235, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:86
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:236.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:87 or the amino acid sequence set forth in SEQ ID NO:237, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:88
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:238.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:89 or the amino acid sequence set forth in SEQ ID NO:239, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:90;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:240.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:91 or the amino acid sequence set forth in SEQ ID NO:241, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:92
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:242.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:93 or the amino acid sequence set forth in SEQ ID NO:243, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:94;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:244.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:95 or the amino acid sequence of SEQ ID NO:245, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:96
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:246.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:97 or the amino acid sequence of SEQ ID NO:247, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:98
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:248.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:99 or the amino acid sequence of SEQ ID NO:249, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:100;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:250.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 101 or the amino acid sequence set forth in SEQ ID NO: 251, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 102;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:252.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 103 or the amino acid sequence set forth in SEQ ID NO: 253, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 104
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:254.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 105 or the amino acid sequence set forth in SEQ ID NO: 255, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 106;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:256.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 107 or the amino acid sequence set forth in SEQ ID NO: 257, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 108;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:258.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 109 or the amino acid sequence set forth in SEQ ID NO: 259, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 110;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:260.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 111 or the amino acid sequence set forth in SEQ ID NO: 261, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 112
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:262.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 113 or the amino acid sequence set forth in SEQ ID NO: 263, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 114;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:264.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 115 or the amino acid sequence set forth in SEQ ID NO: 265, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 116;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:266.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 117 or the amino acid sequence set forth in SEQ ID NO: 267, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 118;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:268.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 119 or the amino acid sequence set forth in SEQ ID NO: 269, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 120
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:270.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 121 or the amino acid sequence set forth in SEQ ID NO: 271, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 122
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:272.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 123 or the amino acid sequence set forth in SEQ ID NO: 273, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 124
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:274.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 125 or the amino acid sequence set forth in SEQ ID NO: 275, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 126
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:276.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 127 or the amino acid sequence set forth in SEQ ID NO: 277, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 128
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:278.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 129 or the amino acid sequence set forth in SEQ ID NO: 279, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 130
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:280.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 131 or the amino acid sequence set forth in SEQ ID NO: 281, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 132
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:282.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 133 or the amino acid sequence set forth in SEQ ID NO: 283, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 134
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:284.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 135 or the amino acid sequence set forth in SEQ ID NO: 285, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 136
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:286.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 137 or the amino acid sequence set forth in SEQ ID NO: 287, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 138;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:288.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 139 or the amino acid sequence set forth in SEQ ID NO: 289, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 140
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:290.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 141 or the amino acid sequence set forth in SEQ ID NO: 291, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 142;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:292.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 143 or the amino acid sequence set forth in SEQ ID NO: 293, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 144
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:294.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 145 or the amino acid sequence set forth in SEQ ID NO: 295, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 146;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:296.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 147 or the amino acid sequence set forth in SEQ ID NO: 297, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 148;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:298.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 149 or the amino acid sequence set forth in SEQ ID NO: 299, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 150
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:300.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 151 or the amino acid sequence set forth in SEQ ID NO: 301, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 152
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:302.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 153 or the amino acid sequence set forth in SEQ ID NO: 303, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 154
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:304.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 155 or the amino acid sequence set forth in SEQ ID NO: 305, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 156
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:306.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 157 or the amino acid sequence set forth in SEQ ID NO: 307, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 158;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:308.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 159 or the amino acid sequence set forth in SEQ ID NO: 309, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 160
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:310.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 161 or the amino acid sequence set forth in SEQ ID NO: 311, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 162
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:312.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 163 or the amino acid sequence set forth in SEQ ID NO: 313, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 164
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:314.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 165 or the amino acid sequence set forth in SEQ ID NO: 315, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 166;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:316.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 167 or the amino acid sequence set forth in SEQ ID NO: 317, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 168;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:318.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 169 or the amino acid sequence set forth in SEQ ID NO: 319, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 170
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:320.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 171 or the amino acid sequence set forth in SEQ ID NO: 321, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 172
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:322.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 173 or the amino acid sequence set forth in SEQ ID NO: 323, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 174
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:324.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 175 or the amino acid sequence set forth in SEQ ID NO: 325, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 176
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:326.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 177 or the amino acid sequence set forth in SEQ ID NO: 327, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 178
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:328.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 179 or the amino acid sequence set forth in SEQ ID NO: 329, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 180
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:330.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 181 or the amino acid sequence set forth in SEQ ID NO: 331, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 182
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:332.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 183 or the amino acid sequence set forth in SEQ ID NO: 333, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 184
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:334.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 185 or the amino acid sequence set forth in SEQ ID NO: 335, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 186
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:336.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 187 or the amino acid sequence set forth in SEQ ID NO: 337, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 188
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:338.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 189 or the amino acid sequence set forth in SEQ ID NO: 339, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 190
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:340.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 191 or the amino acid sequence set forth in SEQ ID NO: 341, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 192
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:342.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 193 or the amino acid sequence set forth in SEQ ID NO: 343, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 194
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:344.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 195 or the amino acid sequence set forth in SEQ ID NO: 345, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 196
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:346.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 197 or the amino acid sequence set forth in SEQ ID NO: 347, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 198
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:348.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 199 or the amino acid sequence set forth in SEQ ID NO: 349, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 200
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:350.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 201 or the amino acid sequence set forth in SEQ ID NO: 351, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 202
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:352.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 203 or the amino acid sequence set forth in SEQ ID NO: 353, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 204
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:354.
