WO2022210830A1 - Anti-sars-cov-2 antibody - Google Patents

Anti-sars-cov-2 antibody Download PDF

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WO2022210830A1
WO2022210830A1 PCT/JP2022/015813 JP2022015813W WO2022210830A1 WO 2022210830 A1 WO2022210830 A1 WO 2022210830A1 JP 2022015813 W JP2022015813 W JP 2022015813W WO 2022210830 A1 WO2022210830 A1 WO 2022210830A1
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seq
amino acid
acid sequence
set forth
sequence set
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龍彦 小澤
裕幸 岸
正治 磯部
信幸 黒澤
芳智 森永
善裕 山本
英樹 仁井見
英樹 谷
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国立大学法人 富山大学
富山県
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    • 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
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the present invention relates to an antibody that binds to the spike protein of the novel coronavirus SARS-CoV-2 and an antigen-binding fragment thereof.
  • SARS-CoV-2 is a type of coronavirus that was first reported in Wuhan, China at the end of 2019 and has since spread widely throughout China and even around the world.
  • the SARS-CoV-2 infection, called COVID-19 is characterized by mild to moderate symptoms in most patients, while severe disease and death in some patients.
  • vaccines have been developed and approved, and vaccination has begun in various countries, but cases of infection after vaccination have been reported (Non-Patent Document 1), and vaccines do not completely suppress viral infections. It is believed that.
  • Patent Document 1 In parallel with the development of vaccines, COVID-19 therapeutic agents are being developed, and therapeutic antibodies have also been reported (Patent Document 1). However, multiple mutant strains of SARS-CoV-2 have been reported, and none of the mutant strains is known to have therapeutic or preventive effects.
  • the present invention is an antibody against SARS-CoV-2 (hereinafter referred to as "anti-SARS-CoV-2 antibody”) that binds to the spike protein of SARS-CoV-2, and in particular, increases infection and transmissibility Antibodies that inhibit the binding of SARS-CoV-2 and ACE2 by binding to SARS-CoV-2 mutants that are concerned about antigenic changes, or those SARS-CoV-2 cells
  • An object of the present invention is to provide an antibody that inhibits internal invasion.
  • the present inventors used plasma obtained from multiple patients with a history of SARS-CoV-2 infection, and found that SARS-CoV-2 spike protein (trimeric structure full-length S protein (ECD)), The presence of antibodies capable of binding to the N-terminal domain (NTD) and receptor binding domain (RBD) was investigated. As a result, it was found that plasma derived from one patient who spontaneously recovered after becoming severe had excellent binding properties to all of ECD, NTD, and RBD. Peripheral blood lymphocytes were isolated from this COVID-19 patient, and the ISAAC method (Immunospot-array assay on a chip) (Jin A et al., Nature medicine (2009) 26: 1088-1092, and Jin A et al. , Nature Protocols (2011) 6:668) were used to purify multiple monoclonal antibodies that specifically bind to the SARS-CoV-2 spike protein.
  • ECD trimeric structure full-length S protein
  • NTD N-terminal domain
  • RBD receptor binding domain
  • SARS-CoV-2 RBD antibody an antibody that binds to SARS-CoV-2 RBD (hereinafter referred to as "SARS-CoV-2 RBD antibody").
  • SARS-CoV-2 RBD antibody an antibody that binds to SARS-CoV-2 RBD
  • SARS-CoV-2 RBD antibody an antibody that binds to SARS-CoV-2 RBD
  • SARS-CoV-2 mutants which are concerned about increased infection and transmissibility and antigenic changes, and found that such mutants RBD We have succeeded in discovering an antibody that binds to and has an infection-suppressing effect.
  • the present invention relates to anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof, therapeutic agents, diagnostic agents, compositions for detecting SARS-CoV-2, etc. containing the antibodies as active ingredients.
  • 1 is a graph showing the results of confirming the binding specificity of an anti-SARS-CoV-2 antibody to a SARS antigen protein.
  • RBD SARS-CoV receptor binding domain
  • ECD trimer SARS-CoV-2 spike protein
  • NTD SARS-CoV-2N- terminal domain (NTD) protein
  • RBD SARS-CoV-2 RBD.
  • the vertical axis indicates absorption (bond strength) at 450 nm. Two or more experiments were performed independently and the data shown are representative.
  • Fig. 3 is a graph showing the results of binding inhibition experiments between SARS-CoV-2 spike protein and ACE2 by anti-SARS-CoV-2 RBD antibody.
  • each anti-SARS-CoV-2 RBD antibody of 1028-05K, 1028-37K, 1028-55K, 1028-61L, and 1028-121K inhibits the binding of SARS-CoV-2 spike protein and ACE2 was verified by ELISA.
  • the horizontal axis is the concentration of anti-SARS-CoV-2 RBD antibody ( ⁇ g/ml), and the vertical axis is the presence of each antibody when the absorbance value measured in the absence of anti-SARS-CoV-2 RBD antibody is set to 100. shows the ratio (%) of absorbance values in . Two or more experiments were performed independently and the data shown are representative. Fig.
  • FIG. 3 is a graph showing the results of an experiment to suppress pseudotype virus infection using an anti-SARS-CoV-2 RBD antibody.
  • the vertical axis shows the logarithmic value of infection reduction ratio (log (% of reduction)) compared to the antibody-free control, and the horizontal axis shows the antibody concentration ( ⁇ g/ml).
  • Black bars (St19pv) show the results for the SARS-CoV-2 spike protein mantle pseudovirus, and white bars show the results for the pseudotyped virus VSVpv used as a control.
  • Fig. 3 is a graph showing the results of binding inhibition experiments between SARS-CoV-2 spike protein and ACE2 by anti-SARS-CoV-2 RBD antibody.
  • Each anti-SARS-CoV-2 RBD antibody 0210-20L, 0210-28K, 0210-30L, 0210-38L, 1028-61L, 1028-55K inhibits the binding of SARS-CoV-2 spike protein and ACE2 or verified by ELISA.
  • the horizontal axis is the concentration of anti-SARS-CoV-2 RBD antibody ( ⁇ g/ml), and the vertical axis is the presence of each antibody when the absorbance value measured in the absence of anti-SARS-CoV-2 RBD antibody is set to 100. (similar to FIGS. 7A and 7B). Two or more experiments were performed independently and the data shown are representative.
  • Fig. 10 is a graph showing the results of an experiment to inhibit the binding of SARS-CoV-2 spike protein and ACE2 by anti-SARS-CoV-2 RBD antibodies 0210-28K and 1028-55K.
  • FIG. 7A shows the results of the same experiment using 0210-30L and 1028-61L.
  • a schematic diagram of the preparation of the SARS-CoV-2 pseudotype virus, a part of the pseudotype virus that envelopes the mutant spike protein used in this experiment, and a virus (VSVpv) that envelopes the envelope protein of VSV as a control are shown.
  • SARS-CoV-2 (Alpha) has a surface spike protein with mutations of HV69-70 deletion, Y144 deletion, N501Y, A570D, P681H, T716I, S982A, and D1118H in the Alpha strain.
  • Fig. 10 is a graph showing the results of an experiment to suppress infection of a pseudotype virus that envelopes a spike protein of a mutant strain using an anti-SARS-CoV-2 RBD antibody.
  • the vertical axis indicates the logarithmic value (log (% of reduction)) or the real value (% of reduction) of the infection reduction rate compared to the control, and the horizontal axis indicates the antibody concentration ( ⁇ g/ml).
  • Bar graphs at each concentration of antibody show, from left to right, results using pseudotyped viruses with spike proteins of Wuhan type (WT), Alpha strain, and Beta strain of SARS-CoV-2.
  • the rightmost bar graph shows the results using a control pseudotyped virus (VSVpv).
  • (Left) shows the results of a binding experiment between the anti-SARS-CoV-2 RBD antibody 0210-28K and the Epsilon strain SARS-CoV-2 RBD (L452R) protein.
  • the vertical axis indicates the absorption (binding strength) at 450 nm
  • the horizontal axis indicates the concentration (ng/ml) of the anti-SARS-CoV-2 RBD antibody.
  • (Right) Graph showing the results of binding inhibition experiments between SARS-CoV2 spike protein and ACE2 by 0210-28K, an anti-SARS-CoV-2 RBD antibody. It was verified by ELISA whether the binding of the Kappa strain SARS-CoV-2 RBD (L452R, E484Q) protein and ACE2 could be inhibited by the anti-SARS-CoV-2 RBD antibody.
  • FIG. Fig. 10 is a graph showing the results of an experiment to suppress infection of a pseudotype virus that envelopes a mutant spike protein by the anti-SARS-CoV-2 RBD antibody 0210-28K.
  • the vertical axis shows the real value (% of reduction) of infection reduction compared to the control, and the horizontal axis shows the antibody concentration (ng/ml).
  • FIG. 10 is a graph showing the results of an experiment to inhibit binding between SARS-CoV-2 spike protein and ACE2 by 0210-28K, an anti-SARS-CoV-2 RBD antibody.
  • Wuhan type (WT) RBD protein Alpha strain (N501Y), Beta strain (K417N, E484K, N501Y), Gamma strain (K417T, E484K, N501Y), Kappa strain (L452R, E484Q), Delta strain (L452R, T478K) , and an Epsilon strain (L452R)-derived mutant RBD protein, and whether 0210-28K can inhibit the binding of ACE2 was verified by ELISA.
  • the vertical and horizontal axes are the same as in FIG. Fig. 10 is a graph showing the results of an experiment to suppress infection of a pseudotype virus that envelopes a mutant spike protein by the anti-SARS-CoV-2 RBD antibody 0210-28K.
  • the vertical axis indicates the rate of infection reduction (% of reduction) compared to the control, and the horizontal axis indicates the antibody concentration (pM).
  • SARS-CoV-2, Wuhan type (WT), Alpha strain, Beta strain, Gamma strain, Kappa strain, Delta strain, and results using pseudotype viruses with spike proteins of Omicron strain, and VSV G protein The results are shown using a pseudotyped virus (control) with Two or more experiments were performed independently and the data shown are representative.
  • Fig. 3 is a graph showing the results of investigating the effects of 0210-28K, an anti-SARS-CoV-2 RBD antibody, on SARS-CoV-2 infection and production.
  • Fig. 4 is a graph showing the results of surface plasmon resonance (SPR) analysis of the affinity of 0210-28K, an anti-SARS-CoV-2 RBD antibody, to Wuhan type (WT) RBD and mutant RBD proteins.
  • SPR surface plasmon resonance
  • the graph shows, from top to bottom, Wuhan type (WT), Alpha strain (N501Y), Beta strain (K417N, E484K, N501Y), Gamma strain (K417T, E484K, N501Y), Kappa strain (L452R, E484Q), and mutant RBD proteins from Delta strains (L452R, T478K).
  • WT Wuhan type
  • Alpha strain N501Y
  • Beta strain K417N, E484K, N501Y
  • Gamma strain K417T, E484K, N501Y
  • Kappa strain L452R, E484Q
  • mutant RBD proteins from Delta strains L452R, T478K.
  • the vertical axis indicates the response (RU) and the horizontal axis indicates the measurement time (s). Two or more experiments were performed independently and the data shown are representative.
  • FIG. 2 shows a luminescence image of luciferase (upper diagram), a graph showing the results of luminescence measurement (lower left diagram), and a graph showing the results of calculating the virus infectivity titer of the whole lung (lower right diagram).
  • the vertical axis indicates the relative luminescence level compared with the control.
  • the horizontal axis indicates the type of pseudotype virus, and from left to right, the pseudotype virus having the G protein of VSV, SARS-CoV-2, Wuhan type (WT), Alpha strain, Beta strain, Gamma strain, Results using pseudotyped viruses with spike proteins of Kappa, Delta, and Omicron strains are shown.
  • the white bar on the left indicates the results of control
  • the black bar on the right indicates the results of administration of 0210-28K.
  • Antibody dosage is 3 mg/hamster for Beta strain and Omicron strain, and 0.3 mg/hamster for others. Two or more experiments were performed independently and the data shown are representative. p-values were calculated by t-test (*, p ⁇ 0.05, **, p ⁇ 0.01).
  • FIG. 18(a) is a cryo-electron microscopy map densified to 3.1 ⁇ resolution with C3 symmetry, showing the heavy chain variable Regions (VH) and light chain variable regions (VL) are indicated.
  • Figures 18(b), (d) to (g) are the results of X-ray crystallographic analysis with a resolution of 3.75 ⁇ , and Figures 18(d) to (g) show the major residues between RBD and UT28K Fab. Interactions are indicated.
  • FIG. 18(c) shows the results of X-ray crystallographic analysis of the binding of 253XL55 (PDB ID: 7BEO) and SARS-CoV-2 RBD (RBD).
  • Fig. 3 is a graph showing the results of ELISA analysis of the binding of 0210-28K, which is an anti-SARS-CoV-2 RBD antibody, to Wuhan type (WT) RBD and RBD proteins derived from mutant strains.
  • the vertical axis indicates absorption (bond strength) at 450 nm, and the horizontal axis indicates the type of RBD protein. Two or more experiments were performed independently and the data shown are representative.
  • the vertical axis indicates the amount of virus increased or decreased (Fold increase) with the amount of virus obtained in the control set to 1, and the horizontal axis indicates the antibody concentration ( ⁇ g/ml).
  • the vertical axis indicates optical density, and the horizontal axis indicates antibody concentration ( ⁇ g/ml).
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV-2 spike protein spike protein is a protein consisting of 1273 amino acids (SEQ ID NO: 629) (of which 1 to 13 are signal sequences) reported as the S protein of SARS-CoV-2.
  • S1 14th to 685th
  • S2 686th to 1273rd
  • the receptor binding domain means a portion consisting of amino acids 319 to 541 contained in the S1 subunit or a peptide consisting of the same portion.
  • the N-terminal domain means a portion consisting of the 14th to 305th amino acids or a peptide consisting of the same portion.
  • amino acid positions refer to amino acid positions in the S protein.
  • SARS-CoV-2 has been reported to have many mutations in addition to the first Wuhan type found (for example, Islam, MR et al., Sci Rep; 10: 14004 (2020). See ), and databases also exist (eg, https://nextstrain.org/sars-cov-2/, etc.).
  • the spikes or RBDs to which the antibodies herein bind may be those possessed by the Wuhan type as well as the mutant forms of SARS-CoV-2 reported therein.
  • a “mutant SARS-CoV-2” may have any mutation that has been reported for SARS-CoV-2, such as mutations in the spike of SARS-CoV-2, particularly RBD Mutations in As used herein, "mutant spike” means a spike having a mutation in the spike of SARS-CoV-2, and “mutant RBD” means an RBD having a mutation in the RBD of SARS-CoV-2. do. For example, 20B/501Y. V1, VOC 202012/01 or B.
  • mutant SARS-CoV-2 is, for example, L18F, T19R, H69 deletion, V70 deletion, D80A, G142D, Y144 deletion, E154K, 157 deletion, 158 deletion, D215G, L242 deletion, A243 deletion, L244 ⁇ ,R246I,G339D,S371L,S373P,S375F,K417N,K417T,N440K,G446S,L452R,S477N,T478K,E484A,E484K,E484Q,Q493R,G496S,Q498R,N501Y,Y505H,A570D,D614G,H655Y,P681H , P681R
  • the mutant SARS-CoV-2 herein may have a mutant RBD, such as G339D, S371L, S373P, S375F, K417N, K417T, N439K, N440K, G446S, One or more, two or more, three or more, four or more selected from L452R, A475V, S477N, T478K, E484A, E484K, E484Q, Q493R, G496S, Q498R, N501Y, and Y505H Above, 5 or more, or 6 or more mutations.
  • RBD such as G339D, S371L, S373P, S375F, K417N, K417T, N439K, N440K, G446S, One or more, two or more, three or more, four or more selected from L452R, A475V, S477N, T478K, E484A, E484K, E484Q,
  • mutant SARS-CoV-2 has include K417N, E484K, and N501Y mutations in RBD; N439K mutation; N440K mutation; A475V mutation; , E484K, and N501Y mutations; L452R and T478K mutations;
  • SARS-CoV-2 will be used to refer to Wuhan-type (non-mutated) SARS-CoV-2 and the mutant SARS-CoV described above, unless it is incompatible to be so construed. -2, or a plurality of SARS-CoV-2 selected from these.
  • SARS-CoV-2 herein refers to one or more selected from Wuhan, Alpha strain, Beta strain, Gamma strain, Epsilon strain, Kappa strain, Delta strain, and Omicron strain, 2 There may be 1 or more types, 3 or more types, 4 or more types, 5 or more types, or 6 or more types, or all of these may be meant.
  • the antibodies of the invention specifically bind to at least one of SARS-CoV-2 spike, NTD and RBD, more preferably SARS-CoV-2 spike and/or RBD. and more preferably, it specifically binds to SARS-CoV-2 RBD.
  • RBD is important for binding to cells in the SARS-CoV-2 spike (Science (2020) Vol.367, Issue6483, pp.1260-1263, and Science (2020) Vol.369 , Issue 6504, pp. 650-655).
  • binding to the RBD may mean binding to the RBD portion in the SARS-CoV-2 spike, or binding to a peptide consisting of an amino acid sequence that constitutes the RBD. good too.
  • the antibody of the present invention specifically binds to spike (preferably SARS-CoV-2 RBD).
  • spike preferably SARS-CoV-2 RBD.
  • an antibody or immunoreactive fragment thereof “specifically” binds (recognizes) means that the antibody or immunoreactive fragment thereof has a specified affinity for other proteins or peptides. It means binding with substantially high affinity to an antigen (eg spike or RBD).
  • binding with substantially high affinity means that the desired measuring device or method can detect the specific protein or peptide of interest by distinguishing it from other proteins or peptides. means high affinity.
  • a substantially high affinity is 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, as the intensity (e.g., fluorescence intensity) detected by ELISA or EIA. It may mean 9 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, or 100 times or more.
  • the antibody of the present invention specifically binds to mutant spike and/or mutant RBD (preferably RBD, in this paragraph, the same shall apply hereinafter). Also preferably, the antibodies of the invention bind to these mutant spikes and/or mutant RBDs and also bind to the spikes and/or RBDs of Wuhan SARS-CoV-2. Furthermore, the antibodies of the present invention may bind to multiple types of mutant spikes and/or mutant RBDs with different mutation patterns, and in this case also preferably Wuhan SARS-CoV-2 spikes and/or Or it also binds to RBD.
  • “having different mutation patterns” means that two or more types of SARS-CoV-2, spikes, or RBDs have different mutations.
  • “different mutation” means that the mutation site and/or the amino acid after mutation are different. If at least one of two types of SARS-CoV-2, spike, or RBD has multiple mutations, even if they have common mutations, the mutation site and/or post-mutation between the two If there are mutations that differ in amino acids, they are considered to have different mutation patterns.
  • the antibody includes Wuhan, K417N, E484K, and N501Y variants, N439K variant, N440K variant, A475V variant, E484K variant, N501Y variant, L452R variant, L452R and E484Q variant, K417T, E484K and N501Y mutants, L452R and T478K mutants, and SARS-H mutants of G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 selected from CoV-2, spike, or RBD or more, or may be combined with all.
  • the antibodies of the present invention are , and Y505H, one or more, two or more, three or more, four or More, 5 or more, 6 or more, or 7 or more may be combined.
  • the antibody of the present invention may be one or more, two or more selected from the above Wuhan type, Alpha strain, Beta strain, Gamma strain, Epsilon strain, Kappa strain, Delta strain, and Omicron strain, May bind to 3 or more, 4 or more, or all SARS-CoV-2, spike, or RBD.
  • the binding strength of the antibody of the present invention to these mutant strains or mutant RBDs is preferably comparable to that of the Wuhan type.
  • the RBD having the K417N, E484K, and N501Y mutations; the N439K mutation; the N440K mutation; the A475V mutation; the E484K mutation; the N501Y mutation; N501Y", “RBD N439K”, “RBD N440K”, “RBD A475V”, “RBD E484K”, “RBD N501Y”, “RBD L452R”, “RBD L452R, E484Q”) binding to the Wuhan type (WT ) may be 94% or greater, 96% or greater, 100% or greater, 101% or greater, 94% or greater, 100% or greater, 100% or greater, 100% or greater, relative to binding to RBD, respectively.
  • These binding forces are preferably binding forces measured by the ELISA method described in the Examples of the present application.
  • whether the test antibody "binds (recognizes)" to the spike or RBD is determined by measuring the binding of the test antibody to the spike and RBD to be examined for binding by the following method. It can be done by Methods for measuring binding between antibodies and proteins or peptides include, for example, EIA method, ELISA method, FACS method, co-immunoprecipitation method, pull-down assay method, Western blotting method, homobifunctional crosslinker or heterobifunctional crosslinker Cross-linking method using linker, label transfer reaction method, interaction mapping method, surface plasmon resonance method, FRET (fluorescence resonance energy transfer) method, BIACORE method, AlphaPPI assay such as AlphaScreen (registered trademark) and AlphaLISA (registered trademark) (PerkinElmer), etc., and various methods are well known in the art, and suitable methods can be employed depending on the desired binding detection.
  • EIA method EIA method
  • ELISA method ELISA method
  • FACS method co-
  • the term “specifically binds (recognizes)” does not bind to substances or parts thereof that are undesirable for antibodies to bind (for example, proteins present in the human body), and does not bind to SARS- It means binding to CoV-2, spike and/or RBD, preferably only to material derived from SARS-CoV-2 or parts thereof (spike, RBD etc.).
  • the term “specifically binds (recognizes)” means that SARS-CoV-2 does not bind to any substance or part thereof (e.g., NTD, etc.), but only to RBD. You can understand.
  • test antibody specifically binds (recognizes) the spike or RBD depends on the binding of the test antibody to the spike and RBD whose binding is to be tested, and the binding of the test antibody to other peptides, proteins or It can be determined by examining the binding of the test antibody to other antigens (for example, substances to which the antibody should not bind).
  • the test antibody specifically binds (recognizes) the spike and/or RBD of interest if it binds to the spike and/or RBD of interest and not to other peptides, proteins, or other antigens. is determined.
  • the antibody of the present invention collects lymphocytes from patients with a history of COVID-19 infection, SARS-CoV-2 spike protein (hereinafter referred to as "spike"), the N-terminal domain in the spike (hereinafter referred to as “NTD” ), or the spike receptor binding domain (herein, referred to as “RBD”) as antigens, select lymphocytes that produce human antibodies that bind to these antigens, and obtain their cDNAs to obtain human anti- SARS-CoV-2 monoclonal antibodies can be produced as recombinant antibodies. That is, all of the antibodies obtained by this method are antibodies that bind to the SARS-CoV-2 spike, NTD, or RBD, and are antibodies capable of binding to these proteins.
  • the antigen is preferably SARS-CoV-2 spike or RBD, more preferably RBD.
  • the antibody of the present invention can be obtained by the ISAAC method.
  • the ISSAC method is a method for rapid and exhaustive screening of antigen-specific antibody-secreting cells (ASC) using a microwell array chip.
  • ASC antigen-specific antibody-secreting cells
  • a microwell array chip having a large number of wells with a size (about 10 to 15 ⁇ m in diameter) in which one antibody-producing cell can be accommodated, and an anti-immunoglobulin antibody (or antibody) on the chip surface around the wells
  • a microwell array chip coated with the antigen for which you are trying to obtain Peripheral blood lymphocytes, spleen cells, or bone marrow cells containing antibody-producing cells are collected from humans or animals that produce antibodies against specific antigens, and the cells are seeded one by one in the wells of a microwell array chip.
  • the antibodies secreted from the cells that produce and secrete the antibodies spread around the wells in which they were stored. Binds to globulin antibodies (or antigens).
  • the antigen labeled with a fluorescent substance or an antibody against the antibody labeled with a fluorescent substance
  • the labeled antigen (or antibody against the antibody) binds to the antibody secreted around the well, and the signal emitted from the labeled substance indicates which well contains the cells that produce and secrete the antigen-specific antibody. Recognize.
  • the cells are harvested using a microcapillary under a fluorescence microscope. Nucleic acids are extracted from cells, antibody cDNA is amplified by RT-PCR or the like, and antibody genes are cloned. A desired antibody can be produced as a recombinant antibody using the gene.
  • an antibody of the invention is preferably an antibody that inhibits binding of SARS-CoV-2, or its spike or RBD, to ACE2 (preferably human ACE2, as throughout the specification).
  • the antibody of the present invention is preferably an antibody that inhibits the binding of mutant SARS-CoV-2, spike, or RBD to ACE2.
  • Antibodies that inhibit binding to ACE2 preferably also inhibit binding of Wuhan SARS-CoV-2, its spike or RBD, to ACE2.
  • the antibodies of the present invention may inhibit the binding of multiple types of SARS-CoV-2, spikes, or RBDs with different mutation patterns to ACE2.
  • the antibody includes Wuhan, K417N, E484K, and N501Y variants, N439K variant, N440K variant, A475V variant, E484K variant, N501Y variant, L452R variant, L452R and E484Q variant, K417T, E484K and N501Y mutants, L452R and T478K mutants, and SARS-H mutants of G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 selected from CoV-2, spike, or RBD Or more, or all, binding to ACE2 may be inhibited.
  • the antibodies of the present invention are , and Y505H, one or more, two or more, three or more, four or Binding of ACE2 to more, 5 or more, 6 or more, or 7 or more may be inhibited.
  • the antibody of the present invention may be one or more, two or more selected from the above Wuhan type, Alpha strain, Beta strain, Gamma strain, Epsilon strain, Kappa strain, Delta strain, and Omicron strain, Binding of 3 or more, 4 or more, or all SARS-CoV-2, spike, or RBD to ACE2 may be inhibited.
  • test antibody inhibits the binding of SARS-CoV-2 to the ACE2 protein can be determined, for example, by adding the test antibody and labeled ACE2 to wells immobilized with SARS-CoV-2 spikes or RBDs to be tested. or a labeled ACE2 solution containing no test antibody is added, washed after standing for a certain period of time, and the ACE2 label bound to the immobilized SARS-CoV-2 spike or RBD is measured. can be determined by If the intensity of labeling with antibodies is weaker than the intensity of labeling without antibodies, the test antibody inhibits the binding of SARS-CoV-2 to ACE2 ( neutralize).
  • the IC50 of the inhibition experiment in vitro is 20 ⁇ g/ml or less, 10 ⁇ g/ml or less, 5 ⁇ g/ml or less, 1 ⁇ g/ml or less, 0.6 ⁇ g/ml or less, 0.5 ⁇ g/ml or less, 0.35 ⁇ g/ml ml or less, 0.2 ⁇ g/ml or less, 0.15 ⁇ g/ml or less, or 0.1 to 20 ⁇ g/ml, 0.2 to 10 ⁇ g/ml, 0.2 to 5 ⁇ g/ml, 0.1 ⁇ g/ml or less. It can be 2-1 ⁇ g/ml.
  • the antibodies of the present invention suppress infection of ACE2 and TMPRSS2 co-expressing cells by SARS-CoV-2 spike-coated pseudotype viruses.
  • the antibodies of the present invention can be used to detect SARS-CoV-2 spike-enveloped pseudotype virus (SARS-CoV-2 St19pv) (Tani H et al., Virology Journal (2021) 18:16). type virus) to TMPRSS2-expressing VeroE6 cells.
  • SARS-CoV-2 St19pv SARS-CoV-2 spike-enveloped pseudotype virus
  • type virus type virus
  • Whether the test antibody suppresses the infection of TMPRSS2-expressing VeroE6 cells by SARS-CoV-2 St19pv can be determined, for example, by the presence of the test antibody in VeroE6 cells (VeroE6/TMPRSS2) overexpressing TMPRSS2.
  • the luminescence value indicating luciferase activity is lower in the presence of antibody than in the case of non-addition of antibody, it is judged that the infection was inhibited by the antibody.
  • the antibody of the present invention preferably exhibits an IC50 value of 500 ng/ml or less in an experiment to suppress infection of ACE2 and TMPRSS2 co-expressing cells with a SARS-CoV-2 spike-enveloped pseudotype virus, more preferably , 100 ng/ml or less, 50 ng/ml or less, or 45 ng/ml or less.
  • the antibody of the present invention is 30 ng/ml or less against Wuhan type (WT), 45 ng/ml or less against Alpha strain, 15 ng/ml or less against Beta strain, and 20 ng/ml or less against Kappa strain. , and/or an antibody that exhibits an IC50 value of 12 ng/ml or less against the Delta strain.
  • the antibody of the present invention is an antibody that suppresses infection of ACE2 and TMPRSS2 co-expressing cells by SARS-CoV-2.
  • Whether the test antibody suppresses the infection of ACE2 and TMPRSS2 co-expressing cells by SARS-CoV-2 is determined by mixing the test antibody and SARS-CoV-2 in a tube and incubating at 37°C for 1 hour. After reacting, it was added to VeroE6/TMPRSS2 cells and allowed to adsorb for 2 hours at 37°C. After that, the mixture containing the unadsorbed test antibody and virus was completely removed, and the culture medium containing the test antibody was added again.
  • test antibody SARS-CoV-2, ACE2 and TMPRSS2 co-expressing cells It is determined that it suppresses the infection of Alternatively, whether the test antibody suppresses the infection of ACE2 and TMPRSS2 co-expressing cells by SARS-CoV-2 is confirmed by adding the test antibody to VeroE6/TMPRSS2 cells to infect SARS-CoV-2.
  • test antibody After culturing at 37°C for 1 hour, remove the culture supernatant, add the test antibody again and culture for 24 hours. It can be determined by measuring. If the amount of viral genomic RNA decreases in the presence of the test antibody, or if it is absent, it is determined that the test antibody suppresses SARS-CoV-2 infection of ACE2 and TMPRSS2 co-expressing cells.
  • the antibody of the present invention is an antibody that suppresses pulmonary infection by SARS-CoV-2 spike-enveloped pseudotype virus by prophylactic administration of the antibody in an in vivo test, such as a hamster model.
  • an in vivo test such as a hamster model.
  • the test antibody suppresses infection can be determined, for example, by inoculating the pseudotype virus directly into the trachea 24 hours after intraperitoneal administration of the test antibody to hamsters, and after 24 hours, extracting the lungs and adding a luciferase substrate solution. can be determined by incubating with and measuring luciferase activity. When the luminescence value, which indicates luciferase activity, is lower after antibody administration than when antibody is not administered, it is determined that infection is inhibited by antibody.
  • inhibitor binding and “inhibit infection” herein refer to 100% inhibition or suppression. is not meant to bring In other words, “inhibit binding” or “inhibit infection” means that binding is inhibited compared to the absence of the antibody or the presence of an antibody that does not bind to SARS-CoV-2. , or that the infection is suppressed.
  • the antibody of the present invention is a human antibody, or an animal in which a part of the human antibody (e.g., constant region, framework region, or part thereof) is replaced with a sequence derived from a non-human animal antibody It may be a modified antibody.
  • non-human animals include antibodies from mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, monkeys, sheep, goats, camels, chickens and ducks.
  • the immunoglobulin class of the antibody of the present invention is not particularly limited, and may be any immunoglobulin class (isotype) of IgG, IgM, IgA, IgE, IgD, or IgY, preferably IgG.
  • the antibody of the present invention when the antibody of the present invention is IgG, it may be of any subclass (IgG1, IgG2, IgG3, or IgG4).
  • the antibody of the present invention is monospecific, bispecific (bispecific antibody), trispecific (trispecific antibody) (for example, WO1991/003493), or multispecific (multispecific antibody) good too.
  • the Fc region may be mutated to improve the functionality of the antibody, for example, in one embodiment, the Fc region is modified with LALA mutations (L234F, L235E mutations) to reduce ADE activity It may be an antibody.
  • the antibody may be an antibody introduced with M428L and N434S mutations, M252Y, S254T and T256E mutations, T25Q and M428L mutations, which are reported to extend antibody half-life in the Fc region.
  • Antibodies are known to have binding properties conferred by their variable regions (especially CDRs), and it is widely known to those skilled in the art that even antibody fragments that are not complete antibodies can utilize these binding properties. It is as used herein, the term "antigen-binding fragment” refers to a protein or peptide containing a portion (partial fragment) of an antibody and retaining the action (antigen-binding) of the antibody on the antigen. .
  • antigen-binding fragments include F(ab′) 2 , Fab′, Fab, Fab 3 , single-chain Fv (hereinafter referred to as “scFv”), (tandem) bispecific single-chain Fv ( sc(Fv) 2 ), single-chain triple body, nanobody, divalent VHH, pentavalent VHH, minibody, (double-stranded) diabodies, tandem diabodies, bispecific tribodies, bispecific bibodies, dual affinity targeting molecules (DART), triabodies (or tribodies), tetrabodies (or [sc(Fv) 2 ] 2 ), or (scFv-SA) 4 ) disulfide-bonded Fv (hereinafter referred to as “dsFv”), compact IgG, heavy chain antibodies, or polymers thereof (Nature Biotechnology, 29(1): 5-6 (2011); Maneesh Jain et al., TRENDS in Biotechnology, 25(7) (2007): 307
  • antigen-binding fragments may be monospecific, bispecific (bispecific), trispecific (trispecific), and multispecific (multispecific).
  • antibody is also intended to include antigen-binding fragments of antibodies, unless otherwise construed as such.
  • the invention has at least one of the heavy chain CDRs and light chain CDRs described below (preferably CDRH3, more preferably a combination of all six), and SARS-CoV-2 An antibody that specifically binds to spike and/or RBD (preferably SARS-CoV-2 RBD).
  • the present invention is an antibody having a combination of heavy chain variable regions and light chain variable regions described below.
  • the present invention provides an antibody having 80% or more identity with any clone in Table 2, that is, 80% or more with the heavy chain variable region (VH) amino acid sequence described for any clone in Table 2 and an amino acid sequence VL having 80% or more identity with the light chain variable region (VL) amino acid sequence listed in Table 2 for the clone, or an antigen thereof Regarding binding fragments.
  • Amino acid sequence identity means the ratio (%) of the number of amino acids of the same type in the range of amino acid sequences to be compared between two proteins, for example, using known programs such as BLAST and FASTA can decide.
  • the identity of the above VH and VL with the sequences listed in Table 2 is 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more. good too.
  • Antibodies with 80% or more identity to any clone in Table 2 preferably have at least one CDR (preferably CDRH3, more preferably all six) of the antibody indicated by the same clone number in Table 1. CDRs). Antibodies with 80% or more identity to any of the clones in Table 2 also preferably bind specifically to the SARS-CoV-2 spike and RBD.
  • an antibody of the invention can be an antibody having a heavy chain with the following heavy chain (HC) amino acid sequence and a light chain with the light chain (LC) amino acid sequence:
  • HC heavy chain
  • LC light chain
  • underlining indicates the signal sequence. Therefore, an antibody as a mature protein may not have the underlined amino acid sequences.
  • amino acids are represented by single-letter codes. Specifically, A is alanine, L is leucine, R is arginine, K is lysine, N is asparagine, M is methionine, D is aspartic acid, F is phenylalanine, C is cysteine, P is proline, Q is glutamine, S is serine, E is glutamic acid, T is threonine, G is glycine, W is tryptophan, H is histidine, Y is tyrosine, I isoleucine and V is valine.
  • "XNY" (X and Y are amino acid one-letter codes; N is a natural number) indicates that amino acid X is substituted with amino acid Y at the N-position.
  • the present invention relates to an antibody that competes with the antibody or antigen-binding fragment thereof for binding to the SARS-CoV2 spike protein or RBD.
  • Whether the test antibody competes with the antibody or antigen-binding fragment thereof for binding to the SARS-CoV2 spike protein or RBD is determined using the labeled antibody or antigen-binding fragment thereof. It can be determined by contacting with an antigen (SARS-CoV2 spike protein or RBD, the same applies hereinafter in this paragraph) immobilized together.
  • an antigen SARS-CoV2 spike protein or RBD, the same applies hereinafter in this paragraph
  • the test antibody After the test antibody is brought into contact with the antigen, it is washed if necessary, and the labeled amount of the labeled antibody or antigen-binding fragment thereof bound to the antigen is compared between the control and the presence of the test antibody.
  • the amount of binding of the labeled antibody or antigen-binding fragment thereof to the antigen when contacted with the antigen in the presence of the test antibody is the same as that of the labeled antibody or antigen-binding fragment thereof in the control. If less than the binding amount, the test antibody can be determined to compete with the labeled antibody or antigen-binding fragment thereof used in the experiment.
  • Antigen binding tests can be performed according to the above-mentioned methods for measuring binding to RBD, etc. as appropriate.
  • measurement of binding between antibody and antigen can be performed by EIA method, ELISA method, FACS method, co-immunoprecipitation method, pull-down assay method, Far-Western blotting method, homobifunctional crosslinker or heterobifunctional crosslinker cross-linking method, label transfer reaction method, interaction mapping method, surface plasmon resonance method, FRET (Fluorescence resonance energy transfer) method, BIACORE method, AlphaScreen (registered trademark) and AlphaPPI assay such as AlphaLISA (registered trademark) ( A variety of methods are well known in the art, such as PerkinElmer), and any suitable method can be employed.
  • the present invention relates to an antibody that recognizes the same epitope as the antibody or antigen-binding fragment thereof.
  • an antibody that recognizes the same epitope as 28K antibody has at least one selected from these as epitopes (e.g., 1, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 ) is an antibody that recognizes the amino acid residues of
  • the epitope of the antibody that recognizes the same epitope as the 28K antibody is a portion selected from RBD A475, G476, S477, and T478 (e.g., one or more, two or more, or three or more) or all.
  • antibodies that recognize the same epitope as the 28K antibody bind by interacting with the backbone of RBD A475, G476, S477, and T478.
  • an antibody that recognizes the same epitope as the 28K antibody has at least one amino acid residue selected from S55, N57, D104, C105, S106, and D107 of its VH, A475, G476, S477, and T478 of RBD. , F486, N487, Y489 and Q493.
  • at least one amino acid residue selected from Y33, Y92 and W97 of VL may be involved in binding to the amino acid residue of F486 of RBD.
  • nucleic acid molecule in another aspect, relates to a nucleic acid molecule comprising a polynucleotide encoding an antibody or immunoreactive fragment thereof of the invention as described above.
  • the invention is a nucleic acid molecule having the nucleic acid sequences of the heavy and light chain variable regions set forth in Table 2, supra.
  • the present invention provides SEQ ID NOs: , 443, 465, 487, 509, 531, 553, 575, or 597, or/and SEQ ID NOS: 14, 36, 58, 80, respectively.
  • a nucleic acid molecule comprising DNA encoding a light chain having the nucleic acid sequence described.
  • the present invention encompasses a vector comprising the nucleic acid molecule.
  • a vector is not particularly limited as long as it can be used for antibody expression, and an appropriate viral vector, plasmid vector, or the like can be selected according to the host to be used.
  • the invention relates to a host cell containing said vector.
  • Host cells are not particularly limited as long as they are host cells that can be used to express antibodies, and mammalian cells (mouse cells, rat cells, hamster cells, rabbit cells, human cells, etc.), yeast, microorganisms (E. coli etc.) can be mentioned.
  • the nucleic acid of the present invention is cloned from the antibody-producing hybridoma obtained above, or by designing a nucleic acid sequence as appropriate based on the amino acid sequence of the antibody or immunoreactive fragment thereof obtained above.
  • the vector of the present invention can be obtained by appropriately integrating the obtained nucleic acid into a vector suitable for expression.
  • the vector of the present invention may contain regions necessary for expression (promoter, enhancer, terminator, etc.) in addition to the nucleic acid of the present invention.
  • the host cell of the present invention can be obtained by introducing the vector of the present invention into an appropriate cell strain (eg, animal cells, insect cells, plant cells, yeast, microorganisms such as E. coli).
  • Antibodies of the present invention can be designed and produced as appropriate based on the sequences described herein.
  • an antibody having a variable region (VR) as described herein can be produced by appropriately introducing a nucleic acid sequence encoding the antibody into a production cell.
  • a nucleic acid sequence encoding a heavy chain and a light chain having the sequence is designed, and after preparing the nucleic acid sequence based on the previous report, production of CHO cells etc. After introduction into cells, clones that bind to the SARS-CoV-2 spike can be selected.
  • Antibodies of the present invention can be obtained by culturing selected cells.
  • the obtained antibody can be purified to homogeneity. Separation and purification of antibodies can be carried out using methods commonly used for separation and purification of proteins. For example, antibodies can be separated and purified by appropriately selecting and combining column chromatography such as affinity chromatography, filter, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and the like. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Columns used for affinity chromatography include, for example, protein A columns and protein G columns. Furthermore, SARS-CoV-2-immobilized columns, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, etc. can also be used regardless of the antibody class. Chromatography can also be in mixed mode.
  • the present invention relates to a pharmaceutical composition containing the above-described antibody or immunoreactive fragment thereof of the present invention as an active ingredient.
  • the antibody or immunoreactive fragment thereof of the present invention can be purified, if necessary, and formulated into a pharmaceutical composition according to a conventional method.
  • the target disease of the pharmaceutical composition of the present invention is a coronavirus infection, for example, a coronavirus, in particular, a viral disease caused by SARS-CoV, MERS-CoV, or SARS-CoV-2, or caused by it It is a disease that Therefore, the pharmaceutical composition of the present invention can be used as a prophylactic agent, therapeutic agent, inhibitor of progression, or ameliorative agent for these diseases or disorders.
  • the invention relates to the use of an antibody or immunoreactive fragment thereof of the invention for the manufacture of said pharmaceutical composition.
  • the invention includes the use of the antibodies of the invention, or immunoreactive fragments thereof, for the treatment or prevention of coronavirus infection.
  • the present invention relates to methods of treating or preventing coronavirus infection comprising administering an antibody or immunoreactive fragment thereof of the present invention.
  • a coronavirus infection is preferably a human coronavirus infection.
  • compositions for parenteral administration include, for example, injections, nasal drops, inhalants, eye drops and the like. An injection is preferred. Dosage forms of the pharmaceutical composition of the present invention include, for example, liquid formulations and freeze-dried formulations.
  • solubilizers such as propylene glycol and ethylenediamine
  • buffering agents such as phosphate, tonicity agents such as sodium chloride and glycerin
  • sulfites such as sodium chloride and glycerin
  • Additives such as stabilizers such as phenol, preservatives such as phenol, and soothing agents such as lidocaine (see “Pharmaceutical excipient dictionary” Yakuji Nippo, “Handbook of Pharmaceutical Excipients Fifth Edition” APhA Publications) .
  • storage containers include ampoules, vials, prefilled syringes, pen-type syringe cartridges, and drip bags.
  • the pharmaceutical composition (therapeutic drug or prophylactic drug) of the present invention when used as an injection, it includes dosage forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection. do.
  • Such injections can be prepared according to known methods, for example, by dissolving, suspending, or emulsifying the above-mentioned antibody in a sterile aqueous or oily liquid commonly used for injections.
  • Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose, sucrose, mannitol, and other adjuvants.
  • polyalcohols eg, propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80, polysorbate 20, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)
  • Sesame oil, soybean oil, and the like can be used as the oily liquid
  • benzyl benzoate, benzyl alcohol, and the like can be added as solubilizers.
  • the prepared injection solution is usually filled into a suitable ampoule, vial or syringe.
  • the antibody of the present invention or an immunoreactive fragment thereof may be added to a suitable excipient to prepare a lyophilized formulation, and dissolved in water for injection, physiological saline, etc. at the time of use to prepare an injection solution.
  • a suitable excipient to prepare a lyophilized formulation, and dissolved in water for injection, physiological saline, etc. at the time of use to prepare an injection solution.
  • oral administration of proteins such as antibodies is considered difficult because they are degraded in the digestive system, but oral administration is also possible with antibody fragments, modified antibody fragments, and ingenuity in dosage forms.
  • Oral preparations include, for example, capsules, tablets, syrups, granules and the like.
  • the pharmaceutical composition of the present invention is preferably prepared in a dosage unit form suitable for the dosage of the active ingredient.
  • dosage unit dosage forms include injections (ampoules, vials, prefilled syringes), and usually 5 to 500 mg, 5 to 100 mg, and 10 to 250 mg of the antibody of the present invention or its immunological agent per dosage unit dosage form. It may contain a reactive fragment.
  • the administration of the antibody or pharmaceutical composition (therapeutic or prophylactic) of the present invention may be local or systemic.
  • the administration method is not particularly limited, and is administered parenterally or orally as described above.
  • Parenteral routes of administration include intraocular, subcutaneous, intraperitoneal, blood (intravenous or intraarterial) injection or infusion into spinal fluid, preferably intraocular or blood administration.
  • the pharmaceutical composition (therapeutic or prophylactic) of the present invention may be administered temporarily, continuously or intermittently. For example, administration can be sustained for 1 minute to 2 weeks.
  • the antibody of the present invention may be administered alone or in combination with other drugs.
  • the dosage of the pharmaceutical composition of the present invention is not particularly limited as long as the desired therapeutic or preventive effect can be obtained, and can be appropriately determined according to symptoms, sex, age, and the like.
  • the dosage of the pharmaceutical composition of the present invention can be determined, for example, using the therapeutic effect or preventive effect of diseases or disorders in which angiogenesis contributes to the onset or exacerbation of angiogenesis.
  • the active ingredient of the pharmaceutical composition of the present invention is usually added at a dose of 0.2 to 0.5 g/day.
  • 01 to 20 mg/kg body weight preferably 0.1 to 10 mg/kg body weight, more preferably 0.1 to 5 mg/kg body weight, about 1 to 10 times a month, preferably 1 to 5 times a month
  • the amount according to this can be administered. If the symptoms are particularly severe, the dose or frequency of administration may be increased according to the symptoms.
  • Diagnostic/Detective Composition Diagnosis of SARS-CoV2 infection and detection of SARS-CoV2 can be performed by ELISA, radioimmunoassay, immunochromatography, immunohistochemistry using the antibody or antigen-binding fragment thereof. An antibody or immunoreactive fragment thereof may optionally be labeled. Attachment of the title can be carried out by methods common in the art. For example, in the case of fluorescent labeling, after washing the protein or peptide with a phosphate buffer, a dye adjusted with DMSO, a buffer, etc. is added, mixed, and allowed to stand at room temperature for 10 minutes for binding.
  • labeling kits include a biotin labeling kit (Biotin Labeling Kit-NH2, Biotin Labeling Kit-SH: Dojindo Laboratories), an alkaline phosphatase labeling kit (Alkaline Phosphatase Labeling Kit-NH2, Alkaline Phosphatase Labeling Kit- SH: Dojindo Laboratories), Peroxidase Labeling Kit-NH 2 , Peroxidase Labeling Kit-NH 2 : Dojindo Laboratories), Phycobiliprotein Labeling Kit (Allophycocyanin Labeling Kit-NH 2 , Allophycocyanin Labeling Kit-SH, B-Phycoerythrin Labeling Kit-NH 2 , B-Phycoerythrin Labeling Kit-SH, R-Phycoerythrin Labeling Kit-NH 2 , R-Phycoerythrin Labeling Kit-SH: Dojindo Laboratories), fluorescent labeling kit (Fluorescein Labeling Kit
  • Diagnosis and detection using the diagnostic composition or detection composition can be performed by methods such as ELISA and immunochromatography. Detection and measurement herein may be quantitative, semi-quantitative or qualitative.
  • Samples used in diagnostic/detection methods can be, for example, tissue samples or fluids taken from a subject as a biopsy.
  • the specimen to be used is not particularly limited as long as it is the object of the measurement of the present invention. Fluids, saliva, oral/nasal washes or fractions or treatments thereof may be mentioned.
  • the patient-derived sample used in the method of the present invention may be pretreated prior to the measurement test, or the specimen collected from the patient may be used as it is.
  • the diagnostic method may be a method of providing information for diagnosis as appropriate, or a method of monitoring the state or progression of SARS-CoV2 infection.
  • the kit of the present invention comprises a carrier selected from the group consisting of a solid phase, a hapten, magnetic beads and an insoluble carrier on which a first antibody that specifically binds to SARS-CoV2 is immobilized. It is a measurement kit.
  • the kit of the present invention may also contain an appropriately labeled antibody that binds to SARS-CoV2.
  • Example 1 Acquisition of anti-SARS-CoV-2 spike protein-binding antibody Peripheral blood lymphocytes were isolated from one COVID-19 patient who was strongly positive for anti-SARS-CoV-2 spike protein antibody titer, and ISAAC (Immunospot -array assay on a chip) method was used to attempt to generate human monoclonal antibodies that bind to the SARS-CoV-2 spike protein.
  • ISAAC Immunospot -array assay on a chip
  • the separated peripheral blood lymphocytes were mixed with 5 ⁇ g/ml R-848 (EnzoLifeSciences), 5 ⁇ g/ml anti-human CD40 antibody (R & D), 100 ⁇ g/ml human IL-21 (PEPROTECH), 100 ng/ml Human BAFF (PEPROTECH), 2 ng/ml Human IL-17 (PEPROTECH), 10 ng/ml Human IL-4 (ProSpec), 1000 IU/ml Human IL-2 (ProSpec), 2.5 ⁇ g/ml CpG2006 (5′-TCGTCGTTTTGTCGTTTTGTCGT -3′) (SEQ ID NO: 617), cultured in RPMI medium containing 5 ⁇ 10 ⁇ 5 M mercaptoethanol and 10% FCS at 37° C. for 6 days.
  • CD138 + cells were isolated using anti-human CD138-specific antibody-conjugated microbeads (Miltenyi Biotec) and LS columns (Miltenyi Biotec) according to the manufacturer's instructions. Separated cells were subjected to the ISAAC method.
  • the lymphocyte chip and ISAAC method were performed with reference to the above-mentioned literature. Briefly, the surface of the lymphocyte chip was treated with 10 ⁇ g/ml SARS-CoV-2 spike protein (provided by the National Institute of Infectious Diseases), 10 ⁇ g/ml SARS-CoV-2 N-terminal domain (NTD) protein (ACRO Biosystems) or 10 ⁇ g/ml SARS-CoV-2 RBD (ACRO Biosystems) in phosphate-buffered saline (PBS) and incubated overnight at 4°C to extract SARS-CoV-2 spike protein, NTD , or RBD was immobilized on the surface of the lymphocyte chip. After removing the antigen solution, the surface of the lymphocyte chip was blocked with 0.01% Biolipidure (NOF Corporation) at room temperature for 30 minutes. After that, the surface of the lymphocyte chip was washed with RPMI medium containing 10% FCS.
  • SARS-CoV-2 spike protein provided by the National Institute of Infectious
  • the separated CD138+ cells were arranged on the lymphocyte chip, and the cells remaining outside the well were removed by gentle washing using RPMI medium containing 10% FCS.
  • the cells on the lymphocyte chip were cultured in RPMI medium containing 10% FCS at 37°C for 3 hours.
  • the antibody gene amplification method for the VH and VL fragments from the collected single cells was performed according to the method described in the above-mentioned literature. Briefly, 0.25 pmol each of Ig ⁇ -RT (5′-GCGAGTAGAGGCCTGAGGAC-3′) (SEQ ID NO: 618), Ig ⁇ -RT (5′-ACTGTCTTCTCCACGGTGCTCC-3′) (SEQ ID NO: 618) was obtained by 15 U SuperScriptTM III (Invitrogen).
  • Ig ⁇ -RT (5′-GATGCCAGTTGTTTGGGTGGT-3′) (SEQ ID NO: 620) primer, 1U RNase inhibitor (New England Biolabs), 0.5 mM each dNTP, 5 mM DTT, 0.2% Triton- Reverse transcription was performed at 55° C. for 1 hour using X100 and 1 ⁇ 1st strand cDNA buffer (Invitrogen). 3′-tailing of the cDNA was performed by 20 U terminal deoxynucleotide transferase (Roche) in 4 mM MgCl 2 , 0.5 mM dGTP, 50 mM PK buffer (25 mM K 2 HPO 4 and 25 mM KH 2 PO 4 ).
  • the first PCR was performed with a dC adapter primer (5′-AGCAGTAGCAGCAGTTCGATAACTTCGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCCCCCCCCCCDN-3′) (SEQ ID NO: 621) and ⁇ chain (Ig ⁇ -1st: 5′-CGAGTTCCAAGTCACGGTCA-3′) (SEQ ID NO: 622), ⁇ chain (Ig ⁇ -1st: 5′-CTTCTCCACGGTGCTCCCTTCAT-3′) (SEQ ID NO: 623), and a mixture of the constant region of the kappa chain (Ig ⁇ -1st: 5′-CTCCCAGGTGACGGTGACAT-3′) (SEQ ID NO: 624).
  • a dC adapter primer 5′-AGCAGTAGCAGCAGTTCGATAACTTCGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCCCCCCCCCCCCCCDN-3′
  • ⁇ chain Ig ⁇ -1st: 5′-
  • Amplification of each product was performed using primeSTAR DNA polymerase (TaKaRa) according to the manufacturer's instructions: 30 cycles of denaturation at 94°C for 15 seconds, annealing at 60°C for 5 seconds, and 72 Extension for 1 minute 30 seconds at °C.
  • the resulting PCR products were then diluted 4-fold with water and 2 ⁇ l of each was used for nested PCR.
  • Nested PCR was performed using an adapter primer (5'-AGCAGTAGCAGCAGTTCGATAA-3') (SEQ ID NO: 625) and ⁇ chain (Ig ⁇ -nest: 5'-GCCTTTGACCAAGCAGCCCAA-3') (SEQ ID NO: 626), ⁇ chain (Ig ⁇ -nest: 5'-AGTGTGGCCTTGTTGGCTTG-3') (SEQ ID NO: 627), or the kappa chain constant region (Ig ⁇ -nest: 5'-CGGGAAAGTATTATTCGCCACA-3') (SEQ ID NO: 628) using primeSTAR DNA polymerase with respective nested primers implemented.
  • an adapter primer 5'-AGCAGTAGCAGCAGTTCGATAA-3'
  • ⁇ chain Ig ⁇ -nest: 5'-GCCTTTGACCAAGCAGCCCAA-3'
  • ⁇ chain Ig ⁇ -nest: 5'-AGTGTGGCCTTGTTGGCTTG-3'
  • PCR products were inserted into expression vectors containing the complete constant region cDNAs for human ⁇ , ⁇ , or ⁇ chains.
  • Expi293F® cells (Thermo Fisher Scientific) were then co-transfected with the ⁇ chain encoding the complete antibody molecule and both the ⁇ or ⁇ chain expression vectors using the Expi293 Expression System (Thermo Fisher Scientific).
  • the supernatant of the cultured cells was collected after 7 days, and then the recombinant monoclonal antibody was purified using a protein G column (GE Healthcare).
  • nucleic acid sequences of these heavy chains are SEQ ID NOS: 1, 23, 45, 67, 89, 111, 133, 155, 177, 199, 221, 243, 265, 287, 309, 331, respectively. , 353, 375, 397, 419, 441, 463, 485, 507, 529, 551, 573, and 595.
  • nucleic acid sequences of these light chains are SEQ ID NOs: 12, 34, 56, 78, 100, 122, 144, 166, 188, 210, 232, 254, 276, 298, 320, respectively.
  • amino acid sequences of these heavy chains are SEQ ID NOs: 2, 24, 46, 68, 90, 112, 133, 156, 178, 200, 222, 244, 266, 288, and 310, respectively. , 332, 354, 376, 398, 420, 442, 464, 486, 508, 530, 552, 574, and 596.
  • amino acid sequences of these light chains are SEQ ID NOs: 13, 35, 57, 79, 101, 123, 145, 167, 189, 211, 233, 255, 277, 299, and 321, respectively. , 343, 365, 387, 409, 431, 453, 475, 497, 519, 541, 563, 585, and 607.
  • Example 2 SARS-CoV-2 spike protein binding assay of obtained antibodies , 1028-61L, 1028-62L, 1028-65K, 1028-68L, 1028-84K, 1028-87L, 1028-106K, 1028-121K, 1028-137K, 1028-164L for SARS-CoV-2 spike protein and It was analyzed by ELISA for binding. Specifically, PBS containing 1 ⁇ g / ml SARS-CoV-2 spike protein is incubated overnight at 4 ° C. in Maxisorp 96 well plate (Nunc) to solidify the SARS-CoV-2 spike protein on the bottom of the well.
  • the cells were blocked with PBS containing 3% bovine serum albumin (BSA) and 0.1% Tween-20 for 1 hour. Next, it was washed with PBS containing 0.1% Tween-20 and incubated with 200 ng/ml of the prepared antibody for 1 hour. Then, the cells were washed with PBS containing 0.1% Tween-20 and incubated with 200 ng/ml peroxidase-labeled anti-human IgG-Fc F(ab)' 2 fragment (sigma) for 1 hour. Finally, the cells were washed with PBS containing 0.1% Tween-20, developed with TMB substrate (SeraCare Life Sciences), and absorbance at 450 nm was measured using a plate reader FLUOstar OMEGA (BMG LABTECH).
  • BSA bovine serum albumin
  • Example 1 Of the antibodies produced in Example 1, the results of 17 types of antibodies are shown in Figure 1 (ECD trimer). Moreover, all of the 28 types of antibodies produced in Example 1 were confirmed to bind to the SARS-CoV-2 spike protein.
  • Example 3 Binding domain analysis of anti-SARS-CoV-2 spike protein antibody Which domains in the SARS-CoV-2 spike protein do the 17 types of antibodies bound to the spike protein in Example 2 bind to? Analyzed by ELISA. Specifically, PBS containing 1 ⁇ g / ml SARS-CoV Receptor binding domain (RBD) protein (ACRO Biosystems), PBS containing 1 ⁇ g / ml SARS-CoV-2 N-terminal domain (NTD) protein (ACRO Biosystems), and 1 ⁇ g PBS containing /ml SARS-CoV-2 RBD (ACRO Biosystems) was incubated overnight at 4 ° C.
  • RBD SARS-CoV Receptor binding domain
  • NTD N-terminal domain
  • 1028-25K, 1028-30K, 1028-50K, 1028-65K, 1028-68L, 1028-84K, 1028-87L bind to SARS-CoV-2 spike protein, but SARS-CoVRBD, SARS-CoV -2 It did not bind to either NTD or SARS-CoV-2 RBD.
  • ACE2 binding inhibition by anti-SARS-CoV-2 RBD antibodies 1028-55K, 1028-61L, 1028-121K) inhibited the binding of SARS-CoV-2 spike protein to ACE2.
  • PBS containing 1 ⁇ g/ml SARS-CoV-2 spike protein ACRO Biosystems was incubated overnight at 4°C in a Maxisorp 96 well plate to solidify the SARS-CoV-2 spike protein on the bottom of the well. did.
  • the cells were washed with PBS containing 0.1% Tween-20, developed using a TMB substrate, and absorbance at 450 nm was measured using a plate reader. Taking the absorbance without antibody as 100, the absorbance with each concentration of antibody was calculated.
  • the cells were blocked with PBS containing 3% BSA and 0.1% Tween-20 for 1 hour. Next, the cells were washed with PBS containing 0.1% Tween-20, and 200 ng/ml of prepared antibody was incubated for 1 hour. After washing with PBS containing 0.1% Tween-20, 200 ng/ml peroxidase-labeled anti-human IgG-Fc F(ab)' 2 fragment was incubated for 1 hour. Finally, the cells were washed with PBS containing 0.1% Tween-20, developed with a TMB substrate, and absorbance at 450 nm was measured using a plate reader. Taking the absorbance obtained by binding to WT as 100, the absorbance obtained by binding to each mutant-derived RBD was calculated.
  • Binding to RBD K417N, E484K, N501Y was reduced to approximately 33 compared to binding to 1028-61L, WT. No reduction in binding was observed for RBD N439K, RBD N440K, RBD A475V, RBD E484K, RBD N501Y. 1028-121K decreased binding to RBD K417N, E484K, N501Y, RBD N439K, RBD N440K, RBD E484K to approximately 62-72 compared to binding to WT. No reduction in binding to RBD A475V, RBD N501Y was observed.
  • 0210-20L did not decrease the binding to any of the mutant-derived RBDs.
  • 0210-28K did not show decreased binding to any of the mutant-derived RBDs.
  • the binding to RBD K417N, E484K, and N501Y decreased to approximately 56 compared to the binding to WT.
  • Binding to RBD E484K decreased to approximately 75.
  • No decrease in binding to RBD N439K, RBD N440K, RBD A475V, and RBD N501Y was observed.
  • 0210-38L reduced the binding to RBD N439K and RBD E484K to approximately 82-90 compared to the binding to WT. No decrease in binding to RBD K417N, E484K, N501Y, RBD N440K, RBD A475V and RBD N501Y was observed.
  • FIG. 10 shows the results of analyzing the binding of the 0210-28K antibody to RBD L452R.
  • the 0210-28K antibody showed binding activity to RBD L452R equivalent to binding to Wuhan-type RBD and RBD K417N, E484K, and N501Y.
  • FIG. 11 shows the results of analyzing the binding of the 0210-28K antibody to RBD L452R and E484Q.
  • the 0210-28K antibody showed binding activity to RBD L452R and E484Q equivalent to binding to Wuhan type RBD, RBD N501Y, and RBD K417N, E484K and N501Y.
  • Example 6 Inhibition of ACE2 binding by anti-SARS-CoV-2 RBD antibodies to mutant antigen-derived RBD Analysis of whether five types of anti-SARS-CoV-2 RBD antibodies inhibit binding between mutant-derived RBD and ACE2 did.
  • PBS containing 1 ⁇ g/ml SARS-CoV-2 RBD WT
  • PBS containing 1 ⁇ g/ml RBD K417N, E484K, N501Y PBS containing 1 ⁇ g/ml RBD N439K
  • PBS containing 1 ⁇ g/ml RBD N440K PBS with 1 ⁇ g/ml SARS-CoV-2 RBD A475V
  • PBS with 1 ⁇ g/ml RBD E484K PBS with 1 ⁇ g/ml RBD N501Y
  • PBS containing 1 ⁇ g/ml SARS-CoV-2 RBD K417T, E484K, N501Y or PBS containing 1 ⁇ g/ml SARS-CoV-2 RBD L452R, T478
  • the cells were washed with PBS containing 0.1% Tween-20, developed using a TMB substrate, and absorbance at 450 nm was measured using a plate reader. Taking the absorbance without antibody as 100, the absorbance with each concentration of antibody was calculated.
  • Beta strain SARS-CoV-2 RBD protein (K417N, E484K, N501Y mutations). It was analyzed whether it inhibits the binding of ACE2 to ACE2. In addition, whether the 0210-28K antibody inhibits the binding of RBD L452R, RBD N501Y, RBD K417T, E484K, N501Y, RBD L452R, T478K, RBD L452R, E484Q, or RBD K417N, E484K, N501Y to ACE2 was examined in a similar manner. analyzed by the method.
  • Table 5 shows the results of 1028-05K, 1028-37K, 1028-55K, 1028-61L, and 1028-121K.
  • 1028-05K hardly suppressed the binding of WT and ACE2 to about 90, and the binding of RBD derived from each mutant to ACE2 was about 90 to 96, which was hardly suppressed like WT.
  • 1028-37K suppressed the binding between WT and ACE2 to about 63, whereas the binding between RBD derived from any of the mutant strains and ACE2 was suppressed to about 53-70, which is the same level as WT.
  • 1028-55K suppresses the coupling between WT and ACE2 to about 22, while RBD N439K, RBD N440K, RBD A475V, RBD E484, and RBD N501Y reduce the coupling between WT and ACE2 to about 25-35. reduced to the same extent.
  • the binding between RBD K417N, E484K, N501Y and ACE2 was about 90, which was hardly suppressed.
  • 1028-61L suppresses the coupling between WT and ACE2 to about 20, while RBD N439K, RBD N440K, RBD A475V, RBD E484, and RBD N501Y suppress the coupling between WT and ACE2 to about 18-33. reduced to the same extent.
  • the binding between RBD K417N, E484K, N501Y and ACE2 was about 78, which was hardly suppressed.
  • the binding between WT and ACE2 was hardly suppressed at about 80, and the binding between RBD derived from each mutant strain and ACE2 was also hardly suppressed at about 80-90, similar to WT.
  • Figures 7A and B show the results of inhibition of binding of 0210-28K, 1028-55K, 0210-30L, and 1028-61L to the RBD proteins (RBD K417N, E484K, N501Y) of Beta strain SARS-CoV-2.
  • the IC50 for the RBD protein of 0210-28K Beta strain SARS-CoV-2 was calculated to be 350 ng/ml
  • the IC50 for the RBD protein of Wuhan type SARS-CoV-2 was calculated to be 600 ng/ml.
  • the IC50 for the RBD protein of 1028-55K Beta strain SARS-CoV-2 was calculated to be 10,000 ng/ml or more, and the IC50 for the RBD protein of Wuhan-type SARS-CoV-2 was calculated to be 400 ng/ml. .
  • 0210-30L dose-dependently inhibited the binding of ACE2 to the RBD protein of Beta strain SARS-CoV-2. Its inhibitory effect was weaker than that of the Wuhan-type SARS-CoV-2 RBD protein.
  • the IC50 for the RBD protein of 0210-30L Beta strain SARS-CoV-2 was calculated to be 2,000 ng/ml
  • the IC50 for the RBD protein of Wuhan type SARS-CoV-2 was calculated to be 500 ng/ml.
  • 1028-61L dose-dependently inhibited the binding of ACE2 to the RBD protein of Beta strain SARS-CoV-2. Its inhibitory effect was weaker than that of the Wuhan-type SARS-CoV-2 RBD protein.
  • the IC50 for the RBD protein of 1028-61L Beta strain SARS-CoV-2 was calculated to be 10,000 ng/ml or more, and the IC50 for the RBD protein of Wuhan type SARS-CoV-2 was calculated to be 700 ng/ml. .
  • Fig. 10 shows the results of analyzing whether 0210-28K inhibits the binding of RBD L452R and ACE2.
  • the 0210-28K antibody showed concentration-dependent binding inhibitory activity to RBD L452R equivalent to that of the Wuhan type.
  • Fig. 11 shows the results of analyzing whether the 0210-28K antibody inhibits the binding of RBD K417N, E484K, N501Y, and RBD L452R, E484Q to ACE2.
  • the 0210-28K antibody showed concentration-dependent binding inhibitory activity equivalent to that of the Wuhan type against RBD K417N, E484K, N501Y and RBD L452R, E484Q.
  • FIG. 13 shows the binding of RBD derived from six types of mutants (RBD 501Y, RBD K417N, E484K, N501Y, RBD K417T, E484K, N501Y, RBD L452R, E484Q, RBD L452R, T478K, RBD L452R) to ACE2.
  • RBD 501Y, RBD K417N, E484K, N501Y, RBD K417T, E484K, N501Y, RBD L452R, E484Q, RBD L452R, T478K, RBD L452R concentration-dependent binding inhibitory activity equivalent to that of Wuhan type (WT) against RBD derived from any of the mutant strains.
  • Example 7 Infection suppression experiment using a pseudotype virus enveloped with the spike protein of the new coronavirus
  • the pseudotype virus (SARS-CoV-2 St19pv) enveloped with the spike protein of the new coronavirus is published in the following paper. It was prepared according to the method described. Tani H et al. , Virology Journal (2021) 18:16.
  • VeroE6 cells overexpressing TMPRSS2 (VeroE6/TMPRSS2) were seeded in a 96-well plate, and the next day, when the cells were 90-100% confluent, each concentration of purified antibody was added. Subsequently, SARS-CoV-2 St19pv and VSV-G-encapsulated control pseudotype virus (VSVpv) were subsequently added. After culturing for one day, a luciferase substrate solution containing a cell lysate was added to the cells, and luciferase activity was measured. If the pseudotype virus can infect cells, luciferase activity is observed and quantified as a luminescence value. Since the luminescence value of luciferase would decrease if the infection was inhibited by the antibody, the infection inhibition rate was calculated based on that value.
  • VSVpv VSV-G-encapsulated control pseudotype virus
  • Example 8 Infection control experiment using new coronavirus
  • the new coronavirus uses a virus strain (TIH/SARS2/2020-1) isolated at the Toyama Prefectural Institute of Health from a patient with a new coronavirus infection in Toyama Prefecture. board.
  • Purified antibodies and infectious titers (Infectious Unit; IU) was mixed with 100 IU of virus in a microtube and allowed to react at 37° C. for 1 hour. After that, it was added to VeroE6/TMPRSS2 cells seeded in a 24-well plate and adsorbed at 37° C. for 2 hours.
  • the mixture containing the unadsorbed antibody and virus was completely removed, and the culture medium containing the antibody at each concentration was added again, and culture was continued for 2 days.
  • the culture supernatant of the cells cultured for 2 days was collected, and the virus infectivity titer was measured by plaque assay when each antibody concentration was added.
  • Figure 5 shows the 50% and 90% inhibitory concentrations calculated by statistical analysis based on the numerical values in Figure 4.
  • 1028-61L showed an IC50 of 157 nM and an IC90 of 159 nM
  • 1028-55K showed an IC50 of 102 nM and an IC90 of 106 nM.
  • Example 9 Infection suppression experiment using a pseudotype virus enveloped with a mutant spike protein of the new coronavirus
  • spike proteins with mutations of Wuhan type (WT) WT
  • Alpha strain and Beta strain
  • SARS-CoV-2 St19pv A pseudotyped virus (SARS-CoV-2 St19pv) enveloping was generated (see FIG. 8).
  • Alpha strains are B.
  • Fig. 9 shows the results of infection inhibition experiments conducted using the pseudotype virus of each mutant strain.
  • Each of the four tested antibodies showed concentration-dependent inhibitory effects, with 0210-28K exhibiting the highest effect, and sufficient inhibition of infection was observed even at 0.2 ⁇ g/ml.
  • these antibodies were found to have an inhibitory effect on not only the Wuhan type, but also the Alpha and Beta strains.
  • Pseudotype viruses enveloped with spike proteins with mutations of the Kappa and Delta strains were similarly prepared, and the results of two rounds of infection inhibition experiments on 0210-28K were performed using the pseudotype viruses of each mutant strain. 12. Concentration-dependent inhibitory effects were observed for each of the five pseudotype viruses.
  • the IC50 values for inhibition of Wuhan-type and mutant SARS-CoV-2 St19pv pseudotype virus infection were respectively as follows.
  • pseudotype viruses enveloped with spike proteins having mutations of the Gamma strain and the Omicron strain were prepared, and seven types of pseudotype viruses (Wuhan type (WT), Alpha strain, Beta strain, Gamma, Kappa, Delta, and Omicron strains) were used to perform infection inhibition experiments for 0210-28K.
  • VeroE6/TMPRSS2 was seeded on a 96-well plate, the next day, 0210-28K at each concentration was added in a state of 90-100% confluency, and then the above-mentioned 7 types of pseudotype viruses or VSV-G were coated.
  • a pseudotyped virus (VSVpv) was added. After culturing at 37°C for 24 hours, luciferase activity was measured using a PicaGene Luminescence Kit (Fujifilm Wako) equipped with a GloMax Navigator Microplate Luminometer (Promega).
  • the results are shown in Fig. 14.
  • the vertical axis indicates the rate of infection reduction (% of reduction) compared to the control.
  • Example 10 Infection suppression experiment using a mutated new coronavirus
  • the new coronavirus is the Wuhan type (WK-521/2020) and Alpha strain (QHN002/2020) isolated at the National Institute of Infectious Diseases in Japan.
  • Beta strain TY8-612/2021
  • Gamma strain TY7-503/2021
  • Kappa strain TY11-330/2021
  • Dleta strain TY11-927/2021
  • Omicron strain TY38-873.
  • VeroE6/TMPRSS2 cells were seeded in a 96-well plate at 2 ⁇ 10 4 cells per well, infected with the above viruses at 0.001 moi per cell, and 0210-28K antibody adjusted to each concentration was added.
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • Example 11 Surface Plasmon Resonance (SPR) Analysis To confirm, the affinity of anti-SARS-CoV-2 RBD antibodies to Wuhan-type RBD or mutant-derived RBD was measured using a Biacore T100 (GE Healthcare). were analyzed by SPR.
  • SPR Surface Plasmon Resonance
  • the 0210-28K antibody was immobilized on a protein A sensor chip (GE Healthcare), and each RBD in the range of 0 nM to 30 nM (Wuhan type RBD (WT), RBD derived from Alpha strain (N501Y), Beta strain RBD derived (K417N, E484K, N501Y), RBD derived from Gamma strain (K417T, E484K, N501Y), RBD derived from Kappa strain (L452R, E484Q), and RBD derived from Delta strain (L452R, T478K)) at 30 ⁇ l/ Injection was performed for 1 minute at a flow rate of min, followed by washing with HBS-P buffer for 5 minutes. Biacore Evaluation Software (GE Healthcare) was used to determine the association and dissociation rate constants, and the affinity was calculated from the ratio of the two constants.
  • Example 12 Infection suppression experiment by preventive administration using a hamster model, it was tested whether preventive administration of antibodies inhibits infection by the novel coronavirus and its pseudotype virus enveloped with its mutant spike protein. . Specifically, 0.3 mg/kg per animal for male hamsters aged 6 to 10 weeks (hamsters purchased from Japan SLC Co., Ltd. and bred in a pathogen-free environment within the Department of Animal Resources Development, University of Toyama). Alternatively, 3 mg/kg of an antibody recognizing West Nile virus (Ozawa et al., Antiviral Research 2018, 154, 58) or 0210-28K antibody was administered intraperitoneally.
  • a control antibody that recognizes West Nile virus was used as an antibody that does not recognize SARS-CoV-2. Twenty-four hours after the intraperitoneal administration, each of the following pseudotype viruses was directly inoculated into the trachea by the method described below. Lungs were harvested from hamsters 24 hours after infection and incubated with 1 mg/mL D-luciferin for 5 minutes. Luminescence of luciferase was measured with an In vivo Imaging System (IVIS Lumina II, PerkinElmer), and luminescence from the front and back of the lung was measured to calculate the virus infectivity titer of the whole lung.
  • IVIS Lumina II In vivo Imaging System
  • Pseudotype virus Spike proteins with mutations of Wuhan type (WT), Alpha strain, Beta strain, Gamma strain, Kappa strain, Delta strain, and Omicron strain, prepared in the same manner as in Examples 7 and 9, and VSV- Pseudotype virus that coats G Inoculation method: see bioRxiv doi (https://doi.org/10.1101/2021.09.17.460745)
  • FIG. 17 shows the results when the antibody dose was 3 mg/kg hamster for Beta strain and Omicron strain, and 0.3 mg/kg hamster for others.
  • the results showed that luciferase luminescence was clearly lower when the 0210-28K antibody was pre-administered compared to the control. From this, it was clarified that the prophylactic administration of the antibody can inhibit infection by the new coronavirus and its pseudotype virus enveloping its mutant spike protein.
  • Example 13 Structural Analysis of Antibody Bound to SARS-CoV-2 Spike Protein
  • cryo-electron microscopy and X-ray crystallography were used. Analytical method was used.
  • the DNA fragment of the 0210-28K VH gene was subjected to expression composed of elements starting from the N-terminus (human IgG CH1 constant region, flexible linker GSSG, decahistidine His10 tag). inserted into the vector.
  • Expi293F cells were then co-transfected with both the Fab and the light chain expression vector encoding the Fab molecule using the Expi293 Expression System and the cultured cell supernatant was harvested and applied to dialysis membranes (Spectrum Laboratories, Collinso Dominguez). dialyzed using The membrane was immersed in a native buffer (20 mM sodium phosphate, 0.5 M NaCl, pH 7.4) contained in a covered container and shaken at 4°C for 72 hours on a shaker. Fabs were then purified by affinity chromatography using Ni Sepharose 6 Fast Flow (Cytiva).
  • the pretreated supernatant was loaded onto a Poly-Prep column (Bio-Rad, Hercules) filled with Ni Sepharose 6 Fast Flow mix, and after washing, the Fab molecules were eluted with an elution buffer (20 mM sodium phosphate, 0.05%). It was eluted with 5M NaCl, 500 mM imidazole, pH 7.4).
  • the protein of SARS-CoV-2 spike 6P was expressed and purified by a reported procedure (Onodera et al., 2021), and the SARS-CoV-2 spike 6P solution was isolated from 37 After incubation for 1 hour at 18°C, the purified 0210-28K Fab was incubated with SARS-CoV-2 spiked 6P solution at a molar ratio of 1:2 for 1 hour at 18°C.
  • a grid was prepared by placing the sample on a Quantifoil R1.2/1.3 Cu 300 mesh grid (Quantifoil Micro Tools GmbH) that had been glow discharged using a PIB-10 (Vacuum Device) at 10 mA for 120 seconds.
  • FIG. 18(a) is a cryo-electron microscope (cryo-EM) map densified to a resolution of 3.1 ⁇ with C3 symmetry. , was recognized on the left shoulder side of the SARS-CoV-2 RBD shown in the figure.
  • cryo-EM cryo-electron microscope
  • Crystals were cryocooled in liquid nitrogen and X-ray diffraction experiments were performed at beamline BL32XU at SPring-8 (Harima). X-ray diffraction data sets were processed with XDS and scaled using Aimless in the CCP4 program package (see, e.g., Evans, P. Acta Crystallogr D Biol Crystallogr 62, 72-82, doi: 10.1107/S0907444905036693. ). The structure was solved by the molecular replacement method using the Phaser program in the PHENIX package, using the RBD structure (PDB ID: 7jmp) and the 0210-28K Fab fragment generated at the alphafold2 colab website as search probes for molecular replacement.
  • FIG. 18(b) is the result of X-ray crystal structure analysis with a resolution of 3.75 ⁇ , and the 0210-28K Fab (UT28K Fab) was recognized in the same region as the cryo-electron microscope result.
  • the 0210-28K antibody recognized F486 of RBD as a core interacting amino acid residue and interacted with the hydrophobic pocket of the antibody.
  • This binding mode resembled the binding of 253XL55 (PDB ID: 7BEO) and SARS-CoV-2 RBD shown in Figure 18(c).
  • Figures 18(d)-(g) show the key residue interactions between the 0210-28K antibody Fab and the SARS-CoV-2 RBD.
  • the side chain of Q493 of RBD formed hydrogen bonds with S55 and N57 of VH of 0210-28K antibody Fab.
  • the side chain of N487 of RBD not only forms a hydrogen bond with the side chain of D107 of VH of antibody Fab, but also the oxygen atoms of the main chains of C105 and S106 of VH of antibody Fab.
  • the side chain of D107 of the VH of the antibody Fab was hydrogen-bonded to the side chains of S477 and T478 in addition to N487 of RBD, and was also bonded to the nitrogen of these main chains.
  • FIG. 18(f) the side chain of D107 of the VH of the antibody Fab was hydrogen-bonded to the side chains of S477 and T478 in addition to N487 of RBD, and was also bonded to the nitrogen of these main chains.
  • the hydrogen atom in the side chain of D104 of VH of antibody Fab formed a bond with the nitrogen atom of the main chain of S477 of RBD and the oxygen atom of the main chains of A475 and G476 of RBD.
  • N487 of RBD and D104 and D107 of VH of 0210-28K antibody Fab are important residues for antigen-antibody interaction, and they recognize the main chains of interacting molecules. It became clear.
  • the 0210-28K antibody had at least four backbone interactions (RBD A475, G476, S477, and T478) and at least three side chain interactions (RBD F486, N487, and Q493) with RBD.
  • Example 14 Epitope Mapping Analysis The binding of the 0210-28K antibody to the mutant-derived RBD protein was analyzed by ELISA. Specifically 1 ⁇ g/ml SARS-CoV-2 RBD (WT), RBD N439K, RBD N440K, RBD G446V, RBD L452R, RBD Y453F, RBD L455F, RBD F456L, RBD K458R, RBD E471Q, RBD I472V, RBD A472V ,RBD G476S,RBD S477N,RBD P479S,RBD N481D,RBD G482S,RBD V483A,RBD E484K,RBD G485S,RBD F486S,RBD N487R,RBD F490S,RBD S494P,RBD P499R,RBD N501Y,RBD Y505C,RBD N417N,E484K ,
  • the cells were blocked with PBS containing 3% BSA and 0.1% Tween-20 for 1 hour. Then, it was washed with PBS containing 0.1% Tween-20 and incubated with 100 ng/ml of the produced antibody for 1 hour. After washing with PBS containing 0.1% Tween-20, 200 ng/ml peroxidase-labeled anti-human IgG-Fc F(ab)' 2 fragment was incubated for 1 hour. Finally, the cells were washed with PBS containing 0.1% Tween-20, developed with a TMB substrate, and absorbance at 450 nm was measured using a plate reader.
  • the results are shown in FIG.
  • the 0210-28K antibody did not significantly decrease binding to RBD proteins other than F486S and N487R, but decreased binding to F486S and N487R.
  • These results demonstrate that at least the RBD proteins F486 and N487 are essential for the binding of the 0210-28K antibody.
  • Culture supernatants were harvested 72 hours after infection and passaged again with twice the amount of antibody for subsequent passages. After three passages, culture supernatants were harvested and viral RNA was isolated using the QIAamp Viral RNA Mini Kit (Qiagen). Whole genome amplification was performed using a modified version of ARTIC Network's protocol for SARS-CoV-2 genome sequencing (Itokawa et al., 2020). Next-generation sequencing (NGS) libraries were constructed using the QIAseq FX DNA Library kit (Qiagen) and sequenced using the iSeq platform (Illumina). NGS readings were performed using the bwa mem algorithm (version 0.7.13-r1126) (see Li, H. & Durbin, R.
  • NGS Next-generation sequencing
  • N487 and Y489 of RBD support the orientation of F486 of RBD that interacts with the hydrophobic pocket of the 0210-28K antibody by causing amino acid substitutions to escape antibody recognition. It is thought that there are In addition, the E484 amino acid residue of RBD, which reduces the reactivity of neutralizing antibodies against SARS-CoV-2 mutants, is located outside the antibody binding site, and the 0210-28K antibody is independent of the E484 amino acid substitution.
  • Example 15 Evaluation of antibody-dependent enhancement of infection (ADE) activity ADE activity was confirmed for 0210-28K prepared in Example 1 and newly prepared 0210-28K-LALA.
  • the 0210-28K-LALA antibody in which the L234A and L235A mutations are introduced into the 0210-28K Fc region, the 0210-28K variable region and the full-length constant region containing the L234A and L235A mutations are DNA-synthesized into a vector. , and antibodies were produced and purified in the same manner as in Example 1.
  • the vertical axis indicates the virus fold increase, with the virus amount obtained in the control being set to 1.
  • the virus amount was low at 1.0 ⁇ 10 ⁇ 1 ⁇ g/ml or higher, but the virus amount was high at 1.0 ⁇ 10 ⁇ 2 ⁇ g/ml, indicating ADE activity.
  • the virus amount did not increase even at 1.0 ⁇ 10 ⁇ 2 ⁇ g/ml or less, and no ADE activity was observed.
  • Example 16 Evaluation of activity to enhance interleukin-6 (IL-6) production It was also confirmed that the 0210-28K and 0210-28K-LALA described above have activity to enhance IL-6 production. Specific evaluation was entrusted to Mican Technologies Inc., cMylc-2-K alpha cells under the conditions of the absence of antibody (control), the presence of 0210-28K, and the presence of 0210-28K-LALA was infected with the novel coronavirus (WK-521/2020) and cultured for 72 hours, the culture supernatant was collected, and the amount of IL-6 produced was measured by ELISA. The above antibody was used after preparing a 10-fold dilution series from a final concentration of 1.0 ⁇ g/ml.

