WO2023145874A1 - Nouvel anticorps monoclonal contre le coronavirus - Google Patents

Nouvel anticorps monoclonal contre le coronavirus Download PDF

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WO2023145874A1
WO2023145874A1 PCT/JP2023/002626 JP2023002626W WO2023145874A1 WO 2023145874 A1 WO2023145874 A1 WO 2023145874A1 JP 2023002626 W JP2023002626 W JP 2023002626W WO 2023145874 A1 WO2023145874 A1 WO 2023145874A1
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amino acid
acid sequence
seq
variable region
chain variable
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PCT/JP2023/002626
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Japanese (ja)
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諭志 永田
春彦 鎌田
知子 伊勢
健太郎 古川
竜也 高橋
貴雄 宍戸
圭太 深尾
舞 吉川
剛 大田
真裕 高山
陽司 津川
圭司 小倉
昂亮 前田
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国立研究開発法人医薬基盤・健康・栄養研究所
塩野義製薬株式会社
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Publication of WO2023145874A1 publication Critical patent/WO2023145874A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the present invention relates to a novel monoclonal antibody against the S2 subunit of the spike protein of SARS-CoV-2 and a pharmaceutical composition for preventing or treating coronavirus infection containing the antibody.
  • SARS-CoV-2 The novel coronavirus (SARS-CoV-2, sometimes referred to herein as "SARS-CoV2”), which was first reported in China at the end of 2019, is an RNA virus belonging to the family Coronaviridae of the order Nidoviridae. It is known that infection with this virus causes acute respiratory symptoms, etc., and if it worsens, it develops pneumonia, leading to death.
  • a combination of casilibimab and imdevimab hereinafter, "imdevimab” is sometimes referred to as "REGN10987"
  • Imdevimab is sometimes referred to as "REGN10987”
  • Administration brand name: Lonaprive
  • sotrovimab brand name: Zebudy
  • Casilibimab, imdevimab, and sotrovimab are all known to bind to the receptor binding domain (RBD) present on the S1 domain of the spike protein and exhibit in vitro antiviral activity against SARS-CoV-2 (non Patent Documents 1 and 2).
  • Non-Patent Document 3 As an antibody that binds to the S2 subunit of the spike protein of SARS-CoV-2 and exhibits antiviral activity in vivo, CV3-25, a monoclonal antibody against the S2 subunit, is described in Non-Patent Document 3. It has been shown to exhibit antiviral activity in vitro and to have a virus-reducing effect and an effect of delaying lethality due to SARS-CoV-2 virus infection in an infected mouse system in vivo. In addition, Non-Patent Document 5 indicates that the epitope of CV3-25 is amino acid residues 1149-1167 of the S2 subunit.
  • Non-Patent Document 4 the monoclonal antibody S2P6, which has an epitope at amino acid residues 1148-1156 of the S2 subunit of the spike protein of SARS-CoV-2, demonstrated in vitro antiviral activity and SARS-CoV-2-infected hamster system. It has been shown to have antiviral effects in In Non-Patent Document 5, hMab5.17, a monoclonal antibody having an epitope at amino acid residues 1161-1175 of the S2 subunit, showed in vitro antiviral activity, and also in the SARS-CoV-2 infected hamster system. It has been shown to have antiviral effects. However, the epitopes of these antibodies are distinctly different from those of the antibodies of the present invention.
  • Antibodies that bind to the S2 subunit of the spike protein of SARS-CoV-2 but do not exhibit antiviral activity in vitro have also been shown to reduce viral load, which is antiviral activity in SARS-CoV-2 infected individuals. There are no known cases of efficacy, suppression of mortality due to viral infection, or improvement of disease conditions such as weight loss due to viral infection.
  • the problem to be solved by the present invention is to provide a new pharmaceutical composition containing an antibody capable of treating or preventing coronavirus infection with a mechanism of action different from that of conventional antibodies against the spike protein of SARS-CoV-2. to provide.
  • the present inventors produced various antibodies that recognize the S2 subunit of the spike protein of SARS-CoV-2 as an antigen, and found the antibody of the present invention, thereby completing the present invention. reached.
  • the present invention relates to the following.
  • a pharmaceutical composition for the prevention or treatment of infectious diseases. (2) The pharmaceutical composition according to (1), wherein said antibody binds to one or more amino acid residues of the amino acid sequence of SEQ ID NO:48 or SEQ ID NO:49.
  • the antibody is combined with one or more amino acid residues of one or more peptides represented by any of SEQ ID NOS: 50 to 85 contained in the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 49;
  • the group wherein said antibody consists of L962A mutant, E988A mutant, Q992A mutant, L996A mutant, R1000A mutant, L1001A mutant, L1004A mutant, Y1007A mutant and I1018A mutant of SEQ ID NO:86;
  • the pharmaceutical composition of (1) which does not bind to the one or more mutants selected from.
  • the antibody is SARS-CoV-1, MERS, Human coronavirus HKU1, Human coronavirus OC43, Bat-CoV (RaTG13), Bat-HpCoV, Bat-HKU4, Bovine-L9, PCoV GX-P1E, Bat- HKU3-3, BtRs-CoV, Swine acute diarrhea syndrome coronavirus, Betacoronavirus HKU24, Rousettus bat coronavirus, Eidolon bat coronavirus/Kenya/KY24/2006, C haerephon bat coronavirus/Kenya/KY22/2006, Rodent coronavirus, BtVs-BetaCoV/SC2013, Rhinolophus bat coronavirus HKU2, Wigeon coronavirus HKU20, Porcine coronavirus HKU15, Munia coronavirus HKU13-3514 and Avian coronavirus IA1162/2020 binds to the S2 subunit of one or more viral strains selected
  • effector activity is one or more effector activities selected from ADCC activity, ADCP activity and CDC activity.
  • effector activity is Fc receptor dependent effector activity on effector cells.
  • a method of preventing or treating coronavirus infection characterized by: (2A) The preventive or therapeutic method according to (1A), wherein the antibody binds to one or more amino acid residues of the amino acid sequence of SEQ ID NO:48 or SEQ ID NO:49.
  • the antibody is combined with one or more amino acid residues of one or more peptides represented by any of SEQ ID NOs: 50 to 85 contained in the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 49;
  • (4A) the group wherein said antibody consists of L962A, E988A, Q992A, L996A, R1000A, L1001A, L1004A, Y1007A and I1018A variants of SEQ ID NO:86
  • the prophylactic or therapeutic method according to (1A) which does not bind to the one or more mutants selected from.
