WO2023199955A1 - 抗ＳＡＲＳ－ＣｏＶ－２抗体 - Google Patents

抗ＳＡＲＳ－ＣｏＶ－２抗体 Download PDF

Info

Publication number
WO2023199955A1
WO2023199955A1 PCT/JP2023/014930 JP2023014930W WO2023199955A1 WO 2023199955 A1 WO2023199955 A1 WO 2023199955A1 JP 2023014930 W JP2023014930 W JP 2023014930W WO 2023199955 A1 WO2023199955 A1 WO 2023199955A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
sequence shown
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2023/014930
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
佑太 松村
峻亮 稲浦
大貴 西口
重文 熊地
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kao Corp
Epsilon Molecular Engineering Inc
Original Assignee
Kao Corp
Epsilon Molecular Engineering Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kao Corp, Epsilon Molecular Engineering Inc filed Critical Kao Corp
Publication of WO2023199955A1 publication Critical patent/WO2023199955A1/ja
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • 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

Definitions

  • the present invention relates to antibodies that bind to the nucleocapsid protein of SARS-CoV-2.
  • SARS coronavirus-2 (Severe acute respiratory syndrome coronavirus 2,; SARS-CoV-2) belongs to the betacoronavirus genus, like SARS coronavirus (SARS-CoV) and MERS coronavirus (MERS-CoV). belongs to acute respiratory It is a SARS-related coronavirus that causes COVID-19. The outbreak was first confirmed in 2019 near Wuhan, Hubei City, China, and has since caused the global COVID-19 pandemic.
  • SARS-CoV-2 has a viral genome of approximately 29,903 bases, and is a single-stranded positive-strand RNA virus. Furthermore, virus particles (virions) have a size of about 50 to 200 nm. Like common coronaviruses, it is composed of four proteins known as spike protein, nucleocapsid protein, integral membrane protein, and envelope protein, and RNA. Among these, the nucleocapsid protein binds to RNA to form a nucleocapsid, which is surrounded by spike protein, integral membrane protein, and envelope protein bound to lipids to form an envelope (Non-Patent Documents 2, 3).
  • N protein The nucleocapsid protein of SARS-CoV-2 (hereinafter referred to as N protein) is a protein that binds to the RNA genome of SARS-CoV-2. It has been reported that in addition to being involved in virus particle formation such as folding of the viral genome, assembly of viral proteins, and virus budding, it also has the function of suppressing host cell responses (Non-Patent Document 4).
  • Virological diagnosis of COVID-19 mainly involves gene detection of SARS-CoV-2 using gene amplification method (PCR).
  • PCR tests are said to be the most sensitive virus tests currently available.
  • antigen test that detects fragments of proteins on the surface of the virus (eg, spike protein, etc.).
  • detection kits based on immunochromatography and ELISA are already commercially available, and although they generally have lower detection sensitivity than PCR tests, they are superior in terms of short time required and simplicity.
  • Virus testing requires a testing method that does not produce false positives or false negatives. For false positives, it is important that the measurement principle has high specificity for the target molecule, and for false negatives, the detection sensitivity of the test method is important. In addition, some virus structures are present, albeit at low concentrations, in body fluids (such as saliva and pharyngeal mucus) and sewage derived from SARS-CoV-2 infected people, and in order to detect these viruses, Existing PCR and antigen testing methods may not be sufficient. Therefore, while PCR tests and antigen tests for SARS-CoV-2 have already become general-purpose technologies, there is a strong need for technological development to detect SARS-CoV-2 more specifically and with high sensitivity.
  • Non-Patent Document 4 It has been reported that the N protein of SARS-CoV-2 is one of the most abundant proteins among the proteins constituting the virus (Non-Patent Document 4). Since the number of N proteins is far greater than the spike protein exposed on the surface of SARS-CoV-2, it is a preferred protein as a target molecule for SARS-CoV-2 antigen testing, especially in terms of detection sensitivity. be. Furthermore, the N protein exists in a state bound to the RNA genome of SARS-CoV-2 as a nucleocapsid. Therefore, for example, if we can develop an antibody that can selectively and strongly bind to the N protein and enrich the N protein-RNA genome complex of SARS-CoV-2, it will be possible to use it in PCR tests as well.
  • Non-Patent Document 5 the ELISA method for the N protein of SARS-CoV, which has been prevalent since November 2002, is highly specific and can diagnose patients who appear to be in the early stages of infection.
  • VHH Very domain of heavy chain of heavy chain antibody
  • IgG antibodies immunoglobulin fragments that can bind to antigens.
  • Non-patent Document 6 The functional characteristics of VHH antibodies are: 1) They have a binding activity equal to or higher than that of normal antibodies, 2) They can penetrate into areas that normal antibodies cannot access, and 3) They are easy to form and regenerate physiological structures.
  • VHH antibodies Since it is a single-chain peptide, it has a very simple structure.
  • VHH antibodies have extremely high stability against heat and pressure, and it is also possible to mass-produce functional antibodies using prokaryotes (E. coli, Bacillus subtilis, Corynebacterium, etc.), which was previously difficult. It is.
  • prokaryotes E. coli, Bacillus subtilis, Corynebacterium, etc.
  • ADC antibody-drug conjugate
  • VHH antibodies have many advantages over ordinary antibodies (such as IgG antibodies) in terms of properties and production. Therefore, detection technology using a VHH antibody targeting the N protein of SARS-CoV-2 is expected to be more advantageous in terms of performance and cost than technology using conventional IgG antibodies.
  • Non-patent Document 1 A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020 Mar;579(7798):270-273.
  • Non-patent Document 2 A new coronavirus associated with human respiratory disease in China. Nature. 2020 Mar;579(7798):265-269.
  • Non-patent Document 3 The Coronavirus Nucleocapsid Is a Multifunctional Protein. Viruses. Volume 6, Issue 8, August 2014, Pages 2991-3018.
  • Non-patent Document 4 The SARS-CoV nucleocapsid protein: A protein with multifarious activities. Infection, Genetics and Evolution. Volume 8, July 2008, Pages 397-405.
