WO2022092181A1 - 重症急性呼吸器症候群コロナウイルス２のヌクレオカプシドタンパク質に特異的に結合する抗体又はその断片、及びその用途 - Google Patents

重症急性呼吸器症候群コロナウイルス２のヌクレオカプシドタンパク質に特異的に結合する抗体又はその断片、及びその用途 Download PDF

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WO2022092181A1
WO2022092181A1 PCT/JP2021/039754 JP2021039754W WO2022092181A1 WO 2022092181 A1 WO2022092181 A1 WO 2022092181A1 JP 2021039754 W JP2021039754 W JP 2021039754W WO 2022092181 A1 WO2022092181 A1 WO 2022092181A1
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amino acid
acid sequence
antibody
fragment
ceremony
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PCT/JP2021/039754
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English (en)
French (fr)
Japanese (ja)
Inventor
隆司 川畑
宇洋 枡屋
光佑 柚原
研吾 西村
宏明 北澤
敏弘 黒板
正治 磯部
信幸 黒澤
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Toyobo Co Ltd
University of Toyama NUC
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Toyobo Co Ltd
University of Toyama NUC
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Priority to EP21886298.5A priority Critical patent/EP4239068A4/en
Priority to JP2022559219A priority patent/JP7832430B2/ja
Priority to US18/251,081 priority patent/US20240288427A1/en
Priority to CN202180074073.3A priority patent/CN116437931A/zh
Publication of WO2022092181A1 publication Critical patent/WO2022092181A1/ja
Anticipated expiration legal-status Critical
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    • 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
    • G01N33/56983—Viruses
    • 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • C07K16/102—Coronaviridae (F)
    • C07K16/104—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2]
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/6857—Antibody fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • SARS CoV2 Severe Acute Respiratory Syndrome coronavirus 2
  • SARS CoV2 is the causative virus of the new coronavirus infection (COVID-19) and has been rapidly spreading worldwide since the beginning of 2020.
  • SARS CoV2 is composed of a nucleocapsid and an envelope surrounding the nucleocapsid, like a general corona virus, and the nucleocapsid contains a viral genome (RNA) and a nucleocapsid protein (N protein) that binds to the viral genome.
  • the envelope includes a lipid and a peaplomer protein (S protein), a membrane protein (M protein), and an envelope protein (E protein) that bind to the lipid.
  • the N protein is a protein involved in the formation of the viral core, packaging and transcription of the viral genome, and is an N-terminal domain (NTD) via a linker having a serine (S) / arginine (R) rich region. And has a structure in which the C-terminal domain (CTD) is bound.
  • NTD N-terminal domain
  • CTD C-terminal domain
  • a linker with an S / R-rich region (a large amount of phosphorylation site) of the N protein of SARSCoV2 was self-degraded by a cysteine protease that induces apoptosis (eg, caspase 3, caspase 6). Since it can be cleaved by action, it has been found that even if an antibody that specifically binds to the N protein of SARS CoV2 is used, the N protein of SARS CoV2 may not be detected depending on the site to which the antibody binds.
  • a first object of the present invention is to provide an antibody or a fragment thereof that specifically binds to NTD or CTD of the N protein of SARSCoV2, and its use.
  • the present inventors in a method for detecting the N protein of SARSCoV2 by forming a sandwich structure with two types of antibodies that specifically bind to the N protein, the present inventors, for example, in NTD as two types of antibodies.
  • NTD as two types of antibodies.
  • the sandwich structure cannot be formed when the linker having an S / R rich region is cleaved, so that the detection sensitivity of the N protein of SARS CoV2 is high. Found to reduce.
  • a second object of the present invention is to provide a kit and a method for detecting the N protein of SARSCoV2 with high sensitivity.
  • the present inventors have found many types of antibodies or fragments thereof that specifically bind to NTD or CTD of the N protein of SARS CoV2.
  • N protein can be detected with high sensitivity by using two or more antibodies or fragments thereof that recognize epitopes.
  • the present invention has been completed as a result of further diligent studies based on these findings.
  • the present invention includes the following aspects.
  • Item 1 An antibody or fragment thereof (antigen-binding fragment) that specifically binds to the amino acid sequence represented by SEQ ID NO: 1 or 2.
  • Item 2. The following formula (A-1) or (A-2): GFX A31 X A41 X A51 X A61 X A71 X A81 (A-1) [During the ceremony, X A31 is T or S X A41 is F, L, or I, X A51 is D, S, N, or T, X A61 is D, N, S, I, or T, X A71 is Y, F, or N, X A81 is G, W, Y, S or T] GFX A33 X A43 X A53 X A63 X A73 X A83 X A93 X A103 (A-2) [During the ceremony, X A33 is T or S X A43 is F, L, or I, X A53 is D, S, N, or T,
  • Heavy chain CDR1 consisting of the amino acid sequence represented by the formula (A-1)
  • Heavy chain CDR2 consisting of the amino acid sequence represented by the formula (B-1) or (B-2)
  • a heavy chain CDR3 consisting of an amino acid sequence represented by any of the formulas (C-1), (C-2), (C-4), and (C-6) to (C-8)
  • Light chain CDR1 consisting of the amino acid sequence represented by the formula (D-1)
  • the light chain CDR2 consisting of the amino acid sequence represented by any of the formulas (E-1) to (E-3) and Item 2.
  • the antibody or fragment thereof according to Item 2 which comprises a light chain CDR3 consisting of an amino acid sequence represented by any of the formulas (F-1) to (F-5).
  • a heavy chain CDR1 consisting of the amino acid sequence shown in any of SEQ ID NOs: 3 to 11 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences
  • a heavy chain CDR2 consisting of the amino acid sequence shown in any of SEQ ID NOs: 12 to 25 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences
  • a heavy chain CDR3 consisting of the amino acid sequence shown in any of SEQ ID NOs: 26 to 41 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences
  • the light chain CDR1 consisting of the amino acid sequence shown in any of SEQ ID NOs: 42 to 50 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences, From the amino acid sequence shown in any of SEQ ID NOs: 51 to 58, the amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences, or the amino acid sequence represented by either KAN, SHN
  • Light chain CDR2 and Item 2 The antibody or fragment thereof according to Item 2, which comprises the amino acid sequence shown in any of SEQ ID NOs: 59 to 73 or the light chain CDR3 consisting of an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences. .. Item 5a.
  • a heavy chain CDR1 consisting of the amino acid sequence shown in any of SEQ ID NOs: 3 to 9 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences
  • a heavy chain CDR2 consisting of the amino acid sequence shown in any of SEQ ID NOs: 12 to 19 and 25 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences
  • a heavy chain CDR3 consisting of the amino acid sequence shown in any of SEQ ID NOs: 26 to 29, 32, 33, 37, 40, and 41 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences.
  • the light chain CDR1 consisting of the amino acid sequence shown in any of SEQ ID NOs: 42 to 48 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences
  • Item 5 The antibody or fragment thereof according to Item 5, wherein the mutation is a conservative substitution.
  • Item 6a The antibody or fragment thereof according to Item 5. The antibody or fragment thereof according to Item 5a, wherein the mutation is a conservative substitution.
  • Item 7. Item 6. The antibody or fragment thereof according to any one of Items 2 to 6, which specifically binds to the amino acid sequence shown in SEQ ID NO: 1.
  • Item 7a The antibody or fragment thereof according to Item 5a or 6a, which specifically binds to the amino acid sequence shown in SEQ ID NO: 1.
  • Heavy chain CDR1 consisting of the amino acid sequence represented by the formula (G-1)
  • Heavy chain CDR2 consisting of the amino acid sequence represented by the formula (H-1) or (H-2)
  • Heavy chain CDR3 consisting of amino acid sequences represented by any of the formulas (I-1) to (I-3) and (I-5) to (I-9)
  • the light chain CDR1 consisting of the amino acid sequence represented by any of the formulas (J-1) to (J-3)
  • Light chain CDR2 consisting of the amino acid sequence represented by the formula (K-1), Item 2.
  • the antibody according to Item 8 which comprises a light chain CDR3 consisting of an amino acid sequence represented by any of the formulas (L-1) to (L-3), (L-5), and (L-6). That fragment.