  • Fab, Fab', F(ab') 2 or scFV the monoclonal antibody or antigen-binding fragment thereof according to any one of the preceding items 2 to 7.
  • a host cell comprising the nucleic acid according to 10 above.
  • An anti-inflammatory composition comprising the monoclonal antibody or antigen-binding fragment thereof according to 2 above as an active ingredient.
  • a therapeutic or prophylactic agent for coronavirus comprising the anti-inflammatory composition according to 12 above.
  • the present invention provides a monoclonal antibody that binds to the ORF8 protein of SARS-CoV-2 and an anti-inflammatory composition containing the antibody.
  • the subject of the present invention is a monoclonal antibody or an antigen-binding fragment thereof that specifically recognizes a dimeric ORF8 protein having three intramolecular disulfide bonds and one intermolecular disulfide bond of SARS-CoV-2, SARS - an isolated monoclonal antibody or antigen-binding fragment thereof that binds to the ORF8 protein of CoV-2, a nucleic acid encoding these monoclonal antibodies or antigen-binding fragments thereof, a host cell (hybridoma) comprising said nucleic acid, a monoclonal antibody thereof Or an anti-inflammatory composition containing an antigen-binding fragment thereof as an active ingredient, a coronavirus therapeutic or prophylactic agent containing the composition, and ORF8 by a sandwich method using these monoclonal antibodies or antigen-binding fragments thereof (especially wild-type ORF8) on detection methods.
  • the present invention will be described in detail below.
  • polynucleotide (DNA molecule) includes not only double-stranded DNA but also single-stranded DNA of the sense strand and antisense strand that constitute it, and is not limited in length. .
  • the polynucleotide (DNA molecule) in this specification can contain at least one of an expression suppressing region, a coding region, a leader sequence, an exon and an intron, regardless of the functional region.
  • Polynucleotides also include RNA and DNA.
  • Polynucleotide variants (mutant DNA) also include naturally occurring allelic variants; non-naturally occurring variants; deletion, substitution, addition and insertion variants. However, these mutants shall encode a polypeptide having substantially the same function as the polypeptide encoded by the polynucleotide before mutation.
  • Polypeptide or Polynucleotide Identity or homology can be analyzed by measurement using the FASTA program ⁇ Clustal, V., Methods Mol. Biol., 25, 307-318 (1994) ⁇ . As the most preferred and simple method for identity/homology analysis, the following known sequence databases can be used.
  • SARS-CoV-2 is classified as a single-stranded positive-strand RNA virus, and its genome has been sequenced.
  • the genome size of SARS-CoV-2 is approximately 30,000 bases, encoding 11 genes (Open Reading Frame, ORF).
  • Each gene contains one nonstructural protein (orf1ab), four structural proteins (spike (S) protein, envelope (E), membrane (M), nucleocapsid protein (N)) and six attachment proteins. It encodes proteins (ORF3a, ORF6, ORF7a, ORF7b, ORF8 and ORF10).
  • the mature ORF8 protein of SARS-CoV-2 (FHQECSLQSCTQHQPYVVDDPCPIHFYSKWYIRVGARKSAPLIELCVDEAGSKSPIQYIDIGNYTVSCLPFTINCQEPKLGSLVVRCSFYEDFLEYHDVRVVLDFI: SEQ ID NO: 54) is known to have three intramolecular disulfide bonds and to be a dimer due to intermolecular disulfide bonds (https:/ /doi.org/10.1073/pnas.2021785118).
  • ORF8 The ORF8 protein has been reported to mediate immune evasion by downregulating MHC-1 (Ref: PNAS June 8, 2021 118 (23) e2024202118; https://doi.org/10.1073/pnas .2024202118). It has also been reported that ORF8 inhibits various steps of the interferon (IFN) signaling pathway. In addition, ORF8 has also been reported to be associated with the cytokine storm associated with COVID-19 as a result of its interaction with the interleukin (IL)-17 receptor (https://www.news- medical.net/news/20210818/SARS-CoV-2-ORF8-and-the-Expression-of-Cytokine-in-Human-Macrophages.aspx).
  • IL-17 receptor https://www.news- medical.net/news/20210818/SARS-CoV-2-ORF8-and-the-Expression-of-Cytokine-in-Human-Macrophages.
  • ORF8 protein is considered to be involved in exacerbation of COVID-19.
  • Antibodies and the like that can inhibit ORF8-induced inflammatory effects are therefore effective ingredients for treating or preventing coronaviruses (particularly SARS-CoV-2).