Abstract

The present invention addresses the problem of providing a novel and effective antibody that binds to the spike protein of SARS-CoV-2, and in particular addresses the problem of providing an antibody that inhibits binding between SARS-CoV-2 and ACE2 or an antibody that inhibits the entry of SARS-CoV-2 into the cell. The present invention provides, for example, an antibody that has the following combination of complementarity-determining regions (CDRs) and that binds to the spike protein of SARS-CoV-2, or an antigen-binding fragment from said antibody: CDRH1, 2, and 3 and CDRL1, 2, and 3 composed of the amino acid sequences of SEQ ID NOS: 9, 10, 11, 20, 21, and 22.

Description

抗ＳＡＲＳ－ＣｏＶ－２抗体Anti-SARS-CoV-2 antibody
　本発明は，新型コロナウイルスＳＡＲＳ－ＣｏＶ－２のスパイクタンパク質に結合する抗体及びその抗原結合性断片に関する。 The present invention relates to an antibody that binds to the spike protein of the novel coronavirus SARS-CoV-2 and an antigen-binding fragment thereof.
　ＳＡＲＳ－ＣｏＶ－２は，２０１９年末に中国の武漢で初めて報告され，その後中国全土，さらには世界中に広く拡大したコロナウイルスの一種である。ＣＯＶＩＤ－１９と呼ばれるＳＡＲＳ－ＣｏＶ－２感染症は，患者のほとんどおいて軽度から中等度の症状である一方で，一部の患者においては重症化して死亡に至るという特徴を有する。現在までに，ワクチンが開発及び承認され，各国で接種が始まっているが，ワクチン接種後の感染事例も報告されており（非特許文献１），ワクチンが完全にウイルス感染を抑制するわけではないと考えられている。 SARS-CoV-2 is a type of coronavirus that was first reported in Wuhan, China at the end of 2019 and has since spread widely throughout China and even around the world. The SARS-CoV-2 infection, called COVID-19, is characterized by mild to moderate symptoms in most patients, while severe disease and death in some patients. To date, vaccines have been developed and approved, and vaccination has begun in various countries, but cases of infection after vaccination have been reported (Non-Patent Document 1), and vaccines do not completely suppress viral infections. It is believed that.
　ワクチンの開発と並行して，ＣＯＶＩＤ－１９治療薬の開発も進められており，治療用抗体についても報告されている（特許文献１）。しかし，ＳＡＲＳ－ＣｏＶ－２には複数の変異株が報告されており，広く変異株にまで治療・予防効果を奏するものは知られていない。 In parallel with the development of vaccines, COVID-19 therapeutic agents are being developed, and therapeutic antibodies have also been reported (Patent Document 1). However, multiple mutant strains of SARS-CoV-2 have been reported, and none of the mutant strains is known to have therapeutic or preventive effects.
国際公開ＷＯ２０２１／０４５８３６号International publication WO2021/045836
　本発明は，ＳＡＲＳ－ＣｏＶ－２に対する抗体（以下，「抗ＳＡＲＳ－ＣｏＶ－２抗体」という）であって，ＳＡＲＳ－ＣｏＶ－２のスパイクタンパク質に結合し，特には，感染・伝播性の増加や抗原性の変化が懸念されるＳＡＲＳ－ＣｏＶ－２の変異株に結合することで，それらのＳＡＲＳ－ＣｏＶ－２とＡＣＥ２との結合を阻害する抗体，又はそれらのＳＡＲＳ－ＣｏＶ－２の細胞内への侵入を阻害する抗体を提供することを課題とする。 The present invention is an antibody against SARS-CoV-2 (hereinafter referred to as "anti-SARS-CoV-2 antibody") that binds to the spike protein of SARS-CoV-2, and in particular, increases infection and transmissibility Antibodies that inhibit the binding of SARS-CoV-2 and ACE2 by binding to SARS-CoV-2 mutants that are concerned about antigenic changes, or those SARS-CoV-2 cells An object of the present invention is to provide an antibody that inhibits internal invasion.
　本発明者らは，ＳＡＲＳ－ＣｏＶ－２感染履歴のある複数の患者から得られた血漿を用いて，血漿中にＳＡＲＳ－ＣｏＶ－２スパイクタンパク（三量体構造全長Ｓタンパク質（ＥＣＤ）），Ｎ末端ドメイン（ＮＴＤ），及び受容体結合ドメイン（ＲＢＤ）への結合能を有する抗体が存在するかを調べた。その結果，重症化後に奇跡的に回復した１名の患者由来の血漿がＥＣＤ，ＮＴＤ，及びＲＢＤの全てに対して優れた結合性を有することを見出した。このＣＯＶＩＤ－１９患者より末梢血リンパ球を分離し，既にＩＳＡＡＣ法（Ｉｍｍｕｎｏｓｐｏｔ－ａｒｒａｙ　ａｓｓａｙ　ｏｎ　ａ　ｃｈｉｐ）（Ｊｉｎ　Ａ　ｅｔ　ａｌ．，Ｎａｔｕｒｅ　ｍｅｄｉｃｉｎｅ（２００９）２６：１０８８－１０９２，及びＪｉｎ　Ａ　ｅｔ　ａｌ．，Ｎａｔｕｒｅ　Ｐｒｏｔｏｃｏｌｓ（２０１１）６：６６８）として報告された手法を用いて，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質に特異的に結合する複数のモノクローナル抗体を精製した。 The present inventors used plasma obtained from multiple patients with a history of SARS-CoV-2 infection, and found that SARS-CoV-2 spike protein (trimeric structure full-length S protein (ECD)), The presence of antibodies capable of binding to the N-terminal domain (NTD) and receptor binding domain (RBD) was investigated. As a result, it was found that plasma derived from one patient who miraculously recovered after becoming severe had excellent binding properties to all of ECD, NTD, and RBD. Peripheral blood lymphocytes were isolated from this COVID-19 patient, and the ISAAC method (Immunospot-array assay on a chip) (Jin A et al., Nature medicine (2009) 26: 1088-1092, and Jin A et al. , Nature Protocols (2011) 6:668) were used to purify multiple monoclonal antibodies that specifically bind to the SARS-CoV-2 spike protein.
　また，本発明者らは，それらのモノクローナル抗体について解析を進めた結果，ＳＡＲＳ－ＣｏＶ－２　ＲＢＤに結合する抗体（以下，「ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体」という）を見出すことに成功した。得られたモノクローナル抗体について，シュードタイプウイルス及びＳＡＲＳ－ＣｏＶ－２による感染抑制実験等を行ったところ，これらの抗体が感染抑制能を有することを見出した。更に，本発明者らは，感染・伝播性の増加や抗原性の変化が懸念されるＳＡＲＳ－ＣｏＶ－２の変異株へのそれらの抗体の効果を調べたところ，そのような変異株のＲＢＤへの結合性を有し，かつ感染抑制効果を有する抗体を見出すことに成功した。 In addition, as a result of analyzing these monoclonal antibodies, the present inventors succeeded in finding an antibody that binds to SARS-CoV-2 RBD (hereinafter referred to as "SARS-CoV-2 RBD antibody"). When the obtained monoclonal antibodies were subjected to infection suppression experiments with pseudotype virus and SARS-CoV-2, it was found that these antibodies have the ability to suppress infection. Furthermore, the present inventors examined the effect of these antibodies on SARS-CoV-2 mutants, which are concerned about increased infection and transmissibility and antigenic changes, and found that such mutants RBD We have succeeded in discovering an antibody that binds to and has an infection-suppressing effect.
　よって，本発明は，抗ＳＡＲＳ－ＣｏＶ－２抗体又はその抗原結合性断片及び当該抗体を有効成分として含有する治療薬，診断薬，及びＳＡＲＳ－ＣｏＶ－２検出用組成物等に関する。 Therefore, the present invention relates to anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof, therapeutic agents, diagnostic agents, compositions for detecting SARS-CoV-2, etc. containing the antibodies as active ingredients.
抗ＳＡＲＳ－ＣｏＶ－２抗体のＳＡＲＳ抗原タンパク質に対する結合特異性を確認した結果を示すグラフである。１０２８－０５Ｋ，１０２８－２５Ｋ，１０２８－２９Ｋ，１０２８－３０Ｋ，１０２８－３７Ｋ，１０２８－５０Ｋ，１０２８－５５Ｋ，１０２８－６１Ｌ，１０２８－６２Ｌ，１０２８－６５Ｋ，１０２８－６８Ｌ，１０２８－８４Ｋ，１０２８－８７Ｌ，１０２８－１０６Ｋ，１０２８－１２１Ｋ，１０２８－１３７Ｋ，および１０２８－１６４Ｌの各抗ＳＡＲＳ－ＣｏＶ－２抗体が，ＳＡＲＳのどの抗原タンパク質と結合するかをＥＬＩＳＡにて検証した。抗原タンパクとして以下を使用した：「ＲＢＤ（ＳＡＲＳ）」：ＳＡＲＳ－ＣｏＶ　Ｒｅｃｅｐｔｏｒ　ｂｉｎｄｉｎｇ　ｄｏｍａｉｎ（ＲＢＤ）タンパク質；「ＥＣＤ　ｔｒｉｍｅｒ」：ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質；「ＮＴＤ」：ＳＡＲＳ－ＣｏＶ－２　Ｎ－ｔｅｒｍｉｎａｌ　ｄｏｍａｉｎ（ＮＴＤ）タンパク質；「ＲＢＤ」：ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ。縦軸は，４５０ｎｍにおける吸収（結合強度）を示す。独立して２回以上の実験を行い，図示しているデータはその代表例を示している。1 is a graph showing the results of confirming the binding specificity of an anti-SARS-CoV-2 antibody to a SARS antigen protein. 1028-05K, 1028-25K, 1028-29K, 1028-30K, 1028-37K, 1028-50K, 1028-55K, 1028-61L, 1028-62L, 1028-65K, 1028-68L, 1028-84K, 1028- It was verified by ELISA which antigen proteins of SARS each of the 87L, 1028-106K, 1028-121K, 1028-137K, and 1028-164L anti-SARS-CoV-2 antibodies binds. The following antigen proteins were used: "RBD (SARS)": SARS-CoV receptor binding domain (RBD) protein; "ECD trimer": SARS-CoV-2 spike protein; "NTD": SARS-CoV-2N- terminal domain (NTD) protein; "RBD": SARS-CoV-2 RBD. The vertical axis indicates absorption (bond strength) at 450 nm. Two or more experiments were performed independently and the data shown are representative. 抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体による，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質とＡＣＥ２との結合阻害実験の結果を示すグラフである。ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質とＡＣＥ２との結合を，１０２８－０５Ｋ，１０２８－３７Ｋ，１０２８－５５Ｋ，１０２８－６１Ｌ，および１０２８－１２１Ｋの各抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体が阻害するか否かを，ＥＬＩＳＡにて検証した。横軸は抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体の濃度（μｇ／ｍｌ），縦軸は抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体非存在下で測定された吸光度の値を１００とした場合の各抗体存在下における吸光度の値の割合（％）を示している。独立して２回以上の実験を行い，図示しているデータはその代表例を示している。Fig. 3 is a graph showing the results of binding inhibition experiments between SARS-CoV-2 spike protein and ACE2 by anti-SARS-CoV-2 RBD antibody. Whether each anti-SARS-CoV-2 RBD antibody of 1028-05K, 1028-37K, 1028-55K, 1028-61L, and 1028-121K inhibits the binding of SARS-CoV-2 spike protein and ACE2 was verified by ELISA. The horizontal axis is the concentration of anti-SARS-CoV-2 RBD antibody (μg/ml), and the vertical axis is the presence of each antibody when the absorbance value measured in the absence of anti-SARS-CoV-2 RBD antibody is set to 100. shows the ratio (%) of absorbance values in . Two or more experiments were performed independently and the data shown are representative. 抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体による，シュードタイプウイルス感染抑制実験の結果を表すグラフである。縦軸は抗体非添加のコントロールと比較した感染低下割合の対数値（ｌｏｇ（％　ｏｆ　ｒｅｄｕｃｔｉｏｎ））を示し，横軸は抗体濃度（μｇ／ｍｌ）を示す。黒バー（Ｓｔ１９ｐｖ）はＳＡＲＳ－ＣｏＶ－２スパイクタンパク質外套シュードウイルスの結果を示し，白色のバーはコントロールとして用いたシュードタイプウイルスＶＳＶｐｖの結果を示す。Fig. 3 is a graph showing the results of an experiment to suppress pseudotype virus infection using an anti-SARS-CoV-2 RBD antibody. The vertical axis shows the logarithmic value of infection reduction ratio (log (% of reduction)) compared to the antibody-free control, and the horizontal axis shows the antibody concentration (μg/ml). Black bars (St19pv) show the results for the SARS-CoV-2 spike protein mantle pseudovirus, and white bars show the results for the pseudotyped virus VSVpv used as a control. 抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体である１０２８－６１Ｌ，１０２８－５５Ｋ，及び１０２８－０５ＫのＳＡＲＳ－ＣｏＶ－２感染及び産生に与える影響を調べた手順の模式図（上図）及びその結果を示すグラフ（下図）である。グラフ中，縦軸は，左グラフではコントロールに対する低下割合の対数値（ｌｏｇ（％　ｏｆ　ｒｅｄｕｃｔｉｏｎ））を示し，右グラフではコントロールに対する低下割合（％　ｏｆ　ｒｅｄｕｃｔｉｏｎ）を示す。横軸は抗体濃度（μｇ／ｍｌ）を示す。＊はウイルスプラークが認められなかったことを示す。各抗体濃度のデータにおいて，左の黒バーは１０２８－６１Ｌ，真ん中のグレーバーは１０２８－５５Ｋ，右の白バーは１０２８－０５Ｋの結果を示す。A schematic diagram (upper figure) of the procedure for investigating the effects of anti-SARS-CoV-2 RBD antibodies 1028-61L, 1028-55K, and 1028-05K on SARS-CoV-2 infection and production, and the results. It is a graph (below). In the graph, the vertical axis indicates the logarithmic value of the reduction ratio (log (% of reduction)) relative to the control in the left graph, and the reduction ratio (% of reduction) relative to the control in the right graph. The horizontal axis indicates antibody concentration (μg/ml). * indicates that no viral plaques were observed. In the data for each antibody concentration, the black bar on the left indicates results for 1028-61L, the gray bar in the middle indicates results for 1028-55K, and the white bar on the right indicates results for 1028-05K. 各抗体の図４の実験結果を，コントロールに対する低下割合の対数値（ｌｏｇ（％　ｏｆ　ｒｅｄｕｃｔｉｏｎ））（縦軸）に対する抗体濃度の対数値（ｌｏｇ（濃度（ｎＭ）））（横軸）でプロットしたグラフである。The experimental results of FIG. 4 for each antibody are plotted against the logarithm of the reduction ratio (log (% of reduction)) (vertical axis) of the antibody concentration (log (concentration (nM))) (horizontal axis). is a graph. 抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体による，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質とＡＣＥ２との結合阻害実験の結果を示すグラフである。ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質とＡＣＥ２との結合を，０２１０－２０Ｌ，０２１０－２８Ｋ，０２１０－３０Ｌ，０２１０－３８Ｌ，１０２８－６１Ｌ，１０２８－５５Ｋの各抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体が阻害するか，ＥＬＩＳＡにて検証した。横軸は抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体の濃度（μｇ／ｍｌ），縦軸は抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体非存在下で測定された吸光度の値を１００とした場合の各抗体存在下における吸光度の値の割合（％）を示している（図７Ａと図７Ｂにおいて同様）。独立して２回以上の実験を行い，図示しているデータはその代表例を示している。Fig. 3 is a graph showing the results of binding inhibition experiments between SARS-CoV-2 spike protein and ACE2 by anti-SARS-CoV-2 RBD antibody. Each anti-SARS-CoV-2 RBD antibody 0210-20L, 0210-28K, 0210-30L, 0210-38L, 1028-61L, 1028-55K inhibits the binding of SARS-CoV-2 spike protein and ACE2 or verified by ELISA. The horizontal axis is the concentration of anti-SARS-CoV-2 RBD antibody (μg/ml), and the vertical axis is the presence of each antibody when the absorbance value measured in the absence of anti-SARS-CoV-2 RBD antibody is set to 100. (similar to FIGS. 7A and 7B). Two or more experiments were performed independently and the data shown are representative. 抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体である０２１０－２８Ｋ，及び１０２８－５５Ｋによる，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質とＡＣＥ２との結合阻害実験の結果を示すグラフである。Ｂｅｔａ株ＳＡＲＳ－ＣｏＶ－２の変異を有するＲＢＤタンパク質（Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ）とＡＣＥ２との結合を，各抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体により阻害できるか，ＥＬＩＳＡにて検証した。縦軸と横軸は図２と同様である（図７Ｂにおいて同じ）。Fig. 10 is a graph showing the results of an experiment to inhibit the binding of SARS-CoV-2 spike protein and ACE2 by anti-SARS-CoV-2 RBD antibodies 0210-28K and 1028-55K. It was verified by ELISA whether each anti-SARS-CoV-2 RBD antibody can inhibit the binding of ACE2 to RBD proteins (K417N, E484K, N501Y) having mutations of Beta strain SARS-CoV-2. The vertical and horizontal axes are the same as in FIG. 2 (same in FIG. 7B). 図７Ａと同じ実験を０２１０－３０Ｌ，及び１０２８－６１Ｌを用いて行った結果を示す。FIG. 7A shows the results of the same experiment using 0210-30L and 1028-61L. ＳＡＲＳ－ＣｏＶ－２シュードタイプウイルスの作製に関する模式図と本実験で用いる変異株スパイクタンパク質を外套するシュードタイプウイルスの一部およびコントロールとなるＶＳＶのエンベロープタンパク質を外套するウイルス（ＶＳＶｐｖ）を示す。ＳＡＲＳ－ＣｏＶ－２（Ａｌｐｈａ）は，Ａｌｐｈａ株における，ＨＶ６９－７０　ｄｅｌｅｔｉｏｎ，Ｙ１４４　ｄｅｌｅｔｉｏｎ，Ｎ５０１Ｙ，Ａ５７０Ｄ，Ｐ６８１Ｈ，Ｔ７１６Ｉ，Ｓ９８２Ａ，Ｄ１１１８Ｈの変異を有するスパイクタンパク質を表面に有する。ＳＡＲＳ－ＣｏＶ－２（Ｂｅｔａ）は，Ｂｅｔａ株における，Ｄ８０Ａ，Ｄ２１５Ｇ，Ｋ４１７Ｎ，Ａ７０１Ｖ，Ｎ５０１Ｙ，Ｅ４８４Ｋの変異を有するスパイクタンパク質を表面に有する。A schematic diagram of the preparation of the SARS-CoV-2 pseudotype virus, a part of the pseudotype virus that envelopes the mutant spike protein used in this experiment, and a virus (VSVpv) that envelopes the envelope protein of VSV as a control are shown. SARS-CoV-2 (Alpha) has a surface spike protein with mutations of HV69-70 deletion, Y144 deletion, N501Y, A570D, P681H, T716I, S982A, and D1118H in the Alpha strain. SARS-CoV-2 (Beta) has a surface spike protein with mutations D80A, D215G, K417N, A701V, N501Y, E484K in the Beta strain. 抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体による，変異株のスパイクタンパク質を外套するシュードタイプウイルスの感染抑制実験の結果を表すグラフである。縦軸はコントロールと比較した感染低下割合の対数値（ｌｏｇ（％　ｏｆ　ｒｅｄｕｃｔｉｏｎ））もしくは実数値（％　ｏｆ　ｒｅｄｕｃｔｉｏｎ）を示し，横軸は抗体濃度（μｇ／ｍｌ）を示す。抗体の各濃度における棒グラフは，左から右へと順に，ＳＡＲＳ－ＣｏＶ－２の，武漢型（ＷＴ），Ａｌｐｈａ株，及びＢｅｔａ株のスパイクタンパク質を有するシュードタイプウイルスを用いた結果を示す。一番右の棒グラフは，コントロールシュードタイプウイルス（ＶＳＶｐｖ）を用いた結果を示す。Fig. 10 is a graph showing the results of an experiment to suppress infection of a pseudotype virus that envelopes a spike protein of a mutant strain using an anti-SARS-CoV-2 RBD antibody. The vertical axis indicates the logarithmic value (log (% of reduction)) or the real value (% of reduction) of the infection reduction rate compared to the control, and the horizontal axis indicates the antibody concentration (μg/ml). Bar graphs at each concentration of antibody show, from left to right, results using pseudotyped viruses with spike proteins of Wuhan type (WT), Alpha strain, and Beta strain of SARS-CoV-2. The rightmost bar graph shows the results using a control pseudotyped virus (VSVpv). （左）抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体である０２１０－２８ＫとＥｐｓｉｌｏｎ株ＳＡＲＳ－ＣｏＶ－２のＲＢＤ（Ｌ４５２Ｒ）タンパク質との結合実験の結果を示す。武漢型（ＷＴ）のＲＢＤ，Ｂｅｔａ株（Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ）変異ＲＢＤとの結合実験の結果も併せて示す。縦軸は，４５０ｎｍにおける吸収（結合強度）を，横軸は抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体の濃度（ｎｇ／ｍｌ）を示す。（右）抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体である０２１０－２８Ｋによる，ＳＡＲＳ－ＣｏＶ２スパイクタンパク質とＡＣＥ２との結合阻害実験の結果を示すグラフである。Ｅｐｓｉｌｏｎ株ＳＡＲＳ－ＣｏＶ－２のＲＢＤ（Ｌ４５２Ｒ）タンパク質とＡＣＥ２の結合を，抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体により阻害できるか，ＥＬＩＳＡにて検証した。武漢型（ＷＴ）のＲＢＤ，Ｂｅｔａ株（Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ）変異ＲＢＤと，ＡＣＥ２との結合阻害実験の結果も併せて示す。縦軸と横軸は図２と同様である。(Left) shows the results of a binding experiment between the anti-SARS-CoV-2 RBD antibody 0210-28K and the Epsilon strain SARS-CoV-2 RBD (L452R) protein. The results of binding experiments with Wuhan type (WT) RBD, Beta strain (K417N, E484K, N501Y) mutant RBD are also shown. The vertical axis indicates the absorption (binding strength) at 450 nm, and the horizontal axis indicates the concentration (ng/ml) of the anti-SARS-CoV-2 RBD antibody. (Right) Graph showing the results of binding inhibition experiments between SARS-CoV2 spike protein and ACE2 by 0210-28K, an anti-SARS-CoV-2 RBD antibody. It was verified by ELISA whether the binding between the Epsilon strain SARS-CoV-2 RBD (L452R) protein and ACE2 could be inhibited by the anti-SARS-CoV-2 RBD antibody. The results of the binding inhibition experiment between Wuhan type (WT) RBD, Beta strain (K417N, E484K, N501Y) mutant RBD and ACE2 are also shown. The vertical and horizontal axes are the same as in FIG. （左）抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体である０２１０－２８ＫとＫａｐｐａ株ＳＡＲＳ－ＣｏＶ－２のＲＢＤ（Ｌ４５２Ｒ，Ｅ４８４Ｑ）タンパク質との結合実験の結果を示す。武漢型（ＷＴ）のＲＢＤ，Ｂｅｔａ株（Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ），及びＡｌｐｈａ株（Ｎ５０１Ｙ）由来の変異ＲＢＤとの結合実験の結果も併せて示す。縦軸は，４５０ｎｍにおける吸収（結合強度）を，横軸は抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体の濃度（ｎｇ／ｍｌ）を示す。（右）抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体である０２１０－２８Ｋによる，ＳＡＲＳ－ＣｏＶ２スパイクタンパク質とＡＣＥ２との結合阻害実験の結果を示すグラフである。Ｋａｐｐａ株ＳＡＲＳ－ＣｏＶ－２のＲＢＤ（Ｌ４５２Ｒ，Ｅ４８４Ｑ）タンパク質とＡＣＥ２の結合を，抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体により阻害できるか，ＥＬＩＳＡにて検証した。武漢型（ＷＴ）のＲＢＤ，Ｂｅｔａ株（Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ），及びＡｌｐｈａ株（Ｎ５０１Ｙ）由来の変異ＲＢＤと，ＡＣＥ２との結合阻害実験の結果も併せて示す。縦軸と横軸は図２と同様である。(Left) shows the results of a binding experiment between the anti-SARS-CoV-2 RBD antibody 0210-28K and the Kappa strain SARS-CoV-2 RBD (L452R, E484Q) proteins. The results of binding experiments with Wuhan type (WT) RBD, beta strains (K417N, E484K, N501Y), and mutated RBDs derived from Alpha strain (N501Y) are also shown. The vertical axis indicates the absorption (binding strength) at 450 nm, and the horizontal axis indicates the concentration (ng/ml) of the anti-SARS-CoV-2 RBD antibody. (Right) Graph showing the results of binding inhibition experiments between SARS-CoV2 spike protein and ACE2 by 0210-28K, an anti-SARS-CoV-2 RBD antibody. It was verified by ELISA whether the binding of the Kappa strain SARS-CoV-2 RBD (L452R, E484Q) protein and ACE2 could be inhibited by the anti-SARS-CoV-2 RBD antibody. The results of binding inhibition experiments between Wuhan type (WT) RBD, Beta strains (K417N, E484K, N501Y), and mutant RBDs derived from Alpha strain (N501Y) and ACE2 are also shown. The vertical and horizontal axes are the same as in FIG. 抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体である０２１０－２８Ｋによる，変異株のスパイクタンパク質を外套するシュードタイプウイルスの感染抑制実験の結果を表すグラフである。縦軸はコントロールと比較した感染低下割合の実数値（％　ｏｆ　ｒｅｄｕｃｔｉｏｎ）を示し，横軸は抗体濃度（ｎｇ／ｍｌ）を示す。抗体の各濃度における棒グラフは，左から右へと順に，ＳＡＲＳ－ＣｏＶ－２の，武漢型（ＷＴ），Ａｌｐｈａ株（Ａｌｐｈａ），Ｂｅｔａ株（Ｂｅｔａ），Ｋａｐｐａ株（Ｋａｐｐａ），及びＤｅｌｔａ株（Ｄｅｌｔａ）のスパイクタンパク質を有するシュードタイプウイルスを用いた結果を示す。一番右の棒グラフは，ＶＳＶのＧタンパク質を有するシュードタイプウイルス（コントロール）を用いた結果を示す。Fig. 10 is a graph showing the results of an experiment to suppress infection of a pseudotype virus that envelopes a mutant spike protein by the anti-SARS-CoV-2 RBD antibody 0210-28K. The vertical axis shows the real value (% of reduction) of infection reduction compared to the control, and the horizontal axis shows the antibody concentration (ng/ml). Bar graphs at each concentration of antibody show, from left to right, SARS-CoV-2 Wuhan strain (WT), Alpha strain (Alpha), Beta strain (Beta), Kappa strain (Kappa), and Delta strain ( Delta) shows results using pseudotyped viruses with the spike protein. The rightmost bar graph shows the results using a pseudotyped virus (control) with the VSV G protein. 抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体である０２１０－２８Ｋによる，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質とＡＣＥ２との結合阻害実験の結果を示すグラフである。武漢型（ＷＴ）のＲＢＤタンパク質，Ａｌｐｈａ株（Ｎ５０１Ｙ），Ｂｅｔａ株（Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ），Ｇａｍｍａ株（Ｋ４１７Ｔ，Ｅ４８４Ｋ，Ｎ５０１Ｙ），Ｋａｐｐａ株（Ｌ４５２Ｒ，Ｅ４８４Ｑ），Ｄｅｌｔａ株（Ｌ４５２Ｒ，Ｔ４７８Ｋ），及びＥｐｓｉｌｏｎ株（Ｌ４５２Ｒ）由来の変異ＲＢＤタンパク質と，ＡＣＥ２との結合を，０２１０－２８Ｋにより阻害できるかＥＬＩＳＡにて検証した。縦軸と横軸は図２と同様である。FIG. 10 is a graph showing the results of an experiment to inhibit binding between SARS-CoV-2 spike protein and ACE2 by 0210-28K, an anti-SARS-CoV-2 RBD antibody. FIG. Wuhan type (WT) RBD protein, Alpha strain (N501Y), Beta strain (K417N, E484K, N501Y), Gamma strain (K417T, E484K, N501Y), Kappa strain (L452R, E484Q), Delta strain (L452R, T478K) , and an Epsilon strain (L452R)-derived mutant RBD protein, and whether 0210-28K can inhibit the binding of ACE2 was verified by ELISA. The vertical and horizontal axes are the same as in FIG. 抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体である０２１０－２８Ｋによる，変異株のスパイクタンパク質を外套するシュードタイプウイルスの感染抑制実験の結果を表すグラフである。縦軸はコントロールと比較した感染低下割合（％　ｏｆ　ｒｅｄｕｃｔｉｏｎ）を示し，横軸は抗体濃度（ｐＭ）を示す。ＳＡＲＳ－ＣｏＶ－２の，武漢型（ＷＴ），Ａｌｐｈａ株，Ｂｅｔａ株，Ｇａｍｍａ株，Ｋａｐｐａ株，Ｄｅｌｔａ株，及びＯｍｉｃｒｏｎ株のスパイクタンパク質を有するシュードタイプウイルスを用いた結果と，ＶＳＶのＧタンパク質を有するシュードタイプウイルス（コントロール）を用いた結果を示す。独立して２回以上の実験を行い，図示しているデータはその代表例を示している。Fig. 10 is a graph showing the results of an experiment to suppress infection of a pseudotype virus that envelopes a mutant spike protein by the anti-SARS-CoV-2 RBD antibody 0210-28K. The vertical axis indicates the rate of infection reduction (% of reduction) compared to the control, and the horizontal axis indicates the antibody concentration (pM). SARS-CoV-2, Wuhan type (WT), Alpha strain, Beta strain, Gamma strain, Kappa strain, Delta strain, and results using pseudotype viruses with spike proteins of Omicron strain, and VSV G protein The results are shown using a pseudotyped virus (control) with Two or more experiments were performed independently and the data shown are representative. 抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体である０２１０－２８ＫのＳＡＲＳ－ＣｏＶ－２感染及び産生に与える影響を調べた結果を示すグラフである。縦軸はウイルス産生阻害率（％　ｏｆ　ｙｉｅｌｄ　ｒｅｄｕｃｔｉｏｎ）を示し，横軸は抗体濃度（ｐＭ）を示す。ＳＡＲＳ－ＣｏＶ－２の，武漢型（ＷＴ），Ａｌｐｈａ株，Ｂｅｔａ株，Ｇａｍｍａ株，Ｋａｐｐａ株，Ｄｅｌｔａ株及びＯｍｉｃｒｏｎ株を用いた結果を示す。独立して２回以上の実験を行い，図示しているデータはその代表例を示している。Fig. 3 is a graph showing the results of investigating the effects of 0210-28K, an anti-SARS-CoV-2 RBD antibody, on SARS-CoV-2 infection and production. The vertical axis indicates the virus production inhibition rate (% of yield reduction), and the horizontal axis indicates the antibody concentration (pM). Results using Wuhan type (WT), Alpha strain, Beta strain, Gamma strain, Kappa strain, Delta strain and Omicron strain of SARS-CoV-2 are shown. Two or more experiments were performed independently and the data shown are representative. 抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体である０２１０－２８Ｋについて，武漢型（ＷＴ）ＲＢＤ及び変異ＲＢＤタンパク質への親和性を，それぞれ表面プラズモン共鳴法（ＳＰＲ）解析した結果を表すグラフである。グラフは，上から下へと順に，武漢型（ＷＴ），Ａｌｐｈａ株（Ｎ５０１Ｙ），Ｂｅｔａ株（Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ），Ｇａｍｍａ株（Ｋ４１７Ｔ，Ｅ４８４Ｋ，Ｎ５０１Ｙ），Ｋａｐｐａ株（Ｌ４５２Ｒ，Ｅ４８４Ｑ），及びＤｅｌｔａ株（Ｌ４５２Ｒ，Ｔ４７８Ｋ）由来の変異ＲＢＤタンパク質の結果を示す。グラフ中，縦軸はレスポンス（ＲＵ）を示し，横軸は測定時間（ｓ）を示す。独立して２回以上の実験を行い，図示しているデータはその代表例を示している。Fig. 4 is a graph showing the results of surface plasmon resonance (SPR) analysis of the affinity of 0210-28K, an anti-SARS-CoV-2 RBD antibody, to Wuhan type (WT) RBD and mutant RBD proteins. The graph shows, from top to bottom, Wuhan type (WT), Alpha strain (N501Y), Beta strain (K417N, E484K, N501Y), Gamma strain (K417T, E484K, N501Y), Kappa strain (L452R, E484Q), and mutant RBD proteins from Delta strains (L452R, T478K). In the graph, the vertical axis indicates the response (RU) and the horizontal axis indicates the measurement time (s). Two or more experiments were performed independently and the data shown are representative. ハムスターモデルを用いた，抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体である０２１０－２８Ｋの予防的投与による，ＳＡＲＳ－ＣｏＶ２及びその変異株のスパイクタンパク質を外套するシュードタイプウイルスの感染抑制実験の結果を示し，ルシフェラーゼの発光画像（上図）と，発光測定結果を示すグラフ（左下図）と，肺全体のウイルス感染価を算出した結果を示すグラフ（右下図）である。グラフ中，縦軸はコントロールと比較した相対発光レベルを示す。横軸はシュードタイプウイルスの種類を示し，左から右へと順に，ＶＳＶのＧタンパク質を有するシュードタイプウイルス，ＳＡＲＳ－ＣｏＶ－２の，武漢型（ＷＴ），Ａｌｐｈａ株，Ｂｅｔａ株，Ｇａｍｍａ株，Ｋａｐｐａ株，Ｄｅｌｔａ株，及びＯｍｉｃｒｏｎ株のスパイクタンパク質を有するシュードタイプウイルスを用いた結果を示す。各シュードタイプウイルスのデータにおいて，左の白バーはコントロール，右の黒バーは０２１０－２８Ｋ投与の結果を示す。抗体投与量はＢｅｔａ株，及びＯｍｉｃｒｏｎ株が３ｍｇ／ハムスター，その他は０．３ｍｇ／ハムスターである。独立して２回以上の実験を行い，図示しているデータはその代表例を示している。ｐ値はｔ検定により算出した（＊，ｐ＜０．０５，＊＊，ｐ＜０．０１）。Using a hamster model, prophylactic administration of the anti-SARS-CoV-2 RBD antibody 0210-28K shows the results of an experiment to suppress the infection of a pseudotype virus that envelopes the spike protein of SARS-CoV2 and its mutants, FIG. 2 shows a luminescence image of luciferase (upper diagram), a graph showing the results of luminescence measurement (lower left diagram), and a graph showing the results of calculating the virus infectivity titer of the whole lung (lower right diagram). In the graph, the vertical axis indicates the relative luminescence level compared with the control. The horizontal axis indicates the type of pseudotype virus, and from left to right, the pseudotype virus having the G protein of VSV, SARS-CoV-2, Wuhan type (WT), Alpha strain, Beta strain, Gamma strain, Results using pseudotyped viruses with spike proteins of Kappa, Delta, and Omicron strains are shown. In the data for each pseudotype virus, the white bar on the left indicates the results of control, and the black bar on the right indicates the results of administration of 0210-28K. Antibody dosage is 3 mg/hamster for Beta strain and Omicron strain, and 0.3 mg/hamster for others. Two or more experiments were performed independently and the data shown are representative. p-values were calculated by t-test (*, p<0.05, **, p<0.01). ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質（ＳＡＲＳ－ＣｏＶ－２　ｓｐｉｋｅ）に結合した抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体である０２１０－２８Ｋ（図においては「ＵＴ２８Ｋ」と表記する）の構造解析結果を表す図である。図１８（ａ）は，Ｃ３対称性で３．１Åの解像度に緻密化したクライオ電子顕微鏡マップであり，ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ（ＲＢＤ）と０２１０－２８ＫのＦａｂ（ＵＴ２８Ｋ　Ｆａｂ）の重鎖可変領域（ＶＨ）及び軽鎖可変領域（ＶＬ）について示す。図１８（ｂ），（ｄ）～（ｇ）は，３．７５Åの解像度のＸ線結晶構造解析結果であり，図１８（ｄ）～（ｇ）にＲＢＤとＵＴ２８Ｋ　Ｆａｂ間の主要残基の相互作用について示す。なお，比較データとして，図１８（ｃ）に２５３ＸＬ５５（ＰＤＢ　ＩＤ：７ＢＥＯ）とＳＡＲＳ－ＣｏＶ－２　ＲＢＤ（ＲＢＤ）の結合についてのＸ線結晶構造解析結果を示す。A diagram showing the structural analysis results of 0210-28K (denoted as "UT28K" in the figure), which is an anti-SARS-CoV-2 RBD antibody bound to the SARS-CoV-2 spike protein (SARS-CoV-2 spike). be. Figure 18(a) is a cryo-electron microscopy map densified to 3.1 Å resolution with C3 symmetry, showing the heavy chain variable Regions (VH) and light chain variable regions (VL) are indicated. Figures 18(b), (d) to (g) are the results of X-ray crystallographic analysis with a resolution of 3.75 Å, and Figures 18(d) to (g) show the major residues between RBD and UT28K Fab. Interactions are indicated. As comparative data, FIG. 18(c) shows the results of X-ray crystallographic analysis of the binding of 253XL55 (PDB ID: 7BEO) and SARS-CoV-2 RBD (RBD). 抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体である０２１０－２８Ｋについて，武漢型（ＷＴ）のＲＢＤ及び変異株由来のＲＢＤタンパク質への結合をＥＬＩＳＡにて解析した結果を表すグラフである。縦軸は４５０ｎｍにおける吸収（結合強度）を示し、横軸はＲＢＤタンパク質の種類を示す。独立して２回以上の実験を行い，図示しているデータはその代表例を示している。Fig. 3 is a graph showing the results of ELISA analysis of the binding of 0210-28K, which is an anti-SARS-CoV-2 RBD antibody, to Wuhan type (WT) RBD and RBD proteins derived from mutant strains. The vertical axis indicates absorption (bond strength) at 450 nm, and the horizontal axis indicates the type of RBD protein. Two or more experiments were performed independently and the data shown are representative. 抗体非存在下（コントロール），抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体である０２１０－２８Ｋ又は０２１０－２８Ｋ－ＬＡＬＡ存在下において、新型コロナウイルスを感染させたｃＭｙｌｃ－２－Ｋ　ａｌｐｈａ細胞におけるウイルス量の測定結果を表すグラフである。縦軸はコントロールで得られるウイルス量を１として，ウイルスの増減量（Ｆｏｌｄ　ｉｎｃｒｅａｓｅ）を示し，横軸は抗体濃度（μｇ／ｍｌ）を示す。Measurement of viral load in cMylc-2-Kalpha cells infected with the new coronavirus in the absence of antibodies (control) and in the presence of anti-SARS-CoV-2 RBD antibodies 0210-28K or 0210-28K-LALA It is a graph showing a result. The vertical axis indicates the amount of virus increased or decreased (Fold increase) with the amount of virus obtained in the control set to 1, and the horizontal axis indicates the antibody concentration (μg/ml). 抗体非存在下（コントロール），抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体である０２１０－２８Ｋ又は０２１０－２８Ｋ－ＬＡＬＡ存在下において、新型コロナウイルスを感染させたｃＭｙｌｃ－２－Ｋ　ａｌｐｈａ細胞におけるＩＬ－６産生量を、ＥＬＩＳＡにて測定した結果を表すグラフである。縦軸は光学濃度（Ｏｐｔｉｃａｌ　Ｄｅｎｓｉｔｙ）を示し，横軸は抗体濃度（μｇ／ｍｌ）を示す。IL-6 production in cMylc-2-Kalpha cells infected with the new coronavirus in the absence of antibodies (control) and in the presence of anti-SARS-CoV-2 RBD antibodies 0210-28K or 0210-28K-LALA It is a graph showing the result of measuring the amount by ELISA. The vertical axis indicates optical density, and the horizontal axis indicates antibody concentration (μg/ml).
　ＳＡＲＳ－ＣｏＶ－２（Ｓｅｖｅｒｅ　ａｃｕｔｅ　ｒｅｓｐｉｒａｔｏｒｙ　ｓｙｎｄｒｏｍｅ　ｃｏｒｏｎａｖｉｒｕｓ　２）は，中国武漢で発生した肺炎の原因ウイルス（当初の名称は２０１９－ｎＣｏＶ）として報告された（Ｚｈｏｕ，Ｐ．ら，　Ｎａｔｕｒｅ　５７９，２７０－２７３（２０２０）．及びＷｕ，Ｆ．ら，Ｎａｔｕｒｅ　５７９，２６５－２６９（２０２０）．）コロナウイルスであり，最初に見つかった武漢型のゲノムが解析され，報告されている（ＮＣＢＩ　Ｒｅｆｅｒｅｎｃｅ　Ｓｅｑｕｅｎｃｅ：ＮＣ＿０４５５１２．２）。本明細書において，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質（スパイク）は，ＳＡＲＳ－ＣｏＶ－２のＳタンパク質として報告されている１２７３アミノ酸（配列番号６２９）（うち１～１３番目はシグナル配列）からなるタンパク質から構成され，Ｓ１（１４～６８５番目）とＳ２（６８６～１２７３番目）の二つのサブユニットからなる（Ｌａｎ，　Ｊ．ら，Ｎａｔｕｒｅ　５８１，２１５－２２０（２０２０）；Ｈｕａｎｇ，Ｙ．ら，Ａｃｔａ　Ｐｈａｒｍａｃｏｌ　Ｓｉｎ　４１，１１４１－１１４９（２０２０）参照）。本明細書における「スパイク」は，好ましくは，スパイクタンパク質全長を意味し，かつ／又はスパイクタンパク質の三量体を意味してもよい。受容体結合ドメイン（ＲＢＤ）は，Ｓ１サブユニットに含まれる３１９～５４１番目のアミノ酸からなる部分または同部分からなるペプチドを意味する。また，Ｎ末端ドメイン（ＮＴＤ）は，１４～３０５番目のアミノ酸からなる部分又は同部分からなるペプチドを意味する。本明細書における変異の記載において，アミノ酸の位置はこのＳタンパク質におけるアミノ酸の位置を示す。 SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) was reported as the causative virus (originally named 2019-nCoV) of pneumonia that occurred in Wuhan, China (Zhou, P. et al., Nature 579, 270-273 (2020).and Wu, F. et al., Nature 579, 265-269 (2020).) It is a coronavirus, and the genome of the first Wuhan type found was analyzed and reported (NCBI Reference Sequence: NC_045512. 2). As used herein, the SARS-CoV-2 spike protein (spike) is a protein consisting of 1273 amino acids (SEQ ID NO: 629) (of which 1 to 13 are signal sequences) reported as the S protein of SARS-CoV-2. consists of two subunits, S1 (14th to 685th) and S2 (686th to 1273rd) (Lan, J. et al., Nature 581, 215-220 (2020); Huang, Y. et al., Acta Pharmacol Sin 41, 1141-1149 (2020)). "Spike" herein preferably refers to the full-length spike protein and/or may refer to trimers of the spike protein. The receptor binding domain (RBD) means a portion consisting of amino acids 319 to 541 contained in the S1 subunit or a peptide consisting of the same portion. Also, the N-terminal domain (NTD) means a portion consisting of the 14th to 305th amino acids or a peptide consisting of the same portion. In describing mutations herein, amino acid positions refer to amino acid positions in the S protein.
　ＳＡＲＳ－ＣｏＶ－２には，最初に見つかった武漢型の他，多くの変異型が報告されており（例えば，Ｉｓｌａｍ，Ｍ．Ｒ．ｅｔ　ａｌ．，Ｓｃｉ　Ｒｅｐ；１０：１４００４（２０２０）．参照），かつ，データベースも存在する（例えば，ｈｔｔｐｓ：／／ｎｅｘｔｓｔｒａｉｎ．ｏｒｇ／ｓａｒｓ－ｃｏｖ－２／など）。本明細書における抗体が結合する，スパイク又はＲＢＤは，武漢型の他，これらにおいて報告された変異型ＳＡＲＳ－ＣｏＶ－２が有するものであってもよい。「変異型ＳＡＲＳ－ＣｏＶ－２」は，ＳＡＲＳ－ＣｏＶ－２について報告されている変異であればいかなる変異を有していてもよく，例えば，ＳＡＲＳ－ＣｏＶ－２のスパイクにおける変異，特にはＲＢＤにおける変異が挙げられる。本明細書において「変異型スパイク」とは，ＳＡＲＳ－ＣｏＶ－２のスパイクにおける変異を有するスパイクを意味し，「変異型ＲＢＤ」とは，ＳＡＲＳ－ＣｏＶ－２のＲＢＤにおける変異を有するＲＢＤを意味する。例えば，２０Ｂ／５０１Ｙ．Ｖ１，ＶＯＣ　２０２０１２／０１又はＢ．１．１．７系統として知られるＡｌｐｈａ株（いわゆる英国型変異株）では，スパイクにおける，Ｈ６９欠損，Ｖ７０欠損，Ｙ１４４欠損，Ｎ５０１Ｙ，Ａ５７０Ｄ，Ｄ６１４Ｇ，Ｐ６８１Ｈ，Ｔ７１６Ｉ，Ｓ９８２Ａ，及びＤ１１１８Ｈの変異が報告されている。また，２０Ｃ／５０１Ｙ．Ｖ２又はＢ．１．３５１系統として知られるＢｅｔａ株（いわゆる南アフリカ型変異株）では，Ｌ１８Ｆ，Ｄ８０Ａ，Ｄ２１５Ｇ，Ｌ２４２欠損，Ａ２４３欠損，Ｌ２４４欠損，Ｒ２４６Ｉ，Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ，Ｄ６１４Ｇ，Ａ７０１Ｖの変異が報告されている。また，２０J／５０１Ｙ．Ｖ３系統として知られるＧａｍｍａ株（いわゆるブラジル型変異株）では，スパイクにおける，Ｌ１８Ｆ，Ｋ４１７Ｔ，Ｅ４８４Ｋ，Ｎ５０１Ｙ，Ｈ６５５Ｙの変異が報告されている。また，２０Ｃ／Ｓ．４５２Ｒ又はＢ．１．４２７／９系統として知られるＥｐｓｉｌｏｎ株（いわゆるカリフォルニア型変異株）では，スパイクにおける，Ｌ４５２Ｒ，Ｄ６１４Ｇの変異が報告されている。また，Ｂ．１．６１７．１系統として知られるＫａｐｐａ株及びＢ．１．６１７．２系統として知られるＤｅｌｔａ株（いわゆるインド型変異株）では，スパイクにおける，Ｇ１４２Ｄ，Ｅ１５４Ｋ，Ｌ４５２Ｒ，Ｅ４８４Ｑ，Ｄ６１４Ｇ，Ｐ６８１Ｒ，及びＱ１０７１Ｈ（以上，Ｂ１．６１７．１系統），あるいは，Ｔ１９Ｒ，Ｇ１４２Ｄ，１５７／１５８欠損，Ｌ４５２Ｒ，Ｔ４７８Ｋ，Ｄ６１４Ｇ，Ｐ６８１Ｒ，及びＤ９５０Ｎ（Ｂ．１．６１７．２系統）の変異が報告されている。更に，Ｂ．１．１．５２９系統として知られるＯｍｉｃｒｏｎ株では，スパイクにおける，例えばＧ３３９Ｄ，Ｓ３７１Ｌ，Ｓ３７３Ｐ，Ｓ３７５Ｆ，Ｋ４１７Ｎ，Ｎ４４０Ｋ，Ｇ４４６Ｓ，Ｓ４７７Ｎ，Ｔ４７８Ｋ，Ｅ４８４Ａ，Ｑ４９３Ｒ，Ｇ４９６Ｓ，Ｑ４９８Ｒ，Ｎ５０１Ｙ，及びＹ５０５Ｈの変異が報告されている。 SARS-CoV-2 has been reported to have many mutations in addition to the first Wuhan type found (for example, Islam, MR et al., Sci Rep; 10: 14004 (2020). See ), and databases also exist (eg, https://nextstrain.org/sars-cov-2/, etc.). The spikes or RBDs to which the antibodies herein bind may be those possessed by the Wuhan type as well as the mutant forms of SARS-CoV-2 reported therein. A "mutant SARS-CoV-2" may have any mutation that has been reported for SARS-CoV-2, such as mutations in the spike of SARS-CoV-2, particularly RBD Mutations in As used herein, "mutant spike" means a spike having a mutation in the spike of SARS-CoV-2, and "mutant RBD" means an RBD having a mutation in the RBD of SARS-CoV-2. do. For example, 20B/501Y. V1, VOC 202012/01 or B. In the Alpha strain (so-called British mutant strain) known as 1.1.7 strain, H69 deletion, V70 deletion, Y144 deletion, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H mutations in the spike have been reported. It is Also, 20C/501Y. V2 or B. In the Beta strain known as 1.351 strain (so-called South African mutant strain), mutations of L18F, D80A, D215G, L242 deletion, A243 deletion, L244 deletion, R246I, K417N, E484K, N501Y, D614G, and A701V have been reported. there is Also, 20J/501Y. In the Gamma strain known as the V3 strain (so-called Brazilian mutant strain), mutations L18F, K417T, E484K, N501Y, and H655Y in the spike have been reported. Also, 20C/S. 452R or B. 1. In the Epsilon strain known as line 427/9 (the so-called California mutant strain), L452R, D614G mutations in the spike have been reported. Also, B. Kappa strain known as lineage 1.617.1 and B. In the Delta strain (the so-called Indian variant), known as the 1.617.2 strain, G142D, E154K, L452R, E484Q, D614G, P681R, and Q1071H (all B1.617.1 strains) in the spike, or Mutations of T19R, G142D, 157/158 deletion, L452R, T478K, D614G, P681R, and D950N (B.1.617.2 strain) have been reported. Furthermore, B. In the Omicron strain known as line 1.1.529, mutations such as G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H in the spike It has been reported.
　本明細書における変異型ＳＡＲＳ－ＣｏＶ－２が有する変異は，これらの変異株において報告されている全て若しくは一部の変異，又はこれらの変異株において報告された変異から選択された任意の２種類以上の組み合わせの変異であってもよい。すなわち，本明細書における変異型ＳＡＲＳ－ＣｏＶ－２は，例えば，Ｌ１８Ｆ，Ｔ１９Ｒ，Ｈ６９欠損，Ｖ７０欠損，Ｄ８０Ａ，Ｇ１４２Ｄ，Ｙ１４４欠損，Ｅ１５４Ｋ，１５７欠損，１５８欠損，Ｄ２１５Ｇ，Ｌ２４２欠損，Ａ２４３欠損，Ｌ２４４欠損，Ｒ２４６Ｉ，Ｇ３３９Ｄ，Ｓ３７１Ｌ，Ｓ３７３Ｐ，Ｓ３７５Ｆ，Ｋ４１７Ｎ，Ｋ４１７Ｔ，Ｎ４４０Ｋ，Ｇ４４６Ｓ，Ｌ４５２Ｒ，Ｓ４７７Ｎ，Ｔ４７８Ｋ，Ｅ４８４Ａ，Ｅ４８４Ｋ，Ｅ４８４Ｑ，Ｑ４９３Ｒ，Ｇ４９６Ｓ，Ｑ４９８Ｒ，Ｎ５０１Ｙ，Ｙ５０５Ｈ，Ａ５７０Ｄ，Ｄ６１４Ｇ，Ｈ６５５Ｙ，Ｐ６８１Ｈ，Ｐ６８１Ｒ，Ａ７０１Ｖ，Ｔ７１６Ｉ，Ｄ９５０Ｎ，Ｓ９８２Ａ，Ｑ１０７１Ｈ，及びＤ１１１８Ｈから選択される１種類又はそれ以上，２種類又はそれ以上，３種類又はそれ以上，４種類又はそれ以上，５種類又はそれ以上，あるいは，６種類又はそれ以上の変異を有していてもよい。 Mutations possessed by mutant SARS-CoV-2 herein are all or part of the mutations reported in these mutant strains, or any two types selected from the mutations reported in these mutant strains The mutation may be a combination of the above. That is, mutant SARS-CoV-2 in the present specification is, for example, L18F, T19R, H69 deletion, V70 deletion, D80A, G142D, Y144 deletion, E154K, 157 deletion, 158 deletion, D215G, L242 deletion, A243 deletion, Ｌ２４４欠損，Ｒ２４６Ｉ，Ｇ３３９Ｄ，Ｓ３７１Ｌ，Ｓ３７３Ｐ，Ｓ３７５Ｆ，Ｋ４１７Ｎ，Ｋ４１７Ｔ，Ｎ４４０Ｋ，Ｇ４４６Ｓ，Ｌ４５２Ｒ，Ｓ４７７Ｎ，Ｔ４７８Ｋ，Ｅ４８４Ａ，Ｅ４８４Ｋ，Ｅ４８４Ｑ，Ｑ４９３Ｒ，Ｇ４９６Ｓ，Ｑ４９８Ｒ，Ｎ５０１Ｙ，Ｙ５０５Ｈ，Ａ５７０Ｄ，Ｄ６１４Ｇ，Ｈ６５５Ｙ，Ｐ６８１Ｈ , P681R, A701V, T716I, D950N, S982A, Q1071H, and D1118H, one or more, two or more, three or more, four or more, five or more, or , may have 6 or more mutations.
