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 PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1002—Coronaviridae
- C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
-
- 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
-
- 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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Abstract
Description
項1.
配列番号1又は2で示されるアミノ酸配列に特異的に結合する抗体又はその断片(抗原結合断片)。
項2.
下記式(A-1)又は(A-2):
GFXA31XA41XA51XA61XA71XA81 (A-1)
[式中、
XA31はT又はSであり、
XA41はF、L、又はIであり、
XA51はD、S、N、又はTであり、
XA61はD、N、S、I、又はTであり、
XA71はY、F、又はNであり、
XA81はG、W、Y、S又はTである]
GFXA33XA43XA53XA63XA73XA83XA93XA103 (A-2)
[式中、
XA33はT又はSであり、
XA43はF、L、又はIであり、
XA53はD、S、N、又はTであり、
XA63はD、N、S、又はTであり、
XA73はN又はSであり、
XA83はG又はYであり、
XA93はY、F、又はNであり、
XA103はT、G、W、又はYである]
で表されるアミノ酸配列からなる重鎖CDR1と、
下記式(B-1)又は(B-2):
IXB21XB31XB41XB51XB61XB71XB81 (B-1)
[式中、
XB21はS、N、D、W、又はHであり、
XB31はY、G、P、又はSであり、
XB41はS、D、又はGであり、
XB51はG、S、T、又はAであり、
XB61はG、D、T、N、又はRであり、
XB71はR、K、T、S、N、G、Y、又はDであり、
XB81はT、I、又はMである]
IXB23XB33XB43XB53XB63XB73 (B-2)
[式中、
XB23はS、N、D、W、又はHであり、
XB33はY、G、P、又はSであり、
XB43はS、D、又はGであり、
XB53はG、S、T、又はAであり、
XB63はR、K、T、S、N、G、Y、又はDであり、
XB73はT、I、又はMである]
で表されるアミノ酸配列からなる重鎖CDR2と、
下記式(C-1)~(C-8):
XC11XC21XC31XC41XC51XC61XC71XC81XC91XC101XC111XC121XC131XC141XC151 (C-1)
[式中、
XC11はA又はSであり、
XC21はR、T、又はKであり、
XC31はD、N、S、又はYであり、
XC41はG、S、又はYであり、
XC51はG、I、又はDであり、
XC61はS又はIであり、
XC71はY、V、又はAであり、
XC81はY、H、S、T、K、又はEであり、
XC91はS、I、又はDであり、
XC101はP又はYであり、
XC111はI、Y、G、P、T、A、又はVであり、
XC121はN、S、Y、P、G、又はFであり、
XC131はF、C、M、又はLであり、
XC141はQ又はDであり、
XC151はF、Y、V、又はSである]
XC13XC23XC33XC43XC53XC63XC73XC83 (C-2)
[式中、
XC13はA又はSであり、
XC23はR、T、又はKであり、
XC33はD、N、S、又はYであり、
XC43はI、Y、G、P、T、A、又はVであり、
XC53はN、S、Y、P、G、又はFであり、
XC63はF、C、M、又はLであり、
XC73はQ又はDであり、
XC83はF、Y、V、又はSである]
XC15XC25XC35XC45XC55XC65XC75XC85XC95TIFTFXC155XC165XC175XC185XC195 (C-3)
[式中、
XC15はA又はSであり、
XC25はR、T、又はKであり、
XC35はG、S、又はYであり、
XC45はG、I、又はDであり、
XC55はS又はIであり、
XC65はY、V、又はAであり、
XC75はY、H、S、T、K、又はEであり、
XC85はS、I、又はDであり、
XC95はP又はYであり、
XC155はI、Y、G、P、T、A、又はVであり、
XC165はN、S、Y、P、G、又はFであり、
XC175はF、C、M、又はLであり、
XC185はQ又はDであり、
XC195はF、Y、V、又はSである]
ARLWSGYALDI (C-4)
XC16XC26XC36XC46XC56XC66XC76XC86XC96XC106XC116XC126XC136 (C-5)
[式中、
XC16はA又はSであり、
XC26はR、T、又はKであり、
XC36はD、N、S、又はYであり、
XC46はG、S、又はYであり、
XC56はG、I、又はDであり、
XC66はY、H、S、T、K、又はEであり、
XC76はS、I、又はDであり、
XC86はP又はYであり、
XC96はI、Y、G、P、T、A、又はVであり、
XC106はN、S、Y、P、G、又はFであり、
XC116はF、C、M、又はLであり、
XC126はQ又はDであり、
XC136はF、Y、V、又はSである]
VXC27PLLVXC77HGXC107XC117XC127XC137XC147 (C-6)
[式中、
XC27はS又はKであり、
XC77はV、Y、又はLであり、
XC107はI、Y、G、P、T、A、又はVであり、
XC117はN、S、Y、P、G、又はFであり、
XC127はF、C、M、又はLであり、
XC137はQ又はDであり、
XC147はF、Y、V、又はSである]
VSPSXC58XC68XC78XC88XC98XC108 (C-7)
[式中、
XC58はG又はAであり、
XC68はI、Y、G、P、T、A、又はVであり、
XC78はN、S、Y、P、G、又はFであり、
XC88はF、C、M、又はLであり、
XC98はQ又はDであり、
XC108はF、Y、V、又はSである]
SPSXC49XC59XC69XC79XC89XC99 (C-8)
[式中、
XC49はG又はAであり、
XC59はI、Y、G、P、T、A、又はVであり、
XC69はN、S、Y、P、G、又はFであり、
XC79はF、C、M、又はLであり、
XC89はQ又はDであり、
XC99はF、Y、V、又はSである]
のいずれかで表されるアミノ酸配列からなる重鎖CDR3と、
下記式(D-1)~(D-3):
SXD21XD31XD41XD51XD61XD71 (D-1)
[式中、
XD21はE、D、Q、又はGであり、
XD31はH、Y、G、又はSであり、
XD41はR、S、I、K、又はLであり、
XD51はS、H、N、又はFであり、
XD61はY又はKであり、
XD71はY、S、W、又はNである]
SXD23XD33XD43XD53XD63XD73GKNKDL (D-2)
[式中、
XD23はE、D、Q、又はGであり、
XD33はH、Y、G、又はSであり、
XD43はR、S、I、K、又はLであり、
XD53はS、H、N、又はFであり、
XD63はY又はKであり、
XD73はY、S、W、又はNである]
NIGGKT (D-3)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR1と、
下記式(E-1)~(E-3):
XE11XE21SXE41GSXE71 (E-1)
[式中、
XE11はL、V、又はIであり、
XE21はK、N、又はEであり、
XE41はD、Y、又はKであり、
XE71はH又はLである]
VGTSDIVG (E-2)
XE12XE22XE32 (E-3)
[式中、
XE12はK、S、又はWであり、
XE22はA又はHであり、
XE32はN又はSである]
のいずれかで表されるアミノ酸配列からなる軽鎖CDR2と、
下記式(F-1)~(F-7):
GXF21XF31XF41XF51XF61XF71XF81XF91XF101 (F-1)
[式中、
XF21はV又はAであり、
XF31はS、D、N、G、又はKであり、
XF41はY、F、V、L、又はHであり、
XF51はS、K、又はNであり、
XF61はG又はNであり、
XF71はG、A、E、又はKであり、
XF81はH、S、Y、又はVであり、
XF91はN、G、又はSであり、
XF101はI、V、L、又はYである]
GXF23XF33XF43XF53XF63XF73XF83XF93YXF113 (F-2)
[式中、
XF23はV又はAであり、
XF33はS、D、N、又はGであり、
XF43はY、F、V、L、又はHであり、
XF53はS、K、又はNであり、
XF63はG又はNであり、
XF73はG、A、E、又はKであり、
XF83はH、S、又はYであり、
XF93はN、G、又はSであり、
XF113はI、V、L、又はYである]
GXF24XF34XF44XF54IXF74XF84XF94YXF114YXF134 (F-3)
[式中、
XF24はV又はAであり、
XF34はS、D、N、又はGであり、
XF44はY、F、V、L、又はHであり、
XF54はS、K、又はNであり、
XF74はG又はNであり、
XF84はG、A、E、又はKであり、
XF94はH、S、又はYであり、
XF114はN、G、又はSであり、
XF134はI、V、L、又はYである]
GXF25XF35XF45XF55XF65XF75XF85XF95 (F-4)
[式中、
XF25はV又はAであり、
XF35はS、D、N、G、又はKであり、
XF45はY、F、V、L、又はHであり、
XF55はS、K、又はNであり、
XF65はG又はNであり、
XF75はG、A、E、又はKであり、
XF85はH、S、Y、又はVであり、
XF105はI、V、L、又はYである]
QYDSTPPLT (F-5)
QVWDSYTYV (F-6)
QFINGPLT (F-7)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR3と
を含む、抗体又はその断片(抗原結合断片)。
項3.
