WO2022102744A1 - Anticorps visant la protéine de spicule du sars-cov-2 - Google Patents

Anticorps visant la protéine de spicule du sars-cov-2 Download PDF

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WO2022102744A1
WO2022102744A1 PCT/JP2021/041726 JP2021041726W WO2022102744A1 WO 2022102744 A1 WO2022102744 A1 WO 2022102744A1 JP 2021041726 W JP2021041726 W JP 2021041726W WO 2022102744 A1 WO2022102744 A1 WO 2022102744A1
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chain variable
variable region
seq
monoclonal antibody
antibody molecule
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Japanese (ja)
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光 園田
ちひろ 岡田
悦子 池田
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株式会社ハカレル
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • G01N33/531—Production of immunochemical test materials
    • 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 the fields of detection and quantification of SARS-CoV-2, the so-called new coronavirus, and treatment or prevention of Covid-19 caused by SARS-CoV-2.
  • the present invention is based on a monoclonal antibody specific for the SARS-CoV-2 peplomer, the detection and quantification of SARS-CoV-2, and Covid-19 due to SARS-CoV-2.
  • the present invention relates to a pharmaceutical composition for treating or preventing the disease.
  • SARS-CoV-2 identified as the causative virus of Covid-19, is a type of coronavirus that has an RNA with a total length of about 30,000 base pairs as a gene, and is a membrane protein expressed on the surface of the virus, the S gene in the virus genome. Has a spiked protein encoded by the virus. Peplomers are composed of two domains, S1 and S2.
  • SARS-CoV-2 When SARS-CoV-2 infects cells, it has been clarified that the S1 domain of the spike protein first binds to angiotensin converting enzyme 2 (ACE2) on the cell surface, and then the virus is taken up into the cells. Is considered to be a receptor for SARS-CoV-2. Furthermore, for spike proteins, a narrow 372-531 region (receptor binding region, RBD region) that binds to ACE2 has been identified by structural analysis of the S1 domain (Non-Patent Document 1).
  • ACE2 angiotensin converting enzyme 2
  • viruses are present in blood and saliva in persons infected with SARS-CoV-2, and the presence or absence of infection can be inspected by detecting the virus in their body fluids. That is, by preparing a monoclonal antibody against the S1 domain of the spike protein expressed on the surface of the virus, an immunoassay against the virus can be constructed, and the monoclonal antibody binds to the spike protein to suppress the infection of the virus into cells. It may be a drug.
  • SARS-CoV-2 has a diameter of 100 to 200 nm and is characterized by expressing a spike protein on its surface. This shape is similar to exosomes, which are 30-120 nm sized microvesicles secreted into body fluids such as blood via the exocytosis pathway.
  • the present invention includes the following forms. ⁇ Monclonal antibody molecule> [1] MAb1A: (i) H-chain variable region CDR1 amino acid sequence of SEQ ID NO: 14, H-chain variable region CDR2 amino acid sequence of SEQ ID NO: 15, H-chain variable region of SEQ ID NO: 16, H-chain variable region containing CDR3 amino acid sequence, and (ii) sequence. L-chain variable region CDR1 amino acid sequence of No.
  • L-chain variable region CDR2 amino acid sequence of SEQ ID NO: 23 and L-chain variable region of SEQ ID NO: 24 L-chain variable region containing CDR3 amino acid sequence, Monoclonal antibody molecule containing; MAb1B: (i) H-chain variable region containing the H-chain variable region amino acid sequence of SEQ ID NO: 12, and (ii) L-chain variable region containing the L-chain variable region amino acid sequence of SEQ ID NO: 20.
  • MAb1C-1 Monoclonal antibody molecule containing; MAb1C-1: (i) H-chain variable region containing an amino acid sequence that is at least 80% identical to the H-chain variable region amino acid sequence of SEQ ID NO: 12, and (ii) at least 80% identical to the L-chain variable region amino acid sequence of SEQ ID NO: 20.
  • a mutant monoclonal antibody molecule comprising the above, wherein the mutant monoclonal antibody molecule has binding identification and function equivalent to that of the monoclonal antibody molecule MAb1A or MAb1B; and MAb1C-2: (i) In the H chain variable region amino acid sequence of SEQ ID NO: 12, at least one framework (FW) containing an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions as compared to it. ) H-chain variable region containing the region; and (ii) an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions in the L-chain variable region amino acid sequence of SEQ ID NO: 20.
  • the mutant monoclonal antibody molecule comprising the above, wherein the mutant monoclonal antibody molecule has binding identification and function equivalent to that of the monoclonal antibody molecule MAb1A or MAb1B;
  • MAb2A (i) H chain variable region CDR1 amino acid sequence of SEQ ID NO: 31, H chain variable region CDR2 amino acid sequence of SEQ ID NO: 32, H chain variable region including H chain variable region CDR3 amino acid sequence of SEQ ID NO: 33, and (ii) sequence. L-chain variable region CDR1 amino acid sequence of No.
  • MAb2C-1 Monoclonal antibody molecule containing; MAb2C-1: (i) H-chain variable region containing an amino acid sequence that is at least 80% identical to the H-chain variable region amino acid sequence of SEQ ID NO: 29, and (ii) at least 80% identical to the L-chain variable region amino acid sequence of SEQ ID NO: 37.
  • the mutant monoclonal antibody molecule comprising the above, wherein the mutant monoclonal antibody molecule has binding identification and function equivalent to that of the monoclonal antibody molecule MAb2A or MAb2B; MAb2C-2: (i) In the H chain variable region amino acid sequence of SEQ ID NO: 29, at least one framework (FW) containing an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions as compared to it. ) H-chain variable region containing the region; and (ii) an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions in the L-chain variable region amino acid sequence of SEQ ID NO: 37.
  • the mutant monoclonal antibody molecule comprising the above, wherein the mutant monoclonal antibody molecule has binding identification and function equivalent to that of the monoclonal antibody molecule MAb2A or MAb2B;
  • MAb3A (i) H-chain variable region CDR1 amino acid sequence of SEQ ID NO: 47, H-chain variable region CDR2 amino acid sequence of SEQ ID NO: 48 and H-chain variable region CDR3 amino acid sequence of SEQ ID NO: 49, and (ii) sequence.
  • Monoclonal antibody molecule containing; MAb3B (i) H-chain variable region containing the H-chain variable region amino acid sequence of SEQ ID NO: 45, and (ii) L-chain variable region containing the L-chain variable region amino acid sequence of SEQ ID NO: 53.
  • MAb3C-1 (i) H-chain variable region containing an amino acid sequence that is at least 80% identical to the H-chain variable region amino acid sequence of SEQ ID NO: 45, and (ii) at least 80% identical to the L-chain variable region amino acid sequence of SEQ ID NO: 53.
  • the mutant monoclonal antibody molecule comprising the above, wherein the mutant monoclonal antibody molecule has binding identification and function equivalent to that of the monoclonal antibody molecule MAb3A or MAb3B; MAb3C-2: (i) In the H chain variable region amino acid sequence of SEQ ID NO: 45, at least one framework (FW) containing an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions as compared to it. ) H-chain variable region containing the region; and (ii) an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions in the L-chain variable region amino acid sequence of SEQ ID NO: 53.
  • FW In the H chain variable region amino acid sequence of SEQ ID NO: 45, at least one framework (FW) containing an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions as compared to it.
  • H-chain variable region containing the region and (ii) an amino acid sequence having
  • L chain variable region including at least one framework region containing A mutant monoclonal antibody molecule or a derivative thereof comprising the above, wherein the mutant monoclonal antibody molecule has binding identification and function equivalent to that of the monoclonal antibody molecule MAb3A or MAb3B;
  • the monoclonal antibody molecule or the derivative of the mutant monoclonal antibody molecule according to any one of [1] to [3], which is Fab, F (ab') 2, Fv or a single chain Fv fragment (scFv).
  • ⁇ Pharmaceutical composition> [5] Fever, malaise, respiratory failure, acute inflammation or thrombosis associated with Covid-19, which contains an effective amount of the monoclonal antibody molecule, mutant monoclonal antibody molecule or derivative thereof according to any one of [1] to [4].
  • a pharmaceutical composition for treatment or prevention. [6] [5] The pharmaceutical composition according to [5], which contains an effective amount of the monoclonal antibody molecule, the mutant monoclonal antibody molecule or a derivative thereof according to 1. [7] The pharmaceutical composition according to [5] or [6] for treating or preventing fever, malaise or respiratory failure associated with Covid-19.
  • ⁇ Method of detecting and / or quantifying SARS-CoV-2> [8] (i) Obtain a biological sample from the subject and (ii) The resulting biological sample is contacted with the isolated monoclonal antibody molecule, mutant monoclonal antibody molecule or derivative thereof according to any one of [1] to [4], and (iii) SARS-.
  • a method for detecting and / or quantifying SARS-CoV-2 in a biological sample comprising detecting or quantifying the formation of a complex between CoV-2 and a monoclonal antibody molecule, a mutant monoclonal antibody molecule or a derivative thereof.