  • Non-patent Document 5 Diagnosis of Severe Acute Respiratory Syndrome (SARS) by Detection of SARS Coronavirus Nucleocapsid Antibodies in an Antigen-Capturing Enzyme-Linked Immunosorbent Assay. Journal of Clinical Microbiology. Volume 41, No. 12, December 2003, Pages 5781 -5782.
  • Non-patent Document 6 The Therapeutic Potential of Nanobodies. BioDrugs. Volume 34, November 2019, Pages 11-26.
  • the present invention relates to the following 1) to 8).
  • SARS-CoV-2 having one or more structural domains containing CDRs shown in the following (a), (b), (c), (d), (e), (f) or (g) Antibodies that bind to.
  • CDR1 consisting of the amino acid sequence shown by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence shown by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence shown by PWGY (SEQ ID NO: 3)
  • CDR1 consisting of the amino acid sequence shown in GFTFSHYFMH (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence shown in TIGSSSTF (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence shown in TLQYYTGLHLNIVGT (SEQ ID NO: 6)
  • CDR1 consisting of the amino acid sequence shown by RSISRFNVWA (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence shown by SITKSGDTN (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence shown by AVFRGITLYTY (SEQ ID NO: 9)
  • CDR1 consisting of the amino acid sequence shown by G
  • SARS-CoV-2 having one or more structural domains containing CDRs shown in (a), (b), (c), (d), (e), (f) or (g) below:
  • a method for detecting SARS-CoV-2 in a sample comprising the step of contacting a test sample with an antibody that binds to SARS-CoV-2.
  • CDR1 consisting of the amino acid sequence shown by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence shown by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence shown by PWGY (SEQ ID NO: 3)
  • CDR1 consisting of the amino acid sequence shown in GFTFSHYFMH (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence shown in TIGSSSTF (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence shown in TLQYYTGLHLNIVGT (SEQ ID NO: 6)
  • CDR1 consisting of the amino acid sequence shown by RSISRFNVWA (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence shown by SITKSGDTN (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence shown by AVFRGITLYTY (SEQ ID NO: 9)
  • CDR1 consisting of the amino acid sequence shown by G
  • the present invention relates to providing antibodies that bind to the N protein of SARS-CoV-2 and methods for using the same.
  • the present inventors investigated to obtain a VHH antibody that binds to the N protein of SARS-CoV-2.
  • the present inventors conducted screening using a cDNA display method from a VHH antibody library whose construct includes CDR1 to CDR3 having a specific number of amino acids. succeeded in obtaining a clone highly reactive to the N protein of SARS-CoV-2.
  • highly reactive anti-SARS-CoV-2 N protein antibodies can be provided. Since the antibody of the present invention can universally bind to the N protein of mutant strains, it is possible to detect SARS-CoV-2 including mutant strains by using the antibody, that is, to detect SARS-CoV-2 infection. This enables rapid detection of acute respiratory disease (COVID-19).
  • antibodies that bind to SARS-CoV-2 of the present invention include the following (a), (b), (c), (d), (e), and (f). Or, it is an anti-SARS-CoV-2 antibody having one or more structural domains including CDR1 to CDR3 shown in (g).
  • CDR1 consisting of the amino acid sequence shown by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence shown by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence shown by PWGY (SEQ ID NO: 3)
  • CDR1 consisting of the amino acid sequence shown in GFTFSHYFMH (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence shown in TIGSSSTF (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence shown in TLQYYTGLHLNIVGT (SEQ ID NO: 6)
  • CDR1 consisting of the amino acid sequence shown by RSISRFNVWA (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence shown by SITKSGDTN (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence shown by AVFRGITLYTY (SEQ ID NO: 9)
  • CDR1 consisting of the amino acid sequence shown by G
  • SARS-CoV-2 is a SARS-related coronavirus that causes acute respiratory disease (COVID-19), and its viral genome is a single-stranded positive-strand RNA virus of approximately 29,903 bases.
  • the antibody of the present invention is an antibody that binds to SARS-CoV-2, and specifically, an antibody that binds to the N protein of SARS-CoV-2.
  • the structural domain of the antibody of the present invention has three CDRs: CDR1, CDR2, and CDR3.
  • CDR Complementarity Determining Region
  • CDR includes an antigen recognition site or random sequence region with variable sequence, and is also referred to as a hypervariable region.
  • three CDRs exist in the order of CDR1, CDR2, and CDR3 from the N-terminal side.
  • the binding ability to SARS-CoV-2 can be evaluated by a method known to those skilled in the art. Specifically, as shown in the Examples described below, the evaluation can be made by determining the equilibrium dissociation constant KD using the surface plasmon resonance method. In addition, evaluation can also be made by methods using, for example, ELISA, immunochromatography, isothermal titration calorimetry, and biolayer interferometry.
  • the structural domain of the antibody of the present invention may have framework regions at both ends of CDR1, CDR2, and CDR3.
  • the framework region refers to a highly conserved region in the variable region of an antibody molecule, excluding the complementarity determining region. That is, in one aspect, the structural domain of the present invention includes a first framework region (FR1), CDR1, a second framework region (FR2), CDR2, a third framework region (FR3), CDR3, and a fourth framework. Examples include those having regions (FR4) in this order.
  • the amino acid sequence of the framework region in the structural domain includes the amino acid sequence shown below or an amino acid sequence having 80% or more identity with the amino acid sequence.
  • FR1 EVQLVESGGGLVQAGGSQKLSCTTS (SEQ ID NO: 19) EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 20) EVQLVESGGGSVQAGGSLRLSCAAS (SEQ ID NO: 21) EVQLVESGGGLVTAGGSLSLSCAAS (SEQ ID NO: 22) EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 23) EVQLVESGGGLVQPGGSLRLTCVAS (SEQ ID NO: 24) FR2: WFRQAPGKEREFVA (SEQ ID NO: 25) WVRQVPGKGLEWVS (SEQ ID NO: 26) WYRQAPGKQRELVA (SEQ ID NO: 27) WVRQAPGKGLEAVS (SEQ ID NO: 28) FR3: YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRH (SEQ ID NO: 29) YADSVKG