式(A-1)で表されるアミノ酸配列からなる重鎖CDR1と、
式(B-1)又は(B-2)で表されるアミノ酸配列からなる重鎖CDR2と、
式(C-1)、(C-2)、(C-4)、及び(C-6)~(C-8)のいずれかで表されるアミノ酸配列からなる重鎖CDR3と、
式(D-1)で表されるアミノ酸配列からなる軽鎖CDR1と、
式(E-1)~(E-3)のいずれかで表されるアミノ酸配列からなる軽鎖CDR2と、
式(F-1)~(F-5)のいずれかで表されるアミノ酸配列からなる軽鎖CDR3と
を含む、項2に記載の抗体又はその断片。
項4.
下記式(A-1-1):
GFXA32XA42XA52XA62XA72XA82 (A-1-1)
[式中、
XA32はT又はSであり、
XA42はF又はLであり、
XA52はD、S、N、又はTであり、
XA62はD、S、又はNであり、
XA72はY又はFであり、
XA82はG、W、S、又はYである]
で表されるアミノ酸配列からなる重鎖CDR1と、
下記式(B-1-1)及び(B-2-1):
IXB22XB32XB42XB52XB62XB72XB82 (B-1-1)
[式中、
XB22はS、N、D、又はHであり、
XB32はY、G、又はPであり、
XB42はS又はDであり、
XB52はG又はSであり、
XB62はG、D、T、又はNであり、
XB72はR、K、T、S、G、又はYであり、
XB82はT又はIである]
IWXB34XB44XB54XB64XB74 (B-2-1)
[式中、
XB34はY又はSであり、
XB44はD又はGであり、
XB54はG又はAであり、
XB64はR又はDであり、
XB74はT又はIである]
のいずれかで表されるアミノ酸配列からなる重鎖CDR2と、
下記式(C-1-1)、(C-2-1)、(C-6-1)、(C-7-1)、及び(C-8-1):
XC12RDGGSYXC82SPIXC122XC132QXC152 (C-1-1)
[式中、
XC12はA又はSであり、
XC82はY、H、又はSであり、
XC122はN又はSであり、
XC132はF又はCであり、
XC152はF又はYである]
ATXC34XC44XC54XC64XC74XC84 (C-2-1)
[式中、
XC34はN又はSであり、
XC44はY又はGであり、
XC54はN又はPであり、
XC64はF又はMであり、
XC74はQ又はDであり、
XC84はY又はVである]
VSPLLVVHGPFFQY (C-6-1)
VSPSGAGLDV (C-7-1)
SPSAVGFDV (C-8-1)
のいずれかで表されるアミノ酸配列からなる重鎖CDR3と、
下記式(D-1-1)~(D-1-7):
SEYKHYN (D-1-1)
SEHRSYY (D-1-2)
SDYSHYS (D-1-3)
SQGINKW (D-1-4)
SGHSSYY (D-1-5)
SEHSSYY (D-1-6)
SDHSSYY (D-1-7)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR1と、
下記式(E-1-1)、(E-2)、及び(E-3-1):
XE12XE22SDGSXE72 (E-1-1)
[式中、
XE12はL、V、又はIであり、
XE22はK、又はEであり、
XE72はH、又はLである]
VGTSDIVG (E-2)
KAN (E-3-1)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR2と、
下記式(F-1-1)、(F-2-1)、(F-3-1)、(F-4-1)、及び(F-5):
GVSXF42SGGHXF92XF102 (F-1-1)
[式中、
XF42はY又はFであり、
XF92はN又はGであり、
XF102はI又はVである]
GADYSGASSYV (F-2-1)
GANHNIGESYGYV (F-3-1)
GVXF36YXF56XF66XF76XF86XF96 (F-4-1)
[式中、
XF36はS、G、又はKであり、
XF56はS又はNであり、
XF66はG又はNであり、
XF76はG又はKであり、
XF86はY又はVであり、
XF96はV又はYである]
QYDSTPPLT (F-5)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR3と
を含む、抗体又はその断片(抗原結合断片)。
項5.
配列番号3~11のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる重鎖CDR1と、
配列番号12~25のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる重鎖CDR2と、
配列番号26~41のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる重鎖CDR3と、
配列番号42~50のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる軽鎖CDR1と、
配列番号51~58のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列、或いはKAN、SHN、又はWASのいずれかで表されるアミノ酸配列からなる軽鎖CDR2と、
配列番号59~73のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる軽鎖CDR3と
を含む、項2に記載の抗体又はその断片。
項5a.
配列番号3~9のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる重鎖CDR1と、
配列番号12~19及び25のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる重鎖CDR2と、
配列番号26~29、32、33、37、40、及び41のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる重鎖CDR3と、
配列番号42~48のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる軽鎖CDR1と、
配列番号51~54及び57のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列、或いはKANで表されるアミノ酸配列からなる軽鎖CDR2と、
配列番号59~61、64~68、及び71のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる軽鎖CDR3と
を含む、項2に記載の抗体又はその断片。
項6.
前記変異が保存的置換である、項5に記載の抗体又はその断片。
項6a.
前記変異が保存的置換である、項5aに記載の抗体又はその断片。
項7.
配列番号1で示されるアミノ酸配列に特異的に結合する、項2~6のいずれかに記載の抗体又はその断片。
項7a.
配列番号1で示されるアミノ酸配列に特異的に結合する、項5a又は6aに記載の抗体又はその断片。
項8.
下記式(G-1)又は(G-2):
GXG21XG31XG41XG51XG61XG71XG81 (G-1)
[式中、
XG21はF又はLであり、
XG31はI、T、S、又はFであり、
XG41はF又はLであり、
XG51はS、N、T、R、又はDであり、
XG61はN、T、又はDであり、
XG71はY、S、又はHであり、
XG81はF、T、Y、W、又はGである]
KLSERY (G-2)
で表されるアミノ酸配列からなる重鎖CDR1と、
下記式(H-1)~(H-3):
IXH21XH31XH41XH51XH61XH71XH81 (H-1)
[式中、
XH21はS、G、K、又はNであり、
XH31はG、N、S、Y、T、又はPであり、
XH41はD、S、T、又はAであり、
XH51はS、G、又はDであり、
XH61はT、S、A、G、又はRであり、
XH71はY、K、N、T、I、又はSであり、
XH81はI、T、P、又はVである]
MWSDGDT (H-2)
NDS (H-3)
のいずれかで表されるアミノ酸配列からなる重鎖CDR2と、
下記式(I-1)~(I-11):
XI11XI21XI31XI41XI51XI61XI71 (I-1)
[式中、
XI11はS又はTであり、
XI21はI又はTであり、
XI31はN、L、D、又はTであり、
XI41はW又はGであり、
XI51はG、V、F、又はNであり、
XI61はD、G、F、又はNであり、
XI71はV又はLである]
ARTSRIVADLVTMGYALDF (I-2)
ARDVVKTGTTGLPFDL (I-3)
AREGVITVGTWGDV (I-4)
ATSEY (I-5)
TTATDV (I-6)
TPATDV (I-7)
SPMTIHGLDT (I-8)
GSGWV (I-9)
GSGWY (I-10)
LSSESDDARV (I-11)
のいずれかで表されるアミノ酸配列からなる重鎖CDR3と、
下記式(J-1)~(J-3):
XJ11LXJ31XJ41XJ51Y (J-1)
[式中、
XJ11はK又はAであり、
XJ31はS又はPであり、
XJ41はN、E、K、T、又はDであり、
XJ51はQ、R、K、又はEである]
SXJ23SLXJ53XJ63XJ73DGXJ103TXJ123 (J-2)
[式中、
XJ23はE又はQであり、
XJ53はL又はVであり、
XJ63はH、D、又はYであり、
XJ73はS又はGであり、
XJ103はN、K、又はYであり、
XJ123はY又はFである]
NIGGKA (J-3)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR1と、
下記式(K-1):
XK11XK21XK31 (K-1)
[式中、
XK11はK、N、L、又はEであり、
XK21はD、G、V、又はIであり、
XK31はS、N、又はDである]
で表されるアミノ酸配列からなる軽鎖CDR2と、
下記式(L-1)~(L-6):
LSXL31XL41XL51XL61XL71XL81XL91XL101XL111 (L-1)[式中、
XL31はR、S、A、又はGであり、
XL41はQ、E、又はDであり、
XL51はS又はGであり、
XL61はD、S、又はNであり、
XL71はD、G、又はTであり、
XL81はA、T、又はYであり、
XL91はA、T、又はYであり、
XL101はS、R、又はWであり、
XL111はV、L、又はYである]
LSXL33XL43XL53XL63XL73XL83XL93XL103 (L-2)
[式中、
XL33はR、S、A、又はGであり、
XL43はQ、E、又はDであり、
XL53はS又はGであり、
XL63はD、S、又はNであり、
XL73はD、G、又はTであり、
XL83はA、T、又はYであり、
XL93はS、R、又はWであり、
XL103はV、L、又はYである]
XL14QXL34XL44XL54XL64PXL84XL94 (L-3)
[式中、
XL14はL又はVであり、
XL34はA又はTであり、
XL44はT又はSであり、
XL54はH又はNであり、
XL64はD、N、又はVであり、
XL84はY、V、I、又はLであり、
XL94はT又はSである]
QXL27XL37XL47XL57PXL77XL87 (L-4)
[式中、
XL27はA又はTであり、
XL37はT又はSであり、
XL47はH又はNであり、
XL57はD、N、又はVであり、
XL77はY、V、I、又はLであり、
XL87はT又はSである]
QVYDSSSFV (L-5)
WQGTDFPR (L-6)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR3と
を含む、抗体又はその断片(抗原結合断片)。
項9.