  • SARS-CoV-2 is one of the SARS-CoV-2 amino acid variants selected from VOC-202012 / 01 (UK type), 501Y.V2 (South African type) and 501Y.V3 (Brazilian type), [ The method according to any one of 8] to [10].
  • a kit for detecting and / or quantifying SARS-CoV-2 which comprises the isolated monoclonal antibody molecule according to any one of [1] to [4] or a derivative thereof.
  • SARS-CoV in the sample -2 is a method comprising determining 501Y.V2 (South African type) or 501Y.V3 (Brazilian type).
  • nucleic acid encoding the monoclonal antibody molecule according to any one of [1] to [4] or a derivative thereof.
  • MAb1A nucleic acid (i) Nucleic acid encoding an H chain variable region comprising the H chain variable region CDR1 nucleotide sequence of SEQ ID NO: 17, the H chain variable region CDR2 nucleotide sequence of SEQ ID NO: 18, and the H chain variable region CDR3 nucleotide sequence of SEQ ID NO: 19.
  • nucleic acid encoding an L-chain variable region comprising the L-chain variable region CDR1 nucleotide acid sequence of SEQ ID NO: 25, the L-chain variable region CDR2 nucleotide sequence of SEQ ID NO: 26, and the L-chain variable region CDR3 nucleotide sequence of SEQ ID NO: 27.
  • MAb1B nucleic acid (i) H-chain variable region of SEQ ID NO: 13 H-chain variable region containing a nucleotide sequence which is a nucleotide sequence, and (ii) L-chain variable region containing an L-chain variable region nucleotide sequence of SEQ ID NO: 21. Nucleic acid encoding the monoclonal antibody molecule MAb1B or a derivative thereof according to [1].
  • MAb1C-1 nucleic acid (i) H-chain variable region containing at least 80% identical to the H-chain variable region nucleotide sequence of SEQ ID NO: 13, and (ii) at least 80% identical to the L-chain variable region nucleotide sequence of SEQ ID NO: 21.
  • L-chain variable region containing nucleotide sequence Nucleic acid encoding a mutant monoclonal antibody molecule or derivative thereof having binding identification and function equivalent to that of the monoclonal antibody molecule MAb1A or MAb1B, including MAb1C-2 nucleic acid: (i) In the H chain variable region nucleotide sequence of SEQ ID NO: 13, at least one framework (FW) region containing a nucleotide sequence having one or several nucleotide substitutions, deletions, insertions or additions relative to the nucleotide sequence.
  • FW framework
  • the L chain variable region containing, and (ii) the L chain variable region nucleotide sequence of SEQ ID NO: 21, at least one comprising a nucleotide sequence having one or several nucleotide substitutions, deletions, insertions or additions relative to it.
  • L-chain variable region containing 15 framework regions; Nucleic acid encoding a mutant monoclonal antibody molecule or derivative thereof having binding identification and function equivalent to that of the monoclonal antibody molecule MAb1A or MAb1B, including The nucleic acid according to [14].
  • MAb2A nucleic acid (i) H chain variable region CDR1 nucleotide sequence of SEQ ID NO: 34, H chain variable region CDR2 nucleotide sequence of SEQ ID NO: 35 and H chain variable region CDR3 nucleotide sequence of SEQ ID NO: 36, and (ii) sequence.
  • L-chain variable region CDR1 nucleotide sequence of No. 42, L-chain variable region CDR2 nucleotide sequence of SEQ ID NO: 43 and L-chain variable region CDR3 nucleotide sequence of SEQ ID NO: 44 Nucleic acid encoding the monoclonal antibody molecule MAb2A or a derivative thereof according to [1].
  • MAb2B nucleic acid (i) H-chain variable region of SEQ ID NO: 30 H-chain variable region containing a nucleotide sequence which is a nucleotide sequence, and (ii) L-chain variable region containing an L-chain variable region nucleotide sequence of SEQ ID NO: 38. Nucleic acid encoding the monoclonal antibody molecule MAb2A or a derivative thereof according to [1].
  • MAb2C-1 nucleic acid (i) H-chain variable region containing at least 80% identical to the H-chain variable region nucleotide sequence of SEQ ID NO: 30 and (ii) at least 80% identical to the L-chain variable region nucleotide sequence of SEQ ID NO: 38.
  • L-chain variable region containing nucleotide sequence Nucleic acid encoding a mutant monoclonal antibody molecule or derivative thereof having binding identification and function equivalent to that of the monoclonal antibody molecule MAb2A or MAb2B, including MAb2C-2 molecule: (i) In the H chain variable region nucleotide sequence of SEQ ID NO: 30, at least one framework (FW) region containing a nucleotide sequence having one or several nucleotide substitutions, deletions, insertions or additions relative thereto.
  • FW framework
  • the L chain variable region containing, and (ii) the L chain variable region nucleotide sequence of SEQ ID NO: 38, at least one comprising a nucleotide sequence having one or several nucleotide substitutions, deletions, insertions or additions relative to it.
  • L-chain variable region containing 15 framework regions [14] The nucleic acid according to [14], which is a nucleic acid encoding a mutant monoclonal antibody molecule or a derivative thereof having binding identification and function equivalent to that of the monoclonal antibody molecule MAb2A or MAb2B.
  • MAb3A nucleic acid (i) H chain variable region CDR1 nucleotide sequence of SEQ ID NO: 58, H chain variable region CDR2 nucleotide sequence of SEQ ID NO: 59 and H chain variable region CDR3 nucleotide sequence of SEQ ID NO: 60, and (ii) sequence.
  • MAb3B nucleic acid (i) H-chain variable region of SEQ ID NO: 46 An H-chain variable region containing a nucleotide sequence which is a nucleotide sequence, and (ii) an L-chain variable region containing a nucleotide sequence which is an L-chain variable region of SEQ ID NO: 54; Nucleic acid encoding the monoclonal antibody molecule MAb3A or a derivative thereof according to [1].
  • MAb3C-1 nucleic acid (i) H-chain variable region containing at least 80% identical to the H-chain variable region nucleotide sequence of SEQ ID NO: 46, and (ii) at least 80% identical to the L-chain variable region nucleotide sequence of SEQ ID NO: 54.
  • L-chain variable region containing nucleotide sequence Nucleic acid encoding a mutant monoclonal antibody molecule or derivative thereof having binding identification and function equivalent to that of the monoclonal antibody molecule MAb3A or MAb3B, including MAb3C-2 nucleic acid: (i) In the H chain variable region nucleotide sequence of SEQ ID NO: 46, at least one framework (FW) region containing a nucleotide sequence having one or several nucleotide substitutions, deletions, insertions or additions relative to the nucleotide sequence.
  • FW framework
  • L chain variable region containing, and (ii) the L chain variable region nucleotide sequence of SEQ ID NO: 54, at least one comprising a nucleotide sequence having one or several nucleotide substitutions, deletions, insertions or additions relative to it.
  • L-chain variable region containing 15 framework regions; A nucleic acid encoding a mutant monoclonal antibody molecule or a derivative thereof having binding identification and function equivalent to that of the monoclonal antibody molecule MAb3A or MAb3B, which comprises the nucleic acid according to [14].
  • An expression vector containing the nucleic acid according to any one of [14] to [17].
  • [19] [18] A host cell containing the vector described.
  • a method for producing a monoclonal antibody molecule or a derivative thereof which comprises culturing the host cell according to [19] under conditions suitable for gene expression.
  • a method for preparing antibodies against highly pathogenic viruses 1) Prepare positive exosomes of a membrane protein or a part thereof expressed on the virus surface of the highly pathogenic virus. 2) An antibody is prepared by performing an immune reaction using a membrane protein or a part thereof expressed on the virus surface of the highly pathogenic virus. 3) An antibody against a highly pathogenic virus is prepared, which comprises examining the reactivity of the antibody prepared in step 2 to the positive exosome prepared in step 1) and selecting an antibody having a desired reactivity. how to. [22] The method according to [21], wherein the antibody is a monoclonal antibody.
  • an immunoassay for SARS-CoV-2 can be constructed by utilizing a monoclonal antibody against the S1 domain of a peaplomer protein expressed on the surface of SARS-CoV-2, and the monoclonal antibody is the spike protein S1 domain. It is possible to provide a drug that suppresses the infection of SARS-CoV-2 into cells by inhibiting the binding between SARS-CoV-2 and ACE2.
  • FIG. 1 is a graph showing the results of comparison of binding reactivity for specifically recognizing RBD region proteins in seven monoclonal antibodies obtained by ELISA.
  • FIG. 2 shows the results of Western blotting with forced expression of peplomer proteins in Expi293 cells and their exosomes.
  • FIG. 3 is a graph showing the reactivity of seven monoclonal antibodies specifically recognizing the SARS-CoV-2 peplomer with peplomer-positive exosomes (Exo-S).