  • G-1-1 GX G22 X G32 X G42 X G52 X G62 X G72 X G82 (G-1-1) [During the ceremony, X G22 is F or L X G32 is I, T, or S, X G42 is F or L, X G52 is S, N, T, or R, X G62 is N or T X G72 is Y, S, or H, X G82 is F, T, Y, or W] Heavy chain CDR1 consisting of the amino acid sequence represented by The following formula (H-1-1) or (H-2): IX H22 X H32 X H42 X H52 X H62 X H72 X H82 (H-1-1) [During the ceremony, X H22 is S, G, K, or N, X H32 is G, N, S, T, or P, X H42 is D, S, or A, X H52 is S, G, or D, X H62 is T
  • a heavy chain CDR1 consisting of the amino acid sequence shown in any of SEQ ID NOs: 74 to 84 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences
  • a heavy chain CDR3 consisting of the amino acid sequence shown in any of SEQ ID NOs: 102 to 118 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences
  • a light chain CDR1 consisting of the amino acid sequence shown in any of SEQ ID NOs: 119 to 133 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences.
  • Light chain CDR2 consisting of an amino acid sequence represented by either NDD, KDS, NGN, NDS, KVS, LIS, or EVS, Item 8.
  • the antibody or fragment thereof according to Item 8 which comprises the amino acid sequence shown in any of SEQ ID NOs: 134 to 149 or the light chain CDR3 consisting of an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences. .. Item 11a.
  • a heavy chain CDR1 consisting of the amino acid sequence shown in any of SEQ ID NOs: 74 to 81 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences
  • Heavy chain CDR2 consisting of the amino acid sequence shown in any of SEQ ID NOs: 85 to 90, 92 to 95, and 101 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences.
  • a heavy chain CDR3 consisting of the amino acid sequence shown in any of SEQ ID NOs: 102 to 106, 109, 110, and 112 to 116 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences
  • a light chain CDR1 consisting of the amino acid sequence shown in any of SEQ ID NOs: 119 to 121, 125 to 129, and 133 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences.
  • Light chain CDR2 consisting of an amino acid sequence represented by either NDD, KDS, NGN, NDS, KVS, LIS, or EVS, Containing the amino acid sequence set forth in any of SEQ ID NOs: 134-136, 140-142, 148, and 149 or the light chain CDR3 consisting of an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences.
  • Item 8 The antibody or fragment thereof.
  • Item 12. The antibody or fragment thereof according to Item 11, wherein the mutation is a conservative substitution.
  • Item 12a The antibody or fragment thereof according to Item 11a, wherein the mutation is a conservative substitution.
  • Item 13. Item 6.
  • Item 13a The antibody or fragment thereof according to any one of Items 8 to 12, which specifically binds to the amino acid sequence shown in SEQ ID NO: 2.
  • Item 13a The antibody or fragment thereof according to Item 6a, which specifically binds to the amino acid sequence shown in SEQ ID NO: 2.
  • Item 14. Severe Acute Respiratory Syndrome An antibody against the nucleocapsid protein of coronavirus 2 (SARS CoV2) or a fragment thereof, wherein the epitope is present in the region consisting of the amino acid sequence set forth in any of SEQ ID NOs: 150 to 152 on the nucleocapsid protein. , Antibodies or fragments thereof (antigen-binding fragments).
  • Item 15a The antibody or fragment thereof according to any one of Items 1 to 14, wherein the antibody is a monoclonal antibody.
  • Item 15a The antibody or fragment thereof according to any one of Items 5a, 6a, 11a, and 12a, wherein the antibody is a monoclonal antibody.
  • Item 16. A polynucleotide comprising the coding sequence of the antibody or fragment thereof according to any one of Items 1 to 15.
  • Item 16a A polynucleotide comprising the coding sequence of the antibody or fragment thereof according to any one of Items 5a, 6a, 11a, 12a, and 15a.
  • Item 17a A cell containing the polynucleotide according to Item 16. Item 17a.
  • Item 18a The reagent or pharmaceutical containing the antibody or fragment thereof according to any one of Items 1 to 15, the polynucleotide according to Item 16, or the cell according to Item 17.
  • Item 18a Reagents or pharmaceuticals comprising the antibody or fragment thereof according to any one of Items 5a, 6a, 11a, 12a, and 15a, the polynucleotide according to Item 16a, or the cell according to Item 17a.
  • Item 19a A kit for detecting the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS CoV2), which comprises the antibody according to any one of Items 1 to 15 or a fragment thereof.
  • SARS CoV2 severe acute respiratory syndrome coronavirus 2
  • a kit for detecting the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 which comprises the antibody or fragment thereof according to any one of Items 5a, 6a, 11a, 12a, and 15a.
  • Item 20 Two or more antibodies or fragments thereof that specifically bind to the amino acid sequence set forth in SEQ ID NO: 1 and recognize different epitopes in the amino acid sequence set forth in SEQ ID NO: 1, respectively, and fragments thereof.
  • / Or Includes two or more antibodies or fragments thereof that specifically bind to the amino acid sequence set forth in SEQ ID NO: 2 and recognize different epitopes in the amino acid sequence set forth in SEQ ID NO: 2, respectively.
  • Severe Acute Respiratory Syndrome A kit for detecting the nucleocapsid protein of coronavirus 2 (SARS CoV2). Item 21.
  • Two or more antibodies or fragments thereof that specifically bind to the amino acid sequence represented by SEQ ID NO: 1 are added to the antibody or fragment thereof according to any one of Items 2 to 7 or the amino acid sequence of the antibody or fragment thereof.
  • Two or more antibodies or fragments thereof that specifically bind to the amino acid sequence represented by SEQ ID NO: 2 are added to the antibody or fragment thereof according to any one of Items 8 to 14 or the amino acid sequence of the antibody or fragment thereof.
  • An antibody or fragment thereof consisting of an amino acid sequence having 90% or more sequence identity Two or more antibodies or fragments thereof that specifically bind to the amino acid sequence represented by SEQ ID NO: 1 are added to the antibody or fragment thereof according to any one of Items 2 to 7 or the amino acid sequence of the antibody or fragment thereof.
  • the kit according to item 20 Item 21a.
  • the antibody or fragment thereof according to Item 5a or 6a is the two or more antibodies or fragments thereof that specifically bind to the amino acid sequence represented by SEQ ID NO: 1.
  • the antibody or fragment thereof according to Item 11a or 12a is the two or more antibodies or fragments thereof that specifically bind to the amino acid sequence represented by SEQ ID NO: 2.
  • the kit according to item 20 Item 22. Severe acute respiratory syndrome coronavirus by immunochromatography, enzyme immunoassay, chemiluminescence immunoassay, chemiluminescence enzyme immunoassay, radioimmunoassay, electrochemical luminescence immunoassay, immunoturbidimetric method, or latex agglutination Item 2.
  • Item 22a Severe acute respiratory syndrome coronavirus by immunochromatography, enzyme immunoassay, chemiluminescence immunoassay, chemiluminescence enzyme immunoassay, radioimmunoassay, electrochemical luminescence immunoassay, immunoturbidimetric method, or latex agglutination Item 2.
  • Item 23 Severe acute respiratory syndrome coronavirus by immunochromatography, enzyme immunoassay, chemiluminescence immunoassay, chemiluminescence enzyme immunoassay, radioimmunoassay, electrochemical luminescence immunoassay, immunoturbidimetric method, or latex agglutination Item 2.
  • Severe acute respiratory syndrome This is a method for detecting the nucleocapsid protein of coronavirus 2 (SARS CoV2), and the following steps (1) to (3): (1) A step of immobilizing a first antibody or a fragment thereof that specifically binds to the amino acid sequence represented by SEQ ID NO: 1 or 2 to a solid phase. (2) A second antibody or a fragment thereof that specifically binds to the nucleocapsid protein of SARS CoV2 and the amino acid sequence represented by SEQ ID NO: 1 or 2 on the solid phase (where, the second antibody or a fragment thereof is The step of recognizing a different epitope in the amino acid sequence to which the first antibody or fragment thereof specifically binds and labeling with a labeling substance), and applying.
  • SARS CoV2 coronavirus 2
  • a method comprising a step of detecting a label derived from a labeling substance of a second antibody or a fragment thereof bound to the solid phase via the nucleocapsid protein.
  • Item 25 A sequence in which the first antibody or fragment thereof and the second antibody or fragment thereof are 90% or more of the amino acid sequence of the antibody or fragment thereof according to any one of Items 2 to 15 and the antibody or fragment thereof.
  • Item 6. The method according to Item 24, which is selected from the group consisting of an antibody consisting of an amino acid sequence having the same identity or a fragment thereof.
  • the first antibody or fragment thereof and the second antibody or fragment thereof are added to the antibody or fragment thereof according to any one of Items 5a, 6a, 11a, 12a, and 15a, and the amino acid sequence of the antibody or fragment thereof.
  • Item 6 The method according to Item 24, which is selected from the group consisting of an antibody consisting of an amino acid sequence having 90% or more sequence identity or a fragment thereof.
  • the present invention provides an antibody or a fragment thereof that specifically binds to NTD or CTD of the N protein of SARSCoV2.
