WO2024019110A1 - Anticorps contre le sars-cov-2 - Google Patents

Anticorps contre le sars-cov-2 Download PDF

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Publication number
WO2024019110A1
WO2024019110A1 PCT/JP2023/026565 JP2023026565W WO2024019110A1 WO 2024019110 A1 WO2024019110 A1 WO 2024019110A1 JP 2023026565 W JP2023026565 W JP 2023026565W WO 2024019110 A1 WO2024019110 A1 WO 2024019110A1
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Prior art keywords
amino acid
acid sequence
seq
set forth
sequence set
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PCT/JP2023/026565
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English (en)
Japanese (ja)
Inventor
宜聖 高橋
彩野 森山
航平 湯本
大祐 黒田
悠 安達
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国立感染症研究所長が代表する日本国
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Priority claimed from JP2023086002A external-priority patent/JP2024014723A/ja
Application filed by 国立感染症研究所長が代表する日本国 filed Critical 国立感染症研究所長が代表する日本国
Publication of WO2024019110A1 publication Critical patent/WO2024019110A1/fr

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    • 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
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • the present invention relates to antibodies against SARS-CoV-2, and more particularly, antibodies that bind to the spike glycoprotein receptor binding site (RBD) of SARS-CoV-2, and prevention or treatment of COVID-19 using the same.
  • the present invention relates to therapeutic compositions, compositions for immunologically detecting SARS-CoV-2, and methods for immunologically detecting SARS-CoV-2.
  • coronavirus severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2
  • COVID-19 severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2
  • mRNA vaccines and DNA vaccines have begun worldwide, but since vaccines are generally preventive drugs, there is a strong desire for useful therapeutic drugs.
  • functional inhibitors such as remdesivir, an RNA-dependent RNA polymerase inhibitor, and anti-inflammatory agents such as dexamethasone are known as treatments for COVID-19, but these drugs have serious side effects. tends to appear more easily.
  • therapeutic antibodies As a therapeutic drug that has both high efficacy and low side effects, therapeutic antibodies have been used in the medical field to treat various diseases such as infectious diseases, cancer, and rheumatoid arthritis. Research into antibodies as therapeutic agents is also actively conducted. For example, in Japan, "antibody cocktail therapy" in which two types of anti-SARS-CoV-2 antibodies are administered to patients was approved in 2021 as a treatment method for mild to moderate cases of COVID-19. - It has been reported that the combination of two or more types of CoV-2 antibodies can suppress the generation of antibody escape mutant viruses (escape viruses) (Baum et al., Science 369, p. 1014-1018, 2020 (non-patent literature) 1).
  • escape viruses escape mutant viruses
  • SARS-CoV-2 is known to infect the host cell by the spike glycoprotein (S protein) on the surface of the virus recognizing angiotensin converting enzyme 2 (ACE-2, S protein receptor) on the host cell.
  • S protein spike glycoprotein
  • ACE-2 angiotensin converting enzyme 2
  • Many of the current antibodies for treating COVID-19 exert their infection-neutralizing effects by blocking the binding of the spike glycoprotein to ACE-2.
  • SARS-CoV-2 has undergone numerous mutations, and mutant strains that have mutations in the base sequence of the viral genome and amino acid substitutions or deletions in the viral protein often have poor immune escape ability. It is known that such mutant strains can acquire growth advantages and replace previously prevalent strains. Therefore, when a mutant strain with a mutation in the spike glycoprotein receptor binding site (RBD), which is the ACE-2 binding region, replaces a new epidemic strain, the strain that was previously developed for the previous epidemic strain is replaced. The therapeutic effect of antibodies for treating COVID-19 will no longer be exerted.
  • RBD spike glycoprotein receptor binding site
  • the present invention was made in view of the above-mentioned problems of the conventional technology, and has a binding affinity for not only the conventional SARS-CoV-2 (Wuhan strain) but also other mutant strains including the Omicron strain. and novel antibodies with high infection-neutralizing activity, compositions for preventing or treating COVID-19 using the same, compositions for immunologically detecting SARS-CoV-2, and SARS-CoV-2.
  • the purpose of this invention is to provide a method for immunologically detecting.
  • the present inventors have conducted intensive research to achieve the above-mentioned objective, and have determined that the SARS-CoV-2 spike glycoprotein receptor binding site (RBD ) and that recognize serine at position 443 or tyrosine at position 489 of the amino acid sequence of the spike glycoprotein.
  • RBD SARS-CoV-2 spike glycoprotein receptor binding site
  • These antibodies have binding affinity not only for the conventional SARS-CoV-2 (Wuhan strain) but also for other mutant strains including the Delta strain and Omicron strains (BA.1, BA.2, etc.). It was also found that the infection neutralizing activity was high. Furthermore, by further improving such antibodies, we have also improved the binding affinity and infection resistance for the later Omicron strains (BA.5, BA.2.75, BQ.1.1, XBB, XBB.1.5), etc. We have developed an antibody with higher anti-oxidation activity and completed the present invention.
  • the present invention provides an antibody against SARS-CoV-2, a composition for preventing or treating COVID-19 using the same, a composition for immunologically detecting SARS-CoV-2, and a composition for immunologically detecting SARS-CoV-2.
  • a composition for immunologically detecting SARS-CoV-2 a composition for immunologically detecting SARS-CoV-2.
  • the method of immunologically detecting -2 more specifically, the following is provided.
  • An antibody against SARS-CoV-2 which binds to the spike glycoprotein receptor binding site (RBD) of SARS-CoV-2, and is an antibody that binds to the spike glycoprotein receptor binding site (RBD), and is an antibody that binds to the 443rd serine or 489th amino acid sequence of the spike glycoprotein.
  • An anti-SARS-CoV-2 antibody that recognizes tyrosine at position.
  • [2] The anti-SARS-CoV-2 according to [1], which binds to the spike glycoprotein receptor binding site (RBD) of SARS-CoV-2 with a dissociation constant (K D ) of 1.0 ⁇ 10 ⁇ 6 M or less antibody.
  • the amino acid sequence set forth in SEQ ID NO: 2, or the amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 2 is used as the heavy chain complementarity determining region 3-1. and hold each as The amino acid sequence at positions 27 to 32 of the amino acid sequence set forth in SEQ ID NO: 31, or the substitution or deletion of one or several amino acids in the amino acid sequence at positions 27 to 32 of the amino acid sequence set forth in SEQ ID NO: 31.
  • the deleted, added, and/or inserted amino acid sequence is the light chain complementarity determining region 1-1; the amino acid sequence at positions 50 to 52 of the amino acid sequence set forth in SEQ ID NO: 31, or The amino acid sequence in which one or several amino acids are substituted, deleted, added and/or inserted in the amino acid sequence at positions 50 to 52 of the amino acid sequence is described in light chain complementarity determining region 2-1; SEQ ID NO: 3. or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 3, respectively, as light chain complementarity determining region 3-1.
  • Antibodies (2) The amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 32, or one or several amino acids in the amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 32.
  • the substituted, deleted, added and/or inserted amino acid sequence is the heavy chain complementarity determining region 1-2; the amino acid sequence at positions 51 to 58 of the amino acid sequence set forth in SEQ ID NO: 32, or SEQ ID NO: 32.
  • the amino acid sequence set forth in SEQ ID NO: 4, or the amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 4, is used as the heavy chain complementarity determining region 3-2. and hold each as The amino acid sequence at positions 26 to 34 of the amino acid sequence set forth in SEQ ID NO: 33, or the substitution or deletion of one or several amino acids in the amino acid sequence at positions 26 to 34 of the amino acid sequence set forth in SEQ ID NO: 33.
  • the deleted, added, and/or inserted amino acid sequence is the light chain complementarity determining region 1-2; the amino acid sequence at positions 52 to 54 of the amino acid sequence set forth in SEQ ID NO: 33, or The amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence at positions 52 to 54 of the amino acid sequence is described in light chain complementarity determining region 2-2; SEQ ID NO: 5. or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 5, respectively, as light chain complementarity determining region 3-2.
  • Antibodies (3) The amino acid sequence at positions 19 to 28 of the amino acid sequence set forth in SEQ ID NO: 34, or one or several amino acids in the amino acid sequence at positions 19 to 28 of the amino acid sequence set forth in SEQ ID NO: 34.
  • the substituted, deleted, added and/or inserted amino acid sequence is the heavy chain complementarity determining region 1-3; the amino acid sequence at positions 46 to 52 of the amino acid sequence set forth in SEQ ID NO: 34, or SEQ ID NO: 34.
  • the amino acid sequence set forth in SEQ ID NO: 6, or the amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 6, is used as the heavy chain complementarity determining region 3-3. and hold each as In the amino acid sequence at positions 26 to 34 of the amino acid sequence set forth in SEQ ID NO: 35, or in the amino acid sequence at positions 26 to 34 of the amino acid sequence set forth in SEQ ID NO: 35, one or several amino acids are substituted or deleted.
  • the deleted, added, and/or inserted amino acid sequence is the light chain complementarity determining region 1-3; the amino acid sequence at positions 52 to 54 of the amino acid sequence set forth in SEQ ID NO: 35, or The amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence at positions 52 to 54 of the amino acid sequence is described in light chain complementarity determining region 2-3; SEQ ID NO: 7. or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 7, respectively, as light chain complementarity determining region 3-3.
  • Antibodies (4) The amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 36, or one or several amino acids in the amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 36.
  • the substituted, deleted, added and/or inserted amino acid sequence is the heavy chain complementarity determining region 1-4; the amino acid sequence at positions 51 to 60 of the amino acid sequence set forth in SEQ ID NO: 36, or SEQ ID NO: 36.
  • Heavy chain complementarity determining region 2-4 SEQ ID NO: The amino acid sequence set forth in SEQ ID NO: 8, or the amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 8, is used as the heavy chain complementarity determining region 3-4. and hold each as In the amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 37, or in the amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 37, one or several amino acids are substituted or deleted.
  • the deleted, added, and/or inserted amino acid sequence is the light chain complementarity determining region 1-4; the amino acid sequence at positions 51 to 53 of the amino acid sequence set forth in SEQ ID NO: 37, or The amino acid sequence in which one or several amino acids are substituted, deleted, added and/or inserted in the amino acid sequence at positions 51 to 53 of the amino acid sequence is described in light chain complementarity determining region 2-4; SEQ ID NO: 9. or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 9, respectively, as light chain complementarity determining regions 3-4.