  • FIG. 4 shows a calibration curve of Exo-S measurements by ELISA using the anti-peplomer monoclonal antibody clone A2-5A12.
  • FIG. 5 is a graph showing the results of comparison of binding inhibition between the spike protein RBD region and ACE2 in the anti-peplomer monoclonal antibody clone A2-5A12 and a commercially available antibody (Sinobiological).
  • FIG. 6 is a graph showing the results of comparison of the binding inhibition of Exo-S and ACE2 in the anti-peplomer monoclonal antibody clone A2-5A12 and the commercially available antibody (Sinobiological).
  • FIG. 7 shows the structure of the H chain variable region of the anti-peplomer monoclonal antibody clone A2-5A12.
  • FIG. 8 shows the structure of the L chain variable region of the anti-peplomer monoclonal antibody clone A2-5A12.
  • FIG. 9 is a graph comparing the antibody titers against the RBD region protein in 7 clones of the anti-peplomer monoclonal monoclonal antibody.
  • FIG. 10 is a graph showing the results of examining the reactivity of 7 clones of anti-spiked protein monoclonal antibody to RBD region protein in ELISA under each antibody combination.
  • FIG. 11 is a graph showing the results of examining the reactivity of 7 clones of the anti-peplomer monoclonal antibody against Exo-S in ELISA under each antibody combination.
  • FIG. 12 is a graph showing ACE2 / peplomer binding inhibition using 7 clones of anti-peplomer monoclonal antibody.
  • FIG. 13 is a graph showing ACE2 / Exo-S binding inhibition using 7 clones of an anti-peplomer monoclonal antibody.
  • FIG. 14 shows the structure of the H chain variable region of the anti-peplomer monoclonal antibody clone A1-1C7.
  • FIG. 15 shows the structure of the L chain variable region of the anti-peplomer monoclonal antibody clone A1-1C7.
  • FIG. 16 shows the structure of the H chain variable region of the anti-peplomer monoclonal antibody clone A1-5C3.
  • FIG. 17 shows the structure of the L chain variable region of the anti-peplomer monoclonal antibody clone A1-5C3.
  • FIG. 18 is a schematic diagram showing amino acid mutations in three SARS-CoV-2 mutants, VOC-202012 / 01 (UK type), 501Y.V2 (South Africa type), and 501Y.V3 (Brazil type). ..
  • FIG. 19 shows amino acid mutations in the RBD regions of three SARS-CoV-2 mutants, VOC-202012 / 01 (UK type), 501Y.V2 (South Africa type), and 501Y.V3 (Brazil type). It is a schematic diagram.
  • FIG. 20 is a graph showing the results of examining the reactivity of 7 clones of the anti-peplomer monoclonal antibody to the RBD region protein having the N501Y mutation in ELISA under each antibody combination.
  • FIG. 21 is a graph showing the results of examining the reactivity of 7 clones of anti-spiked protein monoclonal antibody to RBD region protein having E484K mutation and N501Y mutation in ELISA under each antibody combination.
  • the present invention relates, as one form, to an isolated antibody molecule that specifically recognizes the SARS-CoV-2 peaplomer, or a derivative thereof.
  • viruses are present in biological samples of blood, saliva, and nasal mucosa in SARS-CoV-2 infected persons, and infection is performed by detecting the virus in those samples. Can be inspected for the presence of virus.
  • As a detection method it is possible to prepare a monoclonal antibody against the S1 domain of the spike protein expressed on the surface of the virus and construct an immunoassay against the virus.
  • the monoclonal antibody binds to the spike protein and the binding between the spike protein S1 domain and ACE2 is inhibited, it may become a drug that suppresses virus infection to cells. From these situations, we prepared a monoclonal antibody with high specificity and affinity for the RBD region of the SARS-CoV-2 spike protein S1 domain.
  • the narrow 372-531 region that binds to ACE2 in the S1 domain of the spike protein the anti-peplomer monoclonal antibody clone A2- that strongly reacts with the RBD region protein.
  • 5A12, 4 clones that react strongly like this (A1-1C7, A1-1F6, A2-2B6, A2-7G6), and 2 clones that react slightly strongly (A1-5C3, A2-9D2) were created (Fig.). 9).
  • These seven anti-spiked protein monoclonal antibodies consist of clones A2-5A12, A1-1F6, A2-2B6 and A2-7G6, clones A1-1C7 and A2-9D2 due to differences in reaction specificity to epitopes. It can be divided into three groups: groups and clones A1-5C3.
  • MAb1A (related to clone A2-5A12): (i) H-chain variable region CDR1 amino acid sequence of SEQ ID NO: 14 (58-GYTFIDYT-65), H-chain variable region CDR2 amino acid sequence of SEQ ID NO: 15 (83-FNPKFSDT-90) and H-chain variable region CDR3 of SEQ ID NO: 16. H chain variable region containing the amino acid sequence (128-CARDGYPYYYALDYW-142), (ii) L chain variable region CDR1 amino acid sequence of SEQ ID NO: 22 (51-QNVDGN-56), L chain variable region CDR2 amino acid sequence of SEQ ID NO: 23.
  • L-chain variable region containing the CDR3 amino acid sequence (112-CHQYKSYPYTF-122), An isolated antibody molecule, or derivative thereof, that specifically recognizes the SARS-CoV-2 spike protein, including MAb2A (related to clone A1-1C7): (i) H-chain variable region CDR1 amino acid sequence of SEQ ID NO: 31 (45-GFNIKDTY-52), H-chain variable region CDR2 amino acid sequence of SEQ ID NO: 32 (70-IDPASDNT-77), and H-chain variable region CDR3 of SEQ ID NO: 33.
  • L-chain variable region containing the CDR3 amino acid sequence (107-CQNVLSVPWTF-117), An isolated antibody molecule, or derivative thereof, that specifically recognizes the SARS-CoV-2 peaplomer, including MAb3A (related to clone A1-5C3): (i) H-chain variable region CDR1 amino acid sequence of SEQ ID NO: 47 (45-GFNIKDAY-52), H-chain variable region CDR2 amino acid sequence of SEQ ID NO: 48 (70-IDPANGNT-77) and H-chain variable region CDR3 of SEQ ID NO: 49.
  • MAb3A related to clone A1-5C3
  • H chain variable region containing the amino acid sequence (115-CARDSNPYYYALDFWSQ-131), and (ii) L chain variable region CDR1 amino acid sequence of SEQ ID NO: 55 (49-QDINNY-54), L chain variable region CDR2 amino acid sequence of SEQ ID NO: 56. (72-RAN-74) and L-chain variable region of SEQ ID NO: 57
  • the L-chain variable region containing the CDR3 amino acid sequence 110-CLQYDEFPWTF-120
  • the present invention also comprises another embodiment of the above embodiment.
  • MAb1B (related to clone A2-5A12): (i) H-chain variable region containing the H-chain variable region amino acid sequence of SEQ ID NO: 12, and (ii) L-chain variable region containing the L-chain variable region amino acid sequence of SEQ ID NO: 20.
  • An isolated antibody molecule, or derivative thereof, that specifically recognizes the SARS-CoV-2 peaplomer including MAb3B (related to clone A1-5C3): (i) H-chain variable region containing the H-chain variable region amino acid sequence of SEQ ID NO: 45, and (ii) L-chain variable region containing the L-chain variable region amino acid sequence of SEQ ID NO: 53.
  • isolated antibody molecules, or derivatives thereof, that specifically recognize the SARS-CoV-2 peaplomer including.
  • a "monoclonal antibody molecule” is a tetrapeptide chain structure in which two identical H chains (heavy chains) and two identical L chains (light chains) are interconnected by disulfide bonds. Means the immunoglobulin molecule of.
  • the approximately 110 amino acid sequences flanking the N-terminus of the antibody H and L chains are highly variable and are known as variable regions (Fv regions).
  • the rest of the amino acid sequence near the C-terminus is relatively stable and is known as the constant region.
  • the variable region comprises three hypervariable regions (HVR) and four framework regions (FR) that are relatively conserved.
  • the three hypervariable regions determine the specificity of the antibody and are also known as complementarity determining regions (CDRs).
  • Each L-chain variable region (LCVR) and each H-chain variable region (HCVR) consists of three CDR regions and, from the amino terminus to the carboxyl terminus, FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 in a contiguous order. It consists of four FR regions.
  • the three L-chain CDRs refer to LCDR1, LCDR2 and LCDR3, and the three H-chain CDRs refer to HCDR1, HCDR2 and HCDR3.
  • isolated means a sample isolated from its natural environment.
  • a naturally occurring polynucleotide or polypeptide present in a living animal is the same polynucleotide or polypeptide that has not been isolated but has been artificially isolated from some or all of the coexisting samples in a natural system. The peptide has been isolated.
  • the "derivative of a monoclonal antibody molecule” means a diabody, a single chain molecule, and an “antigen-binding fragment”.
  • Antigen-binding fragment refers to a Fab fragment having antigen-binding activity, a Fab'fragment, an F (ab') 2 fragment, and an Fv fragment and scFv fragment that bind to SARS-CoV-2, as defined in the present invention. It can contain one or more CDR regions in an antibody.