  • the present invention also provides a kit and a method for detecting the N protein of SARSCoV2 with high sensitivity.
  • the amino acid may be a natural amino acid or an unnatural amino acid.
  • unnatural amino acids include citrulline, ornithine, ⁇ -acetyl-lysine, ⁇ -alanine, aminobenzoic acid, 6-aminocaproic acid, aminobutyric acid, hydroxyproline, mercaptopropionic acid, 3-nitrotyrosine, norleucine, pyroglutamic acid and the like.
  • the amino acid may be, for example, L-amino acid, D-amino acid, or DL-amino acid.
  • the amino acid sequence identity refers to the degree of amino acid matching when two or more amino acid sequences to be compared are optimally aligned.
  • the identity of the amino acid sequence can be calculated using commercially available or analysis tools available through the telecommunications line (Internet), for example using the commercially available software GENETYX (Genetics, Inc.) or National Biotechnology Information Systems. Calculated using the default (default) parameters in the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) (http://www.ncbi.nlm.nih.gov/BLAST/). Can be done.
  • NCBI National Center for Biotechnology Information
  • amino acid sequences disclosed herein are deleted, substituted, or modified with one or more (eg, 1, 2, or 3) amino acids in the sequence as long as they do not inhibit the binding of SARS CoV2 to the N protein.
  • one or more (eg, 1, 2, or 3) amino acids may be inserted or added to the sequence.
  • amino acid substitution is preferably a substitution (conservative substitution) with another amino acid having a similar structure and / or property.
  • Conservative substitutions include, for example, substitutions within the group shown in Table 1.
  • amino acid modifications include modification of functional groups such as amino groups, carboxyl groups, hydroxyl groups, and sulfhydryl (SH) groups.
  • Modifications of the functional groups include, for example, glycosylation; methylation; esterification; amidation; PEGylation; phosphorylation; hydroxylation; t-butoxycarbonyl (Boc) group, 9-fluorenylmethyloxycarbonyl (Fmoc) group.
  • Binding to a protecting group such as; biotinylation; binding to a fluorescent dye such as fluorescein isothiocyanate (FITC); binding to an enzyme such as peroxidase (HRP), alkali phosphatase (ALP), or the like.
  • the antibody may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
  • the antibody can be any isotype, such as IgG, IgA, IgD, IgE, IgM and the like.
  • the antibody may be a human antibody or a non-human antibody. Examples of non-human antibodies include, but are not limited to, mouse antibodies, rat antibodies, guinea pig antibodies and the like.
  • the antibody may be a chimeric antibody such as a mouse-human chimeric antibody.
  • the antibody can be a partially or fully humanized antibody.
  • the antibody fragment is not particularly limited as long as it contains heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3, and is not particularly limited, for example, Fv, Fab, Fab', (Fab') 2 , scFv, scFv-Fc. , Diabody, triabody, tetrabody, minibody and the like.
  • the antibody fragment is preferably a fragment having an antigen-binding ability (antigen-binding fragment).
  • heavy chain CDR1 to 3 and light chain CDR1 to 3 are defined based on the IMGT method.
  • nucleotides such as DNA and RNA may be subjected to known chemical modifications as exemplified below.
  • the phosphate residue (phosphate) of each nucleotide can be replaced with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate.
  • PS phosphorothioate
  • methylphosphonate methylphosphonate
  • phosphorodithionate can.
  • the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide is converted into -OR (R is, for example, -CH 3 , -CH 2 CH 2 OCH 3 , -CH 2 CH 2 NHC (NH) NH 2 , -CH.
  • the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, substitution of the carbonyl group at the 2-position with thiocarbonyl, etc. Can be mentioned. Further, examples thereof include those in which the phosphoric acid moiety and the hydroxyl moiety are modified with biotin, an amino group, a lower alkylamine group, an acetyl group and the like, but the present invention is not limited thereto.
  • the N protein (antigen) of SARSCoV2 has the amino acid sequence disclosed in GenBank Accession No. (MN908947).
  • the SARSCoV N protein has the amino acid sequence disclosed in GenBank Accession No. (AY278741).
  • NTD an antibody that specifically binds to the NTD of the N protein of SARS CoV2 or a fragment thereof
  • the NTD of the N protein of SARS CoV2 (sometimes simply referred to as NTD) is the 1st to 172nd from the N-terminal of the N-protein. It means the amino acid region and has the following amino acid sequence: MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQ
  • the antibody or fragment thereof that specifically binds to NTD of SARSCoV2 does not bind to NTD of SARSCoV or has low binding to NTD of SARSCoV.
  • the amino acid sequence shown in SEQ ID NO: 1 for example, the 63rd D, the 65th K, the 79th S, the 94th I, the 103rd D, the 120th G, the 128th D, and the 131st I. , 152nd A, and 157th I are different from the corresponding amino acids of NTD in SARSCoV, respectively.
  • the antibody or fragment thereof that specifically binds to NTD of SARS CoV2 is the 63rd D, 65th K, 79th S, 94th I, 103rd D, 120th G in SEQ ID NO: 1. , 128th D, 131st I, 152nd A, and 157th I. It is preferable to bind to a region (or epitope) containing one or more amino acids selected from the group.
  • the antibody or fragment thereof that specifically binds to NTD of SARSCoV2 may bind to the amino acid region 1 to 180 from the N-terminal of the N protein, and binds to the RNA binding domain of NTD. Is preferable, and it is preferable to bind to the amino acid region at the 44th to 180th position or the 44th to 172nd position from the N-terminal of the N protein.
  • An antibody or fragment thereof that specifically binds to NTD of SARS CoV2 can be produced by combining known methods, and can be produced, for example, by a method including the following steps (P1) to (P6): (P1) Antibodies of animals immunized with SARS CoV2 N protein using fluorescently labeled SARS CoV2 N protein, which has higher staining selectivity for endoplasmic reticulum than that for cell organs other than endoplasmic reticulum.
  • the process of obtaining cells producing an antibody that specifically binds to the N protein of SARS CoV2 from the producing cells (P2) A step of obtaining an antibody variable region gene fragment from the cells obtained in the above step (P1), (P3) A step of obtaining a gene expression unit from the antibody variable region gene fragment obtained in the step (P2). (P4) A step of introducing the gene expression unit obtained in the above step (P3) into cells to express an antibody that binds to the N protein of SARS CoV2. (P5) The step of recovering the antibody expressed in the step (P4), and (P6) A step of obtaining an antibody that specifically binds to NTD from the antibody recovered in the step (P5).
  • the process (P1) can be carried out, for example, according to the method (ERIAA method) described in US Patent Application Publication No. 2013/0029325 (which is incorporated herein by reference in its entirety).
  • the step (P2) can be carried out according to the method (MAGrahd method) described in the literature (Rapid production of antigen-specific monoclonal antibody from a variety of animals, BMC Biology 2012, 10:80), for example.
  • the process (P3) can be carried out, for example, according to the method (TS-jPCT method) described in US Patent Application Publication No. 2013/0023009 (which is incorporated herein by reference in its entirety).
  • the step (P4) can be carried out by a known gene transfer method, for example, a calcium phosphate method, a lipofection method, a DEAE dextran method, an electroporation method, a microinjection method, or the like.
  • the cells into which the gene expression unit is introduced are not particularly limited as long as the antibody can be transiently expressed, and are, for example, mammalian cells such as CHO cells, HEK293 cells, Expi293 cells, 293F cells, 293T cells, and 293FT cells. Can be mentioned.
  • the step (P6) can be carried out by selecting an antibody that specifically binds to NTD by a known screening method, for example, an enzyme-linked immunosorbent assay (ELISA), a Western blotting method, an immunostaining method, or the like.
  • ELISA enzyme-linked immunosorbent assay
  • Western blotting method an immunostaining method, or the like.
  • the antibody may be prepared by a known method, for example, a salting out method using ammonium sulfate, sodium sulfate or the like; an affinity chromatography method using protein A or the like; an ion exchange chromatography method using DEAE cellulose or the like; Gel filtration; Purification may be performed by a purification method using His-tag, FLAG-tag, or the like.
  • the heavy chain CDR1 of an antibody or fragment thereof that specifically binds to NTD of SARS CoV2 preferably consists of the following amino acid sequence: -Amino acid sequences represented by any of the formulas (A-1) and (A-2) shown in Table 2.
  • An amino acid sequence in which an amino acid is mutated preferably a conservative substitution).
  • X A41 is preferably F or L
  • X A51 is preferably D, S, N, or T
  • X A61 is preferably D, N, S, or I.
  • X A71 is preferably Y or F
  • X A81 is preferably G, W, S, or Y.