  • the framework region consisting of an amino acid sequence having 80% or more identity with the amino acid sequences shown in SEQ ID NOs: 19 to 39 is preferably 85% or more, more preferably 90% or more, more preferably 95% or more, or Examples include framework regions consisting of amino acid sequences having an identity of preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and still more preferably 99% or more.
  • sequence identity refers to the ratio (%) of the number of positions where the same amino acid residue is present in both sequences to the total number of amino acid residues when two amino acid sequences are aligned.
  • Sequence identity can be analyzed using, for example, the BLAST (Basic Local Alignment Search Tool) of the National Center for Biotechnology Information (NCBI). It can be calculated by
  • CDR1 is the amino acid sequence shown by SEQ ID NO: 1
  • CDR2 is the amino acid sequence shown by SEQ ID NO: 2
  • CDR3 is the amino acid sequence shown by SEQ ID NO: 2.
  • FR1 is the amino acid sequence shown in SEQ ID NO: 19
  • FR2 is the amino acid sequence shown in SEQ ID NO: 25
  • FR3 is the amino acid sequence shown in SEQ ID NO: 29, and
  • FR4 is the amino acid sequence shown in SEQ ID NO: 36.
  • An antibody having a structural domain (SEQ ID NO: 40) whose CDR1 is the amino acid sequence shown by SEQ ID NO: 4, CDR2 is the amino acid sequence shown by SEQ ID NO: 5, and CDR3 is the amino acid sequence shown by SEQ ID NO: 6.
  • FR1 is the amino acid sequence shown by SEQ ID NO: 20
  • FR2 is the amino acid sequence shown by SEQ ID NO: 26
  • FR3 is the amino acid sequence shown by SEQ ID NO: 30
  • FR4 is the amino acid sequence shown by SEQ ID NO: 37.
  • CDR1 is the amino acid sequence shown by SEQ ID NO: 7
  • CDR2 is the amino acid sequence shown by SEQ ID NO: 8
  • CDR3 is the amino acid sequence shown by SEQ ID NO: 9
  • FR1 is the amino acid sequence shown by SEQ ID NO: 9.
  • FR2 has the amino acid sequence shown in SEQ ID NO: 27,
  • FR3 has the amino acid sequence shown in SEQ ID NO: 31, and
  • FR4 has the amino acid sequence shown in SEQ ID NO: 38 (SEQ ID NO: 42).
  • CDR1 is the amino acid sequence shown by SEQ ID NO: 10
  • CDR2 is the amino acid sequence shown by SEQ ID NO: 11
  • CDR3 is the amino acid sequence shown by SEQ ID NO: 12
  • FR1 is the amino acid sequence shown by SEQ ID NO: 22
  • CDR2 is the amino acid sequence shown by SEQ ID NO: 13
  • CDR2 is the amino acid sequence shown by SEQ ID NO: 15
  • FR1 is the amino acid sequence shown by SEQ ID NO: 23
  • FR2 is shown by SEQ ID NO: 28.
  • FR4 is the amino acid sequence shown by SEQ ID NO: 36
  • CDR2 is the amino acid sequence shown in SEQ ID NO: 17
  • CDR3 is the amino acid sequence shown in SEQ ID NO: 18
  • FR1 is the amino acid sequence shown in SEQ ID NO: 24
  • FR2 is the amino acid sequence shown in SEQ ID NO: 25
  • FR3 is the amino acid sequence shown in SEQ ID NO: 34.
  • CDR3 is the amino acid sequence shown by SGT
  • FR1 is the amino acid sequence shown by SEQ ID NO: 19
  • FR2 is the amino acid sequence shown by SEQ ID NO: 25
  • FR3 is the amino acid sequence shown by SEQ ID NO: 35
  • FR4 is SEQ ID NO:
  • Each antibody having a structural domain (SEQ ID NO: 46) having the amino acid sequence shown by 39 is a clone (SEQ ID NO: 46) with high reactivity to SARS-CoV-2 obtained by screening using the cDNA display method in the Examples described below.
  • SEQ ID NO: 40 is EN2, SEQ ID NO: 41 is EN3, SEQ ID NO: 42 is EN4, SEQ ID NO: 43 is EN5, SEQ ID NO: 44 is EN6, SEQ ID NO: 45 is EN11, and SEQ ID NO: 46 is EN12. ), which is a preferred anti-SARS-CoV-2 antibody.
  • the form of the antibody of the present invention is not limited as long as it has at least one of the above structural domains, and includes single domain antibodies (also called nanobodies) such as VHH antibodies, single chain antibodies, heavy chain antibodies, etc. It may be a multimer, such as a dimer, in which an antibody or a variable region fragment is linked with a peptide linker or the like, or a multimer in which a variable region fragment and one or more variable region fragments having different antigen specificities are linked. . Furthermore, the antibodies of the present invention may be humanized. Since humanized antibodies can be administered to humans, they can be applied as medicines.
  • the method for producing the antibody of the present invention is not particularly limited, and it can be easily produced using techniques known in the technical field.
  • peptides can be produced by a combination of solid-phase peptide synthesis and native chemical ligation (NCL) methods, or by genetic engineering;
  • NCL native chemical ligation
  • a method of designing an artificial gene that is optimized for the expression of the target antibody in host cells through processes such as optimization, inserting it into an appropriate vector, introducing this into host cells, and producing it as a recombinant antibody. is preferred.
  • Host cells used in production as recombinant antibodies include, for example, E. coli, Bacillus subtilis, fungi, animal cells, plant cells, baculovirus/insect cells, or yeast cells.
  • expression vectors for expressing antibodies vectors suitable for various host cells can be used.
  • Expression vectors include, for example, vectors derived from Escherichia coli such as pBR322, pBR325, pUC12, and pUC13; vectors derived from Bacillus subtilis such as pUB110, pTP5, and pC194; shuttle vectors that can be used in common with Escherichia coli and Bacillus subtilis such as pHY300PLK; Yeast-derived vectors such as pSH19 and pSH15; bacteriophages such as ⁇ phage; viruses such as adenovirus, adeno-associated virus, lentivirus, vaccinia virus, and baculovirus; and vectors modified therewith can be used.
  • expression vectors have replication origins, selection markers, and promoters suitable for each vector, and as necessary, enhancers, transcription assembly sequences (terminators), ribosome binding sites, polyadenylation signals, etc. may have. Furthermore, in order to facilitate the purification of the expressed polypeptide, the expression vector may contain a base sequence inserted therein for expression by fusing FLAG tag, His tag, HA tag, GST tag, etc.
  • the expressed antibody of the present invention When extracting the expressed antibody of the present invention from cultured cells or cells, after culturing, collect the cells or cultured cells by a known method, suspend them in an appropriate buffer, and inject them with ultrasound, lysozyme, etc. After destroying the bacterial bodies or cells by freezing and/or thawing, a soluble extract is obtained by centrifugation or filtration. From the obtained extract, the antibody of interest can be obtained by appropriately combining known separation and purification methods.