  • the Fv fragment is the smallest antibody fragment containing all antigen binding sites that do not have an H chain variable region, an L chain variable region, and a constant region.
  • Fv antibodies further include a polypeptide linker between the H-chain variable region and the L-chain variable region domains and can form the structures required for antigen binding.
  • different linkers are used to link the variable regions of the two antibodies to form a single chain antibody or a polypeptide named single chain Fv (scFv).
  • the "antigen binding site" of the invention refers to a discontinuous three-dimensional site on an antigen recognized by the monoclonal antibody or antigen binding fragment of the invention.
  • scFv single chain Fv
  • the "antigen binding site” of the invention refers to a discontinuous three-dimensional site on an antigen recognized by the monoclonal antibody or antigen binding fragment of the invention.
  • SARS-CoV-2 spike protein means having the ability to bind to a SARS-CoV-2 spike protein, which means “the spike protein”. It is synonymous with “monoclonal antibody that binds to the spike protein” or “monoclonal antibody against the spike protein”.
  • the present invention is an embodiment of the above embodiment.
  • MAb1C-1 (related to clone A2-5A12): (i) H-chain variable region containing an amino acid sequence that is at least 80% identical to the H-chain variable region amino acid sequence of SEQ ID NO: 12, and (ii) at least 80% identical to the L-chain variable region amino acid sequence of SEQ ID NO: 20.
  • MAb2C-1 (related to clone A1-1C7): (i) H-chain variable region containing an amino acid sequence that is at least 80% identical to the H-chain variable region amino acid sequence of SEQ ID NO: 29, and (ii) at least 80% identical to the L-chain variable region amino acid sequence of SEQ ID NO: 37.
  • a monoclonal antibody molecule having a mutation that is, a mutant monoclonal antibody molecule or a derivative thereof, has binding characteristics and functions equivalent to those of the original monoclonal antibody molecule.
  • the present invention is a further embodiment of the above embodiment.
  • MAb1C-2 (related to clone A2-5A12): (i) In the H chain variable region amino acid sequence of SEQ ID NO: 12, at least one framework (FW) containing an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions as compared to it. ) H-chain variable region containing the region; and (ii) an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions in the L-chain variable region amino acid sequence of SEQ ID NO: 20.
  • L chain variable region including at least one framework region containing A mutant monoclonal antibody molecule or a derivative thereof comprising the above, wherein the mutant monoclonal antibody molecule has the same binding characteristics and functions as those of the monoclonal antibody molecules MAb1A or MAb1B, or the mutant monoclonal antibody molecule thereof.
  • Derivatives; MAb2C-2 (related to clone A1-1C7): (i) In the H chain variable region amino acid sequence of SEQ ID NO: 29, at least one framework (FW) containing an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions as compared to it.
  • L chain variable region including at least one framework region containing A mutant monoclonal antibody molecule or a derivative thereof comprising the above, wherein the mutant monoclonal antibody molecule has the same binding characteristics and functions as those of the monoclonal antibody molecule MAb2A or MAb2B, or the mutant monoclonal antibody molecule thereof.
  • L chain variable region including at least one framework region containing A mutant monoclonal antibody molecule or a derivative thereof comprising the above, wherein the mutant monoclonal antibody molecule has the same binding characteristics and functions as those of the monoclonal antibody molecule MAb3A or MAb3B, or the mutant monoclonal antibody molecule thereof.
  • a monoclonal antibody molecule having a mutation that is, a mutant monoclonal antibody molecule or a derivative thereof, has binding characteristics and functions equivalent to those of the original monoclonal antibody molecule.
  • Anti-peplomer monoclonal antibody clone A2-5A12 H chain variable region amino acid sequence: SEQ ID NO: 12 1-MSSPQTLNTLTLTMGWSWIFLFLLSGTAGVLSEVQLQQSGPELLKPGASVKISCKASGYTFIDYTMYWVKQSHGKSLEWIGGFNPKFSDTIYNEKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYFCARDGYPYYYALDYWGQGTSVTVSS-152 L chain variable region amino acid sequence: SEQ ID NO: 20 1-MGIKMESQTQVFVYMLLWLSGIDGDIVMTQSQKFMSTSVGDRVSVTCKASQNVDGNVAWYQQNPGQSPKALIYSASSRYSGVPDRFTGSGTAFTLTISNVQSEDLAEYFCHQYKSYPYTFGGGTRLEIK-131
  • Anti-Peplomer Monoclonal Antibody Clone A1-1C7 H chain variable region amino acid sequence: SEQ ID NO: 29 1-MKCSWVIFFLMAVVTGVTSEVQLQQSGTELVTPGASVKLSCTASGFNIKDTYVQWVKQRPEQGLEWIGRIDPASDNTFYASKFQGKASLSSDTSSNTAYMQLSSLTSGDTAVYYCSTYFSNFYYWGQGTPLTVSS-135 L chain variable region amino acid sequence: SEQ ID NO: 37 1-MGVPTQLLLLWLTVVVVRCDIQMTQSPASLSASVGDTVTITCGASENIYGALNWYQRKKGKSPQLLIYGATNLADGMSSRFSGRGSGRQYFLKISSLHPDDVATYYCQNVLSVPWTFGGGTKLEIK-126
  • Anti-peplomer monoclonal antibody clone A1-5C3 H chain variable region amino acid sequence: SEQ ID NO: 45 1-MKCSWVIFFLMAVVTGVNSEVQLQQSGAELVKPGASVKLSCTASGFNIKDAYMHWVKQRPEQGLEWIGRIDPANGNTIYASKFQDKATITADTSSNTAYMQLSSLTSGDTAVYYCARDSNPYYYALDFWSQGTSVTVSS-139 L chain variable region amino acid sequence: SEQ ID NO: 53 1-MDMRTPAQFLGILLLWFPGIKCDIKMTQSPSSMEASLGERVTITCKASQDINNYLTWFQQKPGKSPKTLISRANRLVDGVPSRFSGSGSGQDYSLTISSLEFEDMGIYYCLQYDEFPWTFGGGTNLEIK-129
  • MAb1B (related to clone A2-5A12), MAb2B (related to clone A1-1C7) and MAb3B (related to clone A1-5C3) are H-chain variable region amino acid sequences and L-chain variable.
  • the region amino acid sequence can have an amino acid sequence that is at least 80% identical to the specific amino acid sequence. 80% is preferably 85%, more preferably 90%, and even more preferably 95%.
  • H-chain variable regions and L-chain variable regions are frequently named "complementarity determining regions" (CDRs), which are interspersed with more conserved regions than named “framework regions” (FR or FW). It is a variant because it can be subdivided into regions of variability.
  • CDRs complementarity determining regions
  • the L-chain or H-chain variable framework of the monoclonal antibody molecule against the SARS-CoV-2 peaplomer is at least 80%, 85%, of the L-chain or H-chain variable framework residues derived from the human consensus sequence.
  • An L-chain or H-chain variable framework containing 87%, 90%, 93%, 95%, 97%, 99% can be included.
  • the present invention is intended to treat or prevent fever, malaise, respiratory failure, acute inflammation or thrombosis associated with Covid-19, which comprises, in one form, an effective amount of the monoclonal antibody molecule of the invention or a derivative thereof.
  • an effective amount of the monoclonal antibody molecule of the invention or a derivative thereof With respect to the pharmaceutical composition of.
  • SARS-CoV-2 when SARS-CoV-2 infects a cell, the spike protein S1 domain of SARS-CoV-2 first binds to ACE2 on the cell surface, and then SARS-CoV-2 is taken up into the cell. It has become clear.
  • the monoclonal antibody against the S1 domain of the peplomer expressed on the virus surface inhibits the binding between the peplomer S1 domain and ACE2 by binding to the spike protein of SARS-CoV-2, and infects the virus cells. May be suppressed.
  • the preferred antibody molecule or derivative thereof is MAb1A (related to clone A2-5A12): (i) H-chain variable region CDR1 amino acid sequence of SEQ ID NO: 14, H-chain variable region CDR2 amino acid sequence of SEQ ID NO: 15, H-chain variable region of SEQ ID NO: 16, H-chain variable region containing CDR3 amino acid sequence, and (ii) sequence.
  • SARS-CoV-2 comprising the L-chain variable region CDR1 amino acid sequence of No. 22, the L-chain variable region CDR2 amino acid sequence of SEQ ID NO: 23, and the L-chain variable region of SEQ ID NO: 24; the L-chain variable region containing the CDR3 amino acid sequence.
  • MAb1B An isolated monoclonal antibody molecule or derivative thereof that specifically recognizes spike proteins; MAb1B (related to clone A2-5A12): (i) H-chain variable region of SEQ ID NO: 12 H-chain variable region containing the amino acid sequence, and (ii) L-chain variable region of SEQ ID NO: 20 Spike of SARS-CoV-2 containing the L-chain variable region containing the amino acid sequence.