  • the formula (A-1) is preferably one selected from the group consisting of the formula (A-1-1) or the formulas (A-1-2) to (A-1-9), and more preferably. It is one selected from the group consisting of formulas (A-1-2) to (A-1-8).
  • X A33 is preferably S
  • X A43 is preferably I
  • X A53 is preferably T
  • X A63 is preferably T
  • X A73 is preferably N.
  • S is preferably G or Y
  • X A93 is preferably Y or F, more preferably Y
  • X A103 is preferably T, G, or W, even more preferred. Is T or G.
  • the formula (A-2) is preferably the formula (A-2-1), and more preferably the formula (A-2-2).
  • the heavy chain CDR1 of the antibody or fragment thereof that specifically binds to NTD of SARS CoV2 is preferably the formula (A-1), more preferably the formula (A-1-1) or the formula (A-1-2) to.
  • the heavy chain CDR2 of an antibody or fragment thereof that specifically binds to NTD of SARS CoV2 preferably consists of the following amino acid sequence: -Amino acid sequences represented by any of the formulas (B-1) and (B-2) shown in Table 3.
  • An amino acid sequence in which an amino acid is mutated preferably a conservative substitution).
  • X B21 is preferably S, N, D, or H
  • X B31 is preferably Y, G, or P
  • X B41 is preferably S or D
  • B51 is preferably G or S
  • X B61 is preferably G, D, T, or N
  • X B71 is preferably R, K, T, S, G, or Y
  • X B81 is preferred. Is T or I.
  • the formula (B-1) is preferably one selected from the group consisting of the formula (B-1-1) or the formulas (B-1-2) to (B-1-13), and more preferably. It is one selected from the group consisting of the formulas (B-1-2) to (B-1-10), and more preferably the group consisting of the formulas (B-1-2) to (B-1-9). It is one of the more selected.
  • X B23 is preferably S, N, D, or W, more preferably W, and X B33 is preferably Y, G, or S, even more preferably Y or.
  • S, X B43 is preferably D or G
  • X B53 is preferably G, S, or A, more preferably G or A
  • X B63 is preferably R, K, T, or.
  • the formula (B-2) is preferably the formula (B-2-1), more preferably the formula (B-2-2) or (B-2-3), and further preferably the formula (B-2-1). 2-3).
  • the heavy chain CDR2 of the antibody or fragment thereof that specifically binds to NTD of SARS CoV2 is preferably a group consisting of the formulas (B-1-1) and (B-2-1), or the formula (B-1-). 2)-(B-1-13), (B-2-2), and (B-2-3) groups, more preferably equations (B-1-2)-(B-1-10). , (B-2-2) and (B-2-3), particularly preferably the group consisting of formulas (B-1-2) to (B-1-9) and (B-2-3). It consists of an amino acid sequence selected from the above, or an amino acid sequence having 90% or more identity with respect to the amino acid sequence. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.
  • the heavy chain CDR3 of an antibody or fragment thereof that specifically binds to NTD in SARS CoV2 preferably consists of the following amino acid sequence: -Amino acid sequence represented by any of the formulas (C-1) to (C-8) shown in Table 4.
  • An amino acid sequence in which an amino acid is mutated preferably a conservative substitution).
  • X C21 is preferably R
  • X C31 is preferably D
  • X C41 is preferably G
  • X C51 is preferably G
  • X C61 is preferably S.
  • X C71 is preferably Y or V, more preferably Y
  • X C81 is preferably Y, H, S, T, or K, more preferably Y, H, or S.
  • X C91 is preferably S
  • X C101 is preferably P
  • X C111 is preferably I
  • X C121 is preferably N, S, or Y, and even more preferably N or S.
  • X C131 is preferably F or C
  • X C141 is preferably Q
  • X C151 is preferably F, Y, or V, and even more preferably F or Y.
  • the formula (C-1) is preferably one selected from the group consisting of the formula (C-1-1) or the formulas (C-1-2) to (C-1-7), and more preferably. It is one selected from the group consisting of equations (C-1-2) to (C-1-4).
  • X C13 is preferably A
  • X C23 is preferably T
  • X C33 is preferably N or S
  • X C43 is preferably Y or G
  • X C53 is preferably N, S, or P, more preferably N or P
  • X C63 is preferably F, C, or M, still more preferably F or M
  • X C73 is preferably Q.
  • X C83 is preferably F, Y, or V, and more preferably Y or V.
  • the formula (C-2) is preferably the formula (C-2-1), and more preferably the formula (C-2-2) or (C-2-3).
  • X C15 is preferably A
  • X C25 is preferably R
  • X C35 is preferably S
  • X C45 is preferably I
  • X C55 is preferably I
  • X C65 is preferably A
  • X C75 is preferably E
  • X C85 is preferably I
  • X C95 is preferably P
  • X C155 is preferably P
  • X C165 is preferably N, S, or P, more preferably P
  • X C175 is preferably F, C, or M, even more preferably F
  • X C185 is preferably Q or D.
  • X C195 is preferably F, Y, or V, and even more preferably V.
  • Equation (C-3) is preferably Equation (C-3-1).
  • X C16 is preferably A, X C26 is preferably K, X C36 is preferably Y, X C46 is preferably Y, and X C56 is preferably D.
  • X C66 is preferably Y, X C76 is preferably D, X C86 is preferably Y, X C96 is preferably T, X C106 is preferably G, and X C116 .
  • the formula (C-5) is preferably the formula (C-5-1).
  • X C107 is preferably P
  • X C117 is preferably F
  • X C127 is preferably F, C, or M, more preferably F
  • X C137 is. It is preferably Q or D, more preferably Q
  • X C147 is preferably Y or S.
  • Equation (C-6) is preferably one selected from the group consisting of equations (C-6-1) to (C-6-3).
  • X C58 is preferably G
  • X C68 is preferably A
  • X C78 is preferably G
  • X C88 is preferably L
  • X C98 is preferably Q.
  • D more preferably D
  • X C108 is preferably F, Y, or V, even more preferably V.
  • the formula (C-7) is preferably the formula (C-7-1).
  • X C49 is preferably A
  • X C59 is preferably V
  • X C69 is preferably G
  • X C79 is preferably F, C, or M, and further. It is preferably F
  • X C89 is preferably Q or D, more preferably D
  • X C99 is preferably F, Y, or V, even more preferably V.
  • the formula (C-8) is preferably the formula (C-8-1).
  • the heavy chain CDR3 of the antibody or fragment thereof that specifically binds to NTD of SARS CoV2 is preferably the formulas (C-1), (C-2), (C-4), and (C-6) to (C-6) to (C).
  • a group consisting of -8 more preferably a group consisting of formulas (C-1-1), (C-2-1), and (C-6) to (C-8), or a group consisting of formula (C-1).
  • the group consisting of (C-7-1) and (C-8-1 more preferably the formulas (C-1-2) to (C-1-5), (C-2-2), (C).
  • the light chain CDR1 of an antibody or fragment thereof that specifically binds to NTD of SARS CoV2 preferably consists of the following amino acid sequence: -Amino acid sequence represented by any of the formulas (D-1) to (D-3) shown in Table 5.
  • An amino acid sequence in which an amino acid is mutated preferably a conservative substitution).
  • Equation (D-1) is preferably one selected from the group consisting of equations (D-1-1) to (D-1-7).
  • X D23 is preferably Q
  • X D33 is preferably S
  • X D43 is preferably L
  • X D53 is preferably F
  • X D63 is preferably Y.
  • K more preferably Y
  • X D73 preferably Y, S, or W, even more preferably S.
  • Equation (D-2) is preferably Equation (D-2-1).
  • the light chain CDR1 of an antibody or fragment thereof that specifically binds to NTD of SARS CoV2 preferably comprises the formula (D-1), and more preferably the formulas (D-1-1) to (D-1-7). It consists of an amino acid sequence selected from the group or an amino acid sequence having 90% or more identity with respect to the amino acid sequence. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.
  • the light chain CDR2 of an antibody or fragment thereof that specifically binds to NTD of SARS CoV2 preferably consists of the following amino acid sequence: -Amino acid sequence represented by any of the formulas (E-1) to (E-3) shown in Table 6.
  • An amino acid sequence in which an amino acid is mutated preferably a conservative substitution).
  • X E21 is preferably K or E, and X E41 is preferably D.
  • the formula (E-1) is preferably one selected from the group consisting of the formula (E-1-1) or the formulas (E-1-2) to (E-1-8), and more preferably. It is one selected from the group consisting of equations (E-1-2) to (E-1-5).
  • X E13 is preferably K
  • X E23 is preferably A
  • X E33 is preferably N.
  • Formula (E-3) is preferably one selected from the group consisting of formulas (E-3-1) to (E-3-3), and more preferably formula (E-3-1). be.