  • Known separation and purification methods include methods that utilize solubility such as salting out and solvent precipitation, methods that primarily utilize differences in molecular weight such as dialysis, ultrafiltration, gel filtration, SDS-PAGE, and ion Methods that utilize differences in charge such as exchange chromatography, methods that utilize specific affinity such as affinity chromatography, methods that utilize differences in hydrophobicity such as reversed-phase high performance liquid chromatography, or isoelectric focusing A method that utilizes the difference in isoelectric points, etc., is used.
  • the antibody of the present invention binds to the N protein of SARS-CoV-2
  • the sample can be The presence or absence of SARS-CoV-2 can be confirmed.
  • detection of SARS-CoV-2 using the antibody of the present invention involves contacting the antibody of the present invention with a test sample, and detecting SARS-CoV-2 between the antibody of the present invention and SARS-CoV-2 in the test sample. and detecting SARS-CoV-2 in the conjugate.
  • the antibody of the present invention can be used to detect SARS-CoV-2 against anti-SARS-CoV-2 antibodies (e.g., serum antibodies) contained in an immobilized test sample (serum). It can also be used as an antibody to confirm the presence of the SARS-CoV-2 antigen in the conjugate state by adding and binding a portion of the antigen containing the N-2 protein.
  • anti-SARS-CoV-2 antibodies e.g., serum antibodies
  • Test samples include tracheal swabs, nasal swabs, throat swabs, nasal washes, nasal aspirates, nasal discharges, saliva, sputum, blood, serum, urine, feces, tissues, cells, and crushed tissue or cells.
  • biological samples such as, samples collected from solid surfaces where viruses may be attached, such as doorknobs and toilet bowls, etc.
  • the virus in the test sample be dissolved in a solution containing a surfactant or the like, and then bound to the antibody in the solution.
  • the antibody may or may not be immobilized on a solid phase.
  • the above-mentioned conjugate is reacted with an anti-SARS-CoV-2 antibody that recognizes a different epitope from the antibody of the present invention in the conjugate.
  • an anti-SARS-CoV-2 antibody that recognizes a different epitope from the antibody of the present invention in the conjugate. This can be done by letting Alternatively, SARS-CoV-2 in the above conjugate may be detected in the liquid phase by a homogeneous assay.
  • the antibody of the present invention can be a component of a kit for detecting SARS-CoV-2.
  • the kit can be used as a diagnostic agent for SARS-CoV-2 infection (COVID-19) and as a tool for developing a therapeutic agent for SARS-CoV-2 infection.
  • the detection kit includes, in addition to the anti-SARS-CoV-2 antibody of the present invention, reagents and instruments necessary for detection, such as an antibody that recognizes the antibody of the present invention or an N protein antibody that recognizes an epitope different from the antibody of the present invention. , a solid phase carrier, a buffer solution, an enzyme reaction stop solution, a microplate reader, and the like.
  • the antibody of the present invention may be immobilized on a solid phase. Examples of the solid phase include beads, membranes, side surfaces and bottom surfaces of reaction vessels, plate-like substrates such as glass slides, well substrates such as immunoplates, etc., on which the antibody of the present invention is directly or indirectly immobilized. Ru.
  • the antibody of the present invention can bind to the N protein of SARS-CoV-2 and inhibit the replication of the virus within the cell by translocating it into the cell by introducing a cell membrane-penetrating peptide or the like. Conceivable. Therefore, the antibody of the present invention can also be administered to mammals and used as a medicament for preventing or treating SARS-CoV-2 infection. Examples of mammals include humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cows, horses, and pigs, with humans being preferred.
  • the antibody of the present invention When used as a medicine, it may be administered either orally or parenterally, and may be used in combination with well-known pharmaceutically acceptable non-toxic carriers and diluents as appropriate.
  • Parenteral administration typically includes injections, but administration by inhalation with sprays and the like is also possible.
  • the content of the antibody of the present invention in the composition can be adjusted as appropriate.
  • a medicine can be applied as the antibody of the present invention by administering an effective amount to a patient at intervals of once to several times a week.
  • preferred methods of administration include intravenous injection and drip drip.
  • SARS-CoV- which has one or more structural domains containing CDRs shown in (a), (b), (c), (d), (e), (f) or (g) below.
  • CDR1 consisting of the amino acid sequence shown by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence shown by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence shown by PWGY (SEQ ID NO: 3)
  • CDR1 consisting of the amino acid sequence shown in GFTFSHYFMH (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence shown in TIGSSSTF (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence shown in TLQYYTGLHLNIVGT (SEQ ID NO: 6)
  • CDR1 consisting of the amino acid sequence shown by RSISRFNVWA (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence shown by SITKSGDTN (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence shown by AVFRGITLYTY (SEQ ID NO: 9)
  • CDR1 consisting of the amino acid sequence shown by G
  • ⁇ 3> The antibody of ⁇ 1> or ⁇ 2>, which is a VHH antibody, a heavy chain antibody, or a VHH antibody multimer.
  • ⁇ 4> The antibody of ⁇ 3>, wherein the VHH antibody multimer is a multimer in which a plurality of the structural domains are linked.
  • ⁇ 5> The antibody of ⁇ 3>, wherein the VHH antibody multimer is a dimer in which two of the structural domains are linked.
  • ⁇ 6> The antibody of ⁇ 3>, wherein the VHH antibody multimer is a multimer in which one or more of the structural domains and one or more of the structural domains having different antigen specificities from the structural domain are linked.
  • the structural domain includes the first framework region (FR1), CDR1, second framework region (FR2), CDR2, third framework region (FR3), CDR3, and fourth framework region (FR4).