  • Mutant monoclonal antibody molecule or derivative thereof; and MAb1C-2 (related to clone A2-5A12): (i) In the H chain variable region amino acid sequence of SEQ ID NO: 12, at least one framework (FW) containing an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions as compared to it. ) H-chain variable region containing the region; and (ii) an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions in the L-chain variable region amino acid sequence of SEQ ID NO: 20.
  • L chain variable region containing at least one framework region containing; A mutant monoclonal antibody molecule or a derivative thereof, wherein the mutant monoclonal antibody molecule has binding identification and function equivalent to that of the monoclonal antibody molecule MAb1A; An isolated monoclonal antibody molecule, a mutant monoclonal antibody molecule, or a derivative thereof that specifically recognizes the SARS-CoV-2 spike protein selected from the above.
  • Covid-19 is synonymous with SARS-CoV-2 infection, and droplets containing SARS-CoV-2 emitted from the infected person's lungs, respiratory tract, or oral cavity float in the air, and these droplets are discharged from non-infected persons. It is transmitted and transmitted by a person inhaling it. It can also be transmitted by touching the mouth, nose, eyes, etc. with the hand that touches the virus-attached object. Most cases of infection in children are considered to be subclinical infections, but the incidence increases with age, and symptoms such as fever, malaise, and cough appear in respiratory disorders. When an elderly person is infected, the severity of pneumonia increases and treatment with a ventilator is required, but death is not uncommon.
  • the pharmaceutical composition of the present invention is a pharmaceutical composition for treating or preventing respiratory insufficiency such as fever, malaise, or cough associated with Covid-19.
  • a "pharmaceutical composition” is one or more containing one or more other chemical components such as stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients.
  • a monoclonal antibody against the SARS-CoV-2 spike protein containing one or more other chemical components such as stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients.
  • the pharmaceutical composition facilitates the administration of a monoclonal antibody against the peaplomer of SARS-CoV-2 to an organism.
  • a plurality of techniques for administering a monoclonal antibody against the spike protein of SARS-CoV-2 are known, and include, for example, oral administration or parenteral administration such as local administration, intravenous administration, aerosol administration, and ophthalmic administration.
  • dilute a desired drug eg, a monoclonal antibody against the spike protein of SARS-CoV-2
  • Diluents can also be used for stabilizers as they can provide a more stable environment. Salts dissolved in buffers capable of controlling or maintaining pH are useful as diluents, including phosphate buffered saline.
  • a “effective amount” or “therapeutically effective amount” is a monoclonal antibody to the spike protein of SARS-CoV-2 of the invention administered to relieve one or more degrees of symptoms of the disease, disorder or condition being treated. Means a sufficient amount of antibody. As a result, it is possible to alleviate and / or alleviate the signs, symptoms or causes of the disease.
  • an "effective amount” in a therapeutic application is a spike of SARS-CoV-2 required to provide the desired pharmacological effect, therapeutic improvement or clinically significant reduction of disease symptoms without excessive side effects. The amount of monoclonal antibody against the protein.
  • Effective amount is the metabolism, genetics, combination, or age, weight, general condition, condition to be treated, severity of condition to be treated, and prescription of a monoclonal antibody against the spike protein of SARS-CoV-2. It may differ from subject to subject due to differences in the judgment of the doctor.
  • an “effective amount” includes a “preventive effective amount”, which is the SARS-CoV- of the invention applied to a patient to alleviate one or more degrees of symptoms of the disease, condition or disorder being treated. 2 means the amount of monoclonal antibody against the spike protein. In such prophylactic applications, such amounts may vary with subject health, body weight, etc.
  • subject or “patient” means an animal, which is the subject of treatment, observation or testing.
  • the subject may be a mammal, including a human.
  • Monoclonal antibodies against the SARS-CoV-2 peaplomer can be provided alone or as a pharmaceutical composition, preferably for humans.
  • the compounds may be prepared in a variety of pharmaceutical dosage forms according to prophylactic or therapeutic purposes. Examples of pharmaceutical dosage forms are oral preparations, injections, suppositories, ointments, ointments and the like. Such dosage forms are well known to those of skill in the art and can be formulated by conventional methods.
  • the amount of monoclonal antibody against the SARS-CoV-2 peaplomer incorporated into each unit dosage form may vary depending on the health condition of the subject or the type of formulation.
  • the preferred amount per unit dosage form is about 1-1000 mg for oral administration and about 0.1-500 mg for injection.
  • the daily dose depends on the patient's symptoms, weight, age, gender and other factors, and the usual range is about 0.1-5000 mg, preferably about 1-1000 mg per day for adults.
  • the formulation is preferably administered in a single dose or in 2-4 divided doses.
  • Excipients and, if necessary, binders, disintegrants, lubricants, colorants, corrective agents, fragrances, etc. are added to the anti-PD-1 preparations to formulate solid preparations for oral administration. Is formulated into tablets, coated tablets, granules, powders, capsules and the like by a usual method. Such additives are well known in the art and useful examples are excipients such as lactose, sucrose, sodium chloride, starch, calcium carbonate, kaolin, crystalline cellulose and silicic acid, water, ethanolpropanol, simple.
  • Excipients such as syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate and polyvinylpyrrolidone, dried starch, sodium alginate, agar powder, sodium hydrogen carbonate, Disintegrants such as calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride and lactose, lubricants such as purified talc, stearate, borosand and polyethylene glycol, and corrective agents such as sucrose, orange peel, citric acid and tartrate acid.
  • Disintegrants such as calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride and lactose
  • lubricants such as purified talc, stearate, borosand and polyethylene glycol
  • corrective agents such as sucrose, orange peel, citric acid
  • a corrective agent for the formulation of a liquid formulation for oral administration, a corrective agent, a buffer, a stabilizer, a fragrance, etc. are added to the compound, and the mixture thereof is formulated into an oral solution formulation, a syrup agent, an elixir agent, etc. by a conventional method.
  • a syrup agent for the formulation of a liquid formulation for oral administration, a corrective agent, a buffer, a stabilizer, a fragrance, etc.
  • a conventional method for the formulation of a liquid formulation for oral administration, a corrective agent, a buffer, a stabilizer, a fragrance, etc.
  • a conventional method can be done.
  • useful corrective agents are as described above.
  • buffers are sodium citrate and the like.
  • stabilizers are tragacanth, gum arabic, gelatin and the like.
  • the syrup can be prepared in a conventional manner with the following proportions of the ingredients.
  • Injections can be made subcutaneously, intramuscularly or by the conventional method by adding a pH regulator, buffer, stabilizer, isotonic agent, local anesthetic, etc. to the monoclonal antibody against the spike protein of SARS-CoV-2.
  • pH regulators and buffers are sodium citrate, sodium acetate, sodium phosphate and the like.
  • stabilizers are sodium metabisulfite, EDTA, thioglycolic acid and the like.
  • local anesthetics are procalin hydrochloride, lidocaine hydrochloride and the like.
  • isotonic agents are sodium chloride, glucose and the like.
  • the injection can be prepared in a conventional manner with the proportions of the ingredients shown below.
  • treatment means (1) delaying or preventing the onset of a disease or condition; (2) slowing or stopping the progression, exacerbation, or exacerbation of symptoms of the disease or condition; (3) disease or condition. It results in remission of the symptoms of; or (4) means a method or process aimed at curing a disease or condition. Treatment may be given as a precautionary measure before the onset of the disease or condition, or treatment may be given after the onset of the disease.
  • prevention means preventing the onset of an inflammatory and / or fibrotic state.
  • the pharmaceutical composition usually means a drug for treating, treating or preventing a disease or pathological condition, or for testing / diagnosis.
  • the present invention is, as a further form, (i) Obtain a biological sample from the subject and (ii) The resulting biological sample is contacted with the isolated monoclonal antibody molecule, mutant monoclonal antibody molecule or derivative thereof of the invention, and (iii) SARS-CoV-2 and monoclonal antibody molecule, mutation. It relates to a method for detecting and / or quantifying SARS-CoV-2 in a biological sample, which comprises detecting or quantifying the formation of a complex between a monoclonal antibody molecule or a derivative thereof.
  • the preferred antibody molecule of the invention is -One monoclonal antibody selected from the monoclonal antibody molecules MAb1A and MAb1B, and the mutant monoclonal antibody molecules MAb1C-1 and MAb1C-2 (related to clone A2-5A12). • Monoclonal antibody molecules MAb2A and MAb2B, as well as one monoclonal antibody selected from the mutant monoclonal antibody components MAb2C-1 and MAb2C-2 (related to clone A1-1C7), and • Monoclonal antibody molecules MAb3A and MAb3B, as well as mutant monoclonal antibodies. Minutes Selected from one monoclonal antibody selected from MAb3C-1 and MAb3C-2 (related to clones A1-5C3).