  • ORF8-recognizing antibody of the present invention The ORF8-recognizing antibody of the present invention, "a monoclonal antibody or an antigen-binding fragment thereof that specifically recognizes a dimeric ORF8 protein having three intramolecular disulfide bonds and one intermolecular disulfide bond" or "SARS- An isolated monoclonal antibody or antigen-binding fragment thereof that binds to the ORF8 protein of CoV-2 has the following light and heavy chain variable regions.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:3 or the amino acid sequence of SEQ ID NO:23, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:4
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:24.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:5 or the amino acid sequence of SEQ ID NO:25, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:6;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:26.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:7 or the amino acid sequence set forth in SEQ ID NO:27, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:8;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:28.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 9 or the amino acid sequence set forth in SEQ ID NO: 29, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 10;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:30.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 11 or the amino acid sequence set forth in SEQ ID NO: 31, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 12;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:32.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 13 or the amino acid sequence set forth in SEQ ID NO: 33, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 14
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:34.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 15 or the amino acid sequence set forth in SEQ ID NO: 35, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 16
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:36.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 17 or the amino acid sequence set forth in SEQ ID NO: 37, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 18;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:38.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 19 or the amino acid sequence set forth in SEQ ID NO: 39, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 20;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:40.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:21 or the amino acid sequence set forth in SEQ ID NO:41, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:22
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:42.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:55 or the amino acid sequence set forth in SEQ ID NO:205, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:56
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:206.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:57 or the amino acid sequence set forth in SEQ ID NO:207, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:58;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:208.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:59 or the amino acid sequence set forth in SEQ ID NO:209, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:60;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:210.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:61 or the amino acid sequence set forth in SEQ ID NO:211, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:62
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:212.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 63 or the amino acid sequence set forth in SEQ ID NO: 213, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 64;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:214.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:65 or the amino acid sequence set forth in SEQ ID NO:215, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:66;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:216.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:67 or the amino acid sequence of SEQ ID NO:217, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:68
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:218.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:69 or the amino acid sequence set forth in SEQ ID NO:219, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:70;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:220.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 71 or the amino acid sequence set forth in SEQ ID NO: 221, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 72
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:222.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:73 or the amino acid sequence set forth in SEQ ID NO:223, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:74;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:224.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:75 or the amino acid sequence set forth in SEQ ID NO:225, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:76
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:226.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:77 or the amino acid sequence set forth in SEQ ID NO:227, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:78;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:228.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:79 or the amino acid sequence set forth in SEQ ID NO:229, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:80;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:230.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:81 or the amino acid sequence set forth in SEQ ID NO:231, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:82;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:232.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:83 or the amino acid sequence set forth in SEQ ID NO:233, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:84;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:234.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:85 or the amino acid sequence set forth in SEQ ID NO:235, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:86
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:236.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:87 or the amino acid sequence set forth in SEQ ID NO:237, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:88
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:238.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:89 or the amino acid sequence set forth in SEQ ID NO:239, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:90;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:240.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:91 or the amino acid sequence set forth in SEQ ID NO:241, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:92
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:242.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:93 or the amino acid sequence set forth in SEQ ID NO:243, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO:94;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:244.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:95 or the amino acid sequence of SEQ ID NO:245, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:96
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:246.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:97 or the amino acid sequence of SEQ ID NO:247, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:98
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:248.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:99 or the amino acid sequence of SEQ ID NO:249, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:100;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:250.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 101 or the amino acid sequence set forth in SEQ ID NO: 251, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 102;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:252.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 103 or the amino acid sequence set forth in SEQ ID NO: 253, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 104
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:254.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 105 or the amino acid sequence set forth in SEQ ID NO: 255, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 106;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:256.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 107 or the amino acid sequence set forth in SEQ ID NO: 257, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 108;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:258.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 109 or the amino acid sequence set forth in SEQ ID NO: 259, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 110;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:260.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 111 or the amino acid sequence set forth in SEQ ID NO: 261, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 112
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:262.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 113 or the amino acid sequence set forth in SEQ ID NO: 263, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 114;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:264.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 115 or the amino acid sequence set forth in SEQ ID NO: 265, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 116;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:266.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 117 or the amino acid sequence set forth in SEQ ID NO: 267, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 118;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:268.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 119 or the amino acid sequence set forth in SEQ ID NO: 269, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 120;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:270.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 121 or the amino acid sequence set forth in SEQ ID NO: 271, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 122
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:272.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 123 or the amino acid sequence set forth in SEQ ID NO: 273, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 124
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:274.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 125 or the amino acid sequence set forth in SEQ ID NO: 275, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 126
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:276.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 127 or the amino acid sequence set forth in SEQ ID NO: 277, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 128
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:278.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 129 or the amino acid sequence set forth in SEQ ID NO: 279, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 130
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:280.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 131 or the amino acid sequence set forth in SEQ ID NO: 281, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 132