  • Antibodies (5) The amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 38, or one or several amino acids in the amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 38.
  • the substituted, deleted, added and/or inserted amino acid sequence is the heavy chain complementarity determining region 1-5; the amino acid sequence at positions 51 to 58 of the amino acid sequence set forth in SEQ ID NO: 38, or SEQ ID NO: 38.
  • the amino acid sequence set forth in SEQ ID NO: 10 or the amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 10, is used as the heavy chain complementarity determining region 3-5. and hold each as The amino acid sequence at positions 26 to 34 of the amino acid sequence set forth in SEQ ID NO: 39, or the substitution or deletion of one or several amino acids in the amino acid sequence at positions 26 to 34 of the amino acid sequence set forth in SEQ ID NO: 39.
  • the deleted, added, and/or inserted amino acid sequence is the light chain complementarity determining region 1-5; the amino acid sequence at positions 52 to 54 of the amino acid sequence set forth in SEQ ID NO: 39, or An amino acid sequence in which one or several amino acids are substituted, deleted, added and/or inserted in the amino acid sequence at positions 52 to 54 of the amino acid sequence is described as light chain complementarity determining region 2-5; SEQ ID NO: 11 or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 11, respectively, as light chain complementarity determining regions 3-5.
  • Antibodies (6) The amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 40, or one or several amino acids in the amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 40.
  • the substituted, deleted, added and/or inserted amino acid sequence is the heavy chain complementarity determining region 1-6; the amino acid sequence at positions 51 to 58 of the amino acid sequence set forth in SEQ ID NO: 40, or SEQ ID NO: 40.
  • SEQ ID NO: 12 The amino acid sequence set forth in SEQ ID NO: 12, or the amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 12, is used as the heavy chain complementarity determining region 3-6. and hold each as The amino acid sequence at positions 27 to 33 of the amino acid sequence set forth in SEQ ID NO: 41, or the substitution or deletion of one or several amino acids in the amino acid sequence at positions 27 to 33 of the amino acid sequence set forth in SEQ ID NO: 41.
  • the deleted, added, and/or inserted amino acid sequence is the light chain complementarity determining region 1-6; the amino acid sequence at positions 51 to 53 of the amino acid sequence set forth in SEQ ID NO: 41, or The amino acid sequence in which one or several amino acids have been substituted, deleted, added and/or inserted in the amino acid sequence at positions 51 to 53 of the amino acid sequence is described in light chain complementarity determining region 2-6; SEQ ID NO: 13. or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 13, respectively, as light chain complementarity determining regions 3-6.
  • Antibodies (7) The amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 42, or one or several amino acids in the amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 42.
  • the substituted, deleted, added and/or inserted amino acid sequence is the heavy chain complementarity determining region 1-7; the amino acid sequence at positions 51 to 58 of the amino acid sequence set forth in SEQ ID NO: 42, or SEQ ID NO: 42.
  • Heavy chain complementarity determining region 2-7 SEQ ID NO: The amino acid sequence set forth in SEQ ID NO: 14, or the amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 14, is used as the heavy chain complementarity determining region 3-7. and hold each as In the amino acid sequence at positions 26 to 31 of the amino acid sequence set forth in SEQ ID NO: 43, or in the amino acid sequence at positions 26 to 31 of the amino acid sequence set forth in SEQ ID NO: 43, one or several amino acids are substituted or deleted.
  • the deleted, added, and/or inserted amino acid sequence is the light chain complementarity determining region 1-7; the amino acid sequence at positions 49 to 51 of the amino acid sequence set forth in SEQ ID NO: 43, or The amino acid sequence in which one or several amino acids are substituted, deleted, added and/or inserted in the amino acid sequence at positions 49 to 51 of the amino acid sequence is described in light chain complementarity determining region 2-7; SEQ ID NO: 15. or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 15, respectively, as light chain complementarity determining regions 3-7.
  • Antibodies (8) The amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 44, or one or several amino acids in the amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 44.
  • the substituted, deleted, added and/or inserted amino acid sequence is the heavy chain complementarity determining region 1-8; the amino acid sequence at positions 51 to 57 of the amino acid sequence set forth in SEQ ID NO: 44, or SEQ ID NO: 44.
  • Heavy chain complementarity determining region 2-8 SEQ ID NO: The amino acid sequence set forth in SEQ ID NO: 16, or the amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 16, is used as the heavy chain complementarity determining region 3-8. and hold each as In the amino acid sequence at positions 27 to 32 of the amino acid sequence set forth in SEQ ID NO: 45, or in the amino acid sequence at positions 27 to 32 of the amino acid sequence set forth in SEQ ID NO: 45, one or several amino acids are substituted or deleted.
  • the deleted, added, and/or inserted amino acid sequence is the light chain complementarity determining region 1-8; the amino acid sequence at positions 50 to 52 of the amino acid sequence set forth in SEQ ID NO: 45, or The amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence at positions 50 to 52 of the amino acid sequence is described in light chain complementarity determining region 2-8; SEQ ID NO: 17. or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 17, respectively, as light chain complementarity determining regions 3-8.
  • Antibodies (9) The amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 46, or one or several amino acids in the amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 46.
  • the substituted, deleted, added and/or inserted amino acid sequence is the heavy chain complementarity determining region 1-9; the amino acid sequence at positions 51 to 57 of the amino acid sequence set forth in SEQ ID NO: 46, or SEQ ID NO: 46.
  • the amino acid sequence set forth in SEQ ID NO: 18, or the amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 18, is used as the heavy chain complementarity determining region 3-9. and hold each as The amino acid sequence at positions 27 to 32 of the amino acid sequence set forth in SEQ ID NO: 47, or the substitution or deletion of one or several amino acids in the amino acid sequence at positions 27 to 32 of the amino acid sequence set forth in SEQ ID NO: 47.
  • the deleted, added, and/or inserted amino acid sequence is the light chain complementarity determining region 1-9; the amino acid sequence at positions 50 to 52 of the amino acid sequence set forth in SEQ ID NO: 47, or The amino acid sequence in which one or several amino acids are substituted, deleted, added and/or inserted in the amino acid sequence at positions 50 to 52 of the amino acid sequence is described in light chain complementarity determining region 2-9; SEQ ID NO: 19. or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 19, respectively, as light chain complementarity determining regions 3-9.
  • Antibodies (10) The amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 48, or one or several amino acids in the amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 48.
  • the substituted, deleted, added and/or inserted amino acid sequence is the heavy chain complementarity determining region 1-10; the amino acid sequence at positions 51 to 58 of the amino acid sequence set forth in SEQ ID NO: 48, or SEQ ID NO: 48.
  • the amino acid sequence set forth in SEQ ID NO: 20, or the amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 20, is used as the heavy chain complementarity determining region 3-10. and hold each as The amino acid sequence at positions 27 to 37 of the amino acid sequence set forth in SEQ ID NO: 49, or the substitution or deletion of one or several amino acids in the amino acid sequence at positions 27 to 37 of the amino acid sequence set forth in SEQ ID NO: 49.
  • the deleted, added, and/or inserted amino acid sequence is the light chain complementarity determining region 1-10; the amino acid sequence at positions 55 to 57 of the amino acid sequence set forth in SEQ ID NO: 49, or The amino acid sequence in which one or several amino acids are substituted, deleted, added and/or inserted in the amino acid sequence at positions 55 to 57 of the amino acid sequence is described in light chain complementarity determining region 2-10; SEQ ID NO: 21. or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 21, respectively, as light chain complementarity determining regions 3-10.
  • Antibodies (11) The amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 50, or one or several amino acids in the amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 50.
  • the substituted, deleted, added and/or inserted amino acid sequence is the heavy chain complementarity determining region 1-11; the amino acid sequence at positions 51 to 58 of the amino acid sequence set forth in SEQ ID NO: 50, or SEQ ID NO: 50.
  • Heavy chain complementarity-determining region 2-11 SEQ ID NO: The amino acid sequence set forth in SEQ ID NO: 22, or the amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 22, is used as the heavy chain complementarity determining region 3-11. and hold each as The amino acid sequence at positions 27 to 33 of the amino acid sequence set forth in SEQ ID NO: 51, or the substitution or deletion of one or several amino acids in the amino acid sequence at positions 27 to 33 of the amino acid sequence set forth in SEQ ID NO: 51.
  • the deleted, added, and/or inserted amino acid sequence is the light chain complementarity determining region 1-11; the amino acid sequence at positions 51 to 53 of the amino acid sequence set forth in SEQ ID NO: 51, or The amino acid sequence in which one or several amino acids are substituted, deleted, added and/or inserted in the amino acid sequence at positions 51 to 53 of the amino acid sequence is described in light chain complementarity determining region 2-11; SEQ ID NO: 23. or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 23, respectively, as light chain complementarity determining regions 3-11.
  • Antibodies (12) The amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 52, or the amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 52, in which one or several amino acids are The substituted, deleted, added and/or inserted amino acid sequence is the heavy chain complementarity determining region 1-12; the amino acid sequence at positions 51 to 57 of the amino acid sequence set forth in SEQ ID NO: 52, or SEQ ID NO: 52.
  • Heavy chain complementarity determining region 2-12 SEQ ID NO:
  • the deleted, added, and/or inserted amino acid sequence is the light chain complementarity determining region 1-12; the amino acid sequence at positions 50 to 52 of the amino acid sequence set forth in SEQ ID NO: 53, or The amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence at positions 50 to 52 of the amino acid sequence is described in light chain complementarity determining region 2-12; SEQ ID NO: 25. or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 25, respectively, as light chain complementarity determining regions 3-12.
  • Antibodies (13) The amino acid sequence at positions 26 to 34 of the amino acid sequence set forth in SEQ ID NO: 54, or one or several amino acids in the amino acid sequence at positions 26 to 34 of the amino acid sequence set forth in SEQ ID NO: 54.
  • the substituted, deleted, added and/or inserted amino acid sequence is the heavy chain complementarity determining region 1-13; the amino acid sequence at positions 52 to 58 of the amino acid sequence set forth in SEQ ID NO: 54, or SEQ ID NO: 54.