  • the light chain CDR2 of an antibody or fragment thereof that specifically binds to NTD of SARS CoV2 is preferably a group consisting of the formulas (E-1-1), (E-2), and (E-3), or a formula thereof.
  • the identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.
  • the light chain CDR3 of an antibody or fragment thereof that specifically binds to NTD of SARS CoV2 preferably consists of the following amino acid sequence: -Amino acid sequence represented by any of the formulas (F-1) to (F-7) shown in Table 7. An amino acid sequence having 90% or more (preferably 95% or more) identity to the amino acid sequence, or 1 to 3 (preferably 1 or 2 or even more preferably 1) of the amino acid sequence. An amino acid sequence in which an amino acid is mutated (preferably a conservative substitution).
  • X F21 is preferably V
  • X F31 is preferably S
  • X F41 is preferably Y, F, V, or L
  • X F51 is preferably S or K
  • X F61 is preferably G
  • X F71 is preferably G
  • X F81 is preferably H
  • X F91 is preferred.
  • X F101 is preferably I, V, or L, and more preferably I or V.
  • the formula (F-1) is preferably one selected from the group consisting of the formula (F-1-1) or the formulas (F-1-2) to (F-1-6), and more preferably. Equations (F-1-2) to (F-1-4).
  • X F23 is preferably A
  • X F33 is preferably S or D, more preferably D
  • X F43 is preferably Y, F, V, or L.
  • X F53 is preferably S or K, even more preferably S
  • X F63 is preferably G
  • X F73 is preferably G.
  • A, more preferably A, X F83 preferably H or S, preferably S, X F93 preferably S, and X F113 preferably I, V, or L. Yes, more preferably I or V, still more preferably V.
  • the formula (F-2) is preferably the formula (F-2-1).
  • X F24 is preferably A
  • X F34 is preferably S, D, or N, more preferably N
  • X F44 is preferably Y, F, or H.
  • X F54 is preferably S or N, even more preferably N
  • X F74 is preferably G
  • X F84 is preferably G or E, even more preferably.
  • E, X F94 is preferably H or S, preferably S
  • X F114 is preferably G
  • X F134 is preferably I, V, or L, more preferably I or V. And more preferably V.
  • Equation (F-3) is preferably Equation (F-3-1).
  • X F25 is preferably V
  • X F35 is preferably S, G, or K
  • X F45 is preferably Y
  • X F55 is preferably S or N
  • X F65 is preferably G or N
  • X F75 is preferably G or K
  • X F85 is preferably Y or V
  • X F95 is preferably V or Y.
  • the formula (F-4) is preferably the formula (F-4-1), and more preferably one selected from the group consisting of the formulas (F-4-2) to (F-4-6). Yes, more preferably one selected from the group consisting of formulas (F-4-2) to (F-4-4).
  • the light chain CDR3 of the antibody or fragment thereof that specifically binds to NTD of SARS CoV2 is preferably a group consisting of formulas (F-1) to (F-5), more preferably formula (F-1-1), The group consisting of (F-2-1), (F-3-1), (F-4-1), and (F-5), more preferably the formulas (F-1-2) to (F-1).
  • the identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.
  • Table 8 shows suitable examples of combinations of heavy chain CDR1 to 3 and light chain CDR1 to 3 of an antibody or fragment thereof that specifically binds to NTD of SARSCoV2.
  • 1 to 3 amino acids are mutated (preferably conservative substitutions) in the amino acid sequence of at least one of the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3. It is also preferable to have one.
  • the CTD of the N protein of SARS CoV2 (sometimes simply referred to as CTD) is the 247th to 419th from the N-terminal of the N-protein. It means the amino acid region and has the following amino acid sequence: TKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTAAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQTVTLLPAADDFSK
  • the antibody or fragment thereof that specifically binds to the CTD of SARSCoV2 does not bind to the CTD of SARSCoV or has low binding to the CTD of SARSCoV.
  • the amino acid sequence shown in SEQ ID NO: 2 for example, the 267th A, 290th E, 334th T, 345th N, and 349th Q from the N-terminus of the N protein are CTDs of SARS CoV, respectively. Different from the corresponding amino acids in.
  • An antibody against the N protein of SARS CoV2 or a fragment thereof has the A at the 267th A and the E at the 290th position from the N-terminal of the N protein in SEQ ID NO: 2. It is preferred to bind to a region (or epitope) containing one or more amino acids selected from the group consisting of T at 334, N at 345, and Q at 349.
  • an antibody against the N protein of SARS CoV2 or a fragment thereof binds to the amino acid region 247 to 419 from the N-terminal of the N protein. It may bind to the dimerized domain of CTD, and preferably to the amino acid region at positions 247 to 364 or 256 to 364 from the N-terminal of the N protein.
  • an antibody against the N protein of SARS CoV2 or a fragment thereof is located at positions 250 to 257 and 374 to 382 from the N-terminus of the N protein.
  • the epitope is present in the region that binds to the amino acid region at positions 394 to 405, that is, the region consisting of the amino acid sequence set forth in any of SEQ ID NOs: 150 to 152 on the N protein.
  • An antibody or a fragment thereof that specifically binds to the CTD of SARS CoV2 can be produced by combining known methods, for example, by a method including the above steps (P1) to (P5) and the following steps (P6'). Can be manufactured.
  • P6' A step of obtaining an antibody that specifically binds to CTD from the antibody recovered in the step (P5).
  • an antibody that specifically binds to CTD is selected by a known screening method, for example, an enzyme-linked immunosorbent assay (ELISA method), a Western blotting method, an immunostaining method, or the like. It can be carried out by.
  • ELISA method enzyme-linked immunosorbent assay
  • Western blotting method Western blotting method
  • immunostaining method or the like. It can be carried out by.
  • the heavy chain CDR1 of an antibody or fragment thereof that specifically binds to the CTD of SARS CoV2 preferably consists of the following amino acid sequence: -Amino acid sequences represented by any of the formulas (G-1) and (G-2) shown in Table 9. An amino acid sequence having 90% or more (preferably 95% or more) identity to the amino acid sequence, or 1 to 3 (preferably 1 or 2 or even more preferably 1) of the amino acid sequence. An amino acid sequence in which an amino acid is mutated (preferably a conservative substitution).
  • X G31 is preferably I, T, or S
  • X G51 is preferably S, N, T, or R
  • X G61 is preferably N or T
  • G71 is preferably Y, S, or H
  • X G81 is preferably F, T, Y, or W.
  • the formula (G-1) is preferably the formula (G-1-1), and more preferably one selected from the group consisting of the formulas (G-1-2) to (G-1-11). Yes, more preferably one selected from the group consisting of formulas (G-1-2) to (G-1-9).
  • the heavy chain CDR1 of the antibody or fragment thereof that specifically binds to the CTD of SARS CoV2 is preferably the formula (G-1-1), more preferably the formulas (G-1-2) to (G-1-11).
  • the identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.
  • the heavy chain CDR2 of an antibody or fragment thereof that specifically binds to the CTD of SARS CoV2 preferably consists of the following amino acid sequence: -Amino acid sequence represented by any of the formulas (H-1) to (H-3) shown in Table 10.
  • An amino acid sequence in which an amino acid is mutated preferably a conservative substitution).
  • X H31 is preferably G, N, S, T, or P
  • X H41 is preferably D, S, or A
  • X H61 is preferably T, S, G. , Or R.
  • the formula (H-1) is preferably one selected from the group consisting of the formula (H-1-1) or the formulas (H-1-2) to (H-1-17), and more preferably. It is one selected from the group consisting of the formulas (H-1-2) to (H-1-15), and more preferably the formulas (H-1-2) to (H-1-7) and (H). It is one selected from the group consisting of -1-9) to (H-1-12).
  • the heavy chain CDR2 of the antibody or fragment thereof that specifically binds to the CTD of SARS CoV2 is preferably a group consisting of the formulas (H-1) and (H-2), more preferably the formula (H-1-1) and A group consisting of (H-2) or a group consisting of formulas (H-1-2) to (H-1-17) and (H-2), more preferably formulas (H-1-2) to ( The group consisting of H-1-15) and (H-2), particularly preferably the formulas (H-1-2) to (H-1-7), (H-1-9) to (H-1-12). ), And an amino acid sequence selected from the group consisting of (H-2), or an amino acid sequence having 90% or more identity with respect to the amino acid sequence.
  • the identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.
  • the heavy chain CDR3 of an antibody or fragment thereof that specifically binds to the CTD of SARS CoV2 preferably consists of the following amino acid sequence: -Amino acid sequence represented by any of the formulas (I-1) to (I-11) shown in Table 11. An amino acid sequence having 90% or more (preferably 95% or more) identity to the amino acid sequence, or 1 to 3 (preferably 1 or 2 or even more preferably 1) of the amino acid sequence. An amino acid sequence in which an amino acid is mutated (preferably a conservative substitution).