  • FR1> to ⁇ 9> which have the following in order:
  • FR1 to FR4 consist of the following amino acid sequences.
  • FR1 Amino acid sequence shown by SEQ ID NO: 19, 20, 21, 22, 23 or 24 or an amino acid sequence having 80% or more identity with the amino acid sequence
  • FR2 Amino acid sequence shown by SEQ ID NO: 25, 26, 27 or 28
  • FR3 an amino acid sequence represented by SEQ ID NO: 29, 30, 31, 32, 33, 34 or 35, or an amino acid sequence having 80% or more identity with the amino acid sequence
  • Amino acid sequence having FR4 Amino acid sequence shown by SEQ ID NO: 36, 37, 38 or 39 or an amino acid sequence having 80% or more identity with the amino acid sequence ⁇ 9> FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are connected in this order, CDR1 is the amino acid sequence shown by SEQ ID NO: 1, CDR2 is the amino acid sequence shown by SEQ ID NO: 2, CDR3 is the amino acid sequence shown by SEQ ID NO: 3, and FR1 is shown by SEQ ID NO: 19.
  • FR2 is the amino acid sequence shown by SEQ ID NO: 25
  • FR3 is the amino acid sequence shown by SEQ ID NO: 29
  • FR4 is the amino acid sequence shown by SEQ ID NO: 36.
  • CDR1 has the amino acid sequence shown by SEQ ID NO: 4
  • CDR2 has the amino acid sequence shown by SEQ ID NO: 5
  • CDR3 has the amino acid sequence shown by SEQ ID NO: 6.
  • FR1 is the amino acid sequence shown by SEQ ID NO: 20
  • FR2 is the amino acid sequence shown by SEQ ID NO: 26
  • FR3 is the amino acid sequence shown by SEQ ID NO: 30,
  • FR4 is the amino acid sequence shown by SEQ ID NO: 37.
  • the antibody of ⁇ 7> which has a structural domain (SEQ ID NO: 41).
  • FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are connected in this order, CDR1 has the amino acid sequence shown by SEQ ID NO: 7,
  • CDR2 has the amino acid sequence shown by SEQ ID NO: 8
  • CDR3 has the amino acid sequence shown by SEQ ID NO: 9.
  • FR1 is the amino acid sequence shown by SEQ ID NO: 21
  • FR2 is the amino acid sequence shown by SEQ ID NO: 27
  • FR3 is the amino acid sequence shown by SEQ ID NO: 31
  • FR4 is the amino acid sequence shown by SEQ ID NO: 38.
  • the antibody of ⁇ 7> which has a structural domain having the sequence (SEQ ID NO: 42).
  • FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are connected in this order, CDR1 has the amino acid sequence shown in SEQ ID NO: 10, CDR2 has the amino acid sequence shown in SEQ ID NO: 11, and CDR3 has the amino acid sequence shown in SEQ ID NO: 12.
  • FR1 is the amino acid sequence shown by SEQ ID NO: 22
  • FR2 is the amino acid sequence shown by SEQ ID NO: 25
  • FR3 is the amino acid sequence shown by SEQ ID NO: 32
  • FR4 is the amino acid sequence shown by SEQ ID NO: 36.
  • FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are connected in this order, CDR1 has the amino acid sequence shown in SEQ ID NO: 13, CDR2 has the amino acid sequence shown in SEQ ID NO: 14, and CDR3 has the amino acid sequence shown in SEQ ID NO: 15.
  • FR1 is the amino acid sequence shown by SEQ ID NO: 23
  • FR2 is the amino acid sequence shown by SEQ ID NO: 28
  • FR3 is the amino acid sequence shown by SEQ ID NO: 33
  • FR4 is the amino acid sequence shown by SEQ ID NO: 36.
  • the antibody of ⁇ 7> which has a structural domain having the sequence (SEQ ID NO: 44).
  • FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are connected in this order, CDR1 has the amino acid sequence shown in SEQ ID NO: 16, CDR2 has the amino acid sequence shown in SEQ ID NO: 17, and CDR3 has the amino acid sequence shown in SEQ ID NO: 18.
  • FR1 is the amino acid sequence shown by SEQ ID NO: 24, FR2 is the amino acid sequence shown by SEQ ID NO: 25, FR3 is the amino acid sequence shown by SEQ ID NO: 34, and FR4 is the amino acid sequence shown by SEQ ID NO: 36.
  • the antibody of ⁇ 7> which has a structural domain having the sequence (SEQ ID NO: 45).
  • FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are connected in this order, CDR1 is the amino acid sequence shown by SEQ ID NO: 1, CDR2 is the amino acid sequence shown by SEQ ID NO: 2, and CDR3 is shown by SGT.
  • FR1 is the amino acid sequence shown in SEQ ID NO: 19
  • FR2 is the amino acid sequence shown in SEQ ID NO: 25
  • FR3 is the amino acid sequence shown in SEQ ID NO: 35
  • FR4 is the amino acid sequence shown in SEQ ID NO: 39.
  • the antibody of ⁇ 7> which has a certain structural domain (SEQ ID NO: 46).
  • ⁇ 16> A nucleic acid encoding the antibody of ⁇ 1>.
  • SARS-CoV- which has one or more structural domains containing the CDRs shown in (a), (b), (c), (d), (e), (f) or (g) below:
  • a method for detecting SARS-CoV-2 in a sample comprising the step of contacting a test sample with an antibody that binds to SARS-CoV-2.
  • CDR1 consisting of the amino acid sequence shown by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence shown by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence shown by PWGY (SEQ ID NO: 3)
  • CDR1 consisting of the amino acid sequence shown in GFTFSHYFMH (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence shown in TIGSSSTF (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence shown in TLQYYTGLHLNIVGT (SEQ ID NO: 6)
  • CDR1 consisting of the amino acid sequence shown by RSISRFNVWA (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence shown by SITKSGDTN (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence shown by AVFRGITLYTY (SEQ ID NO: 9)
  • CDR1 consisting of the amino acid sequence shown by G
  • ⁇ 21> Use of any antibody of ⁇ 1> to ⁇ 15> for producing a medicament.
  • ⁇ 22> The use of ⁇ 21>, wherein the medicament is for the prevention or treatment of SARS-CoV-2 infection.
  • ⁇ 23> The antibody according to any one of ⁇ 1> to ⁇ 15> for use as a medicine.
  • ⁇ 24> The antibody of ⁇ 23>, wherein the medicament is a medicament for preventing or treating SARS-CoV-2 infection.
  • a method for preventing or treating SARS-CoV-2 infection which comprises administering any of the antibodies of ⁇ 1> to ⁇ 15> to a subject in need thereof.
  • Example 1 Production of cDNA for display adjustment of VHH presentation cDNA Production of a DNA library encoding a single variable domain of the heavy-chain antibody (VHH) of a full-length alpaca-derived antibody RePHA Provided by GEN S-hinge VHH-specific primer pair (SEQ ID NO: 47, 48) and L-hinge VHH-specific primer pair (SEQ ID NO: 47, After amplification by PCR in 49), overlapping was performed to adjust to a DNA fragment consisting of the T7 promoter, omega ( ⁇ ) enhancer, Kozak consensus sequence, VHH gene, His tag, and linker hybridization region (Y tag). Extension PCR was performed using PCR primers (SEQ ID NOs: 50 and 51) to produce a full-length VHH-encoding DNA library.