式(G-1)で表されるアミノ酸配列からなる重鎖CDR1と、
式(H-1)又は(H-2)で表されるアミノ酸配列からなる重鎖CDR2と、
式(I-1)~(I-3)及び(I-5)~(I-9)のいずれかで表されるアミノ酸配列からなる重鎖CDR3と、
式(J-1)~(J-3)のいずれかで表されるアミノ酸配列からなる軽鎖CDR1と、
式(K-1)で表されるアミノ酸配列からなる軽鎖CDR2と、
式(L-1)~(L-3)、(L-5)、及び(L-6)のいずれかで表されるアミノ酸配列からなる軽鎖CDR3と
を含む、項8に記載の抗体又はその断片。
項10.
下記式(G-1-1):
GXG22XG32XG42XG52XG62XG72XG82 (G-1-1)
[式中、
XG22はF又はLであり、
XG32はI、T、又はSであり、
XG42はF又はLであり、
XG52はS、N、T、又はRであり、
XG62はN又はTであり、
XG72はY、S、又はHであり、
XG82はF、T、Y、又はWである]
で表されるアミノ酸配列からなる重鎖CDR1と、
下記式(H-1-1)又は(H-2):
IXH22XH32XH42XH52XH62XH72XH82 (H-1-1)
[式中、
XH22はS、G、K、又はNであり、
XH32はG、N、S、T、又はPであり、
XH42はD、S、又はAであり、
XH52はS、G、又はDであり、
XH62はT、S、G、又はRであり、
XH72はY、K、N、T、I、又はSであり、
XH82はI、T、P、又はVである]
MWSDGDT (H-2)
で表されるアミノ酸配列からなる重鎖CDR2と、
下記式(I-1-1)、(I-2)、(I-3)、及び(I-5)~(I-9):
XI12XI22XI32XI42XI52XI62XI72 (I-1-1)
[式中、
XI12はS又はTであり、
XI22はI又はTであり、
XI32はN、L、又はTであり、
XI42はW又はGであり、
XI52はG、V、F、又はNであり、
XI62はD、F、又はGであり、
XI72はV又はLである]
ARTSRIVADLVTMGYALDF (I-2)
ARDVVKTGTTGLPFDL (I-3)
ATSEY (I-5)
TTATDV (I-6)
TPATDV (I-7)
SPMTIHGLDT (I-8)
GSGWV (I-9)
のいずれかで表されるアミノ酸配列からなる重鎖CDR3と、
下記式(J-1-1)、(J-2-1)、及び(J-3):
KLSXJ42XJ52Y (J-1-1)
[式中、
XJ42はN、E、又はKであり、
XJ52はQ、R、又はEである]
SXJ24SLXJ54XJ64SDGXJ104TXJ124 (J-2-1)
[式中、
XJ24はE又はQであり、
XJ54はL又はVであり、
XJ64はH、D、又はYであり、
XJ104はN、K、又はYであり、
XJ124はY又はFである]
NIGGKA (J-3)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR1と、
下記式(K-1-1)~(K-1-7):
NDD (K-1-1)
KDS (K-1-2)
NGN (K-1-3)
NDS (K-1-4)
KVS (K-1-5)
LIS (K-1-6)
EVS (K-1-7)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR2と、
下記式(L-1-1)、(L-2-1)、(L-3-1)、(L-5)、及び(L-6):
LSXL32XL42SXL62DAAXL102V (L-1-1)
[式中、
XL32はR又はSであり、
XL42はQ又はEであり、
XL62はD又はNであり、
XL102はS、R、又はWである]
LSGESDDAWV (L-2-1)
XL15QXL35THDPYT (L-3-1)
[式中、
XL15はL又はVであり、
XL35はA又はTである]
QVYDSSSFV (L-5)
WQGTDFPR (L-6)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR3と
を含む、抗体又はその断片(抗原結合断片)。
項11.
配列番号74~84のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる重鎖CDR1と、
配列番号85~101のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列、或いはNDSで表されるアミノ酸配列からなる重鎖CDR2と、
配列番号102~118のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる重鎖CDR3と、
配列番号119~133のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる軽鎖CDR1と、
NDD、KDS、NGN、NDS、KVS、LIS、又はEVSのいずれかで表されるアミノ酸配列からなる軽鎖CDR2と、
配列番号134~149のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる軽鎖CDR3と
を含む、項8に記載の抗体又はその断片。
項11a.
配列番号74~81のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる重鎖CDR1と、
配列番号85~90、92~95、及び101のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる重鎖CDR2と、
配列番号102~106、109、110、及び112~116のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる重鎖CDR3と、
配列番号119~121、125~129、及び133のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる軽鎖CDR1と、
NDD、KDS、NGN、NDS、KVS、LIS、又はEVSのいずれかで表されるアミノ酸配列からなる軽鎖CDR2と、
配列番号134~136、140~142、148、及び149のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる軽鎖CDR3と
を含む、項8に記載の抗体又はその断片。
項12.
前記変異が保存的置換である、項11に記載の抗体又はその断片。
項12a.
前記変異が保存的置換である、項11aに記載の抗体又はその断片。
項13.
配列番号2で示されるアミノ酸配列に特異的に結合する、項8~12のいずれかに記載の抗体又はその断片。
項13a.
配列番号2で示されるアミノ酸配列に特異的に結合する、項11a又は12aに記載の抗体又はその断片。
項14.
重症急性呼吸器症候群コロナウイルス2(SARS CoV2)のヌクレオカプシドタンパク質に対する抗体又はその断片であって、エピトープが前記ヌクレオカプシドタンパク質上の配列番号150~152のいずれかに示されるアミノ酸配列からなる領域に存在する、抗体又はその断片(抗原結合断片)。
項15.
前記抗体がモノクローナル抗体である、項1~14のいずれかに記載の抗体又はその断片。
項15a.
前記抗体がモノクローナル抗体である、項5a、6a、11a、及び12aのいずれかに記載の抗体又はその断片。
項16.
項1~15のいずれかに記載の抗体又はその断片のコード配列を含むポリヌクレオチド。
項16a.
項5a、6a、11a、12a、及び15aのいずれかに記載の抗体又はその断片のコード配列を含むポリヌクレオチド。
項17.
項16に記載のポリヌクレオチドを含む細胞。
項17a.
項16aに記載のポリヌクレオチドを含む細胞。
項18.
項1~15のいずれかに記載の抗体又はその断片、項16に記載のポリヌクレオチド、又は項17に記載の細胞を含む試薬又は医薬。
項18a.
項5a、6a、11a、12a、及び15aのいずれかに記載の抗体又はその断片、項16aに記載のポリヌクレオチド、又は項17aに記載の細胞を含む試薬又は医薬。
項19.
項1~15のいずれかに記載の抗体又はその断片を含む、重症急性呼吸器症候群コロナウイルス2(SARS CoV2)のヌクレオカプシドタンパク質を検出するためのキット。
項19a.
項5a、6a、11a、12a、及び15aのいずれかに記載の抗体又はその断片を含む、重症急性呼吸器症候群コロナウイルス2(SARS CoV2)のヌクレオカプシドタンパク質を検出するためのキット。
項20.
配列番号1で示されるアミノ酸配列に特異的に結合する2以上の抗体又はその断片であって、それぞれ配列番号1で示されるアミノ酸配列中の異なるエピトープを認識する2以上の抗体又はその断片、及び/又は、
配列番号2で示されるアミノ酸配列に特異的に結合する2以上の抗体又はその断片であって、それぞれ配列番号2で示されるアミノ酸配列中の異なるエピトープを認識する2以上の抗体又はその断片
を含む、重症急性呼吸器症候群コロナウイルス2(SARS CoV2)のヌクレオカプシドタンパク質を検出するためのキット。
項21.
前記配列番号1で示されるアミノ酸配列に特異的に結合する2以上の抗体又はその断片が、項2~7のいずれかに記載の抗体又はその断片、或いは、当該抗体又はその断片のアミノ酸配列に対して90%以上の配列同一性を有するアミノ酸配列からなる抗体又はその断片であり、
前記配列番号2で示されるアミノ酸配列に特異的に結合する2以上の抗体又はその断片が、項8~14のいずれかに記載の抗体又はその断片、或いは、当該抗体又はその断片のアミノ酸配列に対して90%以上の配列同一性を有するアミノ酸配列からなる抗体又はその断片である、
項20に記載のキット。
項21a.
前記配列番号1で示されるアミノ酸配列に特異的に結合する2以上の抗体又はその断片が、項5a又は6aに記載の抗体又はその断片であり、
前記配列番号2で示されるアミノ酸配列に特異的に結合する2以上の抗体又はその断片が、項11a又は12aに記載の抗体又はその断片である、
項20に記載のキット。
項22.