　一例において，本明細書における変異型ＳＡＲＳ－ＣｏＶ－２は，変異型ＲＢＤを有していてもよく，例えば，ＲＢＤにおける，Ｇ３３９Ｄ，Ｓ３７１Ｌ，Ｓ３７３Ｐ，Ｓ３７５Ｆ，Ｋ４１７Ｎ，Ｋ４１７Ｔ，Ｎ４３９Ｋ，Ｎ４４０Ｋ，Ｇ４４６Ｓ，Ｌ４５２Ｒ，Ａ４７５Ｖ，Ｓ４７７Ｎ，Ｔ４７８Ｋ，Ｅ４８４Ａ，Ｅ４８４Ｋ，Ｅ４８４Ｑ，Ｑ４９３Ｒ，Ｇ４９６Ｓ，Ｑ４９８Ｒ，Ｎ５０１Ｙ，及びＹ５０５Ｈから選択される１種類又はそれ以上，２種類又はそれ以上，３種類又はそれ以上，４種類又はそれ以上，５種類又はそれ以上，あるいは，６種類又はそれ以上の変異を有する。変異型ＳＡＲＳ－ＣｏＶ－２が有する変異の一例としては，ＲＢＤにおける，Ｋ４１７Ｎ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異；Ｎ４３９Ｋ変異；Ｎ４４０Ｋ変異；Ａ４７５Ｖ変異；Ｅ４８４Ｋ変異；Ｎ５０１Ｙ変異；Ｌ４５２Ｒ変異；Ｌ４５２Ｒ及びＥ４８４Ｑ変異；Ｋ４１７Ｔ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異；Ｌ４５２Ｒ及びＴ４７８Ｋ変異；及び，Ｇ３３９Ｄ，Ｓ３７１Ｌ，Ｓ３７３Ｐ，Ｓ３７５Ｆ，Ｋ４１７Ｎ，Ｎ４４０Ｋ，Ｇ４４６Ｓ，Ｓ４７７Ｎ，Ｔ４７８Ｋ，Ｅ４８４Ａ，Ｑ４９３Ｒ，Ｇ４９６Ｓ，Ｑ４９８Ｒ，Ｎ５０１Ｙ，及びＹ５０５Ｈ変異である。 In one example, the mutant SARS-CoV-2 herein may have a mutant RBD, such as G339D, S371L, S373P, S375F, K417N, K417T, N439K, N440K, G446S, One or more, two or more, three or more, four or more selected from L452R, A475V, S477N, T478K, E484A, E484K, E484Q, Q493R, G496S, Q498R, N501Y, and Y505H Above, 5 or more, or 6 or more mutations. Examples of mutations that mutant SARS-CoV-2 has include K417N, E484K, and N501Y mutations in RBD; N439K mutation; N440K mutation; A475V mutation; , E484K, and N501Y mutations; L452R and T478K mutations;
　本明細書全体にわたって，「ＳＡＲＳ－ＣｏＶ－２」の語は，そのように解することが不適合である場合を除き，武漢型（非変異）ＳＡＲＳ－ＣｏＶ－２及び上述の変異型ＳＡＲＳ－ＣｏＶ－２から選択されるいずれか，または，これらから選択される複数のＳＡＲＳ－ＣｏＶ－２を意味してもよい。例えば，本明細書における，ＳＡＲＳ－ＣｏＶ－２は，武漢型，Ａｌｐｈａ株，Ｂｅｔａ株，Ｇａｍｍａ株，Ｅｐｓｉｌｏｎ株，Ｋａｐｐａ株，Ｄｅｌｔａ株，及びＯｍｉｃｒｏｎ株から選択される，１種類又はそれ以上，２種類又はそれ以上，３種類又はそれ以上，４種類又はそれ以上，５種類又はそれ以上，あるいは，６種類又はそれ以上であってもよく，あるいは，これらの全てを意味していてもよい。 Throughout this specification, the term "SARS-CoV-2" will be used to refer to Wuhan-type (non-mutated) SARS-CoV-2 and the mutant SARS-CoV described above, unless it is incompatible to be so construed. -2, or a plurality of SARS-CoV-2 selected from these. For example, SARS-CoV-2 herein refers to one or more selected from Wuhan, Alpha strain, Beta strain, Gamma strain, Epsilon strain, Kappa strain, Delta strain, and Omicron strain, 2 There may be 1 or more types, 3 or more types, 4 or more types, 5 or more types, or 6 or more types, or all of these may be meant.
　一態様において，本発明の抗体は，ＳＡＲＳ－ＣｏＶ－２スパイク，ＮＴＤ，及びＲＢＤの少なくとも一つと特異的に結合し，より好ましくは，ＳＡＲＳ－ＣｏＶ－２スパイク及び／又はＲＢＤと特異的に結合し，更に好ましくは，ＳＡＲＳ－ＣｏＶ－２　ＲＢＤと特異的に結合する。ＳＡＲＳ－ＣｏＶ－２スパイクの中でＲＢＤが細胞との結合に重要であることが報告されている（Ｓｃｉｅｎｃｅ（２０２０）Ｖｏｌ．３６７，Ｉｓｓｕｅ６４８３，ｐｐ．１２６０－１２６３，及びＳｃｉｅｎｃｅ（２０２０）Ｖｏｌ．３６９，Ｉｓｓｕｅ６５０４，ｐｐ．６５０－６５５参照）。本明細書において，ＲＢＤに結合するとは，ＳＡＲＳ－ＣｏＶ－２スパイク中のＲＢＤ部分に結合することを意味してもよいし，ＲＢＤを構成するアミノ酸配列からなるペプチドに結合することを意味してもよい。 In one embodiment, the antibodies of the invention specifically bind to at least one of SARS-CoV-2 spike, NTD and RBD, more preferably SARS-CoV-2 spike and/or RBD. and more preferably, it specifically binds to SARS-CoV-2 RBD. It has been reported that RBD is important for binding to cells in the SARS-CoV-2 spike (Science (2020) Vol.367, Issue6483, pp.1260-1263, and Science (2020) Vol.369 , Issue 6504, pp. 650-655). As used herein, binding to the RBD may mean binding to the RBD portion in the SARS-CoV-2 spike, or binding to a peptide consisting of an amino acid sequence that constitutes the RBD. good too.
　本発明の抗体は，スパイク（好ましくは，ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ）と特異的に結合する。本明細書において，抗体又はその免疫反応性断片が「特異的に」結合する（認識する）とは，その抗体又はその免疫反応性断片が他のタンパク質やペプチドに対する親和性よりも，特定された抗原（例えば，スパイク又はＲＢＤ）に対して実質的に高い親和性で結合することを意味する。ここで，「実質的に高い親和性で結合する」とは，所望の測定装置又は方法によって，目的とする特定のタンパク質やペプチドを他のタンパク質やペプチドから区別して検出することが可能な程度に高い親和性を意味する。例えば，実質的に高い親和性は，ＥＬＩＳＡ又はＥＩＡにより検出された強度（例えば，蛍光強度）として，３倍以上，４倍以上，５倍以上，６倍以上，７倍以上，８倍以上，９倍以上，１０倍以上，２０倍以上，３０倍以上，４０倍以上，５０倍以上，１００倍以上であることを意味していてもよい。 The antibody of the present invention specifically binds to spike (preferably SARS-CoV-2 RBD). As used herein, an antibody or immunoreactive fragment thereof "specifically" binds (recognizes) means that the antibody or immunoreactive fragment thereof has a specified affinity for other proteins or peptides. It means binding with substantially high affinity to an antigen (eg spike or RBD). Here, "bind with substantially high affinity" means that the desired measuring device or method can detect the specific protein or peptide of interest by distinguishing it from other proteins or peptides. means high affinity. For example, a substantially high affinity is 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, as the intensity (e.g., fluorescence intensity) detected by ELISA or EIA. It may mean 9 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, or 100 times or more.
　ある態様において，本発明の抗体は，変異型スパイク及び／又は変異型ＲＢＤ（好ましくはＲＢＤ，本段落において，以下同様）と特異的に結合する。また，好ましくは，本発明の抗体は，これらの変異型スパイク及び／又は変異型ＲＢＤと結合し，かつ，武漢型ＳＡＲＳ－ＣｏＶ－２のスパイク及び／又はＲＢＤとも結合する。更に，本発明の抗体は，異なる変異パターンを有する複数種類の変異型スパイク及び／又は変異型ＲＢＤと結合してもよく，この場合も，好ましくは，武漢型ＳＡＲＳ－ＣｏＶ－２のスパイク及び／又はＲＢＤとも結合する。 In some embodiments, the antibody of the present invention specifically binds to mutant spike and/or mutant RBD (preferably RBD, in this paragraph, the same shall apply hereinafter). Also preferably, the antibodies of the invention bind to these mutant spikes and/or mutant RBDs and also bind to the spikes and/or RBDs of Wuhan SARS-CoV-2. Furthermore, the antibodies of the present invention may bind to multiple types of mutant spikes and/or mutant RBDs with different mutation patterns, and in this case also preferably Wuhan SARS-CoV-2 spikes and/or Or it also binds to RBD.
　本明細書において，「異なる変異パターンを有する」とは，２種類以上のＳＡＲＳ－ＣｏＶ－２，スパイク，若しくはＲＢＤがそれぞれ異なる変異を有することを意味する。ここで，「異なる変異」とは，変異部位及び／又は変異後のアミノ酸が異なることを意味する。２種類のＳＡＲＳ－ＣｏＶ－２，スパイク，若しくはＲＢＤの少なくとも一方が複数の変異を有する場合，それらが共通する変異を有していても，それらの２種類の間で変異部位及び／又は変異後のアミノ酸が異なる変異が存在する場合には，異なる変異パターンを有すると解される。 As used herein, "having different mutation patterns" means that two or more types of SARS-CoV-2, spikes, or RBDs have different mutations. Here, "different mutation" means that the mutation site and/or the amino acid after mutation are different. If at least one of two types of SARS-CoV-2, spike, or RBD has multiple mutations, even if they have common mutations, the mutation site and/or post-mutation between the two If there are mutations that differ in amino acids, they are considered to have different mutation patterns.
　例えば，本抗体は，武漢型，Ｋ４１７Ｎ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異型，Ｎ４３９Ｋ変異型，Ｎ４４０Ｋ変異型，Ａ４７５Ｖ変異型，Ｅ４８４Ｋ変異型，Ｎ５０１Ｙ変異型，Ｌ４５２Ｒ変異型，Ｌ４５２Ｒ及びＥ４８４Ｑ変異型，Ｋ４１７Ｔ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異型，Ｌ４５２Ｒ及びＴ４７８Ｋ変異型，及び，Ｇ３３９Ｄ，Ｓ３７１Ｌ，Ｓ３７３Ｐ，Ｓ３７５Ｆ，Ｋ４１７Ｎ，Ｎ４４０Ｋ，Ｇ４４６Ｓ，Ｓ４７７Ｎ，Ｔ４７８Ｋ，Ｅ４８４Ａ，Ｑ４９３Ｒ，Ｇ４９６Ｓ，Ｑ４９８Ｒ，Ｎ５０１Ｙ，及びＹ５０５Ｈ変異型のＳＡＲＳ－ＣｏＶ－２，スパイク，若しくはＲＢＤから選択される１種類又はそれ以上，２種類又はそれ以上，３種類又はそれ以上，４種類又はそれ以上，５種類又はそれ以上，６種類又はそれ以上，７種類又はそれ以上，あるいは全てに結合してもよい。これに加えて，本発明の抗体は，Ｇ３３９Ｄ，Ｓ３７１Ｌ，Ｓ３７３Ｐ，Ｓ３７５Ｆ，Ｋ４１７Ｎ，Ｋ４１７Ｔ，Ｎ４３９Ｋ，Ｎ４４０Ｋ，Ｇ４４６Ｓ，Ｌ４５２Ｒ，Ａ４７５Ｖ，Ｓ４７７Ｎ，Ｔ４７８Ｋ，Ｅ４８４Ａ，Ｅ４８４Ｋ，Ｅ４８４Ｑ，Ｑ４９３Ｒ，Ｇ４９６Ｓ，Ｑ４９８Ｒ，Ｎ５０１Ｙ，及びＹ５０５Ｈから選択される１種類又はそれ以上の変異を有する，ＳＡＲＳ－ＣｏＶ－２，スパイク，若しくはＲＢＤの，１種類又はそれ以上，２種類又はそれ以上，３種類又はそれ以上，４種類又はそれ以上，５種類又はそれ以上，６種類又はそれ以上，あるいは７種類又はそれ以上に結合してもよい。あるいは，本発明の抗体は，上述の武漢型，Ａｌｐｈａ株，Ｂｅｔａ株，Ｇａｍｍａ株，Ｅｐｓｉｌｏｎ株，Ｋａｐｐａ株，Ｄｅｌｔａ株，及びＯｍｉｃｒｏｎ株から選択される１種類又はそれ以上，２種類又はそれ以上，３種類又はそれ以上，４種類又はそれ以上，又は全てのＳＡＲＳ－ＣｏＶ－２，スパイク，若しくはＲＢＤに結合してもよい。 For example, the antibody includes Wuhan, K417N, E484K, and N501Y variants, N439K variant, N440K variant, A475V variant, E484K variant, N501Y variant, L452R variant, L452R and E484Q variant, K417T, E484K and N501Y mutants, L452R and T478K mutants, and SARS-H mutants of G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 selected from CoV-2, spike, or RBD or more, or may be combined with all. Additionally, the antibodies of the present invention are , and Y505H, one or more, two or more, three or more, four or More, 5 or more, 6 or more, or 7 or more may be combined. Alternatively, the antibody of the present invention may be one or more, two or more selected from the above Wuhan type, Alpha strain, Beta strain, Gamma strain, Epsilon strain, Kappa strain, Delta strain, and Omicron strain, May bind to 3 or more, 4 or more, or all SARS-CoV-2, spike, or RBD.
　本発明の抗体のこれらの変異株又は変異型ＲＢＤへの結合力は，好ましくは武漢型と同程度である。例えば，本抗体の，Ｋ４１７Ｎ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異；Ｎ４３９Ｋ変異；Ｎ４４０Ｋ変異；Ａ４７５Ｖ変異；Ｅ４８４Ｋ変異；Ｎ５０１Ｙ変異；Ｌ４５２Ｒ変異；Ｌ４５２Ｒ及びＥ４８４Ｑ変異を有するＲＢＤ（以下，それぞれ，「ＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ」，「ＲＢＤ　Ｎ４３９Ｋ」，「ＲＢＤ　Ｎ４４０Ｋ」，「ＲＢＤ　Ａ４７５Ｖ」，「ＲＢＤ　Ｅ４８４Ｋ」，「ＲＢＤ　Ｎ５０１Ｙ」，「ＲＢＤ　Ｌ４５２Ｒ」，「ＲＢＤ　Ｌ４５２Ｒ，Ｅ４８４Ｑ」という）への結合は，武漢型（ＷＴ）ＲＢＤへの結合に対して，それぞれ，９４％以上，９６％以上，１００％以上，１０１％以上，９４％以上，１００％以上，１００％以上，１００％以上であってもよい。これらの結合力は，好ましくは本願実施例に記載のＥＬＩＳＡ法により測定された結合力である。 The binding strength of the antibody of the present invention to these mutant strains or mutant RBDs is preferably comparable to that of the Wuhan type. For example, the RBD having the K417N, E484K, and N501Y mutations; the N439K mutation; the N440K mutation; the A475V mutation; the E484K mutation; the N501Y mutation; N501Y", "RBD N439K", "RBD N440K", "RBD A475V", "RBD E484K", "RBD N501Y", "RBD L452R", "RBD L452R, E484Q") binding to the Wuhan type (WT ) may be 94% or greater, 96% or greater, 100% or greater, 101% or greater, 94% or greater, 100% or greater, 100% or greater, 100% or greater, relative to binding to RBD, respectively. These binding forces are preferably binding forces measured by the ELISA method described in the Examples of the present application.
　本明細書において，被検抗体が，スパイク又はＲＢＤと「結合する（認識する）」か否かは，結合を調べようとするスパイク及びＲＢＤへの当該被検抗体の結合を以下の方法により測定することにより行うことができる。抗体とタンパク質又はペプチドとの結合の測定方法としては，例えば，ＥＩＡ法，ＥＬＩＳＡ法，ＦＡＣＳ法，共免疫沈降法，プルダウンアッセイ法，ウェスタンブロッティング法，ホモバイファンクショナルクロスリンカー若しくはヘテロバイファンクショナルクロスリンカーを利用するクロスリンク法，ラベル転移反応法，相互作用マッピング法，表面プラズモン共鳴法，ＦＲＥＴ（Ｆｌｕｏｒｅｓｃｅｎｃｅ　ｒｅｓｏｎａｎｃｅ　ｅｎｅｒｇｙ　ｔｒａｎｓｆｅｒ）法，ＢＩＡＣＯＲＥ法，ＡｌｐｈａＳｃｒｅｅｎ（登録商標）やＡｌｐｈａＬＩＳＡ（登録商標）などのＡｌｐｈａＰＰＩアッセイ（ＰｅｒｋｉｎＥｌｍｅｒ）など，多様な方法が本技術分野において周知であり，目的とする結合の検出に応じて適宜好ましい方法を採用することができる。 In this specification, whether the test antibody "binds (recognizes)" to the spike or RBD is determined by measuring the binding of the test antibody to the spike and RBD to be examined for binding by the following method. It can be done by Methods for measuring binding between antibodies and proteins or peptides include, for example, EIA method, ELISA method, FACS method, co-immunoprecipitation method, pull-down assay method, Western blotting method, homobifunctional crosslinker or heterobifunctional crosslinker Cross-linking method using linker, label transfer reaction method, interaction mapping method, surface plasmon resonance method, FRET (fluorescence resonance energy transfer) method, BIACORE method, AlphaPPI assay such as AlphaScreen (registered trademark) and AlphaLISA (registered trademark) (PerkinElmer), etc., and various methods are well known in the art, and suitable methods can be employed depending on the desired binding detection.
　本明細書において，「特異的に結合する（認識する）」とは，抗体が結合することが望ましくない物質又はその部分（例えば，人体内に存在するタンパク質等）には結合せず，ＳＡＲＳ－ＣｏＶ－２，スパイク，及び／又はＲＢＤに結合することを意味し，好ましくは，ＳＡＲＳ－ＣｏＶ－２に由来する物質又はその部分（スパイク，ＲＢＤなど）のみに結合することを意味する。必要に応じて，「特異的に結合する（認識する）」の語は，ＳＡＲＳ－ＣｏＶ－２が有する任意の物質又はその部分（例えば，ＮＴＤなど）には結合せず，ＲＢＤのみに結合すると解してもよい。被検抗体が，スパイク又はＲＢＤと「特異的に結合する（認識する）」か否かは，結合を調べようとするスパイク及びＲＢＤへの当該被検抗体の結合と，その他のペプチド，タンパク質又はその他の抗原（例えば，抗体が結合することが望ましくない物質）への当該被検抗体の結合とを調べることにより判定することができる。目的とするスパイク及び／又はＲＢＤと結合し，その他のペプチド，タンパク質，又はその他の抗原と結合しない場合，当該被検抗体は当該目的のスパイク及び／又はＲＢＤと特異的に結合する（認識する）と判定される。 As used herein, the term "specifically binds (recognizes)" does not bind to substances or parts thereof that are undesirable for antibodies to bind (for example, proteins present in the human body), and does not bind to SARS- It means binding to CoV-2, spike and/or RBD, preferably only to material derived from SARS-CoV-2 or parts thereof (spike, RBD etc.). Optionally, the term "specifically binds (recognizes)" means that SARS-CoV-2 does not bind to any substance or part thereof (e.g., NTD, etc.), but only to RBD. You can understand. Whether the test antibody "specifically binds (recognizes)" the spike or RBD depends on the binding of the test antibody to the spike and RBD whose binding is to be tested, and the binding of the test antibody to other peptides, proteins or It can be determined by examining the binding of the test antibody to other antigens (for example, substances to which the antibody should not bind). The test antibody specifically binds (recognizes) the spike and/or RBD of interest if it binds to the spike and/or RBD of interest and not to other peptides, proteins, or other antigens. is determined.
　本発明の抗体は，ＣＯＶＩＤ－１９感染履歴のある患者からリンパ球を採取し，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質（以下，「スパイク」という。），スパイクにおけるＮ末端ドメイン（以下，「ＮＴＤ」という），もしくはスパイクの受容体結合ドメイン（本明細書において，「ＲＢＤ」という）を抗原として，これらの抗原に結合するヒト抗体を産生するリンパ球を選択し，そのｃＤＮＡを得ることで，ヒト抗ＳＡＲＳ－ＣｏＶ－２モノクローナル抗体をリコンビナント抗体として作製することができる。すなわち，本方法により取得された抗体はいずれもＳＡＲＳ－ＣｏＶ－２スパイク，ＮＴＤ，もしくはＲＢＤに結合する抗体として取得されたものであり，これらのタンパク質に結合可能な抗体である。本段落において抗原として，好ましくは，ＳＡＲＳ－ＣｏＶ－２スパイクもしくはＲＢＤであり，より好ましくは，ＲＢＤである。 The antibody of the present invention collects lymphocytes from patients with a history of COVID-19 infection, SARS-CoV-2 spike protein (hereinafter referred to as "spike"), the N-terminal domain in the spike (hereinafter referred to as "NTD" ), or the spike receptor binding domain (herein, referred to as "RBD") as antigens, select lymphocytes that produce human antibodies that bind to these antigens, and obtain their cDNAs to obtain human anti- SARS-CoV-2 monoclonal antibodies can be produced as recombinant antibodies. That is, all of the antibodies obtained by this method are antibodies that bind to the SARS-CoV-2 spike, NTD, or RBD, and are antibodies capable of binding to these proteins. In this paragraph, the antigen is preferably SARS-CoV-2 spike or RBD, more preferably RBD.
　例えば，本発明の抗体は，ＩＳＡＡＣ法により取得することができる。ＩＳＳＡＣ法は，マイクロウェルアレイチップを用いて抗原特異的抗体分泌細胞（ＡＳＣ）を迅速かつ網羅的にスクリーニングする方法である。該方法においては，抗体産生細胞が１個入る程度の大きさ（直径１０～１５μｍ程度）のウエルを多数有するマイクロウェルアレイチップであって，ウエルの周辺のチップ表面を抗免疫グロブリン抗体（又は抗体を得ようとする抗原）で被覆したマイクロウェルアレイチップを用いる。特定の抗原に対する抗体を産生するヒトや動物から抗体産生細胞を含む末梢血リンパ球，脾臓細胞，または骨髄細胞を採取し，マイクロウェルアレイチップのウエルに細胞を１個ずつ播種する。培養液中にチップを浸し，分泌された抗体がウエルから前記被覆層に拡散する条件で培養することにより，抗体を産生分泌する細胞から分泌された抗体が，格納されたウエルの周囲の抗免疫グロブリン抗体（又は抗原）に結合する。次いで，蛍光物質で標識した前記抗原（又は蛍光物質で標識した抗体に対する抗体）をマイクロウェルアレイチップに添加する。標識した前記抗原（又は抗体に対する抗体）がウエルの周囲に分泌された抗体に結合し，標識物質から発せられるシグナルにより，どのウエル内に抗原特異的抗体を産生分泌する細胞が格納されているかがわかる。この細胞を蛍光顕微鏡下でマイクロキャピラリーを用いて回収する。細胞から核酸を抽出し，ＲＴ－ＰＣＲ等により抗体のｃＤＮＡを増幅し，抗体遺伝子をクローニングする。該遺伝子を用いて，所望の抗体をリコンビナント抗体として作製することができる。 For example, the antibody of the present invention can be obtained by the ISAAC method. The ISSAC method is a method for rapid and exhaustive screening of antigen-specific antibody-secreting cells (ASC) using a microwell array chip. In this method, a microwell array chip having a large number of wells with a size (about 10 to 15 μm in diameter) in which one antibody-producing cell can be accommodated, and an anti-immunoglobulin antibody (or antibody) on the chip surface around the wells Use a microwell array chip coated with the antigen for which you are trying to obtain Peripheral blood lymphocytes, spleen cells, or bone marrow cells containing antibody-producing cells are collected from humans or animals that produce antibodies against specific antigens, and the cells are seeded one by one in the wells of a microwell array chip. By immersing the chip in a culture medium and culturing under conditions in which the secreted antibodies diffuse from the wells to the coating layer, the antibodies secreted from the cells that produce and secrete the antibodies spread around the wells in which they were stored. Binds to globulin antibodies (or antigens). Next, the antigen labeled with a fluorescent substance (or an antibody against the antibody labeled with a fluorescent substance) is added to the microwell array chip. The labeled antigen (or antibody against the antibody) binds to the antibody secreted around the well, and the signal emitted from the labeled substance indicates which well contains the cells that produce and secrete the antigen-specific antibody. Recognize. The cells are harvested using a microcapillary under a fluorescence microscope. Nucleic acids are extracted from cells, antibody cDNA is amplified by RT-PCR or the like, and antibody genes are cloned. A desired antibody can be produced as a recombinant antibody using the gene.
　ＳＡＲＳ－ＣｏＶ－２は，ＲＢＤとヒト細胞表面のＡＣＥ２との結合を介してヒト細胞内に侵入する。よって，本発明の抗体は，好ましくは，ＳＡＲＳ－ＣｏＶ－２，又はそのスパイク若しくはＲＢＤと，ＡＣＥ２（好ましくは，ヒトＡＣＥ２，本明細書全体にわたって同様）との結合を阻害する抗体である。また，本発明の抗体は，好ましくは，変異型ＳＡＲＳ－ＣｏＶ－２，スパイク，若しくはＲＢＤと，ＡＣＥ２との結合を阻害する抗体である。ＡＣＥ２との結合を阻害する抗体は，好ましくは，武漢型ＳＡＲＳ－ＣｏＶ－２，そのスパイク若しくはＲＢＤと，ＡＣＥ２との結合も阻害する。また，本発明の抗体は，異なる変異パターンを有する複数種類のＳＡＲＳ－ＣｏＶ－２，スパイク，若しくはＲＢＤと，ＡＣＥ２との結合を阻害してもよい。 SARS-CoV-2 enters human cells through the binding of RBD to ACE2 on the surface of human cells. Thus, an antibody of the invention is preferably an antibody that inhibits binding of SARS-CoV-2, or its spike or RBD, to ACE2 (preferably human ACE2, as throughout the specification). Also, the antibody of the present invention is preferably an antibody that inhibits the binding of mutant SARS-CoV-2, spike, or RBD to ACE2. Antibodies that inhibit binding to ACE2 preferably also inhibit binding of Wuhan SARS-CoV-2, its spike or RBD, to ACE2. Also, the antibodies of the present invention may inhibit the binding of multiple types of SARS-CoV-2, spikes, or RBDs with different mutation patterns to ACE2.
　例えば，本抗体は，武漢型，Ｋ４１７Ｎ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異型，Ｎ４３９Ｋ変異型，Ｎ４４０Ｋ変異型，Ａ４７５Ｖ変異型，Ｅ４８４Ｋ変異型，Ｎ５０１Ｙ変異型，Ｌ４５２Ｒ変異型，Ｌ４５２Ｒ及びＥ４８４Ｑ変異型，Ｋ４１７Ｔ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異型，Ｌ４５２Ｒ及びＴ４７８Ｋ変異型，及び，Ｇ３３９Ｄ，Ｓ３７１Ｌ，Ｓ３７３Ｐ，Ｓ３７５Ｆ，Ｋ４１７Ｎ，Ｎ４４０Ｋ，Ｇ４４６Ｓ，Ｓ４７７Ｎ，Ｔ４７８Ｋ，Ｅ４８４Ａ，Ｑ４９３Ｒ，Ｇ４９６Ｓ，Ｑ４９８Ｒ，Ｎ５０１Ｙ，及びＹ５０５Ｈ変異型のＳＡＲＳ－ＣｏＶ－２，スパイク，若しくはＲＢＤから選択される１種類又はそれ以上，２種類又はそれ以上，３種類又はそれ以上，４種類又はそれ以上，５種類又はそれ以上，６種類又はそれ以上，７種類又はそれ以上，あるいは全てとＡＣＥ２との結合を阻害してもよい。これに加えて，本発明の抗体は，Ｇ３３９Ｄ，Ｓ３７１Ｌ，Ｓ３７３Ｐ，Ｓ３７５Ｆ，Ｋ４１７Ｎ，Ｋ４１７Ｔ，Ｎ４３９Ｋ，Ｎ４４０Ｋ，Ｇ４４６Ｓ，Ｌ４５２Ｒ，Ａ４７５Ｖ，Ｓ４７７Ｎ，Ｔ４７８Ｋ，Ｅ４８４Ａ，Ｅ４８４Ｋ，Ｅ４８４Ｑ，Ｑ４９３Ｒ，Ｇ４９６Ｓ，Ｑ４９８Ｒ，Ｎ５０１Ｙ，及びＹ５０５Ｈから選択される１種類又はそれ以上の変異を有する，ＳＡＲＳ－ＣｏＶ－２，スパイク，若しくはＲＢＤの，１種類又はそれ以上，２種類又はそれ以上，３種類又はそれ以上，４種類又はそれ以上，５種類又はそれ以上，６種類又はそれ以上，あるいは７種類又はそれ以上とＡＣＥ２との結合を阻害してもよい。あるいは，本発明の抗体は，上述の武漢型，Ａｌｐｈａ株，Ｂｅｔａ株，Ｇａｍｍａ株，Ｅｐｓｉｌｏｎ株，Ｋａｐｐａ株，Ｄｅｌｔａ株，及びＯｍｉｃｒｏｎ株から選択される１種類又はそれ以上，２種類又はそれ以上，３種類又はそれ以上，４種類又はそれ以上，又は全てのＳＡＲＳ－ＣｏＶ－２，スパイク，若しくはＲＢＤとＡＣＥ２との結合を阻害してもよい。 For example, the antibody includes Wuhan, K417N, E484K, and N501Y variants, N439K variant, N440K variant, A475V variant, E484K variant, N501Y variant, L452R variant, L452R and E484Q variant, K417T, E484K and N501Y mutants, L452R and T478K mutants, and SARS-H mutants of G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 selected from CoV-2, spike, or RBD Or more, or all, binding to ACE2 may be inhibited. Additionally, the antibodies of the present invention are , and Y505H, one or more, two or more, three or more, four or Binding of ACE2 to more, 5 or more, 6 or more, or 7 or more may be inhibited. Alternatively, the antibody of the present invention may be one or more, two or more selected from the above Wuhan type, Alpha strain, Beta strain, Gamma strain, Epsilon strain, Kappa strain, Delta strain, and Omicron strain, Binding of 3 or more, 4 or more, or all SARS-CoV-2, spike, or RBD to ACE2 may be inhibited.
　被検抗体がＳＡＲＳ－ＣｏＶ－２のＡＣＥ２タンパク質への結合を阻害するか否かは，例えば，試験しようとするＳＡＲＳ－ＣｏＶ－２スパイク又はＲＢＤを固層化したウエルに被検抗体と標識ＡＣＥ２との混合溶液，もしくは被検抗体を含まない標識ＡＣＥ２溶液を添加し，一定時間静置後に洗浄し，固層化された前記ＳＡＲＳ－ＣｏＶ－２スパイク又はＲＢＤに結合したＡＣＥ２の標識を測定することにより決定することができる。抗体を含まない場合の標識の強度等と比較して，抗体を含んだ場合の標識の強度が弱い場合には，当該被検抗体は当該ＳＡＲＳ－ＣｏＶ－２のＡＣＥ２への結合を阻害する（中和する）と判定することができる。例えば，ｉｎ　ｖｉｔｒｏにおける当該阻害実験のＩＣ５０は，２０μｇ／ｍｌ以下，１０μｇ／ｍｌ以下，５μｇ／ｍｌ以下，１μｇ／ｍｌ以下，０．６μｇ／ｍｌ以下，０．５μｇ／ｍｌ以下，０．３５μｇ／ｍｌ以下，０．２μｇ／ｍｌ以下，０．１５μｇ／ｍｌ以下とすることができ，あるいは，０．１～２０μｇ／ｍｌ，０．２～１０μｇ／ｍｌ，０．２～５μｇ／ｍｌ，０．２～１μｇ／ｍｌとすることができる。 Whether the test antibody inhibits the binding of SARS-CoV-2 to the ACE2 protein can be determined, for example, by adding the test antibody and labeled ACE2 to wells immobilized with SARS-CoV-2 spikes or RBDs to be tested. or a labeled ACE2 solution containing no test antibody is added, washed after standing for a certain period of time, and the ACE2 label bound to the immobilized SARS-CoV-2 spike or RBD is measured. can be determined by If the intensity of labeling with antibodies is weaker than the intensity of labeling without antibodies, the test antibody inhibits the binding of SARS-CoV-2 to ACE2 ( neutralize). For example, the IC50 of the inhibition experiment in vitro is 20 μg/ml or less, 10 μg/ml or less, 5 μg/ml or less, 1 μg/ml or less, 0.6 μg/ml or less, 0.5 μg/ml or less, 0.35 μg/ml ml or less, 0.2 μg/ml or less, 0.15 μg/ml or less, or 0.1 to 20 μg/ml, 0.2 to 10 μg/ml, 0.2 to 5 μg/ml, 0.1 μg/ml or less. It can be 2-1 μg/ml.
　好ましくは，本発明の抗体は，ＳＡＲＳ－ＣｏＶ－２のスパイクを外套したシュードタイプウイルスによる，ＡＣＥ２及びＴＭＰＲＳＳ２共発現細胞への感染を抑制する。例えば，本発明の抗体は，ＳＡＲＳ－ＣｏＶ－２のスパイクを外套したシュードタイプウイルス（ＳＡＲＳ－ＣｏＶ－２　Ｓｔ１９ｐｖ）（Ｔａｎｉ　Ｈ　ｅｔ　ａｌ．，Ｖｉｒｏｌｏｇｙ　Ｊｏｕｒｎａｌ（２０２１）１８：１６．に記載されたシュードタイプウイルス）による，ＴＭＰＲＳＳ２発現ＶｅｒｏＥ６細胞への感染を抑制する。被検抗体が，ＳＡＲＳ－ＣｏＶ－２　Ｓｔ１９ｐｖによる，ＴＭＰＲＳＳ２発現ＶｅｒｏＥ６細胞への感染を抑制するか否かは，例えば，ＴＭＰＲＳＳ２を過剰発現しているＶｅｒｏＥ６細胞（ＶｅｒｏＥ６／ＴＭＰＲＳＳ２）に被検抗体の存在下又は非存在下でＳＡＲＳ－ＣｏＶ－２　Ｓｔ１９ｐｖを添加し，１日培養後，細胞に細胞溶解液を含むルシフェラーゼ基質溶液を添加し，ルシフェラーゼの活性を測定することにより決定することができる。抗体非添加の場合と比較して，抗体添加においてルシフェラーゼ活性を示す発光値が低い場合には，抗体により感染が阻害されたと判定される。 Preferably, the antibodies of the present invention suppress infection of ACE2 and TMPRSS2 co-expressing cells by SARS-CoV-2 spike-coated pseudotype viruses. For example, the antibodies of the present invention can be used to detect SARS-CoV-2 spike-enveloped pseudotype virus (SARS-CoV-2 St19pv) (Tani H et al., Virology Journal (2021) 18:16). type virus) to TMPRSS2-expressing VeroE6 cells. Whether the test antibody suppresses the infection of TMPRSS2-expressing VeroE6 cells by SARS-CoV-2 St19pv can be determined, for example, by the presence of the test antibody in VeroE6 cells (VeroE6/TMPRSS2) overexpressing TMPRSS2. It can be determined by adding SARS-CoV-2 St19pv in the presence or absence of SARS-CoV-2 St19pv, culturing for 1 day, adding a luciferase substrate solution containing a cell lysate to the cells, and measuring luciferase activity. When the luminescence value indicating luciferase activity is lower in the presence of antibody than in the case of non-addition of antibody, it is judged that the infection was inhibited by the antibody.
　本発明の抗体は，好ましくは，ＳＡＲＳ－ＣｏＶ－２のスパイクを外套したシュードタイプウイルスによる，ＡＣＥ２及びＴＭＰＲＳＳ２共発現細胞への感染抑制実験において，５００ｎｇ／ｍｌ以下のＩＣ５０値を示し，より好ましくは，１００ｎｇ／ｍｌ以下，５０ｎｇ／ｍｌ以下，又は４５ｎｇ／ｍｌ以下のＩＣ５０値を示す抗体であってもよい。例えば，本発明の抗体は，武漢型（ＷＴ）に対して３０ｎｇ／ｍｌ以下，Ａｌｐｈａ株に対して４５ｎｇ／ｍｌ以下，Ｂｅｔａ株に対して１５ｎｇ／ｍｌ以下，Ｋａｐｐａ株に対して２０ｎｇ／ｍｌ以下，及び／又は，Ｄｅｌｔａ株に対して１２ｎｇ／ｍｌ以下のＩＣ５０値を示す抗体であってもよい。 The antibody of the present invention preferably exhibits an IC50 value of 500 ng/ml or less in an experiment to suppress infection of ACE2 and TMPRSS2 co-expressing cells with a SARS-CoV-2 spike-enveloped pseudotype virus, more preferably , 100 ng/ml or less, 50 ng/ml or less, or 45 ng/ml or less. For example, the antibody of the present invention is 30 ng/ml or less against Wuhan type (WT), 45 ng/ml or less against Alpha strain, 15 ng/ml or less against Beta strain, and 20 ng/ml or less against Kappa strain. , and/or an antibody that exhibits an IC50 value of 12 ng/ml or less against the Delta strain.
　また，好ましくは，本発明の抗体は，ＳＡＲＳ－ＣｏＶ－２による，ＡＣＥ２及びＴＭＰＲＳＳ２共発現細胞への感染を抑制する抗体である。被検抗体が，ＳＡＲＳ－ＣｏＶ－２による，ＡＣＥ２及びＴＭＰＲＳＳ２共発現細胞への感染を抑制するか否かは，被検抗体とＳＡＲＳ－ＣｏＶ－２をチューブ内で混合し，３７℃で１時間反応させた後，ＶｅｒｏＥ６／ＴＭＰＲＳＳ２細胞に添加し，３７℃で２時間吸着させ，その後，吸着しなかった被検抗体とウイルスを含む混合液を完全に除き，再び被検抗体を含む培養培地を添加し，２日間培養した細胞の培養上清のウイルス感染価をプラークアッセイ法により測定することにより決定することができる。被検抗体非存在下と比較して，被検抗体存在下においてプラークが減少するか，又はプラークが存在しない場合には，当該被検抗体はＳＡＲＳ－ＣｏＶ－２による，ＡＣＥ２及びＴＭＰＲＳＳ２共発現細胞への感染を抑制すると判定される。あるいは，被検抗体が，ＳＡＲＳ－ＣｏＶ－２による，ＡＣＥ２及びＴＭＰＲＳＳ２共発現細胞への感染を抑制するか否かは，ＶｅｒｏＥ６／ＴＭＰＲＳＳ２細胞に被検抗体を添加してＳＡＲＳ－ＣｏＶ－２を感染させ，３７℃で１時間の培養後に培養上清を除き，再び被検抗体を添加して２４時間培養し，培養した細胞の培養上清のウイルス感染価をリアルタイムＰＣＲ法によりウイルスゲノムＲＮＡ量を測定することにより決定することができる。被検抗体存在下においてウイルスゲノムＲＮＡ量が減少するか，又は存在しない場合には，当該被検抗体はＳＡＲＳ－ＣｏＶ－２による，ＡＣＥ２及びＴＭＰＲＳＳ２共発現細胞への感染を抑制すると判定される。 Also, preferably, the antibody of the present invention is an antibody that suppresses infection of ACE2 and TMPRSS2 co-expressing cells by SARS-CoV-2. Whether the test antibody suppresses the infection of ACE2 and TMPRSS2 co-expressing cells by SARS-CoV-2 is determined by mixing the test antibody and SARS-CoV-2 in a tube and incubating at 37°C for 1 hour. After reacting, it was added to VeroE6/TMPRSS2 cells and allowed to adsorb for 2 hours at 37°C. After that, the mixture containing the unadsorbed test antibody and virus was completely removed, and the culture medium containing the test antibody was added again. It can be determined by measuring the virus infectivity titer of the culture supernatant of the cells added and cultured for 2 days by plaque assay. Compared to the absence of the test antibody, plaques are reduced in the presence of the test antibody, or if no plaques are present, the test antibody is SARS-CoV-2, ACE2 and TMPRSS2 co-expressing cells It is determined that it suppresses the infection of Alternatively, whether the test antibody suppresses the infection of ACE2 and TMPRSS2 co-expressing cells by SARS-CoV-2 is confirmed by adding the test antibody to VeroE6/TMPRSS2 cells to infect SARS-CoV-2. After culturing at 37°C for 1 hour, remove the culture supernatant, add the test antibody again and culture for 24 hours. It can be determined by measuring. If the amount of viral genomic RNA decreases in the presence of the test antibody, or if it is absent, it is determined that the test antibody suppresses SARS-CoV-2 infection of ACE2 and TMPRSS2 co-expressing cells.
　さらに，好ましくは，本発明の抗体は，Ｉｎ　ｖｉｖｏの試験，例えばハムスターモデルにおいて，抗体の予防的投与によりＳＡＲＳ－ＣｏＶ－２のスパイクを外套したシュードタイプウイルスによる肺への感染を抑制する抗体である。被検抗体が感染を抑制するか否かは，例えばハムスターに被検抗体を腹腔内投与した２４時間後に，シュードタイプウイルスを気管内に直接接種し，２４時間後に肺を採取してシフェラーゼ基質溶液とインキュベートし，ルシフェラーゼの活性を測定することにより決定することができる。抗体非投与の場合と比較して，抗体投与においてルシフェラーゼ活性を示す発光値が低い場合には，抗体により感染が阻害されたと判定される。 Further, preferably, the antibody of the present invention is an antibody that suppresses pulmonary infection by SARS-CoV-2 spike-enveloped pseudotype virus by prophylactic administration of the antibody in an in vivo test, such as a hamster model. be. Whether or not the test antibody suppresses infection can be determined, for example, by inoculating the pseudotype virus directly into the trachea 24 hours after intraperitoneal administration of the test antibody to hamsters, and after 24 hours, extracting the lungs and adding a luciferase substrate solution. can be determined by incubating with and measuring luciferase activity. When the luminescence value, which indicates luciferase activity, is lower after antibody administration than when antibody is not administered, it is determined that infection is inhibited by antibody.
　通常は，抗体による阻害や抑制は完全な阻害や抑制をもたらすものではなく，よって，本明細書における，「結合を阻害する」や「感染を抑制する」の語は，１００％の阻害や抑制をもたらすことを意味するものではない。すなわち，「結合を阻害する」や「感染を抑制する」とは，当該抗体が存在しない場合又はＳＡＲＳ－ＣｏＶ－２とは結合しない抗体が存在する場合と比較して，結合が阻害されること，又は感染が抑制されることを意味するものである。 Generally, inhibition or suppression by antibodies does not result in complete inhibition or suppression, therefore, the terms "inhibit binding" and "inhibit infection" herein refer to 100% inhibition or suppression. is not meant to bring In other words, "inhibit binding" or "inhibit infection" means that binding is inhibited compared to the absence of the antibody or the presence of an antibody that does not bind to SARS-CoV-2. , or that the infection is suppressed.
　本発明の抗体は，本発明の抗体はヒト抗体の他，該ヒト抗体の一部（例えば，定常領域，フレームワーク領域，又はその一部）を非ヒト動物の抗体由来の配列と置き換えた動物化抗体であってもよい。非ヒト動物としては，例えば，マウス，ラット，ハムスター，モルモット，ラビット，イヌ，ネコ，サル，ヒツジ，ヤギ，ラクダ，ニワトリ，アヒル等の抗体を挙げることができる。 The antibody of the present invention is a human antibody, or an animal in which a part of the human antibody (e.g., constant region, framework region, or part thereof) is replaced with a sequence derived from a non-human animal antibody It may be a modified antibody. Examples of non-human animals include antibodies from mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, monkeys, sheep, goats, camels, chickens and ducks.
　本発明の抗体のイムノグロブリンクラスは特に限定されるものではなく，ＩｇＧ，ＩｇＭ，ＩｇＡ，ＩｇＥ，ＩｇＤ，又はＩｇＹのいずれのイムノグロブリンクラス（アイソタイプ）であってもよく，好ましくはＩｇＧである。また，本発明の抗体がＩｇＧの場合，いずれのサブクラス（ＩｇＧ１，ＩｇＧ２，ＩｇＧ３，又はＩｇＧ４）であってもよい。また，本発明の抗体は，モノスペシフィック，バイスペシフィック（二重特異性抗体），トリスペシフィック（三重特異性抗体）（例えば，ＷＯ１９９１／００３４９３号），又はマルチスペシフィック（多重特異性抗体）であってもよい。また，Ｆｃ領域は抗体の機能性を向上させるための変異が導入されていてもよく，例えば，一態様において，ＡＤＥ活性を低下させるためＦｃ領域にＬＡＬＡ変異（Ｌ２３４Ｆ，Ｌ２３５Ｅ変異）が修飾された抗体であってもよい。さらに，一態様において，Ｆｃ領域に抗体の半減期を伸ばすことが報告されているＭ４２８Ｌ及びＮ４３４Ｓ変異や，Ｍ２５２Ｙ，Ｓ２５４Ｔ及びＴ２５６Ｅ変異，Ｔ２５Ｑ及びＭ４２８Ｌ変異が導入された抗体であってもよい。 The immunoglobulin class of the antibody of the present invention is not particularly limited, and may be any immunoglobulin class (isotype) of IgG, IgM, IgA, IgE, IgD, or IgY, preferably IgG. Moreover, when the antibody of the present invention is IgG, it may be of any subclass (IgG1, IgG2, IgG3, or IgG4). In addition, the antibody of the present invention is monospecific, bispecific (bispecific antibody), trispecific (trispecific antibody) (for example, WO1991/003493), or multispecific (multispecific antibody) good too. In addition, the Fc region may be mutated to improve the functionality of the antibody, for example, in one embodiment, the Fc region is modified with LALA mutations (L234F, L235E mutations) to reduce ADE activity It may be an antibody. Furthermore, in one embodiment, the antibody may be an antibody introduced with M428L and N434S mutations, M252Y, S254T and T256E mutations, T25Q and M428L mutations, which are reported to extend antibody half-life in the Fc region.
　抗体はその可変領域（特には，ＣＤＲｓ）が結合特性を付与していることが知られており，完全抗体でない抗体断片であってもその結合特性を利用可能であることが当業者に広く知られている。本明細書において，「抗原結合性断片」とは，抗体の一部分（部分断片）を含むタンパク質又はペプチドであって，抗体の抗原への作用（抗原結合性）を保持するタンパク質又はペプチドを意味する。このような抗原結合性断片としては，例えば，Ｆ（ａｂ’）２，Ｆａｂ’，Ｆａｂ，Ｆａｂ３，一本鎖Ｆｖ（以下，「ｓｃＦｖ」という），（タンデム）バイスペシフィック一本鎖Ｆｖ（ｓｃ（Ｆｖ）２），一本鎖トリプルボディ，ナノボディ，ダイバレントＶＨＨ，ペンタバレントＶＨＨ，ミニボディ，（二本鎖）ダイアボディ，タンデムダイアボディ，バイスペシフィックトリボディ，バイスペシフィックバイボディ，デュアルアフィニティリターゲティング分子（ＤＡＲＴ），トリアボディ（又はトリボディ），テトラボディ（又は［ｓｃ（Ｆｖ）２］２），若しくは（ｓｃＦｖ－ＳＡ）４）ジスルフィド結合Ｆｖ（以下，「ｄｓＦｖ」という），コンパクトＩｇＧ，重鎖抗体，又はそれらの重合体を挙げることができる（Ｎａｔｕｒｅ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，　２９（１）：５－６　（２０１１）；Ｍａｎｅｅｓｈ　Ｊａｉｎ　ｅｔ　ａｌ．，　ＴＲＥＮＤＳ　ｉｎ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，　２５（７）（２００７）：３０７－３１６；及び，Ｃｈｒｉｓｔｏｐｈ　Ｓｔｅｉｎら，Ａｎｔｉｂｏｄｉｅｓ（１）：８８－１２３（２０１２）参照）。本明細書において，抗原結合性断片は，モノスペシフィック，バイスペシフィック（二重特異性），トリスペシフィック（三重特異性），及びマルチスペシフィック（多重特異性）のいずれであってもよい。本明細書において，特にそのように解釈することが不整合である場合を除き，「抗体」の語は抗体の抗原結合性断片をも含むことを意図している。 Antibodies are known to have binding properties conferred by their variable regions (especially CDRs), and it is widely known to those skilled in the art that even antibody fragments that are not complete antibodies can utilize these binding properties. It is As used herein, the term "antigen-binding fragment" refers to a protein or peptide containing a portion (partial fragment) of an antibody and retaining the action (antigen-binding) of the antibody on the antigen. . Examples of such antigen-binding fragments include F(ab′) 2 , Fab′, Fab, Fab 3 , single-chain Fv (hereinafter referred to as “scFv”), (tandem) bispecific single-chain Fv ( sc(Fv) 2 ), single-chain triple body, nanobody, divalent VHH, pentavalent VHH, minibody, (double-stranded) diabodies, tandem diabodies, bispecific tribodies, bispecific bibodies, dual affinity targeting molecules (DART), triabodies (or tribodies), tetrabodies (or [sc(Fv) 2 ] 2 ), or (scFv-SA) 4 ) disulfide-bonded Fv (hereinafter referred to as “dsFv”), compact IgG, heavy chain antibodies, or polymers thereof (Nature Biotechnology, 29(1): 5-6 (2011); Maneesh Jain et al., TRENDS in Biotechnology, 25(7) (2007): 307- 316; and Christoph Stein et al., Antibodies (1):88-123 (2012)). As used herein, antigen-binding fragments may be monospecific, bispecific (bispecific), trispecific (trispecific), and multispecific (multispecific). As used herein, the term "antibody" is also intended to include antigen-binding fragments of antibodies, unless otherwise construed as such.
　一態様において，本発明は，以下に記載する重鎖ＣＤＲ及び軽鎖ＣＤＲの少なくとも１つ，（好ましくは，ＣＤＲＨ３，更に好ましくは６つ全ての組み合わせ）を有し，かつ，ＳＡＲＳ－ＣｏＶ－２スパイク及び／又はＲＢＤ（好ましくはＳＡＲＳ－ＣｏＶ－２　ＲＢＤ）に特異的に結合する抗体である。 In one aspect, the invention has at least one of the heavy chain CDRs and light chain CDRs described below (preferably CDRH3, more preferably a combination of all six), and SARS-CoV-2 An antibody that specifically binds to spike and/or RBD (preferably SARS-CoV-2 RBD).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　一態様において，本発明は，以下に記載する重鎖可変領域及び軽鎖可変領域の組み合わせを有する抗体である。 In one aspect, the present invention is an antibody having a combination of heavy chain variable regions and light chain variable regions described below.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
　また，本発明は，表２のいずれかのクローンと８０％以上の同一性を有する抗体，すなわち，表２のいずれかのクローンについて記載された重鎖可変領域（ＶＨ）アミノ酸配列と８０％以上の同一性を有するアミノ酸配列のＶＨ，及び，当該クローンについて表２に記載された軽鎖可変領域（ＶＬ）アミノ酸配列と８０％以上の同一性を有するアミノ酸配列のＶＬを有する抗体，あるいはその抗原結合性断片に関する。アミノ酸配列の同一性は，２種類のタンパク質間において，比較対象とするアミノ酸配列範囲における種類が同一なアミノ酸数の割合（％）を意味し，例えば，ＢＬＡＳＴ，ＦＡＳＴＡ等の公知のプログラムを用いて決定することができる。上述のＶＨ及ＶＬが表２に記載の配列との間で有する同一性は，８０％以上の他，８５％以上，９０％以上，９５％以上，９８％以上，又は９９％以上であってもよい。 In addition, the present invention provides an antibody having 80% or more identity with any clone in Table 2, that is, 80% or more with the heavy chain variable region (VH) amino acid sequence described for any clone in Table 2 and an amino acid sequence VL having 80% or more identity with the light chain variable region (VL) amino acid sequence listed in Table 2 for the clone, or an antigen thereof Regarding binding fragments. Amino acid sequence identity means the ratio (%) of the number of amino acids of the same type in the range of amino acid sequences to be compared between two proteins, for example, using known programs such as BLAST and FASTA can decide. The identity of the above VH and VL with the sequences listed in Table 2 is 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more. good too.
　表２のいずれかのクローンと８０％以上の同一性を有する抗体は，好ましくは，表１において同一クローン番号で示された抗体の少なくとも一つのＣＤＲ（好ましくはＣＤＲＨ３，更に好ましくは６つの全てのＣＤＲ）を有する。また，表２のいずれかのクローンと８０％以上の同一性を有する抗体は，好ましくは，ＳＡＲＳ－ＣｏＶ－２スパイク及びＲＢＤに特異的に結合する。 Antibodies with 80% or more identity to any clone in Table 2 preferably have at least one CDR (preferably CDRH3, more preferably all six) of the antibody indicated by the same clone number in Table 1. CDRs). Antibodies with 80% or more identity to any of the clones in Table 2 also preferably bind specifically to the SARS-CoV-2 spike and RBD.
　例えば，本発明の抗体は以下の重鎖（ＨＣ）アミノ酸配列の重鎖，及び軽鎖（ＬＣ）アミノ酸配列の軽鎖を有する抗体であってもよい。以下において，下線はシグナル配列を示す。よって，成熟タンパク質としての抗体は，下線部分のアミノ酸配列は有さなくてもよい。
 