  • the antibody is SARS-CoV-1, MERS, Human coronavirus HKU1, Human coronavirus OC43, Bat-CoV (RaTG13), Bat-HpCoV, Bat-HKU4, Bovine-L9, PCoV GX-P1E, Bat- HKU3-3, BtRs-CoV, Swine acute diarrhea syndrome coronavirus, Betacoronavirus HKU24, Rousettus bat coronavirus, Eidolon bat coronavirus/Kenya/KY24/2006, Chaerephon bat coronavirus/Kenya/KY22/2006,Rodent coronavirus,BtVs-BetaCoV/SC2013, Rhinolophus bat coronavirus HKU2, Wigeon coronavirus HKU20, Porcine coronavirus HKU15, Munia coronavirus HKU13-3514 and Avian coronavirus IA1162/2020 binds to the S2 subunit of one or more viral strain
  • (1B) binds to one or more amino acid residues in the region from positions 960 to 1018 of the amino acid sequence of SEQ ID NO:86 and is mediated by the Fc domain of an antibody for use in the prevention or treatment of coronavirus infection A monoclonal antibody having effector activity.
  • (2B) The monoclonal antibody of (1B), which binds to one or more amino acid residues of the amino acid sequence of SEQ ID NO:48 or SEQ ID NO:49. (3B) binds to one or more amino acid residues of one or more peptides represented by any of SEQ ID NOs: 50 to 85 contained in the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 49, (1B ) or the monoclonal antibody according to (2B).
  • (4B) 1 selected from the group consisting of L962A mutant, E988A mutant, Q992A mutant, L996A mutant, R1000A mutant, L1001A mutant, L1004A mutant, Y1007A mutant and I1018A mutant of SEQ ID NO:86
  • the monoclonal antibody of (1B) which does not bind to one or more variants.
  • (6B) The monoclonal antibody of any of (1B)-(5B), wherein said antibody binds to the S2 subunit of the spike protein of a related coronavirus genus other than SARS-CoV-2.
  • (13) 1 selected from the group consisting of L962A mutant, E988A mutant, Q992A mutant, L996A mutant, R1000A mutant, L1001A mutant, L1004A mutant, Y1007A mutant and I1018A mutant of SEQ ID NO:86
  • the monoclonal antibody of (10) which does not bind to one or more variants.
  • CDR1 consisting of an amino acid sequence in which 1 to 2 amino acid residues may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 2, 1 to 2 amino acids in the amino acid sequence of SEQ ID NO: 4 and CDR2 consisting of an amino acid sequence in which 1 to 2 amino acid residues may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 3
  • a heavy chain variable region comprising a CDR3 consisting of an amino acid sequence in which residues may be deleted, substituted, inserted and/or added, and deletion or substitution of 1 to 2 amino acid residues in the amino acid sequence of SEQ ID NO: 6
  • CDR1 consisting of an amino acid sequence that may be inserted and/or added
  • CDR2 consisting of an amino acid sequence in which 1 to 2 amino acid residues may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 7 and 1 to 2 amino acids in the amino acid sequence of SEQ ID NO: 8
  • a heavy chain variable region comprising an amino acid sequence, wherein an antibody having the amino acid sequence of SEQ ID NO: 43 as a heavy chain exhibits an activity equivalent to the binding activity to the S2 subunit of the spike protein of SARS-CoV-2. and an amino acid sequence in which one to several amino acid residues may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 47, and an antibody having the amino acid sequence of SEQ ID NO: 47 as a light chain
  • An amino acid sequence in which one to several amino acid residues may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 88, having the amino acid sequence of SEQ ID NO: 88 as a heavy chain 1 to several amino acid residues in the heavy chain variable region consisting of the amino acid sequence and the amino acid sequence of SEQ ID NO:
  • a heavy chain variable region comprising an amino acid sequence, wherein an antibody having the amino acid sequence of SEQ ID NO: 93 as a heavy chain exhibits an activity equivalent to the binding activity to the S2 subunit of the spike protein of SARS-CoV-2. and an amino acid sequence in which one to several amino acid residues may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 47, and an antibody having the amino acid sequence of SEQ ID NO: 47 as a light chain
  • An amino acid sequence in which one to several amino acid residues may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 94, having the amino acid sequence of SEQ ID NO: 94 as a heavy chain 1 to several amino acid residues in the heavy chain variable region consisting of the amino acid sequence and the amino acid sequence of SEQ ID NO
  • a heavy chain variable region comprising an amino acid sequence, wherein an antibody having the amino acid sequence of SEQ ID NO: 32 as a heavy chain exhibits an activity equivalent to the binding activity to the S2 subunit of the SARS-CoV-2 spike protein. and an amino acid sequence in which one to several amino acid residues may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO:36, and an antibody having the amino acid sequence of SEQ ID NO:36 as a light chain
  • a light chain variable region consisting of the amino acid sequence that exhibits an activity equivalent to the binding activity to the S2 subunit of the spike protein of SARS-CoV-2;
  • the monoclonal antibody according to (15) or (19) comprising (25) the antibody is any of the L962A, E988A, Q992A, L996A, R1000A, L1001A, L1004A, Y1007A and I1018A variants of SEQ ID NO:86;
  • the pharmaceutical composition according to (1) which does not bind.
  • the antibody is SARS-CoV-1, MERS, Human coronavirus HKU1, Human coronavirus OC43, Bat-CoV (RaTG13), Bat-HpCoV, Bat-HKU4, Bovine-L9, PCoV GX-P1E, Bat- HKU3-3, BtRs-CoV, Swine acute diarrhea syndrome coronavirus, Betacoronavirus HKU24, Rousettus bat coronavirus, Eidolon bat coronavirus/Kenya/KY24/2006, Chaerephon bat coronavirus/Kenya/KY22/2006,Rodent coronavirus,BtVs-BetaCoV/SC2013, Any of Rhinolophus bat coronavirus HKU2, Wigeon coronavirus HKU20, Porcine coronavirus HKU15, Munia coronavirus HKU13-3514 and Avian coronavirus IA1162/2020 (1), which also binds to the S2 subunit of
  • the antibody is SARS-CoV-1, MERS, Human coronavirus HKU1, Human coronavirus OC43, Bat-CoV (RaTG13), Bat-HpCoV, Bat-HKU4, Bovine-L9, PCoV GX-P1E, Bat- HKU3-3, BtRs-CoV, Swine acute diarrhea syndrome coronavirus, Betacoronavirus HKU24, Rousettus bat coronavirus, Eidolon bat coronavirus/Kenya/KY24/2006, Chaerephon bat coronavirus/Kenya/KY22/2006,Rodent coronavirus,BtVs-BetaCoV/SC2013, Any of Rhinolophus bat coronavirus HKU2, Wigeon coronavirus HKU20, Porcine coronavirus HKU15, Munia coronavirus HKU13-3514 and Avian coronavirus IA1162/2020.
  • a pharmaceutical composition containing the antibody of the present invention is useful as a drug for treating or preventing coronavirus infections.
  • FIG. 4 is a graph showing the reactivity of CV804 antibody to SARS-CoV2 S2 protein (monomer), S2 protein (trimer), and full-length spike protein (trimer).
  • the name of the antibody is shown in the title of the figure, the vertical axis shows the absorbance indicating the degree of reaction with each protein, and the horizontal axis shows the antigen concentration (ng/ml).
  • FIG. 4 is a graph showing the reactivity of CV801 antibody to SARS-CoV2 S2 protein (monomer), S2 protein (trimer), and full-length spike protein (trimer).
