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WO2022210830A1 - Anticorps anti-sars-cov-2 - Google Patents

Anticorps anti-sars-cov-2 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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amino acid
acid sequence
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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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Toyama Prefecture
University of Toyama NUC
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University of Toyama NUC
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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
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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.

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Abstract

La présente invention aborde le problème de la fourniture d'un nouvel anticorps efficace qui se lie à la protéine de spicule du SARS-CoV-2, et en particulier aborde le problème de la fourniture d'un anticorps qui inhibe la liaison entre le SARS-CoV-2 et ACE2 ou un anticorps qui inhibe l'entrée du SARS-CoV-2 dans la cellule. La présente invention concerne, par exemple, un anticorps qui a la combinaison suivante de régions déterminant la complémentarité (CDR) et qui se lie à la protéine de spicule du SARS-CoV-2, ou un fragment de liaison à l'antigène dudit anticorps : CDRH1, 2, et 3 et CDRL1, 2, et 3 composées des séquences d'acides aminés de SEQ ID NOS : 9, 10, 11, 20, 21 et 22.
PCT/JP2022/015813 2021-03-30 2022-03-30 Anticorps anti-sars-cov-2 Ceased WO2022210830A1 (fr)

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