  • X I31 is preferably N, L, or T, and X I61 is preferably D, F, or G.
  • Formula (I-1) is preferably one selected from the group consisting of formula (I-1-1) or formulas (I-1-2) to (I-1-8), and more preferably. It is one selected from the group consisting of the formulas (I-1-2) to (I-1-7), and more preferably the group consisting of the formulas (I-1-2) to (I-1-6). It is one of the more selected.
  • the heavy chain CDR3 of an antibody or fragment thereof that specifically binds to the CTD of SARS CoV2 is preferably a group consisting of formulas (I-1) to (I-3) and (I-5) to (I-9). More preferably, the group consisting of the formulas (I-1-1), (I-2), (I-3), and (I-5) to (I-9), or the formula (I-1-2).
  • amino acid sequence selected from the group consisting of 1-6), (I-2), (I-3), and (I-5) to (I-9), or 90% or more of the amino acid sequence. It consists of an amino acid sequence having the same identity. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.
  • the light chain CDR1 of an antibody or fragment thereof that specifically binds to the CTD of SARS CoV2 preferably consists of the following amino acid sequence: -Amino acid sequence represented by any of the formulas (J-1) to (J-3) shown in Table 12.
  • An amino acid sequence in which an amino acid is mutated preferably a conservative substitution).
  • X J11 is preferably K
  • X J31 is preferably S
  • X J41 is preferably N
  • E or K
  • X J51 is preferably Q, R, or. E.
  • the formula (J-1) is preferably one selected from the group consisting of the formula (J-1-1) or the formulas (J-1-2) to (I-1-7), and more preferably. It is one selected from the group consisting of equations (J-1-2) to (J-1-4).
  • X J73 is preferably S.
  • Formula (J-2) is preferably one selected from the group consisting of formula (J-2-1) or formulas (J-2-2) to (J-2-9), and more preferably. It is one selected from the group consisting of the formulas (J-2-2) to (J-2-8), and more preferably the group consisting of the formulas (J-2-2) to (J-2-6). It is one of the more selected.
  • the heavy chain CDR3 of the antibody or fragment thereof that specifically binds to the CTD of SARS CoV2 is preferably a group consisting of the formulas (J-1-1), (J-2-1), and (J-3), or , Formulas (J-1-2) to (J-1-7), (J-2-2) to (J-2-9), and (J-3), more preferably formula (J).
  • the light chain CDR2 of an antibody or fragment thereof that specifically binds to the CTD of SARS CoV2 preferably consists of the following amino acid sequence: -Amino acid sequence represented by the formula (K-1) shown in Table 13, An amino acid sequence having 90% or more (preferably 95% or more) identity to the amino acid sequence, or 1 to 3 (preferably 1 or 2 or even more preferably 1) of the amino acid sequence. An amino acid sequence in which an amino acid is mutated (preferably a conservative substitution).
  • Equation (K-1) is preferably one selected from the group consisting of equations (K-1-1) to (K-1-7).
  • the light chain CDR2 of the antibody or fragment thereof that specifically binds to the CTD of SARS CoV2 is preferably an amino acid sequence selected from the group consisting of the formulas (K-1-1) to (K-1-7), or the amino acid sequence thereof. It consists of an amino acid sequence having 90% or more identity with respect to the amino acid sequence. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.
  • the light chain CDR3 of an antibody or fragment thereof that specifically binds to the CTD of SARS CoV2 preferably consists of the following amino acid sequence: -Amino acid sequence represented by any of the formulas (L-1) to (L-6) shown in Table 14. An amino acid sequence having 90% or more (preferably 95% or more) identity to the amino acid sequence, or 1 to 3 (preferably 1 or 2 or even more preferably 1) of the amino acid sequence. An amino acid sequence in which an amino acid is mutated (preferably a conservative substitution).
  • XL31 is preferably R or S
  • XL41 is preferably Q or E
  • XL51 is preferably S
  • XL61 is preferably D or N
  • X L71 is preferably D
  • X L81 is preferably A
  • X L91 is preferably A
  • X L101 is preferably S, R, or W
  • X L111 is preferably V.
  • the formula (L-1) is preferably one selected from the group consisting of the formula (L-1-1) or the formulas (L-1-2) to (L-1-7), and more preferably. It is one selected from the group consisting of equations (L-1-2) to (L-1-4).
  • X L33 is preferably R, S, or G, more preferably G, X L43 is preferably Q or E, still more preferably E, and X L53 . It is preferably S, X L63 is preferably D, X L73 is preferably D, X L83 is preferably A, X L93 is preferably W, and X L103 is preferably V. ..
  • the formula (L-2) is preferably the formula (L-2-1).
  • X L54 is preferably H
  • X L64 is preferably D or N, or D or V
  • X L84 is preferably Y, V, or I, or Y or. It is L.
  • the formula (L-3) is preferably one selected from the group consisting of the formula (L-3-1) or the formulas (L-3-2) to (L-3-7), and more preferably. It is the formula (L-3-2) or (L-3-3).
  • X L27 is preferably T
  • X L37 is preferably T
  • X L47 is preferably N
  • X L57 is preferably D
  • X L77 is preferably Y.
  • XL87 is preferably T.
  • Equation (L-4) is preferably Equation (L-4-1).
  • the light chain CDR3 of an antibody or fragment thereof that specifically binds to the CTD of SARS CoV2 is preferably a group consisting of the formulas (L-1) to (L-3), (L-5), and (L-6). , More preferably the group consisting of the formulas (L-1-1), (L-2-1), (L-3-1), (L-5), and (L-6), or (L-).
  • Table 15 shows suitable examples of combinations of heavy chain CDR1 to 3 and light chain CDR1 to 3 of an antibody or fragment thereof that specifically binds to CTD of SARS CoV2.
  • 1 to 3 amino acids are mutated (preferably conservative substitutions) in the amino acid sequence of at least one of the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3. It is also preferable to have one.
  • the regions other than the heavy chain CDR1 to 3 and the light chain CDR1 to 3 are not particularly limited as long as they can be used for an antibody, and can be any amino acid sequence.
  • the constant region for example, the constant regions of IgG1, IgG2, IgG3, IgA1, IgA2, IgM and the like can be used, but the constant region is not limited thereto.
  • the binding property of the antibody or fragment thereof that specifically binds to NTD or CTD of SARSCoV2 to the N protein of SARSCoV2 is high, and the absorbance measured by the enzyme-linked immunosorbent assay (ELISA) described in Examples described later is used as an index.
  • ELISA enzyme-linked immunosorbent assay
  • it is 0.2 or more, preferably 0.3 or more, more preferably 0.4 or more, and further preferably 0.5 or more.
  • the binding affinity (Kd) of an antibody or fragment thereof that specifically binds to NTD or CTD of SARSCoV2 to the N protein of SARSCoV2 is, for example, 50 nM or less, preferably 10 nM or less, for example, 1 pM or more.
  • Kd can be measured using Biacore TM T200 (Cytiva). Specifically, by an amine coupling reaction using N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (NHS) and N-hydroxysuccinimide (EDC), the sensor chip CM5 was subjected to an anti-guinea pig to carboxymethyl dextran.
  • NHS N'-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • EDC N-hydroxysuccinimide
  • the IgG antibody is immobilized by covalent binding, an antibody that specifically binds to NTD or CTD of SARSCoV2 or a fragment thereof is injected into the sensor chip for binding, and then the N protein of SARSCoV2 is injected into the sensor chip to inject SARSCoV2.
  • Kd can be calculated by reacting with an antibody or a fragment thereof that specifically binds to NTD or CTD and analyzing the reaction signal.
  • polynucleotide of the invention preferably comprises the coding sequence of an antibody or fragment thereof that specifically binds to NTD or CTD of SARS CoV2.
  • the coding sequence include, but are not limited to, a base sequence encoding an antibody or a fragment thereof containing the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3 described in 3 above.
  • the polynucleotide of the invention preferably comprises an expression cassette of an antibody or fragment thereof that specifically binds to NTD or CTD of SARS CoV2.
  • the expression cassette is not particularly limited as long as it allows expression in the host cell, and includes, for example, a promoter and a coding sequence arranged under the control of the promoter.
  • the promoter is not particularly limited and can be appropriately selected according to the type of host cell.
  • various pol II promoters can be used.
  • the polII promoter is not particularly limited, and examples thereof include a CMV promoter, an EF1 promoter, an SV40 promoter, and an MSCV promoter.