イムノクロマト法、酵素免疫測定法、化学発光免疫測定法、化学発光酵素免疫測定法、放射免疫測定法、電気化学発光免疫測定法、免疫比濁法、又はラテックス凝集法で重症急性呼吸器症候群コロナウイルス2(SARS CoV2)のヌクレオカプシドタンパク質を検出する、項19~21のいずれかに記載のキット。
項22a.
イムノクロマト法、酵素免疫測定法、化学発光免疫測定法、化学発光酵素免疫測定法、放射免疫測定法、電気化学発光免疫測定法、免疫比濁法、又はラテックス凝集法で重症急性呼吸器症候群コロナウイルス2(SARS CoV2)のヌクレオカプシドタンパク質を検出する、項19a又は21aに記載のキット。
項23.
前記配列番号1又は2で示されるアミノ酸配列に特異的に結合する2以上の抗体又はその断片の一部が固相化されており、残部が標識化されている、項19~22のいずれかに記載のキット。
項23a.
前記配列番号1又は2で示されるアミノ酸配列に特異的に結合する2以上の抗体又はその断片の一部が固相化されており、残部が標識化されている、項19a、21a、及び22aのいずれかに記載のキット。
項24.
重症急性呼吸器症候群コロナウイルス2(SARS CoV2)のヌクレオカプシドタンパク質を検出する方法であって、下記工程(1)~(3):
(1) 配列番号1又は2で示されるアミノ酸配列に特異的に結合する第1の抗体又はその断片を固相に固定化する工程、
(2) 前記固相に、SARS CoV2のヌクレオカプシドタンパク質及び配列番号1又は2で示されるアミノ酸配列に特異的に結合する第2の抗体又はその断片(ここで、第2の抗体又はその断片は、第1の抗体又はその断片が特異的に結合するアミノ酸配列中の異なるエピトープを認識し、標識物質により標識化されている)を適用する工程、及び
(3) 前記固相に、前記ヌクレオカプシドタンパク質を介して結合した第2の抗体又はその断片の標識物質に由来する標識を検出する工程
を含む、方法。
項25.
第1の抗体又はその断片及び第2の抗体又はその断片が、項2~15のいずれかに記載の抗体又はその断片、及び、当該抗体又はその断片のアミノ酸配列に対して90%以上の配列同一性を有するアミノ酸配列からなる抗体又はその断片からなる群より選択される、項24に記載の方法。
項25a.
第1の抗体又はその断片及び第2の抗体又はその断片が、項5a、6a、11a、12a、及び15aのいずれかに記載の抗体又はその断片、及び、当該抗体又はその断片のアミノ酸配列に対して90%以上の配列同一性を有するアミノ酸配列からなる抗体又はその断片からなる群より選択される、項24に記載の方法。
本明細書において、アミノ酸は、天然アミノ酸であっても非天然アミノ酸であってもよい。非天然アミノ酸としては、例えばシトルリン、オルニチン、ε-アセチル-リジン、β-アラニン、アミノ安息香酸、6-アミノカプロン酸、アミノ酪酸、ヒドロキシプロリン、メルカプトプロピオン酸、3-ニトロチロシン、ノルロイシン、ピログルタミン酸等が挙げられるが、これらに限定されない。また、アミノ酸は、例えばL-アミノ酸、D-アミノ酸、又はDL-アミノ酸であってもよい。
SARS CoV2のNタンパク質のNTD(単にNTDと表記する場合がある)は、当該Nタンパク質のN末端から1~172番目のアミノ酸領域を意味し、下記のアミノ酸配列を有する:
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFY(配列番号1)
(P1) 小胞体に対する染色選択性が小胞体以外の細胞小器官に対する染色選択性に比べて高い蛍光標識で標識したSARS CoV2のNタンパク質を用いて、SARS CoV2のNタンパク質で免疫した動物の抗体産生細胞からSARS CoV2のNタンパク質に特異的に結合する抗体を産生する細胞を得る工程、
(P2) 前記工程(P1)で得た細胞から抗体可変領域遺伝子断片を得る工程、
(P3) 前記工程(P2)で得た抗体可変領域遺伝子断片から遺伝子発現ユニットを得る工程、
(P4) 前記工程(P3)で得た遺伝子発現ユニットを細胞に導入してSARS CoV2のNタンパク質に結合する抗体を発現させる工程、
(P5) 前記工程(P4)で発現した抗体を回収する工程、及び
(P6) 前記工程(P5)で回収した抗体からNTDに特異的に結合する抗体を得る工程。
・表2に示す式(A-1)及び(A-2)のいずれかで表されるアミノ酸配列、
・当該アミノ酸配列に対して90%以上(好ましくは95%以上)の同一性を有するアミノ酸配列、又は
・当該アミノ酸配列の1~3個(好ましくは1又は2個、さらに好ましくは1個)のアミノ酸が変異(好ましくは保存的置換)しているアミノ酸配列。
・表3に示す式(B-1)及び(B-2)のいずれかで表されるアミノ酸配列、
・当該アミノ酸配列に対して90%以上(好ましくは95%以上)の同一性を有するアミノ酸配列、又は
・当該アミノ酸配列の1~3個(好ましくは1又は2個、さらに好ましくは1個)のアミノ酸が変異(好ましくは保存的置換)しているアミノ酸配列。
・表4に示す式(C-1)~(C-8)のいずれかで表されるアミノ酸配列、
・当該アミノ酸配列に対して90%以上(好ましくは95%以上)の同一性を有するアミノ酸配列、又は
・当該アミノ酸配列の1~3個(好ましくは1又は2個、さらに好ましくは1個)のアミノ酸が変異(好ましくは保存的置換)しているアミノ酸配列。
・表5に示す式(D-1)~(D-3)のいずれかで表されるアミノ酸配列、
・当該アミノ酸配列に対して90%以上(好ましくは95%以上)の同一性を有するアミノ酸配列、又は
・当該アミノ酸配列の1~3個(好ましくは1又は2個、さらに好ましくは1個)のアミノ酸が変異(好ましくは保存的置換)しているアミノ酸配列。
・表6に示す式(E-1)~(E-3)のいずれかで表されるアミノ酸配列、
・当該アミノ酸配列に対して90%以上(好ましくは95%以上)の同一性を有するアミノ酸配列、又は
・当該アミノ酸配列の1~3個(好ましくは1又は2個、さらに好ましくは1個)のアミノ酸が変異(好ましくは保存的置換)しているアミノ酸配列。
・表7に示す式(F-1)~(F-7)のいずれかで表されるアミノ酸配列、
・当該アミノ酸配列に対して90%以上(好ましくは95%以上)の同一性を有するアミノ酸配列、又は
・当該アミノ酸配列の1~3個(好ましくは1又は2個、さらに好ましくは1個)のアミノ酸が変異(好ましくは保存的置換)しているアミノ酸配列。
SARS CoV2のNタンパク質のCTD(単にCTDと表記する場合がある)は、当該Nタンパク質のN末端から247~419番目のアミノ酸領域を意味し、下記のアミノ酸配列を有する:
TKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTAAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA(配列番号2)
(P6’) 前記工程(P5)で回収した抗体からCTDに特異的に結合する抗体を得る工程。
・表9に示す式(G-1)及び(G-2)のいずれかで表されるアミノ酸配列、
・当該アミノ酸配列に対して90%以上(好ましくは95%以上)の同一性を有するアミノ酸配列、又は
・当該アミノ酸配列の1~3個(好ましくは1又は2個、さらに好ましくは1個)のアミノ酸が変異(好ましくは保存的置換)しているアミノ酸配列。
・表10に示す式(H-1)~(H-3)のいずれかで表されるアミノ酸配列、
・当該アミノ酸配列に対して90%以上(好ましくは95%以上)の同一性を有するアミノ酸配列、又は
・当該アミノ酸配列の1~3個(好ましくは1又は2個、さらに好ましくは1個)のアミノ酸が変異(好ましくは保存的置換)しているアミノ酸配列。
・表11に示す式(I-1)~(I-11)のいずれかで表されるアミノ酸配列、
・当該アミノ酸配列に対して90%以上(好ましくは95%以上)の同一性を有するアミノ酸配列、又は
・当該アミノ酸配列の1~3個(好ましくは1又は2個、さらに好ましくは1個)のアミノ酸が変異(好ましくは保存的置換)しているアミノ酸配列。
・表12に示す式(J-1)~(J-3)のいずれかで表されるアミノ酸配列、
・当該アミノ酸配列に対して90%以上(好ましくは95%以上)の同一性を有するアミノ酸配列、又は
・当該アミノ酸配列の1~3個(好ましくは1又は2個、さらに好ましくは1個)のアミノ酸が変異(好ましくは保存的置換)しているアミノ酸配列。
・表13に示す式(K-1)で表されるアミノ酸配列、
・当該アミノ酸配列に対して90%以上(好ましくは95%以上)の同一性を有するアミノ酸配列、又は
・当該アミノ酸配列の1~3個(好ましくは1又は2個、さらに好ましくは1個)のアミノ酸が変異(好ましくは保存的置換)しているアミノ酸配列。
・表14に示す式(L-1)~(L-6)のいずれかで表されるアミノ酸配列、
・当該アミノ酸配列に対して90%以上(好ましくは95%以上)の同一性を有するアミノ酸配列、又は
・当該アミノ酸配列の1~3個(好ましくは1又は2個、さらに好ましくは1個)のアミノ酸が変異(好ましくは保存的置換)しているアミノ酸配列。
本発明のポリヌクレオチドは、SARS CoV2のNTD又はCTDに特異的に結合する抗体又はその断片のコード配列を含むことが好ましい。当該コード配列としては、例えば上記3に記載の重鎖CDR1~3及び軽鎖CDR1~3を含む抗体又はその断片をコードする塩基配列が挙げられるが、これに限定されない。