  • Heavy chain complementarity determining region 2-13 SEQ ID NO:
  • the amino acid sequence set forth in SEQ ID NO: 26, or the amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 26, is used as the heavy chain complementarity determining region 3-13. and hold each as In the amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 55, or in the amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 55, one or several amino acids are substituted or deleted.
  • the deleted, added, and/or inserted amino acid sequence is the light chain complementarity determining region 1-13; the amino acid sequence at positions 51 to 53 of the amino acid sequence set forth in SEQ ID NO: 55, or The amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence at positions 51 to 53 of the amino acid sequence is described in light chain complementarity determining region 2-13; SEQ ID NO: 27. or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 27, respectively, as light chain complementarity determining regions 3-13.
  • Antibodies (14) The amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 56, or one or several amino acids in the amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 56.
  • the substituted, deleted, added and/or inserted amino acid sequence is the heavy chain complementarity determining region 1-14; the amino acid sequence at positions 51 to 58 of the amino acid sequence set forth in SEQ ID NO: 56, or SEQ ID NO: 56.
  • Heavy chain complementarity determining region 2-14 is an amino acid sequence in which one or several amino acids are substituted, deleted, added and/or inserted in the amino acid sequence at positions 51 to 58 of the amino acid sequence described in The amino acid sequence set forth in SEQ ID NO: 28, or the amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 28, is used as the heavy chain complementarity determining region 3-14. and hold each as In the amino acid sequence at positions 26 to 34 of the amino acid sequence set forth in SEQ ID NO: 57, or in the amino acid sequence at positions 26 to 34 of the amino acid sequence set forth in SEQ ID NO: 57, one or several amino acids are substituted or deleted.
  • the deleted, added, and/or inserted amino acid sequence is the light chain complementarity determining region 1-14; the amino acid sequence at positions 52 to 54 of the amino acid sequence set forth in SEQ ID NO: 57, or The amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence at positions 52 to 54 of the amino acid sequence is described as light chain complementarity determining region 2-14; SEQ ID NO: 29. or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 29, respectively, as light chain complementarity determining regions 3-14.
  • Antibodies (15) The amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 65, or one or several amino acids in the amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 65.
  • the substituted, deleted, added and/or inserted amino acid sequence is the heavy chain complementarity determining region 1-15; the amino acid sequence at positions 51 to 58 of the amino acid sequence set forth in SEQ ID NO: 65, or SEQ ID NO: 65.
  • Heavy chain complementarity determining region 2-15 SEQ ID NO: The amino acid sequence set forth in SEQ ID NO: 63, or the amino acid sequence in which one or several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 63, is used as the heavy chain complementarity determining region 3-15. and hold each as In the amino acid sequence at positions 23 to 31 of the amino acid sequence set forth in SEQ ID NO: 66, or in the amino acid sequence at positions 23 to 31 of the amino acid sequence set forth in SEQ ID NO: 66, one or several amino acids are substituted or deleted.
  • the deleted, added, and/or inserted amino acid sequence is the light chain complementarity determining region 1-15; the amino acid sequence at positions 49 to 51 of the amino acid sequence set forth in SEQ ID NO: 66, or The amino acid sequence in which one or several amino acids are substituted, deleted, added and/or inserted in the amino acid sequence at positions 49 to 51 of the amino acid sequence is described in light chain complementarity determining region 2-15; SEQ ID NO: 64. or an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and/or inserted in the amino acid sequence set forth in SEQ ID NO: 64, respectively, as light chain complementarity determining regions 3-15.
  • Antibodies The anti-SARS-CoV-2 antibody according to [1] or [2], which is selected from the group consisting of.
  • the amino acid sequence set forth in SEQ ID NO: 54 an amino acid sequence having 80% or more homology to the amino acid sequence set forth in SEQ ID NO: 54, or 1 or 1 in the amino acid sequence set forth in SEQ ID NO: 54.
  • the anti-SARS-CoV-2 antibody according to any one of [1] to [3], selected from the group consisting of.
  • composition for preventing or treating COVID-19 which contains the antibody according to any one of [1] to [4].
  • composition for immunologically detecting SARS-CoV-2 which contains the antibody according to any one of [1] to [4].
  • [7] A method for immunologically detecting SARS-CoV-2 using the antibody according to any one of [1] to [4].
  • [8] A method for preventing or treating COVID-19, which comprises administering to a subject the antibody according to any one of [1] to [4] or the composition according to [6].
  • the present invention in addition to the conventional SARS-CoV-2 (Wuhan strain), a novel antibody with high binding affinity and infection-neutralizing activity against other mutant strains including the Omicron strain, as well as its It is possible to provide a composition for preventing or treating COVID-19 using the present invention, a composition for immunologically detecting SARS-CoV-2, and a method for immunologically detecting SARS-CoV-2. It becomes possible.
  • Antibody No. 2 against SARS-CoV-2 (Wuhan strain, Delta strain, Omicron strain (BA.1), Omicron strain (BA.2)). 1 is a heat map showing the infection neutralizing activity (log 10 IC 50 (ng/mL)) of recombinant monoclonal antibodies 1 to 14 and the sotrovimab parent antibody (S309). Antibody No. against each mutant RBD. Binding properties of recombinant monoclonal antibodies 1 to 14 and sotrovimab parent antibody (S309) (Relative binding amount when the amount of recombinant monoclonal antibody bound to Wuhan strain RBD (Ancestral) is taken as 100 (%) (%)).
  • Hamsters infected with the Wuhan strain of SARS-CoV-2 were given antibody No. Average change in body weight from the day of infection (change when the body weight on the day of infection (day 0) is set as 100) when recombinant monoclonal antibodies 5 to 7, PBS, or sotrovimab parent antibody (S309) were administered. It is a graph showing values. Hamsters infected with the Wuhan strain of SARS-CoV-2 were given antibody No.
  • FIG. 7 is a graph showing the weights (g) of the right posterior lobe of the right lung on day 6 from the day of infection and their average values when recombinant monoclonal antibodies 5 to 7, PBS, or sotrovimab parent antibody (S309) were administered.
  • FIG. 7 is a graph showing the number of virus copies in the nasal wash fluid and their average values on the third day from the day of infection when recombinant monoclonal antibodies 5 to 7, PBS, or sotrovimab parent antibody (S309) were administered.
  • Hamsters infected with SARS-CoV-2 Omicron strain (BA.2) were given antibody No.
  • FIG. 7 is a graph showing the number of virus copies in the nasal wash fluid and their average values on the third day from the day of infection when recombinant monoclonal antibodies 5 to 7, PBS, or sotrovimab parent antibody (S309) were administered.
  • a heat map showing the binding properties of 15 recombinant monoclonal antibodies (Relative to Ancestral (%) when the amount of recombinant monoclonal antibodies bound to Wuhan strain RBD (Ancestral) is taken as 100 (%)).
  • the anti-SARS-CoV-2 antibody of the present invention is an antibody against SARS-CoV-2 that binds to the spike glycoprotein receptor binding site (RBD) of SARS-CoV-2, and is an antibody that binds to the spike glycoprotein receptor binding site (RBD) of SARS-CoV-2.
  • This is an antibody that recognizes serine at position 443 or tyrosine at position 489 of the amino acid sequence of (hereinafter simply referred to as "the antibody of the present invention” in some cases).
  • SARS-CoV-2 refers to severe acute respiratory syndrome coronavirus 2, which is also referred to as the "new coronavirus.”
  • the size of the SARS-CoV-2 virus particle is approximately 50 to 200 nm in diameter, and it consists of spike glycoprotein (S protein), nucleocapsid protein (N protein), membrane protein (M protein), and envelope protein (E protein). It is composed of four proteins and RNA.
  • S protein spike glycoprotein
  • N protein nucleocapsid protein
  • M protein membrane protein
  • E protein envelope protein
  • SARS-CoV-2 has spread to various mutant strains since the conventional strain (referred to herein as the "Wuhan strain” in some cases) that was first confirmed to occur near Wuhan City, Hubei province, People's Republic of China in 2019. have been confirmed, and the prevalent strains change depending on the country or region.
  • the main variants of concern that have occurred to date include alpha strains, beta strains, gamma strains, delta strains, and omicron strains, which are named by the World Health Organization (WHO).
  • these mutant strains are further divided into strains, for example, Omicron strains include BA. 1. BA. A second class system has been confirmed. Furthermore, in recent years, BA. 2.75, BA. 4. BA. 5. BQ.
  • the SARS-CoV-2 according to the present invention is not particularly limited as long as it has the following RBD, but is preferably at least one selected from the group consisting of the Wuhan strain, Delta strain, and Omicron strain, for example.
  • SARS-CoV-2 is a single-stranded positive-strand RNA virus with a total length of the viral genome of approximately 29,900 bases. Analysis of the nucleotide sequence of the SARS-CoV-2 viral genome and the amino acid sequence encoded by it, including the above-mentioned mutant strains, is progressing all over the world, and can be obtained from known databases such as GenBank (NCBI), for example. It is.
  • RBD refers to the spike glycoprotein receptor binding domain within the S protein, and typically consists of 223 amino acids.
  • SARS-CoV-2 this RBD is known to recognize and bind to angiotensin-converting enzyme 2 (ACE-2) on host cells, thereby infecting the host. Therefore, inhibiting (neutralizing) this binding is important in the prevention and treatment of COVID-19.
  • ACE-2 angiotensin-converting enzyme 2
  • the amino acid sequence of the RBD and the nucleotide sequence encoding it, including the mutant strain can be obtained from known databases such as GenBank (NCBI).
  • NCBI GenBank
  • a typical amino acid sequence of the RBD of SARS-CoV-2 according to the present invention includes the amino acid sequence at positions 319 to 541 of the amino acid sequence shown in SEQ ID NO: 1.
  • the amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of the spike glycoprotein of the Wuhan strain of SARS-CoV-2 (NCBI Reference Sequence: YP_009724390.1).
  • the "RBD amino acid sequence" according to the present invention includes the serine at position 443 and / or tyrosine at position 489 (including the amino acid corresponding to serine at position 443 and the amino acid corresponding to tyrosine at position 489 below), but are not limited to this. do not have.