  • the biological sample means a body fluid, particularly blood, plasma, serum, saliva, nasal mucosa, and may be abdominal fluid, brain fluid, bone marrow, urine, feces, or bronchoalveolar lavage fluid.
  • Preferred biological samples are blood, plasma, serum, saliva, or nasal mucosa.
  • the method for detecting the formation of a complex between SARS-CoV-2 and a monoclonal antibody molecule or a derivative thereof is a plastic plate or magnetic beads.
  • a monoclonal antibody that recognizes SARS-CoV-2 to a carrier such as SARS-CoV-2 and contacting it with a biological sample containing SARS-CoV-2, SARS-CoV-2 becomes a monoclonal antibody on the carrier.
  • a luminescent substrate is allowed to act on a complex in which a monoclonal antibody against the spike protein of SARS-CoV-2, which is bound to SARS-CoV-2 and labeled with a labeled substance such as horse radish peroxidase, is bound to the SARS-CoV-2.
  • the complex is quantified by detecting the emission intensity with.
  • the monoclonal antibody and the labeled antibody to be bound to the carrier can be carried out by a combination of any two anti-spiked protein monoclonal antibodies, but in one embodiment, each antibody is SARS-CoV-2. It is preferable to recognize different sites, i.e., epitopes.
  • the antibody to be bound to the carrier and the labeled antibody are selected from different groups. For example, anti-spike protein monoclonal antibody clone A2-5A12 (eg, one antibody molecule selected from the monoclonal antibody molecules MAb1A and MAb1B, and the mutant monoclonal antibody molecules MAb1C-1 and MAb1C-2), A1-1F6, A2-2B6.
  • the monoclonal antibody and labeled antibody to be bound to the carrier is one antibody selected from Group 1 consisting of anti-peplomer monoclonal antibody clones A2-5A12, A1-1F6, A2-2B6 and A2-7G6. Any of different groups from one antibody molecule selected from Group 2 consisting of the molecule, clones A1-1C7 and A2-9D2, and one antibody molecule selected from Group 3 of clone A1-5C3. Use the two.
  • the invention in another form, relates to a kit for detecting and / or quantifying SARS-CoV-2, which comprises the isolated monoclonal antibody molecule of the invention or a derivative thereof.
  • the kit of the present invention may include one or more other elements such as: Instructions for use; agents for labeling other reagents, such as labels, or antibodies.
  • the kit of the present invention preferably contains a standard reagent. Examples of the standard reagent include SARS-CoV-2 spike protein-positive exosomes (Exo-S).
  • the "SARS-CoV-2 spike protein-positive exosome” means an exosome expressing a spike protein on its surface. Peplomer-positive exosomes can be readily prepared by the techniques described herein (Example 2-1).
  • SARS-CoV-2 mutant strain the amino acid mutations of VOC-202012 / 01 (UK type), 501Y.V2 (South Africa type), and 501Y.V3 (Brazil type), which are said to have strong infectivity, are shown in Fig. 18. , The mutations of amino acids in the RBD region of these mutants are shown in FIG. Commonly present in the three mutant strains is N501Y, in which the 501st amino acid asparagine is mutated to tyrosine, and the mutation common to both the South African type and the Brazilian type is the 484th glutamic acid mutated to lysine. It is E484K. The 417th lysine is mutated to asparagine in the South African type and to tyrosine in the Brazilian type.
  • all of the monoclonal antibodies of the invention have the reactivity of each antibody with the RBD region protein with the N501Y mutation as the reactivity with the RBD region protein without the mutation (FIG. 10). Similarly, the N501Y mutation showed that each antibody was unaffected by reactivity (FIG. 20).
  • the ability of the monoclonal antibody of the invention to recognize peplomers without being affected by the N501Y mutation commonly present in the three mutants means that the monoclonal antibody of the invention can detect and / or detect all three mutants. It means that it can be used for an assay for quantification.
  • the present invention is a further embodiment.
  • (i) Obtain a biological sample from the subject and (ii) The resulting biological sample is contacted with the isolated monoclonal antibody molecule, mutant monoclonal antibody molecule or derivative thereof of the invention, and (iii) VOC-202012 / 01 (UK type), 501Y.
  • the monoclonal antibody molecule, the mutant monoclonal antibody molecule, or the biological sample of the present invention is as described above.
  • A1-1C7 is unaffected by the N501Y mutation, but loses reactivity with the E484K and N501Y mutations, so that the E484K mutation reduces the binding force of the A1-1C7 to the RBD region protein. Is shown.
  • the sandwich ELISA using the monoclonal antibody of the present invention is a labeled antibody when measuring a biological sample containing a new coronavirus mutant strain, for example, when A2-5A12 is used as a solid phase antibody.
  • Biological samples were measured using A1-1C7 and A1-5C3, and if a signal was detected in any combination, no mutation or British type, signal was detected only in combination with A1-5C3. In that case, it was shown that it could be determined that it was a South African type or a Brazilian type.
  • an immunoassay for determining SARS-CoV-2 amino acid variants in biological samples (i) Obtain a biological sample from the subject and (ii) The obtained biological sample is used as one antibody molecule or antibody selected from the isolated antibody molecules MAb1A, MAb1B, MAb1C-1 and MAb1C-2 (related to clone A2-5A12) of the present invention.
  • One antibody molecule selected from the molecules MAb2A, MAb2B, MAb2C-1 and MAb2C-2 (related to clone A1-1C7), and antibody molecules MAb3A, MAb3B, MAb3C-1 and MAb3C-2 (related to clone A1-5C3).
  • SARS-CoV-2 in the sample is determined to be no mutation or VOC-202012 / 01 (UK type), or one antibody related to clone A2-5A12. If only the combination of the molecule and one antibody molecule associated with clone A1-5C3 was positive, SARS-CoV-2 in the sample was 501Y.V2 (South African type) or 501Y.V3 (Brazilian type). With respect to methods, including determining that there is.
  • the immunoassay is a general term for antigen testing methods using antibodies, and is preferably a sandwich ELISA method, an immunochromatography method, or the like.
  • the immunochromatography method the presence or absence of an antigen in a sample is detected by dropping the sample onto a reagent device and visually detecting the presence or absence of a product due to an antigen-antibody reaction in the device by moving a cellulose membrane by capillarity. This is a quick and easy method for determining.
  • the invention in another form, relates to an isolated nucleic acid encoding a monoclonal antibody molecule, a mutant monoclonal antibody molecule or a derivative thereof of the invention.
  • the invention comprises an isolated monoclonal antibody molecule or derivative thereof that specifically recognizes a SARS-CoV-2 peaplomer, including MAb1A, MAb2A or MAb3A; including MAb1B, MAb2B or MAb3B.
  • the present invention relates to an isolated nucleic acid encoding a mutant monoclonal antibody molecule thereof or a derivative thereof.
  • the present invention relates to the nucleic acids shown below: MAb1A nucleic acid (related to clone A2-5A12): (i) Nucleic acid encoding an H chain variable region comprising the H chain variable region CDR1 nucleotide sequence of SEQ ID NO: 17, the H chain variable region CDR2 nucleotide sequence of SEQ ID NO: 18, and the H chain variable region CDR3 nucleotide sequence of SEQ ID NO: 19.
  • L-chain variable region CDR2 nucleotide sequence of SEQ ID NO: 43 and L-chain variable region CDR3 nucleotide sequence of SEQ ID NO: 44 Nucleic acid encoding the monoclonal antibody molecule MAb2A or its derivatives, including; MAb3A nucleic acid (related to clone A1-5C3): (i) H chain variable region CDR1 nucleotide sequence of SEQ ID NO: 58, H chain variable region CDR2 nucleotide sequence of SEQ ID NO: 59 and H chain variable region CDR3 nucleotide sequence of SEQ ID NO: 60, and (ii) sequence.
  • L-chain variable region containing the L-chain variable region CDR1 nucleotide sequence of No. 55, the L-chain variable region CDR2 nucleotide sequence of SEQ ID NO: 56, and the L-chain variable region CDR3 nucleotide sequence of SEQ ID NO: 57; Nucleic acid encoding the monoclonal antibody molecule MAb3A or its derivatives, including A nucleic acid encoding an isolated monoclonal antibody molecule or derivative thereof that specifically recognizes the SARS-CoV-2 spike protein.
  • MAb1B nucleic acid (related to clone A2-5A12): (i) H-chain variable region of SEQ ID NO: 13 H-chain variable region containing a nucleotide sequence which is a nucleotide sequence, and (ii) L-chain variable region containing an L-chain variable region nucleotide sequence of SEQ ID NO: 21.
  • Nucleic acid encoding the monoclonal antibody molecule MAb2B or its derivatives including; MAb3B nucleic acid (related to clone A1-5C3): (i) H-chain variable region of SEQ ID NO: 46 An H-chain variable region containing a nucleotide sequence which is a nucleotide sequence, and (ii) an L-chain variable region containing a nucleotide sequence which is an L-chain variable region of SEQ ID NO: 54; Nucleic acid encoding the monoclonal antibody molecule MAb3B or its derivatives, including The present invention relates to a nucleic acid encoding a monoclonal antibody molecule of the present invention or a derivative thereof.