本発明の細胞は、上記4に記載したポリヌクレオチドを含むことが好ましい。細胞としては、例えばEscherichia coli K12等の大腸菌、Bacillus subtilis MI114等のバチルス属細菌、Saccharomyces cerevisiae AH22等の酵母、Spodoptera frugiperda由来のSf細胞系もしくはTrichoplusia ni由来のHighFive細胞系、嗅神経細胞等の昆虫細胞、動物細胞等を挙げることができる。動物細胞としては、好ましくは哺乳動物由来の培養細胞、具体的には、COS7細胞、CHO細胞、HEK293細胞、Expi293細胞、293F細胞、293T細胞、293FT細胞、Hela細胞、PC12細胞、N1E-115細胞、SH-SY5Y細胞等が挙げられる。
本発明の試薬又は医薬は、SARS CoV2のNTD又はCTDに特異的に結合する抗体又はその断片、当該抗体又はその断片のコード配列を含むポリヌクレオチド、又は当該ポリヌクレオチドを含む細胞を含むことが好ましい。本発明の試薬又は医薬は、さらに薬学的に許容可能な賦形剤もしくは担体及び/又は添加剤を含むことが好ましい。
本発明のSARS CoV2のNタンパク質を検出するためのキットは、SARS CoV2のNTD又はCTDに特異的に結合する抗体又はその断片を含むことが好ましい。当該抗体又はその断片は、例えば上記6に記載した試薬の形態であってもよい。
・SARS CoV2のNTDに特異的に結合する2以上の抗体又はその断片であって、それぞれNTDの異なるエピトープを認識する2以上の抗体又はその断片、及び/又は
・SARS CoV2のCTDに特異的に結合する2以上の抗体又はその断片であって、それぞれCTDの異なるエピトープを認識する2以上の抗体又はその断片
を含むことが好ましい。
SARS CoV2のNタンパク質を検出する方法は、下記の工程(1)~(3):
(1) NTD又はCTDに特異的に結合する第1の抗体又はその断片を固相に固定化する工程、
(2) 前記固相に、SARS CoV2のNタンパク質及び配列番号1又は2で示されるアミノ酸配列に特異的に結合する第2の抗体又はその断片(ここで、第2の抗体又はその断片は、第1の抗体又はその断片が特異的に結合するアミノ酸配列中の異なるエピトープを認識し、標識物質により標識化されている)を適用する工程、及び
(3) 前記固相に、前記ヌクレオカプシドタンパク質を介して結合した第2の抗体又はその断片の標識物質に由来する標識を検出する工程
を含むことが好ましい。
初回免疫として、PBSに懸濁した195μgのSARS-CoV2 Nタンパク質を、アジュバントであるTiterMAX Gold(フナコシ株式会社)と混合し、モルモットの左右後肢筋肉内に投与した。初回免疫より14日後に、PBSに懸濁した100μgのSARS-CoV2 Nタンパク質をTiterMAX Goldと混合し、モルモットの左右後肢筋肉内に追加免疫した。さらに、追加免疫より31日後に、PBSに懸濁した100μgのSARS-CoV2 Nタンパク質をモルモットの左右後肢筋肉内に再度追加免疫した。2度目の追加免疫より8日後に、2匹のモルモットを麻酔下全採血および安楽死後、腰部リンパ節を摘出した。
「形質細胞同定及び単離用蛍光プローブ並びにこのプローブを用いた形質細胞の同定又は単離方法」(米国特許出願公開2013/0029325号)の方法(ERIAA法)に準じて実施した。
ソーティングによって取得した各細胞について、Kurosawaらの方法(MAGrahd法)に準じてmRNAの抽出、cDNAの合成、および抗体可変領域遺伝子断片の増幅を行った(Rapid production of antigen-specific monoclonal antibodies from a variety of animals, BMC Biology 2012, 10:80)。
増幅した抗体可変領域遺伝子断片から、「標的遺伝子由来配列を含む連結DNA断片の特異的作製方法」(米国特許出願公開第2013/0023009号)の方法(TS-jPCR法)に準じて抗体遺伝子発現ユニットを作製した。
作製した抗体遺伝子発現ユニットとFuGene HD Transfection Reagent(商標)とOpti-MEM(商標)とを1:1:50の液量比で混合して20分間常温で放置したのち、10%(w/v) FBS/IMDM培地で懸濁した293FT細胞に添加することで抗体遺伝子発現ユニットを導入した。抗体遺伝子発現ユニットを導入した293FT細胞を、24ウェルプレートに播種し、37℃、8%(v/v) CO2環境下で2日間インキュベートしたのち、培養上清を回収した。培養上清中に放出された抗体を利用して、以降のスクリーニングを実施した。
各抗体のSARS-CoV2 Nタンパク質との反応強度をELISAによって評価することによって、スクリーニングを行った。ELISAはSARS-CoV2 Nタンパク質15ng/ウェルとなるように96穴プレート底面に固相化し、抗体を一過性に発現させた293FT細胞の培養上清を100μL加えて、室温にて2時間抗原抗体反応を行った。抗原に結合したモルモット抗体を、西洋ワサビペルオキシダーゼが結合したヤギ抗モルモット抗体を用いて検出し、マイクロプレートリーダーを用いて450nmの吸光度を測定した。
取得した抗体がSARS-CoV2 Nタンパク質のN末端領域(NTD)およびC末端領域(CTD)のいずれを認識するのかを確認するため、293FT細胞中でNTDおよびCTDをそれぞれコードするDNA断片を一過性に発現させ、取得した抗体を添加して免疫染色を行うことで、抗体とNTDおよびCTDをそれぞれコードするDNA断片との反応性を評価した。
ELISAによる評価の結果、反応性が良好であった抗体について、作製した遺伝子発現ユニットをExpi293細胞にトランスフェクションすることによって、抗体を作製した。トランスフェクションにあたっては、Expi293 Expression Medium(GIBCO社)およびExpi Fectamine 293 Transfection Kit(GIBCO社)を利用した。E250培養フラスコ1本あたり、作製した各抗体の遺伝子発現ユニット50μgとOptiMEM I 3mLとExpi Fectamine 293 0.16mLとを混和して10分間静置後、Expi293細胞へ添加することで遺伝子導入した。1日間培養後、Expi Fectamine 293 Transfection Enhancerをフィード添加しさらに4日間培養した後、上清を回収しプロテインAカラムで精製することにより抗体を取得した。
ビオチン標識した各クローン(固相化抗体)をTBSで5 μg/mLに希釈し、96穴マイクロプレートに50 μL/well加え、室温で2時間コーティングさせた。1%(w/v) BSA-TBS-Tでブロッキングし、TBS-Tで洗浄後、0.25 μg/mLアルカリホスファターゼ標識した各クローン(検出抗体)および10 ng/mL 抗原溶液(1%(w/v)TBS-T希釈)を50 μL/wellずつ添加し、室温で2時間反応させた。なお、抗原溶液としては、全長のSARS-CoV2 Nタンパク質の試料1に加えて、SARS-CoV2 Nタンパク質の分解が生じた検体を模してSARS-CoV2 Nタンパク質の全量を等mol量のNTDおよびCTDで置換した試料2を用いた。TBS-Tで洗浄後、発色基質としてpNPPを100μL/well添加し、室温で15分間反応させた後、マイクロプレートリーダーを用いて405 nmの吸光度を測定した。結果を表20に示す。なお、表20の%は試料1の吸光度測定値に対する相対吸光度値を表す。
ビオチン標識した複数のクローンを混合した固相化抗体をTBSで5 μg/mLに希釈し、96穴マイクロプレートに50 μL/well加え、室温で2時間コーティングさせた。1%(w/v)BSA-TBS-Tでブロッキングし、TBS-Tで洗浄後、アルカリホスファターゼ標識した複数のクローンを混合した0.25 μg/mL検出抗体および10 ng/mL 抗原溶液(1%(w/v)TBS-T希釈)を50 μL/wellずつ添加し、室温で2時間反応させた。なお、2種類の固相化抗体を使用する場合、各固相化抗体の添加量は1/2にして総量が50μL/wellとなるように添加した。2種類の検出抗体を使用する場合も同様である。抗原溶液としては、全長のSARS-CoV2 Nタンパク質の試料1に加えて、SARS-CoV2 Nタンパク質の分解が生じた検体を模してSARS-CoV2 Nタンパク質の全量を等mol量のNTDおよびCTDで置換した試料2を用いた。TBS-Tで洗浄後、発色基質としてpNPPを100uL/well添加し、室温で15分間反応させた後、マイクロプレートリーダーを用いて405 nmの吸光度を測定した。結果を表21に示す。
エピトープマッピングはPEPperPRINT社のConformational Epitope Mappingsサービスを利用して決定した。SARS CoV 2のNタンパク質のN末端から200~419番目のアミノ酸領域の内、7、10、13アミノ酸からなるペプチドを6、9、12アミノ酸ずつ重複するように1アミノ酸ずつずらしてペプチドアレイ上に合成し、抗体の反応性すなわち検出シグナルにより、どのペプチドが抗体結合のエピトープであるかを特定した。
モノクローナル抗体のエピトープマッピングにより、
表18に示されるクローンのうち、3つのクローンのエピトープが、それぞれ、配列番号150:LLPAADLDDFSK (SARS CoV 2のNタンパク質のN末端から394~405番目)、配列番号151:KKADETQAL (SARS CoV 2のNタンパク質のN末端から374~382番目)、配列番号152:SAAEASKK (SARS CoV 2のNタンパク質のN末端から250~257番目)の領域にあることが確認された。
Claims (25)