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:282.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 133 or the amino acid sequence set forth in SEQ ID NO: 283, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 134
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:284.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 135 or the amino acid sequence set forth in SEQ ID NO: 285, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 136
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:286.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 137 or the amino acid sequence set forth in SEQ ID NO: 287, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 138;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:288.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 139 or the amino acid sequence set forth in SEQ ID NO: 289, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 140
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:290.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 141 or the amino acid sequence set forth in SEQ ID NO: 291, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 142;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:292.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 143 or the amino acid sequence set forth in SEQ ID NO: 293, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 144
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:294.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 145 or the amino acid sequence set forth in SEQ ID NO: 295, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 146;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:296.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 147 or the amino acid sequence set forth in SEQ ID NO: 297, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 148;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:298.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 149 or the amino acid sequence set forth in SEQ ID NO: 299, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 150
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:300.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 151 or the amino acid sequence set forth in SEQ ID NO: 301, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 152
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:302.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 153 or the amino acid sequence set forth in SEQ ID NO: 303, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 154
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:304.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 155 or the amino acid sequence set forth in SEQ ID NO: 305, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 156
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:306.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 157 or the amino acid sequence set forth in SEQ ID NO: 307, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 158;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:308.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 159 or the amino acid sequence set forth in SEQ ID NO: 309, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 160
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:310.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 161 or the amino acid sequence set forth in SEQ ID NO: 311, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 162
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:312.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 163 or the amino acid sequence set forth in SEQ ID NO: 313, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 164
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:314.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 165 or the amino acid sequence set forth in SEQ ID NO: 315, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 166;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:316.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 167 or the amino acid sequence set forth in SEQ ID NO: 317, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 168;
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:318.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 169 or the amino acid sequence set forth in SEQ ID NO: 319, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 170
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:320.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 171 or the amino acid sequence set forth in SEQ ID NO: 321, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 172
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:322.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 173 or the amino acid sequence set forth in SEQ ID NO: 323, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 174
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:324.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 175 or the amino acid sequence set forth in SEQ ID NO: 325, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 176
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:326.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 177 or the amino acid sequence set forth in SEQ ID NO: 327, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 178
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:328.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 179 or the amino acid sequence set forth in SEQ ID NO: 329, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 180
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:330.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 181 or the amino acid sequence set forth in SEQ ID NO: 331, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 182
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:332.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 183 or the amino acid sequence set forth in SEQ ID NO: 333, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 184
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:334.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 185 or the amino acid sequence set forth in SEQ ID NO: 335, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 186
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:336.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 187 or the amino acid sequence set forth in SEQ ID NO: 337, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 188
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:338.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 189 or the amino acid sequence set forth in SEQ ID NO: 339, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 190
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:340.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 191 or the amino acid sequence set forth in SEQ ID NO: 341, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 192
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:342.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 193 or the amino acid sequence set forth in SEQ ID NO: 343, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 194
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:344.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 195 or the amino acid sequence set forth in SEQ ID NO: 345, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 196
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:346.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 197 or the amino acid sequence set forth in SEQ ID NO: 347, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 198
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:348.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 199 or the amino acid sequence set forth in SEQ ID NO: 349, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 200
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:350.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 201 or the amino acid sequence set forth in SEQ ID NO: 351, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 202
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:352.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 203 or the amino acid sequence set forth in SEQ ID NO: 353, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 204
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:354.
  • a light chain variable region and/or a heavy chain variable region comprising a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence in which one or more bases have been deleted, inserted, substituted or added antibody.
  • a plurality of amino acids have been deleted, inserted, substituted or added means that 2 or more to 20 or less amino acids have been deleted, inserted, substituted or added.
  • the plurality of amino acids is preferably 2 or more and 10 or less, more preferably 2 or more and 7 or less, even more preferably 2 or more and 5 or less.
  • this modified amino acid sequence has an identity of, for example, about 95% or more, preferably about 96% or more, more preferably about 97% or more, and even more preferably about 98% identity with the amino acid sequence described in SEQ ID NO. % or more.
  • Means for introducing mutations such as deletion, substitution, addition or insertion are known per se, and for example, Ulmer's technique (K. M.
  • a plurality of bases have been deleted, inserted, substituted or added means that 2 or more to 20 or less bases have been deleted, inserted, substituted or added.
  • the plurality of bases is preferably 2 or more and 20 or less, more preferably 2 or more and 10 or less, even more preferably 2 or more and 5 or less.
  • this modified base sequence preferably has, for example, about 95% or more identity, preferably about 98% or more identity, and more preferably about 99% or more identity with the nucleotide sequence described in SEQ ID NO.
  • stringent conditions refers to conditions in which hybridization occurs in 2 ⁇ SSC containing 0.1% SDS at 50° C. and no detachment occurs even after washing in 1 ⁇ SSC containing 0.1% SDS at 60° C. can be mentioned.
  • the ORF8-recognizing antibody of the present invention is IgG. According to Example 2 below, it was confirmed that the ORF8-recognizing antibodies of the present invention (especially clone numbers: 4A3, 11G3, 31E2) have an inhibitory effect on inflammation (especially an inhibitory effect on inflammation by ORF8 protein).
  • 31E2 a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 17 or the amino acid sequence set forth in SEQ ID NO: 37, and encoded by the nucleotide sequence set forth in SEQ ID NO: 18
  • Heavy chain variable region comprising an amino acid sequence or a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:38 11G3: an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:13 or a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:33 and a heavy chain variable region 4A3 comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 14 or the amino acid sequence set forth in SEQ ID NO: 34: set forth in SEQ ID NO: 7
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucle
  • 3A11 a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:9 or the amino acid sequence of SEQ ID NO:29, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:10
  • a heavy chain variable region comprising a polypeptide consisting of an amino acid sequence or the amino acid sequence set forth in SEQ ID NO: 30
  • the ORF8-recognizing antibody of the present invention has a higher dimeric ORF8 recognition ability than a monomeric ORF8 protein. is high.