  • the "RBD amino acid sequence" preferably includes, for example, 70% or more, preferably 80% or more, 90% or more of the amino acid sequence at positions 319 to 541 of the amino acid sequence shown in SEQ ID NO: 1, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) homology (preferably identity), and the following serine at position 443 and/or tyrosine at position 489
  • Amino acid sequence having one or several amino acids (within 70 amino acids, preferably within 45 amino acids, within 25 amino acids, within 20 amino acids, within 10 amino acids, within 5
  • the anti-SARS-CoV-2 antibody of the present invention recognizes serine at position 443 or tyrosine at position 489 of the amino acid sequence of the spike glycoprotein in the RBD.
  • the amino acid positions indicated as “position 443” and “position 489” are based on the amino acid sequence of the spike glycoprotein of SARS-CoV-2 (typically, SEQ ID NO: 1). The number of amino acid residues from the N-terminus in the shown amino acid sequence) is shown.
  • SARS-CoV-2 serine at position 443 and tyrosine at position 489 of the amino acid sequence are highly conserved.
  • nucleotide sequence of a virus changes due to mutations, etc.
  • amino acid sequence of the protein encoded by it can also be changed accordingly.
  • tyrosine at position 489 of the amino acid sequence of the spike glycoprotein each include the corresponding amino acids (preferably serine and tyrosine, respectively).
  • an amino acid that "corresponds" to a specific amino acid in an amino acid sequence is defined as an amino acid that is defined using amino acid sequence analysis software (e.g., GENETYX-MAC, Sequencher, etc.) or ClustalW, etc. (e.g., parameters: default values (i.e., initial settings). Value))
  • amino acid sequence analysis software e.g., GENETYX-MAC, Sequencher, etc.
  • ClustalW e.g., parameters: default values (i.e., initial settings). Value)
  • select the amino acid that is the same as the reference amino acid i.e., for example, serine at position 443 and/or tyrosine at position 489 of the amino acid sequence set forth in SEQ ID NO: 1). show.
  • Amino acids that "correspond" to a particular amino acid in an amino acid sequence include amino acids with chemically similar amino acid side chains (e.g., amino acids with hydroxy groups (serine, threonine), amino acids with aromatic groups (phenylalanine, tyrosine), etc. ⁇ Tryptophan)) is preferable, and the amino acid corresponding to "serine at position 443 of the amino acid sequence of spike glycoprotein" is more preferably serine, and "tyrosine at position 489 of the amino acid sequence of spike glycoprotein" Tyrosine is more preferred as the corresponding amino acid.
  • amino acids with chemically similar amino acid side chains e.g., amino acids with hydroxy groups (serine, threonine), amino acids with aromatic groups (phenylalanine, tyrosine), etc. ⁇ Tryptophan)
  • amino acid corresponding to "serine at position 443 of the amino acid sequence of spike glycoprotein" is more preferably serine
  • antibody includes not only complete antibodies but also functional fragments thereof.
  • functional fragment refers to a part (partial fragment) of a complete antibody that recognizes the RBD, specifically, Fab, F(ab')2, Fab' , variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc(Fv)2, diabody, and polymers thereof.
  • Fab refers to a monovalent antigen-binding fragment of an immunoglobulin consisting of one light chain and part of a heavy chain, and can be obtained, for example, by papain digestion of an antibody or by recombinant methods.
  • F(ab')2 means a bivalent antigen-binding fragment of an immunoglobulin consisting of portions of both light chains and both heavy chains, which can be obtained, for example, by pepsin digestion of antibodies or by recombinant methods. Can be done.
  • Fab' can be obtained, for example, by reducing F(ab')2, including one or more cysteines in the hinge region of the antibody, and only a few at the carboxy terminus of the heavy chain CH1 domain. It differs from Fab by the addition of a residue.
  • variable region fragment refers to the smallest antibody fragment that has a complete antigen recognition and binding site.
  • Fv is a dimer in which a heavy chain variable region and a light chain variable region are tightly linked by non-covalent bonds.
  • a "single chain Fv (scFv)” includes the heavy and light chain variable regions of an antibody, which regions are present in a single polypeptide chain.
  • sc(Fv)2 is a single chain made by linking two heavy chain variable regions and two light chain variable regions with a linker or the like.
  • a “diabody” is a small antibody fragment with two antigen-binding sites, which contains a heavy chain variable region joined to a light chain variable region in the same polypeptide chain, each region having a distinct It forms a pair with the complementary region of the strand.
  • antibody includes all classes and subclasses of immunoglobulin, and also includes polyclonal antibodies and monoclonal antibodies.
  • Polyclonal antibody refers to an antibody preparation that includes different antibodies directed against different epitopes
  • monoclonal antibody refers to antibodies (including antibody fragments) that are obtained from a substantially homogeneous population of antibodies.
  • the antibody of the present invention is preferably a monoclonal antibody.
  • the antibody of the present invention may be any antibody that recognizes serine at position 443 or tyrosine at position 489 of the amino acid sequence of the spike glycoprotein, and its origin, type, shape, etc. are not particularly limited. Specific examples include antibodies derived from humans, antibodies derived from non-human animals (e.g. rabbit antibodies, mouse antibodies, rat antibodies, camel antibodies), chimeric antibodies, humanized antibodies, and functional fragments of these antibodies. When used for the purpose of a therapeutic antibody, it is preferably a human-derived antibody or a humanized antibody, and more preferably a human-derived antibody.
  • the antibody of the present invention recognizes serine at position 443 or tyrosine at position 489 of the amino acid sequence of the spike glycoprotein.
  • "recognizing" each of the above amino acids means that the presence of at least one of these amino acids on the RBD increases the binding affinity for the antigen RBD and the infection neutralizing activity for SARS-CoV-2. It means to show.
  • the binding affinity can be evaluated by a person skilled in the art using a well-known immunological method or a method similar thereto.
  • a part for example, a protein consisting of the amino acid sequence from positions 331 to 529 of the amino acid sequence of SEQ ID NO: 1 (Ancestral)
  • RBD of Delta strain or a part thereof for example, L452R and T478K mutations in the amino acid sequence of Ancestral
  • the number indicates the position in the amino acid sequence of SEQ ID NO: 1, hereinafter the same applies to the Omicron strain.
  • the dissociation constant (K D ) between the RBD and the antibody is, for example, the dissociation constant (K D ) between the RBD of Omicron strain (BA.1) and the antibody, for example, 1.0 ⁇ It is even more preferably 10 ⁇ 7 M or less, and particularly preferably 1.0 ⁇ 10 ⁇ 8 M or less.
  • the infection neutralizing activity can also be evaluated by a person skilled in the art using a well-known immunological method or a method similar thereto.
  • SARS-CoV-2 e.g., Wuhan strain, Delta strain, Omicron strain
  • VeroE6/TMPRSS2 cells e.g., Wuhan strain, Delta strain, Omicron strain
  • Absorbance, luminescence intensity, etc. is 50% of the number of infected cells when SARS-CoV-2 alone (no antibody) is added to VeroE6/TMPRSS2 cells (IC 50 ) at an antibody concentration (IC 50 ) of 1 ⁇ g/mL or less. It is preferable that there be.
  • the antibody recognizes serine at position 443 or tyrosine at position 489 of the amino acid sequence of the spike glycoprotein is determined, for example, as shown in the Examples below. This can be evaluated by measuring the amount of binding to a protein consisting of an amino acid sequence in which the S443N or Y489S mutation has been introduced. More specifically, if the amount of binding to the mutant RBD is lower than that to the RBD before mutation introduction (for example, a protein (Ancestral) consisting of the amino acid sequence of positions 331 to 529 of the RBD of the Wuhan strain), the Preferably, the spike It can be determined that the antibody recognizes serine at position 443 or tyrosine at position 489 of the amino acid sequence of glycoprotein.
  • antibodies of the present invention include antibodies that retain the following variable regions.
  • an antibody that has a heavy chain variable region 3 comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain variable region 3 comprising the amino acid sequence set forth in SEQ ID NO: 35;
  • the complementarity determining regions can also be determined based on the sequences. Can be done. Accordingly, the present invention also provides antibodies that retain each of the following CDRs 1 to 3.
  • Light chain variable region 1 retaining heavy chain CDRs 1 to 3 determined from heavy chain variable region 1 containing the amino acid sequence set forth in SEQ ID NO: 30, and containing the amino acid sequence set forth in SEQ ID NO: 31 an antibody that retains light chain CDRs 1-3 determined from;
  • Light chain variable region 2 retaining heavy chain CDRs 1 to 3 determined from heavy chain variable region 2 comprising the amino acid sequence set forth in SEQ ID NO: 32 and comprising the amino acid sequence set forth in SEQ ID NO: 33 an antibody that retains light chain CDRs 1-3 determined from;
  • Light chain variable region 3 retaining heavy chain CDRs 1 to 3 determined from heavy chain variable region 3 containing the amino acid sequence set forth in SEQ ID NO: 34, and containing the amino acid sequence set forth in SEQ ID NO: 35 an antibody that retains light chain CDRs 1-3 determined from;
  • Light chain variable region 4 retaining heavy chain CDRs 1 to 3 determined from heavy chain variable region 4 comprising the amino acid sequence set forth in SEQ ID NO: 36, and comprising the amino
  • the amino acid sequence set forth in SEQ ID NO: 2 is retained as heavy chain CDR3 (HV CDR3-1), and the amino acid sequence set forth in SEQ ID NO: 3 is retained as light chain CDR3 (LV CDR3-1). ,antibody;
  • the amino acid sequence set forth in SEQ ID NO: 4 is retained as heavy chain CDR3 (HV CDR3-2), and the amino acid sequence set forth in SEQ ID NO: 5 is retained as light chain CDR3 (LV CDR3-2).
  • the amino acid sequence set forth in SEQ ID NO: 12 is retained as heavy chain CDR3 (HV CDR3-6), and the amino acid sequence set forth in SEQ ID NO: 13 is retained as light chain CDR3 (LV CDR3-6).
  • the amino acid sequence set forth in SEQ ID NO: 14 is retained as heavy chain CDR3 (HV CDR3-7), and the amino acid sequence set forth in SEQ ID NO: 15 is retained as light chain CDR3 (LV CDR3-7).
  • HV CDR3-1, LV CDR3-1 Heavy chain CDR1 (HV CDR1-1): Amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 30.
  • Heavy chain CDR2 (HV CDR2-1): amino acid sequence at position 51 of the amino acid sequence set forth in SEQ ID NO: 30.