  • FIG. 4 is a graph showing the reactivity of CV820 antibody to SARS-CoV2 S2 protein (monomer), S2 protein (trimer), and full-length spike protein (trimer).
  • the name of the antibody is shown in the title of the figure, the vertical axis shows the absorbance indicating the degree of reaction with each protein, and the horizontal axis shows the antigen concentration (ng/ml).
  • FIG. 4 is a graph showing the reactivity of CV820 antibody to SARS-CoV2 S2 protein (monomer), S2 protein (trimer), and full-length spike protein (trimer).
  • the name of the antibody is shown in the title of the figure, the vertical axis shows the absorbance indicating the degree of reaction with each protein, and the horizontal axis shows the antigen concentration (ng/ml).
  • FIG. 4 is a graph showing the reactivity of CV820 antibody to SARS-CoV2 S2 protein (monomer), S2 protein (trimer), and full-length spike protein (trimer
  • FIG. 4 is a graph showing the reactivity of CV925 antibody to SARS-CoV2 S2 protein (monomer), S2 protein (trimer), and full-length spike protein (trimer).
  • the name of the antibody is shown in the title of the figure, the vertical axis shows the absorbance indicating the degree of reaction with each protein, and the horizontal axis shows the antigen concentration (ng/ml).
  • FIG. 4 is a graph showing the reactivity of CV1117 antibody to SARS-CoV2 S2 protein (monomer), S2 protein (trimer), and full-length spike protein (trimer).
  • FIG. 4 shows the reactivity of each antibody to SARS-CoV2 spike protein and S2 protein-expressing cells.
  • the vertical axis indicates the intensity of the fluorescent signal indicating the amount of antigen expression
  • the horizontal axis indicates the intensity of the fluorescent signal indicating the amount of antibody binding.
  • FIG. 2 shows the ADCC activity of each antibody.
  • the vertical axis indicates fluorescence signals indicating cytotoxic activity
  • the horizontal axis indicates antibody concentration.
  • FIG. 2 shows the ADCC activity of each antibody.
  • the vertical axis indicates fluorescence signals indicating cytotoxic activity, and the horizontal axis indicates antibody concentration.
  • CV804 (WT) in FIG. 8 means CV804, and "CV804 (LALA)” means CV804-LALA.
  • Fig. 2 shows an in vitro viral infection inhibition experiment of CV804 antibody.
  • the vertical axis indicates cell viability (%), and the horizontal axis indicates antibody concentration (nM).
  • Fig. 3 shows experimental results showing the in vivo therapeutic effect of the CV804 antibody.
  • the vertical axis indicates body weight change rate (%), and the horizontal axis indicates the number of days after infection. Note that "CV804 (WT)" in FIG.
  • FIG. 10 means CV804, and "CV804 (LALA)” means CV804-LALA.
  • Fig. 3 shows experimental results showing the in vivo therapeutic effect of the CV804 antibody. The vertical axis indicates the mouse survival rate (%), and the horizontal axis indicates the number of days after infection.
  • CV804 (WT) in FIG. 11 means CV804, and "CV804 (LALA)” means CV804-LALA.
  • the antibody of the present invention binds to amino acid residues in the region from positions 960 to 1018 of the amino acid sequence shown in the SARS-CoV-2 spike protein (Genbank ACC No. QHD43416.1 (SEQ ID NO: 86)).
  • Antibodies of the invention also bind to the relevant region in mutant strains of SARS-CoV-2 (eg, the ⁇ (Omicron) strain).
  • the portions corresponding to positions 960 to 1018 in the S2 subunit of various coronaviruses, not limited to SARS-CoV-2 mutant strains, have high homology among different coronavirus species.
  • the antibodies of the present invention bind to a variety of coronaviruses, including coronaviruses currently only hosted in wild animals.
  • Antibodies of the present invention are expected to exert therapeutic and preventive effects on various coronavirus infections. Even if coronaviruses whose hosts are only animals acquire the ability to infect humans, the antibodies of the present invention are expected to exert therapeutic and preventive effects on various coronavirus infections in humans. .
  • an antibody binds to one or more amino acid residues in the region from positions 960 to 1018 of the amino acid sequence of SEQ ID NO: 86" can be determined by known methods. .
  • Known methods include mass spectrometry (e.g., hydrogen/deuterium exchange mass spectrometry (HDX-MS) as described in Example 11), amino acid mutagenesis (e.g., alanine scanning method as described in Example 16). , crystallography, peptide competition, antibody competition, cryo-electron microscopy, and other methods well known to those skilled in the art.
  • an antibody “binds to one or more amino acid residues in the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 49” can be determined by known methods.
  • Known methods include mass spectrometry (e.g., hydrogen/deuterium exchange mass spectrometry (HDX-MS) as described in Example 11), amino acid mutagenesis (e.g., alanine scanning method as described in Example 16). , crystallography, peptide competition, and other methods well known to those skilled in the art.
  • the antibody of the present invention is an antibody determined by HDX-MS to bind to one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:48 or SEQ ID NO:49.
  • an antibody is defined as "one or more amino acid residues of one or more peptides represented by any of SEQ ID NOS: 50 to 85 contained in the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 49 and Whether or not it "binds" can be measured by a known method.
  • Known methods include mass spectrometry (e.g., hydrogen/deuterium exchange mass spectrometry (HDX-MS) as described in Example 11), amino acid mutagenesis (e.g., alanine scanning method as described in Example 16). , crystallography, peptide competition, and other methods well known to those skilled in the art.
  • the antibody of the present invention is an antibody that is determined by HDX-MS as binding to one or more peptides set forth in any of SEQ ID NOs:50-85, more preferably SEQ ID NOs:50-85. Antibodies measured by HDX-MS as binding to any of the indicated peptides.
  • L962A mutant of SEQ ID NO: 86 the 962nd wild-type SARS-CoV2 spike protein (Genbank ACC No. QHD43416.1 (SEQ ID NO: 86)) means an amino acid variant in which the amino acid residue of is substituted from L to A, and the same applies to other variants.
  • “L962A mutant” may be simply referred to as “L962A” in the examples.
  • the antibody is defined as "the group consisting of the L962A variant, the E988A variant, the Q992A variant, the L996A variant, the R1000A variant, the L1001A variant, the L1004A variant, the Y1007A variant and the I1018A variant of SEQ ID NO: 86.
  • "Does not bind to one or more mutants selected from” means that the antibody does not bind to any one mutant selected from the above group, or 2 arbitrarily selected from the above group It means that it either does not bind to the above mutants. Whether an antibody binds to each mutant can be determined by a known method.
  • the antibody of the invention is an antibody that binds to SEQ ID NO:86 and does not bind to any of the above variants.
  • an epitope refers to a portion on an antigen to which an antibody targeting the antigen binds.
  • An epitope is a specific portion of an antigen, which, when the antigen is a protein, can be expressed as specific amino acid residues, or ranges thereof, in the amino acid sequence of the antigen that directly contact an antibody. That is, the epitope does not have to be contiguous amino acid residues as long as it is composed of amino acid residues that contact the antibody, and amino acid residues at different locations may form an epitope sterically.