1028-05K　HC（配列番号２）
MDTLCYTLLLLTTPSWVLSQVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVSWIRQPPGKALEWLAHIFSNDEKSYSTSLKRRLTISKDTPKSQVVLTMTNMDPVDTATYYCARTLGFWSGEYYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-05K　LC（配列番号１３）
MEAPAQLLFLLLLWLPDTTGEIVMTQSPATLSVSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQHYNNWPPLFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
1028-25K　HC（配列番号２４）
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASEFTFSGYAMHWVRQAPGKGLEWVAVISYDGINKYYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARDPDYGDEGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-25K　LC（配列番号３５）
MRLPAQLLGLLMLWVPGSSGDVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQGTHWPPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
1028-29K　HC（配列番号４６）
MDWTWRVFCLLAVAPGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGTPDIVVVPAAPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-29K　LC（配列番号５７）
MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDVSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPVLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
1028-30K　HC（配列番号６８）
MDWTWRFLFVVAAATGVQSQVQLVQSGAEVKKPGSSVKVSCKASGGTFNTYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGEPGYCSGGNCYSSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-30K　LC（配列番号７９）
MRLPAQLLGLLMLWVPGSSGDVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
1028-37K　HC（配列番号９０）
MSVSFLIFLPVLGLPWGVLSQVQLQQSGPGLVKPSQTLSLTCDISGDSVSSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCASGGYQLLYDDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-37K　LC（配列番号１０１）
MEAPAQLLFLLLLWLPDTTGEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYSNWLLYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
1028-50K　HC（配列番号１１２）
MELGLRWVFLVAILEGVQCEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYIMNWVRQAPGKGLEWVSSISSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARIVGATSYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-50K　LC（配列番号１２３）
MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
1028-55K　HC（配列番号１３４）
MEFGLSWVFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFIVSSNYMSWVRQAPGKGLEWVSLIYSGGSTYYVDSVKGRFTISRDNTKNTLYLQMNSLRAEDTAVYYCARDLDVRGALDIWGQGTMVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-55K　LC（配列番号１４５）
METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYFCQQYGSSPQTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
1028-61L　HC（配列番号１５６）
MEFWLSWVFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGIDVSSNYMSWVRQAPGKGLEWVSVIYSGGSTYYADAVRGRFTISRHNSKNTLYLQMNSLRAEDAAVYYCARDAQRYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-61L　LC（配列番号１６７）
MAWTFLLLGLLSHCTASVTSYVLTQPPSVSVAPGKTARITCGGNNIGSNTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQLWDSSSDHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
 
1028-62L　HC（配列番号１７８）
MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSNNQFSLKLSSVTAADTAVYYCARGWNYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-62L　LC（配列番号１８９）
MAWALLLLTLLTQDTGSWAQSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSSTWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
 
1028-65K　HC（配列番号２００）
MDWTWRVFCLLAVAPGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFSSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYFCAREGRAAAETDNNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-65K　LC（配列番号２１１）
MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
1028-68L　HC（配列番号２２２）
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARADGSGWNYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-68L　LC（配列番号２３３）
MAWTFLLLGLLTLCTGSEASYELTQPPSVSVSPGQTARITCSGDPLPKQYAYWYQQKPGQAPVLVIYKDSERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQSADSSGTYGWEFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
 
1028-84K　HC（配列番号２４４）
MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYAMHWVFQAPGMGLEWVAVISYDGINKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHRDDYVDSADAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-84K　LC（配列番号２５５）
MRLPAQLLGLLMLWVPGSSGDVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
1028-87L　HC（配列番号２６６）
MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLASFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-87L　LC（配列番号２７７）
MPWALLLLTLLTHSAVSVVQAGLTQPPSVSKGLRQTATLTCTGNSNNVGNQGAAWLQQHQGHPPKLLSYRNNNRPSGISERLSASRSGNTASLTITGLQPEDEADYYCSAWDSSLSAQVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
 
1028-106K　HC（配列番号２８８）
MDLMCKKMKHLWFFLLLVAAPRWVLSQLQLQESGPGLVKPSETLSLTCTVSGGSISSSYHYWGWIRQPPGKGLEWIGTIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRGYSYDSSGDGVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-106K　LC（配列番号２９９）
MDMRVPAQLLGLLLLWFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
1028-121K　HC（配列番号３１０）
MDTLCYTLLLLTTPSWVLSQVTLKESGPVLVKPTETLTLTCTVSGFSLSDARMGVSWIRQPPGKALEWLAHIFSNDEKSYNTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARALGFWSGYYYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-121K　LC（配列番号３２１）
MEAPAQLLFLLLLWLPDTTGEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPPLFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
1028-137K　HC（配列番号３３２）
MEFGLSWVFLVALLRGVQCQVHLVESGGGVVQPGRSLRLSCAASGFTFNSYAMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDDSGSYLYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-137K　LC（配列番号３４３）
MRLPAQLLGLLMLWVPGSSGDVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLTWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
1028-164L　HC（配列番号３５４）
MDILCSTLLLLTVPSWVLSQVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMCVSWIRQPPGKALEWLALIDWDDDKYYSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYYCARIPVEMATIGDYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1028-164L　LC（配列番号３６５）
MAGFPLLLTLLTHCAGSWAQSVLTQPPSESGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
 
0210-19K　HC（配列番号３７６）
MSVSFLIFLPVLGLPWGVLSQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQLSLQLNSVTPEDTAVYYCARSPSYQLLYPYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
0210-19K　LC（配列番号３８７）
MDMRVPAQLLGLLLLWLPGTRCDIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPELLLYAASRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYYSTPYTFGQGTKLEIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
0210-20L　HC（配列番号３９８）
MDWTWRFLFVVAAATGVQSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARALGYSGYGSTYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
0210-20L　LC（配列番号４０９）
MAWSPLLLTLLAHCTGSWAQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
 
0210-28K　HC（配列番号４２０）
MDWIWRILFLVGAATGAHSQMQLVQSGPEVKKPGTSVKVSCKASGFTFSISAVQWVRQARGQRLEWIGWIVVGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRFEDTAVYYCAAPYCGGDCSDGFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
0210-28K　LC（配列番号４３１）
METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGNSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
0210-30L　HC（配列番号４４２）
MDWTWRFLFVVAAATGVQSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGRIIPILGRANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNPEEFSSSYVYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
0210-30L　LC（配列番号４５３）
MAWALLLLSLLTQGTGSWAQSALTQPRSVSGSPGQSVTISCTGTSSDVGGYSYVSWYQQHPDKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSYTSWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
 
0210-31L　HC（配列番号４６４）
MEFGLSWVFLVAILKGVQCEVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWVRQAPGKGLEWVGFIRSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDSITMVQGVIIRLDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
0210-31L　LC（配列番号４７５）
MAWAPLLLTLLAHCTGSWANFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNKVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
 
0210-35K　HC（配列番号４８６）
MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIFYSGSANYNPSLRSRVTISVDTSKNHFSLKLSSVTAADTAVYYCARDIGYDSSGPDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
0210-35K　LC（配列番号４９７）
MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPGTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
0210-38L　HC（配列番号５０８）
MELGLSWVFLVAILEGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFNSYWMSWVRQAPGKGLEWVADIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYYGPIDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
0210-38L　LC（配列番号５１９）
MAWAPLLLTLLAHCTGSWANFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSNNRVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
 
0210-52L　HC（配列番号５３０）
MGSTAILALLLAVLQGVCAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKFISTAYLQWSSLKASDTAMYYCARRSGYDLASSHNWLDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
0210-52L　LC（配列番号５４１）
MAWSPLLLTLLAHCTGSWAQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDNTLSGSLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
 
0210-58K　HC（配列番号５５２）
MDTLCYTLLLLTTPSWVLSQVTLKESGPVLVKPTETLTLTCTVSGFSLSNAKMGVSWIRQPPGKALEWLAHIFSGDEKSYSTSLKSRLTISKDTSKSQVVVTMTNMDPVDTATYYCARILWFRSYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
0210-58K　LC（配列番号５６３）
MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
1-15　HC（配列番号５７４）
MELGLSWVFLVAILEGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHWVRQATGKGLEWVSTIGTAGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARGIQQQLALRYYYYLDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1-15　LC（配列番号５８５）
MRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSNPTWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 
1-44　HC（配列番号５９６）
MEFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLLMNSLRAEDTAVYYCAKDGEWYYDFWSGPVYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
 
1-44　LC（配列番号６０７）
MDMRVPAQLLGLLLLWLPGAKCDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC For example, an antibody of the invention can be an antibody having a heavy chain with the following heavy chain (HC) amino acid sequence and a light chain with the light chain (LC) amino acid sequence: In the following, underlining indicates the signal sequence. Therefore, an antibody as a mature protein may not have the underlined amino acid sequences.

1028-05K HC (SEQ ID NO:2)
MDTLCYTLLLLTTPSWVLS QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVSWIRQPPGKALEWLAHIFSNDEKSYSTSLKRRLTISKDTPKSQVVLTMTNMDPVDTATYYCARTLGFWSGEYYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-05K LC (SEQ ID NO: 13)
MEAPAQLLFLLLLWLPDTTG EIVMTQSPATLSVSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQHYNNWPPLFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGCSPVTKQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTGLGENRHQ

1028-25K HC (SEQ ID NO:24)
MEFGLSWVFLVALLRGVQC QVQLVESGGGVVQPGRSLRLSCAASEFTFSGYAMHWVRQAPGKGLEWVAVISYDGINKYYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARDPDYGDEGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-25K LC (SEQ ID NO:35)
MRLPAQLLGLLMLWVPGSSG DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQGTHWPPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKKTKVDNALQSGNSQESVTEQDSKDSTYSLSVSSTLGSGLACEDYVTSFCPVSSTLKAHQHQ

1028-29K HC (SEQ ID NO:46)
MDWTWRVFCLLAVAPGAHS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGTPDIVVVPAAPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-29K LC (SEQ ID NO:57)
MEAPAQLLFLLLLWLPDTTG EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDVSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPVLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSSSGTASVVCLLNNFYPREAKVQWKVDNALQSGPVNSTKQESVTEQDSKDSTYSLSSTLGENRCSFVTEKHKVYACENRCSFHQ

1028-30K HC (SEQ ID NO:68)
MDWTWRFLFVVAAATGVQS QVQLVQSGAEVKKPGSSVKVSCKASGGTFNTYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGEPGYCSGGNCYSSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-30K LC (SEQ ID NO:79)
MRLPAQLLGLLMLWVPGSSG DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKTKKVDNALQSGNSQESVTEQDSKDSTYSLSSTLSVTCPVSSTLGSPVACEDGEHQHK

1028-37K HC (SEQ ID NO:90)
MSVSFLIFLPVLGLPWGVLS QVQLQQSGPGLVKPSQTLSLTCDISGDSVSSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCASGGYQLLYDDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-37K LC (SEQ ID NO: 101)
MEAPAQLLFLLLLWLPDTTG EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYSNWLLYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSPVTKQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTGLGENRHQ

1028-50K HC (SEQ ID NO: 112)
MELGLRWVFLVAILEGVQC EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYIMNWVRQAPGKGLEWVSSISSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARIVGATSYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-50K LC (SEQ ID NO: 123)
MDMRVPAQLLGLLLLWLRGARC DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGPVNSTKQESVTEQDSKDSTYSLSSTLGENRCSFVTEKHKVYACE

1028-55K HC (SEQ ID NO: 134)
MEFGLSWVFLVAILKGVQC EVQLVESGGGLVQPGGSLRLSCAASGFIVSSNYMSWVRQAPGKGLEWVSLIYSGGSTYYVDSVKGRFTISRDNTKNTLYLQMNSLRAEDTAVYYCARDLDVRGALDIWGQGTMVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-55K LC (SEQ ID NO: 145)
METPAQLLFLLLLWLPDTTG EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYFCQQYGSSPQTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGPVNSQESVTEQDSKDSTYSLGLSSTLTLSKASFKDSTYSLGSTLTLSKASFKHKGSSTKVYHQ

1028-61L HC (SEQ ID NO: 156)
MEFWLSWVFLVAILKGVQC EVQLVESGGGLVQPGGSLRLSCAASGIDVSSNYMSWVRQAPGKGLEWVSVIYSGGSTYYADAVRGRFTISRHNSKNTLYLQMNSLRAEDAAVYYCARDAQRYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-61L LC (SEQ ID NO: 167)
MAWTFLLLGLLSHCTASVT SYVLTQPPSVVSVAPGKTARITCGGNNIGSNTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQLWDSSSDHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCTAGSTECSVTHETV

1028-62L HC (SEQ ID NO: 178)
MKHLWFFLLLVAAPRWVLS QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSNNQFSLKLSSVTAADTAVYYCARGWNYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-62L LC (SEQ ID NO: 189)
MAWALLLLTLLTQDTGSWA QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSSTWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQTEGSTVVTHE

1028-65K HC (SEQ ID NO:200)
MDWTWRVFCLLAVAPGAHS QVQLVQSGAEVKKPGASVKVSCKASGYTFSSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYFCAREGRAAAETDNNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-65K LC (SEQ ID NO:211)
MVLQTQVFISLLLWISGAYG DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKTKVDNALQSGNSQESVTEQDSKDSSFCPVSSKGLSSTLGSKACEDYVTSFCPVSSTLKAACEDY

1028-68L HC (SEQ ID NO:222)
MEFGLSWVFLVALLRGVQC QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARADGSGWNYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-68L LC (SEQ ID NO:233)
MAWTFLLLGLLTLCTGSEA SYELTQPPSVSVSPGQTARITCSGDPLPKQYAYWYQQKPGQAPVLVIYKDSERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQSADSSGTYGWEFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTTVQWKSTVGSHREKCSAPTECSTVGSHREKCQVTHE

1028-84K HC (SEQ ID NO:244)
MEFGLSWVFLVALLRGVQC QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYAMHWVFQAPGMGLEWVAVISYDGINKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHRDDYVDSADAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-84K LC (SEQ ID NO:255)
MRLPAQLLGLLMLWVPGSSG DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQPVTKKVDNALQSGNSQESVTEQDSKDSSFHSLSSTLGSGLSTLKAACEDY

1028-87L HC (SEQ ID NO:266)
MDWTWRILFLVAAATGAHS QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCLASFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-87L LC (SEQ ID NO:277)
MPWALLLLTLLTHSAVSVV QAGLTQPPSVSKGLRQTATLTCTGNSNNVGNQGAAWLQQHQGHPPKLLSYRNNNRPSGISERLSASRSGNTASLTITGLQPEDEADYYCSAWDSSLSAQVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRTECSTVEKVVTECSTVEKQ

1028-106K HC (SEQ ID NO:288)
MDLMCKKMKHLWFFLLLVAAPRWVLS QLQLQESGPGLVKPSETLSLTCTVSGGSISSSYHYWGWIRQPPGKGLEWIGTIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRGYSYDSSGDGVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-106K LC (SEQ ID NO:299)
MDMRVPAQLLGLLLLWFPGARC DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSPVTKQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTGLGENRHQ

1028-121K HC (SEQ ID NO:310)
MDTLCYTLLLLTTPSWVLS QVTLKESGPVLVKPTETLTLTCTVSGFSLSDARMGVSWIRQPPGKALEWLAHIFSNDEKSYNTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARALGFWSGYYYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-121K LC (SEQ ID NO:321)
MEAPAQLLFLLLLWLPDTTG EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPPLFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGCSPVTKQESVTEQDSKDSTYSLSSTLTLSKADYEKHKSSSFGLVTGLGENRHQ

1028-137K HC (SEQ ID NO:332)
MEFGLSWVFLVALLRGVQC QVHLVESGGGVVQPGRSLRLSCAASGFTFNSYAMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDDSGSYLYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-137K LC (SEQ ID NO:343)
MRLPAQLLGLLMLWVPGSSG DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLTWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKKTKVDNALQSGNSQESVTEQDSKDSTYSLVSSTLKAACEDYVTSFCPVSSTLKAACEDKHQ

1028-164L HC (SEQ ID NO:354)
MDILCSTLLLLTVPSWVLS QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMCVSWIRQPPGKALEWLALIDWDDDKYYSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYYCARIPVEMATIGDYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1028-164L LC (SEQ ID NO:365)
MAGFPLLLTLLTHCAGSWA QSVLTQPPSESGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSTECSTVSYSCQVTHEGSTECSTVEKQ

0210-19K HC (SEQ ID NO:376)
MSVSFLIFLPVLGLPWGVLS QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQLSLQLNSVTPEDTAVYYCARSPSYQLLYPYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

0210-19K LC (SEQ ID NO:387)
MDMRVPAQLLGLLLLWLPGTRC DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPELLLYAASRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYYSTPYTFGQGTKLEIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGPVNSQESVTEQDSKDSTYSLSSTLTLSKASFKDSTYSLGSTLTLSKASFTK

0210-20L HC (SEQ ID NO:398)
MDWTWRFLFVVAAATGVQS QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARALGYSGYGSTYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

0210-20L LC (SEQ ID NO: 409)
MAWSPLLLTLLAHCTGSWA QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSTVECTVAPTECVTHE

0210-28K HC (SEQ ID NO:420)
MDWIWRILFLVGAATGAHS QMQLVQSGPEVKKPGTSVKVSCKASGFTFSISAVQWVRQARGQRLEWIGWIVVGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRFEDTAVYYCAAPYCGGDCSDGFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

0210-28K LC (SEQ ID NO:431)
METPAQLLFLLLLWLPDTTG EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGNSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGPVNSQESVTEQDSKDSTYSLGLSSTLTLSKASFKDSTYSLSSTLTLSKASFKHKGSSTKVYACE

0210-30L HC (SEQ ID NO:442)
MDWTWRFLFVVAAATGVQS QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGRIIPILGRANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNPEEFSSSYVYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

0210-30L LC (SEQ ID NO:453)
MAWALLLLSLLTQGTGSWA QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYSYVSWYQQHPDKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSYTSWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSTVEKSTVTECTVAPQVTHE

0210-31L HC (SEQ ID NO:464)
MEFGLSWVFLVAILKGVQC EVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWVRQAPGKGLEWVGFIRSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDSITMVQGVIIRLDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

0210-31L LC (SEQ ID NO:475)
MAWAPLLLTLLAHCTGSWA NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNKVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQGSEKSTVTHETV

0210-35K HC (SEQ ID NO:486)
MKHLWFFLLLVAAPRWVLS QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIFYSGSANYNPSLRSRVTISVDTSKNHFSLKLSSVTAADTAVYYCARDIGYDSSGPDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

0210-35K LC (SEQ ID NO:497)
MEAPAQLLFLLLLWLPDTTG EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPGTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSPVTKQESVTEQDSKDSTYSLSSTLTLSKADYEKHKSSTLTLSKADYEKHKSSTLTLSVTGLGENRHQ

0210-38L HC (SEQ ID NO:508)
MELGLSWVFLVAILEGVQC EVQLVESGGGLVQPGGSLRLSCAASGFTFNSYWMSWVRQAPGKGLEWVADIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYYGPIDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

0210-38L LC (SEQ ID NO:519)
MAWAPLLLTLLAHCTGSWA NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSNNRVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQGSKAPTSTHETV

0210-52L HC (SEQ ID NO:530)
MGSTAILALLLAVLQGVCA EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKFISTAYLQWSSLKASDTAMYYCARRSGYDLASSHNWLDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

0210-52L LC (SEQ ID NO: 541)
MAWSPLLLTLLAHCTGSWA QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDNTLSGSLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQTECSTVVTHE

0210-58K HC (SEQ ID NO:552)
MDTLCYTLLLLTTPSWVLS QVTLKESGPVLVKPTETLTLTCTVSGFSLSNAKMGVSWIRQPPGKALEWLAHIFSGDEKSYSTSLKSRLTISKDTSKSQVVVTMTNMDPVDTATYYCARILWFRSYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

0210-58K LC (SEQ ID NO:563)
MDMRVPAQLLGLLLLWFPGSRC DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSSSGTASVVCLLNNFYPREAKVQWKVDNALQSGPVTKQESVTEQDSKDSTYSLSSTLGENRCSFVTEKHKVYACENRCSF

1-15HC (SEQ ID NO:574)
MELGLSWVFLVAILEGVQC EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHWVRQATGKGLEWVSTIGTAGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARGIQQQLALRYYYYLDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1-15 LC (SEQ ID NO: 585)
MRVPAQLLGLLLLWLRGARC DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSNPTWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSSSGTASVVCLLNNFYPREAKVQWKVDNALQSGPVTKQESVTEQDSKDSTYSLSSTLGENRCSFVTEKHKVYACE VTKDSTYSLSSTLGENRKASF