- 配列番号1又は2で示されるアミノ酸配列に特異的に結合する抗体又はその断片。
- 下記式(A-1)又は(A-2):
GFXA31XA41XA51XA61XA71XA81 (A-1)
[式中、
XA31はT又はSであり、
XA41はF、L、又はIであり、
XA51はD、S、N、又はTであり、
XA61はD、N、S、I、又はTであり、
XA71はY、F、又はNであり、
XA81はG、W、Y、S又はTである]
GFXA33XA43XA53XA63XA73XA83XA93XA103 (A-2)
[式中、
XA33はT又はSであり、
XA43はF、L、又はIであり、
XA53はD、S、N、又はTであり、
XA63はD、N、S、又はTであり、
XA73はN又はSであり、
XA83はG又はYであり、
XA93はY、F、又はNであり、
XA103はT、G、W、又はYである]
で表されるアミノ酸配列からなる重鎖CDR1と、
下記式(B-1)又は(B-2):
IXB21XB31XB41XB51XB61XB71XB81 (B-1)
[式中、
XB21はS、N、D、W、又はHであり、
XB31はY、G、P、又はSであり、
XB41はS、D、又はGであり、
XB51はG、S、T、又はAであり、
XB61はG、D、T、N、又はRであり、
XB71はR、K、T、S、N、G、Y、又はDであり、
XB81はT、I、又はMである]
IXB23XB33XB43XB53XB63XB73 (B-2)
[式中、
XB23はS、N、D、W、又はHであり、
XB33はY、G、P、又はSであり、
XB43はS、D、又はGであり、
XB53はG、S、T、又はAであり、
XB63はR、K、T、S、N、G、Y、又はDであり、
XB73はT、I、又はMである]
で表されるアミノ酸配列からなる重鎖CDR2と、
下記式(C-1)~(C-8):
XC11XC21XC31XC41XC51XC61XC71XC81XC91XC101XC111XC121XC131XC141XC151 (C-1)
[式中、
XC11はA又はSであり、
XC21はR、T、又はKであり、
XC31はD、N、S、又はYであり、
XC41はG、S、又はYであり、
XC51はG、I、又はDであり、
XC61はS又はIであり、
XC71はY、V、又はAであり、
XC81はY、H、S、T、K、又はEであり、
XC91はS、I、又はDであり、
XC101はP又はYであり、
XC111はI、Y、G、P、T、A、又はVであり、
XC121はN、S、Y、P、G、又はFであり、
XC131はF、C、M、又はLであり、
XC141はQ又はDであり、
XC151はF、Y、V、又はSである]
XC13XC23XC33XC43XC53XC63XC73XC83 (C-2)
[式中、
XC13はA又はSであり、
XC23はR、T、又はKであり、
XC33はD、N、S、又はYであり、
XC43はI、Y、G、P、T、A、又はVであり、
XC53はN、S、Y、P、G、又はFであり、
XC63はF、C、M、又はLであり、
XC73はQ又はDであり、
XC83はF、Y、V、又はSである]
XC15XC25XC35XC45XC55XC65XC75XC85XC95TIFTFXC155XC165XC175XC185XC195 (C-3)
[式中、
XC15はA又はSであり、
XC25はR、T、又はKであり、
XC35はG、S、又はYであり、
XC45はG、I、又はDであり、
XC55はS又はIであり、
XC65はY、V、又はAであり、
XC75はY、H、S、T、K、又はEであり、
XC85はS、I、又はDであり、
XC95はP又はYであり、
XC155はI、Y、G、P、T、A、又はVであり、
XC165はN、S、Y、P、G、又はFであり、
XC175はF、C、M、又はLであり、
XC185はQ又はDであり、
XC195はF、Y、V、又はSである]
ARLWSGYALDI (C-4)
XC16XC26XC36XC46XC56XC66XC76XC86XC96XC106XC116XC126XC136 (C-5)
[式中、
XC16はA又はSであり、
XC26はR、T、又はKであり、
XC36はD、N、S、又はYであり、
XC46はG、S、又はYであり、
XC56はG、I、又はDであり、
XC66はY、H、S、T、K、又はEであり、
XC76はS、I、又はDであり、
XC86はP又はYであり、
XC96はI、Y、G、P、T、A、又はVであり、
XC106はN、S、Y、P、G、又はFであり、
XC116はF、C、M、又はLであり、
XC126はQ又はDであり、
XC136はF、Y、V、又はSである]
VXC27PLLVXC77HGXC107XC117XC127XC137XC147 (C-6)
[式中、
XC27はS又はKであり、
XC77はV、Y、又はLであり、
XC107はI、Y、G、P、T、A、又はVであり、
XC117はN、S、Y、P、G、又はFであり、
XC127はF、C、M、又はLであり、
XC137はQ又はDであり、
XC147はF、Y、V、又はSである]
VSPSXC58XC68XC78XC88XC98XC108 (C-7)
[式中、
XC58はG又はAであり、
XC68はI、Y、G、P、T、A、又はVであり、
XC78はN、S、Y、P、G、又はFであり、
XC88はF、C、M、又はLであり、
XC98はQ又はDであり、
XC108はF、Y、V、又はSである]
SPSXC49XC59XC69XC79XC89XC99 (C-8)
[式中、
XC49はG又はAであり、
XC59はI、Y、G、P、T、A、又はVであり、
XC69はN、S、Y、P、G、又はFであり、
XC79はF、C、M、又はLであり、
XC89はQ又はDであり、
XC99はF、Y、V、又はSである]
のいずれかで表されるアミノ酸配列からなる重鎖CDR3と、
下記式(D-1)~(D-3):
SXD21XD31XD41XD51XD61XD71 (D-1)
[式中、
XD21はE、D、Q、又はGであり、
XD31はH、Y、G、又はSであり、
XD41はR、S、I、K、又はLであり、
XD51はS、H、N、又はFであり、
XD61はY又はKであり、
XD71はY、S、W、又はNである]
SXD23XD33XD43XD53XD63XD73GKNKDL (D-2)
[式中、
XD23はE、D、Q、又はGであり、
XD33はH、Y、G、又はSであり、
XD43はR、S、I、K、又はLであり、
XD53はS、H、N、又はFであり、
XD63はY又はKであり、
XD73はY、S、W、又はNである]
NIGGKT (D-3)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR1と、
下記式(E-1)~(E-3):
XE11XE21SXE41GSXE71(E-1)
[式中、
XE11はL、V、又はIであり、
XE21はK、N、又はEであり、
XE41はD、Y、又はKであり、
XE71はH又はLである]
VGTSDIVG (E-2)
XE12XE22XE32 (E-3)
[式中、
XE12はK、S、又はWであり、
XE22はA又はHであり、
XE32はN又はSである]
のいずれかで表されるアミノ酸配列からなる軽鎖CDR2と、
下記式(F-1)~(F-7):
GXF21XF31XF41XF51XF61XF71XF81XF91XF101 (F-1)
[式中、
XF21はV又はAであり、
XF31はS、D、N、G、又はKであり、
XF41はY、F、V、L、又はHであり、
XF51はS、K、又はNであり、
XF61はG又はNであり、
XF71はG、A、E、又はKであり、
XF81はH、S、Y、又はVであり、
XF91はN、G、又はSであり、
XF101はI、V、L、又はYである]
GXF23XF33XF43XF53XF63XF73XF83XF93YXF113 (F-2)
[式中、
XF23はV又はAであり、
XF33はS、D、N、又はGであり、
XF43はY、F、V、L、又はHであり、
XF53はS、K、又はNであり、
XF63はG又はNであり、
XF73はG、A、E、又はKであり、
XF83はH、S、又はYであり、
XF93はN、G、又はSであり、
XF113はI、V、L、又はYである]
GXF24XF34XF44XF54IXF74XF84XF94YXF114YXF134 (F-3)
[式中、
XF24はV又はAであり、
XF34はS、D、N、又はGであり、
XF44はY、F、V、L、又はHであり、
XF54はS、K、又はNであり、
XF74はG又はNであり、
XF84はG、A、E、又はKであり、
XF94はH、S、又はYであり、
XF114はN、G、又はSであり、
XF134はI、V、L、又はYである]
GXF25XF35XF45XF55XF65XF75XF85XF95(F-4)
[式中、
XF25はV又はAであり、
XF35はS、D、N、G、又はKであり、
XF45はY、F、V、L、又はHであり、
XF55はS、K、又はNであり、
XF65はG又はNであり、
XF75はG、A、E、又はKであり、
XF85はH、S、Y、又はVであり、
XF105はI、V、L、又はYである]
QYDSTPPLT (F-5)
QVWDSYTYV (F-6)
QFINGPLT (F-7)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR3と
を含む、抗体又はその断片。 - 式(A-1)で表されるアミノ酸配列からなる重鎖CDR1と、
式(B-1)又は(B-2)で表されるアミノ酸配列からなる重鎖CDR2と、
式(C-1)、(C-2)、(C-4)、及び(C-6)~(C-8)のいずれかで表されるアミノ酸配列からなる重鎖CDR3と、