  • the antibody of the present invention does not have SARS-CoV-2 virus infection-blocking activity in vitro.
  • Viral infection inhibitory activity in vitro is an index of viral infection such as viral invasion into cells, viral proliferation, and cell death due to viral proliferation by infecting SARS-CoV-2-susceptible cultured cells with the virus. means that it has no inhibitory activity against Specifically, it can be measured by the method described in Examples below. In the present specification, "having no inhibitory activity” does not necessarily mean that the inhibitory activity is "0 (zero)". can also be interpreted as not superior.
  • the monoclonal antibody against the SARS-CoV-2 spike protein of the present invention is an antibody that binds to the epitope located at positions 960 to 1018 of the SARS-CoV-2 spike protein, it is a human-derived antibody, a mouse-derived antibody, Antibodies may be rat-, rabbit-, or goat-derived antibodies, and may be native, chimerized, humanized, or fully human antibodies. Preferred are humanized or fully human antibodies.
  • the antibody of the present invention is characterized by having effector activity mediated by the Fc fragment.
  • Effector activities mediated by Fc fragments include effector activities mediated by Fc receptors present on the surface of effector cells such as natural killer cells and macrophages, and effector activities not mediated by Fc receptors.
  • Effector activities mediated by Fc receptors include ADCC activity, ADCP activity, and effector activities not mediated by Fc receptors include CDC activity.
  • a preferred effector activity of the antibodies of the invention is ADCC activity.
  • the antibodies of the present invention are preferably antibody isotypes having an antibody Fc domain capable of inducing ADCC activity.
  • ADCC Antibody-dependent cell-mediated cytotoxicity; antibody-dependent cytotoxicity
  • the antibody bound to the antigen on the cell surface, the Fc receptor present on the Fc region and effector cell surface of the antibody and means the activity of recruiting and activating effector cells and injuring target cells through the binding of Effector cells include natural killer cells, activated macrophages, and the like.
  • the antibody of the present invention is preferably an antibody that has ADCC activity against cells expressing the spike protein of coronavirus (eg, SARS-CoV-2) because it can eliminate virus-infected cells. Whether or not the antibody of the present invention has such ADCC activity can be determined, for example, by the method described in Example 8 of the present specification.
  • ADCP antibody-dependent cell-mediated phagocytosis; antibody-dependent cell phagocytosis
  • ADCP activity means that an antibody recruits macrophages, etc. to its vicinity, and the recruited macrophages for cells or biomolecules present in the vicinity of the antibody It is exemplified as an activity that exerts phagocytosis such as
  • the method for measuring ADCP activity is not particularly limited, for example, Journal of Bioscience and Bioengineering. VOL. 111 no. 4, 391-396, 2011, etc. can be measured by appropriately modifying the method described in known literature.
  • CDC activity means that antibodies bind to surface antigens of target cells (cells infected with bacteria or viruses, etc.) and are complemented by complement proteins bound to these antibodies. Refers to the activity in which a somatic pathway is activated and the target cell is lysed. CDC activity can be measured by a known method. For example, Current Protocols in Immunology, Chapter 7. It can be measured by the method described in Immunological studies in humans, edited by Coligan et al. (1993).
  • the antibody of the present invention can be produced according to a known antibody production method using an antigen protein containing the full-length coronavirus spike protein, the S2 subunit protein, or a partial protein thereof.
  • the antibody of the present invention preferably binds to an epitope located at positions 960-1018 of the coronavirus spike protein and has effector activity mediated by the Fc domain, such as ADCC activity.
  • These antigens can be prepared by known protein expression and purification methods.
  • Screening for hybridoma clones producing monoclonal antibodies is carried out by culturing hybridoma cells, for example, in a microtiter plate, collecting antibodies in the culture supernatant of wells in which growth is observed, and subjecting the obtained antibodies to the aforementioned mouse immunization.
  • Reactivity to the antigen of the present invention used in sensitization is measured by assay methods such as RIA, ELISA, FACS, etc., and clones producing monoclonal antibodies that exhibit specific binding to the antigen or hapten are selected. It can be carried out.
  • a method in which the antigen is immobilized and the antibody in the culture supernatant that binds thereto is detected with a secondary antibody labeled with a radioactive substance, a fluorescent substance, an enzyme, or the like.
  • a hybridoma culture supernatant is added to the cells, followed by reaction with a fluorescently-labeled secondary antibody.
  • a monoclonal antibody that can bind to the antigen of the present invention on the cell membrane can be detected by measuring fluorescence intensity.
  • Monoclonal antibodies can be produced from selected hybridomas by culturing the hybridomas in vitro or in ascitic fluid of mice, rats, guinea pigs, hamsters or rabbits, preferably mice or rats, more preferably mice. It can be performed by isolating from the culture supernatant obtained from the culture, or the ascites fluid of mammals.
  • hybridomas are grown, maintained and preserved according to various conditions such as the characteristics of the cell type to be cultured, the purpose of the test research and the culture method, and monoclonal antibodies are produced in the culture supernatant. Any nutrient medium derived from a known nutrient medium or a known basal medium, such as that used for the method, can be used.
  • basal medium examples include D-MEM medium, RPMI1640 medium, IMDM medium, ASF104 medium and the like. can contain
  • Isolation and purification of monoclonal antibodies can be carried out by subjecting the above-mentioned culture supernatant or ascites to saturated ammonium sulfate, ion exchange chromatography (DEAE or DE52, etc.), affinity column chromatography such as anti-immunoglobulin column or protein A column, etc. It can be done by
  • a recombinant antibody produced by cloning an antibody gene from an antibody-producing cell such as a hybridoma, inserting it into an appropriate vector, introducing it into a host cell, and producing it using genetic recombination technology is used.
  • an antibody-producing cell such as a hybridoma
  • an appropriate vector e.g, Carl et al., THERAPEUTIC MONOCLONAL ANTIBODIES, 1990.
  • the antibody variable region is encoded from a hybridoma that produces the antibody of interest or an antibody-producing immune cell such as a sensitized lymphocyte that has been immortalized by an oncogene or the like. Isolate the mRNA. Isolation of mRNA is carried out by a known method, for example, guanidine ultracentrifugation (Chirgwin, J.M. et al., Biochemistry (1979) 18, 5294-5299), etc., to prepare total RNA and use mRNA Purification Kit (manufactured by Pharmacia), etc. Prepare mRNA using.
  • cDNA of the antibody V region is synthesized using reverse transcriptase.
  • cDNA synthesis can be performed using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit or the like.
  • AMV Reverse Transcriptase First-strand cDNA Synthesis Kit for synthesis and amplification of cDNA, 5'-Ampli FINDER RACE Kit (manufactured by Clontech) and 5'-RACE method using PCR (Frohman, MA et al., Proc. Natl. Acad. Sci. USA 1988, Vol. 85, p. 8998, etc.) can be used.
  • a target DNA is purified from the resulting PCR product and ligated with vector DNA to prepare a recombinant vector.