1-44HC (SEQ ID NO:596)
MEFGLSWLFLVAILKGVQC EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLLMNSLRAEDTAVYYCAKDGEWYYDFWSGPVYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1-44 LC (SEQ ID NO: 607)
MDMRVPAQLLGLLLLWLPGAKC DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGPVTKQESVTEQDSKDSTYSLSSTLGENRCSFDEKHKVYACENRCSF
　本明細書において，アミノ酸は一文字表記で表される。具体的には，Ａはアラニン，Ｌはロイシン，Ｒはアルギニン，Ｋはリシン，Ｎはアスパラギン，Ｍはメチオニン，Ｄはアスパラギン酸，Ｆはフェニルアラニン，Ｃはシステイン，Ｐはプロリン，Ｑはグルタミン，Ｓはセリン，Ｅはグルタミン酸，Ｔはトレオニン，Ｇはグリシン，Ｗはトリプトファン，Ｈはヒスチジン，Ｙはチロシン，Ｉはイソロイシン，Ｖはバリンを表す。本明細書において，「ＸＮＹ」（Ｘ及びＹはアミノ酸一文字表記；Ｎは自然数）は，Ｎ位のアミノ酸Ｘがアミノ酸Ｙに置換されていることを示す。 In this specification, amino acids are represented by single-letter codes. Specifically, A is alanine, L is leucine, R is arginine, K is lysine, N is asparagine, M is methionine, D is aspartic acid, F is phenylalanine, C is cysteine, P is proline, Q is glutamine, S is serine, E is glutamic acid, T is threonine, G is glycine, W is tryptophan, H is histidine, Y is tyrosine, I isoleucine and V is valine. As used herein, "XNY" (X and Y are amino acid one-letter codes; N is a natural number) indicates that amino acid X is substituted with amino acid Y at the N-position.
　別の態様において，本発明は，前記抗体又はその抗原結合断片と，ＳＡＲＳ－ＣｏＶ２スパイクタンパク質又はＲＢＤへの結合において競合する抗体に関する。被検抗体が，前記抗体又はその抗原結合断片と，ＳＡＲＳ－ＣｏＶ２スパイクタンパク質又はＲＢＤへの結合において競合するか否かは，標識化された前記抗体又はその抗原結合断片を用いて，被検抗体と共に固相化された抗原（ＳＡＲＳ－ＣｏＶ２スパイクタンパク質又はＲＢＤ，本段落において以下同様）に接触させることにより決定することができる。コントロールとして，標識化された前記抗体又はその抗原結合断片を単独で（被検抗体非存在下で）抗原に接触させる。被検抗体を抗原に接触させた後，必要に応じて洗浄し，抗原に結合した標識化された前記抗体又はその抗原結合断片の標識量をコントロールと，被検抗体存在下とで比較する。被検抗体の存在下で抗原に接触させた場合の標識化された前記抗体又はその抗原結合断片の抗原への結合量が，コントロールにおける標識化された前記抗体又はその抗原結合断片の抗原への結合量よりも少ない場合には，当該被検抗体は，当該実験において用いられた標識化された前記抗体又はその抗原結合断片と競合すると決定することができる。 In another aspect, the present invention relates to an antibody that competes with the antibody or antigen-binding fragment thereof for binding to the SARS-CoV2 spike protein or RBD. Whether the test antibody competes with the antibody or antigen-binding fragment thereof for binding to the SARS-CoV2 spike protein or RBD is determined using the labeled antibody or antigen-binding fragment thereof. It can be determined by contacting with an antigen (SARS-CoV2 spike protein or RBD, the same applies hereinafter in this paragraph) immobilized together. As a control, the labeled antibody or antigen-binding fragment thereof alone (in the absence of the antibody to be tested) is brought into contact with the antigen. After the test antibody is brought into contact with the antigen, it is washed if necessary, and the labeled amount of the labeled antibody or antigen-binding fragment thereof bound to the antigen is compared between the control and the presence of the test antibody. The amount of binding of the labeled antibody or antigen-binding fragment thereof to the antigen when contacted with the antigen in the presence of the test antibody is the same as that of the labeled antibody or antigen-binding fragment thereof in the control. If less than the binding amount, the test antibody can be determined to compete with the labeled antibody or antigen-binding fragment thereof used in the experiment.
　抗原への結合試験は，適宜上述のＲＢＤ等への結合を測定する方法に準じて行うことができる。例えば，抗体と抗原との結合の測定は，例えば，ＥＩＡ法，ＥＬＩＳＡ法，ＦＡＣＳ法，共免疫沈降法，プルダウンアッセイ法，ファーウェスタンブロッティング法，ホモバイファンクショナルクロスリンカー若しくはヘテロバイファンクショナルクロスリンカーを利用するクロスリンク法，ラベル転移反応法，相互作用マッピング法，表面プラズモン共鳴法，ＦＲＥＴ（Ｆｌｕｏｒｅｓｃｅｎｃｅ　ｒｅｓｏｎａｎｃｅ　ｅｎｅｒｇｙ　ｔｒａｎｓｆｅｒ）法，ＢＩＡＣＯＲＥ法，ＡｌｐｈａＳｃｒｅｅｎ（登録商標）やＡｌｐｈａＬＩＳＡ（登録商標）などのＡｌｐｈａＰＰＩアッセイ（ＰｅｒｋｉｎＥｌｍｅｒ）など，多様な方法が本技術分野において周知であり，適宜好ましい方法を採用することができる。 Antigen binding tests can be performed according to the above-mentioned methods for measuring binding to RBD, etc. as appropriate. For example, measurement of binding between antibody and antigen can be performed by EIA method, ELISA method, FACS method, co-immunoprecipitation method, pull-down assay method, Far-Western blotting method, homobifunctional crosslinker or heterobifunctional crosslinker cross-linking method, label transfer reaction method, interaction mapping method, surface plasmon resonance method, FRET (Fluorescence resonance energy transfer) method, BIACORE method, AlphaScreen (registered trademark) and AlphaPPI assay such as AlphaLISA (registered trademark) ( A variety of methods are well known in the art, such as PerkinElmer), and any suitable method can be employed.
　更に別の態様において，本発明は，前記抗体又はその抗原結合断片と，同じエピトープを認識する抗体に関する。例えば、０２１０－２８Ｋ（２８Ｋ）抗体の場合、ＳＡＲＳ－ＣｏＶ－２　ＲＢＤのＡ４７５，Ｇ４７６，Ｓ４７７，Ｔ４７８，Ｆ４８６，Ｎ４８７，Ｙ４８９及びＱ４９３と相互作用することから、２８Ｋ抗体と同じエピトープを認識する抗体は、エピトープとしてこれらから選択される少なくとも１個（例えば、１個、２個またはそれ以上、３個またはそれ以上、４個またはそれ以上、５個またはそれ以上、６個またはそれ以上、あるいは７個）のアミノ酸残基を認識する抗体である。特に、２８Ｋ抗体と同じエピトープを認識する抗体のエピトープは、ＲＢＤのＡ４７５，Ｇ４７６，Ｓ４７７，及びＴ４７８から選択される一部（例えば、１個またはそれ以上、２個またはそれ以上、あるいは、３個またはそれ以上）又は全てを含む。好ましくは、２８Ｋ抗体と同じエピトープを認識する抗体は、ＲＢＤのＡ４７５，Ｇ４７６，Ｓ４７７，及びＴ４７８の主鎖と相互作用することにより結合する。例えば，２８Ｋ抗体と同じエピトープを認識する抗体は、そのＶＨのＳ５５，Ｎ５７，Ｄ１０４，Ｃ１０５，Ｓ１０６，及びＤ１０７から選択される少なくとも１個のアミノ酸残基が，ＲＢＤのＡ４７５，Ｇ４７６，Ｓ４７７，Ｔ４７８，Ｆ４８６，Ｎ４８７，Ｙ４８９及びＱ４９３から選択される少なくとも１個のアミノ酸残基と結合してもよい。また，ＶＬのＹ３３，Ｙ９２，Ｗ９７から選択される少なくとも１個のアミノ酸残基が，ＲＢＤのＦ４８６のアミノ酸残基との結合に関与していてもよい。 In yet another aspect, the present invention relates to an antibody that recognizes the same epitope as the antibody or antigen-binding fragment thereof. For example, in the case of 0210-28K (28K) antibody, since it interacts with A475, G476, S477, T478, F486, N487, Y489 and Q493 of SARS-CoV-2 RBD, an antibody that recognizes the same epitope as 28K antibody has at least one selected from these as epitopes (e.g., 1, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 ) is an antibody that recognizes the amino acid residues of In particular, the epitope of the antibody that recognizes the same epitope as the 28K antibody is a portion selected from RBD A475, G476, S477, and T478 (e.g., one or more, two or more, or three or more) or all. Preferably, antibodies that recognize the same epitope as the 28K antibody bind by interacting with the backbone of RBD A475, G476, S477, and T478. For example, an antibody that recognizes the same epitope as the 28K antibody has at least one amino acid residue selected from S55, N57, D104, C105, S106, and D107 of its VH, A475, G476, S477, and T478 of RBD. , F486, N487, Y489 and Q493. In addition, at least one amino acid residue selected from Y33, Y92 and W97 of VL may be involved in binding to the amino acid residue of F486 of RBD.
（核酸分子）
　別の態様において，本発明は，上述の本発明の抗体又はその免疫反応性断片をコードするポリヌクレオチドを有する核酸分子に関する。一態様において，本発明は，上述の表２に記載された重鎖可変領域及び軽鎖可変領域の核酸配列を有する核酸分子である。また，一例において，本発明は，配列番号３，２５，４７，６９，９１，１１３，１３５，１５７，１７９，２０１，２２３，２４５，２６７，２８９，３１１，３３３，３５５，３７７，３９９，４２１，４４３，４６５，４８７，５０９，５３１，５５３，５７５，又は５９７に記載の核酸配列を有する重鎖をコードするＤＮＡを含む核酸分子，あるいは／並びに，それぞれ，配列番号１４，３６，５８，８０，１０２，１２４，１４６，１６８，１９０，２１２，２３４，２５６，２７８，３００，３２２，３４４，３６６，３８８，４１０，４３２，４５４，４７６，４９８，５２０，５４２，５６４，５８６，又は６０８に記載の核酸配列を有する軽鎖をコードするＤＮＡを含む核酸分子である。 (nucleic acid molecule)
In another aspect, the invention relates to a nucleic acid molecule comprising a polynucleotide encoding an antibody or immunoreactive fragment thereof of the invention as described above. In one aspect, the invention is a nucleic acid molecule having the nucleic acid sequences of the heavy and light chain variable regions set forth in Table 2, supra. Also, in one example, the present invention provides SEQ ID NOs: , 443, 465, 487, 509, 531, 553, 575, or 597, or/and SEQ ID NOS: 14, 36, 58, 80, respectively. , 102,124,146,168,190,212,234,256,278,300,322,344,366,388,410,432,454,476,498,520,542,564,586, or 608 A nucleic acid molecule comprising DNA encoding a light chain having the nucleic acid sequence described.
　更に，本発明は，前記核酸分子を有するベクターを包含する。このようなベクターとしては，抗体の発現に利用可能なベクターであれば特に制限されるものではなく，適切なウイルスベクター又はプラスミドベクターなどを使用する宿主に応じて選択することができる。別の態様において，本発明は，前記ベクターを含有する宿主細胞に関する。宿主細胞としては，抗体の発現に利用可能な宿主細胞であれば特に制限されるものではなく，哺乳類細胞（マウス細胞，ラット細胞，ハムスター細胞，ウサギ細胞，ヒト細胞など），酵母，微生物（大腸菌など）を挙げることができる。 Furthermore, the present invention encompasses a vector comprising the nucleic acid molecule. Such a vector is not particularly limited as long as it can be used for antibody expression, and an appropriate viral vector, plasmid vector, or the like can be selected according to the host to be used. In another aspect, the invention relates to a host cell containing said vector. Host cells are not particularly limited as long as they are host cells that can be used to express antibodies, and mammalian cells (mouse cells, rat cells, hamster cells, rabbit cells, human cells, etc.), yeast, microorganisms (E. coli etc.) can be mentioned.
　本発明の核酸は，上述において得られた抗体を産生するハイブリドーマからクローニングするか，あるいは，上述において得られた抗体又はその免疫反応性断片のアミノ酸配列を基に，適宜核酸配列を設計することにより得ることができる。本発明のベクターは，得られた核酸を適宜発現に適したベクターに組み込むことにより得ることができる。本発明のベクターは，本発明の核酸の他，発現に必要な領域（プロモーター，エンハンサー，ターミネーター等）を含んでいてもよい。また，本発明の宿主細胞は，本発明のベクターを適切な細胞株（例えば，動物細胞，昆虫細胞，植物細胞，酵母，大腸菌等の微生物）に導入することにより得ることができる。 The nucleic acid of the present invention is cloned from the antibody-producing hybridoma obtained above, or by designing a nucleic acid sequence as appropriate based on the amino acid sequence of the antibody or immunoreactive fragment thereof obtained above. Obtainable. The vector of the present invention can be obtained by appropriately integrating the obtained nucleic acid into a vector suitable for expression. The vector of the present invention may contain regions necessary for expression (promoter, enhancer, terminator, etc.) in addition to the nucleic acid of the present invention. Also, the host cell of the present invention can be obtained by introducing the vector of the present invention into an appropriate cell strain (eg, animal cells, insect cells, plant cells, yeast, microorganisms such as E. coli).
（抗体の製造）
　本発明の抗体は，本明細書記載の配列に基づき適宜設計して作成することができる。例えば，本明細書記載の可変領域（ＶＲ）を有する抗体は，当該抗体をコードする核酸配列を適宜産生細胞に導入して抗体を産生させることができる。また，例えば，本明細書記載のＣＤＲを有する抗体の場合，当該配列を有する重鎖及び軽鎖をコードする核酸配列を設計し，当該核酸配列を既報に基づいて調製後，ＣＨＯ細胞などの産生細胞に導入した後，ＳＡＲＳ－ＣｏＶ－２スパイクに結合するクローンを選択することができる。具体的には，当該細胞を培養後，培養上清を採取し，上清中の抗体のＳＡＲＳ－ＣｏＶ－２スパイクへの結合を測定することによりＳＡＲＳ－ＣｏＶ－２スパイクへ結合するクローンを選択する。選択された細胞を培養して本発明の抗体を取得することができる。 (Manufacturing of antibody)
Antibodies of the present invention can be designed and produced as appropriate based on the sequences described herein. For example, an antibody having a variable region (VR) as described herein can be produced by appropriately introducing a nucleic acid sequence encoding the antibody into a production cell. In addition, for example, in the case of an antibody having the CDRs described herein, a nucleic acid sequence encoding a heavy chain and a light chain having the sequence is designed, and after preparing the nucleic acid sequence based on the previous report, production of CHO cells etc. After introduction into cells, clones that bind to the SARS-CoV-2 spike can be selected. Specifically, after culturing the cells, the culture supernatant is collected and the binding of antibodies in the supernatant to the SARS-CoV-2 spike is measured to select clones that bind to the SARS-CoV-2 spike. do. Antibodies of the present invention can be obtained by culturing selected cells.
　得られた抗体は，均一にまで精製することができる。抗体の分離，精製は通常のタンパク質で使用されている分離，精製方法を使用することができる。例えば，アフィニティークロマトグラフィ等のカラムクロマトグラフィ，フィルター，限外濾過，塩析，透析，ＳＤＳポリアクリルアミドゲル電気泳動，等電点電気泳動等を適宜選択，組み合わせることにより，抗体を分離，精製することができる（Ａｎｔｉｂｏｄｉｅｓ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ．Ｅｄ　Ｈａｒｌｏｗ　ａｎｄ　Ｄａｖｉｄ　Ｌａｎｅ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ，１９８８）。アフィニティークロマトグラフィに用いるカラムとしては，例えば，プロテインＡカラム，プロテインＧカラムを挙げることができる。更に，抗体のクラスによらず，ＳＡＲＳ－ＣｏＶ－２固相化カラム，サイズ排除クロマトグラフィ，イオン交換クロマトグラフィ，疎水相互作用クロマトグラフィ等を用いることもできる。また，クロマトグラフィをミックスモードとすることもできる。 The obtained antibody can be purified to homogeneity. Separation and purification of antibodies can be carried out using methods commonly used for separation and purification of proteins. For example, antibodies can be separated and purified by appropriately selecting and combining column chromatography such as affinity chromatography, filter, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and the like. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Columns used for affinity chromatography include, for example, protein A columns and protein G columns. Furthermore, SARS-CoV-2-immobilized columns, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, etc. can also be used regardless of the antibody class. Chromatography can also be in mixed mode.
（医薬組成物（治療薬，予防薬）及び治療方法）
　また，別の態様において，本発明は，上述の本発明の抗体又はその免疫反応性断片を有効成分として含有する医薬組成物に関する。本発明の抗体又はその免疫反応性断片は，必要により精製した後，常法に従って製剤化して，医薬組成物とすることができる。本発明の医薬組成物の対象疾患は，コロナウイルス感染症であり，例えば，コロナウイルス，特には，ＳＡＲＳ－ＣｏＶ，ＭＥＲＳ－ＣｏＶ，若しくはＳＡＲＳ－ＣｏＶ－２が原因のウイルス性疾患，又はそれに起因する疾患である。よって，本発明の医薬組成物は，これらの疾患又は障害の予防薬，治療薬，進行抑制剤又は改善薬とすることができる。あるいは，本発明は，前記医薬組成物を製造するための，本発明の抗体又はその免疫反応性断片の使用に関する。あるいは，本発明は，本発明の抗体又はその免疫反応性断片の，コロナウイルス感染症の治療若しくは予防のための使用を含む。更に，本発明は，本発明の抗体又はその免疫反応性断片を投与することを含む，コロナウイルス感染症の治療方法若しくは予防方法に関する。コロナウイルス感染症は，好ましくは，ヒトコロナウイルス感染症である。 (Pharmaceutical composition (therapeutic drug, prophylactic drug) and treatment method)
In another aspect, the present invention relates to a pharmaceutical composition containing the above-described antibody or immunoreactive fragment thereof of the present invention as an active ingredient. The antibody or immunoreactive fragment thereof of the present invention can be purified, if necessary, and formulated into a pharmaceutical composition according to a conventional method. The target disease of the pharmaceutical composition of the present invention is a coronavirus infection, for example, a coronavirus, in particular, a viral disease caused by SARS-CoV, MERS-CoV, or SARS-CoV-2, or caused by it It is a disease that Therefore, the pharmaceutical composition of the present invention can be used as a prophylactic agent, therapeutic agent, inhibitor of progression, or ameliorative agent for these diseases or disorders. Alternatively, the invention relates to the use of an antibody or immunoreactive fragment thereof of the invention for the manufacture of said pharmaceutical composition. Alternatively, the invention includes the use of the antibodies of the invention, or immunoreactive fragments thereof, for the treatment or prevention of coronavirus infection. Additionally, the present invention relates to methods of treating or preventing coronavirus infection comprising administering an antibody or immunoreactive fragment thereof of the present invention. A coronavirus infection is preferably a human coronavirus infection.
　本発明の医薬組成物は，患者に投与することができる製剤であれば経口又は非経口のいかなる製剤を採用してもよい。非経口投与のための組成物としては，例えば，注射剤，点鼻剤，吸入剤，点眼剤等を挙げることができる。好ましくは，注射剤である。本発明の医薬組成物の剤形は，例えば，液剤又は凍結乾燥製剤を挙げることができる。本発明の医薬組成物を注射剤として使用する場合，必要に応じて，プロピレングリコール，エチレンジアミン等の溶解補助剤，リン酸塩等の緩衝材，塩化ナトリウム，グリセリン等の等張化剤，亜硫酸塩等の安定剤，フェノール等の保存剤，リドカイン等の無痛化剤等の添加物（「医薬品添加物事典」薬事日報社，「Ｈａｎｄｂｏｏｋ　ｏｆ　Ｐｈａｒｍａｃｅｕｔｉｃａｌ　Ｅｘｃｉｐｉｅｎｔｓ　Ｆｉｆｔｈ　Ｅｄｉｔｉｏｎ」ＡＰｈＡ　Ｐｕｂｌｉｃａｔｉｏｎｓ社参照）を加えることができる。また，本発明の医薬組成物を注射剤として使用する場合，保存容器としては，アンプル，バイアル，プレフィルドシリンジ，ペン型注射器用カートリッジ，及び，点滴用バッグ等を挙げることができる。 The pharmaceutical composition of the present invention may adopt any oral or parenteral formulation as long as it can be administered to a patient. Compositions for parenteral administration include, for example, injections, nasal drops, inhalants, eye drops and the like. An injection is preferred. Dosage forms of the pharmaceutical composition of the present invention include, for example, liquid formulations and freeze-dried formulations. When the pharmaceutical composition of the present invention is used as an injection, if necessary, solubilizers such as propylene glycol and ethylenediamine, buffering agents such as phosphate, tonicity agents such as sodium chloride and glycerin, and sulfites. Additives such as stabilizers such as phenol, preservatives such as phenol, and soothing agents such as lidocaine (see "Pharmaceutical excipient dictionary" Yakuji Nippo, "Handbook of Pharmaceutical Excipients Fifth Edition" APhA Publications) . When the pharmaceutical composition of the present invention is used as an injection, storage containers include ampoules, vials, prefilled syringes, pen-type syringe cartridges, and drip bags.
　例えば，本発明の医薬組成物（治療薬若しくは予防薬）は，注射剤として利用する場合，静脈注射剤，皮下注射剤，皮内注射剤，筋肉注射剤，点滴注射剤などの剤形を包含する。このような注射剤は，公知の方法に従って，例えば，上記抗体等を通常注射剤に用いられる無菌の水性もしくは油性液に溶解，懸濁又は乳化することによって調製することができる。注射用の水性液としては，例えば，生理食塩水，ブドウ糖，ショ糖，マンニトール，その他の補助薬を含む等張液等が用いることができ，適当な溶解補助剤，例えば，アルコール（例，エタノール），ポリアルコール（例，プロピレングリコール，ポリエチレングリコール），非イオン界面活性剤〔例，ポリソルベート８０，ポリソルベート２０，ＨＣＯ－５０（ｐｏｌｙｏｘｙｅｔｈｙｌｅｎｅ（５０ｍｏｌ）ａｄｄｕｃｔ　ｏｆ　ｈｙｄｒｏｇｅｎａｔｅｄ　ｃａｓｔｏｒ　ｏｉｌ）〕等を添加することができる。油性液としては，例えば，ゴマ油，大豆油などを用いることができ，溶解補助剤として安息香酸ベンジル，ベンジルアルコール等を添加することができる。調製された注射液は，通常，適当なアンプル，バイアル，シリンジに充填される。また，本発明の抗体又はその免疫反応性断片に適当な賦形剤を添加することにより，凍結乾燥製剤を調製し，用時，注射用水，生理食塩水などで溶解して注射液とすることもできる。なお，一般的に抗体などのタンパク質の経口投与は消化器により分解されるため困難とされるが，抗体断片や修飾した抗体断片と剤形の創意工夫により，経口投与の可能性もある。経口投与の製剤としては，例えば，カプセル剤，錠剤，シロップ剤，顆粒剤等を挙げることができる。 For example, when the pharmaceutical composition (therapeutic drug or prophylactic drug) of the present invention is used as an injection, it includes dosage forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection. do. Such injections can be prepared according to known methods, for example, by dissolving, suspending, or emulsifying the above-mentioned antibody in a sterile aqueous or oily liquid commonly used for injections. Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose, sucrose, mannitol, and other adjuvants. ), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, polysorbate 20, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. can be added. . Sesame oil, soybean oil, and the like can be used as the oily liquid, and benzyl benzoate, benzyl alcohol, and the like can be added as solubilizers. The prepared injection solution is usually filled into a suitable ampoule, vial or syringe. Alternatively, the antibody of the present invention or an immunoreactive fragment thereof may be added to a suitable excipient to prepare a lyophilized formulation, and dissolved in water for injection, physiological saline, etc. at the time of use to prepare an injection solution. can also In general, oral administration of proteins such as antibodies is considered difficult because they are degraded in the digestive system, but oral administration is also possible with antibody fragments, modified antibody fragments, and ingenuity in dosage forms. Oral preparations include, for example, capsules, tablets, syrups, granules and the like.
　本発明の医薬組成物は，活性成分の投与量に適合するような投薬単位の剤形に調製されることが好適である。このような投薬単位の剤形としては，注射剤（アンプル，バイアル，プレフィルドシリンジ）が例示され，投薬単位剤形当たり通常５～５００ｍｇ，５～１００ｍｇ，１０～２５０ｍｇの本発明の抗体又はその免疫反応性断片を含有していても良い。 The pharmaceutical composition of the present invention is preferably prepared in a dosage unit form suitable for the dosage of the active ingredient. Examples of such dosage unit dosage forms include injections (ampoules, vials, prefilled syringes), and usually 5 to 500 mg, 5 to 100 mg, and 10 to 250 mg of the antibody of the present invention or its immunological agent per dosage unit dosage form. It may contain a reactive fragment.
　本発明の抗体や医薬組成物（治療薬若しくは予防薬）の投与は，局所的であってもよく，全身的であってもよい。投与方法には特に制限はなく，上述のとおり非経口的又は経口的に投与される。非経口的投与経路としては，眼内，皮下，腹腔内，血中（静脈内，若しくは動脈内）又は脊髄液への注射又は点滴等が挙げられ，好ましくは，眼内又は血中への投与である。本発明の医薬組成物（治療薬若しくは予防薬）は，一時的に投与してもよいし，持続的又は断続的に投与してもよい。例えば，投与は，１分間～２週間の持続投与することもできる。また，本発明の抗体は，単独で投与されてもよいし，他の薬剤と共に併用により投与されてもよい。 The administration of the antibody or pharmaceutical composition (therapeutic or prophylactic) of the present invention may be local or systemic. The administration method is not particularly limited, and is administered parenterally or orally as described above. Parenteral routes of administration include intraocular, subcutaneous, intraperitoneal, blood (intravenous or intraarterial) injection or infusion into spinal fluid, preferably intraocular or blood administration. is. The pharmaceutical composition (therapeutic or prophylactic) of the present invention may be administered temporarily, continuously or intermittently. For example, administration can be sustained for 1 minute to 2 weeks. Moreover, the antibody of the present invention may be administered alone or in combination with other drugs.
　本発明の医薬組成物の投与量は，所望の治療効果又は予防効果が得られる投与量であれば特に限定は無く，症状，性別，年齢等により適宜決定することができる。本発明の医薬組成物の投与量は，例えば，血管新生が発症又は増悪化に寄与する疾患又は障害の治療効果又は予防効果を指標として決定することができる。例えば，血管新生が発症又は増悪化に寄与する疾患又は障害の患者の予防及び／又は治療のために使用する場合には，本発明の医薬組成物の有効成分の１回量として，通常０．０１～２０ｍｇ／ｋｇ体重程度，好ましくは０．１～１０ｍｇ／ｋｇ体重程度，さらに好ましくは０．１～５ｍｇ／ｋｇ体重程度を，１月１～１０回程度，好ましくは１月１～５回程度，静脈注射により投与するのが好都合である。他の非経口投与及び経口投与の場合もこれに準ずる量を投与することができる。症状が特に重い場合には，その症状に応じて増量又は投与回数を増加させてもよい。 The dosage of the pharmaceutical composition of the present invention is not particularly limited as long as the desired therapeutic or preventive effect can be obtained, and can be appropriately determined according to symptoms, sex, age, and the like. The dosage of the pharmaceutical composition of the present invention can be determined, for example, using the therapeutic effect or preventive effect of diseases or disorders in which angiogenesis contributes to the onset or exacerbation of angiogenesis. For example, when used for the prevention and/or treatment of a patient with a disease or disorder in which angiogenesis contributes to the onset or exacerbation of angiogenesis, the active ingredient of the pharmaceutical composition of the present invention is usually added at a dose of 0.2 to 0.5 g/day. 01 to 20 mg/kg body weight, preferably 0.1 to 10 mg/kg body weight, more preferably 0.1 to 5 mg/kg body weight, about 1 to 10 times a month, preferably 1 to 5 times a month To some extent, it is convenient to administer by intravenous injection. In the case of other parenteral administration and oral administration, the amount according to this can be administered. If the symptoms are particularly severe, the dose or frequency of administration may be increased according to the symptoms.
（診断用・検出用組成物）
　ＳＡＲＳ－ＣｏＶ２感染症の診断及びＳＡＲＳ－ＣｏＶ２の検出は，前記抗体又はその抗原結合性断片を用いて，ＥＬＩＳＡ，ラジオイムノアッセイ，イムノクロマト，免疫組織化学的方法により行うことができる。抗体又はその免疫反応性断片は，必要に応じて標識されていてもよい。標記の結合は当分野において一般的な方法により行うことができる。例えば，蛍光標識する場合，タンパク質又はペプチドをリン酸緩衝液で洗浄した後，ＤＭＳＯ，緩衝液等で調整した色素を加え，混合した後室温で１０分間静置することにより結合させることができる。また，市販の標識キットとして，ビオチン標識キット（Ｂｉｏｔｉｎ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＮＨ２，Ｂｉｏｔｉｎ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＳＨ：株式会社同仁化学研究所），アルカリフォスファターゼ標識用キット（Ａｌｋａｌｉｎｅ　Ｐｈｏｓｐｈａｔａｓｅ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＮＨ２，Ａｌｋａｌｉｎｅ　Ｐｈｏｓｐｈａｔａｓｅ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＳＨ：株式会社同仁化学研究所），ペルオキシダーゼ標識キット（Ｐｅｒｏｘｉｄａｓｅ　Ｌａｂｅｒｉｎｇ　Ｋｉｔ－ＮＨ２，Ｐｅｒｏｘｉｄａｓｅ　Ｌａｂｅｒｉｎｇ　Ｋｉｔ－ＮＨ２：株式会社同仁化学研究所），フィコビリプロテイン標識キット（Ａｌｌｏｐｈｙｃｏｃｙａｎｉｎ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＮＨ２，Ａｌｌｏｐｈｙｃｏｃｙａｎｉｎ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＳＨ，Ｂ－Ｐｈｙｃｏｅｒｙｔｈｒｉｎ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＮＨ２，Ｂ－Ｐｈｙｃｏｅｒｙｔｈｒｉｎ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＳＨ，Ｒ－Ｐｈｙｃｏｅｒｙｔｈｒｉｎ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＮＨ２，Ｒ－Ｐｈｙｃｏｅｒｙｔｈｒｉｎ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＳＨ：株式会社同仁化学研究所），蛍光標識キット（Ｆｌｕｏｒｅｓｃｅｉｎ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＮＨ２，ＨｉＬｙｔｅ　Ｆｌｕｏｒ（登録商標）　５５５　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＮＨ２，ＨｉＬｙｔｅ　Ｆｌｕｏｒ（登録商標）　６４７　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＮＨ２：株式会社同仁化学研究所），ＤｙＬｉｇｈｔ５４７，ＤｙＬｉｇｈｔ６４７（テクノケミカル株式会社），Ｚｅｎｏｎ（登録商標）Ａｌｅｘａ　Ｆｌｕｏｒ（登録商標）抗体標識キット，Ｑｄｏｔ（登録商標）抗体標識キット（インビトロゲン社），ＥＺ－Ｌａｂｅｌ　Ｐｒｏｔｅｉｎ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ（フナコシ株式会社）等を用いて標識することもできる。 (Diagnostic/Detective Composition)
Diagnosis of SARS-CoV2 infection and detection of SARS-CoV2 can be performed by ELISA, radioimmunoassay, immunochromatography, immunohistochemistry using the antibody or antigen-binding fragment thereof. An antibody or immunoreactive fragment thereof may optionally be labeled. Attachment of the title can be carried out by methods common in the art. For example, in the case of fluorescent labeling, after washing the protein or peptide with a phosphate buffer, a dye adjusted with DMSO, a buffer, etc. is added, mixed, and allowed to stand at room temperature for 10 minutes for binding. In addition, commercially available labeling kits include a biotin labeling kit (Biotin Labeling Kit-NH2, Biotin Labeling Kit-SH: Dojindo Laboratories), an alkaline phosphatase labeling kit (Alkaline Phosphatase Labeling Kit-NH2, Alkaline Phosphatase Labeling Kit- SH: Dojindo Laboratories), Peroxidase Labeling Kit-NH 2 , Peroxidase Labeling Kit-NH 2 : Dojindo Laboratories), Phycobiliprotein Labeling Kit (Allophycocyanin Labeling Kit-NH 2 , Allophycocyanin Labeling Kit-SH, B-Phycoerythrin Labeling Kit-NH 2 , B-Phycoerythrin Labeling Kit-SH, R-Phycoerythrin Labeling Kit-NH 2 , R-Phycoerythrin Labeling Kit-SH: Dojindo Laboratories), fluorescent labeling kit (Fluorescein Labeling Kit-NH 2 , HiLyte Fluor (registered trademark) 555 Labeling Kit-NH 2 , HiLyte Fluor (registered trademark) 647 Labeling Kit-NH 2 : Dojindo Laboratories), DyLight 547, DyLight 647 (Techno Chemical Co., Ltd.) ), Zenon (registered trademark) Alexa Fluor (registered trademark) antibody labeling kit, Qdot (registered trademark) antibody labeling kit (Invitrogen), EZ-Label Protein Labeling Kit (Funakoshi Co., Ltd.), etc. can.
　前記診断用組成物又は検出用組成物を用いた診断及び検出は，ＥＬＩＳＡ，イムノクロマトなどの方法で行うことができる。本明細書における検出や測定は，定量的，反定量的，定性的であってもよい。診断／検出方法において使用するサンプルとしては，例えば，生検として被験者から採取した組織試料または液体を使用することができる。使用される検体は，本発明の測定の対象となるものであれば特に限定はなく，例えば，組織，血液，血漿，血清，リンパ液，尿，漿液，髄液，関節液，眼房水，涙液，唾液，口腔内／鼻腔内洗浄液またはそれらの分画物若しくは処理物を挙げることができる。また，本発明の方法において使用する患者由来の試料は，測定試験に先立ち前処理されたものであってもよいし，患者から採取した検体をそのまま使用してもよい。診断方法は，適宜診断ための情報を提供する方法，又は，ＳＡＲＳ－ＣｏＶ２感染症の状態又は進行をモニターする方法であってもよい。 Diagnosis and detection using the diagnostic composition or detection composition can be performed by methods such as ELISA and immunochromatography. Detection and measurement herein may be quantitative, semi-quantitative or qualitative. Samples used in diagnostic/detection methods can be, for example, tissue samples or fluids taken from a subject as a biopsy. The specimen to be used is not particularly limited as long as it is the object of the measurement of the present invention. Fluids, saliva, oral/nasal washes or fractions or treatments thereof may be mentioned. In addition, the patient-derived sample used in the method of the present invention may be pretreated prior to the measurement test, or the specimen collected from the patient may be used as it is. The diagnostic method may be a method of providing information for diagnosis as appropriate, or a method of monitoring the state or progression of SARS-CoV2 infection.
（検出用キット）
　本発明のキットは，ＳＡＲＳ－ＣｏＶ２と特異的に結合する第一抗体が固定化した，固相，ハプテン，磁気ビーズ及び不溶性担体からなる群より選択される担体を含むことを特徴とする免疫化学測定のキットである。また，本発明のキットは，適宜標識されたＳＡＲＳ－ＣｏＶ２と結合する抗体を含んでいてもよい。 (Detection kit)
The kit of the present invention comprises a carrier selected from the group consisting of a solid phase, a hapten, magnetic beads and an insoluble carrier on which a first antibody that specifically binds to SARS-CoV2 is immobilized. It is a measurement kit. The kit of the present invention may also contain an appropriately labeled antibody that binds to SARS-CoV2.
　以下，実施例に基づいて本発明をより具体的に説明する。ただし，本発明はこれらの実施例に限定されるものではない。なお，本願全体を通して引用される全文献は参照によりそのまま本願に組み込まれる。 The present invention will be described more specifically below based on examples. However, the present invention is not limited to these examples. All documents cited throughout this application are hereby incorporated by reference in their entirety.
（実施例１）抗ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質結合抗体の取得
　抗ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質抗体価強陽性である１名のＣＯＶＩＤ－１９患者より末梢血リンパ球を分離し，ＩＳＡＡＣ（Ｉｍｍｕｎｏｓｐｏｔ－ａｒｒａｙ　ａｓｓａｙ　ｏｎ　ａ　ｃｈｉｐ）法を用いて，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質に結合するヒトモノクローナル抗体の作製を試みた。ＩＳＡＡＣ法については，Ｊｉｎ　Ａ　ｅｔ　ａｌ．，Ｎａｔｕｒｅ　ｍｅｄｉｃｉｎｅ（２００９）２６：１０８８－１０９２，及びＪｉｎ　Ａ　ｅｔ　ａｌ．，Ｎａｔｕｒｅ　Ｐｒｏｔｏｃｏｌｓ（２０１１）６：６６８，日本国特許第４１４８３６７号及び第６２９３４０９号，並びに，日本国特許公開公報第２０１４－１６２７７２号，特開２０１８－３８３６４，特開２０１８－７７１９４，及び特開２０１８－１２７４４３参照。 (Example 1) Acquisition of anti-SARS-CoV-2 spike protein-binding antibody Peripheral blood lymphocytes were isolated from one COVID-19 patient who was strongly positive for anti-SARS-CoV-2 spike protein antibody titer, and ISAAC (Immunospot -array assay on a chip) method was used to attempt to generate human monoclonal antibodies that bind to the SARS-CoV-2 spike protein. For the ISAAC method, see Jin A et al. , Nature medicine (2009) 26:1088-1092, and Jin A et al. , Nature Protocols (2011) 6:668, Japanese Patent Nos. 4148367 and 6293409, and Japanese Patent Publication Nos. 2014-162772, JP 2018-38364, JP 2018-77194, and JP 2018 See -127443.
　具体的には，まず，分離した末梢血リンパ球を，５μｇ／ｍｌ　Ｒ－８４８（ＥｎｚｏＬｉｆｅＳｃｉｅｎｃｅｓ），５μｇ／ｍｌ抗ヒトＣＤ４０抗体（Ｒ＆Ｄ），１００μｇ／ｍｌ　ヒトＩＬ－２１（ＰＥＰＲＯＴＥＣＨ），１００ｎｇ／ｍｌ　ヒトＢＡＦＦ（ＰＥＰＲＯＴＥＣＨ），２ｎｇ／ｍｌ　ヒトＩＬ－１７（ＰＥＰＲＯＴＥＣＨ），１０ｎｇ／ｍｌ　ヒトＩＬ－４（ＰｒｏＳｐｅｃ），１０００ＩＵ／ｍｌ　ヒトＩＬ－２（ＰｒｏＳｐｅｃ），２．５μｇ／ｍｌ　ＣｐＧ２００６（５’－ＴＣＧＴＣＧＴＴＴＴＧＴＣＧＴＴＴＴＧＴＣＧＴ－３’）（配列番号６１７），５×１０－５Ｍ　メルカプトエタノール，１０％　ＦＣＳを含むＲＰＭＩ培地にて３７℃で６日間培養した。 Specifically, first, the separated peripheral blood lymphocytes were mixed with 5 μg/ml R-848 (EnzoLifeSciences), 5 μg/ml anti-human CD40 antibody (R & D), 100 μg/ml human IL-21 (PEPROTECH), 100 ng/ml Human BAFF (PEPROTECH), 2 ng/ml Human IL-17 (PEPROTECH), 10 ng/ml Human IL-4 (ProSpec), 1000 IU/ml Human IL-2 (ProSpec), 2.5 μg/ml CpG2006 (5′-TCGTCGTTTTGTCGTTTTGTCGT -3′) (SEQ ID NO: 617), cultured in RPMI medium containing 5×10 −5 M mercaptoethanol and 10% FCS at 37° C. for 6 days.
　培養後，ＣＤ１３８＋細胞を，抗ヒトＣＤ１３８特異的抗体コンジュゲートマイクロビーズ（Ｍｉｌｔｅｎｙｉ　Ｂｉｏｔｅｃ）と，ＬＳカラム（Ｍｉｌｔｅｎｙｉ　Ｂｉｏｔｅｃ）を用いて，製造者の説明書にしたがって分離した。分離した細胞をＩＳＡＡＣ法に供した。 After culturing, CD138 + cells were isolated using anti-human CD138-specific antibody-conjugated microbeads (Miltenyi Biotec) and LS columns (Miltenyi Biotec) according to the manufacturer's instructions. Separated cells were subjected to the ISAAC method.
　リンパ球チップ及びＩＳＡＡＣ法は上掲の文献を参照して実施した。簡潔には，リンパ球チップの表面を１０μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質（感染研より供与いただいたもの），１０μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　Ｎ－ｔｅｒｍｉｎａｌ　ｄｏｍａｉｎ（ＮＴＤ）タンパク質（ＡＣＲＯ　Ｂｉｏｓｙｓｔｅｍｓ）を含むＰＢＳ，もしくは，１０μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ（ＡＣＲＯ　Ｂｉｏｓｙｓｔｅｍｓ）を含むリン酸緩衝生理食塩水（ＰＢＳ）で浸し，４℃で一夜保温することでＳＡＲＳ－ＣｏＶ－２スパイクタンパク質，ＮＴＤ，もしくはＲＢＤをリンパ球チップの表面に固層化した。その抗原溶液を除去した後，そのリンパ球チップの表面を０．０１％　Ｂｉｏｌｉｐｉｄｕｒｅ（ＮＯＦ　Ｃｏｒｐｏｒａｔｉｏｎ）で室温において３０分間ブロッキングした。その後リンパ球チップの表面を，１０％ＦＣＳを含むＲＰＭＩ培地で洗浄した。 The lymphocyte chip and ISAAC method were performed with reference to the above-mentioned literature. Briefly, the surface of the lymphocyte chip was treated with 10 μg/ml SARS-CoV-2 spike protein (provided by the National Institute of Infectious Diseases), 10 μg/ml SARS-CoV-2 N-terminal domain (NTD) protein (ACRO Biosystems) or 10 μg/ml SARS-CoV-2 RBD (ACRO Biosystems) in phosphate-buffered saline (PBS) and incubated overnight at 4°C to extract SARS-CoV-2 spike protein, NTD , or RBD was immobilized on the surface of the lymphocyte chip. After removing the antigen solution, the surface of the lymphocyte chip was blocked with 0.01% Biolipidure (NOF Corporation) at room temperature for 30 minutes. After that, the surface of the lymphocyte chip was washed with RPMI medium containing 10% FCS.
　洗浄後，分離したＣＤ１３８＋細胞をそのリンパ球チップに配列し，ウエルの外側に残った細胞を１０％ＦＣＳを含むＲＰＭＩ培地を用い穏やかな洗浄により除去した。そのリンパ球チップ上の細胞を，１０％ＦＣＳを含むＲＰＭＩ培地にて３７℃で３時間培養した。 After washing, the separated CD138+ cells were arranged on the lymphocyte chip, and the cells remaining outside the well were removed by gentle washing using RPMI medium containing 10% FCS. The cells on the lymphocyte chip were cultured in RPMI medium containing 10% FCS at 37°C for 3 hours.
　培養後ＰＢＳにて穏やかに洗浄した後，そのチップ上にＣｙ３コンジュゲート抗ヒトＩｇＧ－Ｆｃ抗体（Ｓｉｇｍａ）を添加して３０分間保温した。ＰＢＳにて緩やかな洗浄後，最後に１μＭのＣｅｌｌ　Ｔｒａｃｅ　Ｏｒｅｇｏｎ　Ｇｒｅｅｎ（Ｍｏｌｅｃｕｌａｒ　Ｐｒｏｂｅｓ）を用いて室温で５分間染色した。単一の細胞から放出された抗原特異的抗体を蛍光顕微鏡（ＢＸ５１ＷＩ，Ｏｌｙｍｐｕｓ）の下で観察した。抗原特異的抗体産生細胞を個々のウエルから蛍光顕微鏡下で毛細管（Ｐｒｉｍｅｔｅｃｈ）を取り付けたマイクロマニピュレーター（ＴｒａｎｓｆｅｒＭａｎ　４ｒ，　Ｅｐｐｅｎｄｏｒｆ）を用いて回収し，次いでそれらを逆転写のためにマイクロチューブに吐出した。 After culturing and gently washing with PBS, a Cy3-conjugated anti-human IgG-Fc antibody (Sigma) was added to the chip and incubated for 30 minutes. After gentle washing with PBS, the cells were finally stained with 1 μM Cell Trace Oregon Green (Molecular Probes) at room temperature for 5 minutes. Antigen-specific antibodies released from single cells were observed under a fluorescence microscope (BX51WI, Olympus). Antigen-specific antibody-producing cells were collected from individual wells under a fluorescence microscope using a micromanipulator (TransferMan 4r, Eppendorf) fitted with a capillary tube (Primetech), and then ejected into microtubes for reverse transcription.
　回収した単一細胞からのＶＨ及びＶＬ断片に関する抗体遺伝子の増幅法は，上掲の文献に記載された方法に準じて行った。簡潔には，１５Ｕ　ＳｕｐｅｒＳｃｒｉｐｔＴＭ　ＩＩＩ（Ｉｎｖｉｔｒｏｇｅｎ）により，それぞれ０．２５ｐｍｏｌのＩｇγ－ＲＴ（５’－ＧＣＧＡＧＴＡＧＡＧＧＣＣＴＧＡＧＧＡＣ－３’）（配列番号６１８），Ｉｇλ－ＲＴ（５’－ＡＣＴＧＴＣＴＴＣＴＣＣＡＣＧＧＴＧＣＴＣＣ－３’）（配列番号６１９），及びＩｇκ－ＲＴ（５’－ＧＡＴＧＣＣＡＧＴＴＧＴＴＴＧＧＧＴＧＧＴ－３’）（配列番号６２０）プライマー，１Ｕ　ＲＮａｓｅ阻害剤（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ），０．５ｍＭのそれぞれのｄＮＴＰ，５ｍＭ　ＤＴＴ，０．２％　Ｔｒｉｔｏｎ－Ｘ１００，及び１×１ｓｔ　ｓｔｒａｎｄ　ｃＤＮＡ緩衝液（Ｉｎｖｉｔｒｏｇｅｎ）を用いて，５５℃で１時間逆転写を実施した。ｃＤＮＡの３’テイリング（３’－ｔａｉｌｉｎｇ）を，２０Ｕ　末端デオキシヌクレオチドトランスフェラーゼ（Ｒｏｃｈｅ）により，４ｍＭ　ＭｇＣｌ２，０．５ｍＭ　ｄＧＴＰ，５０ｍＭ　Ｐ－Ｋ緩衝液　（２５ｍＭ　Ｋ２ＨＰＯ４及び２５ｍＭ　ＫＨ２ＰＯ４，ｐＨ７．０）及び１Ｕ　ＲＮａｓｅ阻害剤を用いて，３７℃で１時間実施した。第１ＰＣＲを，ｄＣアダプタープライマー（５’－ＡＧＣＡＧＴＡＧＣＡＧＣＡＧＴＴＣＧＡＴＡＡＣＴＴＣＧＡＡＴＴＣＴＧＣＡＧＴＣＧＡＣＧＧＴＡＣＣＧＣＧＧＧＣＣＣＧＧＧＡＴＣＣＣＣＣＣＣＣＣＣＣＣＣＤＮ－３’）（配列番号６２１）ならびにγ鎖（Ｉｇγ－１ｓｔ：５’－ＣＧＡＧＴＴＣＣＡＡＧＴＣＡＣＧＧＴＣＡ－３’）（配列番号６２２），λ鎖（Ｉｇλ－１ｓｔ：５’－ＣＴＴＣＴＣＣＡＣＧＧＴＧＣＴＣＣＣＴＴＣＡＴ－３’）（配列番号６２３），及びκ鎖の定常領域（Ｉｇκ－１ｓｔ：５’－ＣＴＣＣＣＡＧＧＴＧＡＣＧＧＴＧＡＣＡＴ－３’）（配列番号６２４）の混合物を用いて実施した。それぞれの産生物の増幅を，ｐｒｉｍｅＳＴＡＲ　ＤＮＡポリメラーゼ（ＴａＫａＲａ）を用いて，製造業者の説明書にしたがって実施した：３０サイクルの，９４℃で１５秒間の変性，６０℃で５秒間のアニーリング，及び７２℃で１分３０秒間の伸長。次に，結果として得られたＰＣＲ産物を水で４倍希釈し，それぞれ２μｌをネステッドＰＣＲのために用いた。ネステッドＰＣＲをアダプタープライマー（５’－ＡＧＣＡＧＴＡＧＣＡＧＣＡＧＴＴＣＧＡＴＡＡ－３’）（配列番号６２５）及びγ鎖（Ｉｇγ－ｎｅｓｔ：５’－ＧＣＣＴＴＴＧＡＣＣＡＡＧＣＡＧＣＣＣＡＡ－３’）（配列番号６２６），λ鎖（Ｉｇλ－ｎｅｓｔ：５’－ＡＧＴＧＴＧＧＣＣＴＴＧＴＴＧＧＣＴＴＧ－３’）（配列番号６２７），又はκ鎖定常領域（Ｉｇκ－ｎｅｓｔ：５’－ＣＧＧＧＡＡＡＧＴＡＴＴＴＡＴＴＣＧＣＣＡＣＡ－３’）（配列番号６２８）に特異的なそれぞれのネステッドプライマーにより，ｐｒｉｍｅＳＴＡＲ　ＤＮＡポリメラーゼを用いて実施した。そのＰＣＲ産物を，ヒトγ鎖，λ鎖，又はκ鎖に関する完全な定常領域ｃＤＮＡを含有する発現ベクターの中に挿入した。その後，Ｅｘｐｉ２９３Ｆ（登録商標）細胞（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）をその完全な抗体分子をコードするγ鎖及び，λ鎖もしくはκ鎖発現ベクター両方をＥｘｐｉ２９３　Ｅｘｐｒｅｓｓｉｏｎ　Ｓｙｓｔｅｍ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）を用いて同時形質移入し，培養した細胞の上清を７日後に集め，次いでリコンビナントモノクローナル抗体をプロテインＧカラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）を用いて精製した。 The antibody gene amplification method for the VH and VL fragments from the collected single cells was performed according to the method described in the above-mentioned literature. Briefly, 0.25 pmol each of Igγ-RT (5′-GCGAGTAGAGGCCTGAGGAC-3′) (SEQ ID NO: 618), Igλ-RT (5′-ACTGTCTTCTCCACGGTGCTCC-3′) (SEQ ID NO: 618) was obtained by 15 U SuperScript™ III (Invitrogen). 619), and Igκ-RT (5′-GATGCCAGTTGTTTGGGTGGT-3′) (SEQ ID NO: 620) primer, 1U RNase inhibitor (New England Biolabs), 0.5 mM each dNTP, 5 mM DTT, 0.2% Triton- Reverse transcription was performed at 55° C. for 1 hour using X100 and 1×1st strand cDNA buffer (Invitrogen). 3′-tailing of the cDNA was performed by 20 U terminal deoxynucleotide transferase (Roche) in 4 mM MgCl 2 , 0.5 mM dGTP, 50 mM PK buffer (25 mM K 2 HPO 4 and 25 mM KH 2 PO 4 ). , pH 7.0) and 1 U RNase inhibitor at 37°C for 1 hour. The first PCR was performed with a dC adapter primer (5′-AGCAGTAGCAGCAGTTCGATAACTTCGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCCCCCCCCCCCCDN-3′) (SEQ ID NO: 621) and γ chain (Igγ-1st: 5′-CGAGTTCCAAGTCACGGTCA-3′) (SEQ ID NO: 622), λ chain (Igλ-1st: 5′-CTTCTCCACGGTGCTCCCTTCAT-3′) (SEQ ID NO: 623), and a mixture of the constant region of the kappa chain (Igκ-1st: 5′-CTCCCAGGTGACGGTGACAT-3′) (SEQ ID NO: 624). Amplification of each product was performed using primeSTAR DNA polymerase (TaKaRa) according to the manufacturer's instructions: 30 cycles of denaturation at 94°C for 15 seconds, annealing at 60°C for 5 seconds, and 72 Extension for 1 minute 30 seconds at °C. The resulting PCR products were then diluted 4-fold with water and 2 μl of each was used for nested PCR. Nested PCR was performed using an adapter primer (5'-AGCAGTAGCAGCAGTTCGATAA-3') (SEQ ID NO: 625) and γ chain (Igγ-nest: 5'-GCCTTTGACCAAGCAGCCCAA-3') (SEQ ID NO: 626), λ chain (Igλ-nest: 5'-AGTGTGGCCTTGTTGGCTTG-3') (SEQ ID NO: 627), or the kappa chain constant region (Igκ-nest: 5'-CGGGAAAGTATTATTCGCCACA-3') (SEQ ID NO: 628) using primeSTAR DNA polymerase with respective nested primers implemented. The PCR products were inserted into expression vectors containing the complete constant region cDNAs for human γ, λ, or κ chains. Expi293F® cells (Thermo Fisher Scientific) were then co-transfected with the γ chain encoding the complete antibody molecule and both the λ or κ chain expression vectors using the Expi293 Expression System (Thermo Fisher Scientific). , the supernatant of the cultured cells was collected after 7 days, and then the recombinant monoclonal antibody was purified using a protein G column (GE Healthcare).