  • examples of the promoter include tryptophan promoters such as trc and tac; lac promoter; T7 promoter; T5 promoter; T3 promoter; SP6 promoter; arabinose-induced promoter; cold shock promoter; tetracycline-induced promoter and the like.
  • the expression cassette may contain other elements, if necessary.
  • Other elements include, for example, multicloning sites (MCS), drug resistance genes, origins of replication, enhancer sequences, repressor sequences, insulator sequences, reporter protein coding sequences, drug resistance gene coding sequences and the like. These may be one kind alone or a combination of two or more kinds.
  • the polynucleotide of the present invention can be, for example, in the form of a vector.
  • An appropriate vector is selected according to the purpose of use, the type of host cell, and the like.
  • Escherichia coli-hosted vectors include M13 phage or its variants, ⁇ phage or its variants, pBR322 or its variants (eg pB325, pAT153, pUC8), and yeast-hosted vectors include pYepSec1, pMFa, etc.
  • vectors such as pYES2 and pPIC3.5K are pAc and pVL as vectors having insect cells as hosts, and pcDNA, pCDM8, pMT2PC and the like can be exemplified as vectors using mammalian cells as hosts.
  • Cell The cell of the present invention preferably contains the polynucleotide described in 4 above.
  • Examples of cells include Escherichia coli such as Escherichia coli K12, Bacillus bacteria such as Bacillus subtilis MI114, yeast such as Saccharomyces cerevisiae AH22, Sf cell lineage derived from Spodoptera frugiperda or High Five cell lineage derived from Trichoplusia ni, and insects such as olfactory nerve cells. Examples include cells, animal cells and the like.
  • the animal cells are preferably cultured cells derived from mammals, specifically, COS7 cells, CHO cells, HEK293 cells, Expi293 cells, 293F cells, 293T cells, 293FT cells, Hela cells, PC12 cells, N1E-115 cells. , SH-SY5Y cells and the like.
  • the cells of the present invention preferably express an antibody or fragment thereof that specifically binds to NTD or CTD of SARSCoV2.
  • the cells of the present invention preferably secrete or have on the cell surface an antibody or fragment thereof that specifically binds to NTD or CTD of SARSCoV2.
  • the reagent or medicine of the present invention is an antibody or fragment thereof that specifically binds to NTD or CTD of SARS CoV2, a polynucleotide containing the coding sequence of the antibody or fragment thereof, or a cell containing the polynucleotide. It is preferable to include.
  • the reagent or pharmaceutical of the present invention preferably further comprises a pharmaceutically acceptable excipient or carrier and / or additive.
  • excipient or carrier examples include starch, lactose, crystalline cellulose, sorbitol, calcium hydrogen phosphate, water, ethanol, (poly) ethylene glycol, (poly) propylene glycol, glycerol, vegetable oil and the like. These can be used alone or in combination of two or more.
  • the additive examples include a buffering agent, an tonicity agent, a thickener, a chelating agent, an emulsifier, a coloring agent, a preservative and the like. These can be used alone or in combination of two or more.
  • the reagent or pharmaceutical of the present invention may be an antibody or a fragment thereof that specifically binds to NTD or CTD of SARS CoV2 dissolved or dispersed in a solvent, or may be freeze-dried.
  • Kit for detecting N protein of SARS CoV2 The kit for detecting N protein of SARS CoV2 of the present invention preferably contains an antibody or a fragment thereof that specifically binds to NTD or CTD of SARS CoV2. ..
  • the antibody or fragment thereof may be, for example, in the form of the reagent described in 6 above.
  • the kit is -Two or more antibodies or fragments thereof that specifically bind to NTD of SARS CoV2, and two or more antibodies or fragments thereof that recognize different epitopes of NTD, respectively, and / or-Specifically to CTD of SARS CoV2. It is preferable to include two or more antibodies or fragments thereof that bind to each other and recognize different epitopes of CTD.
  • two or more antibodies or fragments thereof that specifically bind to NTD have different amino acid sequences of at least one of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3, respectively.
  • first anti-NTD antibody or fragment thereof a portion of two or more antibodies or fragments thereof that specifically bind to NTD
  • first anti-NTD antibody or fragment thereof is immobilized and the rest (hereinafter referred to as “1st anti-NTD antibody or fragment thereof").
  • the “second anti-NTD antibody or fragment thereof”) is preferably labeled.
  • the solid phase on which the first anti-NTD antibody or a fragment thereof is immobilized include, but are not limited to, beads, wells, chips, strips, and the like.
  • the method for immobilizing the first anti-NTD antibody or a fragment thereof is not particularly limited, and examples thereof include a method in which a biotinylated first anti-NTD antibody or a fragment thereof is brought into contact with a solid phase surface having avidin. Be done.
  • the labeling substance for labeling the second anti-NTD antibody or a fragment thereof is not particularly limited, and examples thereof include enzymes such as peroxidase (HRP) and alkaline phosphatase (ALP).
  • the first and second anti-NTD antibodies or fragments thereof can be selected from, for example, the antibodies or fragments thereof described in 2 above, preferably from N-1 in Table 8, more preferably Table 8. It can be selected from 8 N-2, more preferably N-3 in Table 8, and particularly preferably the combination of N-4 and N-5 in Table 8, N-4 and N-.
  • the amino acid sequences of the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3 of N-13 may be an amino acid sequence having 90% or more identity with respect to the amino acid sequence, and 1 to 3 of the amino acid sequence may be used. It can be selected from (may be a variation of individual amino acids).
  • two or more antibodies or fragments thereof that specifically bind to CTD differ in the amino acid sequences of at least one of the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3, respectively.
  • a portion of two or more antibodies or fragments thereof that specifically bind to CTD (hereinafter referred to as “first anti-CTD antibody or fragment thereof") is immobilized and the rest (hereinafter referred to as “1st anti-CTD antibody or fragment thereof").
  • the “second anti-CTD antibody or fragment thereof”) is preferably labeled.
  • the solid phase on which the first anti-CTD antibody or a fragment thereof is immobilized include, but are not limited to, beads, wells, chips, strips, and the like.
  • the method for immobilizing the first anti-CTD antibody or a fragment thereof is not particularly limited, and examples thereof include a method in which a biotinylated first anti-CTD antibody or a fragment thereof is brought into contact with a solid phase surface having avidin. Be done.
  • the labeling substance for labeling the second anti-CTD antibody or a fragment thereof is not particularly limited, and examples thereof include enzymes such as peroxidase (HRP) and alkaline phosphatase (ALP).
  • the first and second anti-CTD antibodies or fragments thereof can be selected from, for example, the antibodies or fragments thereof described in 3 above, preferably from C-1 in Table 15, and more preferably from Table 15. It can be selected from 15 C-2, more preferably C-3 in Table 15, and particularly preferably the combination of C-4 and C-5 in Table 15, C-4 and C-.
  • amino acid sequences of 16 heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 may be an amino acid sequence having 90% or more identity with respect to the amino acid sequence, and 1 to 3 amino acids in the amino acid sequence. Can be mutated).
  • the kit is preferably a kit for detecting the N protein of SARSCoV2 in the test sample.
  • the test sample may be a biological sample or a non-biological sample.
  • the inspection sample includes not only the sample as it is collected but also the sample which has been subjected to pretreatment such as removal of impurities.
  • Biological samples include, for example, blood, serum, plasma, bone marrow fluid, lymph, tears, nasal discharge, nasal lavage fluid, nasal swab, saliva, mouthwash, sputum, pharyngeal swab, sweat, bronchial suction fluid, bronchial lavage fluid, pleural effusion, etc.
  • Examples include, but are not limited to, runny nose, sheep water, intestinal lavage fluid, urine, feces, cell extract, tissue extract, organ extract, and the like.
  • non-biological samples include, but are not limited to, samples collected from environmental surfaces such as faucets, handles, handrails, straps, switches, walls, floors, desks, chairs, and toilet seats.
  • the method for detecting the N protein of SARSCoV2 using the kit is not particularly limited, but is, for example, an immunochromatography method, an enzyme immunoassay, a chemiluminescent immunoassay, a chemiluminescent enzyme immunoassay, a radioimmunoassay, or electricity. Chemiluminescent immunoassays, immunoturbidimetric methods, latex agglomeration methods and the like can be mentioned.
  • the kit can be used with other substances (for example, buffer solution, enzyme solution, dilution solution, secondary antibody, etc.), instruments used, etc., depending on the method for preparing the test sample, the method for detecting the N protein of SARSCoV2, etc. Instructions and the like may be included.
  • substances for example, buffer solution, enzyme solution, dilution solution, secondary antibody, etc.