  • the ORF8-recognizing antibody of the present invention recognizes a dimer of wild-type ORF8 protein having three disulfide bonds in the molecule close to the native state and one disulfide bond in the molecule, and has the effect of suppressing the inflammatory action of Additionally, the antibody includes a humanized antibody or humanized antibody fragment, a single chain antibody or single chain antibody fragment, or Fab, Fab', F(ab') 2 or scFV. Furthermore, the antibody can be made into a humanized antibody by a method known per se.
  • the ORF8-recognizing antibodies of the present invention described below have the property of recognizing only dimers without recognizing monomers.
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:3 or the amino acid sequence of SEQ ID NO:23, and a light chain variable region encoded by the nucleotide sequence of SEQ ID NO:4
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO: 24: 2
  • a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 9 or the amino acid sequence set forth in SEQ ID NO: 29, and a light chain variable region encoded by the nucleotide sequence set forth in SEQ ID NO: 10
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention can detect ORF8 (particularly wild-type ORF8) by specifically recognizing ORF8 (particularly wild-type ORF8). Furthermore, the sandwich method using the monoclonal antibody or antigen-binding fragment thereof of the present invention can detect ORF8 (especially wild-type ORF8) with high sensitivity. In the method for detecting ORF8 by the sandwich method of the present invention, a sandwich method known per se can be employed. Moreover, in the present invention, one antibody can be the monoclonal antibody of the present invention, but two antibodies are preferably the monoclonal antibodies of the present invention.
  • 31E2 and 4E6 31E2 and 7C9 31E2 and 11G3 31E2 and 12B7 31E2 and 31E2 4E6: a light chain variable region comprising a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 9 or the amino acid sequence set forth in SEQ ID NO: 29, and encoded by the nucleotide sequence set forth in SEQ ID NO: 10
  • a heavy chain variable region comprising a polypeptide consisting of the amino acid sequence or the amino acid sequence set forth in SEQ ID NO:30
  • a peptide containing all or part of the amino acid sequence of the ORF8 protein can be used as an immunogen for producing the ORF8-recognizing antibody of the present invention.
  • a peptide containing all or part of the amino acid sequence of a wild-type ORF8 protein dimer having one intermolecular disulfide bond is preferable to use.
  • a purified peptide or partial peptide serving as an immunogen as described above is dissolved or suspended in an appropriate buffer such as phosphate buffer (PBS) and used as an antigen solution.
  • the antigen solution may be prepared to a concentration containing about 50 to 500 ⁇ g/mL of the antigenic substance.
  • an appropriate carrier protein such as albumin or keyhole limpet hemocyanin (KLH).
  • animals to be immunized with the antigen include mice, rats, hamsters, horses, goats, and rabbits. Mouse is preferred.
  • the antigen solution can be mixed with an adjuvant and administered.
  • Adjuvants that can be used here are Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Ribi (MPL), Ribi (TDM), Ribi (MPL+TDM).
  • FCA Freund's complete adjuvant
  • FIA Freund's incomplete adjuvant
  • MPL MPL
  • TDM Ribi
  • MPL+TDM Ribi
  • Pertussis vaccine Booredetella pertussis vaccine
  • FCA Freund's complete adjuvant
  • FIA Freund's incomplete adjuvant
  • MPL MPL
  • TDM Ribi
  • MPL+TDM Ribi
  • Pertussis vaccine Booredetella pertussis vaccine
  • MDP muramyl dipeptide
  • ALUM aluminum adjuvant
  • the injection site and schedule can be changed as appropriate depending on the type of antigen used and the presence or absence of adjuvant mixture.
  • 10 to 200 ⁇ g of antigenic substance is injected intraperitoneally, subcutaneously, intramuscularly or (tail) into the vein, followed by 1 to 4 booster immunizations about every 4 to 21 days from the initial immunization, and about 1 to 4 weeks later.
  • the antigen solution can also be administered without an adjuvant by intraperitoneal injection with a large amount of antigen.
  • the antibody titer is determined by collecting blood. The antibody titer can be measured by a commonly used method according to the antibody titer assay described below.
  • splenocytes are isolated from the immunized animal to obtain antibody-producing cells.
  • Monoclonal antibodies (hereinafter sometimes abbreviated as "MoAb") can be obtained by a method known per se, for example, the method of Kohler and Milstein (Kohler G, Milstein C. (1975) Continuous cultures of fused cellssecreting antibody of predefined specificity. Nature 256, 495-497.).
  • tissue containing antibody-producing cells e.g., spleen or lymph node
  • hybridomas are prepared by fusing the antibody-producing cells with known tumor cells (e.g., bone tumor cells). After cloning the hybridomas, the hybridomas producing the desired antibody are selected, and the antibody is recovered from the hybridoma culture medium.
  • myeloma cells those derived from mice, rats, humans, etc. are used.