  • the base sequence of the DNA of interest is confirmed by a known method such as the deoxy method.
  • the DNA encoding the variable region (V region) of the antibody of interest is obtained, it is ligated with the DNA encoding the desired antibody constant region (C region) and incorporated into an expression vector.
  • the DNA encoding the antibody V region may be incorporated into an expression vector containing the antibody C region DNA.
  • antibody genes are incorporated into expression vectors such that they are expressed under the control of expression control regions, eg, enhancers/promoters. This expression vector can then be used to transform a host cell to express the antibody.
  • Antibody gene expression may be carried out by separately inserting antibody heavy chains (H chains) or light chains (L chains) into expression vectors and co-transforming the host, or by co-transforming the host with DNA encoding the H and L chains. may be incorporated into a single expression vector to transform the host (see WO94/11523).
  • H chains antibody heavy chains
  • L chains light chains
  • phage display technology can also be used for methods of producing the antibodies of the present invention other than those described above.
  • screening from an antibody gene library prepared by a known method using B lymphocytes of humans or animals (e.g., rabbits, mice, rats, hamsters, etc.), or germline sequences of humans or animals and the modified and completely synthesized antibody gene library is displayed on the cell surface of bacteriophage, Escherichia coli, yeast, animal cells, etc., or on ribosomes.
  • the forms of antibodies to be displayed on the cell surface include IgG molecules, IgM molecules, Fab fragments, single-chain Fv (scFv) fragments, and the like.
  • the antibody fragment gene thus obtained can be recombined with the corresponding region of the IgG antibody gene by a known method to obtain the antibody gene. Then, the gene thus obtained can be incorporated into an appropriate vector, introduced into a host, and an antibody produced using gene recombination technology (for example, Carl et al., THERAPEUTIC MONOCLONAL ANTIBODYS, 1990). published in 2009).
  • Antibodies of the present invention include antibodies artificially modified for the purpose of reducing heteroantigenicity against humans, such as chimerized antibodies and humanized antibodies.
  • the antibodies of the present invention may be conjugated antibodies bound to various molecules such as polyethylene glycol (PEG), radioactive substances, toxins and sugar chains.
  • PEG polyethylene glycol
  • Such a conjugated antibody can be obtained by chemically modifying the obtained antibody. Methods for modifying antibodies have already been established in this field.
  • Antibodies in the present invention also include these conjugated antibodies.
  • Antibodies of the present invention include antibodies in which an N-glycoside-linked sugar chain is bound to the Fc region of the antibody and fucose is not bound to the N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain.
  • Examples of antibodies in which an N-glycoside-linked sugar chain is bound to the Fc region of the antibody and fucose is not bound to the N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain include, for example, ⁇ 1,6-fucosyltransferase gene and antibodies produced using CHO cells deficient in (WO2005/035586, WO02/31140).
  • the antibody of the present invention in which an N-glycoside-linked sugar chain is bound to the Fc region of the antibody and the N-acetylglucosamine at the reducing terminal of the N-glycoside-linked sugar chain is not bound to fucose, has high ADCC activity.
  • the antibodies of the present invention may also be fused to other proteins at their N-terminus or C-terminus (Clinical Cancer Research, 2004, 10, 1274-1281).
  • a protein to be fused can be appropriately selected by those skilled in the art.
  • severed pieces when there is a description of "one to several pieces” means any one of 2 to 10 pieces. Preferably 5, 6, 7, 8, 9 or 10, more preferably 3 or 4, still more preferably 2.
  • the antibody of the present invention has the effect of suppressing the onset of infections caused by SARS-CoV-2. Specifically, it means the antiviral effect in individuals infected with SARS-CoV-2, specifically, the effect of reducing the amount of virus in infected individuals, the effect of suppressing mortality due to virus infection in infected individuals, and the effect of suppressing infection It means having an effect of improving disease conditions such as weight loss caused by viral infection in individuals.
  • coronavirus infection in the "pharmaceutical composition for preventing or treating coronavirus infection” of the present invention can include all infectious diseases caused by various coronaviruses to which the antibody of the present invention binds. Specific examples include infectious diseases caused by coronaviruses such as SARS-CoV-1, MERS, and Bat-CoV (RaTG13). More preferred are infections caused by SARS-related coronaviruses such as SARS-CoV-1. Infectious diseases caused by SARS-CoV-2 are more preferable.
  • the pharmaceutical composition of the present invention may also be used for suppressing the onset of infections caused by SARS-CoV-2.
  • Effective doses are selected from the range of 0.01 mg to 100 mg per kg body weight per dose.
  • a preferred effective dose is 50 mg per kg body weight.
  • pharmaceutical compositions containing antibodies of the present invention are not limited to these dosages.
  • the pharmaceutical composition of the present invention may contain both pharmaceutically acceptable carriers and additives depending on the route of administration. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, sodium alginate, water-soluble dextran, pectin, methylcellulose, ethylcellulose, casein, diglycerin, propylene glycol.
  • Additives to be used are appropriately selected from the above or in combination according to the dosage form, but are not limited to these.
  • the administration period of the pharmaceutical composition of the present invention can be appropriately selected according to the patient's age, symptoms, and the like. Preferably, it is desirable to administer once or twice or more early (within 0 to 2 days) after infection with coronavirus is found. Administration routes include intravenous injection, subcutaneous injection, and the like.
  • Example 1 Production of anti-spike protein antibody-producing mouse hybridoma Full-length spike protein derived from SARS-CoV2 (Genbank ACC No. QHD43416.1) or S2 subunit (Genbank ACC No. 686 of spike protein described in QHD43416.1)
  • SARS-CoV2 Genbank ACC No. QHD43416.1
  • S2 subunit Genebank ACC No. 686 of spike protein described in QHD43416.1
  • A/J Jms Slc female mice were immunized with an expression vector DNA or protein encoding (position-1213) as antigen. The immunization was repeated 3 or more times at 2-week intervals, and 2-3 weeks after the final immunization, booster immunization was performed with trimerized spike protein or S2 subunit protein.
  • spleen cells and mouse myeloma cells were fused using the PEG method and electrofusion method, and selected in a medium containing hypoxanthine, aminopterin and thymidine.
  • Anti-spike protein antibodies were selected from this culture supernatant by the following method.
  • the culture supernatant was reacted with spike protein-expressing HEK293 cells, and clones that specifically bind to the spike protein expressed on the cell membrane were selected.
  • SARS-CoV2 SARS-CoV2
  • SARS-CoV-1 Genbank ACC No. P5959594
  • Middle East respiratory syndrome-related coronavirus Genbank ACC No.
  • YP_009047204.1 Bat-CoV (Genbank ACC No. QHR63300.1 ) to HEK293 cells expressing the spike protein, clones with high cross-reactivity were selected. Clones selected using the above criteria were cloned to establish hybridomas.