　その結果，クローン番号１０２８－０５Ｋ，１０２８－２５Ｋ，１０２８－２９Ｋ，１０２８－３０Ｋ，１０２８－３７Ｋ，１０２８－５０Ｋ，１０２８－５５Ｋ，１０２８－６１Ｌ，１０２８－６２Ｌ，１０２８－６５Ｋ，１０２８－６８Ｌ，１０２８－８４Ｋ，１０２８－８７Ｌ，１０２８－１０６Ｋ，１０２８－１２１Ｋ，１０２８－１３７Ｋ，１０２８－１６４Ｌ，０２１０－１９Ｋ，０２１０－２０Ｌ，０２１０－２８Ｋ，０２１０－３０Ｌ，０２１０－３１Ｌ，０２１０－３５Ｋ，０２１０－３８Ｌ，０２１０－５２Ｌ，０２１０－５８Ｋ，１－１５，及び１－４４の抗体が得られた。これらの重鎖（シグナルペプチドを含む）の核酸配列は，それぞれ，配列番号１，２３，４５，６７，８９，１１１，１３３，１５５，１７７，１９９，２２１，２４３，２６５，２８７，３０９，３３１，３５３，３７５，３９７，４１９，４４１，４６３，４８５，５０７，５２９，５５１，５７３，及び５９５に記載の通りであった。また，これらの軽鎖（シグナルペプチドを含む）の核酸配列は，それぞれ，配列番号１２，３４，５６，７８，１００，１２２，１４４，１６６，１８８，２１０，２３２，２５４，２７６，２９８，３２０，３４２，３６４，３８６，４０８，４３０，４５２，４７４，４９６，５１８，５４０，５６２，５８４，及び６０６に記載の通りであった。また，これらの重鎖（シグナルペプチドを含む）のアミノ酸配列は，それぞれ，配列番号２，２４，４６，６８，９０，１１２，１３３，１５６，１７８，２００，２２２，２４４，２６６，２８８，３１０，３３２，３５４，３７６，３９８，４２０，４４２，４６４，４８６，５０８，５３０，５５２，５７４，及び５９６に記載の通りであった。また，これらの軽鎖（シグナルペプチドを含む）のアミノ酸配列は，それぞれ，配列番号１３，３５，５７，７９，１０１，１２３，１４５，１６７，１８９，２１１，２３３，２５５，２７７，２９９，３２１，３４３，３６５，３８７，４０９，４３１，４５３，４７５，４９７，５１９，５４１，５６３，５８５，及び６０７に記載の通りであった。 As a result, clone numbers 1028-05K, 1028-25K, 1028-29K, 1028-30K, 1028-37K, 1028-50K, 1028-55K, 1028-61L, 1028-62L, 1028-65K, 1028-68L, 1028 -84K, 1028-87L, 1028-106K, 1028-121K, 1028-137K, 1028-164L, 0210-19K, 0210-20L, 0210-28K, 0210-30L, 0210-31L, 0210-35K, 0210-38L , 0210-52L, 0210-58K, 1-15, and 1-44 antibodies were obtained. The nucleic acid sequences of these heavy chains (including signal peptides) are SEQ ID NOS: 1, 23, 45, 67, 89, 111, 133, 155, 177, 199, 221, 243, 265, 287, 309, 331, respectively. , 353, 375, 397, 419, 441, 463, 485, 507, 529, 551, 573, and 595. Also, the nucleic acid sequences of these light chains (including signal peptides) are SEQ ID NOs: 12, 34, 56, 78, 100, 122, 144, 166, 188, 210, 232, 254, 276, 298, 320, respectively. , 342, 364, 386, 408, 430, 452, 474, 496, 518, 540, 562, 584, and 606. The amino acid sequences of these heavy chains (including signal peptides) are SEQ ID NOs: 2, 24, 46, 68, 90, 112, 133, 156, 178, 200, 222, 244, 266, 288, and 310, respectively. , 332, 354, 376, 398, 420, 442, 464, 486, 508, 530, 552, 574, and 596. The amino acid sequences of these light chains (including signal peptides) are SEQ ID NOs: 13, 35, 57, 79, 101, 123, 145, 167, 189, 211, 233, 255, 277, 299, and 321, respectively. , 343, 365, 387, 409, 431, 453, 475, 497, 519, 541, 563, 585, and 607.
（実施例２）取得抗体のＳＡＲＳ－ＣｏＶ－２スパイクタンパク質結合アッセイ
　作製した抗体のうち，１０２８－０５Ｋ，１０２８－２５Ｋ，１０２８－２９Ｋ，１０２８－３０Ｋ，１０２８－３７Ｋ，１０２８－５０Ｋ，１０２８－５５Ｋ，１０２８－６１Ｌ，１０２８－６２Ｌ，１０２８－６５Ｋ，１０２８－６８Ｌ，１０２８－８４Ｋ，１０２８－８７Ｌ，１０２８－１０６Ｋ，１０２８－１２１Ｋ，１０２８－１３７Ｋ，１０２８－１６４Ｌについて，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質と結合するかＥＬＩＳＡにて解析した。具体的には１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質を含むＰＢＳを，Ｍａｘｉｓｏｒｐ　９６　ｗｅｌｌ　ｐｌａｔｅ（Ｎｕｎｃ）に４℃で一夜保温することで，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質をウエルの底面に固層化した。抗原溶液を除去した後，３％牛血清アルブミン（ＢＳＡ）と０．１％　Ｔｗｅｅｎ－２０を含むＰＢＳで１時間ブロッキングした。次に，０．１％　Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，２００ｎｇ／ｍｌの作製した抗体を１時間インキュベートした。その後０．１％　Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，２００　ｎｇ／ｍｌペルオキシダーゼ標識抗ヒトＩｇＧ－Ｆｃ　Ｆ（ａｂ）’２フラグメント（ｓｉｇｍａ）で１時間インキュベートした。最後に０．１％　Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，ＴＭＢ基質（ＳｅｒａＣａｒｅ　Ｌｉｆｅ　Ｓｃｉｅｎｃｅｓ）を用いて発色させ，プレートリーダーＦＬＵＯｓｔａｒ　ＯＭＥＧＡ　（ＢＭＧ　ＬＡＢＴＥＣＨ）を用いて吸光度４５０ｎｍの測定を行った。 (Example 2) SARS-CoV-2 spike protein binding assay of obtained antibodies , 1028-61L, 1028-62L, 1028-65K, 1028-68L, 1028-84K, 1028-87L, 1028-106K, 1028-121K, 1028-137K, 1028-164L for SARS-CoV-2 spike protein and It was analyzed by ELISA for binding. Specifically, PBS containing 1 μg / ml SARS-CoV-2 spike protein is incubated overnight at 4 ° C. in Maxisorp 96 well plate (Nunc) to solidify the SARS-CoV-2 spike protein on the bottom of the well. turned into After removing the antigen solution, the cells were blocked with PBS containing 3% bovine serum albumin (BSA) and 0.1% Tween-20 for 1 hour. Next, it was washed with PBS containing 0.1% Tween-20 and incubated with 200 ng/ml of the prepared antibody for 1 hour. Then, the cells were washed with PBS containing 0.1% Tween-20 and incubated with 200 ng/ml peroxidase-labeled anti-human IgG-Fc F(ab)' 2 fragment (sigma) for 1 hour. Finally, the cells were washed with PBS containing 0.1% Tween-20, developed with TMB substrate (SeraCare Life Sciences), and absorbance at 450 nm was measured using a plate reader FLUOstar OMEGA (BMG LABTECH).
　実施例１において作製した抗体のうち，１７種類の抗体の結果を図１（ＥＣＤ　ｔｒｉｍｅｒ）に示す。また，実施例１において作製した２８種類の抗体は，いずれもＳＡＲＳ－ＣｏＶ－２スパイクタンパク質との結合を確認した。 Of the antibodies produced in Example 1, the results of 17 types of antibodies are shown in Figure 1 (ECD trimer). Moreover, all of the 28 types of antibodies produced in Example 1 were confirmed to bind to the SARS-CoV-2 spike protein.
（実施例３）抗ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質抗体の結合ドメイン解析
　実施例２でスパイクタンパクに結合した１７種類の抗体が，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質内に存在するどのドメインと結合するかＥＬＩＳＡにて解析した。具体的には１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ　Ｒｅｃｅｐｔｏｒ　ｂｉｎｄｉｎｇ　ｄｏｍａｉｎ（ＲＢＤ）タンパク質（ＡＣＲＯ　Ｂｉｏｓｙｓｔｅｍｓ）含むＰＢＳ，１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　Ｎ－ｔｅｒｍｉｎａｌ　ｄｏｍａｉｎ（ＮＴＤ）タンパク質（ＡＣＲＯ　Ｂｉｏｓｙｓｔｅｍｓ）を含むＰＢＳ，及び１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ（ＡＣＲＯ　Ｂｉｏｓｙｓｔｅｍｓ）含むＰＢＳをそれぞれＭａｘｉｓｏｒｐ　９６　ｗｅｌｌ　ｐｌａｔｅ（Ｎｕｎｃ）に４℃で一夜保温することで，ＳＡＲＳ－ＣｏＶ　ＲＢＤ，ＳＡＲＳ－ＣｏＶ－２　ＮＴＤ，及びＳＡＲＳ－ＣｏＶ－２　ＲＢＤをウエルの底面にそれぞれ固層化した。抗原溶液を除去した後，３％ＢＳＡと０．１％Ｔｗｅｅｎ－２０を含むＰＢＳで１時間ブロッキングした。次に，０．１％Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，２００ｎｇ／ｍｌの作製した抗体を１時間インキュベートした。その後０．１％Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，２００ｎｇ／ｍｌペルオキシダーゼ標識抗ヒトＩｇＧ－Ｆｃ　Ｆ（ａｂ）’２フラグメントを１時間インキュベートした。最後に０．１％Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，ＴＭＢ基質を用いて発色させ，プレートリーダーを用いて吸光度４５０ｎｍの測定を行った。 (Example 3) Binding domain analysis of anti-SARS-CoV-2 spike protein antibody Which domains in the SARS-CoV-2 spike protein do the 17 types of antibodies bound to the spike protein in Example 2 bind to? Analyzed by ELISA. Specifically, PBS containing 1 μg / ml SARS-CoV Receptor binding domain (RBD) protein (ACRO Biosystems), PBS containing 1 μg / ml SARS-CoV-2 N-terminal domain (NTD) protein (ACRO Biosystems), and 1 μg PBS containing /ml SARS-CoV-2 RBD (ACRO Biosystems) was incubated overnight at 4 ° C. in Maxisorp 96 well plate (Nunc) to generate SARS-CoV RBD, SARS-CoV-2 NTD, and SARS-CoV- 2 RBDs were immobilized on the bottom of each well. After removing the antigen solution, the cells were blocked with PBS containing 3% BSA and 0.1% Tween-20 for 1 hour. Next, the cells were washed with PBS containing 0.1% Tween-20, and 200 ng/ml of prepared antibody was incubated for 1 hour. After washing with PBS containing 0.1% Tween-20, 200 ng/ml peroxidase-labeled anti-human IgG-Fc F(ab)' 2 fragment was incubated for 1 hour. Finally, the cells were washed with PBS containing 0.1% Tween-20, developed using a TMB substrate, and absorbance at 450 nm was measured using a plate reader.
　結果を図１（ＲＢＤ（ＳＡＲＳ），ＮＴＤ，及びＲＢＤ）に示す。１０２８－０５Ｋ，１０２８－３７Ｋ，１０２８－１２１Ｋは，ＳＡＲＳ－ＣｏＶ　ＲＢＤとＳＡＲＳ－ＣｏＶ－２　ＲＢＤに結合し，ＳＡＲＳ－ＣｏＶ－２　ＮＴＤには結合しなかった。１０２８－５５Ｋ，１０２８－６１Ｌ，は，ＳＡＲＳ－ＣｏＶ－２　ＲＢＤに結合し，ＳＡＲＳ－ＣｏＶ　ＲＢＤとＳＡＲＳ－ＣｏＶ－２　ＮＴＤには結合しなかった。１０２８－２９Ｋ，１０２８－６２Ｌ，１０２８－１０６Ｋ，１０２８－１３７Ｋ，１０２８－１６４Ｌは，ＳＡＲＳ－ＣｏＶ－２　ＮＴＤに結合し，ＳＡＲＳ－ＣｏＶ　ＲＢＤとＳＡＲＳ－ＣｏＶ－２　ＲＢＤには結合しなかった。１０２８－２５Ｋ，１０２８－３０Ｋ，１０２８－５０Ｋ，１０２８－６５Ｋ，１０２８－６８Ｌ，１０２８－８４Ｋ，１０２８－８７Ｌは，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質には結合するが，ＳＡＲＳ－ＣｏＶ　ＲＢＤ，ＳＡＲＳ－ＣｏＶ－２　ＮＴＤ，及びＳＡＲＳ－ＣｏＶ－２　ＲＢＤのいずれにも結合しなかった。 The results are shown in Figure 1 (RBD (SARS), NTD, and RBD). 1028-05K, 1028-37K, and 1028-121K bound to SARS-CoV RBD and SARS-CoV-2 RBD, but did not bind to SARS-CoV-2 NTD. 1028-55K, 1028-61L, bound to SARS-CoV-2 RBD, but not to SARS-CoV RBD and SARS-CoV-2 NTD. 1028-29K, 1028-62L, 1028-106K, 1028-137K, and 1028-164L bound to SARS-CoV-2 NTD, but not to SARS-CoV RBD and SARS-CoV-2 RBD. 1028-25K, 1028-30K, 1028-50K, 1028-65K, 1028-68L, 1028-84K, 1028-87L bind to SARS-CoV-2 spike protein, but SARS-CoVRBD, SARS-CoV -2 It did not bind to either NTD or SARS-CoV-2 RBD.
（実施例４）抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体によるＡＣＥ２結合阻害
　実施例３でＳＡＲＳ－ＣｏＶ－２　ＲＢＤに結合した５種類の抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体（１０２８－０５Ｋ，１０２８－３７Ｋ，１０２８－５５Ｋ，１０２８－６１Ｌ，１０２８－１２１Ｋ）が，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質とＡＣＥ２との結合を阻害するか解析した。
具体的には１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質（ＡＣＲＯ　Ｂｉｏｓｙｓｔｅｍｓ）含むＰＢＳ　をＭａｘｉｓｏｒｐ　９６　ｗｅｌｌ　ｐｌａｔｅに４℃で一夜保温することで，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質をウエルの底面に固層化した。 (Example 4) ACE2 binding inhibition by anti-SARS-CoV-2 RBD antibodies 1028-55K, 1028-61L, 1028-121K) inhibited the binding of SARS-CoV-2 spike protein to ACE2.
Specifically, PBS containing 1 μg/ml SARS-CoV-2 spike protein (ACRO Biosystems) was incubated overnight at 4°C in a Maxisorp 96 well plate to solidify the SARS-CoV-2 spike protein on the bottom of the well. did.
　抗原溶液を除去した後，３％ＢＳＡと０．１％Ｔｗｅｅｎ－２０を含むＰＢＳで１時間ブロッキングした。次に，０．１％Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，５０ｎｇ／ｍｌビオチン標識ＡＣＥ２（Ｓｉｎｏ　Ｂｉｏｌｉｇｉｃａｌ）と１０，０００～２０ｎｇ／ｍｌ抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体の混合溶液，もしくは抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体を含まない５０ｎｇ／ｍｌビオチン標識ＡＣＥ２溶液を１時間インキュベートした。その後０．１％Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，２００ｎｇ／ｍｌペルオキシダーゼ標識ストレプトアビジン（Ａｂｃａｍ）を１時間インキュベートした。最後に０．１％Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，ＴＭＢ基質を用いて発色させ，プレートリーダーを用いて吸光度４５０ｎｍの測定を行った。抗体を含まない場合の吸光度を１００として，各濃度の抗体を含んだ場合の吸光度を算出した。 After removing the antigen solution, it was blocked with PBS containing 3% BSA and 0.1% Tween-20 for 1 hour. Next, wash with PBS containing 0.1% Tween-20, mixed solution of 50 ng/ml biotin-labeled ACE2 (Sino Biological) and 10,000 to 20 ng/ml anti-SARS-CoV-2 RBD antibody, or anti-SARS -50 ng/ml biotin-labeled ACE2 solution without CoV-2 RBD antibody was incubated for 1 hour. After washing with PBS containing 0.1% Tween-20, 200 ng/ml peroxidase-labeled streptavidin (Abcam) was incubated for 1 hour. Finally, the cells were washed with PBS containing 0.1% Tween-20, developed using a TMB substrate, and absorbance at 450 nm was measured using a plate reader. Taking the absorbance without antibody as 100, the absorbance with each concentration of antibody was calculated.
　結果を図２に示した。５種類の抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体は，いずれも量依存的に，ＡＣＥ２がＳＡＲＳ－ＣｏＶ－２スパイクタンパク質と結合することを阻害していた。本実験系において，１０２８－０５ＫのＩＣ５０は１０，０００ｎｇ／ｍｌ以上，１０２８－３７ＫのＩＣ５０はおおよそ７００ｎｇ／ｍｌ，１０２８－５５ＫのＩＣ５０はおおよそ３００ｎｇ／ｍｌ，１０２８－６１ＬのＩＣ５０はおおよそ２５０ｎｇ／ｍｌ，１０２８－１２１ＫのＩＣ５０はおおよそ７，５００ｎｇ／ｍｌと算出された。 The results are shown in Figure 2. All five types of anti-SARS-CoV-2 RBD antibodies dose-dependently inhibited the binding of ACE2 to the SARS-CoV-2 spike protein. In this experimental system, the IC50 for 1028-05K is 10,000 ng/ml or more, the IC50 for 1028-37K is approximately 700 ng/ml, the IC50 for 1028-55K is approximately 300 ng/ml, and the IC50 for 1028-61L is approximately 250 ng/ml. , 1028-121K was calculated to be approximately 7,500 ng/ml.
　更に４種類の抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体（０２１０－２０Ｌ，０２１０－２８Ｋ，０２１０－３０Ｌ，及び０２１０－３８Ｌ）が，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質とＡＣＥ２との結合を阻害するかについて，同様に解析した。 Furthermore, whether four types of anti-SARS-CoV-2 RBD antibodies (0210-20L, 0210-28K, 0210-30L, and 0210-38L) inhibit the binding of SARS-CoV-2 spike protein and ACE2, analyzed in the same way.
　結果を図６に示した。４種類の抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体のうち，０２１０－２０Ｌ以外は，いずれも量依存的に，ＡＣＥ２がＳＡＲＳ－ＣｏＶ－２スパイクタンパク質と結合することを阻害していた。本実験系において，０２１０－２８ＫのＩＣ５０はおおよそ１５０ｎｇ／ｍｌ，０２１０－３０ＬのＩＣ５０はおおよそ２００ｎｇ／ｍｌ，０２１０－３８ＬのＩＣ５０はおおよそ１，５００ｎｇ／ｍｌと算出された。 The results are shown in Figure 6. Of the four anti-SARS-CoV-2 RBD antibodies, all but 0210-20L inhibited the binding of ACE2 to the SARS-CoV-2 spike protein in a dose-dependent manner. In this experimental system, the IC50 of 0210-28K was calculated to be approximately 150 ng/ml, the IC50 of 0210-30L to approximately 200 ng/ml, and the IC50 of 0210-38L to approximately 1,500 ng/ml.
（実施例５）変異抗原由来ＲＢＤに対する抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体結合解析
　最近報告されている変異株由来のＲＢＤに対して，９種類の抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体が結合するか解析した。具体的には１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ（ＷＴ）（ＡＣＲＯ　Ｂｉｏｓｙｓｔｅｍｓ）を含むＰＢＳ，１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ（ＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ）（ＡＣＲＯ　Ｂｉｏｓｙｓｔｅｍｓ）を含むＰＢＳ，１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ（ＲＢＤ　Ｎ４３９Ｋ）（ＡＣＲＯ　Ｂｉｏｓｙｓｔｅｍｓ）を含むＰＢＳ，１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ（ＲＢＤ　Ｎ４４０Ｋ）（ＡＣＲＯ　Ｂｉｏｓｙｓｔｅｍｓ）を含むＰＢＳ，１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ（ＲＢＤ　Ａ４７５Ｖ）（ＡＣＲＯ　Ｂｉｏｓｙｓｔｅｍｓ）を含むＰＢＳ，１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ（ＲＢＤ　Ｅ４８４Ｋ）（ＡＣＲＯ　Ｂｉｏｓｙｓｔｅｍｓ）を含むＰＢＳ，１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ（ＲＢＤ　Ｎ５０１Ｙ）（ＡＣＲＯ　Ｂｉｏｓｙｓｔｅｍｓ）を含むＰＢＳ，１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ（ＲＢＤ　Ｌ４５２Ｒ）（ＡＣＲＯ　Ｂｉｏｓｙｓｔｅｍｓ）を含むＰＢＳ，又は１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ（ＲＢＤ　Ｌ４５２Ｒ，Ｅ４８４Ｑ）（ＡＣＲＯ　Ｂｉｏｓｙｓｔｅｍｓ）を含むＰＢＳをそれぞれＭａｘｉｓｏｒｐ　９６　ｗｅｌｌ　ｐｌａｔｅに４℃で一夜保温することで，それぞれのタンパク質をウエルの底面にそれぞれ固層化した。 (Example 5) Anti-SARS-CoV-2 RBD antibody binding analysis to mutant antigen-derived RBD Analysis of whether nine types of anti-SARS-CoV-2 RBD antibodies bind to recently reported mutant strain-derived RBD did. Specifically, PBS containing 1 μg/ml SARS-CoV-2 RBD (WT) (ACRO Biosystems), PBS containing 1 μg/ml SARS-CoV-2 RBD (RBD K417N, E484K, N501Y) (ACRO Biosystems), 1 μg /ml SARS-CoV-2 RBD (RBD N439K) (ACRO Biosystems) in PBS, 1 μg/ml SARS-CoV-2 RBD (RBD N440K) (ACRO Biosystems) in PBS, 1 μg/ml SARS-CoV-2 RBD PBS with (RBD A475V) (ACRO Biosystems), 1 μg/ml SARS-CoV-2 RBD (RBD E484K) (ACRO Biosystems) in PBS, 1 μg/ml SARS-CoV-2 RBD (RBD N501Y) (ACRO Biosystems) PBS containing 1 μg/ml SARS-CoV-2 RBD (RBD L452R) (ACRO Biosystems), or PBS containing 1 μg/ml SARS-CoV-2 RBD (RBD L452R, E484Q) (ACRO Biosystems), respectively By incubating the Maxisorp 96 well plate at 4° C. overnight, each protein was immobilized on the bottom surface of the well.
　抗原溶液を除去した後，３％ＢＳＡと０．１％Ｔｗｅｅｎ－２０を含むＰＢＳで１時間ブロッキングした。次に，０．１％Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，２００ｎｇ／ｍｌの作製した抗体を１時間インキュベートした。その後０．１％Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，２００ｎｇ／ｍｌペルオキシダーゼ標識抗ヒトＩｇＧ－Ｆｃ　Ｆ（ａｂ）’２フラグメントを１時間インキュベートした。最後に０．１％　Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，ＴＭＢ基質を用いて発色させ，プレートリーダーを用いて吸光度４５０ｎｍの測定を行った。ＷＴに対する結合で得られた吸光度を１００として，各変異株由来ＲＢＤに対する結合で得られた吸光度を算出した。 After removing the antigen solution, the cells were blocked with PBS containing 3% BSA and 0.1% Tween-20 for 1 hour. Next, the cells were washed with PBS containing 0.1% Tween-20, and 200 ng/ml of prepared antibody was incubated for 1 hour. After washing with PBS containing 0.1% Tween-20, 200 ng/ml peroxidase-labeled anti-human IgG-Fc F(ab)' 2 fragment was incubated for 1 hour. Finally, the cells were washed with PBS containing 0.1% Tween-20, developed with a TMB substrate, and absorbance at 450 nm was measured using a plate reader. Taking the absorbance obtained by binding to WT as 100, the absorbance obtained by binding to each mutant-derived RBD was calculated.
　結果を表３及び表４に示した。１０２８－０５Ｋは，ＷＴに対する結合に比べてＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ，ＲＢＤ　Ｎ４３９Ｋ，ＲＢＤ　Ｎ４４０Ｋ，ＲＢＤ　Ｅ４８４Ｋに対する結合が，おおよそ７５～８８に減少した。ＲＢＤ　Ａ４７５Ｖ，ＲＢＤ　Ｎ５０１Ｙに対する結合の減少は認められなかった。１０２８－３７Ｋは，いずれの各変異株由来ＲＢＤに対する結合の減少は認められなかった。
１０２８－５５Ｋは，ＷＴに対する結合に比べてＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙに対する結合が，おおよそ３に減少した。ＲＢＤ　Ｎ４３９Ｋ，ＲＢＤ　Ｎ４４０Ｋ，ＲＢＤ　Ａ４７５Ｖ，ＲＢＤ　Ｅ４８４Ｋ，ＲＢＤ　Ｎ５０１Ｙに対する結合の減少は認められなかった。１０２８－６１Ｌ，ＷＴに対する結合に比べてＲＢＤ　Ｋ４１７Ｎ，　Ｅ４８４Ｋ，Ｎ５０１Ｙに対する結合が，おおよそ３３に減少した。ＲＢＤ　Ｎ４３９Ｋ，ＲＢＤ　Ｎ４４０Ｋ，ＲＢＤ　Ａ４７５Ｖ，ＲＢＤ　Ｅ４８４Ｋ，ＲＢＤ　Ｎ５０１Ｙに対する結合の減少は認められなかった。１０２８－１２１Ｋは，ＷＴに対する結合に比べてＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ，ＲＢＤ　Ｎ４３９Ｋ，ＲＢＤ　Ｎ４４０Ｋ，ＲＢＤ　Ｅ４８４Ｋに対する結合が，おおよそ６２～７２に減少した。ＲＢＤ　Ａ４７５Ｖ，ＲＢＤ　Ｎ５０１Ｙに対する結合の減少は認められなかった。 The results are shown in Tables 3 and 4. 1028-05K reduced binding to RBD K417N, E484K, N501Y, RBD N439K, RBD N440K, RBD E484K to approximately 75-88 compared to binding to WT. No reduction in binding to RBD A475V, RBD N501Y was observed. 1028-37K did not show decreased binding to any of the mutant-derived RBDs.
1028-55K reduced binding to RBD K417N, E484K, N501Y by approximately 3 compared to binding to WT. No reduction in binding was observed for RBD N439K, RBD N440K, RBD A475V, RBD E484K, RBD N501Y. Binding to RBD K417N, E484K, N501Y was reduced to approximately 33 compared to binding to 1028-61L, WT. No reduction in binding was observed for RBD N439K, RBD N440K, RBD A475V, RBD E484K, RBD N501Y. 1028-121K decreased binding to RBD K417N, E484K, N501Y, RBD N439K, RBD N440K, RBD E484K to approximately 62-72 compared to binding to WT. No reduction in binding to RBD A475V, RBD N501Y was observed.
　また，０２１０－２０Ｌは，いずれの各変異株由来ＲＢＤに対する結合の減少は認められなかった。０２１０－２８Ｋは，いずれの各変異株由来ＲＢＤに対する結合の減少は認められなかった。０２１０－３０Ｌは，ＷＴに対する結合に比べてＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙに対する結合が，おおよそ５６に減少した。ＲＢＤ　Ｅ４８４Ｋに対する結合が，おおよそ７５に減少した。ＲＢＤ　Ｎ４３９Ｋ，ＲＢＤ　Ｎ４４０Ｋ，ＲＢＤ　Ａ４７５Ｖ，ＲＢＤ　Ｎ５０１Ｙに対する結合の減少は認められなかった。０２１０－３８Ｌは，ＷＴに対する結合に比べてＲＢＤ　Ｎ４３９Ｋ，ＲＢＤ　Ｅ４８４Ｋに対する結合が，おおよそ８２～９０に減少したに減少した。ＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ，ＲＢＤ　Ｎ４４０Ｋ，ＲＢＤ　Ａ４７５Ｖ，ＲＢＤ　Ｎ５０１Ｙに対する結合の減少は認められなかった。 In addition, 0210-20L did not decrease the binding to any of the mutant-derived RBDs. 0210-28K did not show decreased binding to any of the mutant-derived RBDs. For 0210-30L, the binding to RBD K417N, E484K, and N501Y decreased to approximately 56 compared to the binding to WT. Binding to RBD E484K decreased to approximately 75. No decrease in binding to RBD N439K, RBD N440K, RBD A475V, and RBD N501Y was observed. 0210-38L reduced the binding to RBD N439K and RBD E484K to approximately 82-90 compared to the binding to WT. No decrease in binding to RBD K417N, E484K, N501Y, RBD N440K, RBD A475V and RBD N501Y was observed.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
　また，ＲＢＤ　Ｌ４５２Ｒに対する０２１０－２８Ｋ抗体の結合を解析した結果を図１０（左図）に示す。０２１０－２８Ｋ抗体は，ＲＢＤ　Ｌ４５２Ｒに対して，武漢型のＲＢＤ，及びＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙへの結合と同等の結合活性を示した。さらに、ＲＢＤ　Ｌ４５２Ｒ，Ｅ４８４Ｑに対する０２１０－２８Ｋ抗体の結合を解析した結果を図１１（左図）に示す。０２１０－２８Ｋ抗体は，ＲＢＤ　Ｌ４５２Ｒ，Ｅ４８４Ｑに対して，武漢型のＲＢＤ，ＲＢＤ　Ｎ５０１Ｙ，及びＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙへの結合と同等の結合活性を示した。 Figure 10 (left figure) shows the results of analyzing the binding of the 0210-28K antibody to RBD L452R. The 0210-28K antibody showed binding activity to RBD L452R equivalent to binding to Wuhan-type RBD and RBD K417N, E484K, and N501Y. Furthermore, FIG. 11 (left) shows the results of analyzing the binding of the 0210-28K antibody to RBD L452R and E484Q. The 0210-28K antibody showed binding activity to RBD L452R and E484Q equivalent to binding to Wuhan type RBD, RBD N501Y, and RBD K417N, E484K and N501Y.
（実施例６）変異抗原由来ＲＢＤに対する抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体によるＡＣＥ２結合阻害
　５種類の抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体が，変異株由来のＲＢＤとＡＣＥ２との結合を阻害するか解析した。具体的には１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ（ＷＴ）含むＰＢＳ，１μｇ／ｍｌ　ＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙを含むＰＢＳ，１μｇ／ｍｌ　ＲＢＤ　Ｎ４３９Ｋを含むＰＢＳ，１μｇ／ｍｌ　ＲＢＤ　Ｎ４４０Ｋを含むＰＢＳ，１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ　Ａ４７５Ｖを含むＰＢＳ，１μｇ／ｍｌ　ＲＢＤ　Ｅ４８４Ｋを含むＰＢＳ，１μｇ／ｍｌ　ＲＢＤ　Ｎ５０１Ｙを含むＰＢＳ，１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ　Ｌ４５２Ｒ，１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ　Ｌ４５２Ｒ，Ｅ４８４Ｑを含むＰＢＳ，１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ　Ｋ４１７Ｔ，Ｅ４８４Ｋ，Ｎ５０１Ｙを含むＰＢＳ，又は１μｇ／ｍｌ　ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ　Ｌ４５２Ｒ，Ｔ４７８Ｋ（いずれもＡＣＲＯ　Ｂｉｏｓｙｓｔｅｍｓ）を含むＰＢＳをそれぞれＭａｘｉｓｏｒｐ　９６ｗｅｌｌ　ｐｌａｔｅに４℃で一夜保温することで，それぞれのタンパク質をウエルの底面にそれぞれ固層化した。 (Example 6) Inhibition of ACE2 binding by anti-SARS-CoV-2 RBD antibodies to mutant antigen-derived RBD Analysis of whether five types of anti-SARS-CoV-2 RBD antibodies inhibit binding between mutant-derived RBD and ACE2 did. Specifically, PBS containing 1 μg/ml SARS-CoV-2 RBD (WT), PBS containing 1 μg/ml RBD K417N, E484K, N501Y, PBS containing 1 μg/ml RBD N439K, PBS containing 1 μg/ml RBD N440K, PBS with 1 μg/ml SARS-CoV-2 RBD A475V, PBS with 1 μg/ml RBD E484K, PBS with 1 μg/ml RBD N501Y, 1 μg/ml SARS-CoV-2 RBD L452R, 1 μg/ml SARS-CoV- 2 PBS containing RBD L452R, E484Q, PBS containing 1 μg/ml SARS-CoV-2 RBD K417T, E484K, N501Y, or PBS containing 1 μg/ml SARS-CoV-2 RBD L452R, T478K (all from ACRO Biosystems) Each protein was immobilized on the bottom of each well by incubating each Maxisorp 96-well plate at 4° C. overnight.
　抗原溶液を除去した後，３％ＢＳＡと０．１％Ｔｗｅｅｎ－２０を含むＰＢＳで１時間ブロッキングした。次に，０．１％Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，５０ｎｇ／ｍｌビオチン標識ＡＣＥ２（Ｓｉｎｏ　Ｂｉｏｌｉｇｉｃａｌ）と１，０００ｎｇ／ｍｌ　抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体の混合溶液，もしくは抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体を含まない５０ｎｇ／ｍｌビオチン標識ＡＣＥ２溶液を１時間インキュベートした。その後０．１％Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，２００ｎｇ／ｍｌペルオキシダーゼ標識ストレプトアビジン（Ａｂｃａｍ）を１時間インキュベートした。最後に０．１％Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，ＴＭＢ基質を用いて発色させ，プレートリーダーを用いて吸光度４５０ｎｍの測定を行った。抗体を含まない場合の吸光度を１００として，各濃度の抗体を含んだ場合の吸光度を算出した。 After removing the antigen solution, it was blocked with PBS containing 3% BSA and 0.1% Tween-20 for 1 hour. Next, wash with PBS containing 0.1% Tween-20, mixed solution of 50 ng/ml biotin-labeled ACE2 (Sino Biological) and 1,000 ng/ml anti-SARS-CoV-2 RBD antibody, or anti-SARS-CoV -2 A 50 ng/ml biotin-labeled ACE2 solution containing no RBD antibody was incubated for 1 hour. After washing with PBS containing 0.1% Tween-20, 200 ng/ml peroxidase-labeled streptavidin (Abcam) was incubated for 1 hour. Finally, the cells were washed with PBS containing 0.1% Tween-20, developed using a TMB substrate, and absorbance at 450 nm was measured using a plate reader. Taking the absorbance without antibody as 100, the absorbance with each concentration of antibody was calculated.
　同様に４種類の抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体（０２１０－２８Ｋ，０２１０－３０Ｌ，１０２８－５５Ｋ，１０２８－６１Ｌ）が，Ｂｅｔａ株ＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質（Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ変異を有する）とＡＣＥ２との結合を阻害するかを解析した。また，０２１０－２８Ｋ抗体が，ＲＢＤ　Ｌ４５２Ｒ，ＲＢＤ　Ｎ５０１Ｙ，ＲＢＤ　Ｋ４１７Ｔ，Ｅ４８４Ｋ，Ｎ５０１Ｙ，ＲＢＤ　Ｌ４５２Ｒ，Ｔ４７８Ｋ，ＲＢＤ　Ｌ４５２Ｒ，Ｅ４８４Ｑ，又はＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１ＹとＡＣＥ２との結合を阻害するかを同様の方法で解析した。 Similarly, four types of anti-SARS-CoV-2 RBD antibodies (0210-28K, 0210-30L, 1028-55K, 1028-61L) inhibited the Beta strain SARS-CoV-2 RBD protein (K417N, E484K, N501Y mutations). It was analyzed whether it inhibits the binding of ACE2 to ACE2. In addition, whether the 0210-28K antibody inhibits the binding of RBD L452R, RBD N501Y, RBD K417T, E484K, N501Y, RBD L452R, T478K, RBD L452R, E484Q, or RBD K417N, E484K, N501Y to ACE2 was examined in a similar manner. analyzed by the method.
　１０２８－０５Ｋ，１０２８－３７Ｋ，１０２８－５５Ｋ，１０２８－６１Ｌ，及び１０２８－１２１Ｋの結果を表５に示す。１０２８－０５Ｋは，ＷＴとＡＣＥ２との結合を９０程度と殆ど抑えられず，いずれの各変異株由来ＲＢＤとＡＣＥ２との結合も９０－９６程度と，ＷＴと同様に殆ど抑えられなかった。１０２８－３７Ｋは，ＷＴとＡＣＥ２との結合を６３程度までに抑えるのに対し，いずれの各変異株由来ＲＢＤとＡＣＥ２との結合を５３－７０程度と，ＷＴと同程度に抑えた。１０２８－５５Ｋは，ＷＴとＡＣＥ２との結合を２２程度までに抑えるのに対し，ＲＢＤ　Ｎ４３９Ｋ，ＲＢＤ　Ｎ４４０Ｋ，ＲＢＤ　Ａ４７５Ｖ，ＲＢＤ　Ｅ４８４，及びＲＢＤ　Ｎ５０１ＹとＡＣＥ２との結合を２５－３５程度と，ＷＴと同程度に抑えた。一方ＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１ＹとＡＣＥ２との結合は９０程度と，殆ど抑えられなかった。１０２８－６１Ｌは，ＷＴとＡＣＥ２との結合を２０程度までに抑えるのに対し，ＲＢＤ　Ｎ４３９Ｋ，ＲＢＤ　Ｎ４４０Ｋ，ＲＢＤ　Ａ４７５Ｖ，ＲＢＤ　Ｅ４８４，及びＲＢＤ　Ｎ５０１ＹとＡＣＥ２との結合を１８－３３程度と，ＷＴと同程度に抑えた。一方ＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１ＹとＡＣＥ２との結合は７８程度と，殆ど抑えられなかった。１０２８－１２１Ｋは，ＷＴとＡＣＥ２との結合を８０程度と殆ど抑えられず，いずれの各変異株由来ＲＢＤとＡＣＥ２との結合も８０－９０程度と，ＷＴと同様に殆ど抑えられなかった。 Table 5 shows the results of 1028-05K, 1028-37K, 1028-55K, 1028-61L, and 1028-121K. 1028-05K hardly suppressed the binding of WT and ACE2 to about 90, and the binding of RBD derived from each mutant to ACE2 was about 90 to 96, which was hardly suppressed like WT. 1028-37K suppressed the binding between WT and ACE2 to about 63, whereas the binding between RBD derived from any of the mutant strains and ACE2 was suppressed to about 53-70, which is the same level as WT. 1028-55K suppresses the coupling between WT and ACE2 to about 22, while RBD N439K, RBD N440K, RBD A475V, RBD E484, and RBD N501Y reduce the coupling between WT and ACE2 to about 25-35. reduced to the same extent. On the other hand, the binding between RBD K417N, E484K, N501Y and ACE2 was about 90, which was hardly suppressed. 1028-61L suppresses the coupling between WT and ACE2 to about 20, while RBD N439K, RBD N440K, RBD A475V, RBD E484, and RBD N501Y suppress the coupling between WT and ACE2 to about 18-33. reduced to the same extent. On the other hand, the binding between RBD K417N, E484K, N501Y and ACE2 was about 78, which was hardly suppressed. In 1028-121K, the binding between WT and ACE2 was hardly suppressed at about 80, and the binding between RBD derived from each mutant strain and ACE2 was also hardly suppressed at about 80-90, similar to WT.
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
　０２１０－２８Ｋ，１０２８－５５Ｋ，０２１０－３０Ｌ，及び１０２８－６１ＬのＢｅｔａ株ＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質（ＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ）との結合阻害の結果を図７Ａ及びＢに示す。 Figures 7A and B show the results of inhibition of binding of 0210-28K, 1028-55K, 0210-30L, and 1028-61L to the RBD proteins (RBD K417N, E484K, N501Y) of Beta strain SARS-CoV-2.
　０２１０－２８Ｋは，量依存的に，ＡＣＥ２がＢｅｔａ株ＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質（ＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ）と結合することを阻害していた。その阻害効果は，武漢型のＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質よりも強かった。本実験系において，０２１０－２８ＫのＢｅｔａ株ＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質に対するＩＣ５０は３５０ｎｇ／ｍｌ，武漢型のＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質に対するＩＣ５０は６００ｎｇ／ｍｌと算出された。 　0210-28K dose-dependently inhibited the binding of ACE2 to the RBD proteins (RBD K417N, E484K, N501Y) of Beta strain SARS-CoV-2. Its inhibitory effect was stronger than that of the Wuhan-type SARS-CoV-2 RBD protein. In this experimental system, the IC50 for the RBD protein of 0210-28K Beta strain SARS-CoV-2 was calculated to be 350 ng/ml, and the IC50 for the RBD protein of Wuhan type SARS-CoV-2 was calculated to be 600 ng/ml.
　１０２８－５５Ｋは，量依存的に，ＡＣＥ２が武漢型のＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質と結合することを阻害していた。一方で，Ｂｅｔａ株ＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質と結合することを，阻害できなかった。本実験系において，１０２８－５５ＫのＢｅｔａ株ＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質に対するＩＣ５０は１０，０００ｎｇ／ｍｌ以上，武漢型のＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質に対するＩＣ５０は４００ｎｇ／ｍｌと算出された。 　1028-55K dose-dependently inhibited the binding of ACE2 to the RBD protein of Wuhan-type SARS-CoV-2. On the other hand, it failed to inhibit binding to the RBD protein of Beta strain SARS-CoV-2. In this experimental system, the IC50 for the RBD protein of 1028-55K Beta strain SARS-CoV-2 was calculated to be 10,000 ng/ml or more, and the IC50 for the RBD protein of Wuhan-type SARS-CoV-2 was calculated to be 400 ng/ml. .
　０２１０－３０Ｌは，量依存的に，ＡＣＥ２がＢｅｔａ株ＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質と結合することを阻害していた。その阻害効果は，武漢型のＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質よりも弱かった。本実験系において，０２１０－３０ＬのＢｅｔａ株ＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質に対するＩＣ５０は２，０００ｎｇ／ｍｌ，武漢型のＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質に対するＩＣ５０は５００ｎｇ／ｍｌと算出された。 　0210-30L dose-dependently inhibited the binding of ACE2 to the RBD protein of Beta strain SARS-CoV-2. Its inhibitory effect was weaker than that of the Wuhan-type SARS-CoV-2 RBD protein. In this experimental system, the IC50 for the RBD protein of 0210-30L Beta strain SARS-CoV-2 was calculated to be 2,000 ng/ml, and the IC50 for the RBD protein of Wuhan type SARS-CoV-2 was calculated to be 500 ng/ml.
　１０２８－６１Ｌは，量依存的に，ＡＣＥ２がＢｅｔａ株ＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質と結合することを阻害していた。その阻害効果は，武漢型のＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質よりも弱かった。本実験系において，１０２８－６１ＬのＢｅｔａ株ＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質に対するＩＣ５０は１０，０００ｎｇ／ｍｌ以上，武漢型のＳＡＲＳ－ＣｏＶ－２のＲＢＤタンパク質に対するＩＣ５０は７００ｎｇ／ｍｌと算出された。 　1028-61L dose-dependently inhibited the binding of ACE2 to the RBD protein of Beta strain SARS-CoV-2. Its inhibitory effect was weaker than that of the Wuhan-type SARS-CoV-2 RBD protein. In this experimental system, the IC50 for the RBD protein of 1028-61L Beta strain SARS-CoV-2 was calculated to be 10,000 ng/ml or more, and the IC50 for the RBD protein of Wuhan type SARS-CoV-2 was calculated to be 700 ng/ml. .
　また，０２１０－２８ＫがＲＢＤ　Ｌ４５２ＲとＡＣＥ２との結合を阻害するか解析した結果を図１０（右図）に示す。０２１０－２８Ｋ抗体は，ＲＢＤ　Ｌ４５２Ｒに対して，武漢型と同等の濃度依存的結合阻害活性を示した。 Fig. 10 (right figure) shows the results of analyzing whether 0210-28K inhibits the binding of RBD L452R and ACE2. The 0210-28K antibody showed concentration-dependent binding inhibitory activity to RBD L452R equivalent to that of the Wuhan type.
　さらに，０２１０－２８Ｋ抗体がＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ，及びＲＢＤ　Ｌ４５２Ｒ，Ｅ４８４ＱとＡＣＥ２との結合を阻害するか解析した結果を図１１（右図）に示す。０２１０－２８Ｋ抗体は，ＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ，及びＲＢＤ　Ｌ４５２Ｒ，Ｅ４８４Ｑに対して，武漢型と同等の濃度依存的結合阻害活性を示した。 Furthermore, Fig. 11 (right figure) shows the results of analyzing whether the 0210-28K antibody inhibits the binding of RBD K417N, E484K, N501Y, and RBD L452R, E484Q to ACE2. The 0210-28K antibody showed concentration-dependent binding inhibitory activity equivalent to that of the Wuhan type against RBD K417N, E484K, N501Y and RBD L452R, E484Q.
　さらに，図１３に，６種類の変異株由来のＲＢＤ（ＲＢＤ　５０１Ｙ，ＲＢＤ　Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ，ＲＢＤ　Ｋ４１７Ｔ，Ｅ４８４Ｋ，Ｎ５０１Ｙ，ＲＢＤ　Ｌ４５２Ｒ，Ｅ４８４Ｑ，ＲＢＤ　Ｌ４５２Ｒ，Ｔ４７８Ｋ，ＲＢＤ　Ｌ４５２Ｒ）とＡＣＥ２との結合を，０２１０－２８Ｋ抗体が阻害するか解析した結果を示す。いずれの変異株由来のＲＢＤに対しても，武漢型（ＷＴ）と同等の濃度依存的結合阻害活性を示した。 Furthermore, FIG. 13 shows the binding of RBD derived from six types of mutants (RBD 501Y, RBD K417N, E484K, N501Y, RBD K417T, E484K, N501Y, RBD L452R, E484Q, RBD L452R, T478K, RBD L452R) to ACE2. is inhibited by the 0210-28K antibody. It showed concentration-dependent binding inhibitory activity equivalent to that of Wuhan type (WT) against RBD derived from any of the mutant strains.
（実施例７）新型コロナウイルスのスパイクタンパク質を外套したシュードタイプウイルスを用いた感染抑制実験
　新型コロナウイルスのスパイクタンパク質を外套したシュードタイプウイルス（ＳＡＲＳ－ＣｏＶ－２　Ｓｔ１９ｐｖ）は，以下の論文に掲載してある方法で作製した。Ｔａｎｉ　Ｈ　ｅｔ　ａｌ．，Ｖｉｒｏｌｏｇｙ　Ｊｏｕｒｎａｌ（２０２１）１８：１６． (Example 7) Infection suppression experiment using a pseudotype virus enveloped with the spike protein of the new coronavirus The pseudotype virus (SARS-CoV-2 St19pv) enveloped with the spike protein of the new coronavirus is published in the following paper. It was prepared according to the method described. Tani H et al. , Virology Journal (2021) 18:16.
　ＴＭＰＲＳＳ２を過剰発現しているＶｅｒｏＥ６細胞（ＶｅｒｏＥ６／ＴＭＰＲＳＳ２）を９６ｗｅｌｌプレートに播種し，翌日９０～１００％コンフルエントの状態で，各濃度の精製抗体を添加した。その後，続けてＳＡＲＳ－ＣｏＶ－２　Ｓｔ１９ｐｖ及びＶＳＶ－Ｇを外套したコントロールのシュードタイプウイルス（ＶＳＶｐｖ）を添加した。１日培養後，細胞に細胞溶解液を含むルシフェラーゼ基質溶液を添加し，ルシフェラーゼの活性を測定した。シュードタイプウイルスが細胞に感染できれば，ルシフェラーゼ活性が見られ発光値として数値化される。抗体により感染が阻害されればルシフェラーゼの発光値が低くなるため，その数値を元に感染阻止率を計算した。 　VeroE6 cells overexpressing TMPRSS2 (VeroE6/TMPRSS2) were seeded in a 96-well plate, and the next day, when the cells were 90-100% confluent, each concentration of purified antibody was added. Subsequently, SARS-CoV-2 St19pv and VSV-G-encapsulated control pseudotype virus (VSVpv) were subsequently added. After culturing for one day, a luciferase substrate solution containing a cell lysate was added to the cells, and luciferase activity was measured. If the pseudotype virus can infect cells, luciferase activity is observed and quantified as a luminescence value. Since the luminescence value of luciferase would decrease if the infection was inhibited by the antibody, the infection inhibition rate was calculated based on that value.
　その結果，図３に示すように１０２８－５５Ｋ及び１０２８－６１Ｌにおいて顕著に，抗体濃度依存的かつＳＡＲＳ－ＣｏＶ－２　Ｓｔ１９ｐｖ特異的な感染阻害が認められた。また，１０２８－５５Ｋや１０２８－６１Ｌに比べると弱いながらも１０２８－０５Ｋ及び１０２８－１２１ＫでもＳＡＲＳ－ＣｏＶ－２　Ｓｔ１９ｐｖ特異的な感染阻害が認められた。 As a result, as shown in Figure 3, significant antibody concentration-dependent and SARS-CoV-2 St19pv-specific inhibition of infection was observed at 1028-55K and 1028-61L. SARS-CoV-2 St19pv-specific inhibition of infection was also observed with 1028-05K and 1028-121K, although weaker than with 1028-55K and 1028-61L.
（実施例８）新型コロナウイルスを用いた感染抑制実験
　新型コロナウイルスは，富山県内新型コロナウイルス感染症罹患患者より富山県衛生研究所で分離したウイルス株（ＴＩＨ／ＳＡＲＳ２／２０２０－１）を用いた。終濃度０．０６２５，０．１２５，０．２５，０．５，１．０μｇ／ｍｌになるように，それぞれ１０２８－０５Ｋ，１０２８－５５Ｋ及び１０２８－６１Ｌの精製抗体と感染価（Ｉｎｆｅｃｔｉｏｕｓ　Ｕｎｉｔ；ＩＵ）で１００ＩＵのウイルスをマイクロチューブ内で混合し，３７℃で１時間反応させた。その後，２４ｗｅｌｌプレートに播種したＶｅｒｏＥ６／ＴＭＰＲＳＳ２細胞に添加し，３７℃で２時間吸着させた。その後，吸着しなかった抗体とウイルスを含む混合液を完全に除き，再び各濃度の抗体を含む培養培地を添加し，２日間培養を続けた。２日間培養した細胞の培養上清を回収し，各抗体濃度添加時のウイルス感染価をプラークアッセイ法により測定した。 (Example 8) Infection control experiment using new coronavirus The new coronavirus uses a virus strain (TIH/SARS2/2020-1) isolated at the Toyama Prefectural Institute of Health from a patient with a new coronavirus infection in Toyama Prefecture. board. Purified antibodies and infectious titers (Infectious Unit; IU) was mixed with 100 IU of virus in a microtube and allowed to react at 37° C. for 1 hour. After that, it was added to VeroE6/TMPRSS2 cells seeded in a 24-well plate and adsorbed at 37° C. for 2 hours. After that, the mixture containing the unadsorbed antibody and virus was completely removed, and the culture medium containing the antibody at each concentration was added again, and culture was continued for 2 days. The culture supernatant of the cells cultured for 2 days was collected, and the virus infectivity titer was measured by plaque assay when each antibody concentration was added.
　それぞれ３ｗｅｌｌのウイルスプラーク数を数え，平均値を計算し，抗体非存在下におけるウイルス産生量を１００として，抗体存在下におけるウイルス産生量の割合を図４に示す。その結果，１０２８－０５Ｋにおいては，ウイルス産生量は抗体濃度１μｇ／ｍｌでもほとんど低下しなかったが，１０２８－５５Ｋ及び１０２８－６１Ｌでは濃度依存的にウイルス産生量が低下することがわかった。１０２８－６１Ｌでは１μｇ／ｍｌの濃度で完全にウイルス産生を抑えられることがわかった。 The number of viral plaques in each of the 3 wells was counted, the average value was calculated, and the virus production amount in the absence of antibody was set to 100, and the ratio of the virus production amount in the presence of antibody is shown in Figure 4. As a result, it was found that at 1028-05K, the virus yield hardly decreased even at an antibody concentration of 1 μg/ml, but at 1028-55K and 1028-61L, the virus yield decreased in a concentration-dependent manner. With 1028-61L, it was found that virus production could be completely suppressed at a concentration of 1 μg/ml.
　図４の数値を元に統計解析して５０％及び９０％阻害濃度を算出したものを図５に示す。１０２８－６１ＬではＩＣ５０が１５７ｎＭ，ＩＣ９０が１５９ｎＭであることが，１０２８－５５ＫではＩＣ５０が１０２ｎＭ，ＩＣ９０が１０６ｎＭであることが示された。 　Figure 5 shows the 50% and 90% inhibitory concentrations calculated by statistical analysis based on the numerical values in Figure 4. 1028-61L showed an IC50 of 157 nM and an IC90 of 159 nM, while 1028-55K showed an IC50 of 102 nM and an IC90 of 106 nM.
（実施例９）新型コロナウイルスの変異型スパイクタンパク質を外套したシュードタイプウイルスを用いた感染抑制実験
　実施例７と同様にして，武漢型（ＷＴ），Ａｌｐｈａ株，Ｂｅｔａ株の変異を有するスパイクタンパク質を外套したシュードタイプウイルス（ＳＡＲＳ－ＣｏＶ－２　Ｓｔ１９ｐｖ）を作製した（図８参照）。Ａｌｐｈａ株は，Ｂ．１．１．７　ｌｉｎｅａｇｅ（ＨＶ６９－７０ｄｅｌｅｔｉｏｎ，Ｙ１４４　ｄｅｌｅｔｉｏｎ，Ｎ５０１Ｙ，Ａ５７０Ｄ，Ｐ６８１Ｈ，Ｔ７１６Ｉ，Ｓ９８２Ａ，Ｄ１１１８Ｈ）を，Ｂｅｔａ株は，Ｂ．１．３５１（Ｄ８０Ａ，Ｄ２１５Ｇ，Ｋ４１７Ｎ，Ａ７０１Ｖ，Ｎ５０１Ｙ，Ｅ４８４Ｋ）を元にヒトコドンに最適化した配列の挿入されたプラスミドを用いている（大阪大学微生物病研究所松浦善治教授より分与）。 (Example 9) Infection suppression experiment using a pseudotype virus enveloped with a mutant spike protein of the new coronavirus In the same manner as in Example 7, spike proteins with mutations of Wuhan type (WT), Alpha strain, and Beta strain A pseudotyped virus (SARS-CoV-2 St19pv) enveloping was generated (see FIG. 8). Alpha strains are B. 1.1.7 lineage (HV69-70 deletion, Y144 deletion, N501Y, A570D, P681H, T716I, S982A, D1118H); Based on 1.351 (D80A, D215G, K417N, A701V, N501Y, E484K), a plasmid inserted with sequences optimized for human codons is used (provided by Professor Yoshiharu Matsuura, Research Institute for Microbial Diseases, Osaka University).