  • MAb1C-1 nucleic acid (related to clone A2-5A12): (i) H-chain variable region containing at least 80% identical to the H-chain variable region nucleotide sequence of SEQ ID NO: 13, and (ii) at least 80% identical to the L-chain variable region nucleotide sequence of SEQ ID NO: 21.
  • L-chain variable region containing nucleotide sequence containing nucleotide sequence; MAb2C-1 nucleic acid (related to clone A1-1C7): (i) H-chain variable region containing at least 80% identical to the H-chain variable region nucleotide sequence of SEQ ID NO: 30 and (ii) at least 80% identical to the L-chain variable region nucleotide sequence of SEQ ID NO: 38.
  • L chain variable region containing nucleotide sequence or MAb3C-1 nucleic acid (related to clone A1-5C3): (i) H-chain variable region containing at least 80% identical to the H-chain variable region nucleotide sequence of SEQ ID NO: 46, and (ii) at least 80% identical to the L-chain variable region nucleotide sequence of SEQ ID NO: 54.
  • the present invention relates to a nucleic acid encoding a mutant monoclonal antibody molecule of the present invention or a derivative thereof.
  • the nucleic acid encoding a monoclonal antibody molecule having a mutation or a derivative thereof is a nucleic acid encoding a nucleic acid having binding characteristics and functions equivalent to those of the original monoclonal antibody molecule.
  • MAb1C-2 nucleic acid (related to clone A2-5A12): (i) In the H chain variable region nucleotide sequence of SEQ ID NO: 13, at least one framework (FW) region containing a nucleotide sequence having one or several nucleotide substitutions, deletions, insertions or additions relative to the nucleotide sequence.
  • FW framework
  • the L chain variable region nucleotide sequence of SEQ ID NO: 21 at least one comprising a nucleotide sequence having one or several nucleotide substitutions, deletions, insertions or additions relative to it.
  • the present invention relates to a nucleic acid encoding a mutant monoclonal antibody molecule of the present invention or a derivative thereof.
  • the nucleic acid encoding a monoclonal antibody molecule having a mutation or a derivative thereof is a nucleic acid encoding a nucleic acid having binding characteristics and functions equivalent to those of the original monoclonal antibody molecule.
  • nucleic acid means a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof.
  • the invention relates to an expression vector comprising the nucleic acid of the invention and a host cell comprising the vector of the invention.
  • the vector comprises a nucleotide sequence encoding the monoclonal antibody molecule described herein. In certain forms, the vector comprises the nucleotide sequences described herein.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).
  • YACs yeast artificial chromosomes
  • cells in which DNA is stably integrated into the chromosome may be selected by introducing one or more markers that allow selection of the transfected host cell. Markers can confer auxotrophic hosts, for example prototrophic, biocide resistant (eg, antibiotics), or resistance to heavy metals such as copper.
  • the selectable marker gene may be directly linked to the expressed DNA sequence or may be introduced into the same cell by co-transformation. Additional elements may be needed for optimal synthesis of mRNA. These elements may include splice signals, transcriptional promoters, enhancers, and termination signals.
  • the host cell has been genetically modified to contain a nucleic acid encoding a monoclonal antibody molecule.
  • the host cell has been genetically modified by the use of an expression cassette.
  • the expression "expression cassette" refers to a nucleotide sequence that can affect the expression of a gene in a host to which the sequence matches. Such cassettes may include promoters, open reading frames with or without introns, and termination signals. Additional factors required to enable expression, such as inducible promoters, may be used.
  • the cell can be a eukaryotic cell, a bacterial cell, an insect cell or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, CHO cells and the like. Suitable insect cells include, but are not limited to, Sf9 cells.
  • the invention relates to a method of producing a monoclonal antibody molecule or fragment thereof, comprising culturing the host cells of the invention under conditions suitable for gene expression.
  • the present invention is a method for preparing an antibody against a highly pathogenic virus. 1) Prepare positive exosomes of a membrane protein or a part thereof expressed on the virus surface of the highly pathogenic virus. 2) An antibody is prepared by performing an immune reaction using a membrane protein or a part thereof expressed on the virus surface of the highly pathogenic virus. 3) An antibody against a highly pathogenic virus is prepared, which comprises examining the reactivity of the antibody prepared in step 2 to the positive exosome prepared in step 1) and selecting an antibody having a desired reactivity. Regarding how to do it. Preferred antibodies are monoclonal antibodies, but polyclonal antibodies can also be prepared.
  • a hybridoma is produced from splenocytes excised from mice immunized with an antigen, and a hybridoma that secretes an antibody highly reactive with the antigen is screened.
  • a hybridoma that secretes an antibody highly reactive with the antigen is screened.
  • virus particles can be prepared and / or screened using virus particles.
  • highly virulent and highly pathogenic viruses such as SARS-CoV-2 require laboratory facilities with a high level of containment. As a means to avoid this, the idea was to express a virus surface antigen on the surface of exosomes to produce and use virus particle-like vesicles that are not infectious or pathogenic.
  • highly pathogenic virus is synonymous with highly virulent virus, for example, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS) coronavirus (MARS). -CoV), SARS coronavirus 2 (SARS-CoV2), highly pathogenic influenza virus, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), Ebola virus, etc. ..
  • SARS severe acute respiratory syndrome
  • MERS Middle East respiratory syndrome coronavirus
  • SARS-CoV SARS coronavirus 2
  • highly pathogenic influenza virus highly pathogenic influenza virus
  • HAV human immunodeficiency virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • Ebola virus etc. ..
  • the "membrane protein expressed on the surface of a virus” is a membrane that is expressed on the surface of a virus having a structure surrounded by a lipid double membrane and is considered to function when infecting a host cell.
  • Means protein examples include spike proteins in SARS-CoV-2, hemagglutinin (HA) and neurominidase (NA) in influenza virus, env proteins in HIV, HBs in HBV, E1 or E2 enveloped proteins in HCV, and enveloped glycoproteins in Ebola virus. Can be mentioned.
  • the "membrane protein expressed on the surface of a virus or a part thereof” is not particularly limited as long as it is a part of the membrane protein capable of inducing an antibody against the membrane protein together with an exosome. It may be a portion of a membrane protein, or it is a variant membrane protein satisfying this condition in which one or several amino acid residues are substituted, deleted or added in the amino acid sequence constituting the protein. It's okay.
  • the highly pathogenic virus is SARS-CoV-2
  • its diameter is usually 100 to 200 nm
  • spike proteins are expressed on the membrane surface.
  • This shape resembles an exosome, which is a microvesicle with a diameter of 30 to 120 nm. Therefore, we used a technique for producing an antibody against an exosome surface antigen and actually applied it to the production of an antibody against the spike protein of SARS-CoV-2.
  • the spike protein or a part of the gene When the spike protein or a part of the gene was introduced into cultured animal cells, the expression of the spike protein was confirmed in the exosomes purified from the culture supernatant.
  • exosomes are referred to as "the positive exosomes of the SARS-CoV-2 spike protein or a portion thereof". This time, by using the exosome, it was possible to obtain an antibody having higher specificity against the surface antigen expressed on the particulate lipid bilayer membrane.
  • This new screening method using exosomes is considered to be a method for obtaining antibodies with higher specificity for membrane proteins.
  • a membrane protein expressed on the surface of a highly pathogenic virus for example, a SARS-CoV-2 spike protein, or a part thereof is expressed in an exosome to prepare a positive exosome thereof. do.
  • a gene of a membrane protein expressed on the surface of a highly pathogenic virus for example, a peaplomer protein of SARS-CoV-2 is introduced into cultured animal cells and expressed.
  • exosomes can be prepared using conventional genetic engineering techniques as described herein.
  • the exosomes used are prepared by centrifuging the supernatant and body fluid of cultured animal cells in multiple steps.
  • the animal cell used in the present invention is, for example, HEK293 cell.
  • the gene of a part of the spike protein is a gene encoding both a part capable of penetrating the membrane of exosome and a part containing a binding site with ACE2. If it is, it is an arbitrary gene which is not particularly limited. It may be a portion of the spike protein, or it may be a gene encoding a mutant protein lacking a specific moiety that meets the above conditions.
  • the binding site with ACE2 is the S1 domain of the peplomer protein, more preferably the RBD region (Non-Patent Document 1). More preferably, some genes of the spike protein are genes lacking a protease recognition site.
  • protease recognition site examples include a sequence encoding an amino acid (Arg-Arg-Ala-Arg), which is a recognition site for the protein-cleaving enzyme furin.
  • the amino acid (Arg-Arg-Ala-Arg) of the recognition site of the protein-cleaving enzyme Flynn existing between the S1 and S2 region genes constituting the SARS-CoV-2 spike protein is coded. It is a part of the gene excluding all the sequences to be used, and its length is 3807 nt (nucleotide).