  • Example 2 Full length of SARS-CoV2 spike protein and reactivity of antibody to S2 subunit (S2 protein) of spike protein , CV925 and CV1117) were added and allowed to react overnight at 4°C. After washing three times with ELISA washing buffer, S2 protein (Acro BIOSYSTEMS, S2N-C52H5), trimerized S2 protein, and full-length spike protein were added and allowed to react at room temperature for 1 hour. After washing three times with an ELISA washing buffer, an anti-histidine tag antibody was added and allowed to react at room temperature for 1 hour. After washing three times with an ELISA washing buffer, an ALP-labeled anti-human IgG antibody was added and allowed to react at room temperature for 1 hour.
  • S2 protein Acro BIOSYSTEMS, S2N-C52H5
  • trimerized S2 protein trimerized S2 protein
  • full-length spike protein were added and allowed to react at room temperature for 1 hour.
  • an anti-histidine tag antibody was added and allowed to react at room temperature
  • Example 3 Reactivity to SARS-CoV2 full-length spike protein and S2 protein-expressing cells HEK293 cells transiently expressing the full-length SARS-CoV2 spike protein and the S2 subunit and the five types of mouse antibodies obtained The reaction was allowed to proceed at 4°C for 30 minutes. After washing once with FACS buffer, a PE-labeled anti-mouse antibody was added and allowed to react at 4°C for 30 minutes. Thereafter, the cells were washed once with FACS buffer, resuspended in FACS buffer, and the antibody-reacted cells were analyzed by FACS (fluorescence activated cell sorting). As shown in FIG. 6, all antibodies reacted with the S2 subunit (S2 protein) of the spike protein expressed on the cell surface, and also reacted with the full-length spike protein.
  • FACS fluorescence activated cell sorting
  • mice antibodies shown in Table 1 were analyzed for the amino acid sequences of the antibody light chain variable region and heavy chain variable region from hybridoma cells according to a conventional method.
  • Example 5 Evaluation of binding to spike protein-expressing cells of related coronaviruses SARS-CoV2 (Genbank ACC No. QHD43416.1) and SARS-CoV1 (Genbank ACC No. P59594), Middle East respiratory syndrome-related coronavirus (MERS, Genbank ACC No. YP_009047204.1), Human coronavirus 229E (Genbank ACC No. NP_073551.1), Human coronavirus HKU1 (Genbank ACC No. YP_173238.1), Human coronavirus NL63 (Genbank ACC No. YP_003767.1), Bat- CoV RaTG13 (Genbank ACC No.
  • AVM41569.1 Spike protein expression plasmid vectors were prepared, and HEK2 transfected into 93 cells conducted an injection. The transfected cell group and the obtained mouse antibody were each allowed to react at 4°C for 30 minutes. After washing once with FACS buffer, a PE-labeled anti-mouse antibody was added and allowed to react at 4°C for 30 minutes. Thereafter, the cells were washed once with FACS buffer, resuspended in FACS buffer, and the antibody-reacted cells were analyzed by FACS (fluorescence activated cell sorting). Since the spike protein of Human coronavirus OC43 (YP_009555241.1) could not be expressed in cells, antibody reactivity was assessed by ELISA using recombinant protein.
  • ELISA a histidine-tagged spike protein S2 subunit recombinant protein of Human coronavirus OC43 was added to an anti-histidine tag antibody-immobilized plate, and reacted overnight at 4°C. After washing three times with an ELISA washing buffer, each anti-S2 antibody was added and allowed to react at room temperature for 1 hour. After washing three times with an ELISA washing buffer, an ALP-labeled anti-mouse IgG antibody was added and allowed to react at room temperature for 1 hour. After washing four times with an ELISA washing solution, a P-nitrophenyl phosphate solution was added to develop color, and absorbance at 405 nm was measured. Table 2 shows the presence or absence of binding to each virus strain.
  • CV804 responded to over 70% different spike proteins out of 17 different viral spikes.
  • ADCC Antibody-dependent cell-mediated cytotoxicity activity evaluation against spike-expressing cells HEK293 cells expressing spike protein on the cell membrane as target cells were cultured overnight and allowed to adhere.
  • Mouse antibodies (CV801, CV804, CV804-LALA (LALA variants, i.e. L241A and L242A mutations in the Fc amino acid sequence), CV820, CV925, CV1117) obtained against the cells and mFcgRIV, an effector expressing NFAT luciferase reporter Cells (Promega) were added and reacted for 6 hours in a 37°C, 5% CO2 incubator. After that, a coloring reagent was added and allowed to react for 5 minutes, after which fluorescence signals were detected.
  • Example 7 Reactivity to SARS-CoV2 spike protein mutant-expressing cells SARS-CoV2 spike proteins shown in Table 4, specifically SARS-CoV2 wild type (Genbank ACC No. QHD43416.1), SARS-CoV2 Spike protein mutants ⁇ (alpha) strain (GISAID ID: EPI_ISL_768526) and ⁇ (gamma) strain (GISAID ID: EPI_ISL_833366), ⁇ (Omicron) strain (GISAID ID: EPI_ISL_6640917) and each SARS- The following experiment was performed for the purpose of confirming the reactivity of the antibody of the present invention with the amino acid mutants of the CoV2 spike protein.
  • the cells expressing each full-length spike protein shown in Table 4 and the obtained mouse antibodies shown in Table 4 were each reacted at 4° C. for 30 minutes.
  • the notation of the amino acid mutant of the SARS-CoV2 spike protein in Table 4 is, for example, L452R, the 452nd amino acid residue of the wild-type SARS-CoV2 spike protein (Genbank ACC No. QHD43416.1) from L It means that it is mutated to R, and the same applies to other mutants.
  • Amino acid numbers 686 and above are the extracellular region of the S2 subunit, a mutation that has been detected in any of the natural SARS-CoV2 spike protein mutants.
  • Example 8 Cell Infection Inhibition Experiment in Vitro
  • the virus strain used was hCoV-19/Japan/TY/WK-521/2020 (2.32 ⁇ 10 6 TCID50/mL), and VeroE6/TMPRSS2 cells were used as infected cells.
  • the medium used was a Minimum Essential Medium supplemented with 2% FBS and 1 mg/mL Geneticin, and the dilution of the antibody was 500 nM for CV804 and 18.5 nM for REGN10987, each with a maximum concentration of 10 points at a 3-fold common ratio. and the virus was adjusted to 450 TCID50/well.
  • the CV804 antibody of the invention and REGN10987 were measured in this experiment.
  • Equal amounts of the prepared antibody dilution solution and virus solution were mixed and allowed to react at room temperature for 1 hour.
  • the virus-antibody mixture was added to 15000 cells/well of VeroE6/TMPRSS2 cells and cultured at 37°C under 5% CO2 conditions. After 72 hours, CellTiter Glo 2.0 was added and the fluorescence intensity was measured to detect viable cells, and the antibody concentration that inhibited cell death by 50% was defined as IC50.
  • REGN10987 exhibited strong in vitro viral infection-inhibitory activity
  • the CV804 antibody of the present invention did not exhibit any in vitro viral infection-inhibitory activity up to a high concentration (500 nM).