　各変異株のシュードタイプウイルスを用いて行なった感染阻害実験の結果を図９に示す。調べた４種の抗体では，それぞれ濃度依存的に阻害効果が認められたが，０２１０－２８Ｋが最も効果が高く，０.２μｇ／ｍｌでも十分な感染阻害が認められた。また，これらの抗体は，武漢型だけでなく，Ａｌｐｈａ株，Ｂｅｔａ株にも同様に感染阻害効果が認められた。 Fig. 9 shows the results of infection inhibition experiments conducted using the pseudotype virus of each mutant strain. Each of the four tested antibodies showed concentration-dependent inhibitory effects, with 0210-28K exhibiting the highest effect, and sufficient inhibition of infection was observed even at 0.2 μg/ml. In addition, these antibodies were found to have an inhibitory effect on not only the Wuhan type, but also the Alpha and Beta strains.
　同様にＫａｐｐａ株，Ｄｅｌｔａ株の変異を有するスパイクタンパク質を外套したシュードタイプウイルスも作製し，各変異株のシュードタイプウイルスを用いて，０２１０－２８Ｋについて行なった２回の感染阻害実験の結果を図１２に示す。５種類のシュードタイプウイルスで，それぞれ濃度依存的に阻害効果が認められた。武漢型及び変異型ＳＡＲＳ－ＣｏＶ－２　Ｓｔ１９ｐｖシュードタイプウイルス感染阻害におけるＩＣ５０値は，それぞれ以下の通りであった。
１回目
　武漢型（ＷＴ）：８．４ｎｇ／ｍｌ
　Ａｌｐｈａ株：１２．３ｎｇ／ｍｌ
　Ｂｅｔａ株：２３．０ｎｇ／ｍｌ
　Ｋａｐｐａ株：１０．８ｎｇ／ｍｌ
　Ｄｅｌｔａ株：＜７．５ｎｇ／ｍｌ
２回目
　武漢型（ＷＴ）：３０．３±５．７ｎｇ／ｍｌ
　Ａｌｐｈａ株：４５．４±２０．５ｎｇ／ｍｌ
　Ｂｅｔａ株：１５．２±６．３ｎｇ／ｍｌ
　Ｋａｐｐａ株：２０．３±３．５ｎｇ／ｍｌ
　Ｄｅｌｔａ株：１２．１±１．８ｎｇ／ｍｌ Pseudotype viruses enveloped with spike proteins with mutations of the Kappa and Delta strains were similarly prepared, and the results of two rounds of infection inhibition experiments on 0210-28K were performed using the pseudotype viruses of each mutant strain. 12. Concentration-dependent inhibitory effects were observed for each of the five pseudotype viruses. The IC50 values for inhibition of Wuhan-type and mutant SARS-CoV-2 St19pv pseudotype virus infection were respectively as follows.
1st Wuhan type (WT): 8.4 ng/ml
Alpha strain: 12.3ng/ml
Beta strain: 23.0ng/ml
Kappa strain: 10.8 ng/ml
Delta strain: <7.5ng/ml
2nd Wuhan type (WT): 30.3 ± 5.7 ng/ml
Alpha strain: 45.4±20.5ng/ml
Beta strain: 15.2±6.3ng/ml
Kappa strain: 20.3±3.5 ng/ml
Delta strain: 12.1±1.8 ng/ml
　さらに，実施例７と同様にして，Ｇａｍｍａ株，Ｏｍｉｃｒｏｎ株の変異を有するスパイクタンパク質を外套したシュードタイプウイルスを作製し，７種類のシュードタイプウイルス（武漢型（ＷＴ），Ａｌｐｈａ株，Ｂｅｔａ株，Ｇａｍｍａ株，Ｋａｐｐａ株，Ｄｅｌｔａ株，及びＯｍｉｃｒｏｎ株）を用いて，０２１０－２８Ｋについて感染阻害実験を行った。ＶｅｒｏＥ６／ＴＭＰＲＳＳ２を９６ｗｅｌｌプレートに播種し，翌日９０～１００％コンフルエントの状態で，各濃度の０２１０－２８Ｋを添加し，その後，続けて上記７種類のシュードタイプウイルス又はＶＳＶ－Ｇを外套したコントロールのシュードタイプウイルス（ＶＳＶｐｖ）を添加した。３７℃で２４時間培養後，ＧｌｏＭａｘ　Ｎａｖｉｇａｔｏｒ　Ｍｉｃｒｏｐｌａｔｅ　Ｌｕｍｉｎｏｍｅｔｅｒ　（Ｐｒｏｍｅｇａ）を備えたＰｉｃａＧｅｎｅ　Ｌｕｍｉｎｅｓｃｅｎｃｅ　Ｋｉｔ　（Ｆｕｊｉｆｉｌｍ　Ｗａｋｏ）を用いてルシフェラーゼの活性を測定した。　 Furthermore, in the same manner as in Example 7, pseudotype viruses enveloped with spike proteins having mutations of the Gamma strain and the Omicron strain were prepared, and seven types of pseudotype viruses (Wuhan type (WT), Alpha strain, Beta strain, Gamma, Kappa, Delta, and Omicron strains) were used to perform infection inhibition experiments for 0210-28K. VeroE6/TMPRSS2 was seeded on a 96-well plate, the next day, 0210-28K at each concentration was added in a state of 90-100% confluency, and then the above-mentioned 7 types of pseudotype viruses or VSV-G were coated. A pseudotyped virus (VSVpv) was added. After culturing at 37°C for 24 hours, luciferase activity was measured using a PicaGene Luminescence Kit (Fujifilm Wako) equipped with a GloMax Navigator Microplate Luminometer (Promega).　
　結果を図１４に示す。縦軸はコントロールと比較した感染低下割合（％　ｏｆ　ｒｅｄｕｃｔｉｏｎ）を示す。Ｏｍｉｃｒｏｎ株に対する感染阻害効果（ＩＣ５０値：約２００ｐＭ）は，他の６種類のシュードタイプウイルスに対する阻害効果に比べて低下したものの，０２１０－２８Ｋ抗体によって，調べた７種類のシュードタイプウイルスで阻害効果が認められた。 The results are shown in Fig. 14. The vertical axis indicates the rate of infection reduction (% of reduction) compared to the control. Although the inhibitory effect on the Omicron strain (IC50 value: about 200 pM) was lower than the inhibitory effect on the other 6 pseudotype viruses, the 0210-28K antibody inhibited the 7 pseudotype viruses examined. was accepted.
（実施例１０）変異型の新型コロナウイルスを用いた感染抑制実験
　新型コロナウイルスは，日本の国立感染症研究所で分離された武漢型（ＷＫ－５２１／２０２０），Ａｌｐｈａ株（ＱＨＮ００２／２０２０），Ｂｅｔａ株（ＴＹ８－６１２／２０２１），Ｇａｍｍａ株（ＴＹ７－５０３／２０２１），Ｋａｐｐａ株（ＴＹ１１－３３０／２０２１），Ｄｌｅｔａ株（ＴＹ１１－９２７／２０２１），及びＯｍｉｃｒｏｎ株（ＴＹ３８－８７３）を用いた。ＶｅｒｏＥ６／ＴＭＰＲＳＳ２細胞を１ｗｅｌｌあたり２×１０４細胞となるように９６ｗｅｌｌプレートに播種し，上記ウイルスをそれぞれ１細胞あたり０．００１ｍｏｉで感染させ，各濃度に調整した０２１０－２８Ｋ抗体を添加した。３７℃で１時間の培養後，培養上清を除き，再び各濃度の抗体を添加して１０％ウシ胎児血清（ＦＢＳ）を含むダルベッコの改変イーグルミディアム（ＤＭＥＭ）を用いて２４時間培養した。その後，培養した細胞から培養上清を回収し，ＳＡＲＳ－ＣｏＶ－２直接検出ＲＴ－ｑＰＣＲキット（タカラバイオ株式会社）を用いたリアルタイムＰＣＲ法によりウイルスゲノムＲＮＡ量を測定することで，各濃度の抗体を添加した際のウイルス感染価を評価した。 (Example 10) Infection suppression experiment using a mutated new coronavirus The new coronavirus is the Wuhan type (WK-521/2020) and Alpha strain (QHN002/2020) isolated at the National Institute of Infectious Diseases in Japan. , Beta strain (TY8-612/2021), Gamma strain (TY7-503/2021), Kappa strain (TY11-330/2021), Dleta strain (TY11-927/2021), and Omicron strain (TY38-873). Using. VeroE6/TMPRSS2 cells were seeded in a 96-well plate at 2×10 4 cells per well, infected with the above viruses at 0.001 moi per cell, and 0210-28K antibody adjusted to each concentration was added. After culturing at 37° C. for 1 hour, the culture supernatant was removed, antibodies of various concentrations were added again, and the cells were cultured for 24 hours using Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). After that, the culture supernatant was collected from the cultured cells, and the amount of viral genome RNA was measured by real-time PCR using the SARS-CoV-2 direct detection RT-qPCR kit (Takara Bio Inc.). The virus infectivity titer was evaluated when the antibody was added.
　それぞれ３ｗｅｌｌのウイルスゲノム量を測定して平均値を計算し，各濃度の０２１０－２８Ｋ抗体存在下におけるウイルス産生阻害率を図１５に示す。その結果，Ｏｍｉｃｒｏｎ株に対するウイルス産生阻害効果は，他の６種類のシュードタイプウイルスに対する阻害効果に比べて低下したものの，０２１０－２８Ｋ抗体によって，調べた７種類の新型コロナウイルスのウイルス産生阻害効果が認められた。 The virus genome amount of each 3 wells was measured and the average value was calculated, and the virus production inhibition rate in the presence of each concentration of 0210-28K antibody is shown in Figure 15. As a result, although the viral production inhibitory effect on the Omicron strain was lower than the inhibitory effect on the other 6 pseudotype viruses, the 0210-28K antibody inhibited the viral production of the 7 tested novel coronaviruses. Admitted.
（実施例１１）表面プラズモン共鳴法（ＳＰＲ）解析
　確認的に，抗ＳＡＲＳ－ＣｏＶ－２　ＲＢＤ抗体について，武漢型のＲＢＤ又は変異株由来のＲＢＤに対する親和性を、Ｂｉａｃｏｒｅ　Ｔ１００（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）を用いてＳＰＲ解析した。具体的には，０２１０－２８Ｋ抗体をプロテインＡセンサーチップ（ＧＥヘルスケア）に固定化し，０ｎＭから３０ｎＭ範囲の各ＲＢＤ（武漢型のＲＢＤ（ＷＴ），Ａｌｐｈａ株由来のＲＢＤ（Ｎ５０１Ｙ），Ｂｅｔａ株由来のＲＢＤ（Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ），Ｇａｍｍａ株由来のＲＢＤ（Ｋ４１７Ｔ，Ｅ４８４Ｋ，Ｎ５０１Ｙ），Ｋａｐｐａ株由来のＲＢＤ（Ｌ４５２Ｒ，Ｅ４８４Ｑ），及びＤｅｌｔａ株由来のＲＢＤ（Ｌ４５２Ｒ，Ｔ４７８Ｋ））を３０μｌ／ｍｉｎの流速で１分間注入し，その後，ＨＢＳ－Ｐバッファで５分間洗浄した。Ｂｉａｃｏｒｅ　Ｅｖａｌｕａｔｉｏｎ　Ｓｏｆｔｗａｒｅ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）を使用して，結合速度定数と解離速度定数を求め，２つの定数の比から親和性を算出した。 Example 11 Surface Plasmon Resonance (SPR) Analysis To confirm, the affinity of anti-SARS-CoV-2 RBD antibodies to Wuhan-type RBD or mutant-derived RBD was measured using a Biacore T100 (GE Healthcare). were analyzed by SPR. Specifically, the 0210-28K antibody was immobilized on a protein A sensor chip (GE Healthcare), and each RBD in the range of 0 nM to 30 nM (Wuhan type RBD (WT), RBD derived from Alpha strain (N501Y), Beta strain RBD derived (K417N, E484K, N501Y), RBD derived from Gamma strain (K417T, E484K, N501Y), RBD derived from Kappa strain (L452R, E484Q), and RBD derived from Delta strain (L452R, T478K)) at 30 µl/ Injection was performed for 1 minute at a flow rate of min, followed by washing with HBS-P buffer for 5 minutes. Biacore Evaluation Software (GE Healthcare) was used to determine the association and dissociation rate constants, and the affinity was calculated from the ratio of the two constants.
　結果を図１６に示す。０２１０－２８Ｋ抗体の上記ＲＢＤに対する親和性は，それぞれ以下の通りであった。
　武漢型のＲＢＤ（ＷＴ）：１．８２×１０－１０（Ｍ）
　Ａｌｐｈａ株由来のＲＢＤ（Ｎ５０１Ｙ）：１．６７×１０－９（Ｍ）
　Ｂｅｔａ株由来のＲＢＤ（Ｋ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ）：４．０２×１０－９（Ｍ）
　Ｇａｍｍａ株由来のＲＢＤ（Ｋ４１７Ｔ，Ｅ４８４Ｋ，Ｎ５０１Ｙ）：１．５９×１０－９（Ｍ）
　Ｋａｐｐａ株由来のＲＢＤ（Ｌ４５２Ｒ，Ｅ４８４Ｑ）：３．１４×１０－９（Ｍ）
　Ｄｅｌｔａ株由来のＲＢＤ（Ｌ４５２Ｒ，Ｔ４７８Ｋ）：１．４１×１０－９（Ｍ） The results are shown in FIG. The affinities of the 0210-28K antibodies to the RBD were as follows.
　Wuhan type RBD (WT): 1.82×10 −10 (M)
RBD from Alpha strain (N501Y): 1.67×10 −9 (M)
RBD from Beta strain (K417N, E484K, N501Y): 4.02×10 −9 (M)
RBD from Gamma strain (K417T, E484K, N501Y): 1.59×10 −9 (M)
RBD from Kappa strain (L452R, E484Q): 3.14×10 −9 (M)
RBD from Delta strain (L452R, T478K): 1.41×10 −9 (M)
（実施例１２）ハムスターモデルを用いた予防的投与による感染抑制実験
　ハムスターモデルにおいて，抗体の予防的投与により新型コロナウイルス及びその変異型スパイクタンパク質を外套したシュードタイプウイルスによる感染を阻害するか実験した。具体的には６～１０週齢の雄ハムスター（日本エスエルシー株式会社から購入し，富山大学動物資源開発部内で病原体のない環境で飼育したハムスター）に対して，１匹につき０．３ｍｇ／ｋｇ又は３ｍｇ／ｋｇのウエストナイルウイルスを認識する抗体（Ｏｚａｗａ　ｅｔ　ａｌ．，　Ａｎｔｉｖｉｒａｌ　Ｒｅｓｅａｒｃｈ　２０１８，　１５４，　５８），又は０２１０－２８Ｋ抗体を腹腔内に投与した。コントロールであるウエストナイルウイルスを認識する抗体は，ＳＡＲＳ－ＣｏＶ－２を認識しない抗体として用いた。上記腹腔内投与２４時間後に，下記シュードタイプウイルスをそれぞれ，下記方法により気管内に直接接種した。感染から２４時間後にハムスターから肺を採取し，１ｍｇ／ｍＬのＤ－ルシフェリンと５分間インキュベートした。ルシフェラーゼの発光をＩｎ　ｖｉｖｏ　イメージングシステム（ＩＶＩＳ　Ｌｕｍｉｎａ　ＩＩ，パーキンエルマー）で測定し，肺の表と裏からの発光を測定して肺全体のウイルス感染価を算出した。
　シュードタイプウイルス：実施例７，９と同様にして作製した，武漢型（ＷＴ），Ａｌｐｈａ株，Ｂｅｔａ株，Ｇａｍｍａ株，Ｋａｐｐａ株，Ｄｅｌｔａ株，及びＯｍｉｃｒｏｎ株の変異を有するスパイクタンパク質，及びＶＳＶ－Ｇを外套したシュードタイプウイルス
　接種方法：ｂｉｏＲｘｉｖ　ｄｏｉ（ｈｔｔｐｓ：／／ｄｏｉ．ｏｒｇ／１０．１１０１／２０２１．０９．１７．４６０７４５）参照 (Example 12) Infection suppression experiment by preventive administration using a hamster model In a hamster model, it was tested whether preventive administration of antibodies inhibits infection by the novel coronavirus and its pseudotype virus enveloped with its mutant spike protein. . Specifically, 0.3 mg/kg per animal for male hamsters aged 6 to 10 weeks (hamsters purchased from Japan SLC Co., Ltd. and bred in a pathogen-free environment within the Department of Animal Resources Development, University of Toyama). Alternatively, 3 mg/kg of an antibody recognizing West Nile virus (Ozawa et al., Antiviral Research 2018, 154, 58) or 0210-28K antibody was administered intraperitoneally. A control antibody that recognizes West Nile virus was used as an antibody that does not recognize SARS-CoV-2. Twenty-four hours after the intraperitoneal administration, each of the following pseudotype viruses was directly inoculated into the trachea by the method described below. Lungs were harvested from hamsters 24 hours after infection and incubated with 1 mg/mL D-luciferin for 5 minutes. Luminescence of luciferase was measured with an In vivo Imaging System (IVIS Lumina II, PerkinElmer), and luminescence from the front and back of the lung was measured to calculate the virus infectivity titer of the whole lung.
Pseudotype virus: Spike proteins with mutations of Wuhan type (WT), Alpha strain, Beta strain, Gamma strain, Kappa strain, Delta strain, and Omicron strain, prepared in the same manner as in Examples 7 and 9, and VSV- Pseudotype virus that coats G Inoculation method: see bioRxiv doi (https://doi.org/10.1101/2021.09.17.460745)
　その結果を図１７に示す。図１７は，抗体投与量がＢｅｔａ株，及びＯｍｉｃｒｏｎ株は３ｍｇ／ｋｇハムスターで，そのほかは０．３ｍｇ／ｋｇハムスターの場合の結果である。結果から，０２１０－２８Ｋ抗体を事前に投与した場合には，コントロールに比べてルシフェラーゼの発光が明らかに低かった。このことから，新型コロナウイルス及びその変異型スパイクタンパク質を外套したシュードタイプウイルスによる感染を，抗体の予防的投与により阻害できることが明らかとなった。 The results are shown in FIG. FIG. 17 shows the results when the antibody dose was 3 mg/kg hamster for Beta strain and Omicron strain, and 0.3 mg/kg hamster for others. The results showed that luciferase luminescence was clearly lower when the 0210-28K antibody was pre-administered compared to the control. From this, it was clarified that the prophylactic administration of the antibody can inhibit infection by the new coronavirus and its pseudotype virus enveloping its mutant spike protein.
（実施例１３）ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質に結合した抗体の構造解析
　ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質に結合した０２１０－２８Ｋ抗体の構造を解析するために，クライオ電子顕微鏡法とＸ線結晶構造解析法を用いた。
　まず，０２１０－２８ＫのＦａｂを生成するために，０２１０－２８ＫのＶＨ遺伝子のＤＮＡ断片を，Ｎ末端から始まる要素（ヒトＩｇＧ　ＣＨ１の定常領域，フレキシブルリンカーＧＳＳＧ，ｄｅｃａｈｉｓｔｉｄｉｎｅ　Ｈｉｓ１０　ｔａｇ）で構成される発現ベクターに挿入した。その後，Ｅｘｐｉ２９３Ｆ細胞を，ＦａｂとＦａｂ分子をコードする軽鎖発現ベクター両方をＥｘｐｉ２９３　Ｅｘｐｒｅｓｓｉｏｎ　Ｓｙｓｔｅｍを用いて同時形質移入し，培養した細胞の上清を回収して透析膜（Ｓｐｅｃｔｒｕｍ　Ｌａｂｏｒａｔｏｒｉｅｓ，　Ｒａｎｃｈｏ　Ｄｏｍｉｎｇｕｅｚ）を使用して透析した。膜はカバー付き容器に含んだネイティブ緩衝液（２０ｍＭリン酸ナトリウム，０．５Ｍ　ＮａＣｌ，ｐＨ７．４）に浸し，４°Ｃ，７２時間シェーカーで振った。その後，Ｎｉ　Ｓｅｐｈａｒｏｓｅ　６　Ｆａｓｔ　Ｆｌｏｗ（Ｃｙｔｉｖａ）を使用したアフィニティークロマトグラフィによりＦａｂを精製した。具体的には，Ｎｉ　Ｓｅｐｈａｒｏｓｅ　６　Ｆａｓｔ　Ｆｌｏｗミックスを充填したＰｏｌｙ－Ｐｒｅｐカラム（Ｂｉｏ－Ｒａｄ，Ｈｅｒｃｕｌｅｓ）に前処理した上清をロードし，洗浄後にＦａｂ分子を溶出バッファ（２０ｍＭリン酸ナトリウム，０．５Ｍ　ＮａＣｌ，５００ｍＭイミダゾール，ｐＨ７．４）で溶出した。 Example 13 Structural Analysis of Antibody Bound to SARS-CoV-2 Spike Protein To analyze the structure of the 0210-28K antibody bound to the SARS-CoV-2 spike protein, cryo-electron microscopy and X-ray crystallography were used. Analytical method was used.
First, in order to generate the 0210-28K Fab, the DNA fragment of the 0210-28K VH gene was subjected to expression composed of elements starting from the N-terminus (human IgG CH1 constant region, flexible linker GSSG, decahistidine His10 tag). inserted into the vector. Expi293F cells were then co-transfected with both the Fab and the light chain expression vector encoding the Fab molecule using the Expi293 Expression System and the cultured cell supernatant was harvested and applied to dialysis membranes (Spectrum Laboratories, Rancho Dominguez). dialyzed using The membrane was immersed in a native buffer (20 mM sodium phosphate, 0.5 M NaCl, pH 7.4) contained in a covered container and shaken at 4°C for 72 hours on a shaker. Fabs were then purified by affinity chromatography using Ni Sepharose 6 Fast Flow (Cytiva). Specifically, the pretreated supernatant was loaded onto a Poly-Prep column (Bio-Rad, Hercules) filled with Ni Sepharose 6 Fast Flow mix, and after washing, the Fab molecules were eluted with an elution buffer (20 mM sodium phosphate, 0.05%). It was eluted with 5M NaCl, 500 mM imidazole, pH 7.4).
　クライオ電子顕微鏡法で画像化するために，ＳＡＲＳ－ＣｏＶ－２スパイク６Ｐのタンパク質を報告されている手法で発現および精製し（Ｏｎｏｄｅｒａ　ｅｔ　ａｌ．，２０２１）,ＳＡＲＳ－ＣｏＶ－２スパイク６Ｐ溶液を３７℃,１時間インキュベートしてから,精製した０２１０－２８ＫのＦａｂをＳＡＲＳ－ＣｏＶ－２スパイク６Ｐ溶液とモル比１：２で１８℃，１時間インキュベートした。このサンプルをＰＩＢ－１０（Ｖａｃｕｕｍ　Ｄｅｖｉｃｅ）を使用して１０ｍＡで１２０秒間グロー放電処理したＱｕａｎｔｉｆｏｉｌ　Ｒ１．２／１．３　Ｃｕ　３００メッシュグリッド（Ｑｕａｎｔｉｆｏｉｌ　Ｍｉｃｒｏ　Ｔｏｏｌｓ　ＧｍｂＨ）に載せることによりグリッドを作製した。サンプルを１８℃，湿度１００％，５の力で５秒間Ｖｉｔｒｏｂｏｔ　ｍａｒｋ　ＩＶ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）を用いてブロットし，液体エタンに浸した。顕微鏡写真は，Ｋ３直接電子検出器（Ｇａｔａｎ）を備えた３００ｋＶで動作するＫｒｉｏｓ　Ｇ４（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）を使用し，公称倍率１３０，０００（物理ピクセルあたり０．６７），２０ｅＶスリット幅のＧＩＦ－Ｂｉｏｃｏｎｔｉｎｕｕｍエネルギーフィルター（Ｇａｔａｎ）を用いて，５０フレームにわたって総被ばく１．５秒で総線量を５３．１４ｅ／Å２として取得した。
　すべての画像処理ステップはＲｅｌｉｏｎ　３．１（例えば，Ｚｉｖａｎｏｖ，　Ｊ．　ｅｔ　ａｌ．，　ｅＬｉｆｅ　７；　１０．７５５４／ｅＬｉｆｅ．４２１６６　（２０１８）.参照）で実行された。ＭｏｔｉｏｎＣｏｒ２（Ｚｈｅｎｇ，　Ｓ．　Ｑ．　ｅｔ　ａｌ．，　Ｎａｔ　Ｍｅｔｈｏｄｓ　１４，　３３１－３３２，　ｄｏｉ：１０．１０３８／ｎｍｅｔｈ．４１９３　（２０１７）.参照）を用いて電子線誘起性ドリフトを補正し，コントラスト伝達関数の推定はＣＴＦＦＩＮＤ４６５（Ｒｏｈｏｕ，　Ａ．　＆　Ｇｒｉｇｏｒｉｅｆｆ，　Ｎ．，　Ｊ　Ｓｔｒｕｃｔ　Ｂｉｏｌ　１９２，　２１６－２２１，　ｄｏｉ：１０．１０１６／ｊ．ｊｓｂ．２０１５．０８．００８　（２０１５）.参照）を使用して実行された。ＬａＰｌａｃｉａｎ－Ｇａｕｓｓｉａｎフィルターと２Ｄ分類ジョブタイプを用いた粒子ピッキングにより生成されたテンプレートに基づいて，７１１２４２粒子を自動ピッキングし，３Ｄ分類と２Ｄ分類でノイズ粒子を除去したのち，３Ｄ分類ジョブタイプを実行して，３つのＦａｂと１つのスパイクを含む１クラスを識別した。このクラスには，コントラスト伝達関数の補正とＢａｙｅｓｉａｎ　ｐｏｌｉｓｈｉｎｇのあとに，３．１Åの解像度でマップを生成するためにＣ３対称性を使用した最終的な３Ｄ再構成で使用される５６２４７個の粒子が含まれる。なお，解像度はフーリエシェル相関曲線（ＦＳＣ　＝　０．１４３）基準に基づき，図はＣｈｉｍｅｒａとＣｈｉｍｅｒａＸ（Ｐｅｔｔｅｒｓｅｎ，　Ｅ．　Ｆ．　ｅｔ　ａｌ．，　Ｊ　Ｃｏｍｐｕｔ　Ｃｈｅｍ　２５，　１６０５－１６１２，　ｄｏｉ：１０．１００２／ｊｃｃ．２００８４　（２００４）.参照）を用いて作成された。 For imaging by cryo-electron microscopy, the protein of SARS-CoV-2 spike 6P was expressed and purified by a reported procedure (Onodera et al., 2021), and the SARS-CoV-2 spike 6P solution was isolated from 37 After incubation for 1 hour at 18°C, the purified 0210-28K Fab was incubated with SARS-CoV-2 spiked 6P solution at a molar ratio of 1:2 for 1 hour at 18°C. A grid was prepared by placing the sample on a Quantifoil R1.2/1.3 Cu 300 mesh grid (Quantifoil Micro Tools GmbH) that had been glow discharged using a PIB-10 (Vacuum Device) at 10 mA for 120 seconds. Samples were blotted using a Vitrobot mark IV (Thermo Fisher Scientific) at 18° C., 100% humidity, force of 5 for 5 seconds and immersed in liquid ethane. Photomicrographs were obtained using a Krios G4 (Thermo Fisher Scientific) operating at 300 kV with a K3 direct electron detector (Gatan), nominal magnification of 130,000 (0.67 per physical pixel), GIF-scan with 20 eV slit width. A total dose of 53.14 e/Å2 was acquired with a total exposure of 1.5 sec over 50 frames using a Biocontinuum energy filter (Gatan).
All image processing steps were performed in Relion 3.1 (see, eg, Zivanov, J. et al., eLife 7; 10.7554/eLife.42166 (2018).). MotionCor2 (Zheng, S.Q. et al., Nat Methods 14, 331-332, doi: 10.1038/nmeth.4193 (2017).) was used to correct for electron beam-induced drift, and the contrast transfer function was was performed using CTFFIND465 (Rohou, A. & Grigorieff, N., J Struct Biol 192, 216-221, doi: 10.1016/j.jsb.2015.08.008 (2015). See) was done. Based on a template generated by particle picking using a LaPlacian-Gaussian filter and a 2D classification job type, 711242 particles were automatically picked, noise particles were removed by 3D classification and 2D classification, and then the 3D classification job type was run. We identified one class containing 3 Fabs and 1 spike. This class contains 56247 particles that, after contrast transfer function correction and Bayesian polishing, are used in the final 3D reconstruction using C3 symmetry to generate a map with a resolution of 3.1 Å. included. The resolution is based on the Fourier shell correlation curve (FSC = 0.143) standard, and the figure is based on Chimera and ChimeraX (Pettersen, EF et al., J Comput Chem 25, 1605-1612, doi: 10.1002/ Jcc.20084 (2004).
　その結果を図１８（ａ）に示す。図１８（ａ）はＣ３対称性で３．１Åの解像度に緻密化したクライオ電子顕微鏡（ｃｒｙｏ－ＥＭ）マップであり，０２１０－２８ＫのＦａｂ（図１８においては「ＵＴ２８Ｋ　Ｆａｂ」と表記する）は，図で示すＳＡＲＳ－ＣｏＶ－２　ＲＢＤの左肩側に認識された。 The result is shown in FIG. 18(a). FIG. 18(a) is a cryo-electron microscope (cryo-EM) map densified to a resolution of 3.1 Å with C3 symmetry. , was recognized on the left shoulder side of the SARS-CoV-2 RBD shown in the figure.
　次に，精製されたＳＡＲＳ－ＣｏＶ－２　ＲＢＤと０２１０－２８ＫのＦａｂをモル比１：１．３で混合し，２０ｍＭＴｒｉｓ　ｐＨ８．０，１００ｍＭ　ＮａＣｌで平衡化したｓｕｐｅｒｄｅｘ２００　１０／３００（Ｃｙｔｉｖａ）でＲＢＤ／Ｆａｂ複合体を精製し，ＲＢＤ及びＦａｂの両方を含む画分を収集して１３ｍｇ／ｍｌまで濃縮した。結晶化スクリーニングは市販のスクリーニングキットを使用して実施した。ＲＢＤ／Ｆａｂ複合体の結晶はシッティングドロップ蒸気拡散法により２９３Ｋで成長し，最終的な結晶化条件は０．１Ｍクエン酸　ｐＨ５．０，１．０Ｍ塩化リチウム，１０％（ｗ／ｖ）ＰＥＧ６０００であった。
　結晶を液体窒素で低温冷却し，Ｘ線回折実験をＳＰｒｉｎｇ－８（Ｈａｒｉｍａ）のビームラインＢＬ３２ＸＵで実施した。Ｘ線回折データセットはＸＤＳで処理され，ＣＣＰ４プログラムパッケージでＡｉｍｌｅｓｓを使用してスケーリングされた（例えば，Ｅｖａｎｓ，　Ｐ．Ａｃｔａ　Ｃｒｙｓｔａｌｌｏｇｒ　Ｄ　Ｂｉｏｌ　Ｃｒｙｓｔａｌｌｏｇｒ　６２，　７２－８２，　ｄｏｉ：１０．１１０７／Ｓ０９０７４４４９０５０３６６９３.参照）。構造はＰＨＥＮＩＸパッケージのＰｈａｓｅｒプログラムを使用した分子置換法により解析され，ＲＢＤ構造（ＰＤＢ　ＩＤ：７ｊｍｐ）とａｌｐｈａｆｏｌｄ２　ｃｏｌａｂ　Ｗｅｂサイトで生成された０２１０－２８ＫのＦａｂフラグメントを分子置換の検索プローブとして使用した。構造精密化はｐｈｅｎｉｘ．ｒｅｆｉｎｅとＣＯＯＴ（例えば，Ｌｉｅｂｓｃｈｎｅｒ，　Ｄ．　ｅｔ　ａｌ．，　Ａｃｔａ　Ｃｒｙｓｔａｌｌｏｇｒ　Ｄ　Ｓｔｒｕｃｔ　Ｂｉｏｌ　７５，　８６１－８７７，　ｄｏｉ：１０．１１０７／Ｓ２０５９７９８３１９０１１４７１.参照）を使用し，構造の立体化学的特性はＭｏｌＰｒｏｂｉｔｙ（Ｄａｖｉｓ，　Ｉ．　Ｗ．　ｅｔ　ａｌ．，Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ　３５，　Ｗ３７５－３８３，　ｄｏｉ：１０．１０９３／ｎａｒ／ｇｋｍ２１６.参照）を使用して評価した。図はＰｙＭＯＬ（ｈｔｔｐ：／／ｐｙｍｏｌ．ｓｏｕｒｃｅｆｏｒｇｅ．ｎｅｔ）を用いて作成し,分子間接触原子はＣＣＰ４プログラムパッケージのＰＩＳＡおよびＣＯＮＴＡＣＴを使用して分析された（Ｋｒｉｓｓｉｎｅｌ，　Ｅ．　＆　Ｈｅｎｒｉｃｋ，　Ｋ．　Ｊ　Ｍｏｌ　Ｂｉｏｌ　３７２，　７７４－７９７，　ｄｏｉ：１０．１０１６／ｊ．ｊｍｂ．２００７．０５．０２２.参照）。 Purified SARS-CoV-2 RBD and 0210-28K Fabs were then mixed at a molar ratio of 1:1.3 and RBDs were treated with superdex200 10/300 (Cytiva) equilibrated with 20 mM Tris pH 8.0, 100 mM NaCl. The /Fab conjugate was purified and fractions containing both RBD and Fab were collected and concentrated to 13 mg/ml. Crystallization screening was performed using a commercially available screening kit. Crystals of the RBD/Fab complex were grown at 293 K by the sitting drop vapor diffusion method, and the final crystallization conditions were 0.1 M citric acid pH 5.0, 1.0 M lithium chloride, 10% (w/v) PEG6000. there were.
Crystals were cryocooled in liquid nitrogen and X-ray diffraction experiments were performed at beamline BL32XU at SPring-8 (Harima). X-ray diffraction data sets were processed with XDS and scaled using Aimless in the CCP4 program package (see, e.g., Evans, P. Acta Crystallogr D Biol Crystallogr 62, 72-82, doi: 10.1107/S0907444905036693. ). The structure was solved by the molecular replacement method using the Phaser program in the PHENIX package, using the RBD structure (PDB ID: 7jmp) and the 0210-28K Fab fragment generated at the alphafold2 colab website as search probes for molecular replacement. Structural refinement was performed using phenix. Refine and COOT (see, e.g., Liebschner, D. et al., Acta Crystallogr D Struct Biol 75, 861-877, doi: 10.1107/S2059798319011471.) were used, and the stereochemical characterization of structures was performed using MolProbity (Davis, IW et al., Nucleic Acids Res 35, W375-383, doi: 10.1093/nar/gkm216.). Figures were generated using PyMOL (http://pymol.sourceforge.net) and intermolecular contact atoms were analyzed using PISA and CONTACT in the CCP4 program package (Krissinel, E. & Henrick, K.J. Mol Biol 372, 774-797, doi: 10.1016/j.jmb.2007.05.022.).
　その結果を図１８（ｂ），（ｄ）～（ｇ）に示す。
　図１８（ｂ）は，３．７５Åの解像度のＸ線結晶構造解析結果であり，０２１０－２８ＫのＦａｂ（ＵＴ２８Ｋ　Ｆａｂ）がクライオ電子顕微鏡での結果と同様の領域に認識された。０２１０－２８Ｋ抗体は，ＲＢＤのＦ４８６をコア相互作用アミノ酸残基として認識し，抗体の疎水性ポケットと相互作用していた。この結合モードは，図１８（ｃ）に示す２５３ＸＬ５５（ＰＤＢ　ＩＤ：７ＢＥＯ）とＳＡＲＳ－ＣｏＶ－２　ＲＢＤの結合に似ていた。
　図１８（ｄ）～（ｇ）に，０２１０－２８Ｋ抗体ＦａｂとＳＡＲＳ－ＣｏＶ－２　ＲＢＤ間の主要残基の相互作用について示す。図１８（ｄ）からＲＢＤのＱ４９３の側鎖は，０２１０－２８Ｋ抗体ＦａｂのＶＨのＳ５５及びＮ５７と水素結合を形成していた。また，図１８（ｅ）から，ＲＢＤのＮ４８７の側鎖は，抗体ＦａｂのＶＨのＤ１０７の側鎖と水素結合を形成するだけでなく，抗体ＦａｂのＶＨのＣ１０５及びＳ１０６の主鎖の酸素原子と結合していた。また，図１８（ｆ）から，抗体ＦａｂのＶＨのＤ１０７の側鎖はＲＢＤのＮ４８７に加えて，Ｓ４７７及びＴ４７８の側鎖と水素結合し，これらの主鎖の窒素とも結合していた。さらに，図１８（ｇ）から，抗体ＦａｂのＶＨのＤ１０４の側鎖の水素原子は，ＲＢＤのＳ４７７の主鎖の窒素原子及びＡ４７５とＧ４７６の主鎖の酸素原子と結合を形成していた。
　これらの結果から，ＲＢＤのＮ４８７と，０２１０－２８Ｋ抗体ＦａｂのＶＨのＤ１０４及びＤ１０７は，抗原抗体相互作用の重要な残基であり，相互作用する分子の主鎖を認識し合っていることが明らかとなった。０２１０－２８Ｋ抗体はＲＢＤと，少なくとも４つの主鎖相互作用（ＲＢＤのＡ４７５，Ｇ４７６，Ｓ４７７，及びＴ４７８）と，少なくとも３つの側鎖相互作用（ＲＢＤのＦ４８６，Ｎ４８７，及びＱ４９３）があった。 The results are shown in FIGS. 18(b), (d) to (g).
FIG. 18(b) is the result of X-ray crystal structure analysis with a resolution of 3.75 Å, and the 0210-28K Fab (UT28K Fab) was recognized in the same region as the cryo-electron microscope result. The 0210-28K antibody recognized F486 of RBD as a core interacting amino acid residue and interacted with the hydrophobic pocket of the antibody. This binding mode resembled the binding of 253XL55 (PDB ID: 7BEO) and SARS-CoV-2 RBD shown in Figure 18(c).
Figures 18(d)-(g) show the key residue interactions between the 0210-28K antibody Fab and the SARS-CoV-2 RBD. From FIG. 18(d), the side chain of Q493 of RBD formed hydrogen bonds with S55 and N57 of VH of 0210-28K antibody Fab. In addition, from FIG. 18 (e), the side chain of N487 of RBD not only forms a hydrogen bond with the side chain of D107 of VH of antibody Fab, but also the oxygen atoms of the main chains of C105 and S106 of VH of antibody Fab. was connected with Further, from FIG. 18(f), the side chain of D107 of the VH of the antibody Fab was hydrogen-bonded to the side chains of S477 and T478 in addition to N487 of RBD, and was also bonded to the nitrogen of these main chains. Furthermore, from FIG. 18(g), the hydrogen atom in the side chain of D104 of VH of antibody Fab formed a bond with the nitrogen atom of the main chain of S477 of RBD and the oxygen atom of the main chains of A475 and G476 of RBD.
From these results, N487 of RBD and D104 and D107 of VH of 0210-28K antibody Fab are important residues for antigen-antibody interaction, and they recognize the main chains of interacting molecules. It became clear. The 0210-28K antibody had at least four backbone interactions (RBD A475, G476, S477, and T478) and at least three side chain interactions (RBD F486, N487, and Q493) with RBD.
（実施例１４）エピトープマッピング解析
　変異株由来のＲＢＤタンパク質への０２１０－２８Ｋ抗体の結合をＥＬＩＳＡにて解析した。具体的には１μｇ／ｍｌのＳＡＲＳ－ＣｏＶ－２　ＲＢＤ（ＷＴ），ＲＢＤ　Ｎ４３９Ｋ，ＲＢＤ　Ｎ４４０Ｋ，ＲＢＤ　Ｇ４４６Ｖ，ＲＢＤ　Ｌ４５２Ｒ，ＲＢＤ　Ｙ４５３Ｆ，ＲＢＤ　Ｌ４５５Ｆ，ＲＢＤ　Ｆ４５６Ｌ，ＲＢＤ　Ｋ４５８Ｒ，ＲＢＤ　Ｅ４７１Ｑ，ＲＢＤ　Ｉ４７２Ｖ，ＲＢＤ　Ａ４７５Ｖ，ＲＢＤ　Ｇ４７６Ｓ，ＲＢＤ　Ｓ４７７Ｎ，ＲＢＤ　Ｐ４７９Ｓ，ＲＢＤ　Ｎ４８１Ｄ，ＲＢＤ　Ｇ４８２Ｓ，ＲＢＤ　Ｖ４８３Ａ，ＲＢＤ　Ｅ４８４Ｋ，ＲＢＤ　Ｇ４８５Ｓ，ＲＢＤ　Ｆ４８６Ｓ，ＲＢＤ　Ｎ４８７Ｒ，ＲＢＤ　Ｆ４９０Ｓ，ＲＢＤ　Ｓ４９４Ｐ，ＲＢＤ　Ｐ４９９Ｒ，ＲＢＤ　Ｎ５０１Ｙ，ＲＢＤ　Ｙ５０５Ｃ，ＲＢＤ　Ｎ４１７Ｎ，Ｅ４８４Ｋ，Ｎ５０１Ｙ，ＲＢＤ　Ｋ４１７Ｔ，Ｅ４８４Ｋ，Ｎ５０１Ｙ，ＲＢＤ　Ｌ４５２Ｒ，Ｔ４７８Ｋ，及びＲＢＤ　Ｌ４５２Ｒ，Ｅ４８４Ｑをそれぞれ含むＰＢＳを，それぞれＭａｘｉｓｏｒｐ　９６　ｗｅｌｌ　ｐｌａｔｅに４℃で一夜保温することで，それぞれのタンパク質をウエルの底面にそれぞれ固層化した。 (Example 14) Epitope Mapping Analysis The binding of the 0210-28K antibody to the mutant-derived RBD protein was analyzed by ELISA. Specifically 1 μg/ml SARS-CoV-2 RBD (WT), RBD N439K, RBD N440K, RBD G446V, RBD L452R, RBD Y453F, RBD L455F, RBD F456L, RBD K458R, RBD E471Q, RBD I472V, RBD A472V ，ＲＢＤ Ｇ４７６Ｓ，ＲＢＤ Ｓ４７７Ｎ，ＲＢＤ Ｐ４７９Ｓ，ＲＢＤ Ｎ４８１Ｄ，ＲＢＤ Ｇ４８２Ｓ，ＲＢＤ Ｖ４８３Ａ，ＲＢＤ Ｅ４８４Ｋ，ＲＢＤ Ｇ４８５Ｓ，ＲＢＤ Ｆ４８６Ｓ，ＲＢＤ Ｎ４８７Ｒ，ＲＢＤ Ｆ４９０Ｓ，ＲＢＤ Ｓ４９４Ｐ，ＲＢＤ Ｐ４９９Ｒ，ＲＢＤ Ｎ５０１Ｙ，ＲＢＤ Ｙ５０５Ｃ，ＲＢＤ Ｎ４１７Ｎ，Ｅ４８４Ｋ , N501Y, RBD K417T, E484K, N501Y, RBD L452R, T478K, and RBD L452R, E484Q in PBS, respectively. solidified.
　抗原溶液を除去した後，３％ＢＳＡと０．１％　Ｔｗｅｅｎ－２０を含むＰＢＳで１時間ブロッキングした。次に，０．１％　Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，１００ｎｇ／ｍｌの作製した抗体を１時間インキュベートした。その後０．１％　Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，２００　ｎｇ／ｍｌペルオキシダーゼ標識抗ヒトＩｇＧ－Ｆｃ　Ｆ（ａｂ）’２フラグメントを１時間インキュベートした。最後に０．１％　Ｔｗｅｅｎ－２０を含むＰＢＳで洗浄し，ＴＭＢ基質を用いて発色させ，プレートリーダーを用いて吸光度４５０ｎｍの測定を行った。 After removing the antigen solution, the cells were blocked with PBS containing 3% BSA and 0.1% Tween-20 for 1 hour. Then, it was washed with PBS containing 0.1% Tween-20 and incubated with 100 ng/ml of the produced antibody for 1 hour. After washing with PBS containing 0.1% Tween-20, 200 ng/ml peroxidase-labeled anti-human IgG-Fc F(ab)' 2 fragment was incubated for 1 hour. Finally, the cells were washed with PBS containing 0.1% Tween-20, developed with a TMB substrate, and absorbance at 450 nm was measured using a plate reader.
　その結果を図１９に示す。０２１０－２８Ｋ抗体は，Ｆ４８６ＳとＮ４８７Ｒを除くＲＢＤタンパク質に対する結合の有意な減少は認められなかったが，Ｆ４８６ＳとＮ４８７Ｒに対しては結合の減少が認められた。これらの結果から，０２１０－２８Ｋ抗体の結合に，少なくともＲＢＤタンパク質であるＦ４８６とＮ４８７が不可欠であることが明らかとなった。
　これは，０２１０－２８Ｋ抗体が，実施例１３において主鎖相互作用することが明らかとなったＲＢＤのＡ４７５Ｖ，Ｇ４７６Ｓ，Ｓ４７７Ｎ，及びＴ４７８Ｋには反応したが，側鎖相互作用するＦ４８６Ｓ及びＮ４８７Ｒに対する突然変異に反応しなかったことを示し，０２１０－２８Ｋ抗体が主鎖優位抗原抗体相互作用であることが明らかとなった。 The results are shown in FIG. The 0210-28K antibody did not significantly decrease binding to RBD proteins other than F486S and N487R, but decreased binding to F486S and N487R. These results demonstrate that at least the RBD proteins F486 and N487 are essential for the binding of the 0210-28K antibody.
This shows that the 0210-28K antibody reacted with A475V, G476S, S477N, and T478K of RBD, which were shown to interact with the main chain in Example 13, but suddenly reacted with F486S and N487R with side chain interactions. It was shown that the 0210-28K antibody did not react to the mutation, revealing that the 0210-28K antibody is a main chain-dominant antigen-antibody interaction.
　さらに，０２１０－２８Ｋ抗体存在下において，ウイルス増殖中に発生する可能性がある変異について調べた。実験は，日本の国立感染症研究所で分離された武漢型（ＷＫ－５２１／２０２０）を，阻害濃度９０％（ＩＣ９０）の０２１０－２８Ｋ抗体またはウエストナイルウイルスを認識する抗体（Ｏｚａｗａ　ｅｔ　ａｌ．，　Ａｎｔｉｖｉｒａｌ　Ｒｅｓｅａｒｃｈ　２０１８，　１５４，　５８）１．０μｇ／ｍｌとプレインキュベートし，ＶｅｒｏＥ６／ＴＭＰＲＳＳ２細胞に接種した。コントロール抗体であるウエストナイルウイルスを認識する抗体は，ＳＡＲＳ－ＣｏＶ－２を認識しない抗体として用いた。感染後７２時間で培養上清を回収し，その後の継代のために抗体量を２倍にして再び継代した。３回の継代後，培養上清を回収し，ＱＩＡａｍｐ　Ｖｉｒａｌ　ＲＮＡ　Ｍｉｎｉ　Ｋｉｔ（Ｑｉａｇｅｎ）を使用してウイルスＲＮＡを単離した。全ゲノム増幅は，ＳＡＲＳ－ＣｏＶ－２　ｇｅｎｏｍｅ　ｓｅｑｕｅｎｃｉｎｇ（Ｉｔｏｋａｗａ　ｅｔ　ａｌ．，　２０２０）のためのＡＲＴＩＣ　Ｎｅｔｗｏｒｋ’ｓ　ｐｒｏｔｏｃｏｌ修正版を使用して実行した。次世代シーケンシング（ＮＧＳ）ライブラリは，ＱＩＡｓｅｑ　ＦＸ　ＤＮＡ　Ｌｉｂｒａｒｙ　ｋｉｔ　（Ｑｉａｇｅｎ）を使用して構築され，ｉＳｅｑ　ｐｌａｔｆｏｒｍ　（Ｉｌｌｕｍｉｎａ）を使用してシーケンシングした。ＮＧＳ読み取りは，ｂｗａ　ｍｅｍ　ａｌｇｏｒｉｔｈｍ　（ｖｅｒｓｉｏｎ　０．７．１３－ｒ１１２６）（Ｌｉ，　Ｈ．　＆　Ｄｕｒｂｉｎ，　Ｒ．　Ｂｉｏｉｎｆｏｒｍａｔｉｃｓ　２５，　１７５４－１７６０，　ｄｏｉ：１０．１０９３／ｂｉｏｉｎｆｏｒｍａｔｉｃｓ／ｂｔｐ３２４.参照）を使用してＳＡＲＳ－ＣｏＶ－２　Ｗｕｈａｎ－Ｈｕ－１　ｒｅｆｅｒｅｎｃｅ　ｇｅｎｏｍｅ　ｓｅｑｕｅｎｃｅ　（ＧｅｎＢａｎｋ　ａｃｃｅｓｓｉｏｎ　ｎｏ．　ＭＮ９０８９４７．３）にマッピングし，ＶａｒＳｃａｎ　ｖｅｒｓｉｏｎ　２．４．３（Ｋｏｂｏｌｄｔ，　Ｄ．　Ｃ．　ｅｔ　ａｌ．，　Ｇｅｎｏｍｅ　Ｒｅｓ　２２，　５６８－５７６，　ｄｏｉ：１０．１１０１／ｇｒ．１２９６８４．１１１.参照）を使用して変異対立遺伝子頻度を分析した。
　実験の結果，０２１０－２８Ｋ抗体存在下において，独立して３回実験したすべての武漢型ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質にＹ４８９Ｈ変異が含まれていた。一方，コントロール抗体には３株中２株にそれぞれＡ４７５ＶまたはＨ６５５Ｙ変異が含まれていた。 Furthermore, in the presence of the 0210-28K antibody, possible mutations during virus growth were investigated. In the experiment, the Wuhan type (WK-521/2020) isolated at the National Institute of Infectious Diseases in Japan was treated with the 0210-28K antibody with an inhibitory concentration of 90% (IC90) or an antibody that recognizes West Nile virus (Ozawa et al. , Antiviral Research 2018, 154, 58) were preincubated with 1.0 μg/ml and inoculated into VeroE6/TMPRSS2 cells. An antibody that recognizes West Nile virus, which is a control antibody, was used as an antibody that does not recognize SARS-CoV-2. Culture supernatants were harvested 72 hours after infection and passaged again with twice the amount of antibody for subsequent passages. After three passages, culture supernatants were harvested and viral RNA was isolated using the QIAamp Viral RNA Mini Kit (Qiagen). Whole genome amplification was performed using a modified version of ARTIC Network's protocol for SARS-CoV-2 genome sequencing (Itokawa et al., 2020). Next-generation sequencing (NGS) libraries were constructed using the QIAseq FX DNA Library kit (Qiagen) and sequenced using the iSeq platform (Illumina). NGS readings were performed using the bwa mem algorithm (version 0.7.13-r1126) (see Li, H. & Durbin, R. Bioinformatics 25, 1754-1760, doi: 10.1093/bioinformatics/btp324.). Mapped to the SARS-CoV-2 Wuhan-Hu-1 reference genome sequence (GenBank accession no. MN908947.3) and VarScan version 2.4.3 (Koboldt, D.C. 576, doi: 10.1101/gr.129684.111.) was used to analyze mutation allele frequencies.
As a result of the experiment, in the presence of the 0210-28K antibody, all Wuhan-type SARS-CoV-2 spike proteins independently tested three times contained the Y489H mutation. On the other hand, the control antibody contained the A475V or H655Y mutation in two of the three strains, respectively.
　実施例１３，１４の結果から，ＲＢＤのＮ４８７及びＹ４８９は，抗体認識から逃れるためにアミノ酸置換を引き起こすことで，０２１０－２８Ｋ抗体の疎水性ポケットと相互作用するＲＢＤのＦ４８６の配向をサポートしていると考えられる。また，ＳＡＲＳ－ＣｏＶ－２変異株に対する中和抗体の反応性を低下させるＲＢＤのＥ４８４のアミノ酸残基が，抗体結合部位である外側に位置し，Ｅ４８４アミノ酸置換とは無関係に０２１０－２８Ｋ抗体が存在すると考えられ，これらの特徴は０２１０－２８Ｋ抗体がＲＢＤの幅広い主鎖を認識する相互作用とともに，高い中和能力の構造的基盤であると考察している。
　なお，実施例９，１０において，０２１０－２８Ｋ抗体のＯｍｉｃｒｏｎ株に対する反応が他の変異株と比較して低下したのは，Ｏｍｉｃｒｏｎ株の変異の１つであるＱ４９３Ｒ突然変異によって水素結合の消失を引き起こしたためではないかと推測している。また，０２１０－２８Ｋ抗体存在下において，ウイルス増殖中に発生する可能性がある変異について調べたが，循環するＳＡＲＳ－ＣｏＶ－２に対する競争上の優位性を失う可能性が高いため，０２１０－２８Ｋ抗体中和耐性ＳＡＲＳ－ＣｏＶ－２変異体の出現は起こり得ないと考えられる。 From the results of Examples 13 and 14, N487 and Y489 of RBD support the orientation of F486 of RBD that interacts with the hydrophobic pocket of the 0210-28K antibody by causing amino acid substitutions to escape antibody recognition. It is thought that there are In addition, the E484 amino acid residue of RBD, which reduces the reactivity of neutralizing antibodies against SARS-CoV-2 mutants, is located outside the antibody binding site, and the 0210-28K antibody is independent of the E484 amino acid substitution. These features, along with interactions that allow the 0210-28K antibody to recognize a broad backbone of RBD, are the structural basis for its high neutralizing ability.
In Examples 9 and 10, the reaction of the 0210-28K antibody to the Omicron strain was reduced compared to other mutant strains because the Q493R mutation, which is one of the mutations in the Omicron strain, caused the loss of hydrogen bonding. I'm assuming that's what caused it. In addition, in the presence of 0210-28K antibody, we investigated mutations that may occur during virus growth, but since there is a high possibility of losing competitive advantage against circulating SARS-CoV-2, 0210-28K antibody The emergence of antibody-neutralizing resistant SARS-CoV-2 variants is considered unlikely.
（実施例１５）抗体依存性感染増強（ＡＤＥ）活性の評価
　実施例１で作製した０２１０－２８Ｋと，新たに作製した０２１０－２８Ｋ－ＬＡＬＡに関してＡＤＥ活性を確認した。０２１０－２８ＫのＦｃ領域にＬ２３４Ａ，Ｌ２３５Ａ変異を導入した０２１０－２８Ｋ－ＬＡＬＡの抗体を得るために，０２１０－２８Ｋの可変領域とＬ２３４Ａ，Ｌ２３５Ａ変異を含む定常領域全長とをＤＮＡ合成してベクター中に挿入し，実施例１と同様の方法で抗体産生及び精製した。具体的な評価はマイキャン・テクノロジーズ株式会社に委託し，抗体非存在下（コントロール），０２１０－２８Ｋ存在下，及び０２１０－２８Ｋ－ＬＡＬＡ存在下のそれぞれの条件下で，同社が開発した新型コロナウイルス研究用細胞（ｃＭｙｌｃ－２－Ｋ　ａｌｐｈａ細胞）に新型コロナウイルスを感染させて７２時間培養した。新型コロナウイルスは，日本の国立感染症研究所で分離された武漢型（ＷＫ－５２１／２０２０）を用いた。培養した細胞から培養上清を回収し,ウイルス量を測定してＡＤＥ活性を確認した。ウイルス量はＱＩＡａｍｐ（登録商標）ウイルスＲＮＡミニキット（ＱＩＡＧＥＮ）を用いたｑＰＣＲ（ｑｕａｎｔｉｔａｔｉｖｅ　ＰＣＲ）にて測定した。上記抗体は最終濃度を１．０μｇ／ｍｌから１０倍希釈列を調製して用いた。 (Example 15) Evaluation of antibody-dependent enhancement of infection (ADE) activity ADE activity was confirmed for 0210-28K prepared in Example 1 and newly prepared 0210-28K-LALA. In order to obtain the 0210-28K-LALA antibody in which the L234A and L235A mutations are introduced into the 0210-28K Fc region, the 0210-28K variable region and the full-length constant region containing the L234A and L235A mutations are DNA-synthesized into a vector. , and antibodies were produced and purified in the same manner as in Example 1. Specific evaluation was entrusted to Mican Technologies Co., Ltd., and under the conditions of the absence of antibodies (control), the presence of 0210-28K, and the presence of 0210-28K-LALA, the new coronavirus developed by the company Research cells (cMylc-2-K alpha cells) were infected with the novel coronavirus and cultured for 72 hours. For the new coronavirus, the Wuhan type (WK-521/2020) isolated by the National Institute of Infectious Diseases in Japan was used. The culture supernatant was collected from the cultured cells, and the viral load was measured to confirm the ADE activity. The viral load was measured by qPCR (quantitative PCR) using the QIAamp® Viral RNA Mini Kit (QIAGEN). The above antibody was used by preparing a 10-fold dilution series from a final concentration of 1.0 μg/ml.
　その結果を図２０に示す。グラフ中，縦軸はコントロールで得られるウイルス量を１として，ウイルスの増減量（Ｆｏｌｄ　ｉｎｃｒｅａｓｅ）を示している。０２１０－２８Ｋ存在下においては，１．０×１０－１μｇ／ｍｌ以上ではウイルス量が少ないが，１．０×１０－２μｇ／ｍｌではウイルス量が多く，ＡＤＥ活性を示した。一方，０２１０－２８Ｋ－ＬＡＬＡ存在下においては，１．０×１０－２μｇ／ｍｌ以下でもウイルス量が多くならず，ＡＤＥ活性が認められなかった。 The results are shown in FIG. In the graph, the vertical axis indicates the virus fold increase, with the virus amount obtained in the control being set to 1. In the presence of 0210-28K, the virus amount was low at 1.0×10 −1 μg/ml or higher, but the virus amount was high at 1.0×10 −2 μg/ml, indicating ADE activity. On the other hand, in the presence of 0210-28K-LALA, the virus amount did not increase even at 1.0×10 −2 μg/ml or less, and no ADE activity was observed.
（実施例１６）インターロイキン－６（ＩＬ－６）産生増強活性の評価
　上記０２１０－２８Ｋ及び０２１０－２８Ｋ－ＬＡＬＡに関して，ＩＬ－６産生を増強させる活性を有しているかも確認した。具体的な評価はマイキャン・テクノロジーズ株式会社に委託し，抗体非存在下（コントロール），０２１０－２８Ｋ存在下，及び０２１０－２８Ｋ－ＬＡＬＡ存在下のそれぞれの条件下で，ｃＭｙｌｃ－２－Ｋ　ａｌｐｈａ細胞に新型コロナウイルス（ＷＫ－５２１／２０２０）を感染させて７２時間培養し，培養上清を回収してＩＬ－６産生量をＥＬＩＳＡにて測定した。上記抗体は最終濃度を１．０μｇ／ｍｌから１０倍希釈列を調製し用いた。 (Example 16) Evaluation of activity to enhance interleukin-6 (IL-6) production It was also confirmed that the 0210-28K and 0210-28K-LALA described above have activity to enhance IL-6 production. Specific evaluation was entrusted to Mican Technologies Inc., cMylc-2-K alpha cells under the conditions of the absence of antibody (control), the presence of 0210-28K, and the presence of 0210-28K-LALA was infected with the novel coronavirus (WK-521/2020) and cultured for 72 hours, the culture supernatant was collected, and the amount of IL-6 produced was measured by ELISA. The above antibody was used after preparing a 10-fold dilution series from a final concentration of 1.0 μg/ml.
　その結果を図２１に示す。新型コロナウイルスに感染した重症化患者の血清では，ｃＭｙｌｃ－２－Ｋ　ａｌｐｈａ細胞からの高いＩＬ－６産生増強活性が認められることが報告されている（例えば，Ｏｐｔｉｃａｌ　Ｄｅｎｓｉｔｙが約１．１程度）。本実施例の結果から，０２１０－２８Ｋ及び０２１０－２８Ｋ－ＬＡＬＡ存在下においては，抗体の各濃度のいずれでもＩＬ－６産生量に大きな差がみられず，ｃＭｙｌｃ－２－Ｋ　ａｌｐｈａ細胞に対するＩＬ－６の産生増強活性が認められなかった。 The results are shown in FIG. It has been reported that the serum of severely ill patients infected with the new coronavirus shows high IL-6 production enhancement activity from cMylc-2-K alpha cells (for example, Optical Density is about 1.1). . From the results of this example, in the presence of 0210-28K and 0210-28K-LALA, no significant difference in IL-6 production was observed at any of the antibody concentrations, and IL-6 production against cMylc-2-K alpha cells -6 production enhancing activity was not observed.