  • Amino acid sequence at positions 27 to 32 of the amino acid sequence light chain CDR1 (LV CDR1-1): set forth in SEQ ID NO: 31
  • Light chain CDR2 (LV CDR2-1): set forth in SEQ ID NO: 31
  • Heavy chain CDR1 (HV CDR1-2): Amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 32.
  • Heavy chain CDR2 (HV CDR2-2): amino acid sequence at position 51 of the amino acid sequence set forth in SEQ ID NO: 32.
  • Amino acid sequence at positions 26 to 34 of the amino acid sequence light chain CDR1 (LV CDR1-2): set forth in SEQ ID NO: 33
  • Light chain CDR2 (LV CDR2-2): set forth in SEQ ID NO: 33
  • the amino acid sequence of positions 52 to 54 of the amino acid sequence of; (3) HV CDR3-3, LV CDR3-3 Heavy chain CDR1 (HV CDR1-3): Amino acid sequence at positions 19 to 28 of the amino acid sequence set forth in SEQ ID NO: 34 Heavy chain CDR2 (HV CDR2-3): Position 46 of the amino acid sequence set forth in SEQ ID NO: 34
  • Amino acid sequence at positions 26 to 34 of the amino acid sequence light chain CDR1 (LV CDR1-3): set forth in SEQ ID NO: 35
  • Light chain CDR2 (LV CDR2-3): set forth in SEQ ID NO: 35
  • Heavy chain CDR2 (HV CDR2-5): amino acid sequence at position 51 of the amino acid sequence set forth in SEQ ID NO: 38. Amino acid sequence at positions 26 to 34 of the amino acid sequence light chain CDR1 (LV CDR1-5): set forth in SEQ ID NO: 39 Light chain CDR2 (LV CDR2-5): set forth in SEQ ID NO: 39 The amino acid sequence of positions 52 to 54 of the amino acid sequence of; (6) HV CDR3-6, LV CDR3-6 Heavy chain CDR1 (HV CDR1-6): Amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 40 Heavy chain CDR2 (HV CDR2-6): Position 51 of the amino acid sequence set forth in SEQ ID NO: 40 Amino acid sequence at positions 27 to 33 of the amino acid sequence light chain CDR1 (LV CDR1-6): set forth in SEQ ID NO: 41 Light chain CDR2 (LV CDR2-6): set forth in SEQ ID NO: 41 The amino acid sequence of positions 51 to 53 of
  • Heavy chain CDR2 (HV CDR2-8): amino acid sequence at position 51 of the amino acid sequence set forth in SEQ ID NO: 44. Amino acid sequence at positions 27 to 32 of the amino acid sequence light chain CDR1 (LV CDR1-8): set forth in SEQ ID NO: 45 Light chain CDR2 (LV CDR2-8): set forth in SEQ ID NO: 45 The amino acid sequence of positions 50 to 52 of the amino acid sequence of; (9) HV CDR3-9, LV CDR3-9 Heavy chain CDR1 (HV CDR1-9): Amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 46 Heavy chain CDR2 (HV CDR2-9): Position 51 of the amino acid sequence set forth in SEQ ID NO: 46 Amino acid sequence at positions 27 to 32 of the amino acid sequence light chain CDR1 (LV CDR1-9): set forth in SEQ ID NO: 47 Light chain CDR2 (LV CDR2-9): set forth in SEQ ID NO: 47 The amino acid sequence of positions
  • Heavy chain CDR2 (HV CDR2-10): amino acid sequence at position 51 of the amino acid sequence set forth in SEQ ID NO: 48. Amino acid sequence at positions 27 to 37 of the amino acid sequence light chain CDR1 (LV CDR1-10): set forth in SEQ ID NO: 49 Light chain CDR2 (LV CDR2-10): set forth in SEQ ID NO: 49 The amino acid sequence of positions 55 to 57 of the amino acid sequence of; (11) HV CDR3-11, LV CDR3-11 Heavy chain CDR1 (HV CDR1-11): Amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 50.
  • Heavy chain CDR2 (HV CDR2-11): amino acid sequence at position 51 of the amino acid sequence set forth in SEQ ID NO: 50. Amino acid sequence at positions 27 to 33 of the amino acid sequence light chain CDR1 (LV CDR1-11) at positions ⁇ 58: SEQ ID NO:51 Light chain CDR2 (LV CDR2-11): set forth at SEQ ID NO:51 The amino acid sequence of positions 51 to 53 of the amino acid sequence of; (12) HV CDR3-12, LV CDR3-12 Heavy chain CDR1 (HV CDR1-12): Amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 52.
  • Heavy chain CDR2 (HV CDR2-12): amino acid sequence at position 51 of the amino acid sequence set forth in SEQ ID NO: 52. Amino acid sequence at positions 27 to 32 of the amino acid sequence light chain CDR1 (LV CDR1-12): set forth in SEQ ID NO: 53 Light chain CDR2 (LV CDR2-12): set forth in SEQ ID NO: 53 The amino acid sequence of positions 50 to 52 of the amino acid sequence of; (13) HV CDR3-13, LV CDR3-13 Heavy chain CDR1 (HV CDR1-13): Amino acid sequence at positions 26 to 34 of the amino acid sequence set forth in SEQ ID NO: 54.
  • Heavy chain CDR2 (HV CDR2-13): amino acid sequence at position 52 of the amino acid sequence set forth in SEQ ID NO: 54. Amino acid sequence at positions 26 to 33 of the amino acid sequence light chain CDR1 (LV CDR1-13) set forth in SEQ ID NO:55 Light chain CDR2 (LV CDR2-13): set forth in SEQ ID NO:55 The amino acid sequence of positions 51 to 53 of the amino acid sequence of; (14) HV CDR3-14, LV CDR3-14 Heavy chain CDR1 (HV CDR1-14): Amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 56.
  • Heavy chain CDR2 (HV CDR2-14): amino acid sequence at position 51 of the amino acid sequence set forth in SEQ ID NO: 56. Amino acid sequence at positions 26 to 34 of the amino acid sequence light chain CDR1 (LV CDR1-14): set forth in SEQ ID NO: 57 Light chain CDR2 (LV CDR2-14): set forth in SEQ ID NO: 57 The amino acid sequence of positions 52 to 54 of the amino acid sequence of; (15) HV CDR3-15, LV CDR3-15 Heavy chain CDR1 (HV CDR1-15): Amino acid sequence at positions 26 to 33 of the amino acid sequence set forth in SEQ ID NO: 65.
  • Heavy chain CDR2 (HV CDR2-15): amino acid sequence at position 51 of the amino acid sequence set forth in SEQ ID NO: 65.
  • Amino acid sequence at positions 23 to 31 of the amino acid sequence light chain CDR1 (LV CDR1-15): set forth in SEQ ID NO: 66
  • Light chain CDR2 (LV CDR2-15): set forth in SEQ ID NO: 66
  • the antibody of the present invention may also be an antibody that further recognizes amino acids other than these amino acids.
  • the antibodies (6) to (7), (10), (11), and (13) above also recognize phenylalanine at position 486 of the amino acid sequence of the spike glycoprotein; ) also recognizes asparagine at position 487 of the amino acid sequence of the spike glycoprotein.
  • the antibodies of the present invention include the above (2), (5), and (6) from the viewpoint that they tend to have higher binding affinity for the RBD and/or infection neutralizing activity for SARS-CoV-2. ) and (7) are preferred, and the antibodies (5), (6), and (7) are more preferred. Furthermore, from the viewpoint that they tend to have higher binding affinity and/or infection neutralizing activity for mutant strains, the antibodies (5), (6), and (7) above are preferable, and the antibodies (6) and (7) above are preferable. Antibodies are more preferred. Furthermore, from the viewpoint that antibody escape mutant viruses (escape viruses) are particularly unlikely to occur, the antibodies described in (5) and (6) above are preferable.
  • the antibody (15), which is a further improved version of the antibody (5), is preferable from the viewpoint that it tends to have higher binding affinity and/or infection neutralizing activity for RBDs such as (5).
  • the antibodies of the present invention include not only antibodies that retain variable regions and/or CDRs consisting of the above-mentioned specific amino acid sequences, but also antibodies that have desirable reactivity (binding affinity for the above-mentioned RBD, Antibodies may have their amino acid sequences modified without reducing their activity, etc.). Examples of the modification of the amino acid sequence include substitution, deletion, insertion, and/or addition of one or several amino acids within the amino acid sequence.
  • the site where the amino acid sequence is modified may be the constant region of the heavy chain or light chain of the antibody, as long as it has an activity equivalent to that of the antibody before modification, or the variable region.
  • Framework Framework region
  • CDR Modification of amino acids other than CDRs is thought to have relatively little effect on reactivity with antigens, but currently there is a method of modifying amino acids of CDRs to screen for antibodies with increased affinity for antigens.
  • PNAS, 102:8466-8471 (2005), Protein Engineering, Design & Selection, etc.
  • an integrated computational chemistry system for example, Molecular Operating Environment, manufactured by CCG, Canada
  • the number of amino acids modified in the variable region is preferably within 25 amino acids, more preferably within 20 amino acids, even more preferably within 10 amino acids, within 5 amino acids, or within 3 amino acids (for example, within 2 amino acids, 1 amino acid). Furthermore, all such modifications are preferably made in areas other than CDRs, that is, FRs, from the viewpoint of having little effect on reactivity with antigens.
  • "several" when “one or several amino acids have been substituted, deleted, added and/or inserted” is preferably within 5 amino acids, more preferably within 3 amino acids (for example, within 2 amino acids, 1 amino acid).
  • the modification of the amino acid sequence is preferably a conservative substitution.
  • "conservative substitution” means substitution with another amino acid residue having a chemically similar side chain.
  • Groups of amino acid residues having chemically similar amino acid side chains are well known in the art to which this invention pertains.
  • acidic amino acids aspartic acid and glutamic acid
  • basic amino acids lysine, arginine, histidine
  • neutral amino acids include amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), and hydroxy groups.
  • an antibody whose modified amino acid sequence has a variable region containing an amino acid sequence having 80% or more homology at the amino acid sequence level with a variable region consisting of the above-mentioned specific amino acid sequence is also an antibody before modification. It is included in the antibodies of the present invention as long as it has an activity equivalent to that of the antibody. Such homology may be at least 80%, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more (for example, 96% or more, 97% or more, 98% or more, 99% or more). % or more).