  • Example 9 Verification test of therapeutic effect in aged mice (1) Materials and methods (1.1) Antibody administration After transfection, the culture supernatant was prepared by protein G purification and gel filtration purification. C1.18.4 (Isotype control) used was purchased from Bio X cell. These antibodies were prepared using Dulbecco's Phosphate-buffered Saline (DPBS). The dose volume was 0.15 mL per mouse. (1.2) Virus The hCoV-19/Japan/TY7-501/2021 strain of SARS-CoV-2 was obtained from the National Institute of Infectious Diseases. (1.3) Animals Specific pathogen-free female BALB/c mice (CLEA Japan, Inc.) aged approximately 45 weeks were used in this study.
  • DPBS Dulbecco's Phosphate-buffered Saline
  • mice were promptly euthanized according to humane endpoints and considered dead in survival analyses, if their body weight decreased by less than 80% compared to pre-virus infection.
  • mice were anesthetized by intramuscular injection of 100 ⁇ L of anesthetic solution containing 0.03 mg/mL medetomidine hydrochloride, 0.4 mg/mL midazolam, 0.5 mg/mL butorphanol tartrate in saline.
  • anesthetic solution containing 0.03 mg/mL medetomidine hydrochloride, 0.4 mg/mL midazolam, 0.5 mg/mL butorphanol tartrate in saline.
  • Verification of Antiviral Effect in Mouse Model Mice were nasally inoculated with 50 ⁇ L of hCoV-19/Japan/TY7-501/2021 (1.00 ⁇ 10 5 TCID 50 ) under anesthesia.
  • mice treated with CV804-LALA and REGN10987 administration survived 40% and 20%, respectively, but no significant survival extension was observed compared to C1.18.4-administered mice (Fig. 10 and Figure 11).
  • SARS-CoV-2 infection mouse model may be useful for estimating the effects of CV804 administration in severe cases.
  • CV804-LALA inserted with the LALA mutation that attenuates the ADCC activity of CV804 the effect of prolonging survival was attenuated compared to CV804. suggested to have been fulfilled.
  • Example 10 Humanization of Antibody CV804 was humanized by the following method. Numbering according to Kabat definition using antibody sequence analysis software abYsis (Kabat numbering (Wu, TT and Kabat, EA, J Exp. Med. Aug1; 132 (2): 211-50.
  • the maximum signal value of mCV804 (hIgG1 chimera), a mouse-human chimeric antibody whose heavy and light chain variable regions are CV804 and whose constant region is human IgG1, is 100%, and the signal value in the absence of antibody is 0.
  • the strength of binding activity was evaluated by calculating the antibody concentration that gives 50% of the signal as the EC50 value.As a result, 8 types of humanized monoclonal antibodies with binding activity equal to or higher than that of the mouse human chimeric antibody were evaluated.
  • the SEQ ID NOs of the light chain variable regions, each CDR, the heavy chain variable region, and the amino acid sequences of each CDR of these antibodies are shown in Table 5, and the designed humanized light chain variable regions and heavy chain variable regions The amino acid sequences are shown in Tables 6 and 7, respectively, and the binding activities of these antibodies are shown in Table 8.
  • Example 11 Epitope mapping of CV804 antibody binding to SARS-CoV-2 spike protein by hydrogen/deuterium exchange method .QHD43416.1), hydrogen/deuterium exchange epitope mapping using mass spectrometry was performed.
  • a general description of hydrogen/deuterium exchange methods can be found, for example, in Ehring (1999) Analytical Biochemistry 267(2):252-259; and Engen and Smith (2001) Anal. Chem. 73:256A-265A.
  • HDX-PAL system Leap Technologies Inc
  • Enzyme Pepsin Column Waters
  • Hypersil Gold column Thermo Scientific
  • Acclaim PepMap300 C18 T hermo Scientific
  • hydrogen/deuterium exchange mass spectrometry experiments were performed using a system consisting of Orbitrap Eclipses (ThermoFisher) for mass measurement of digested peptides.
  • a recombinant SARS-CoV-2 spike protein expressed in HEK293 cells was used as the target protein for hydrogen/deuterium exchange epitope mapping.
  • the recombinant protein used contains the SARS-CoV-2 spike protein ectodomain (positions 1-1213 of the S protein described in Genbank ACC No. QHD43416.1), positions 986 (K) and 987 (V ) is replaced with proline (P).
  • As an antibody used for hydrogen/deuterium exchange epitope mapping hCV804-8, which is a humanized antibody of CV804 antibody described in Example 10, was used.
  • the deuterated buffer was prepared at pH 7.4 with 10 mmol/L PBS buffer adjusted with D2O. 1.45 ⁇ mol/L of the target protein was reacted at 37° C. for 30 minutes in the presence or absence of 1.45 ⁇ mol/L of hCV804-8 antibody.
  • Deuterium labeling initiation, reaction time control, quench reaction, injection into the UPLC system and digestion time control were performed fully automatically by the HDX-PAL system.
  • Deuterium labeling was initiated by first diluting the target protein/antibody complex or target protein 10-fold in deuterated buffer. Deuterium labeling was carried out at 10° C. for labeling times of 30 seconds, 60 seconds, 120 seconds and 240 seconds.
  • the digested peptides were captured on a trap column (Hypersil Gold column) at 1°C, and 10% to 35% B (mobile phase A: 0.1% formic acid water, mobile phase B: 0.1% formic acid/acetonitrile)
  • a 7 minute gradient separation was used to elute onto an analytical column (Acclaim PepMap300 C18).
  • the mass spectrometer (Orbitrap Eclipse) was set to an electrospray voltage of 4000 V, a scan time of 1 second, and a mass/charge range of 260-2000. A spectrum was acquired.
  • the target protein sequence coverage was 98.5%, and 737 peptides were identified in samples with and without hCV804-8 antibody. Of the 737 identified peptides, 36 peptides satisfy the criterion that the difference in deuterium exchange rate ( ⁇ D%) between samples with and without hCV804-8 antibody is 10% or more.
  • the peptide is contained in 42 amino acid residues (SEQ ID NO: 49) corresponding to amino acid residues 960 to 1001 (SEQ ID NO: 48) of the SARS-CoV-2 spike protein (Genbank ACC No. QHD43416.1) I found out. From this, it was found that the amino acid residues of the SARS-CoV-2 spike protein to which the hCV804-8 antibody binds are positions 960-1001.
  • Tables 9-1 and 9-2 show the 36 SEQ ID NOs, amino acid sequences, amino acid residue positions in the spike protein of SARS-CoV-2, and ⁇ D%. A similar test was performed with the CV804 antibody, with results indicating that the epitope includes positions 960-1001 of the spike protein of SARS-CoV-2, similar to the hCV804-8 antibody.
  • Example 12 Optimization of antibody humanization CV804 was humanized in the following manner. Antibodies were humanized with the aim of improving antibody yield, thermal stability, and binding affinity by in silico analysis based on the Bioluminate module of structural analysis software Maestro. Antibody sequences were subjected to CDR identification according to Example 10. The designed humanized monoclonal antibody was transiently expressed in the culture supernatant using Expi293 cells and prepared by two-step purification: affinity purification using Protein A magnetic beads or a column, followed by purification using a gel filtration column. . The binding activity to the SARS-CoV2 full-length spike protein was measured for these purified antibody solutions.