Claims (34)

  1. 　以下の（ｉ）～（ｘｉｉ）の全てのアミノ酸部分に結合可能な抗体又はその抗原結合性断片：
    （ｉ）配列番号６２９に記載のアミノ酸配列の３１９～５４１番目のアミノ酸からなる部分，
    （ｉｉ）Ｋ４１７Ｎ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異を有する前記（ｉ）のアミノ酸部分，
    （ｉｉｉ）Ｎ４３９Ｋ変異を有する前記（ｉ）に記載のアミノ酸部分，
    （ｉｖ）Ｎ４４０Ｋ変異を有する前記（ｉ）に記載のアミノ酸部分，
    （ｖ）Ａ４７５Ｖ変異を有する前記（ｉ）に記載のアミノ酸部分，
    （ｖｉ）Ｅ４８４Ｋ変異を有する前記（ｉ）に記載のアミノ酸部分，
    （ｖｉｉ）Ｎ５０１Ｙ変異を有する前記（ｉ）に記載のアミノ酸部分，
    （ｖｉｉｉ）Ｌ４５２Ｒ変異を有する前記（ｉ）に記載のアミノ酸部分，
    （ｉｘ）Ｌ４５２Ｒ及びＥ４８４Ｑ変異を有する前記（ｉ）に記載のアミノ酸部分，
    （ｘ）Ｋ４１７Ｔ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異を有する前記（ｉ）に記載のアミノ酸部分，
    （ｘｉ）Ｌ４５２Ｒ及びＴ４７８Ｋ変異を有する前記（ｉ）に記載のアミノ酸部分，及び，
    （ｘｉｉ）Ｇ３３９Ｄ，Ｓ３７１Ｌ，Ｓ３７３Ｐ，Ｓ３７５Ｆ，Ｋ４１７Ｎ，Ｎ４４０Ｋ，Ｇ４４６Ｓ，Ｓ４７７Ｎ，Ｔ４７８Ｋ，Ｅ４８４Ａ，Ｑ４９３Ｒ，Ｇ４９６Ｓ，Ｑ４９８Ｒ，Ｎ５０１Ｙ，及びＹ５０５Ｈ変異を有する前記（ｉ）に記載のアミノ酸部分。     An antibody or an antigen-binding fragment thereof capable of binding to all of the following amino acid moieties (i) to (xii):
    (i) a portion consisting of the 319th to 541st amino acids of the amino acid sequence set forth in SEQ ID NO: 629;
    (ii) the amino acid portion of (i) having K417N, E484K, and N501Y mutations;
    (iii) the amino acid moiety according to (i) above having the N439K mutation;
    (iv) the amino acid moiety according to (i) above, which has an N440K mutation;
    (v) the amino acid moiety of (i) above having the A475V mutation;
    (vi) the amino acid moiety according to (i) above, which has an E484K mutation;
    (vii) the amino acid moiety according to (i) above, which has the N501Y mutation;
    (viii) the amino acid moiety according to (i) above, which has the L452R mutation;
    (ix) the amino acid moiety of (i) above having L452R and E484Q mutations;
    (x) the amino acid moiety of (i) above having K417T, E484K, and N501Y mutations;
    (xi) the amino acid portion of (i) above having L452R and T478K mutations, and
    (xii) The amino acid moiety of (i) above having mutations G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H.
  2. 　以下の（ｉ）～（ｘｉｉ）の全てのペプチドと，ヒトＡＣＥ２との結合を阻害可能な，請求項１に記載の抗体又はその抗原結合性断片：
    （ｉ）配列番号６２９に記載のアミノ酸配列の３１９～５４１番目のアミノ酸からなるペプチド，
    （ｉｉ）Ｋ４１７Ｎ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異を有する前記（ｉ）のペプチド，
    （ｉｉｉ）Ｎ４３９Ｋ変異を有する前記（ｉ）に記載のペプチド，
    （ｉｖ）Ｎ４４０Ｋ変異を有する前記（ｉ）に記載のペプチド，
    （ｖ）Ａ４７５Ｖ変異を有する前記（ｉ）に記載のペプチド，
    （ｖｉ）Ｅ４８４Ｋ変異を有する前記（ｉ）に記載のペプチド，
    （ｖｉｉ）Ｎ５０１Ｙ変異を有する前記（ｉ）に記載のペプチド，
    （ｖｉｉｉ）Ｌ４５２Ｒ変異を有する前記（ｉ）に記載のペプチド，
    （ｉｘ）Ｌ４５２Ｒ及びＥ４８４Ｑ変異を有する前記（ｉ）に記載のペプチド，
    （ｘ）Ｋ４１７Ｔ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異を有する前記（ｉ）に記載のペプチド，
    （ｘｉ）Ｌ４５２Ｒ及びＴ４７８Ｋ変異を有する前記（ｉ）に記載のペプチド，及び，
    （ｘｉｉ）Ｇ３３９Ｄ，Ｓ３７１Ｌ，Ｓ３７３Ｐ，Ｓ３７５Ｆ，Ｋ４１７Ｎ，Ｎ４４０Ｋ，Ｇ４４６Ｓ，Ｓ４７７Ｎ，Ｔ４７８Ｋ，Ｅ４８４Ａ，Ｑ４９３Ｒ，Ｇ４９６Ｓ，Ｑ４９８Ｒ，Ｎ５０１Ｙ，及びＹ５０５Ｈ変異を有する前記（ｉ）に記載のペプチド。     The antibody or antigen-binding fragment thereof according to claim 1, which is capable of inhibiting binding of all of the following peptides (i) to (xii) to human ACE2:
    (i) a peptide consisting of the 319th to 541st amino acids of the amino acid sequence set forth in SEQ ID NO: 629;
    (ii) the peptide of (i) having K417N, E484K, and N501Y mutations;
    (iii) the peptide of (i) above having the N439K mutation;
    (iv) the peptide of (i) above, which has the N440K mutation;
    (v) the peptide of (i) above having the A475V mutation;
    (vi) the peptide according to (i) above, which has an E484K mutation;
    (vii) the peptide of (i) above having the N501Y mutation;
    (viii) the peptide according to (i) above, which has the L452R mutation;
    (ix) the peptide of (i) above having L452R and E484Q mutations;
    (x) the peptide of (i) having K417T, E484K, and N501Y mutations;
    (xi) the peptide according to (i) above having L452R and T478K mutations, and
    (xii) The peptide of (i) above having mutations G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H.
  3. 　以下の（ｉ）～（ｘｉｉ）のタンパク質を有するシュードウイルスのＶｅｒｏ細胞への感染を阻害可能な，請求項１又は請求項２に記載の抗体又はその抗原結合性断片：
    （ｉ）配列番号６２９に記載のアミノ酸配列からなるタンパク質，
    （ｉｉ）Ｋ４１７Ｎ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異を有する前記（ｉ）に記載のタンパク質，
    （ｉｉｉ）Ｎ４３９Ｋ変異を有する前記（ｉ）に記載のタンパク質，
    （ｉｖ）Ｎ４４０Ｋ変異を有する前記（ｉ）に記載のタンパク質，
    （ｖ）Ａ４７５Ｖ変異を有する前記（ｉ）に記載のタンパク質，
    （ｖｉ）Ｅ４８４Ｋ変異を有する前記（ｉ）に記載のタンパク質，
    （ｖｉｉ）Ｎ５０１Ｙ変異を有する前記（ｉ）に記載のタンパク質，
    （ｖｉｉｉ）Ｌ４５２Ｒ変異を有する前記（ｉ）に記載のタンパク質，
    （ｉｘ）Ｌ４５２Ｒ及びＥ４８４Ｑ変異を有する前記（ｉ）に記載のタンパク質，
    （ｘ）Ｋ４１７Ｔ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異を有する前記（ｉ）に記載のタンパク質，
    （ｘｉ）Ｌ４５２Ｒ及びＴ４７８Ｋ変異を有する前記（ｉ）に記載のタンパク質，及び，
    （ｘｉｉ）Ｇ３３９Ｄ，Ｓ３７１Ｌ，Ｓ３７３Ｐ，Ｓ３７５Ｆ，Ｋ４１７Ｎ，Ｎ４４０Ｋ，Ｇ４４６Ｓ，Ｓ４７７Ｎ，Ｔ４７８Ｋ，Ｅ４８４Ａ，Ｑ４９３Ｒ，Ｇ４９６Ｓ，Ｑ４９８Ｒ，Ｎ５０１Ｙ，及びＹ５０５Ｈ変異を有する前記（ｉ）に記載のタンパク質。     The antibody or antigen-binding fragment thereof according to claim 1 or claim 2, which is capable of inhibiting infection of Vero cells with a pseudovirus having the following proteins (i) to (xii):
    (i) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 629;
    (ii) the protein of (i) above having K417N, E484K, and N501Y mutations;
    (iii) the protein of (i) above having the N439K mutation;
    (iv) the protein of (i) above, which has the N440K mutation;
    (v) the protein of (i) above having the A475V mutation;
    (vi) the protein of (i) above, which has the E484K mutation;
    (vii) the protein of (i) above having the N501Y mutation;
    (viii) the protein of (i) above, which has the L452R mutation;
    (ix) the protein of (i) above having L452R and E484Q mutations;
    (x) the protein of (i) above having K417T, E484K, and N501Y mutations;
    (xi) the protein of (i) above having L452R and T478K mutations, and
    (xii) The protein of (i), having mutations G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H.
  4. 　以下から選択されるいずれかの組み合わせの相補性決定領域（ＣＤＲ）を有し，かつ，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質又はＲＢＤに結合する抗体又はその抗原結合性断片：
    （ａ）ＧＦＳＬＳＮＡＲＭＧ（配列番号９）のアミノ酸配列からなる重鎖ＣＤＲ（ＣＤＲＨ）１，
    　　ＩＦＳＮＤＥＫ（配列番号１０）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＴＬＧＦＷＳＧＥＹＹＹＹＹＭＤＶ（配列番号１１）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＳＶＳＳＳＹ（配列番号２０）のアミノ酸配列からなる軽鎖ＣＤＲ（ＣＤＲＬ）１，
    　　ＧＡＳ（配列番号２１）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＨＹＮＮＷＰＰＬＦＴ（配列番号２２）のアミノ酸配列からなるＣＤＲＬ３；
    （ｂ）ＥＦＴＦＳＧＹＡ（配列番号３１）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＳＹＤＧＩＮＫ（配列番号３２）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＤＰＤＹＧＤＥＧＡＦＤＹ（配列番号３３）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＳＬＶＨＳＤＧＮＴＹ（配列番号４２）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＫＶＳ（配列番号４３）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＭＱＧＴＨＷＰＰＷＴ（配列番号４４）のアミノ酸配列からなるＣＤＲＬ３；
    （ｃ）ＧＹＴＦＴＳＹＹ（配列番号５３）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＮＰＳＧＧＳＴ（配列番号５４）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＤＧＴＰＤＩＶＶＶＰＡＡＰＬＤＹ（配列番号５５）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＳＶＳＳＹ（配列番号６４）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＤＶＳ（配列番号６５）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＱＲＳＮＷＰＰＶＬＴ（配列番号６６）のアミノ酸配列からなるＣＤＲＬ３；
    （ｄ）ＧＧＴＦＮＴＹＡ（配列番号７５）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＩＰＩＦＧＴＡ（配列番号７６）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＧＥＰＧＹＣＳＧＧＮＣＹＳＳＤＹ（配列番号７７）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＳＬＶＨＳＤＧＮＴＹ（配列番号８６）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＫＶＳ（配列番号８７）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＭＱＧＴＨＷＰＰＴ（配列番号８８）のアミノ酸配列からなるＣＤＲＬ３；
    （ｅ）ＧＤＳＶＳＳＮＳＶＡ（配列番号９７）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＴＹＹＲＳＫＷＹＮ（配列番号９８）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＳＧＧＹＱＬＬＹＤＤＡＦＤＩ（配列番号９９）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＳＶＳＳＮ（配列番号１０８）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＧＡＳ（配列番号１０９）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＱＹＳＮＷＬＬＹＴ（配列番号１１０）のアミノ酸配列からなるＣＤＲＬ３；
    （ｆ）ＧＦＴＦＳＳＹＩ（配列番号１１９）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＳＳＳＳＹＩ（配列番号１２０）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＩＶＧＡＴＳＹＹＹＹＧＭＤＶ（配列番号１２１）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＳＩＳＳＹ（配列番号１３０）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＡＡＳ（配列番号１３１）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＱＳＹＳＴＰＰＴ（配列番号１３２）のアミノ酸配列からなるＣＤＲＬ３；
    （ｇ）ＧＦＩＶＳＳＮＹ（配列番号１４１）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＹＳＧＧＳＴ配列番号１４２）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＤＬＤＶＲＧＡＬＤＩ（配列番号１４３）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＳＶＳＳＳＹ（配列番号１５２）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＧＡＳ（配列番号１５３）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＱＹＧＳＳＰＱＴ（配列番号１５４）のアミノ酸配列からなるＣＤＲＬ３；
    （ｈ）ＧＩＤＶＳＳＮＹ（配列番号１６３）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＹＳＧＧＳＴ（配列番号１６４）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＤＡＱＲＹＧＭＤＶ（配列番号１６５）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＮＩＧＳＮＴ（配列番号１７４）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＹＤＳ（配列番号１７５）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＬＷＤＳＳＳＤＨＶＶ（配列番号１７６）のアミノ酸配列からなるＣＤＲＬ３；
    （ｉ）ＧＧＳＩＳＳＳＮＷ（配列番号１８５）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＹＨＳＧＳＴ（配列番号１８６）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＧＷＮＹＤＹ（配列番号１８７）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＳＳＤＶＧＳＹＮＬ（配列番号１９６）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＥＧＳ（配列番号１９７）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＣＳＹＡＧＳＳＴＷＶ（配列番号１９８）のアミノ酸配列からなるＣＤＲＬ３；
    （ｊ）ＧＹＴＦＳＳＹＹ（配列番号２０７）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＮＰＳＧＧＳＴ（配列番号２０８）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＥＧＲＡＡＡＥＴＤＮＮＷＦＤＰ（配列番号２０９）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＳＶＬＹＳＳＮＮＫＮＹ（配列番号２１８）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＷＡＳ（配列番号２１９）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＱＹＹＮＴＰＹＴ（配列番号２２０）のアミノ酸配列からなるＣＤＲＬ３；
    （ｋ）ＧＦＴＦＳＳＹＡ（配列番号２２９）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＳＹＤＧＳＮＫ（配列番号２３０）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＡＤＧＳＧＷＮＹ（配列番号２３１）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＰＬＰＫＱＹ（配列番号２４０）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＫＤＳ（配列番号２４１）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＳＡＤＳＳＧＴＹＧＷＥ（配列番号２４２）のアミノ酸配列からなるＣＤＲＬ３；
    （ｌ）ＧＦＴＦＳＮＹＡ（配列番号２５１）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＳＹＤＧＩＮＫ（配列番号２５２）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＳＨＲＤＤＹＶＤＳＡＤＡＦＤＩ（配列番号２５３）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＳＬＶＨＳＤＧＮＴＹ（配列番号２６２）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＫＶＳ（配列番号２６３）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＭＱＧＴＨＷＰＰＷＴ（配列番号２６４）のアミノ酸配列からなるＣＤＲＬ３；
    （ｍ）ＧＹＴＦＴＧＹＹ（配列番号２７３）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＮＰＮＳＧＧＴ（配列番号２７４）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＬＡＳＦＤＹ（配列番号２７５）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＳＮＮＶＧＮＱＧ（配列番号２８４）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＲＮＮ（配列番号２８５）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＳＡＷＤＳＳＬＳＡＱＶ（配列番号２８６）のアミノ酸配列からなるＣＤＲＬ３；
    （ｎ）ＧＧＳＩＳＳＳＹＨＹ（配列番号２９５）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＹＹＳＧＳＴ（配列番号２９６）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＲＧＹＳＹＤＳＳＧＤＧＶＤＹ（配列番号２９７）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＧＩＲＮＤ（配列番号３０６）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＡＡＳ（配列番号３０７）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＬＱＨＮＳＹＰＬＴ（配列番号３０８）のアミノ酸配列からなるＣＤＲＬ３；
    （ｏ）ＧＦＳＬＳＤＡＲＭＧ（配列番号３１７）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＦＳＮＤＥＫ（配列番号３１８）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＡＬＧＦＷＳＧＹＹＹＹＹＹＭＤＶ（配列番号３１９）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＳＶＳＳＮ（配列番号３２８）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＧＡＳ（配列番号３２９）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＱＹＮＮＷＰＰＬＦＴ（配列番号３３０）のアミノ酸配列からなるＣＤＲＬ３；
    （ｐ）ＧＦＴＦＮＳＹＡ（配列番号３３９）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＳＹＤＧＮＮＫ（配列番号３４０）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＤＤＤＳＧＳＹＬＹＹＦＤＹ（配列番号３４１）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＳＬＶＨＳＤＧＮＴＹ（配列番号３５０）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＫＶＳ（配列番号３５１）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＭＱＧＴＨＷＰＰＬＴ（配列番号３５２）のアミノ酸配列からなるＣＤＲＬ３；
    ；
    （ｑ）ＧＦＳＬＳＴＳＧＭＣ（配列番号３６１）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＤＷＤＤＤＫ（配列番号３６２）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＩＰＶＥＭＡＴＩＧＤＹＹＹＹＧＭＤＶ（配列番号３６３）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＳＳＮＩＧＳＮＹ（配列番号３７２）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＲＮＮ（配列番号３７３）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＡＡＷＤＤＳＬＳＧＷＶ（配列番号３７４）のアミノ酸配列からなるＣＤＲＬ３；
    （ｒ）ＧＤＳＶＳＳＮＲＡＡ（配列番号３８３）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＴＹＹＲＳＫＷＹＮ（配列番号３８４）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＳＰＳＹＱＬＬＹＰＹＹＦＤＹ（配列番号３８５）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＧＩＳＮＳ（配列番号３９４）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＡＡＳ（配列番号３９５）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＱＹＹＳＴＰＹＴ（配列番号３９６）のアミノ酸配列からなるＣＤＲＬ３；
    （ｓ）ＧＧＴＦＳＳＹＡ（配列番号４０５）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＩＰＩＬＧＩＡ（配列番号４０６）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＡＬＧＹＳＧＹＧＳＴＹＹＭＤＶ（配列番号４０７）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＳＳＮＩＧＡＧＹＤ（配列番号４１６）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＧＮＳ（配列番号４１７）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＳＹＤＳＳＬＳＧＧＶ（配列番号４１８）のアミノ酸配列からなるＣＤＲＬ３；
    （ｔ）ＧＦＴＦＳＩＳＡ（配列番号４２７）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＶＶＧＳＧＮＴ（配列番号４２８）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＡＰＹＣＧＧＤＣＳＤＧＦＤＩ（配列番号４２９）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＳＶＳＳＳＹ（配列番号４３８）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＧＡＳ（配列番号４３９）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＱＹＧＮＳＰＷＴ（配列番号４４０）のアミノ酸配列からなるＣＤＲＬ３；
    （ｕ）ＧＧＴＦＳＳＹＡ（配列番号４２７）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＩＰＩＬＧＲＡ（配列番号４２８）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＮＰＥＥＦＳＳＳＹＶＹＹＹＹＭＤＶ（配列番号４２９）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＳＳＤＶＧＧＹＳＹ（配列番号４６０）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＤＶＳ（配列番号４６１）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＣＳＹＡＧＳＹＴＳＷＶ（配列番号４６２）のアミノ酸配列からなるＣＤＲＬ３；
    （ｖ）ＧＦＴＦＧＤＹＡ（配列番号４７１）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＲＳＫＡＹＧＧＴＴ（配列番号４７２）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＴＲＤＳＩＴＭＶＱＧＶＩＩＲＬＤＹＦＤＹ（配列番号４７３）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＳＧＳＩＡＳＮＹ（配列番号４８２）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＥＤＮ（配列番号４８３）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＳＹＤＳＳＮＫＶ（配列番号４８４）のアミノ酸配列からなるＣＤＲＬ３；
    （ｗ）ＧＧＳＩＳＳＹＹ（配列番号４９３）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＦＹＳＧＳＡ（配列番号４９４）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＤＩＧＹＤＳＳＧＰＤＡＦＤＩ（配列番号４９５）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＳＶＳＳＹ（配列番号５０４）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＤＡＳ（配列番号５０５）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＱＲＳＮＷＰＧＴ（配列番号５０６）のアミノ酸配列からなるＣＤＲＬ３；
    （ｘ）ＧＦＴＦＮＳＹＷ（配列番号５１５）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＫＱＤＧＳＥＫ（配列番号５１６）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＱＬＹＹＹＧＰＩＤＹ（配列番号５１７）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＳＧＳＩＡＳＮＹ（配列番号５２６）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＥＤＮ（配列番号５２７）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＳＹＤＳＮＮＲＶ（配列番号５２８）のアミノ酸配列からなるＣＤＲＬ３；
    （ｙ）ＧＹＳＦＴＳＹＷ（配列番号５３７）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＹＰＧＤＳＤＴ（配列番号５３８）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＲＳＧＹＤＬＡＳＳＨＮＷＬＤＰ（配列番号５３９）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＳＳＮＩＧＡＧＹＤ（配列番号５４８）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＧＮＳ（配列番号５４９）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＳＹＤＮＴＬＳＧＳＬ（配列番号５５０）のアミノ酸配列からなるＣＤＲＬ３；
    （ｚ）ＧＦＳＬＳＮＡＫＭＧ（配列番号５５９）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＦＳＧＤＥＫ（配列番号５６０）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＩＬＷＦＲＳＹＹＦＤＹ（配列番号５６１）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＧＩＳＳＷ（配列番号５７０）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＡＡＳ（配列番号５７１）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＱＡＮＳＦＰＦＴ（配列番号５７２）のアミノ酸配列からなるＣＤＲＬ３；
    （ａａ）ＧＦＴＦＳＳＹＤ（配列番号５８１）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＧＴＡＧＤＴ（配列番号５８２）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＲＧＩＱＱＱＬＡＬＲＹＹＹＹＬＤＶ（配列番号５８３）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＳＩＳＳＹ（配列番号５９２）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＡＡＳ（配列番号５９３）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＦＧＱＧＴＫＶＥＩＫ（配列番号５９４）のアミノ酸配列からなるＣＤＲＬ３；並びに，
    （ａｂ）ＧＦＴＦＳＳＹＡ（配列番号６０３）のアミノ酸配列からなるＣＤＲＨ１，
    　　ＩＳＧＳＧＧＳＴ（配列番号６０４）のアミノ酸配列からなるＣＤＲＨ２，及び
    　　ＡＫＤＧＥＷＹＹＤＦＷＳＧＰＶＹＦＤＹ（配列番号６０５）のアミノ酸配列からなるＣＤＲＨ３，並びに，
    　　ＱＳＩＳＳＷ（配列番号６１４）のアミノ酸配列からなるＣＤＲＬ１，
    　　ＫＡＳ（配列番号６１５）のアミノ酸配列からなるＣＤＲＬ２，及び
    　　ＱＱＹＮＳＹＳＲＴ（配列番号６１６）のアミノ酸配列からなるＣＤＲＬ３。     An antibody or antigen-binding fragment thereof that has any combination of complementarity determining regions (CDRs) selected from the following and binds to SARS-CoV-2 spike protein or RBD:
    (a) a heavy chain CDR (CDRH) 1 consisting of the amino acid sequence of GFSLSSNARMG (SEQ ID NO: 9);
    CDRH2 consisting of the amino acid sequence of IFSNDEK (SEQ ID NO: 10) and CDRH3 consisting of the amino acid sequence of ARTLGFWSGEYYYYYMDV (SEQ ID NO: 11), and
    a light chain CDR (CDRL) 1 consisting of the amino acid sequence of QSVSSSY (SEQ ID NO: 20),
    CDRL2 consisting of the amino acid sequence of GAS (SEQ ID NO: 21) and CDRL3 consisting of the amino acid sequence of QHYNNWPPLFT (SEQ ID NO: 22);
    (b) CDRH1, consisting of the amino acid sequence of EFTFSGYA (SEQ ID NO: 31);
    CDRH2 consisting of the amino acid sequence of ISYDGINK (SEQ ID NO: 32) and CDRH3 consisting of the amino acid sequence of ARDPDYGDEGAFDY (SEQ ID NO: 33), and
    CDRL1 consisting of the amino acid sequence of QSLVHSDGNTY (SEQ ID NO: 42),
    CDRL2 consisting of the amino acid sequence of KVS (SEQ ID NO: 43) and CDRL3 consisting of the amino acid sequence of MQGTHWPPWT (SEQ ID NO: 44);
    (c) CDRH1, consisting of the amino acid sequence of GYTFTSYY (SEQ ID NO: 53);
    CDRH2 consisting of the amino acid sequence of INPSGGST (SEQ ID NO: 54) and CDRH3 consisting of the amino acid sequence of ARDGTPDIVVVPAAPLDY (SEQ ID NO: 55), and
    CDRL1 consisting of the amino acid sequence of QSVSSY (SEQ ID NO: 64),
    CDRL2 consisting of the amino acid sequence of DVS (SEQ ID NO: 65) and CDRL3 consisting of the amino acid sequence of QQRSNWPPVLT (SEQ ID NO: 66);
    (d) CDRH1, consisting of the amino acid sequence of GGTFNTYA (SEQ ID NO: 75);
    CDRH2 consisting of the amino acid sequence IIIPIFGTA (SEQ ID NO: 76) and CDRH3 consisting of the amino acid sequence ARGEPGYCSGGNCYSSDY (SEQ ID NO: 77), and
    CDRL1 consisting of the amino acid sequence of QSLVHSDGNTY (SEQ ID NO: 86),
    CDRL2 consisting of the amino acid sequence of KVS (SEQ ID NO: 87) and CDRL3 consisting of the amino acid sequence of MQGTHWPPT (SEQ ID NO: 88);
    (e) CDRH1, consisting of the amino acid sequence of GDSVSSNSVA (SEQ ID NO: 97);
    CDRH2 consisting of the amino acid sequence TYYRSKWYN (SEQ ID NO: 98) and CDRH3 consisting of the amino acid sequence ASGGYQLLYDDAFDI (SEQ ID NO: 99), and
    CDRL1 consisting of the amino acid sequence of QSVSSN (SEQ ID NO: 108),
    CDRL2 consisting of the amino acid sequence of GAS (SEQ ID NO: 109) and CDRL3 consisting of the amino acid sequence of QQYSNWLLYT (SEQ ID NO: 110);
    (f) CDRH1, consisting of the amino acid sequence of GFTFSSYI (SEQ ID NO: 119);
    CDRH2 consisting of the amino acid sequence of ISSSSYI (SEQ ID NO: 120) and CDRH3 consisting of the amino acid sequence of ARIVGATSYYYYGMDV (SEQ ID NO: 121), and
    CDRL1 consisting of the amino acid sequence of QSISSY (SEQ ID NO: 130),
    CDRL2 consisting of the amino acid sequence of AAS (SEQ ID NO: 131) and CDRL3 consisting of the amino acid sequence of QQSYSTPPT (SEQ ID NO: 132);
    (g) CDRH1, consisting of the amino acid sequence of GFIVSSNY (SEQ ID NO: 141);
    CDRH2 consisting of the amino acid sequence of IYSGGST SEQ ID NO: 142) and CDRH3 consisting of the amino acid sequence of ARDLDVRGALDI (SEQ ID NO: 143), and
    CDRL1 consisting of the amino acid sequence of QSVSSSY (SEQ ID NO: 152),
    CDRL2 consisting of the amino acid sequence of GAS (SEQ ID NO: 153) and CDRL3 consisting of the amino acid sequence of QQYGSSPQT (SEQ ID NO: 154);
    (h) CDRH1 consisting of the amino acid sequence of GIDVSSNY (SEQ ID NO: 163);
    CDRH2 consisting of the amino acid sequence of IYSGGST (SEQ ID NO: 164) and CDRH3 consisting of the amino acid sequence of ARDAQRYGMDV (SEQ ID NO: 165), and
    CDRL1 consisting of the amino acid sequence of NIGSNT (SEQ ID NO: 174),
    CDRL2 consisting of the amino acid sequence of YDS (SEQ ID NO: 175) and CDRL3 consisting of the amino acid sequence of QLWDSSSDHVV (SEQ ID NO: 176);
    (i) a CDRH1 consisting of the amino acid sequence of GGSISSSNW (SEQ ID NO: 185),
    CDRH2 consisting of the amino acid sequence of IYHSGST (SEQ ID NO: 186) and CDRH3 consisting of the amino acid sequence of ARGWNYDY (SEQ ID NO: 187), and
    CDRL1 consisting of the amino acid sequence of SSDVGSYNL (SEQ ID NO: 196),
    CDRL2 consisting of the amino acid sequence of EGS (SEQ ID NO: 197) and CDRL3 consisting of the amino acid sequence of CSYAGSSTWV (SEQ ID NO: 198);
    (j) CDRH1, consisting of the amino acid sequence of GYTFSSYY (SEQ ID NO: 207);
    CDRH2 consisting of the amino acid sequence of INPSGGST (SEQ ID NO: 208) and CDRH3 consisting of the amino acid sequence of AREGRAAAETDNNWFDP (SEQ ID NO: 209), and
    CDRL1 consisting of the amino acid sequence of QSVLYSSNNKNY (SEQ ID NO: 218),
    CDRL2 consisting of the amino acid sequence of WAS (SEQ ID NO: 219) and CDRL3 consisting of the amino acid sequence of QQYYNTPYT (SEQ ID NO: 220);
    (k) CDRH1, consisting of the amino acid sequence of GFTFSSSYA (SEQ ID NO: 229);
    CDRH2 consisting of the amino acid sequence of ISYDGSNK (SEQ ID NO: 230) and CDRH3 consisting of the amino acid sequence of ARADGSGWNY (SEQ ID NO: 231), and
    CDRL1 consisting of the amino acid sequence PLPKQY (SEQ ID NO: 240),
    CDRL2 consisting of the amino acid sequence of KDS (SEQ ID NO: 241) and CDRL3 consisting of the amino acid sequence of QSADSSGTYGWE (SEQ ID NO: 242);
    (l) CDRH1 consisting of the amino acid sequence of GFTFSNYA (SEQ ID NO: 251),
    CDRH2 consisting of the amino acid sequence of ISYDGINK (SEQ ID NO: 252) and CDRH3 consisting of the amino acid sequence of ASHRDDYVDSADAFDI (SEQ ID NO: 253), and
    CDRL1 consisting of the amino acid sequence of QSLVHSDGNTY (SEQ ID NO: 262),
    CDRL2 consisting of the amino acid sequence of KVS (SEQ ID NO: 263) and CDRL3 consisting of the amino acid sequence of MQGTHWPPWT (SEQ ID NO: 264);
    (m) CDRH1, consisting of the amino acid sequence of GYTFTGYY (SEQ ID NO: 273);
    CDRH2 consisting of the amino acid sequence of INPNSGGT (SEQ ID NO: 274), and CDRH3 consisting of the amino acid sequence of LASFDY (SEQ ID NO: 275), and
    CDRL1 consisting of the amino acid sequence of SNNVGNQG (SEQ ID NO: 284),
    CDRL2 consisting of the amino acid sequence of RNN (SEQ ID NO:285) and CDRL3 consisting of the amino acid sequence of SAWDSSLSAQV (SEQ ID NO:286);
    (n) CDRH1, consisting of the amino acid sequence of GGSISSSYHY (SEQ ID NO: 295);
    CDRH2 consisting of the amino acid sequence of IYYSGST (SEQ ID NO: 296) and CDRH3 consisting of the amino acid sequence of ARRGYSYDSSGDGVDY (SEQ ID NO: 297), and
    CDRL1 consisting of the amino acid sequence of QGIRND (SEQ ID NO: 306),
    CDRL2 consisting of the amino acid sequence of AAS (SEQ ID NO: 307) and CDRL3 consisting of the amino acid sequence of LQHNSYPLT (SEQ ID NO: 308);
    (o) CDRH1 consisting of the amino acid sequence of GFSLSDARMG (SEQ ID NO: 317);
    CDRH2 consisting of the amino acid sequence of IFSNDEK (SEQ ID NO: 318) and CDRH3 consisting of the amino acid sequence of ARALGFWSGYYYYYYMDV (SEQ ID NO: 319), and
    CDRL1 consisting of the amino acid sequence of QSVSSN (SEQ ID NO: 328),
    CDRL2 consisting of the amino acid sequence of GAS (SEQ ID NO:329) and CDRL3 consisting of the amino acid sequence of QQYNNWPPLFT (SEQ ID NO:330);
    (p) CDRH1, consisting of the amino acid sequence of GFTFNSYA (SEQ ID NO: 339);
    CDRH2 consisting of the amino acid sequence of ISYDGNNK (SEQ ID NO: 340) and CDRH3 consisting of the amino acid sequence of ARDDDSGSYLYYFDY (SEQ ID NO: 341), and
    CDRL1 consisting of the amino acid sequence of QSLVHSDGNTY (SEQ ID NO: 350),
    CDRL2 consisting of the amino acid sequence of KVS (SEQ ID NO: 351) and CDRL3 consisting of the amino acid sequence of MQGTHWPPLT (SEQ ID NO: 352);
    ;
    (q) CDRH1, consisting of the amino acid sequence of GFSLSTSGMC (SEQ ID NO: 361);
    CDRH2 consisting of the amino acid sequence IDWDDDK (SEQ ID NO: 362) and CDRH3 consisting of the amino acid sequence ARIPVEMATIGDYYYYGMDV (SEQ ID NO: 363), and
    CDRL1 consisting of the amino acid sequence of SSNIGSNY (SEQ ID NO: 372),
    CDRL2 consisting of the amino acid sequence of RNN (SEQ ID NO: 373) and CDRL3 consisting of the amino acid sequence of AAWDDSLSGWV (SEQ ID NO: 374);
    (r) CDRH1 consisting of the amino acid sequence of GDSVSSNRAA (SEQ ID NO: 383),
    CDRH2 consisting of the amino acid sequence TYYRSKWYN (SEQ ID NO: 384) and CDRH3 consisting of the amino acid sequence ARSPSYQLLYPYYFDY (SEQ ID NO: 385), and
    CDRL1 consisting of the amino acid sequence of QGISNS (SEQ ID NO: 394),
    CDRL2 consisting of the amino acid sequence of AAS (SEQ ID NO:395) and CDRL3 consisting of the amino acid sequence of QQYYSTPYT (SEQ ID NO:396);
    (s) CDRH1 consisting of the amino acid sequence of GGTFSSSYA (SEQ ID NO: 405),
    CDRH2 consisting of the amino acid sequence of IIPILGIA (SEQ ID NO: 406) and CDRH3 consisting of the amino acid sequence of ARALGYSGYGSTYYMDV (SEQ ID NO: 407), and
    CDRL1 consisting of the amino acid sequence of SSNIGAGYD (SEQ ID NO: 416),
    CDRL2 consisting of the amino acid sequence of GNS (SEQ ID NO: 417) and CDRL3 consisting of the amino acid sequence of QSYDSSLSGGV (SEQ ID NO: 418);
    (t) CDRH1, consisting of the amino acid sequence of GFTFSISA (SEQ ID NO: 427);
    CDRH2 consisting of the amino acid sequence IVVGSGNT (SEQ ID NO: 428) and CDRH3 consisting of the amino acid sequence AAPYCGGDCSDGFDI (SEQ ID NO: 429), and
    CDRL1 consisting of the amino acid sequence of QSVSSSY (SEQ ID NO: 438),
    CDRL2 consisting of the amino acid sequence of GAS (SEQ ID NO: 439) and CDRL3 consisting of the amino acid sequence of QQYGNSPWT (SEQ ID NO: 440);
    (u) CDRH1, consisting of the amino acid sequence of GGTFSSSYA (SEQ ID NO: 427);
    CDRH2 consisting of the amino acid sequence of IIPILGRA (SEQ ID NO: 428) and CDRH3 consisting of the amino acid sequence of ARNPEEFSSSSYVYYYYMDV (SEQ ID NO: 429), and
    CDRL1 consisting of the amino acid sequence of SSDVGGYSY (SEQ ID NO: 460),
    CDRL2 consisting of the amino acid sequence of DVS (SEQ ID NO: 461) and CDRL3 consisting of the amino acid sequence of CSYAGSYTSWV (SEQ ID NO: 462);
    (v) CDRH1 consisting of the amino acid sequence of GFTFGDYA (SEQ ID NO: 471);
    CDRH2 consisting of the amino acid sequence IRSKAYGGTT (SEQ ID NO: 472) and CDRH3 consisting of the amino acid sequence TRDSITMVQGVIIRLDYFDY (SEQ ID NO: 473), and
    CDRL1 consisting of the amino acid sequence of SGSIASNY (SEQ ID NO: 482),
    CDRL2 consisting of the amino acid sequence of EDN (SEQ ID NO: 483) and CDRL3 consisting of the amino acid sequence of QSYDSSNKV (SEQ ID NO: 484);
    (w) CDRH1 consisting of the amino acid sequence of GGSISSYY (SEQ ID NO: 493);
    CDRH2 consisting of the amino acid sequence of IFYSGSA (SEQ ID NO: 494) and CDRH3 consisting of the amino acid sequence of ARDIGYDSSGPDAFDI (SEQ ID NO: 495), and
    CDRL1 consisting of the amino acid sequence of QSVSSY (SEQ ID NO: 504),
    CDRL2 consisting of the amino acid sequence of DAS (SEQ ID NO: 505) and CDRL3 consisting of the amino acid sequence of QQRSNWPGT (SEQ ID NO: 506);
    (x) CDRH1 consisting of the amino acid sequence of GFTFNSYW (SEQ ID NO: 515),
    CDRH2 consisting of the amino acid sequence of IKQDGSEK (SEQ ID NO: 516) and CDRH3 consisting of the amino acid sequence of ARQLYYYGPIDY (SEQ ID NO: 517), and
    CDRL1 consisting of the amino acid sequence of SGSIASNY (SEQ ID NO: 526),
    CDRL2 consisting of the amino acid sequence of EDN (SEQ ID NO:527) and CDRL3 consisting of the amino acid sequence of QSYDSNNRV (SEQ ID NO:528);
    (y) CDRH1 consisting of the amino acid sequence of GYSFTSYW (SEQ ID NO: 537);
    CDRH2 consisting of the amino acid sequence of IYPGDSDT (SEQ ID NO: 538) and CDRH3 consisting of the amino acid sequence of ARRSGYDLASSHNWLDP (SEQ ID NO: 539), and
    CDRL1 consisting of the amino acid sequence of SSNIGAGYD (SEQ ID NO: 548),
    CDRL2 consisting of the amino acid sequence of GNS (SEQ ID NO: 549) and CDRL3 consisting of the amino acid sequence of QSYDNTTLSGSL (SEQ ID NO: 550);
    (z) CDRH1, consisting of the amino acid sequence of GFSLSNAKMG (SEQ ID NO: 559);
    CDRH2 consisting of the amino acid sequence of IFSGDEK (SEQ ID NO: 560) and CDRH3 consisting of the amino acid sequence of ARILWFRSYYFDY (SEQ ID NO: 561), and
    CDRL1 consisting of the amino acid sequence of QGISSW (SEQ ID NO: 570),
    CDRL2 consisting of the amino acid sequence of AAS (SEQ ID NO: 571) and CDRL3 consisting of the amino acid sequence of QQANSFPFT (SEQ ID NO: 572);
    (aa) CDRH1, consisting of the amino acid sequence of GFTFSSYD (SEQ ID NO: 581);
    CDRH2 consisting of the amino acid sequence IGTAGDT (SEQ ID NO: 582) and CDRH3 consisting of the amino acid sequence ARGIQQQLALRYYYYLDV (SEQ ID NO: 583), and
    CDRL1 consisting of the amino acid sequence of QSISSY (SEQ ID NO: 592),
    CDRL2 consisting of the amino acid sequence of AAS (SEQ ID NO: 593) and CDRL3 consisting of the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 594);
    (ab) CDRH1, consisting of the amino acid sequence of GFTFSSSYA (SEQ ID NO: 603);
    CDRH2 consisting of the amino acid sequence of ISGSGGST (SEQ ID NO: 604) and CDRH3 consisting of the amino acid sequence of AKDGEWYYDFWSGPVYFDY (SEQ ID NO: 605), and
    CDRL1 consisting of the amino acid sequence of QSISSW (SEQ ID NO: 614),
    CDRL2 consisting of the amino acid sequence of KAS (SEQ ID NO:615) and CDRL3 consisting of the amino acid sequence of QQYNSYSRT (SEQ ID NO:616).
  5. 　以下から選択されるいずれかの組み合わせの可変領域を有し，かつ，ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質に結合する，請求項４に記載の抗体又はその抗原結合性断片：
    （ａ１）請求項４の（ａ）の抗体であって，配列番号８に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号１９に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｂ１）請求項４の（ｂ）の抗体であって，配列番号３０に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号４１に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｃ１）請求項４の（ｃ）の抗体であって，配列番号５２に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号６３に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｄ１）請求項４の（ｄ）の抗体であって，配列番号７４に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号８５に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｅ１）請求項４の（ｅ）の抗体であって，配列番号９６に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号１０７に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｆ１）請求項４の（ｆ）の抗体であって，配列番号１１８に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号１２９に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｇ１）請求項４の（ｇ）の抗体であって，配列番号１４０に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号１５１に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｈ１）請求項４の（ｈ）の抗体であって，配列番号１６２に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号１７３に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｉ１）請求項４の（ｉ）の抗体であって，配列番号１８４に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号１９５に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｊ１）請求項４の（ｊ）の抗体であって，配列番号２０６に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号２１７に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｋ１）請求項４の（ｋ）の抗体であって，配列番号２２８に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号２３９に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｌ１）請求項４の（ｌ）の抗体であって，配列番号２５０に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号２６１に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｍ１）請求項４の（ｍ）の抗体であって，配列番号２７２に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号２８３に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｎ１）請求項４の（ｎ）の抗体であって，配列番号２９４に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号３０５に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｏ１）請求項４の（ｏ）の抗体であって，配列番号３１６に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号３２７に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｐ１）請求項４の（ｐ）の抗体であって，配列番号３３８に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号３４９に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｑ１）請求項４の（ｑ）の抗体であって，配列番号３６０に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号３７１に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｒ１）請求項４の（ｒ）の抗体であって，配列番号３８２に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号３９３に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｓ１）請求項４の（ｓ）の抗体であって，配列番号４０４に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号４１５に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｔ１）請求項４の（ｔ）の抗体であって，配列番号４２６に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号４３７に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｕ１）請求項４の（ｕ）の抗体であって，配列番号４４８に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号４５９に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｖ１）請求項４の（ｖ）の抗体であって，配列番号４７０に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号４８１に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｗ１）請求項４の（ｗ）の抗体であって，配列番号４９２に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号５０３に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｘ１）請求項４の（ｘ）の抗体であって，配列番号５１４に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号５２５に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｙ１）請求項４の（ｙ）の抗体であって，配列番号５３６に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号５４７に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ｚ１）請求項４の（ｚ）の抗体であって，配列番号５５８に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号５６９に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；
    （ａａ１）請求項４の（ａａ）の抗体であって，配列番号５８０に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号５９１に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ；並びに，
    （ａｂ１）請求項４の（ａｂ）の抗体であって，配列番号６０２に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＨ，及び，配列番号６１３に記載されたアミノ酸配列と８０％以上の同一性であるアミノ酸配列を有するＶＬ。     The antibody or antigen-binding fragment thereof according to claim 4, which has any combination of variable regions selected from the following and binds to SARS-CoV-2 spike protein:
    (a1) The antibody of (a) of claim 4, wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 8, and the amino acid set forth in SEQ ID NO: 19 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (b1) the antibody of claim 4 (b), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 30, and the amino acid set forth in SEQ ID NO: 41 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (c1) The antibody of (c) of claim 4, wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 52, and the amino acid set forth in SEQ ID NO: 63 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (d1) The antibody of claim 4 (d), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 74, and the amino acid set forth in SEQ ID NO: 85 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (e1) The antibody of claim 4 (e), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 96 and the amino acid set forth in SEQ ID NO: 107 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (f1) the antibody of claim 4 (f), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 118, and the amino acid set forth in SEQ ID NO: 129 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (g1) The antibody of (g) of claim 4, wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 140, and the amino acid set forth in SEQ ID NO: 151 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (h1) The antibody of claim 4 (h), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 162, and the amino acid set forth in SEQ ID NO: 173 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (i1) The antibody of (i) of claim 4, wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 184, and the amino acid set forth in SEQ ID NO: 195. a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (j1) The antibody of claim 4 (j), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 206, and the amino acid set forth in SEQ ID NO: 217 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (k1) The antibody of claim 4 (k), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 228, and the amino acid set forth in SEQ ID NO: 239 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (l1) The antibody of claim 4 (l), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 250, and the amino acid set forth in SEQ ID NO: 261 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (m1) The antibody of claim 4 (m), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 272, and the amino acid set forth in SEQ ID NO: 283 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (n1) The antibody of claim 4 (n), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 294, and the amino acid set forth in SEQ ID NO: 305 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (o1) The antibody of (o) of claim 4, wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 316, and the amino acid set forth in SEQ ID NO: 327 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (p1) The antibody of (p) of claim 4, wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 338, and the amino acid set forth in SEQ ID NO: 349 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (q1) The antibody of (q) of claim 4, which VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 360, and the amino acid set forth in SEQ ID NO: 371 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (r1) The antibody of claim 4 (r), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 382, and the amino acid set forth in SEQ ID NO: 393 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (s1) The antibody of (s) of claim 4, wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 404, and the amino acid set forth in SEQ ID NO: 415 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (t1) The antibody of claim 4 (t), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 426 and the amino acid set forth in SEQ ID NO: 437 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (u1) The antibody of claim 4 (u), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 448, and the amino acid set forth in SEQ ID NO: 459 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (v1) The antibody of (v) of claim 4, wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 470, and the amino acid set forth in SEQ ID NO: 481 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (w1) The antibody of claim 4 (w), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 492, and the amino acid set forth in SEQ ID NO: 503 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (x1) The antibody of claim 4 (x), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 514, and the amino acid set forth in SEQ ID NO: 525 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (y1) The antibody of claim 4 (y), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 536, and the amino acid set forth in SEQ ID NO: 547 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (z1) The antibody of claim 4 (z), wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 558, and the amino acid set forth in SEQ ID NO: 569 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (aa1) The antibody of (aa) of claim 4, wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 580, and the amino acid set forth in SEQ ID NO: 591 a VL having an amino acid sequence that is 80% or more identical to the sequence;
    (ab1) The antibody of (ab) of claim 4, wherein the VH has an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 602, and the amino acid set forth in SEQ ID NO: 613 A VL having an amino acid sequence that is 80% or more identical to the sequence.
  6. 　以下から選択されるいずれかの組み合わせの可変領域を有する抗体又はその抗原結合性断片：
    （ａ２）配列番号８に記載されたアミノ酸配列を有するＶＨ，及び，配列番号１９に記載されたアミノ酸配列を有するＶＬ；
    （ｂ２）配列番号３０に記載されたアミノ酸配列を有するＶＨ，及び，配列番号４１に記載されたアミノ酸配列を有するＶＬ；
    （ｃ２）配列番号５２に記載されたアミノ酸配列を有するＶＨ，及び，配列番号６３に記載されたアミノ酸配列を有するＶＬ；
    （ｄ２）配列番号７４に記載されたアミノ酸配列を有するＶＨ，及び，配列番号８５に記載されたアミノ酸配列を有するＶＬ；
    （ｅ２）配列番号９６に記載されたアミノ酸配列を有するＶＨ，及び，配列番号１０７に記載されたアミノ酸配列を有するＶＬ；
    （ｆ２）配列番号１１８に記載されたアミノ酸配列を有するＶＨ，及び，配列番号１２９に記載されたアミノ酸配列を有するＶＬ；
    （ｇ２）配列番号１４０に記載されたアミノ酸配列を有するＶＨ，及び，配列番号１５１に記載されたアミノ酸配列を有するＶＬ；
    （ｈ２）配列番号１６２に記載されたアミノ酸配列を有するＶＨ，及び，配列番号１７３に記載されたアミノ酸配列を有するＶＬ；
    （ｉ２）配列番号１８４に記載されたアミノ酸配列を有するＶＨ，及び，配列番号１９５に記載されたアミノ酸配列を有するＶＬ；
    （ｊ２）配列番号２０６に記載されたアミノ酸配列を有するＶＨ，及び，配列番号２１７に記載されたアミノ酸配列を有するＶＬ；
    （ｋ２）配列番号２２８に記載されたアミノ酸配列を有するＶＨ，及び，配列番号２３９に記載されたアミノ酸配列を有するＶＬ；
    （ｌ２）配列番号２５０に記載されたアミノ酸配列を有するＶＨ，及び，配列番号２６１に記載されたアミノ酸配列を有するＶＬ；
    （ｍ２）配列番号２７２に記載されたアミノ酸配列を有するＶＨ，及び，配列番号２８３に記載されたアミノ酸配列を有するＶＬ；
    （ｎ２）配列番号２９４に記載されたアミノ酸配列を有するＶＨ，及び，配列番号３０５に記載されたアミノ酸配列を有するＶＬ；
    （ｏ２）配列番号３１６に記載されたアミノ酸配列を有するＶＨ，及び，配列番号３２７に記載されたアミノ酸配列を有するＶＬ；
    （ｐ２）配列番号３３８に記載されたアミノ酸配列を有するＶＨ，及び，配列番号３４９に記載されたアミノ酸配列を有するＶＬ；
    （ｑ２）配列番号３６０に記載されたアミノ酸配列を有するＶＨ，及び，配列番号３７１に記載されたアミノ酸配列を有するＶＬ；
    （ｒ２）配列番号３８２に記載されたアミノ酸配列を有するＶＨ，及び，配列番号３９３に記載されたアミノ酸配列を有するＶＬ；
    （ｓ２）配列番号４０４に記載されたアミノ酸配列を有するＶＨ，及び，配列番号４１５に記載されたアミノ酸配列を有するＶＬ；
    （ｔ２）配列番号４２６に記載されたアミノ酸配列を有するＶＨ，及び，配列番号４３７に記載されたアミノ酸配列を有するＶＬ；
    （ｕ２）配列番号４４８に記載されたアミノ酸配列を有するＶＨ，及び，配列番号４５９に記載されたアミノ酸配列を有するＶＬ；
    （ｖ２）配列番号４７０に記載されたアミノ酸配列を有するＶＨ，及び，配列番号４８１に記載されたアミノ酸配列を有するＶＬ；
    （ｗ２）配列番号４９２に記載されたアミノ酸配列を有するＶＨ，及び，配列番号５０３に記載されたアミノ酸配列を有するＶＬ；
    （ｘ２）配列番号５１４に記載されたアミノ酸配列を有するＶＨ，及び，配列番号５２５に記載されたアミノ酸配列を有するＶＬ；
    （ｙ２）配列番号５３６に記載されたアミノ酸配列を有するＶＨ，及び，配列番号５４７に記載されたアミノ酸配列を有するＶＬ；
    （ｚ２）配列番号５５８に記載されたアミノ酸配列を有するＶＨ，及び，配列番号５６９に記載されたアミノ酸配列を有するＶＬ；
    （ａａ２）配列番号５８０に記載されたアミノ酸配列を有するＶＨ，及び，配列番号５９１に記載されたアミノ酸配列を有するＶＬ；並びに，
    （ａｂ２）配列番号６０２に記載されたアミノ酸配列を有するＶＨ，及び，配列番号６１３に記載されたアミノ酸配列を有するＶＬ。     An antibody or antigen-binding fragment thereof having any combination of variable regions selected from the following:
    (a2) VH having the amino acid sequence set forth in SEQ ID NO: 8 and VL having the amino acid sequence set forth in SEQ ID NO: 19;
    (b2) VH having the amino acid sequence set forth in SEQ ID NO: 30 and VL having the amino acid sequence set forth in SEQ ID NO: 41;
    (c2) VH having the amino acid sequence set forth in SEQ ID NO: 52 and VL having the amino acid sequence set forth in SEQ ID NO: 63;
    (d2) VH having the amino acid sequence set forth in SEQ ID NO: 74 and VL having the amino acid sequence set forth in SEQ ID NO: 85;
    (e2) VH having the amino acid sequence set forth in SEQ ID NO: 96 and VL having the amino acid sequence set forth in SEQ ID NO: 107;
    (f2) VH having the amino acid sequence set forth in SEQ ID NO: 118 and VL having the amino acid sequence set forth in SEQ ID NO: 129;
    (g2) VH having the amino acid sequence set forth in SEQ ID NO: 140 and VL having the amino acid sequence set forth in SEQ ID NO: 151;
    (h2) VH having the amino acid sequence set forth in SEQ ID NO: 162 and VL having the amino acid sequence set forth in SEQ ID NO: 173;
    (i2) VH having the amino acid sequence set forth in SEQ ID NO: 184 and VL having the amino acid sequence set forth in SEQ ID NO: 195;
    (j2) VH having the amino acid sequence set forth in SEQ ID NO: 206 and VL having the amino acid sequence set forth in SEQ ID NO: 217;
    (k2) VH having the amino acid sequence set forth in SEQ ID NO: 228 and VL having the amino acid sequence set forth in SEQ ID NO: 239;
    (l2) VH having the amino acid sequence set forth in SEQ ID NO: 250 and VL having the amino acid sequence set forth in SEQ ID NO: 261;
    (m2) VH having the amino acid sequence set forth in SEQ ID NO: 272 and VL having the amino acid sequence set forth in SEQ ID NO: 283;
    (n2) VH having the amino acid sequence set forth in SEQ ID NO: 294 and VL having the amino acid sequence set forth in SEQ ID NO: 305;
    (o2) VH having the amino acid sequence set forth in SEQ ID NO: 316 and VL having the amino acid sequence set forth in SEQ ID NO: 327;
    (p2) VH having the amino acid sequence set forth in SEQ ID NO: 338 and VL having the amino acid sequence set forth in SEQ ID NO: 349;
    (q2) VH having the amino acid sequence set forth in SEQ ID NO: 360 and VL having the amino acid sequence set forth in SEQ ID NO: 371;
    (r2) VH having the amino acid sequence set forth in SEQ ID NO: 382 and VL having the amino acid sequence set forth in SEQ ID NO: 393;
    (s2) VH having the amino acid sequence set forth in SEQ ID NO: 404 and VL having the amino acid sequence set forth in SEQ ID NO: 415;
    (t2) VH having the amino acid sequence set forth in SEQ ID NO: 426 and VL having the amino acid sequence set forth in SEQ ID NO: 437;
    (u2) VH having the amino acid sequence set forth in SEQ ID NO: 448 and VL having the amino acid sequence set forth in SEQ ID NO: 459;
    (v2) VH having the amino acid sequence set forth in SEQ ID NO: 470 and VL having the amino acid sequence set forth in SEQ ID NO: 481;
    (w2) VH having the amino acid sequence set forth in SEQ ID NO: 492 and VL having the amino acid sequence set forth in SEQ ID NO: 503;
    (x2) VH having the amino acid sequence set forth in SEQ ID NO: 514 and VL having the amino acid sequence set forth in SEQ ID NO: 525;
    (y2) VH having the amino acid sequence set forth in SEQ ID NO: 536 and VL having the amino acid sequence set forth in SEQ ID NO: 547;
    (z2) VH having the amino acid sequence set forth in SEQ ID NO: 558 and VL having the amino acid sequence set forth in SEQ ID NO: 569;
    (aa2) VH having the amino acid sequence set forth in SEQ ID NO: 580 and VL having the amino acid sequence set forth in SEQ ID NO: 591;
    (ab2) VH having the amino acid sequence set forth in SEQ ID NO:602 and VL having the amino acid sequence set forth in SEQ ID NO:613.
  7. 　以下から選択されるいずれかの組み合わせの重鎖及び軽鎖を有する，請求項６に記載の抗体又はその抗原結合性断片：
    （ａ３）配列番号４に記載されたアミノ酸配列を有する重鎖，及び，配列番号１５に記載されたアミノ酸配列を有する軽鎖；
    （ｂ３）配列番号２６に記載されたアミノ酸配列を有する重鎖，及び，配列番号３７に記載されたアミノ酸配列を有する軽鎖；
    （ｃ３）配列番号４８に記載されたアミノ酸配列を有する重鎖，及び，配列番号５９に記載されたアミノ酸配列を有する軽鎖；
    （ｄ３）配列番号７０に記載されたアミノ酸配列を有する重鎖，及び，配列番号８１に記載されたアミノ酸配列を有する軽鎖；
    （ｅ３）配列番号９２に記載されたアミノ酸配列を有する重鎖，及び，配列番号１０３に記載されたアミノ酸配列を有する軽鎖；
    （ｆ３）配列番号１１４に記載されたアミノ酸配列を有する重鎖，及び，配列番号１２５に記載されたアミノ酸配列を有する軽鎖；
    （ｇ３）配列番号１３６に記載されたアミノ酸配列を有する重鎖，及び，配列番号１４７に記載されたアミノ酸配列を有する軽鎖；
    （ｈ３）配列番号１５８に記載されたアミノ酸配列を有する重鎖，及び，配列番号１６９に記載されたアミノ酸配列を有する軽鎖；
    （ｉ３）配列番号１８０に記載されたアミノ酸配列を有する重鎖，及び，配列番号１９１に記載されたアミノ酸配列を有する軽鎖；
    （ｊ３）配列番号２０２に記載されたアミノ酸配列を有する重鎖，及び，配列番号２１３に記載されたアミノ酸配列を有する軽鎖；
    （ｋ３）配列番号２２４に記載されたアミノ酸配列を有する重鎖，及び，配列番号２３５に記載されたアミノ酸配列を有する軽鎖；
    （ｌ３）配列番号２４６に記載されたアミノ酸配列を有する重鎖，及び，配列番号２５７に記載されたアミノ酸配列を有する軽鎖；
    （ｍ３）配列番号２６８に記載されたアミノ酸配列を有する重鎖，及び，配列番号２７９に記載されたアミノ酸配列を有する軽鎖；
    （ｎ３）配列番号２９０に記載されたアミノ酸配列を有する重鎖，及び，配列番号３０１に記載されたアミノ酸配列を有する軽鎖；
    （ｏ３）配列番号３１２に記載されたアミノ酸配列を有する重鎖，及び，配列番号３２３に記載されたアミノ酸配列を有する軽鎖；
    （ｐ３）配列番号３３４に記載されたアミノ酸配列を有する重鎖，及び，配列番号３４５に記載されたアミノ酸配列を有する軽鎖；
    （ｑ３）配列番号３５６に記載されたアミノ酸配列を有する重鎖，及び，配列番号３６７に記載されたアミノ酸配列を有する軽鎖；
    （ｒ３）配列番号３７８に記載されたアミノ酸配列を有する重鎖，及び，配列番号３８９に記載されたアミノ酸配列を有する軽鎖；
    （ｓ３）配列番号４００に記載されたアミノ酸配列を有する重鎖，及び，配列番号４１１に記載されたアミノ酸配列を有する軽鎖；
    （ｔ３）配列番号４２２に記載されたアミノ酸配列を有する重鎖，及び，配列番号４３３に記載されたアミノ酸配列を有する軽鎖；
    （ｕ３）配列番号４４４に記載されたアミノ酸配列を有する重鎖，及び，配列番号４５５に記載されたアミノ酸配列を有する軽鎖；
    （ｖ３）配列番号４６６に記載されたアミノ酸配列を有する重鎖，及び，配列番号４７７に記載されたアミノ酸配列を有する軽鎖；
    （ｗ３）配列番号４８８に記載されたアミノ酸配列を有する重鎖，及び，配列番号４９９に記載されたアミノ酸配列を有する軽鎖；
    （ｘ３）配列番号５１０に記載されたアミノ酸配列を有する重鎖，及び，配列番号５２１に記載されたアミノ酸配列を有する軽鎖；
    （ｙ３）配列番号５３２に記載されたアミノ酸配列を有する重鎖，及び，配列番号５４３に記載されたアミノ酸配列を有する軽鎖；
    （ｚ３）配列番号５５４に記載されたアミノ酸配列を有する重鎖，及び，配列番号５６５に記載されたアミノ酸配列を有する軽鎖；
    （ａａ３）配列番号５７６に記載されたアミノ酸配列を有する重鎖，及び，配列番号５８７に記載されたアミノ酸配列を有する軽鎖；並びに，
    （ａｂ３）配列番号５９８に記載されたアミノ酸配列を有する重鎖，及び，配列番号６０９に記載されたアミノ酸配列を有する軽鎖。     7. The antibody or antigen-binding fragment thereof according to claim 6, which has any combination of heavy and light chains selected from:
    (a3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 4 and a light chain having the amino acid sequence set forth in SEQ ID NO: 15;
    (b3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 26 and a light chain having the amino acid sequence set forth in SEQ ID NO: 37;
    (c3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 48 and a light chain having the amino acid sequence set forth in SEQ ID NO: 59;
    (d3) a heavy chain having the amino acid sequence set forth in SEQ ID NO:70 and a light chain having the amino acid sequence set forth in SEQ ID NO:81;
    (e3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 92 and a light chain having the amino acid sequence set forth in SEQ ID NO: 103;
    (f3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 114 and a light chain having the amino acid sequence set forth in SEQ ID NO: 125;
    (g3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 136 and a light chain having the amino acid sequence set forth in SEQ ID NO: 147;
    (h3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 158 and a light chain having the amino acid sequence set forth in SEQ ID NO: 169;
    (i3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 180 and a light chain having the amino acid sequence set forth in SEQ ID NO: 191;
    (j3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 202 and a light chain having the amino acid sequence set forth in SEQ ID NO: 213;
    (k3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 224 and a light chain having the amino acid sequence set forth in SEQ ID NO: 235;
    (l3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 246 and a light chain having the amino acid sequence set forth in SEQ ID NO: 257;
    (m3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 268 and a light chain having the amino acid sequence set forth in SEQ ID NO: 279;
    (n3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 290 and a light chain having the amino acid sequence set forth in SEQ ID NO: 301;
    (o3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 312 and a light chain having the amino acid sequence set forth in SEQ ID NO: 323;
    (p3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 334 and a light chain having the amino acid sequence set forth in SEQ ID NO: 345;
    (q3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 356 and a light chain having the amino acid sequence set forth in SEQ ID NO: 367;
    (r3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 378 and a light chain having the amino acid sequence set forth in SEQ ID NO: 389;
    (s3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 400 and a light chain having the amino acid sequence set forth in SEQ ID NO: 411;
    (t3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 422 and a light chain having the amino acid sequence set forth in SEQ ID NO: 433;
    (u3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 444 and a light chain having the amino acid sequence set forth in SEQ ID NO: 455;
    (v3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 466 and a light chain having the amino acid sequence set forth in SEQ ID NO: 477;
    (w3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 488 and a light chain having the amino acid sequence set forth in SEQ ID NO: 499;
    (x3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 510 and a light chain having the amino acid sequence set forth in SEQ ID NO: 521;
    (y3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 532 and a light chain having the amino acid sequence set forth in SEQ ID NO: 543;
    (z3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 554 and a light chain having the amino acid sequence set forth in SEQ ID NO: 565;
    (aa3) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 576 and a light chain having the amino acid sequence set forth in SEQ ID NO: 587;
    (ab3) A heavy chain having the amino acid sequence set forth in SEQ ID NO:598 and a light chain having the amino acid sequence set forth in SEQ ID NO:609.
  8. 　Ｆｃ領域におけるＬ２３４Ａ，Ｌ２３５Ａのアミノ酸変異を有する，請求項１～請求項７のいずれか１項に記載の抗体。 The antibody according to any one of claims 1 to 7, which has L234A and L235A amino acid mutations in the Fc region.
  9. 　以下から選択されるいずれかの組み合わせの重鎖及び軽鎖を有する，請求項６に記載の抗体
    （ａ３’）配列番号６３０に記載されたアミノ酸配列を有する重鎖，及び，配列番号１５に記載されたアミノ酸配列を有する軽鎖；
    （ｂ３’）配列番号６３１に記載されたアミノ酸配列を有する重鎖，及び，配列番号３７に記載されたアミノ酸配列を有する軽鎖；
    （ｃ３’）配列番号６３２に記載されたアミノ酸配列を有する重鎖，及び，配列番号５９に記載されたアミノ酸配列を有する軽鎖；
    （ｄ３’）配列番号６３３に記載されたアミノ酸配列を有する重鎖，及び，配列番号８１に記載されたアミノ酸配列を有する軽鎖；
    （ｅ３’）配列番号６３４に記載されたアミノ酸配列を有する重鎖，及び，配列番号１０３に記載されたアミノ酸配列を有する軽鎖；
    （ｆ３’）配列番号６３５に記載されたアミノ酸配列を有する重鎖，及び，配列番号１２５に記載されたアミノ酸配列を有する軽鎖；
    （ｇ３’）配列番号６３６に記載されたアミノ酸配列を有する重鎖，及び，配列番号１４７に記載されたアミノ酸配列を有する軽鎖；
    （ｈ３’）配列番号６３７に記載されたアミノ酸配列を有する重鎖，及び，配列番号１６９に記載されたアミノ酸配列を有する軽鎖；
    （ｉ３’）配列番号６３８に記載されたアミノ酸配列を有する重鎖，及び，配列番号１９１に記載されたアミノ酸配列を有する軽鎖；
    （ｊ３’）配列番号６３９に記載されたアミノ酸配列を有する重鎖，及び，配列番号２１３に記載されたアミノ酸配列を有する軽鎖；
    （ｋ３’）配列番号６４０に記載されたアミノ酸配列を有する重鎖，及び，配列番号２３５に記載されたアミノ酸配列を有する軽鎖；
    （ｌ３’）配列番号６４１に記載されたアミノ酸配列を有する重鎖，及び，配列番号２５７に記載されたアミノ酸配列を有する軽鎖；
    （ｍ３’）配列番号６４２に記載されたアミノ酸配列を有する重鎖，及び，配列番号２７９に記載されたアミノ酸配列を有する軽鎖；
    （ｎ３’）配列番号６４３に記載されたアミノ酸配列を有する重鎖，及び，配列番号３０１に記載されたアミノ酸配列を有する軽鎖；
    （ｏ３’）配列番号６４４に記載されたアミノ酸配列を有する重鎖，及び，配列番号３２３に記載されたアミノ酸配列を有する軽鎖；
    （ｐ３’）配列番号６４５に記載されたアミノ酸配列を有する重鎖，及び，配列番号３４５に記載されたアミノ酸配列を有する軽鎖；
    （ｑ３’）配列番号６４６に記載されたアミノ酸配列を有する重鎖，及び，配列番号３６７に記載されたアミノ酸配列を有する軽鎖；
    （ｒ３’）配列番号６４７に記載されたアミノ酸配列を有する重鎖，及び，配列番号３８９に記載されたアミノ酸配列を有する軽鎖；
    （ｓ３’）配列番号６４８に記載されたアミノ酸配列を有する重鎖，及び，配列番号４１１に記載されたアミノ酸配列を有する軽鎖；
    （ｔ３’）配列番号６４９に記載されたアミノ酸配列を有する重鎖，及び，配列番号４３３に記載されたアミノ酸配列を有する軽鎖；
    （ｕ３’）配列番号６５０に記載されたアミノ酸配列を有する重鎖，及び，配列番号４５５に記載されたアミノ酸配列を有する軽鎖；
    （ｖ３’）配列番号６５１に記載されたアミノ酸配列を有する重鎖，及び，配列番号４７７に記載されたアミノ酸配列を有する軽鎖；
    （ｗ３’）配列番号６５２に記載されたアミノ酸配列を有する重鎖，及び，配列番号４９９に記載されたアミノ酸配列を有する軽鎖；
    （ｘ３’）配列番号６５３に記載されたアミノ酸配列を有する重鎖，及び，配列番号５２１に記載されたアミノ酸配列を有する軽鎖；
    （ｙ３’）配列番号６５４に記載されたアミノ酸配列を有する重鎖，及び，配列番号５４３に記載されたアミノ酸配列を有する軽鎖；
    （ｚ３’）配列番号６５５に記載されたアミノ酸配列を有する重鎖，及び，配列番号５６５に記載されたアミノ酸配列を有する軽鎖；
    （ａａ３’）配列番号６５６に記載されたアミノ酸配列を有する重鎖，及び，配列番号５８７に記載されたアミノ酸配列を有する軽鎖；並びに，
    （ａｂ３’）配列番号６５７に記載されたアミノ酸配列を有する重鎖，及び，配列番号６０９に記載されたアミノ酸配列を有する軽鎖。     The antibody (a3') according to claim 6, having any combination of heavy and light chains selected from the following: a light chain having the amino acid sequence identified;
    (b3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 631 and a light chain having the amino acid sequence set forth in SEQ ID NO: 37;
    (c3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 632 and a light chain having the amino acid sequence set forth in SEQ ID NO: 59;
    (d3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 633 and a light chain having the amino acid sequence set forth in SEQ ID NO: 81;
    (e3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 634 and a light chain having the amino acid sequence set forth in SEQ ID NO: 103;
    (f3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 635 and a light chain having the amino acid sequence set forth in SEQ ID NO: 125;
    (g3') a heavy chain having the amino acid sequence set forth in SEQ ID NO: 636 and a light chain having the amino acid sequence set forth in SEQ ID NO: 147;
    (h3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 637 and a light chain having the amino acid sequence set forth in SEQ ID NO: 169;
    (i3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 638 and a light chain having the amino acid sequence set forth in SEQ ID NO: 191;
    (j3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 639 and a light chain having the amino acid sequence set forth in SEQ ID NO: 213;
    (k3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 640 and a light chain having the amino acid sequence set forth in SEQ ID NO: 235;
    (l3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 641 and a light chain having the amino acid sequence set forth in SEQ ID NO: 257;
    (m3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO:642 and a light chain having the amino acid sequence set forth in SEQ ID NO:279;
    (n3') a heavy chain having the amino acid sequence set forth in SEQ ID NO: 643 and a light chain having the amino acid sequence set forth in SEQ ID NO: 301;
    (o3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 644 and a light chain having the amino acid sequence set forth in SEQ ID NO: 323;
    (p3') a heavy chain having the amino acid sequence set forth in SEQ ID NO: 645 and a light chain having the amino acid sequence set forth in SEQ ID NO: 345;
    (q3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 646 and a light chain having the amino acid sequence set forth in SEQ ID NO: 367;
    (r3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 647 and a light chain having the amino acid sequence set forth in SEQ ID NO: 389;
    (s3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 648 and a light chain having the amino acid sequence set forth in SEQ ID NO: 411;
    (t3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 649 and a light chain having the amino acid sequence set forth in SEQ ID NO: 433;
    (u3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 650 and a light chain having the amino acid sequence set forth in SEQ ID NO: 455;
    (v3') a heavy chain having the amino acid sequence set forth in SEQ ID NO: 651 and a light chain having the amino acid sequence set forth in SEQ ID NO: 477;
    (w3') a heavy chain having the amino acid sequence set forth in SEQ ID NO: 652 and a light chain having the amino acid sequence set forth in SEQ ID NO: 499;
    (x3') a heavy chain having the amino acid sequence set forth in SEQ ID NO: 653 and a light chain having the amino acid sequence set forth in SEQ ID NO: 521;
    (y3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 654 and a light chain having the amino acid sequence set forth in SEQ ID NO: 543;
    (z3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 655 and a light chain having the amino acid sequence set forth in SEQ ID NO: 565;
    (aa3′) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 656 and a light chain having the amino acid sequence set forth in SEQ ID NO: 587;
    (ab3') A heavy chain having the amino acid sequence set forth in SEQ ID NO:657 and a light chain having the amino acid sequence set forth in SEQ ID NO:609.
  10. 　請求項６，請求項７又は請求項９に記載の抗体又はその抗原結合性断片とＳＡＲＳ－ＣｏＶ－２スパイクタンパク質又はＲＢＤへの結合において競合する抗体又はその抗原結合性断片，あるいは，請求項６，請求項７又は請求項９に記載の抗体又はその抗原結合性断片と同じエピトープを認識する抗体又はその抗原結合性断片。 An antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof of claim 6, claim 7 or claim 9 in binding to SARS-CoV-2 spike protein or RBD, or claim 6 An antibody or antigen-binding fragment thereof that recognizes the same epitope as the antibody or antigen-binding fragment thereof according to claim 7 or claim 9.
  11. 　ＲＢＤのＡ４７５，Ｇ４７６，Ｓ４７７，Ｔ４７８，Ｆ４８６，Ｎ４８７，Ｙ４８９及びＱ４９３から選択される少なくとも１個のアミノ酸残基を認識する，請求項１０に記載の抗体又はその抗原結合性断片。 The antibody or antigen-binding fragment thereof according to claim 10, which recognizes at least one amino acid residue selected from A475, G476, S477, T478, F486, N487, Y489 and Q493 of RBD.
  12. 　請求項１１に記載の抗体又はその抗原結合性断片であって，ＶＨのＳ５５，Ｎ５７，Ｄ１０４，Ｃ１０５，Ｓ１０６，及びＤ１０７から選択される少なくとも１個のアミノ酸残基を有し，かつこれらのアミノ酸残基が，ＲＢＤのＡ４７５，Ｇ４７６，Ｓ４７７，Ｔ４７８，Ｆ４８６，Ｎ４８７，Ｙ４８９及びＱ４９３から選択される少なくとも１個のアミノ酸残基と結合する抗体又はその抗原結合性断片。 12. The antibody or antigen-binding fragment thereof of claim 11, having at least one amino acid residue selected from VH S55, N57, D104, C105, S106, and D107, and An antibody or antigen-binding fragment thereof, wherein residues bind to at least one amino acid residue selected from A475, G476, S477, T478, F486, N487, Y489 and Q493 of RBD.
  13. 　請求項４～請求項９のいずれか１項に記載の抗体の重鎖及び／若しくは軽鎖，又はそれらの抗原結合性断片のアミノ酸配列をコードする核酸分子。 A nucleic acid molecule encoding the amino acid sequence of the heavy chain and/or light chain of the antibody according to any one of claims 4 to 9, or an antigen-binding fragment thereof.
  14. 　以下の群から選択されるいずれか１つのポリヌクレオチドを含有する，請求項１３に記載の核酸分子：
    （ａ４）配列番号５若しくは配列番号７に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号１６若しくは配列番号１８に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｂ４）配列番号２７若しくは配列番号２９に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号３８若しくは配列番号４０に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｃ４）配列番号４９若しくは配列番号５１に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号６０若しくは配列番号６２に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｄ４）配列番号７１若しくは配列番号７３に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号８２若しくは配列番号８４に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｅ４）配列番号９３若しくは配列番号９５に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号１０４若しくは配列番号１０６に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｆ４）配列番号１１５若しくは配列番号１１７に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号１２６若しくは配列番号１２８に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｇ４）配列番号１３７若しくは配列番号１３９に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号１４８若しくは配列番号１５０に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｈ４）配列番号１５９若しくは配列番号１６１に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号１７０若しくは配列番号１７２に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｉ４）配列番号１８１若しくは配列番号１８３に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号１９２若しくは配列番号１９４に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｊ４）配列番号２０３若しくは配列番号２０５に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号２１４若しくは配列番号２１６に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｋ４）配列番号２２５若しくは配列番号２２７に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号２３６若しくは配列番号２３８に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｌ４）配列番号２４７若しくは配列番号２４９に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号２５８若しくは配列番号２６０に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｍ４）配列番号２６９若しくは配列番号２７１に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号２８０若しくは配列番号２８２に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｎ４）配列番号２９１若しくは配列番号２９３に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号３０２若しくは配列番号３０４に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｏ４）配列番号３１３若しくは配列番号３１５に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号３２４若しくは配列番号３２６に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｐ４）配列番号３３５若しくは配列番号３３７に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号３４６若しくは配列番号３４８に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｑ４）配列番号３５７若しくは配列番号３５９に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号３６８若しくは配列番号３７０に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｒ４）配列番号３７９若しくは配列番号３８１に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号３９０若しくは配列番号３９２に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｓ４）配列番号４０１若しくは配列番号４０３に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号４１２若しくは配列番号４１４に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｔ４）配列番号４２３若しくは配列番号４２５に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号４２４若しくは配列番号４３６に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｕ４）配列番号４４５若しくは配列番号４４７に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号４５６若しくは配列番号４５８に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｖ４）配列番号４６７若しくは配列番号４６９に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号４７８若しくは配列番号４８０に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｗ４）配列番号４８９若しくは配列番号４９１に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号５００若しくは配列番号５０２に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｘ４）配列番号５１１若しくは配列番号５１３に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号５２２若しくは配列番号５２４に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｙ４）配列番号５３３若しくは配列番号５３５に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号５４４若しくは配列番号５４６に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ｚ４）配列番号５５５若しくは配列番号５５７に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号５６６若しくは配列番号５６８に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ａａ４）配列番号５７７若しくは配列番号５７９に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号５８８若しくは配列番号５９０に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド；
    （ａｂ４）配列番号５９９若しくは配列番号６０１に記載のヌクレオチド配列を有するＶＨをコードするポリヌクレオチド，及び／又は，配列番号６１０若しくは配列番号６１２に記載のヌクレオチド配列を有するＶＬをコードするポリヌクレオチド。     14. The nucleic acid molecule of claim 13, comprising any one polynucleotide selected from the group of:
    (a4) a VH-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7, and/or a VL-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 16 or SEQ ID NO: 18;
    (b4) a polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO:27 or SEQ ID NO:29 and/or a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO:38 or SEQ ID NO:40;
    (c4) a VH-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 49 or SEQ ID NO: 51 and/or a VL-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 60 or SEQ ID NO: 62;
    (d4) a polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO:71 or SEQ ID NO:73 and/or a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO:82 or SEQ ID NO:84;
    (e4) a polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 93 or SEQ ID NO: 95 and/or a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 104 or SEQ ID NO: 106;
    (f4) a VH-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 117 and/or a VL-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 126 or SEQ ID NO: 128;
    (g4) a VH-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 137 or SEQ ID NO: 139 and/or a VL-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 148 or SEQ ID NO: 150;
    (h4) a VH-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 159 or SEQ ID NO: 161 and/or a VL-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 170 or SEQ ID NO: 172;
    (i4) a VH-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 181 or SEQ ID NO: 183, and/or a VL-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 192 or SEQ ID NO: 194;
    (j4) a polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO:203 or SEQ ID NO:205 and/or a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO:214 or SEQ ID NO:216;
    (k4) a polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO:225 or SEQ ID NO:227 and/or a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO:236 or SEQ ID NO:238;
    (l4) a polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 247 or SEQ ID NO: 249 and/or a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 258 or SEQ ID NO: 260;
    (m4) a polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 269 or SEQ ID NO: 271 and/or a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 280 or SEQ ID NO: 282;
    (n4) a VH-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 291 or SEQ ID NO: 293, and/or a VL-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 302 or SEQ ID NO: 304;
    (o4) a VH-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO:313 or SEQ ID NO:315, and/or a VL-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO:324 or SEQ ID NO:326;
    (p4) a VH-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO:335 or SEQ ID NO:337 and/or a VL-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO:346 or SEQ ID NO:348;
    (q4) a polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 357 or SEQ ID NO: 359 and/or a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 368 or 370;
    (r4) a VH-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO:379 or SEQ ID NO:381 and/or a VL-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO:390 or SEQ ID NO:392;
    (s4) a VH-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 401 or SEQ ID NO: 403 and/or a VL-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 412 or SEQ ID NO: 414;
    (t4) a VH-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 423 or SEQ ID NO: 425 and/or a VL-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 424 or SEQ ID NO: 436;
    (u4) a VH-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 445 or SEQ ID NO: 447 and/or a VL-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 456 or SEQ ID NO: 458;
    (v4) a VH-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 467 or SEQ ID NO: 469 and/or a VL-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 478 or SEQ ID NO: 480;
    (w4) a VH-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 489 or SEQ ID NO: 491 and/or a VL-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 500 or SEQ ID NO: 502;
    (x4) a polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 511 or SEQ ID NO: 513, and/or a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 522 or 524;
    (y4) a polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 533 or 535 and/or a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 544 or 546;
    (z4) a polynucleotide encoding VH having the nucleotide sequence set forth in SEQ ID NO: 555 or SEQ ID NO: 557 and/or a polynucleotide encoding VL having the nucleotide sequence set forth in SEQ ID NO: 566 or 568;
    (aa4) a polynucleotide encoding a VH having the nucleotide sequence set forth in SEQ ID NO: 577 or SEQ ID NO: 579 and/or a polynucleotide encoding a VL having the nucleotide sequence set forth in SEQ ID NO: 588 or 590;
    (ab4) a VH-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO:599 or SEQ ID NO:601, and/or a VL-encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO:610 or SEQ ID NO:612;
  15. 　以下の群から選択されるいずれか１つのポリヌクレオチドを含有する，請求項１４に記載の核酸分子：
    （ａ５）配列番号１若しくは配列番号３に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号１２若しくは配列番号１４に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｂ５）配列番号２３若しくは配列番号２５に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号３４若しくは配列番号３６に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｃ５）配列番号４５若しくは配列番号４７に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号５６若しくは配列番号５８に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｄ５）配列番号６７若しくは配列番号６９に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号７８若しくは配列番号８０に載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｅ５）配列番号８９若しくは配列番号９１に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号１００若しくは配列番号１０２に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｆ５）配列番号１１１若しくは配列番号１１３に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号１２２若しくは配列番号１２４に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｇ５）配列番号１３３若しくは配列番号１３５に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号１４４若しくは配列番号１４６に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｈ５）配列番号１５５若しくは配列番号１５７に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号１６６若しくは配列番号１６８に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｉ５）配列番号１７７若しくは配列番号１７９に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号１８８若しくは配列番号１９０に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｊ５）配列番号１９９若しくは配列番号２０１に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号２１０若しくは配列番号２１２に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｋ５）配列番号２２１若しくは配列番号２２３に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号２３２若しくは配列番号２３４に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｌ５）配列番号２４３若しくは配列番号２４５に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号２５４若しくは配列番号２５６に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｍ５）配列番号２６５若しくは配列番号２６７に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号２７６若しくは配列番号２７８に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｎ５）配列番号２８７若しくは配列番号２８９に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号２９８若しくは配列番号３００に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｏ５）配列番号３０９若しくは配列番号３１１に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号３２０若しくは配列番号３２２に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｐ５）配列番号３３１若しくは配列番号３３３に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号３４２若しくは配列番号３４５に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｑ５）配列番号３５３若しくは配列番号３５５に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号３６４若しくは配列番号３６６に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｒ５）配列番号３７５若しくは配列番号３７７に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号３８６若しくは配列番号３８８に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｓ５）配列番号３９７若しくは配列番号３９９に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号４０８若しくは配列番号４１０に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｔ５）配列番号４１９若しくは配列番号４２１に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号４３０若しくは配列番号４３２に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｕ５）配列番号４４１若しくは配列番号４４３に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号４５２若しくは配列番号４５４に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｖ５）配列番号４６３若しくは配列番号４６５に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号４７４若しくは配列番号４７６に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｗ５）配列番号４８５若しくは配列番号４８７に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号４９６若しくは配列番号４９８に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｘ５）配列番号５０７若しくは配列番号５０９に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号５１８若しくは配列番号５２０に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｙ５）配列番号５２９若しくは配列番号５３１に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号５４０若しくは配列番号５４２に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ｚ５）配列番号５５１若しくは配列番号５５３に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号５６２若しくは配列番号５６４に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ａａ５）配列番号５７３若しくは配列番号５７５に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号５８４若しくは配列番号５８６に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド；
    （ａｂ５）配列番号５９５若しくは配列番号５９７に記載のヌクレオチド配列を有する重鎖をコードするポリヌクレオチド，及び／又は，配列番号６０６若しくは配列番号６０８に記載のヌクレオチド配列を有する軽鎖をコードするポリヌクレオチド。     15. The nucleic acid molecule of claim 14, containing any one polynucleotide selected from the group of:
    (a5) A polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 14 ;
    (b5) A polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 25 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 34 or 36 ;
    (c5) a polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 47 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 56 or 58 ;
    (d5) a polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 67 or SEQ ID NO: 69 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 78 or SEQ ID NO: 80 ;
    (e5) a polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 89 or SEQ ID NO: 91 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 100 or SEQ ID NO: 102 ;
    (f5) a polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 113 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 122 or 124 ;
    (g5) a polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 133 or SEQ ID NO: 135 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 144 or 146 ;
    (h5) a polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 155 or SEQ ID NO: 157 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 166 or 168 ;
    (i5) A polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 177 or SEQ ID NO: 179 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 188 or 190 ;
    (j5) A polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 199 or SEQ ID NO: 201 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 210 or 212 ;
    (k5) a polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 221 or SEQ ID NO: 223 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 232 or 234 ;
    (l5) A polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 243 or SEQ ID NO: 245 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 254 or 256 ;
    (m5) a polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 265 or SEQ ID NO: 267 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 276 or 278 ;
    (n5) A polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 287 or SEQ ID NO: 289 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 298 or 300 ;
    (o5) A polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 309 or SEQ ID NO: 311 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 320 or 322 ;
    (p5) A polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 331 or SEQ ID NO: 333 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 342 or 345 ;
    (q5) A polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 353 or 355 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 364 or 366 ;
    (r5) A polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 375 or SEQ ID NO: 377 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 386 or 388 ;
    (s5) A polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 397 or SEQ ID NO: 399 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 408 or 410 ;
    (t5) A polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 419 or SEQ ID NO: 421 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 430 or 432 ;
    (u5) a polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 441 or SEQ ID NO: 443 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 452 or 454 ;
    (v5) A polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 463 or SEQ ID NO: 465 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 474 or 476 ;
    (w5) a polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 485 or SEQ ID NO: 487 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 496 or 498 ;
    (x5) a polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 507 or SEQ ID NO: 509 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 518 or 520 ;
    (y5) a polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 529 or 531 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 540 or 542 ;
    (z5) a polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 551 or 553 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 562 or 564 ;
    (aa5) a polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 573 or SEQ ID NO: 575 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 584 or 586 ;
    (ab5) a polynucleotide encoding a heavy chain having the nucleotide sequence set forth in SEQ ID NO: 595 or 597 and/or a polynucleotide encoding a light chain having the nucleotide sequence set forth in SEQ ID NO: 606 or 608 .
  16. 　請求項１３～請求項１５のいずれか１項に記載の核酸分子を有するベクター。 A vector comprising the nucleic acid molecule according to any one of claims 13 to 15.
  17. 　請求項１６に記載のベクターを有する宿主細胞。 A host cell having the vector according to claim 16.
  18. 　請求項１７に記載の宿主細胞を培養することを含む，請求項３～請求項９のいずれか１項に記載の抗体又はその抗原結合性断片の製造方法。 A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 3 to 9, comprising culturing the host cell according to claim 17.
  19. 　ＳＡＲＳ－ＣｏＶ－２のスパイクタンパク質又はＲＢＤとＡＣＥ２との結合を阻害するための，請求項１～請求項１２のいずれか１項に記載の抗体又はその抗原結合性断片を含有する組成物。 A composition containing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 for inhibiting the binding of SARS-CoV-2 spike protein or RBD to ACE2.
  20. 　ＳＡＲＳ－ＣｏＶ－２の細胞への侵入を阻害するための，請求項１～請求項１２のいずれか１項に記載の抗体又はその抗原結合性断片を含有する組成物。 A composition containing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 for inhibiting entry of SARS-CoV-2 into cells.
  21. 　ＳＡＲＳ－ＣｏＶ－２と結合させるための，請求項１～請求項１２のいずれか１項に記載の抗体又はその抗原結合性断片を含有する組成物。 A composition containing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 for binding to SARS-CoV-2.
  22. 　請求項１～請求項１２のいずれか１項に記載の抗体又はその抗原結合性断片を有効成分として含有する，医薬組成物。 A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 as an active ingredient.
  23. 　ＳＡＲＳ－ＣｏＶ－２の感染に基づく疾患の治療用又は予防用である，請求項２２に記載の医薬組成物。 The pharmaceutical composition according to claim 22, which is for treatment or prevention of diseases based on SARS-CoV-2 infection.
  24. 　請求項１～請求項１２のいずれか１項に記載の抗体又はその抗原結合性断片を有効成分として含有する，診断用組成物。 A diagnostic composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 as an active ingredient.
  25. 　ＳＡＲＳ　ＣｏＶ－２感染症の診断用である，請求項２４に記載の診断用組成物。 The diagnostic composition according to claim 24, which is used for diagnosing SARS CoV-2 infection.
  26. 　請求項１～請求項１２のいずれか１項に記載の抗体又はその抗原結合性断片を含有する，ＳＡＲＳ　ＣｏＶ－２検出用組成物。 A composition for detecting SARS CoV-2, containing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.
  27. 　前記ＳＡＲＳ－ＣｏＶ－２が，ＲＢＤにおける変異の無いＳＡＲＳ－ＣｏＶ－２，並びに，ＲＢＤにおける，Ｇ３３９Ｄ，Ｓ３７１Ｌ，Ｓ３７３Ｐ，Ｓ３７５Ｆ，Ｋ４１７Ｎ，Ｋ４１７Ｔ，Ｎ４３９Ｋ，Ｎ４４０Ｋ，Ｇ４４６Ｓ，Ｌ４５２Ｒ，Ａ４７５Ｖ，Ｓ４７７Ｎ，Ｔ４７８Ｋ，Ｅ４８４Ａ，Ｅ４８４Ｋ，Ｅ４８４Ｑ，Ｑ４９３Ｒ，Ｇ４９６Ｓ，Ｑ４９８Ｒ，Ｎ５０１Ｙ，及びＹ５０５Ｈから選択される１種類以上の変異を有するＳＡＲＳ－ＣｏＶ－２から選択される１種類以上のＳＡＲＳ－ＣｏＶ－２である，請求項１９～請求項２６のいずれか１項に記載の組成物。 SARS-CoV-2 is SARS-CoV-2 without mutation in RBD, and G339D, S371L, S373P, S375F, K417N, K417T, N439K, N440K, G446S, L452R, A475V, S477N, T478K, E484A, E484K, E484Q, Q493R, G496S, Q498R, N501Y, and one or more SARS-CoV-2 selected from SARS-CoV-2 having one or more mutations selected from Y505H, claims A composition according to any one of claims 19-26.
  28. 　前記ＳＡＲＳ－ＣｏＶ－２が，ＲＢＤにおける変異の無いＳＡＲＳ－ＣｏＶ－２，並びに，ＲＢＤにおける変異であって，Ｇ３３９Ｄ，Ｓ３７１Ｌ，Ｓ３７３Ｐ，Ｓ３７５Ｆ，Ｋ４１７Ｎ，Ｋ４１７Ｔ，Ｎ４３９Ｋ，Ｎ４４０Ｋ，Ｇ４４６Ｓ，Ｌ４５２Ｒ，Ａ４７５Ｖ，Ｓ４７７Ｎ，Ｔ４７８Ｋ，Ｅ４８４Ａ，Ｅ４８４Ｋ，Ｅ４８４Ｑ，Ｑ４９３Ｒ，Ｇ４９６Ｓ，Ｑ４９８Ｒ，Ｎ５０１Ｙ，及びＹ５０５Ｈから選択される１種類以上の変異を有するＳＡＲＳ－ＣｏＶ－２から選択される１種類以上のＳＡＲＳ－ＣｏＶ－２である，請求項１９～請求項２６のいずれか１項に記載の組成物。 The SARS-CoV-2 is SARS-CoV-2 without mutations in RBD, and mutations in RBD, G339D, S371L, S373P, S375F, K417N, K417T, N439K, N440K, G446S, L452R, A475V, At least one SARS-CoV-2 selected from SARS-CoV-2 having one or more mutations selected from S477N, T478K, E484A, E484K, E484Q, Q493R, G496S, Q498R, N501Y, and Y505H The composition of any one of claims 19-26, wherein the composition is
  29. 　前記ＲＢＤにおける変異が，以下の（ｉｉ）～（ｘｉｉ）から選択される１種類以上の変異である，請求項２８に記載の組成物。
    （ｉｉ）Ｋ４１７Ｎ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異，
    （ｉｉｉ）Ｎ４３９Ｋ変異，
    （ｉｖ）Ｎ４４０Ｋ変異，
    （ｖ）Ａ４７５Ｖ変異，
    （ｖｉ）Ｅ４８４Ｋ変異，
    （ｖｉｉ）Ｎ５０１Ｙ変異，
    （ｖｉｉｉ）Ｌ４５２Ｒ変異，
    （ｉｘ）Ｌ４５２Ｒ及びＥ４８４Ｑ変異，
    （ｘ）Ｋ４１７Ｔ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異，
    （ｘｉ）Ｌ４５２Ｒ及びＴ４７８Ｋ変異，及び，
    （ｘｉｉ）Ｇ３３９Ｄ，Ｓ３７１Ｌ，Ｓ３７３Ｐ，Ｓ３７５Ｆ，Ｋ４１７Ｎ，Ｎ４４０Ｋ，Ｇ４４６Ｓ，Ｓ４７７Ｎ，Ｔ４７８Ｋ，Ｅ４８４Ａ，Ｑ４９３Ｒ，Ｇ４９６Ｓ，Ｑ４９８Ｒ，Ｎ５０１Ｙ，及びＹ５０５Ｈ変異。     29. The composition according to claim 28, wherein the mutation in said RBD is one or more mutations selected from (ii) to (xii) below.
    (ii) K417N, E484K, and N501Y mutations,
    (iii) the N439K mutation,
    (iv) the N440K mutation,
    (v) A475V mutation,
    (vi) E484K mutation,
    (vii) N501Y mutation,
    (viii) L452R mutation,
    (ix) L452R and E484Q mutations,
    (x) K417T, E484K, and N501Y mutations,
    (xi) L452R and T478K mutations, and
    (xii) G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H mutations.
  30. 　前記ＳＡＲＳ－ＣｏＶ－２が，
    （ｉ）ＲＢＤにおける変異を有しないＳＡＲＳ－ＣｏＶ－２，
    （ｉｉ）Ｋ４１７Ｎ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異を有するＳＡＲＳ－ＣｏＶ－２，
    （ｉｉｉ）Ｎ４３９Ｋ変異を有するＳＡＲＳ－ＣｏＶ－２，
    （ｉｖ）Ｎ４４０Ｋ変異を有するＳＡＲＳ－ＣｏＶ－２，
    （ｖ）Ａ４７５Ｖ変異を有するＳＡＲＳ－ＣｏＶ－２，
    （ｖｉ）Ｅ４８４Ｋ変異を有するＳＡＲＳ－ＣｏＶ－２，
    （ｖｉｉ）Ｎ５０１Ｙ変異を有するＳＡＲＳ－ＣｏＶ－２，
    （ｖｉｉｉ）Ｌ４５２Ｒ変異を有するＳＡＲＳ－ＣｏＶ－２，
    （ｉｘ）Ｌ４５２Ｒ及びＥ４８４Ｑ変異を有するＳＡＲＳ－ＣｏＶ－２，
    （ｘ）Ｋ４１７Ｔ，Ｅ４８４Ｋ，及びＮ５０１Ｙ変異を有するＳＡＲＳ－ＣｏＶ－２，
    （ｘｉ）Ｌ４５２Ｒ及びＴ４７８Ｋ変異を有するＳＡＲＳ－ＣｏＶ－２，及び，
    （ｘｉｉ）Ｇ３３９Ｄ，Ｓ３７１Ｌ，Ｓ３７３Ｐ，Ｓ３７５Ｆ，Ｋ４１７Ｎ，Ｎ４４０Ｋ，Ｇ４４６Ｓ，Ｓ４７７Ｎ，Ｔ４７８Ｋ，Ｅ４８４Ａ，Ｑ４９３Ｒ，Ｇ４９６Ｓ，Ｑ４９８Ｒ，Ｎ５０１Ｙ，及びＹ５０５Ｈ変異を有するＳＡＲＳ－ＣｏＶ－２である，
    　請求項２９に記載の組成物。     The SARS-CoV-2 is
    (i) SARS-CoV-2 without mutations in RBD,
    (ii) SARS-CoV-2 with K417N, E484K, and N501Y mutations,
    (iii) SARS-CoV-2 with N439K mutation,
    (iv) SARS-CoV-2 with N440K mutation,
    (v) SARS-CoV-2 with A475V mutation,
    (vi) SARS-CoV-2 with the E484K mutation,
    (vii) SARS-CoV-2 with N501Y mutation,
    (viii) SARS-CoV-2 with L452R mutation,
    (ix) SARS-CoV-2 with L452R and E484Q mutations,
    (x) SARS-CoV-2 with K417T, E484K, and N501Y mutations,
    (xi) SARS-CoV-2 with L452R and T478K mutations, and
    (xii) SARS-CoV-2 having the G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H mutations;
    30. A composition according to claim 29.
  31. 　請求項１～請求項１２のいずれか１項に記載の抗体又はその抗原結合性断片を含有する，ＳＡＲＳ－ＣｏＶ－２検出用キット。 A SARS-CoV-2 detection kit containing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.
  32. 　イムノクロマト又はＥＬＩＳＡである，請求項３１に記載の検出用キット。 The detection kit according to claim 31, which is immunochromatography or ELISA.
  33. 　サンプル中のＳＡＲＳ－ＣｏＶ－２の検出方法であって：
    　前記サンプルと請求項１～請求項１２のいずれか１項に記載の抗体又はその抗原結合性断片とを接触させること，
    　前記抗体又はその抗原結合性断片と結合した，前記サンプル中のＳＡＲＳ－ＣｏＶ－２を検出すること，及び，
    　ＳＡＲＳ－ＣｏＶ－２が検出されたサンプルについてＳＡＲＳ－ＣｏＶ－２が存在すると判定することを含む方法。     A method of detecting SARS-CoV-2 in a sample, comprising:
    contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12;
    detecting SARS-CoV-2 in the sample bound to the antibody or antigen-binding fragment thereof; and
    A method comprising determining that SARS-CoV-2 is present for a sample in which SARS-CoV-2 is detected.
  34. 　被験者のＳＡＲＳへの感染を判定する方法であって，
    　被験者由来のサンプルと請求項１～請求項１２のいずれか１項に記載の抗体又はその抗原結合性断片とを接触させること，及び，
    　前記抗体又はその抗原結合性断片と結合したＳＡＲＳ－ＣｏＶ－２が検出された，前記サンプル中に由来する被験者を，ＳＡＲＳ－ＣｏＶ－２に感染していると判定することを含む方法。     A method for determining SARS infection in a subject, comprising:
    Contacting a subject-derived sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, and
    determining that a subject from said sample in whom SARS-CoV-2 bound to said antibody or antigen-binding fragment thereof is detected is infected with SARS-CoV-2.
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