  • Example 2 Preparation of cDNA displays Each cDNA display was prepared according to the following procedures (1) to (5). The buffers used are listed in Table 1.
  • the temperature was lowered to 25°C at a rate of 0.1°C/sec, and then to 10°C at a rate of 2°C/sec, to hybridize the cnvK linker to the 3' end of the mRNA.
  • UVP CrossLinker CL-3000
  • 365 nm 100-115 V (Analytik Jena)
  • 4060 mJ/ cm2 of 365 nm UV was irradiated to photo-crosslink the cnvK linker and mRNA, and the mRNA-linker complex was Obtained.
  • cDNA display 30 ⁇ L of His Mag Sepharose Ni Beads (GE Health Care) was placed in an Eppendorf tube, washed with 200 ⁇ L of His tag binding/washing buffer, and then prepared using the methods (1) to (5) above. cDNA display was added and stirred for 30 minutes at 25°C. After washing with 200 ⁇ L of His tag binding/washing buffer, cDNA display was eluted by adding 30 ⁇ L of His tag elution buffer and stirring at 37° C. for 15 minutes.
  • His Mag Sepharose Ni Beads GE Health Care
  • Example 3 Selection of VHH (1) In vitro selection experiment Selection was performed using the cDNA display prepared from the library obtained in Example 2. The synthesis scale of the VHH presentation cDNA display library used in each selection round is shown in Table 3.
  • the mixture was added to Dynabeads Myone streptavidin C1 and mixed by inversion at 4°C for 30 minutes to immobilize the biotinylated N protein containing the cDNA display-N protein complex onto the beads. After all the beads were combined, they were washed 4 times with PBS-T. Thereafter, the bound cDNA display was collected and purified using AMpure XP (manufactured by Beckman Coulter) according to the attached manual.
  • AMpure XP manufactured by Beckman Coulter
  • the purified solution was subjected to PCR using PrimeSTAR Max (Takara Bio) at an annealing temperature of 65°C and an extension reaction of 10 seconds. After that, the PCR products were analyzed by 4% denaturing PAGE. After the PCR product was purified using AMpure XP according to the attached manual, the obtained purified product R1 library was subjected to the second round of selection.
  • the bound cDNA display was collected and purified using AMpure XP (manufactured by Beckman Coulter) according to the attached manual.
  • the purified solution was subjected to PCR reaction under the same conditions as in Example 3 (1-1), and purified using AMpure XP according to the attached manual to obtain an R2 library.
  • the mixed solution was added to 20 ⁇ L of Dynabeads Myone streptavidin T1, and the biotinylated N protein containing the cDNA display-N protein complex was mixed by inversion at 4° C. for 30 minutes and immobilized on beads.
  • the beads were washed three times with 200 ⁇ L of PBS-T, and then 20 ⁇ L of PBST was added and mixed by inversion at 25° C. for 30 minutes. Thereafter, the bound cDNA display was collected and purified using AMpure XP (manufactured by Beckman Coulter) according to the attached manual.
  • the purified solution was subjected to PCR reaction under the same conditions as in Example 3 (1-1), and purified using AMpure XP according to the attached manual to obtain an R3 library.
  • NGS next-generation sequencer
  • Sequence samples were prepared with reference to the 2-step PCR Amplicon Library Preparation method provided by Illumina.
  • Amplicon PCR was performed using the PCR products after each selection as templates and NGS Fw 1st PCR primer (SEQ ID NO: 54) and NGS Rv 1st PCR primer (SEQ ID NO: 55) as primers.
  • PCR conditions were as follows: 98°C for 1 minute, 15 cycles of 98°C for 10 seconds, 62°C for 5 seconds, 72°C for 35 seconds, and 72°C for 1 minute.
  • the obtained PCR product was purified according to the instructions of Agencourt AMPure XP, and then Index PCR was performed according to the instructions of Illumina.
  • the obtained PCR product was purified according to the instructions of Agencourt AMPure ) was used to perform sequence analysis.
  • the obtained DNA sequence was translated into the amino acid sequence of VHH and ranked by frequency of appearance.
  • Table 4 was selected as a representative VHH with high affinity for N protein.
  • amino acid sequence mutations including substitutions, insertions, and deletions
  • SEQ ID NO: 77-98 as a CDR variant of EN2 (SEQ ID NO: 40), SEQ ID NO: 99-106 as a CDR variant of EN3 (SEQ ID NO: 41), and SEQ ID NO: 107-119 as a CDR variant of EN4 (SEQ ID NO: 42).
  • SEQ ID NO: 127 to 134 as a CDR variant of EN6
  • SEQ ID NO: 135 as a CDR variant of EN11 (SEQ ID NO: 45)
  • SEQ ID NOs: 136 to 150 were selected as CDR variants of EN12 (SEQ ID NO: 46) (FIGS. 1 and 2). Since these CDR variants are also VHHs selected through a selection process, it is considered that there is a high possibility that they exhibit binding activity to the N protein of SARS-CoV-2.
  • Example 4 Production of VHH by protease-deficient recombinant Bacillus subtilis (1) Artificial synthesis of gene
  • the amino acid sequence of the synthesized VHH includes a linker sequence on the C-terminal side of the amino acid sequence of each VHH listed in Table 4.
  • a His tag sequence (SEQ ID NO: 56) was added via The VHHs synthesized by this are His-tagged EN2 (SEQ ID NO: 57), His-tagged EN3 (SEQ ID NO: 58), His-tagged EN4 (SEQ ID NO: 59), His-tagged EN5 (SEQ ID NO: 60), and His-tagged EN5 (SEQ ID NO: 60).
  • EN6 SEQ ID NO: 61
  • His-tagged EN11 SEQ ID NO: 62
  • His-tagged EN12 SEQ ID NO: 63
  • these VHHs shown by SEQ ID NOs: 56 to 63 are collectively referred to as His-tagged VHHs.
  • Artificially synthesized genes for synthesizing these His-tagged VHHs include His-tagged artificially synthesized gene for EN2 (SEQ ID NO: 64), His-tagged EN3 artificially synthesized gene (SEQ ID NO: 65), and His-tagged EN4 artificially synthesized gene.
  • SEQ ID NO: 66 His-tagged artificially synthesized gene for EN5 (SEQ ID NO: 67), His-tagged artificially synthesized gene for EN6 (SEQ ID NO: 68), His-tagged artificially synthesized gene for EN11 (SEQ ID NO: 69), His An artificial synthetic gene for EN12 with a tag (SEQ ID NO: 70) was synthesized and used for experiments.
  • Promoter DNA derived from the oVG gene was amplified. .
  • the obtained promoter DNA was incorporated into a plasmid sequence using In-Fusion HD Cloning Kit (Takara) to construct a VHH expression plasmid linked to the spoVG promoter.
  • the plasmid sequence was amplified by PCR using a primer set of 5'-TGCTGCAAGAGCTGCCGGAAATAAA-3' (SEQ ID NO: 75) and 5'-TCTATTAAAACTAGTTATAGGG-3' (SEQ ID NO: 76) and PrimeSTAR Max DNA polymerase (TaKaRa).