  • Table 10 shows the light chain variable regions, CDRs, heavy chain variable regions, and amino acid sequences of the CDRs of these antibodies. 11, 12 and the binding activities of these antibodies are shown in Table 13.
  • Example 13 Mutant Generation Mutants were designed for CV804, the antibodies generated in Examples 10 and 12. CV804, each of the antibodies listed in Tables 5 and 10, has a heavy chain CDR1 sequence of SEQ ID NO: 2 (SDYAWN), a heavy chain CDR2 sequence of SEQ ID NO: 3 (YMAYSGTTSYNPSLKG) and a heavy chain CDR3 sequence of SEQ ID NO: 4 (RGIYYDFDGSSWFAY); It has a light chain CDR1 sequence of SEQ ID NO: 6 (KSSQSLLSSNNQKNYLA), a light chain CDR2 sequence of SEQ ID NO: 7 (WASTGES), and a light chain CDR3 sequence of SEQ ID NO: 8 (QQFYSYPTT), and is determined by Kabat numbering of the heavy chain variable region sequence.
  • SDYAWN heavy chain CDR1 sequence of SEQ ID NO: 2
  • YMAYSGTTSYNPSLKG a heavy chain CDR3 sequence of SEQ ID NO: 4
  • Table 14 shows the light chain variable region, each CDR, heavy chain variable region, and the amino acid sequence number of each CDR of these antibodies
  • Table 15 shows the amino acid sequences of the designed humanized heavy chain variable regions.
  • Table 16 shows the binding activity of the amino acid mutants in which the amino acid residue G at position 100c was substituted by Kabat numbering of the heavy chain variable region sequence of hCV804-35 measured by the same method as shown in Example 12.
  • the notation of the amino acid mutant of hCV804-35 in Table 16 is, for example, hCV804-35 (G100cN), and the amino acid residue G at position 100c by Kabat numbering of the heavy chain variable region sequence of hCV804-35 is changed to N. means that it has been replaced.
  • hCV804-48 in Table 16 is a mutant in which amino acid residue G at position 100c is replaced with V by Kabat numbering of the heavy chain variable region sequence of hCV804-35.
  • Example 14 Assessment of binding of CV804 to spike protein-expressing cells of related coronaviruses Betacoronavirus HKU24 (Genbank ACC No. YP_009113025), Rousettus bat coronavirus (Genbank ACC No. AOG30822.1), Longquan AA m house corona virus (Genbank ACC No. AID16631.1), Eidolon bat coronavirus/Kenya/KY24/2006 (Genbank ACC No. ADX59466.1), Chaerephon bat coronavirus/Kenya/KY22/2006 (Uniprot ACC No. F1DB20), Rodent corona virus (Genbank ACC No. YP_009755834.
  • YP_002308506. 1 Beluga whale coronavirus SW1 (Genbank ACC No. YP_001876437.1) and Avian coronavirus IA1162/2020 (Genbank ACC No. QOS02275.1) spike protein expression plasmid vectors were prepared and transfected into HEK293 cells. action . The presence or absence of binding between the transfected cell group and CV804 was evaluated in the same manner as in Example 5. Table 17 shows the presence or absence of binding to each virus strain. As shown in Table 17, CV804 responded to 73% or more different spike proteins out of 15 different viral spikes.
  • Example 15 Reactivity of CV804 to SARS-CoV2 Spike Protein Mutant-Expressing Cells SARS-CoV2 spike proteins shown in Tables 18, 19 and 20, specifically SARS-CoV2 wild type (Genbank ACC No. QHD43416.1), SARS-CoV2 spike protein mutants ⁇ (alpha) strain (GISAID ID: EPI_ISL_768526), ⁇ (gamma) strain (GISAID ID: EPI_ISL_833366), ⁇ (Omicron) strain BA1 (GISAID ID: EPI_ISL_6640917 ), ⁇ strain BA2 (GISAID ID: EPI_ISL_9595859), ⁇ strain BA5 (GISAID ID: EPI_ISL_13241867), ⁇ strain BQ1.1 (GISAID ID: EPI_ISL_15579783), ⁇ strain XBB (GISAID ID: EPI_ISL_15669344), ⁇ strain XE (GISAID ID : EPI
  • the amino acid variants of the SARS-CoV2 spike protein are 452 of the wild-type SARS-CoV2 spike protein (Genbank ACC No. QHD43416.1 (SEQ ID NO: 86)), for example, L452R. means that the amino acid residue is substituted from L to R, and if it is D614G_S929I, the 614th amino acid of the wild-type SARS-CoV2 spike protein (Genbank ACC No.
  • Example 16 Epitope mapping of CV804 antibody of SARS-CoV-2 spike protein by alanine scanning method
  • Site-specific mutation in which amino acids at 50 sites are substituted with alanine among positions 960 to 1019 of SARS-CoV2 spike protein We prepared 50 kinds of antibodies and examined the reactivity of the CV804 antibody.
  • the nomenclature for each variant is the same as in Example 15.
  • Reactivity evaluation was performed in the same manner as in Example 15. The results obtained are shown in Tables 21 and 22, with positive binding indicated as "+” and negative binding indicated as "-”.
  • this alanine scanning method by replacing each amino acid with alanine, the entire structure of the protein is not destroyed, and each amino acid residue is replaced with a methyl group that constitutes alanine.
  • each amino acid residue before substitution can be detected.
  • Alanine is used because it is not bulky, has a chemically inert methyl group, and can mimic the secondary structure exhibited by many other amino acids.
  • the reactivity of the CV804 antibody was negative for binding to the L962A, E988A, Q992A, L996A, R1000A, L1001A, L1004A, Y1007A, and I1018A mutants.
  • the amino acid substituted with alanine is an amino acid important for the binding of the CV804 antibody and constitutes the epitope of the CV804 antibody.
  • Reactive deletion of the CV804 antibody therefore included positions 962-1018 of the spike protein of SARS-CoV-2 (Genbank ACC No. QHD43416.1 (SEQ ID NO: 86)) in the epitope sequence of the CV804 antibody.
  • a pharmaceutical composition containing the monoclonal antibody of the present invention is useful as a medicine for treating or preventing coronavirus infections.

Abstract

Le but de la présente invention est de fournir : un nouvel anticorps monoclonal contre la sous-unité S2 d'une protéine spike du SARS-CoV-2 ; et une composition pharmaceutique contenant ledit anticorps pour la prévention ou le traitement de maladies infectieuses à coronavirus. Un anticorps selon la présente invention a été découvert grâce à la production de divers anticorps en utilisant la sous-unité S2 de la protéine spike du SARS-CoV-2 en tant qu'antigène, ce qui a conduit à la finalisation de la présente invention. Cet anticorps monoclonal peut être utilisé pour la prévention ou le traitement de maladies infectieuses à coronavirus.
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