  • the identity is at least 80%, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more). It is. Sequence homology and identity can also be determined using the BLASTP (amino acid level) program (Altschul et al. J. Mol. Biol., 215:403-410, 1990, parameters: default). can. The program is based on the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90:5873-5877, 1993). There is.
  • BLASTP amino acid level
  • “having equivalent activity” means, for example, that the binding affinity for the RBD and/or the infection neutralizing activity for SARS-CoV-2 is higher than that of the target antibody (typically, the above (1) to (15) ), preferably at least any of the antibodies (5) to (7) above, more preferably the antibodies (6) and/or (7) above) (e.g. 70% or more, preferably 80% or more, more preferably 90% or more).
  • the constant region other than the variable region is not particularly limited.
  • ⁇ 1 can be combined with each of the heavy chain variable regions, but is not limited to this, and ⁇ 2 to ⁇ 4 may also be used.
  • ⁇ or ⁇ can be appropriately combined with each of the light chain variable regions as the light chain constant region.
  • the amino acid sequences of these constant regions and the base sequences encoding them can be obtained from known databases such as GenBank (NCBI), for example.
  • the amino acid sequence of ⁇ 1 is the amino acid sequence of SEQ ID NO: 58
  • the amino acid sequence of ⁇ is the amino acid sequence of SEQ ID NO: 60
  • the amino acid sequence of ⁇ is the amino acid sequence of SEQ ID NO: 59.
  • the amino acid sequence is not limited to these, and the amino acid sequence may be modified as appropriate.
  • the antibody of the present invention may be a functional fragment as described above, and in this case, the constant region may be a part thereof or may not include the constant region.
  • the antibody of the present invention can be produced by a conventionally known method or a method analogous thereto, for example, by a hybridoma method or a recombinant DNA method.
  • the hybridoma method typically includes the method of Kohler and Milstein (Kohler & Milstein, Nature, 256:495 (1975)) and methods similar thereto.
  • the recombinant DNA method involves cloning or synthesizing DNA encoding the antibody of the present invention from hybridomas, B cells, etc., inserting it into an appropriate vector, and injecting it into host cells (e.g., mammalian cell lines such as HEK cells). , Escherichia coli, yeast cells, insect cells, plant cells, etc.) to produce the antibody of the present invention as a recombinant antibody (for example, P.J. Delves, Antibody Production: Essential Techniques, 1997 WILEY, P. .Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS, Vandamme A.M.
  • the DNA encoding the heavy chain or light chain may be separately incorporated into an expression vector to transform host cells. It may be integrated into a single expression vector to transform host cells.
  • nucleotide sequences encoding the heavy chain variable region (HV) and light chain variable region (LV) of the antibodies of the present invention include the sequences set forth in SEQ ID NOs: 30-57, 65, and 66.
  • Examples include, but are not limited to, nucleotide sequences encoding amino acid sequences; each sequence may independently correspond to, for example, an antibody in which the above amino acid sequence has been modified; It may also be codon-optimized.
  • the antibody of the present invention can be obtained in a substantially pure and homogeneous form by culturing the above-mentioned host cells, separating and purifying the antibodies within the host cells or from the culture solution. For isolation and purification of antibodies, methods commonly used for purification of polypeptides can be used.
  • transgenic animal cow, goat, sheep, pig, etc.
  • monoclonal antibodies derived from the antibody gene can be produced from the milk of the transgenic animal. It is also possible to obtain large amounts of.
  • the present invention provides a DNA encoding the antibody of the present invention, a vector containing the DNA, a host cell retaining the DNA, and a method for producing an antibody, which includes the steps of culturing the host cell and recovering the antibody. can also be provided.
  • composition for prevention or treatment of COVID-19 also provides a composition for preventing or treating COVID-19 (hereinafter, in some cases, simply referred to as "therapeutic composition of the present invention") containing the anti-SARS-CoV-2 antibody of the present invention. .
  • the antibody of the present invention (anti-SARS-CoV-2 antibody) has high binding affinity with RBD as described above and has high neutralizing activity against SARS-CoV-2.
  • the therapeutic composition of the present invention can be used for the prevention or treatment of COVID-19, a SARS-CoV-2 infection. Therefore, the present invention provides the use of an anti-SARS-CoV-2 antibody of the present invention or a therapeutic composition of the present invention for the prevention or treatment of COVID-19, as well as a composition for the prevention or treatment of COVID-19. Also provided is the use of an anti-SARS-CoV-2 antibody of the invention for the production of a SARS-CoV-2 antibody.
  • the therapeutic composition of the present invention may contain only one type of antibody of the present invention, or may contain a combination of two or more types.
  • the content of the antibody of the present invention includes the form of the composition, dosage, purpose and method of administration or ingestion, etc. Although not particularly limited as it can be adjusted appropriately depending on , 30 to 70% by weight, 40 to 60% by weight, or 40 to 50% by weight.
  • the content of the antibody may be 5 to 10% by mass, 10 to 20% by mass, 20 to 30% by mass, 30 to 40% by mass, 40 to 50% by mass. It may be 50-60% by mass, 60-70% by mass, 70-80% by mass, 80-90% by mass, 90-99% by mass, or 99% by mass or more.
  • the form of the therapeutic composition of the present invention is not particularly limited, but examples include liquid preparations such as general solutions (including oral preparations and injections), suspensions, emulsions, and syrups; suppositories, eye drops, and drops. Examples include external preparations such as nasal preparations.
  • the therapeutic composition of the present invention may further contain other components that are acceptable as therapeutic agents, depending on the form thereof.
  • Such other ingredients include, for example, pharmacologically acceptable carriers or diluents (sterile water, physiological saline, vegetable oil, etc.), excipients, disintegrants, buffers, emulsifiers, suspending agents, stabilizers, etc.
  • agents preservatives, and antiseptics, and these may be used alone or in combination of two or more. Furthermore, it may contain anti-SARS-CoV-2 antibodies other than the antibodies of the present invention.
  • the content of the other components is not particularly limited and can be adjusted as appropriate depending on the form of the composition, dosage, purpose and method of administration, etc.
  • Examples of the excipient include lactose, starch, sorbitol, D-mannitol, and sucrose.
  • Examples of the disintegrant include starch, carboxymethyl cellulose, and calcium carbonate.
  • Examples of the buffer include phosphate, citrate, and acetate.
  • Examples of the emulsifier include gum arabic, sodium alginate, and tragacanth.
  • Examples of the suspending agent include glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate.
  • Examples of the stabilizer include propylene glycol, diethyl sulfite, and ascorbic acid.
  • Examples of the preservative include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben.
  • Examples of the preservative include sodium azide, benzalkonium chloride, paraoxybenzoic acid, and chlorobutanol.
  • the therapeutic composition of the present invention can be produced, for example, by adding one or more of the other components to the antibody of the present invention, as appropriate, by a known method or a method analogous thereto depending on the form of the antibody. be able to.
  • the present invention also provides a method for preventing or treating COVID-19 (hereinafter referred to as the "therapeutic method of the present invention” as the case may be), in which the anti-SARS-CoV-2 antibody of the present invention or the therapeutic composition of the present invention is administered to a subject. ) is also provided.
  • the therapeutic method of the present invention includes the step of administering to a subject an effective amount of the antibody of the present invention.
  • Targets for the treatment method of the present invention include humans or non-human mammals (mouse, sheep, cows, pigs, horses, monkeys, dogs, cats, rabbits, etc.), with humans being preferred.
  • the above-mentioned subjects include those who have not been infected with SARS-CoV-2, and those who have already been infected with SARS-CoV-2 but have not developed COVID-19.
  • it may be for any mild to severe form of COVID-19.
  • the antibody of the present invention can be administered to the subject as it is or as a therapeutic composition of the present invention, and is preferably administered by injection.
  • the effective amount of the antibody of the present invention is appropriately determined depending on each individual case, taking into account the purpose and method of administration; the species, age, weight, sex, and severity of symptoms of the subject.
  • the amount of the antibody of the present invention in the case of two or more types, the total amount thereof may be sufficient to meet the target's needs, although there is no particular limitation.
  • the amount can be 0.0001 to 1000 mg per kg of body weight and per dose, but the present invention is not necessarily limited to these values.
  • the number of administrations can be from once a day to an appropriate number of times.
  • the present invention also provides a method for immunologically detecting SARS-CoV-2 in a sample, which method uses the anti-SARS-CoV-2 antibody of the present invention described above (hereinafter, in some cases, simply referred to as "the present invention”). Detection method).
  • the "sample” to be subjected to the detection method of the present invention includes SARS-CoV-2 (complete virus particles as well as constituent proteins containing at least the above-mentioned RBD).
  • SARS-CoV-2 complete virus particles as well as constituent proteins containing at least the above-mentioned RBD.
  • the detection method and detection composition of the present invention will be described.
  • the sample can contain the same substance (same in the body); for example, body fluids (blood, lymph, tissue fluid, body cavity fluid, cerebrospinal fluid, synovial fluid, nasal mucus, etc.) isolated from living organisms, tissues, etc. It may be a cultured cell or a culture thereof (culture supernatant, etc.). Moreover, it may be diluted or suspended with a diluent as necessary.
  • the diluent include phosphate buffer, Tris buffer, Good's buffer, borate buffer, acetate buffer, citrate buffer, glycine buffer, succinate buffer, phthalate buffer
  • the detection method of the present invention involves bringing SARS-CoV-2 in a sample into contact with the antibody of the present invention, and detecting SARS-CoV-2 in the sample based on an immune complex formed by an antigen-antibody reaction. It is something.
  • Immunological detection methods include, for example, the EIA method (enzyme immunoassay) using an enzyme as a labeling substance, the ELISA method or CLEIA method (chemiluminescent enzyme immunoassay), which is a form of the EIA method, and the use of a labeling substance.
  • Labeled immunoassay using an antigen or antibody labeled with RIA method (radioimmunoassay) using a radioactive isotope as a labeling substance
  • CLIA method chemiluminescent immunoassay
  • immunochromatography examples include, but are not limited to, immunoagglutination methods (latex agglutination method, colloidal gold agglutination method, etc.) that measure by detecting agglutination.
  • labeling substance those generally used in immunoassays and similar assays can be used as appropriate, such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), ⁇ -galactosidase, etc. ( ⁇ -gal), enzymes such as firefly luciferase; fluorescent dyes such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC); fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE); 125 Examples include radioactive isotopes such as I; particles such as latex particles and colloidal gold particles; avidin and biotin.