式(D-1)で表されるアミノ酸配列からなる軽鎖CDR1と、
式(E-1)~(E-3)のいずれかで表されるアミノ酸配列からなる軽鎖CDR2と、
式(F-1)~(F-5)のいずれかで表されるアミノ酸配列からなる軽鎖CDR3と
を含む、請求項2に記載の抗体又はその断片。 - 下記式(A-1-1):
GFXA32XA42XA52XA62XA72XA82 (A-1-1)
[式中、
XA32はT又はSであり、
XA42はF又はLであり、
XA52はD、S、N、又はTであり、
XA62はD、S、又はNであり、
XA72はY又はFであり、
XA82はG、W、S、又はYである]
で表されるアミノ酸配列からなる重鎖CDR1と、
下記式(B-1-1)及び(B-2-1):
IXB22XB32XB42XB52XB62XB72XB82 (B-1-1)
[式中、
XB22はS、N、D、又はHであり、
XB32はY、G、又はPであり、
XB42はS又はDであり、
XB52はG又はSであり、
XB62はG、D、T、又はNであり、
XB72はR、K、T、S、G、又はYであり、
XB82はT又はIである]
IWXB34XB44XB54XB64XB74 (B-2-1)
[式中、
XB34はY又はSであり、
XB44はD又はGであり、
XB54はG又はAであり、
XB64はR又はDであり、
XB74はT又はIである]
のいずれかで表されるアミノ酸配列からなる重鎖CDR2と、
下記式(C-1-1)、(C-2-1)、(C-6-1)、(C-7-1)、及び(C-8-1):
XC12RDGGSYXC82SPIXC122XC132QXC152 (C-1-1)
[式中、
XC12はA又はSであり、
XC82はY、H、又はSであり、
XC122はN又はSであり、
XC132はF又はCであり、
XC152はF又はYである]
ATXC34XC44XC54XC64XC74XC84 (C-2-1)
[式中、
XC34はN又はSであり、
XC44はY又はGであり、
XC54はN又はPであり、
XC64はF又はMであり、
XC74はQ又はDであり、
XC84はY又はVである]
VSPLLVVHGPFFQY (C-6-1)
VSPSGAGLDV (C-7-1)
SPSAVGFDV (C-8-1)
のいずれかで表されるアミノ酸配列からなる重鎖CDR3と、
下記式(D-1-1)~(D-1-7):
SEYKHYN (D-1-1)
SEHRSYY (D-1-2)
SDYSHYS (D-1-3)
SQGINKW (D-1-4)
SGHSSYY (D-1-5)
SEHSSYY (D-1-6)
SDHSSYY (D-1-7)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR1と、
下記式(E-1-1)、(E-2)、及び(E-3-1):
XE12XE22SDGSXE72 (E-1-1)
[式中、
XE12はL、V、又はIであり、
XE22はK、又はEであり、
XE72はH、又はLである]
VGTSDIVG (E-2)
KAN (E-3-1)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR2と、
下記式(F-1-1)、(F-2-1)、(F-3-1)、(F-4-1)、及び(F-5):
GVSXF42SGGHXF92XF102 (F-1-1)
[式中、
XF42はY又はFであり、
XF92はN又はGであり、
XF102はI又はVである]
GADYSGASSYV (F-2-1)
GANHNIGESYGYV (F-3-1)
GVXF36YXF56XF66XF76XF86XF96 (F-4-1)
[式中、
XF36はS、G、又はKであり、
XF56はS又はNであり、
XF66はG又はNであり、
XF76はG又はKであり、
XF86はY又はVであり、
XF96はV又はYである]
QYDSTPPLT (F-5)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR3と
を含む、抗体又はその断片。 - 配列番号3~11のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる重鎖CDR1と、
配列番号12~25のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる重鎖CDR2と、
配列番号26~41のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる重鎖CDR3と、
配列番号42~50のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる軽鎖CDR1と、
配列番号51~58のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列、或いはKAN、SHN、又はWASのいずれかで表されるアミノ酸配列からなる軽鎖CDR2と、
配列番号59~73のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる軽鎖CDR3と
を含む、請求項2に記載の抗体又はその断片。 - 前記変異が保存的置換である、請求項5に記載の抗体又はその断片。
- 配列番号1で示されるアミノ酸配列に特異的に結合する、請求項2~6のいずれかに記載の抗体又はその断片。
- 下記式(G-1)又は(G-2):
GXG21XG31XG41XG51XG61XG71XG81 (G-1)
[式中、
XG21はF又はLであり、
XG31はI、T、S、又はFであり、
XG41はF又はLであり、
XG51はS、N、T、R、又はDであり、
XG61はN、T、又はDであり、
XG71はY、S、又はHであり、
XG81はF、T、Y、W、又はGである]
KLSERY (G-2)
で表されるアミノ酸配列からなる重鎖CDR1と、
下記式(H-1)~(H-3):
IXH21XH31XH41XH51XH61XH71XH81 (H-1)
[式中、
XH21はS、G、K、又はNであり、
XH31はG、N、S、Y、T、又はPであり、
XH41はD、S、T、又はAであり、
XH51はS、G、又はDであり、
XH61はT、S、A、G、又はRであり、
XH71はY、K、N、T、I、又はSであり、
XH81はI、T、P、又はVである]
MWSDGDT (H-2)
NDS (H-3)
のいずれかで表されるアミノ酸配列からなる重鎖CDR2と、
下記式(I-1)~(I-11):
XI11XI21XI31XI41XI51XI61XI71 (I-1)
[式中、
XI11はS又はTであり、
XI21はI又はTであり、
XI31はN、L、D、又はTであり、
XI41はW又はGであり、
XI51はG、V、F、又はNであり、
XI61はD、G、F、又はNであり、
XI71はV又はLである]
ARTSRIVADLVTMGYALDF (I-2)
ARDVVKTGTTGLPFDL (I-3)
AREGVITVGTWGDV (I-4)
ATSEY (I-5)
TTATDV (I-6)
TPATDV (I-7)
SPMTIHGLDT (I-8)
GSGWV (I-9)
GSGWY (I-10)
LSSESDDARV (I-11)
のいずれかで表されるアミノ酸配列からなる重鎖CDR3と、
下記式(J-1)~(J-3):
XJ11LXJ31XJ41XJ51Y (J-1)
[式中、
XJ11はK又はAであり、
XJ31はS又はPであり、
XJ41はN、E、K、T、又はDであり、
XJ51はQ、R、K、又はEである]
SXJ23SLXJ53XJ63XJ73DGXJ103TXJ123 (J-2)
[式中、
XJ23はE又はQであり、
XJ53はL又はVであり、
XJ63はH、D、又はYであり、
XJ73はS又はGであり、
XJ103はN、K、又はYであり、
XJ123はY又はFである]
NIGGKA (J-3)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR1と、
下記式(K-1):
XK11XK21XK31 (K-1)
[式中、
XK11はK、N、L、又はEであり、
XK21はD、G、V、又はIであり、
XK31はS、N、又はDである]
で表されるアミノ酸配列からなる軽鎖CDR2と、
下記式(L-1)~(L-6):
LSXL31XL41XL51XL61XL71XL81XL91XL101XL111 (L-1)[式中、
XL31はR、S、A、又はGであり、
XL41はQ、E、又はDであり、
XL51はS又はGであり、
XL61はD、S、又はNであり、
XL71はD、G、又はTであり、
XL81はA、T、又はYであり、
XL91はA、T、又はYであり、
XL101はS、R、又はWであり、
XL111はV、L、又はYである]
LSXL33XL43XL53XL63XL73XL83XL93XL103 (L-2)
[式中、
XL33はR、S、A、又はGであり、
XL43はQ、E、又はDであり、
XL53はS又はGであり、
XL63はD、S、又はNであり、
XL73はD、G、又はTであり、
XL83はA、T、又はYであり、
XL93はS、R、又はWであり、
XL103はV、L、又はYである]
XL14QXL34XL44XL54XL64PXL84XL94 (L-3)
[式中、
XL14はL又はVであり、
XL34はA又はTであり、
XL44はT又はSであり、
XL54はH又はNであり、
XL64はD、N、又はVであり、
XL84はY、V、I、又はLであり、
XL94はT又はSである]
QXL27XL37XL47XL57PXL77XL87 (L-4)
[式中、
XL27はA又はTであり、