  • Steps (1) to (3) (1) A step of immobilizing a first antibody or a fragment thereof that specifically binds to NTD or CTD to a solid phase. (2) A second antibody or fragment thereof that specifically binds to the N protein of SARS CoV2 and the amino acid sequence represented by SEQ ID NO: 1 or 2 on the solid phase (where, the second antibody or fragment thereof is The step of recognizing a different epitope in the amino acid sequence to which the first antibody or fragment thereof specifically binds and labeling with a labeling substance), and applying. (3) It is preferable to include a step of detecting a label derived from a labeling substance of the second antibody or a fragment thereof bound to the solid phase via the nucleocapsid protein.
  • the first and second antibodies or fragments thereof may be the first and second anti-NTD antibodies or fragments thereof described in 7 above, and / or the first and second anti-CTD antibodies or fragments thereof. preferable.
  • solid phase and the labeling substance for example, those described in 7 above can be selected.
  • the cells obtained from the removed lumbar lymph nodes were fixed on ice for 10 minutes with a formaldehyde PBS solution. Immobilized cells were stained with Dylight488-labeled SARS-CoV2N protein, Dylight550-labeled SARS-CoVN protein, Dylight650-labeled anti-guinea pig IgG antibody, and DAPI (4', 6-diamidino-2-phenylindole) and SARS- Plasma cells showing strong positive for CoV2N protein, negative for SARS-CoVN protein, and strong positive for anti-morphot IgG were sorted by flow cytometry.
  • antibody gene expression unit From the amplified antibody variable region gene fragment, antibody gene expression is performed according to the method (TS-jPCR method) of "Specific preparation method of linked DNA fragment containing sequence derived from target gene" (US Patent Application Publication No. 2013/0023009). The unit was made.
  • Antibody screening by ELISA Screening was performed by evaluating the reaction intensity of each antibody with the SARS-CoV2 N protein by ELISA.
  • the ELISA was immobilized on the bottom of a 96-well plate so that the SARS-CoV2 N protein was 15 ng / well, and 100 ⁇ L of the culture supernatant of 293FT cells transiently expressing the antibody was added, and the antigen-antibody antibody was added at room temperature for 2 hours.
  • the reaction was carried out.
  • Guinea pig antibody bound to the antigen was detected using a goat anti-guinea pig antibody bound to horseradish peroxidase, and the absorbance at 450 nm was measured using a microplate reader.
  • NTD N-terminal region
  • CTD C-terminal region
  • amino acid sequences of NTD and CTD are shown in Table 16.
  • DNA fragments encoding NTD and CTD were prepared by PCR.
  • the prepared DNA fragment 50 ng / ⁇ L
  • FuGene HD Transfection Reagent (trademark) (Promega)
  • Opti-MEM (trademark) (Thermo Fisher) were mixed at a liquid volume ratio of 1: 1: 50 and kept at room temperature for 20 minutes. After leaving it in, it was added to 293FT cells suspended in 10% (w / v) FBS / IMDM medium.
  • 293FT cells introduced with DNA fragments were seeded on collagen-coated 96-well plates and incubated for 2 days at 37 ° C. in an 8% (v / v) CO 2 environment. After that, the medium is removed, the cells are washed with PBS, 100 ⁇ L / well of 3% (w / v) formaldehyde PBS solution is added, and the cells are allowed to stand at room temperature for 20 minutes to fix the cells, and further 0.1% (w / v). 200 ⁇ L / well of Triton PBS solution was added, and the mixture was allowed to stand at room temperature for 5 minutes for cell membrane permeation treatment.
  • the antibody was prepared by transfecting the prepared gene expression unit into Expi293 cells for the antibody having good reactivity.
  • Expi 293 Expression Medium GIBCO
  • Expi Fectamine 293 Transfection Kit GIBCO
  • 50 ⁇ g of the gene expression unit of each prepared antibody 3 mL of OptiMEM I and 0.16 mL of Expi Fectamine 293 were mixed, allowed to stand for 10 minutes, and then added to Expi293 cells for gene transfer.
  • Expi Fectamine 293 Transfection Enhancer was added as a feed, and after culturing for another 4 days, the supernatant was collected and purified by a protein A column to obtain an antibody.
  • each clone (detection antibody) labeled with 0.25 ⁇ g / mL alkaline phosphatase and 10 ng / mL antigen solution (1% (w / w /)) v) TBS-T dilution) was added in increments of 50 ⁇ L / well, and the mixture was reacted at room temperature for 2 hours.
  • the antigen solution in addition to the full-length SARS-CoV2 N protein sample 1, the total amount of SARS-CoV2 N protein is equal to mol of NTD and the sample in which the SARS-CoV2 N protein is degraded.
  • Sample 2 substituted with CTD was used. After washing with TBS-T, 100 ⁇ L / well of pNPP was added as a color-developing substrate, and the mixture was reacted at room temperature for 15 minutes, and then the absorbance at 405 nm was measured using a microplate reader. The results are shown in Table 20. In addition,% in Table 20 represents the relative absorbance value with respect to the absorbance measurement value of the sample 1.
  • the total amount of SARS-CoV2 N protein was used in equal mols of NTD and CTD to imitate the sample in which the SARS-CoV2 N protein was degraded.
  • the substituted sample 2 was used.
  • 100 uL / well of pNPP was added as a color-developing substrate, and the mixture was reacted at room temperature for 15 minutes, and then the absorbance at 405 nm was measured using a microplate reader. The results are shown in Table 21.
  • Epitope mapping was determined using PEPperPRINT's Conformational Epitope Mappings service. Of the 200th to 419th amino acid regions of the N-protein of SARS CoV 2, peptides consisting of 7, 10 and 13 amino acids are shifted by 1 amino acid so as to overlap by 6, 9 and 12 amino acids on the peptide array. Synthesized and the reactivity of the antibody, i.e., the detection signal, identified which peptide was the epitope of the antibody binding.
  • the epitopes of three clones are SEQ ID NO: 150: LLPAADLDDFSK (394-405 from the N-terminus of the N-protein of SARS CoV 2) and SEQ ID NO: 151: KKADETQAL (SARS CoV 2), respectively. It was confirmed to be in the region of SEQ ID NO: 152: SAAEASKK (250 to 257th from the N-terminus of SARS CoV 2), 374-382th from the N-terminus of the N-protein.

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US18/251,081 US20240288427A1 (en) 2020-10-30 2021-10-28 Antibody capable of binding specifically to nucleocapsid protein of severe acute respiratory syndrome coronavirus-2 or fragment thereof, and use of said antibody or said fragment
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SHROCK ELLEN, FUJIMURA ERIC, KULA TOMASZ, TIMMS RICHARD T., LEE I-HSIU, LENG YUMEI, ROBINSON MATTHEW L., SIE BRANDON M., LI MAMIE : "Viral epitope profiling of COVID-19 patients reveals cross-reactivity and correlates of severity", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 370, no. 6520, 27 November 2020 (2020-11-27), US , pages 1 - 18, XP055926664, ISSN: 0036-8075, DOI: 10.1126/science.abd4250 *
TERRY JAMES S., ANDERSON LORAN BR, SCHERMAN MICHAEL S., MCALISTER CARLEY E., PERERA RUSHIKA, SCHOUNTZ TONY, GEISS BRIAN J.: "Development of SARS-CoV2 Nucleocapsid Specific Monoclonal Antibodies", BIORXIV, 3 September 2020 (2020-09-03), XP055794548, [retrieved on 20210413], DOI: 10.1101/2020.09.03.280370 *
WANG HONGYE, WU XIAN, ZHANG XIAOMEI, HOU XIN, LIANG TE, WANG DAN, TENG FEI, DAI JIAYU, DUAN HU, GUO SHUBIN, LI YONGZHE, YU XIAOBO: "SARS-CoV-2 Proteome Microarray for Mapping COVID-19 Antibody Interactions at Amino Acid Resolution", ACS CENTRAL SCIENCE, vol. 6, no. 12, 23 December 2020 (2020-12-23), pages 2238 - 2249, XP055864645, ISSN: 2374-7943, DOI: 10.1021/acscentsci.0c00742 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023167317A1 (ja) * 2022-03-04 2023-09-07 公立大学法人横浜市立大学 変異株を含む、体液中のＳＡＲＳ－ＣｏＶ－２抗原に対する抗ＳＡＲＳ－ＣｏＶ－２抗体、該抗体を用いてＳＡＲＳ－ＣｏＶ－２を検出する方法、および該抗体を含むキット
WO2024166921A1 (ja) * 2023-02-08 2024-08-15 デンカ株式会社 ＳＡＲＳ－ＣｏＶ－２の検出方法及びそのためのキット

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EP4239068A1 (en) 2023-09-06
US20240288427A1 (en) 2024-08-29

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