  • DNA containing the artificially synthesized genes was inserted into the obtained PCR fragment using In-Fusion HD Cloning Kit (Takara) to construct VHH expression plasmids containing each of the artificially synthesized VHH genes.
  • Bacillus subtilis stocked with glycerol was inoculated into 1 mL of LB liquid medium, and cultured with shaking at 30° C. and 210 rpm overnight. The next day, 10 ⁇ L of this culture solution was inoculated into a new 1 mL LB liquid medium, and cultured with shaking at 37° C. and 210 rpm for about 2 hours. The culture solution was collected into a 1.5 mL tube, centrifuged at 1,2000 rpm for 5 minutes, the supernatant was removed, and the pellet was mixed with SMMP (0.5 M sucrose, 20 mM disodium maleate) containing 4 mg/mL of Lysozyme (SIGMA).
  • SMMP 0.5 M sucrose, 20 mM disodium maleate
  • VHH production The recombinant Bacillus subtilis produced in (3) was inoculated into 1 mL of LB medium containing 50 ppm tetracycline, and shaken back and forth at 30° C. overnight to prepare a preculture solution. 1% of the preculture solution was inoculated into 20 mL of 2 ⁇ L-mal medium in a pleated Erlenmeyer flask, and cultured with shaking at 30° C. for 72 hours. At the end of the culture, 1 mL of the culture solution was centrifuged in a microtube at 4° C., 15,000 rpm for 5 minutes, and the supernatant was collected. His-tagged VHH was purified using Ni-NTA agarose beads (Fuji Film Wako Pure Chemical Industries, Ltd.) according to the kit protocol. The purified protein was dissolved in PBS containing 30mM imidazole.
  • the detection antibody was diluted 1/5,000 with PBST (PBS containing 0.05% (v/v) Tween 20). 50 ⁇ L of detection antibody was added to each well and incubated for 1 hour at room temperature. After removing the detection antibody, 200 ⁇ L of PBST (PBS containing 0.05% (v/v) Tween 20) was added and removed immediately. This washing operation was performed three times.
  • the coloring substrate was prepared by dissolving OPD tablets (Thermo Fisher Scientific) in Stable Peroxide Substrate Buffer (Thermo Fisher Scientific).
  • the detection antibody was diluted 1/5,000 with PBST (PBS containing 0.05% (v/v) Tween 20). 50 ⁇ L of detection antibody was added to each well and incubated for 1 hour at room temperature. After removing the detection antibody, 200 ⁇ L of PBST (PBS containing 0.05% (v/v) Tween 20) was added and removed immediately. This washing operation was performed three times.
  • the coloring substrate was prepared by dissolving OPD tablets (Thermo Fisher Scientific) in Stable Peroxide Substrate Buffer (Thermo Fisher Scientific).
  • Example 7 Measurement of binding activity by biolayer interferometry Concentrations of various His-tagged VHHs were determined using bovine serum albumin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) as a standard protein and Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher). Quantification was performed by the BCA method using . The binding activity of various His-tagged VHHs to biotinylated N protein immobilized on a Streptavidin (SA) Biosensor chip (Sartorius) was measured using Octet (trademark) RED384 (Sartorius). Each measurement was performed by adding 60 ⁇ L of each measurement solution to a 384-well plate.
  • SA Streptavidin
  • the sensor chip was hydrated by immersing the tip of the SA Biosensor chip in 200 ⁇ L of PBST for 10 minutes.
  • Baseline step Measurement for 30 seconds in PBST
  • Association step Measurement for 120 seconds with various His-tagged VHHs of 250 nM, 125 nM, 62.6 nM, 31.3 nM, 15.6 nM, and 0 nM diluted with PBST.
  • Dissociation step Measurement for 240 seconds in PBS-T
  • Regeneration step Measurement for 5 seconds in Glycin-HCL (pH 2.2) and 5 seconds in PBS-T, repeated 3 times 1) In steps to 4), adsorption of various His-tagged VHHs to the SA Biosensor chip was measured.
  • biotinylated N protein was immobilized on the SA Biosensor chip until a response of about 6.5 nm was obtained by measuring with PBS-T containing biotinylated N protein for 600 seconds.
  • Steps 1) to 4) above were performed using an SA Biosensor chip on which biotinylated N protein was immobilized.
  • Octet software version (1.2.1.5) manufactured by Molecular Devices
  • the measurement at a concentration of 0 nM of various His-tagged VHHs and the result before immobilizing biotinylated N protein were taken as double references, and the Global Fitting was performed and binding and dissociation rates were calculated (Table 5).
  • Example 8 Evaluation of binding activity to Omicron strain N protein by ELISA method F96 Cert. 50 ⁇ L of 10 ⁇ g/mL SARS-CoV-2 (2019-nCoV) Nucleocapsid-His Recombinant in each well of Maxisorp Nunc-ImmunoTM Plate (Thermo Fisher Scientific) Protein (His-tagged N protein), SARS-CoV-2 B. 1.529 (Omicron) Nucleocapsid Protein (His Tag) (both Sino Biological) was added, and after sealing, the mixture was allowed to stand overnight at 4°C.
  • the detection antibody was Peroxidase-AffiniPure Goat Anti-Alpaca IgG (H+L) (min X Bov, Hu, Ms, Rb, Rat Sr Prot) (Jackson Immuno Research Laboratories) ( 1.0 mg/mL) was used.
  • the detection antibody was diluted 1/5,000 with PBST (PBS containing 0.05% (v/v) Tween 20). 50 ⁇ L of detection antibody was added to each well and incubated for 1 hour at room temperature. After removing the detection antibody, 200 ⁇ L of PBST (PBS containing 0.05% (v/v) Tween 20) was added and removed immediately. This washing operation was performed three times.
  • the coloring substrate was prepared by dissolving OPD tablets (Thermo Fisher Scientific) in Stable Peroxide Substrate Buffer (Thermo Fisher Scientific). After adding 100 ⁇ L of chromogenic substrate to each well and incubating in the dark for 30 minutes, absorbance at 450 nm was immediately measured using Microplate Reader Infinite M1000 PRO (TECAN) (FIG. 7). As a result, it was confirmed that all His-tagged VHHs used in this example also bind to the N protein of Omicron strain of SARS-CoV-2.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Public Health (AREA)
  • Hematology (AREA)
  • Genetics & Genomics (AREA)
  • Urology & Nephrology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biophysics (AREA)
  • Veterinary Medicine (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Physics & Mathematics (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
PCT/JP2023/014930 2022-04-12 2023-04-12 抗ＳＡＲＳ－ＣｏＶ－２抗体 Ceased WO2023199955A1 (ja)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2022065933A JP2023156196A (ja) 2022-04-12 2022-04-12 抗ＳＡＲＳ－ＣｏＶ－２抗体
JP2022-065933 2022-04-12