XL37はT又はSであり、
XL47はH又はNであり、
XL57はD、N、又はVであり、
XL77はY、V、I、又はLであり、
XL87はT又はSである]
QVYDSSSFV (L-5)
WQGTDFPR (L-6)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR3と
を含む、抗体又はその断片。 - 式(G-1)で表されるアミノ酸配列からなる重鎖CDR1と、
式(H-1)又は(H-2)で表されるアミノ酸配列からなる重鎖CDR2と、
式(I-1)~(I-3)及び(I-5)~(I-9)のいずれかで表されるアミノ酸配列からなる重鎖CDR3と、
式(J-1)~(J-3)のいずれかで表されるアミノ酸配列からなる軽鎖CDR1と、
式(K-1)で表されるアミノ酸配列からなる軽鎖CDR2と、
式(L-1)~(L-3)、(L-5)、及び(L-6)のいずれかで表されるアミノ酸配列からなる軽鎖CDR3と
を含む、請求項8に記載の抗体又はその断片。 - 下記式(G-1-1):
GXG22XG32XG42XG52XG62XG72XG82 (G-1-1)
[式中、
XG22はF又はLであり、
XG32はI、T、又はSであり、
XG42はF又はLであり、
XG52はS、N、T、又はRであり、
XG62はN又はTであり、
XG72はY、S、又はHであり、
XG82はF、T、Y、又はWである]
で表されるアミノ酸配列からなる重鎖CDR1と、
下記式(H-1-1)又は(H-2):
IXH22XH32XH42XH52XH62XH72XH82 (H-1-1)
[式中、
XH22はS、G、K、又はNであり、
XH32はG、N、S、T、又はPであり、
XH42はD、S、又はAであり、
XH52はS、G、又はDであり、
XH62はT、S、G、又はRであり、
XH72はY、K、N、T、I、又はSであり、
XH82はI、T、P、又はVである]
MWSDGDT (H-2)
で表されるアミノ酸配列からなる重鎖CDR2と、
下記式(I-1-1)、(I-2)、(I-3)、及び(I-5)~(I-9):
XI12XI22XI32XI42XI52XI62XI72 (I-1-1)
[式中、
XI12はS又はTであり、
XI22はI又はTであり、
XI32はN、L、又はTであり、
XI42はW又はGであり、
XI52はG、V、F、又はNであり、
XI62はD、F、又はGであり、
XI72はV又はLである]
ARTSRIVADLVTMGYALDF (I-2)
ARDVVKTGTTGLPFDL (I-3)
ATSEY (I-5)
TTATDV (I-6)
TPATDV (I-7)
SPMTIHGLDT (I-8)
GSGWV (I-9)
のいずれかで表されるアミノ酸配列からなる重鎖CDR3と、
下記式(J-1-1)、(J-2-1)、及び(J-3):
KLSXJ42XJ52Y (J-1-1)
[式中、
XJ42はN、E、又はKであり、
XJ52はQ、R、又はEである]
SXJ24SLXJ54XJ64SDGXJ104TXJ124 (J-2-1)
[式中、
XJ24はE又はQであり、
XJ54はL又はVであり、
XJ64はH、D、又はYであり、
XJ104はN、K、又はYであり、
XJ124はY又はFである]
NIGGKA (J-3)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR1と、
下記式(K-1-1)~(K-1-7):
NDD (K-1-1)
KDS (K-1-2)
NGN (K-1-3)
NDS (K-1-4)
KVS (K-1-5)
LIS (K-1-6)
EVS (K-1-7)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR2と、
下記式(L-1-1)、(L-2-1)、(L-3-1)、(L-5)、及び(L-6):
LSXL32XL42SXL62DAAXL102V (L-1-1)
[式中、
XL32はR又はSであり、
XL42はQ又はEであり、
XL62はD又はNであり、
XL102はS、R、又はWである]
LSGESDDAWV (L-2-1)
XL15QXL35THDPYT (L-3-1)
[式中、
XL15はL又はVであり、
XL35はA又はTである]
QVYDSSSFV (L-5)
WQGTDFPR (L-6)
のいずれかで表されるアミノ酸配列からなる軽鎖CDR3と
を含む、抗体又はその断片。 - 配列番号74~84のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる重鎖CDR1と、
配列番号85~101のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列、或いはNDSで表されるアミノ酸配列からなる重鎖CDR2と、
配列番号102~118のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる重鎖CDR3と、
配列番号119~133のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる軽鎖CDR1と、
NDD、KDS、NGN、NDS、KVS、LIS、又はEVSのいずれかで表されるアミノ酸配列からなる軽鎖CDR2と、
配列番号134~149のいずれかに示されるアミノ酸配列又はこれらのアミノ酸配列において1~3個のアミノ酸が変異しているアミノ酸配列からなる軽鎖CDR3と
を含む、請求項8に記載の抗体又はその断片。 - 前記変異が保存的置換である、請求項11に記載の抗体又はその断片。
- 配列番号2で示されるアミノ酸配列に特異的に結合する、請求項8~12のいずれかに記載の抗体又はその断片。
- 重症急性呼吸器症候群コロナウイルス2(SARS CoV2)のヌクレオカプシドタンパク質に対する抗体又はその断片であって、エピトープが前記ヌクレオカプシドタンパク質上の配列番号150~152のいずれかに示されるアミノ酸配列からなる領域に存在する、抗体又はその断片。
- 前記抗体がモノクローナル抗体である、請求項1~14のいずれかに記載の抗体又はその断片。
- 請求項1~15のいずれかに記載の抗体又はその断片のコード配列を含むポリヌクレオチド。
- 請求項16に記載のポリヌクレオチドを含む細胞。
- 請求項1~15のいずれかに記載の抗体又はその断片、請求項16に記載のポリヌクレオチド、又は請求項17に記載の細胞を含む試薬又は医薬。
- 請求項1~15のいずれかに記載の抗体又はその断片を含む、重症急性呼吸器症候群コロナウイルス2(SARS CoV2)のヌクレオカプシドタンパク質を検出するためのキット。
- 配列番号1で示されるアミノ酸配列に特異的に結合する2以上の抗体又はその断片であって、それぞれ配列番号1で示されるアミノ酸配列中の異なるエピトープを認識する2以上の抗体又はその断片、及び/又は、
配列番号2で示されるアミノ酸配列に特異的に結合する2以上の抗体又はその断片であって、それぞれ配列番号2で示されるアミノ酸配列中の異なるエピトープを認識する2以上の抗体又はその断片
を含む、重症急性呼吸器症候群コロナウイルス2(SARS CoV2)のヌクレオカプシドタンパク質を検出するためのキット。 - 前記配列番号1で示されるアミノ酸配列に特異的に結合する2以上の抗体又はその断片が、請求項2~7のいずれかに記載の抗体又はその断片、或いは、当該抗体又はその断片のアミノ酸配列に対して90%以上の配列同一性を有するアミノ酸配列からなる抗体又はその断片であり、
前記配列番号2で示されるアミノ酸配列に特異的に結合する2以上の抗体又はその断片が、請求項8~14のいずれかに記載の抗体又はその断片、或いは、当該抗体又はその断片のアミノ酸配列に対して90%以上の配列同一性を有するアミノ酸配列からなる抗体又はその断片である、
請求項20に記載のキット。 - イムノクロマト法、酵素免疫測定法、化学発光免疫測定法、化学発光酵素免疫測定法、放射免疫測定法、電気化学発光免疫測定法、免疫比濁法、又はラテックス凝集法で重症急性呼吸器症候群コロナウイルス2(SARS CoV2)のヌクレオカプシドタンパク質を検出する、請求項19~21のいずれかに記載のキット。
- 前記配列番号1又は2で示されるアミノ酸配列に特異的に結合する2以上の抗体又はその断片の一部が固相化されており、残部が標識化されている、請求項19~22のいずれかに記載のキット。
- 重症急性呼吸器症候群コロナウイルス2(SARS CoV2)のヌクレオカプシドタンパク質を検出する方法であって、下記工程(1)~(3):
(1) 配列番号1又は2で示されるアミノ酸配列に特異的に結合する第1の抗体又はその断片を固相に固定化する工程、
(2) 前記固相に、SARS CoV2のヌクレオカプシドタンパク質及び配列番号1又は2で示されるアミノ酸配列に特異的に結合する第2の抗体又はその断片(ここで、第2の抗体又はその断片は、第1の抗体又はその断片が特異的に結合するアミノ酸配列中の異なるエピトープを認識し、標識物質により標識化されている)を適用する工程、及び
(3) 前記固相に、前記ヌクレオカプシドタンパク質を介して結合した第2の抗体又はその断片の標識物質に由来する標識を検出する工程
を含む、方法。 - 第1の抗体又はその断片及び第2の抗体又はその断片が、請求項2~15のいずれかに記載の抗体又はその断片、及び、当該抗体又はその断片のアミノ酸配列に対して90%以上の配列同一性を有するアミノ酸配列からなる抗体又はその断片からなる群より選択される、請求項24に記載の方法。
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