  • Myeloma cells are exemplified. Some myeloma cells produce an immunoglobulin light chain, and if this is used as a fusion target, the immunoglobulin heavy chain produced by the antibody-producing cell and this light chain may randomly combine. In particular, it is preferable to use myeloma cells that do not produce immunoglobulin light chains, such as P3X63-Ag8 ⁇ 653 and SP2/o-Ag14.
  • Antibody-producing cells and myeloma cells are preferably derived from allogeneic animals, particularly animals of the same strain.
  • Myeloma cells may be preserved according to a method known per se. For example, myeloma cells are subcultured in a common medium supplemented with horse, rabbit or fetal bovine serum and preserved by freezing. Also, it is preferable to use cells in the logarithmic growth phase for cell fusion.
  • Examples of methods for producing hybridomas by fusing antibody-producing cells and myeloma cells include a method using polyethylene glycol (PEG), a method using Sendai virus, and a method using an electrofusion device.
  • PEG polyethylene glycol
  • splenocytes and myeloma cells are mixed at a ratio of 1-10:1, preferably 5-10:1, in an appropriate medium or buffer containing about 30-60% PEG (average molecular weight: 1,000-6,000). and allowed to react for about 30 seconds to 3 minutes at a temperature of about 25 to 37° C. and a pH of 6 to 8.
  • a selective medium is a medium in which the parental cell line is killed and only fused cells can grow, and hypoxanthine-aminopterin-thymidine (HAT) medium is usually used.
  • Selection of hybridomas is usually carried out by exchanging part, preferably about half, of the medium with selective medium 1 to 7 days after the fusion procedure, and culturing while repeating the same medium exchange every 2 or 3 days. Wells in which hybridoma colonies are growing are confirmed by microscopic observation.
  • the culture supernatant may be collected and an antibody titer assay may be performed by a method known per se. Further, single clones are isolated by limiting dilution, soft agar, fluorescence-excited cell sorter, or the like.
  • the immunoglobulin subclass of the antibody produced by the hybridoma is determined by culturing the hybridoma under general conditions and analyzing the antibody secreted in the culture supernatant using a commercially available antibody class/subclass determination kit. can know.
  • the method for obtaining MoAb from hybridomas can be appropriately selected depending on the required amount, properties of hybridomas, and the like. For example, a method of obtaining ascites from a mouse transplanted with the hybridoma, a method of obtaining from a culture supernatant by cell culture, and the like are exemplified. MoAb at a high concentration of several mg/mL can be obtained from the ascites of a hybridoma that can proliferate in the peritoneal cavity of a mouse. Hybridomas that cannot grow in vivo are obtained from cell culture supernatants.
  • Obtaining MoAb by cell culture produces a lower amount of antibody than in vivo, but has the advantage of being less contaminated with immunoglobulins and other contaminants contained in the peritoneal cavity of mice and easier to purify.
  • antibodies are obtained intraperitoneally from hybridoma-implanted mice, for example, intraperitoneally from BALB/c mice pre-administered with a substance having an immunosuppressive effect such as pristane (2, 6, 10, 14-tetramethylpentadecane)
  • Hybridomas approximately 10 6 or more
  • heterologous hybridomas eg mouse and rat
  • nude mice radiation-treated mice.
  • a culture method such as a high-density culture method or a spinner flask culture method is used to culture the hybridoma to contain the antibody.
  • Obtain the culture supernatant for Purification of MoAb from ascites or culture supernatant can be performed by a method known per se.
  • conventional methods for purifying immunoglobulins include fractionation by salting out using ammonium sulfate or sodium sulfate, polyethylene glycol fractionation, ethanol fractionation, DEAE ion exchange chromatography, gel filtration, and the like. is easily achieved.
  • the MoAb is mouse IgG, it can be easily purified by affinity chromatography using a protein A binding substance or an anti-mouse immunoglobulin binding substance.
  • the ORF8-recognizing antibody of the present invention recognizes a dimer of the wild-type ORF8 protein having three intramolecular disulfide bonds in a near-native state and one intermolecular disulfide bond. It has the effect of suppressing the inflammatory action of the dimer. Therefore, an anti-inflammatory composition containing the ORF8-recognizing antibody of the present invention as an active ingredient can suppress ORF8-mediated inflammation. Furthermore, the anti-inflammatory composition can be used for powders, granules, tablets, capsules, enteric agents, liquids, injections (solutions, suspensions) or gene therapy, depending on the purpose of treatment (including prevention). Various forms such as the form used can be prepared according to a conventional method.
  • the dosage or intake of the anti-inflammatory composition is not particularly limited as long as the effects of the present invention can be obtained. , properties of the subject (body weight, age, medical condition, use or non-use of other medicines, etc.), and the judgment of the doctor in charge.
  • the anti-inflammatory composition can be administered or ingested once to several times a day, or intermittently administered or ingested once every several days or several weeks.
  • the orf8-recognizing antibody of the present invention becomes an active ingredient of a coronavirus (especially, SARS-CoV-2) therapeutic or prophylactic agent.
  • the coronavirus therapeutic or prophylactic agent can be powder, granule, tablet, capsule, enteric agent, liquid, injection (liquid, suspension) or gene Various forms, such as those used for therapy, can be prepared according to conventional methods. Additionally, fillers, extenders, binders, wetting agents, disintegrants, lubricants, diluents and excipients may be included.