Publications (1)

Publication Number Publication Date
WO2023199955A1 true WO2023199955A1 (ja) 2023-10-19

Family

ID=88329816

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2023/014930 Ceased WO2023199955A1 (ja) 2022-04-12 2023-04-12 抗ＳＡＲＳ－ＣｏＶ－２抗体

Country Status (2)

Country Link
JP (1) JP2023156196A (https=)
WO (1) WO2023199955A1 (https=)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111153991A (zh) * 2020-02-26 2020-05-15 北京博奥森生物技术有限公司 一种人SARS-CoV-2单克隆抗体及其制备方法和应用
CN112028993A (zh) * 2020-10-15 2020-12-04 武汉华美生物工程有限公司 一种抗sars-cov-2病毒n蛋白的纳米抗体及其制备方法和应用
WO2022181550A1 (ja) * 2021-02-25 2022-09-01 花王株式会社 抗ＳＡＲＳ－ＣｏＶ－２抗体

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111153991A (zh) * 2020-02-26 2020-05-15 北京博奥森生物技术有限公司 一种人SARS-CoV-2单克隆抗体及其制备方法和应用
CN112028993A (zh) * 2020-10-15 2020-12-04 武汉华美生物工程有限公司 一种抗sars-cov-2病毒n蛋白的纳米抗体及其制备方法和应用
WO2022181550A1 (ja) * 2021-02-25 2022-09-01 花王株式会社 抗ＳＡＲＳ－ＣｏＶ－２抗体

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GRANSAGNE MARION, AYMÉ GABRIEL, BRIER SÉBASTIEN, CHAUVEAU-LE FRIEC GAËLLE, MERIAUX VÉRONIQUE, NOWAKOWSKI MIREILLE, DEJARDIN FRANÇO: "Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 298, no. 1, 20 October 2021 (2021-10-20), US , pages 101290, XP055876224, ISSN: 0021-9258, DOI: 10.1016/j.jbc.2021.101290 *
HAGA KEI, TAKAI TODAKA REIKO, MATSUMURA YUTA, SONG CHIHONG, TAKANO TOMOMI, TOJO TAKUTO, NAGAMI ATSUSHI, ISHIDA YUKI, MASAKI HIDEKA: "Nasal delivery of single-domain antibody improves symptoms of SARS-CoV-2 infection in an animal model", PLOS PATHOGENS, PUBLIC LIBRARY OF SCIENCE, US, vol. 17, no. 10, 14 October 2021 (2021-10-14), US , pages e1009542, XP055863369, ISSN: 1553-7366, DOI: 10.1371/journal.ppat.1009542 *
YE QIAOZHEN, LU SHAN, CORBETT KEVIN D.: "Structural Basis for SARS-CoV-2 Nucleocapsid Protein Recognition by Single-Domain Antibodies", FRONTIERS IN IMMUNOLOGY, vol. 12, 1 January 2021 (2021-01-01), pages 1 - 9, XP055961800, DOI: 10.3389/fimmu.2021.719037 *

Also Published As

Publication number Publication date
JP2023156196A (ja) 2023-10-24

Similar Documents

Publication Publication Date Title
CN112010965B (zh) 一种针对新冠病毒SARS-CoV-2棘突蛋白RBD区的单克隆抗体及其应用
JP6215223B2 (ja) マルチプレックス免疫スクリーニングアッセイ
CN111778218A (zh) 噬菌体展示抗体库及基于其淘筛获得的针对新冠病毒SARS-CoV-2的单克隆抗体
KR102671155B1 (ko) Ns1 단백질의 결합 단백질
KR102785081B1 (ko) 항 인간 심근 트로포닌 i의 항체 및 그 사용
KR102792597B1 (ko) Ns1-결합 단백질
US20210301000A1 (en) NS1-Binding Protein and Uses Thereof
EP4299745A1 (en) Anti-sars-cov-2 antibody
KR102785236B1 (ko) 항 인간 심근 트로포닌 i의 항체 및 그 사용
JP7414225B2 (ja) ＳＡＲＳ－ＣｏＶ－２結合ペプチド
WO2023199955A1 (ja) 抗ＳＡＲＳ－ＣｏＶ－２抗体
JP2024151260A (ja) 抗ＳＡＲＳ－ＣｏＶ－２抗体
JP2023156197A (ja) ＳＡＲＳ－ＣｏＶ－２結合ペプチド
JP7038367B2 (ja) 抗ＳＡＲＳ－ＣｏＶ－２抗体
KR102709435B1 (ko) SARS-CoV-2 특이적 신규 항체를 이용한 SARS-CoV-2의 검출방법
JP7174961B2 (ja) 抗ＳＡＲＳ－ＣｏＶ－２抗体を用いた医薬品及び検査キット
CN121537507B (zh) 一种抗mers的纳米抗体、纳米抗体融合蛋白及其应用
TW202204381A (zh) 重組抗體、包含重組抗體之套組及其用途
CA3109433C (en) Ns1-binding protein and uses thereof
JP2024165150A (ja) ＳＡＲＳ－ＣｏＶ－２中和抗体
CN116547004A (zh) SARS-CoV-2结合肽
CN120349397A (zh) 乙型流感n蛋白特异性抗体及其制备方法与应用
CN120209137A (zh) 抗降钙素原纳米抗体及其应用
CN114805562A (zh) 抗新型冠状病毒人源化纳米抗体及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23788373

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 23788373

Country of ref document: EP

Kind code of ref document: A1