  • HRP horseradish peroxidase
  • ALP alkaline phosphatase
  • ⁇ -galactosidase etc.
  • enzymes such as firefly luciferase
  • fluorescent dyes such as fluorescein isothiocyanate (FITC) and
  • the detection method of the present invention it is preferable to detect the presence or absence of SARS-CoV-2 in a sample by detecting the presence or absence of a signal corresponding to the labeling substance.
  • the above-mentioned “signals” include coloration (color development), extinction, reflected light, luminescence, fluorescence, radiation from radioactive isotopes, etc., and in addition to those that can be confirmed with the naked eye, they can also be detected by detection methods and devices depending on the type of signal. This includes things that can be confirmed.
  • the substrate when an enzyme is used as the labeling substance, the substrate may be a chromogenic substrate (for example, 3,3',5,5'-tetramethylbenzidine, which develops color through an oxidation catalytic reaction by HRP in the presence of hydrogen peroxide).
  • TMB chromogenic substrate
  • chemiluminescent substrates e.g. AMPPD (3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-
  • AMPPD 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-
  • the labeling substance may be bound to the antibody of the present invention to directly detect SARS-CoV-2, or the antibody of the present invention may not be bound to the labeling substance, but a secondary antibody bound to the labeling substance may be used.
  • SARS-CoV-2 may be detected indirectly using methods such as SARS-CoV-2.
  • the "secondary antibody” is an antibody that shows reactivity with an antibody (primary antibody, ie, the antibody of the present invention) that directly binds to an antigen.
  • primary antibody ie, the antibody of the present invention
  • protein G, protein A, etc. bound to a labeling substance may be used.
  • the binding of the antibody and the labeling substance conventionally known methods or methods similar thereto can be appropriately employed.
  • the antibody and the labeling substance may be directly bound by an active group, etc.
  • the binding may be performed indirectly via a biotin-avidin system (for example, an antibody is biotinylated, an avidinized labeling substance is applied to the antibody, and the interaction between biotin and avidin is used to bind the antibody to the antibody. (binding of a labeling substance) may also be used.
  • a sandwich method such as the CLEIA method is preferred from the viewpoint that it tends to be more sensitive.
  • a detection target substance SARS-CoV-2
  • a capture antibody capture body
  • a detection antibody labeled body
  • B/F separation washing
  • detection is performed depending on the type of labeling substance.
  • the target substance to be detected is recognized by a labeled body, and while B/F separation is performed, the target substance to be detected is captured by a capture body, and detection is performed according to the type of labeled substance. Good too.
  • those generally used in immunoassay methods and similar assay methods can be used as appropriate, such as particles such as magnetic particles and latex particles, plates such as plastic plates, Fibrous materials such as nitrocellulose fibers can be used.
  • the antibody of the present invention is preferable to use as at least one of the capture antibody and the detection antibody.
  • the other antibody may be an antibody against the RBD, but is preferably an antibody that does not compete with the antibody of the present invention in binding to the RBD (more preferably an antibody at serine at position 443 of the spike glycoprotein or This is an antibody that does not recognize tyrosine at position 489).
  • the other antibody may be a monoclonal antibody or a polyclonal antibody, but from the viewpoint of supply stability, it is preferably a monoclonal antibody.
  • the amount of SARS-CoV-2 When quantifying the amount of SARS-CoV-2 from the measured value of the detected signal, it can generally be done by comparing it with the measured value of a standard specimen. In this case, for example, it is possible to determine the amount of SARS-CoV-2 in the sample by examining where the actual measured value is located on the standard curve created based on the measured value of the standard sample. can.
  • the detection method of the present invention can be used as a method for testing COVID-19, which is an infectious disease caused by SARS-CoV-2. Furthermore, the method of obtaining data regarding the amount of SARS-CoV-2 using the detection method of the present invention includes a method of collecting data regarding the amount of SARS-CoV-2 described above for diagnosis by a doctor, and a method of presenting the data to a doctor. , it can also be expressed as a method for assisting a doctor's diagnosis.
  • the present invention also provides a composition for immunologically detecting SARS-CoV-2 containing the anti-SARS-CoV-2 antibody of the present invention (hereinafter, as the case may be, "detection composition of the present invention”). ).
  • detection composition of the present invention can be suitably used in the detection method of the present invention described above.
  • the antibodies contained in the detection composition of the present invention are as described above, but depending on various immunological techniques, even in the form in which the above-mentioned labeling substance is bound, the antibodies may be immobilized on the solid phase. It may be in any other form.
  • the method for binding such a labeling substance and the method for immobilizing it on a solid phase are as described above.
  • the detection composition of the present invention can further contain other components that are acceptable as reagents.
  • Such other components include, for example, those similar to those listed in the therapeutic composition of the present invention.
  • the antibodies and detection compositions of the present invention include substrates necessary for signal detection, solutions for dissolving sample proteins (protein dissolution reagents), and buffers used for diluting and washing samples (dilution solutions). , washing solution), a reagent for stopping the signal detection reaction (reaction stopper), a positive control (e.g., RBD), a negative control, an isotype control antibody for the antibody of the present invention, etc., as appropriate. You can.
  • Such a kit includes, for example, the antibody of the present invention bound to a labeling substance or the detection composition of the present invention containing the same, a reagent (e.g., a substrate) necessary for detecting a signal according to the labeling substance, and a positive control. , and at least one selected from a negative control.
  • a substance that binds to the antibody for example, a secondary antibody, protein G, protein A, etc.
  • a kit can include instructions for using the kit.
  • Each RBD expression vector was created by inserting each base sequence into a pCAGGS vector together with an N-terminal signal peptide sequence, a C-terminal His tag, and a C-terminal Avitag.
  • the proteins encoded by each base sequence were expressed using each of the prepared RBD expression vectors and Expi293 Expression System (manufactured by Thermo Fisher Scientific), and each protein was extracted from the culture supernatant using a TALON column (manufactured by Takara).
  • RBD antigen protein
  • each biotinylated RBD was prepared in the same manner except that during expression, BirA-Flag plasmid (manufactured by Addgene) and biotin were added to the culture system to add biotin to the RBD.
  • RBD 2 Ancestral (each RBD in which amino acid mutations of each mutant strain were introduced in the amino acid sequence of the spike glycoprotein of the Wuhan strain of SARS-CoV-2 (Wuhan strain RBD, positions 331 to 529 of the amino acid sequence of SEQ ID NO: 1): Alpha strain RBD: RBD with N501Y mutation introduced into the amino acid sequence of Ancestral; Omicron strain (BA.5) RBD: BA. RBD with L452R, F486V, and R493Q mutations introduced into the amino acid sequence of 2; Omicron strain (BA.2.75) RBD: BA.
  • RBD with G339H, G446S, N460K, and R493Q mutations introduced into the amino acid sequence of 2; Omicron strain (BA.2.75.2) RBD: BA. RBD with R346T and F486S mutations introduced into the amino acid sequence of 2; Omicron strain (BQ.1.1) RBD: BA. RBD with R346T, K444T, and N460K mutations introduced into the amino acid sequence of 5; Omicron Stock (XBB) RBD: BA.
  • RBD in which mutations of L368I, V445P, and F490S were introduced into the amino acid sequence of 2.75.2; and Omicron strain (XBB.1.5) RBD: RBD in which mutations of F486P were introduced into the amino acid sequence of XBB.
  • the nucleotide sequences encoding each were synthesized. From each base sequence, each biotinylated RBD was prepared in the same manner as in ⁇ Preparation of RBD 1> above.
  • Preparation example 3 ⁇ Isolation of antigen-specific B cells and culture of single B cells> The mononuclear cells separated in Preparation Example 1 were treated with BUV395-labeled CD19 antibody (HIB19), BV510-labeled CD2 antibody (RPA-2.10), BV510-labeled CD4 antibody (RPA-T4), BV510-labeled CD10 antibody (HI10a), and BV510-labeled CD10 antibody (HI10a).
  • HAB19 BUV395-labeled CD19 antibody
  • RPA-2.10 BV510-labeled CD2 antibody
  • RPA-T4 antibody BV510-labeled CD4 antibody
  • HI10a BV510-labeled CD10 antibody
  • HI10a BV510-labeled CD10 antibody
  • the isolated living cells were cultured on MS40L-low feeder cells for 24 days.
  • the culture medium contained 10% FBS, 55 ⁇ M 2-mercaptoethanol, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 1% MEM non-essential amino acids, 50 ng/mL human IL-2, RPMI1640 medium supplemented with 10 ng/mL human IL-4, 10 ng/mL human IL-21, and 10 ng/mL human BAFF was used.
  • RNA was first extracted from the cells cultured for 24 days in Preparation Example 3 using an RNeasy micro kit (manufactured by Qiagen), and cDNA was synthesized from the extracted RNA by reverse transcription reaction. Using the obtained cDNA as a template, Tiller et al. , Journal of Immunological Methods, 329: p. Using the primers described in 112-124, the heavy chain variable region and light chain variable region of the antibody gene were each amplified by PCR method, and the base sequence was analyzed by Sanger method.
  • the obtained base sequences were analyzed by IgBLAST (https://www.ncbi.nlm.nih.gov/igblast/) to determine the amino acid sequences of the heavy chain variable region and light chain variable region, as well as the complementarity determining region.
  • the amino acid sequences of 1 to 3 were determined.
  • Table 1 shows the amino acid sequences of the heavy chain variable region and light chain variable region of the 14 analyzed antibodies, as well as the sequence number of the amino acid sequence of each complementarity determining region 3 (CDR3).
  • the amino acid sequences of each complementarity determining region 1 to 2 (CDR1 and CDR2) in the heavy chain variable region and light chain variable region of the 14 analyzed antibodies are shown in Table 2 below.
  • the nucleotide sequences encoding the heavy chain variable region and light chain variable region of the 14 antibody genes amplified and analyzed above are the combinations of human IgG1 constant region ( ⁇ 1, sequence A base sequence encoding the constant region of human IgKappa ( ⁇ ) (amino acid sequence of SEQ ID NO: 59), or a constant region of human IgLambda ( ⁇ ) (SEQ ID NO: 60)
  • a heavy chain expression vector and a light chain expression vector were prepared by introducing the nucleotide sequences encoding the amino acid sequence (amino acid sequence) into mammalian expression vectors, respectively.