  • the dosage or intake of the therapeutic or prophylactic agent is not particularly limited as long as the effect of the present invention can be obtained, and the efficacy of the ingredients contained, dosage form, administration route, type of disease , properties of the subject (body weight, age, medical condition, use or non-use of other medicines, etc.), and the judgment of the doctor in charge.
  • the therapeutic or prophylactic agent can be administered or ingested once to several times a day, or intermittently administered or ingested once every several days or several weeks.
  • a dimer of ORF8 protein having three intramolecular disulfide bonds and one intermolecular disulfide bond close to the native state of SARS-CoV-2 was used as an antigen. Characterized by For a method for producing a dimer of ORF8 protein having three intramolecular disulfide bonds and one intermolecular disulfide bond, refer to the document “Plant Cell Reports volume 40, pages 433–436 (2021)”.
  • pBICLBSER-ToMV-SP-His-ORF8 was introduced into the BY-2 strain (pBICHgLBSXVE introduced strain) expressing a transcription factor (XVE gene) that is activated by estradiol using the Agrobacterium method.
  • BY-2 cells transfected with pBICLBSER-ToMV-SP-His-ORF8 were selected with medium containing kanamycin.
  • the selected BY-2 cells were treated with estradiol, and the amount of 8His-ORF8 accumulated in the culture supernatant was evaluated by SDS-PAGE (see Archives of Virology 151, 1075-1084. (2006)). Based on the evaluation results, the BY-2 strain that expresses the most 8His-ORF8 was selected.
  • the obtained BY-2 cells were cultured on a 1 L scale, estradiol was added, and the culture supernatant was collected 7 days after the addition.
  • the recovered culture supernatant was crudely purified using a nickel column that specifically purifies His-tagged proteins. After crude purification, purification was performed using a gel filtration column, and then the buffer was exchanged with PBS by dialysis to obtain 8HIS-ORF8 used for immunization.
  • 3 BALB/c mice (mouse No. 1, No. 2 and No. 3) were immunized three times per mouse by mixing 8HIS-ORF8 with an adjuvant by lymph node method.
  • the second immunization was performed 15 days after the first immunization, and the third immunization was performed 22 days (mouse No. 1 and No. 2) and 25 days (mouse No. 3).
  • mouse spleens and lymph nodes were collected to prepare lymphocytes.
  • the prepared lymphocytes and myeloma cells (P3U1) were fused.
  • the fused cells were cultured in HAT medium to select hybridoma cells.
  • Hybridoma strains were obtained by limiting dilution and culture. Eighty-five lines of antigen-positive hybridomas were selected by the ELISA method (Table) of the obtained hybridoma culture medium. Ten strains were selected from among the 85 strains, and the marginal interpretation method was repeated 2-3 times to establish hybridomas as single clones.
  • RNA was purified from 10 hybridoma strains using a Qiagen RNA extraction kit.
  • cDNA was synthesized with SMARTScribe Reverse Transcriptase using ttgtcgttcactgccatcaatc (SEQ ID NO: 43) for the light chain and agctgggaaggtgtgcacac (SEQ ID NO: 44) for the heavy chain as primers for reverse transcription of the purified RNA.
  • RNA-RNA hybrid primer a DNA-RNA hybrid primer was used as a 2nd synthesis primer (aagcagtggtatcaacgcagagtacatrgrgrg, rg: guanosine monophosphate (RNA), (SEQ ID NO: 45)) for complete cloning of the 5-end.
  • the light chain PCR primer set (aagcagtggtatcaacgcagag (SEQ ID NO: 46), acattgatgtctttggggtagaag (SEQ ID NO: 47)), the heavy chain PCR primer set (aagcagtggtatcaacgcagag (SEQ ID NO: 48), gggatccagagttccaggtc (SEQ ID NO: 49) ), a 40-cycle PCR reaction was performed using PrimeStarGXL as the PCR enzyme to amplify the variable region of each monoclonal antibody gene.
  • T25 flask Raw264.7 cells that had become confluent in a T25 flask were seeded onto a 24-well plate at 1/48 and cultured at 37°C for 36 hours. After replacing the medium with serum- and antibiotic-free Dulbecco's modified Eagle's medium (high glucose), each of the three monoclonal antibodies purified by Spin column based Antibody Purification Kit was added and cultured for 24 hours. Then, using Enzyme-Linked Immuno SorbentAssay, tumor necrosis factor alpha (TNF ⁇ ) in the culture supernatant was quantified.
  • TNF ⁇ tumor necrosis factor alpha
  • ORF8 final concentration 1 ug/ml
  • mouse IgG final concentration 2.5 ug/ml
  • anti-ORF8 mouse IgG clone numbers: 4A3, 11G3, 31E2, final concentration 2.5 ug/ml
  • the ELISA protocol is as follows. ELISA plate: #208004 (BM) Immobilization: 1.0ug/ml (PBS) O/N Blocking: 0.5% skim milk 1st Ab: sup. 50uL/well
  • ALP-Ab ALP Goat-a-mIgG (1030-04 SBI) ⁇ 2500
  • Substrate Kind-King modification Measurement: 492nm
  • Fig. 3 shows the measurement results of TNF ⁇ .
  • isolated monoclonal antibodies that bind to the ORF8 protein of SARS-CoV-2, clone numbers 4A3, 11G3 and 31E2 reduce the elevated TNF ⁇ concentration upon addition of ORF8. It was confirmed that it has an anti-inflammatory effect due to
  • Example 2 The same method as in Example 2 was performed under the following conditions. Antigen without DTT treatment: 1 ug/ml ORF8_WT (diluted with Coating buffer) Antigen with DTT treatment: 1 ug/ml ORF8_WT (diluted with Coating buffer) with 10 mM DTT Block: 1% Block Ace Sample: 50ul/well 2nd Ab: 1/3000 anti-mouse IgG (diluted with 1% Block Ace)
  • Table 1 below shows the results of comparing the dimer ORF8 recognition ability and the monomer ORF8 recognition ability of monoclonal antibodies. As is clear from the results in Table 1, it was confirmed that the 10 types of monoclonal antibodies have a higher ability to recognize dimer ORF8 than monomeric ORF8. As a result, it was confirmed that the monoclonal antibody of the present invention recognizes, as an antigen, a dimer of ORF8 protein having three intramolecular disulfide bonds and one intermolecular disulfide bond in a near-native state.
  • the absorbance of the wild-type ORF8 protein dimer having three intramolecular and one intermolecular disulfide bonds was 2.454, but the absorbance of the mutant L84S dimer was 1.251. there were.
  • the monoclonal antibody of the present invention can specifically recognize a dimer of wild-type ORF8 protein having three intramolecular disulfide bonds and one intermolecular disulfide bond. confirmed.
  • ORF8 dissolved in cracking buffer (25 mM DTT) and ORF8 dissolved in cracking buffer (0 mM DTT) were heat-treated at 95°C for 5 minutes and electrophoresed on a 15% SDS-PAGE gel.
  • 4 ⁇ L of a commercially available molecular weight marker (Precision Plus Protein Dual Color Standards, BIO-RAD, #161-0374) was electrophoresed simultaneously.
  • ORF8 was transferred to Immobilon-P membrane (Merck Millipore) using transfer buffer (2.5 mM Tris, 192 mM glycine, 20% methanol).
  • the membranes onto which the dimer and monomeric ORF8 were transferred were confirmed for their detectability by the following method.
  • (1) The membrane after transfer was immersed in 15 mL of BlockingOne (Nacalai Tesque, 03953-95) and shaken at room temperature for 1 hour.
  • (2) The membrane was gently washed with Milli-Q, immersed in a monoclonal antibody diluted with 3 mL of Can Get Signal (registered trademark) Immunoreaction Enhancer Solution 1 (TOYOBO, NKB-101), and shaken overnight at 4°C.
  • Can Get Signal registered trademark
  • Monoclonal antibody 31E2 was dissolved in 100 ⁇ l of carbonate buffer and immobilized on the plate for 4 ⁇ 16 hours. After fixation, the cells were washed with PBS three times. After that, 300 ⁇ l of Bloking One (Nacalai) diluted 4 times was added to the well, left at room temperature for 45 minutes, and washed 5 times with PBS-T. 100 ⁇ l of ORF8 (0.5 ⁇ g/ml in Can get signal 1 (Toyobo)) was added to the wells.
  • ORF8 0.5 ⁇ g/ml in Can get signal 1 (Toyobo)
  • biotin-labeled monoclonal antibodies 4E6, 7C9, 11G3, 12B7, and 31E2 (1 ⁇ g/ml in Can get signal 1) were added respectively, left at 37°C for 1 hour, and then washed three times with PBS-T.
  • 100 ⁇ l of 10,000-fold diluted streptavidin-added peroxider (Pierce High Sensitivity Streptavidin-HRP) was added and allowed to stand at room temperature for 30 minutes. After washing five times with PBS-T, 100 ⁇ l of TMB solution was added, left at room temperature for 30 minutes, and 100 ⁇ l of 1N sulfuric acid was added.
  • the absorbance (450) was measured to determine the combinations of monoclonal antibodies to which the sand pitch method can be applied.
  • antibodies with the highest binding capacity without non-specific binding were selected.
  • a HIS-tagged marker protein (3-Color Prestained XL-Ladder) that does not bind to the ORF8 monoclonal antibody during Western blotting analysis was blotted.
  • the monoclonal antibody was assessed as having non-specific binding if the marker protein was detected. In addition, when not detected, it was evaluated that there was no non-specific binding.
  • Monoclonal antibody 31C10 confirmed the absence of non-specific binding. Based on this, it was determined that the monoclonal antibody 31C10 has the highest binding capacity without non-specific binding.
  • a monoclonal antibody that binds to ORF8 of SARS-CoV-2 and an anti-inflammatory composition containing the antibody can be provided.

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CN119464223B (zh) * 2025-01-07 2025-04-04 中山大学 分泌抗新型冠状病毒orf8蛋白单克隆抗体的杂交瘤细胞株、单克隆抗体、抗体组合物和应用

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