  • the proteins encoded by each base sequence were expressed using each of the prepared expression vectors and Expi293 Expression System (manufactured by Thermo Fisher Scientific), and the recombinant monoclonal antibody was purified from the culture supernatant using a Protein G column. , 14 (No. 1 to 14) recombinant monoclonal antibodies were obtained.
  • the combinations of each variable region and CDR3 in the heavy chain and light chain of the produced recombinant monoclonal antibody and each constant region are shown in Table 1 below.
  • a complex model (antibody-RBD) of the Fv region of the 5 antibody and Omicron strain (XBB.1.5) RBD was created, and this was improved using the FastRelax protocol (Nivon et al., PloS One 8, e59004, 2013) ( 2,000 complex models were designed using the FastDesign protocol (Maguire et al., Proteins 89, p. 436-449, 2021) using the InterfaceDesign2019 script. Docking calculations (Gray et al., J. Mol. Biol. 331, p.
  • nucleotide sequence encoding the heavy chain variable region and the nucleotide sequence encoding the light chain variable region of the improved antibody selected above are respectively replaced with the nucleotide sequence encoding the human IgG1 constant region ( ⁇ 1, amino acid sequence of SEQ ID NO: 58).
  • a heavy chain expression vector and a light chain expression vector were produced by introducing the vector into a mammalian expression vector containing the base sequence encoding the sequence and the constant region (amino acid sequence of SEQ ID NO: 60) of human IgLambda ( ⁇ ), respectively.
  • the proteins encoded by each base sequence were expressed using each of the prepared expression vectors and Expi293 Expression System (manufactured by Thermo Fisher Scientific), and the recombinant monoclonal antibody was purified from the culture supernatant using a Protein G column. , No. Fifteen recombinant monoclonal antibodies were obtained.
  • the SEQ ID numbers of the amino acid sequences of the heavy chain variable region, light chain variable region, and each complementarity determining region 3 (CDR3) of the produced recombinant monoclonal antibody, and the combination with the constant region are set according to Table 1 above. Shown. Furthermore, the amino acid sequences of each complementarity determining region 1 to 2 (CDR1 and CDR2) in the heavy chain variable region and light chain variable region of the produced recombinant monoclonal antibody are shown in Table 2 above.
  • Example 1 ⁇ Neutralizing activity measurement using virus isolates> This experiment was conducted at the BSL3 experimental facility of the National Institute of Infectious Diseases. First, each recombinant monoclonal antibody (No. 1 to 14) produced in Preparation Example 4 was serially diluted with DMEM culture medium supplemented with 2% FBS, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin, and diluted with 100 TCID 50 . of SARS-CoV-2 (Wuhan strain, Delta strain, Omicron strain (BA.1), Omicron strain (BA.2)) and reacted for 1 hour in a CO 2 incubator at 37°C.
  • SARS-CoV-2 Wang strain, Delta strain, Omicron strain (BA.1), Omicron strain (BA.2)
  • the IC 50 of the antibody concentration required for each test was calculated.
  • a heat map showing the log 10 IC 50 is shown in Figure 1.
  • the IC 50 was also calculated when the sotrovimab parent antibody (S309), which is a conventional anti-SARS-CoV-2 antibody, was used in place of the recombinant monoclonal antibody. Since infection neutralization was not shown under the concentration conditions, it could not be calculated ("X" in FIG. 1).
  • Preparation Example 4 was added to each well (RBD amount: 60 ng/well) of a U-plex plate (manufactured by MSD) on which each biotinylated mutant RBD prepared in ⁇ Preparation of RBD 1> of Preparation Example 2 was immobilized.
  • the recombinant monoclonal antibodies (No. 1 to 14) prepared in the above were diluted with Diluent 100 (manufactured by MSD) and added (antibody amount: 250 pg/well), and reacted for 1 hour at room temperature while shaking at 700 rpm. Ta.
  • the amount of IgG antibody bound to the recombinant monoclonal antibody was determined by electrochemiluminescence method. It was measured.
  • the amount of recombinant monoclonal antibody bound when using Wuhan strain RBD (Ancestral) as RBD is set as 100 (%), and the relative binding amount of recombinant monoclonal antibody bound when using each mutant RBD (Relative to Ancestral) (%)) was calculated.
  • Example 3 [Preparation of recombinant Fab protein] Among the antibody genes amplified in Preparation Example 4, antibody No.
  • the nucleotide sequences encoding each of the heavy chain variable regions of 2, 5 to 7 encode the CH1 region of the constant region of human IgG1 (amino acid sequence at positions 1 to 105 of the amino acid sequence ( ⁇ 1) of SEQ ID NO: 58). It was introduced into a mammalian expression vector containing the nucleotide sequence and a His tag, and used as a heavy chain expression vector.
  • the heavy chain expression vector produced and the antibody No. 1 produced in Preparation Example 4 were used.
  • the proteins encoded by each of the nucleotide sequences were expressed using a light chain expression vector containing the nucleotide sequences encoding each of the light chain variable regions of 2, 5 to 7, and Expi293 Expression System (manufactured by Thermo Fisher Scientific).
  • the recombinant Fab protein was purified from the culture supernatant using a TALON column (manufactured by Takara).
  • Example 4 ⁇ Antibody escape mutant virus (escape virus) analysis> First, the recombinant monoclonal antibodies (No. 2, 5 to 7) prepared in Preparation Example 4 were serially diluted with DMEM culture medium supplemented with 2% FBS, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin, and diluted with 100 TCID. The mixture was mixed with 50 SARS-CoV-2 (Wuhan strain) and reacted for 1 hour in a CO 2 incubator at 37°C. Next, this was added to VeroE6/TMPRSS2 cells cultured overnight on a 12-well plate, and cultured in a CO 2 incubator at 37° C. (Culture 1).
  • the obtained antibody evacuation mutant virus candidate was cloned using the limiting dilution method, then RNA was extracted using a Direct-zol RNA kit (manufactured by ZYMO RESEARCH), and the base sequence encoding its RBD was extracted using the Sanger method. An analysis was conducted.
  • Example 5 ⁇ Evaluation of therapeutic effects in animals> This experiment was conducted at the BSL3 experimental facility of the National Institute of Infectious Diseases. Seven to nine Syrian hamsters were infected by intranasal inoculation with 10,000 TCID 50 of SARS-CoV-2 (Wuhan strain, Omicron strain (BA.1), Omicron strain (BA.2)), and the next day, The recombinant monoclonal antibodies (No. 5 to 7, antibody amount: 5 mg/kg) prepared in Preparation Example 4 were intraperitoneally administered. In addition, as a comparative example, PBS or sotrovimab parent antibody (S309), which is a conventional anti-SARS-CoV-2 antibody, was administered in the same manner.
  • S309 sotrovimab parent antibody
  • FIG. 3 shows the average change in body weight from the day of infection, where the body weight on the day of infection (day 0) is taken as 100.
  • the right lung lobe was collected on the 6th day from the day of infection and its weight was measured.
  • Figure 4 shows the weights (g) of the right lung posterior lobe on the 6th day from the day of infection and their average values (indicated by bars).
  • Figure 5 shows the virus copy numbers and their average values (indicated by bars) in the nasal wash on the third day from the day of infection when infected with Omicron strain (BA.1). The number of virus copies in the nasal wash fluid and their average value (indicated by bars) on the third day from the day of infection when infected with (BA.2) are shown.
  • Example 6 ⁇ Neutralizing activity measurement using pseudotyped virus>
  • a neutralization activity test using pseudotyped virus was conducted.
  • No. prepared in Preparation Example 5 was prepared. 15 recombinant monoclonal antibodies were serially diluted in DMEM medium supplemented with 2% FBS, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin, and 100 TCID 50 of SARS-CoV-2 pseudotyped virus (Omicron strain (XBB .1.5)) and reacted for 1 hour in a CO 2 incubator at 37°C.
  • Example 7 As the biotinylated mutant RBD (antigen protein), Ancestral prepared in Preparation Example 2 ⁇ Preparation of RBD 1>, Beta strain RBD, Delta strain RBD, Omicron strain (BA.1) RBD, Omicron strain (BA.2) RBD In addition to the biotinylated RBD of Y489S, each biotinylated RBD prepared in ⁇ Preparation of RBD 2> was used, and as a recombinant monoclonal antibody, No. Binding analysis between the antigen protein and the monoclonal antibody was performed in the same manner as in Example 2 except that No. 15 recombinant monoclonal antibody was used.
  • a novel antibody with high binding affinity and infection-neutralizing activity against other mutant strains including Omicron strain in addition to the conventional SARS-CoV-2, a novel antibody with high binding affinity and infection-neutralizing activity against other mutant strains including Omicron strain, and , a composition for preventing or treating COVID-19 using the same, a composition for immunologically detecting SARS-CoV-2, and a method for immunologically detecting SARS-CoV-2. becomes possible.

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  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention vise à fournir un nouvel anticorps ayant une affinité de liaison élevée et une activité de neutralisation d'infection élevée contre le SARS-CoV-2 classique (souche Wuhan) et également d'autres variants, y compris la souche omicron. L'invention concerne un anticorps contre le SARS-CoV-2 qui se lie à un domaine de liaison au récepteur (RBD) de glycoprotéine de spicule du SARS-CoV-2, l'anticorps anti-SARS-CoV-2 étant caractérisé en ce qu'il reconnaît la sérine 443 ou la tyrosine 489 de la séquence d'acides aminés de la glycoprotéine de spicule.
PCT/JP2023/026565 2022-07-20 2023-07-20 Anticorps contre le sars-cov-2 WO2024019110A1 (fr)

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JP2023086002A JP2024014723A (ja) 2022-07-20 2023-05-25 ＳＡＲＳ－ＣｏＶ－２に対する抗体
JP2023-086002 2023-05-25

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WO2022003530A1 (fr) * 2020-06-30 2022-01-06 Fondazione Toscana Life Sciences Anticorps neutralisants dirigés contre le coronavirus 2 du sars
WO2022053188A1 (fr) * 2020-09-11 2022-03-17 Alfasigma S.P.A. Nouvelles séquences d'adn encodant des anticorps humains scfv à activité neutralisante contre le sars-coronavirus-2 et leurs utilisations
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