  • step 2) an immune reaction is carried out using a membrane protein expressed on the virus surface of a highly pathogenic virus or a part thereof to prepare various monoclonal antibodies.
  • a method for producing a monoclonal antibody a method well known to those skilled in the art can be used.
  • step 3 the reactivity of the monoclonal antibody prepared in step 2 to the positive exosomes prepared in step 1) is examined.
  • the method for preparing an antibody of the present invention in this form is when a membrane protein expressed on the virus surface of a highly pathogenic virus or a part thereof is immunized into a mouse to measure an antibody titer, or the splenocytes of an immunized mouse.
  • a membrane protein expressed on the virus surface of a highly pathogenic virus is expressed as a substitute for an infectious and pathogenic virus. Examine the reactivity with the virus.
  • the reactivity of the antibody with the exosome is, for example, an anti-mouse IgG antibody that recognizes a membrane protein or a part thereof expressed on the virus surface of a highly pathogenic virus and an anti-CD9 that recognizes the exosome. It can be examined by sandwich ELISA sandwiched between antibodies or anti-CD63 antibodies.
  • an antibody having a desired reactivity is selected.
  • the desired reactivity between the antibody and the exosome means the reactivity based on the index of showing significantly higher absorbance as compared with the non-specific antibody which is a negative control in the above-mentioned sandwich ELISA.
  • antibodies having such desired reactivity are selected.
  • the non-specific antibody serving as a negative control is an antibody whose absorbance due to non-specific binding can be examined.
  • the preparation method of the present invention can be applied to an antibody screening method for selecting a desired antibody from many antibodies. That is, the present invention is a method for screening a desired antibody against a highly pathogenic virus. 1) Prepare positive exosomes of a membrane protein or a part thereof expressed on the virus surface of the highly pathogenic virus. 2) An antibody is prepared by performing an immune reaction using a membrane protein or a part thereof expressed on the virus surface of the highly pathogenic virus. 3) The present invention relates to a method for screening an antibody against a highly pathogenic virus, which examines the reactivity of the antibody prepared in step 2 with respect to the positive exosome prepared in step 1) and selects an antibody having a desired reactivity. The desired reactivity is as described above.
  • the antibody is preferably a monoclonal antibody.
  • Example 1 Preparation of Monoclonal Antibodies to Spike Protein of SARS-CoV-2 (1) 1-1: Preparation of antigen Of the S1 and S2 regions constituting the spike protein of SARS-CoV-2, the expression vector pCMV3-CoV-2-S1-FLAG (manufactured by Shino Biological) of the S1 region was used as a template.
  • S1 protein receptor binding site RBD region, A cDNA (SEQ ID NO: 3) encoding ACWalls et al., Structure, function and antigenicity of the SARS-CoV-2 spike glycoprotein, Cell 180 (2020), 1-12
  • the signal sequence of human immunoglobulin is connected to the 5'side of this cDNA, and the Fc region and histidine tag of rabbit immunoglobulin are connected to the 3'side, and downstream of the cytomegalovirus promoter in the expression vector pD603 (manufactured by ATUM). Incorporated.
  • This plasmid pD603-CoV-2-SB-rFc was introduced into Expi293 cells and transiently expressed by the Expi293 Expression System (manufactured by Thermo Fisher Scientific), and then His Mag Sepharose excel (manufactured by Citiva). ) was used to purify to a purity of about 95% to prepare an antigen for the SARS-CoV-2 spike protein as an Fc fusion protein in the RBD region.
  • Example 1-1 Preparation of monoclonal antibody A fusion protein of SARS-CoV-2 ⁇ RBD region and rabbit Fc protein prepared in Example 1-1 was used, and a complete Freund's adjuvant (manufactured by Becton Dickinson) was used for the initial immunization. From the second time onward, an emulsion as an immunogen was prepared by mixing an equal amount with an incomplete Freund's adjuvant (manufactured by Becton Dickinson).
  • Monoclonal antibodies were prepared by the method described in K. Watanabe et al., Vasohibin as an endothelium-derived negative feedback regulator of angiogenesis, J. Clin. Inves. 114 (2004), 898-907. Specifically, 4-week-old female A / J mice were administered with 100 ⁇ g of Fc fusion protein subcutaneously and intraperitoneally in equal doses per administration. Then, after the fourth immunization, the spleen was removed from the mice subjected to the final booster (3 days before cell fusion) to prepare spleen cells. Cell fusion between splenocytes and myeloma cells (P3U1) is described in K. Watanabe et al., Vasohibin as an endothelium-derived negative feedback regulator of angiogenesis, J. Clin. Inves. 114 (2004), 898-907. The hybridoma was prepared by the above method.
  • the evaluation of the antibody titer (primary evaluation) of the hybridoma culture supernatant was performed by the ELISA method described below. That is, the hybridoma culture supernatant was added to a 96-well microplate on which a goat anti-mouse IgG antibody was immobilized, and the RBD region protein biotinylated by a biotin labeling kit (manufactured by Dojindo) and europium-labeled streptavidin (Perkin) were added. Elmer) was mixed and stirred, and then reacted at room temperature for 2 hours.
  • a biotin labeling kit manufactured by Dojindo
  • Eu europium-labeled streptavidin
  • the mixture was washed 3 times with a washing solution (physiological saline containing 0.01% Tween 20 and 0.1% ProClin 150), 100 ⁇ l enhancing reagent (Delfia Enhancement Solution, manufactured by PerkinElmer) was added, and the mixture was stirred for 5 minutes.
  • the time-resolved fluorescence intensity was measured with Nivo (manufactured by Perkin Elmer), and if the signal intensity significantly exceeded the signal obtained when the unused medium was used alone, it was judged to be positive, and the hybridoma 7 clone (A1-5F4) was judged to be positive.
  • A1-10E3, A2-4D12, A2-4G11, A2-5A12, A2-11H1, and A2-3F8) were selected (Fig. 1).
  • Example 2 Confirmation of reactivity with Exo-S in monoclonal antibody against peaplomer of SARS-CoV-2: Secondary evaluation 2-1: Preparation of Exo-S (peplomer-positive exosome) All of peplomer of SARS-CoV-2
  • the cDNA encoding the regions (S1 and S2 regions) was synthesized by PCR of the S1 and S2 regions, respectively, and joint PCR using these as templates.
  • the S1 region was subjected to PCR reaction using the primer of 5), and the sequence was sequenced using the plasmid pCMV3-CoV-2-S2-His (manufactured by Sino Biological) as a template: 5'-ACCCAGACCAACAGCCCATCTGTGGCAAGCCAGAGC-3'(SEQ ID NO: 6) and Sequence: The S2 region was amplified by PCR reaction using the primer of 5'-CTGATTTTAGAGACCTTAGTGATGGTGGTGATG-3'(SEQ ID NO: 7).
  • the cDNA (SEQ ID NO:) encoding the entire region of the spike protein was subjected to a joint PCR reaction using the fragments of the S1 and S2 regions thus obtained as templates and primers having the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 7. 8) was synthesized.
  • the recognition sequence of the protein-cleaving enzyme furin (Arg-Arg-Ala-Arg) present at the carboquil terminal of the S1 region was excluded for the purpose of stabilizing the expression of the spike protein.
  • the plasmid pD603-CoV-2-S1S2 was constructed by inserting it into an expression vector pD603 (manufactured by ATUM) in which a histidine tag was connected to the 3'side of the full-length spike protein cDNA thus obtained.
  • This plasmid pD603-CoV-2-S1S2 was introduced into Expi293 cells (manufactured by Thermo Fisher Scientific) using the Expi293 Expression System (manufactured by Thermo Fisher Scientific) and was transiently expressed 5 days later. The culture broth was collected.
  • the culture solution was centrifuged at 2000 xg for 10 minutes at 4 ° C, and the supernatant was passed through a 0.22 ⁇ m PVDF filter (manufactured by Millipore), and then exosomes were subjected to ultracentrifugation (centrifugation at 100,000 ⁇ g at 4 ° C for 70 minutes).
  • RVDF filter manufactured by Millipore
  • Exo-S. Extracts were prepared from the cells using RIPA solution (manufactured by Santa Cruz Biotechnology, Inc.).
  • Example 2-2 Evaluation of monoclonal antibody using Exo-S
  • the culture supernatant of the hybridoma 7 clone selected in Example 1-3 was examined for its reactivity with Exo-S prepared in Example 2-1. This is because the reactivity with SARS-CoV-2 should be examined, but it has a similar shape to SARS-CoV-2 in order to reduce the risk to biosafety in experiments using infectious viruses.
  • Spike proteins were artificially expressed on the surface of exosomes, which are vesicles, and their reactivity with non-pathogenic Exo-S was investigated. This aims to safely select antibodies with higher specificity for SARS-CoV-2.
  • Example 2-1 a hybridoma supernatant was added to a 96-well microplate on which a goat anti-mouse IgG antibody was immobilized, and then Exo-S prepared in Example 2-1 or Expi293 cell-derived exosome and biotin-labeled as a negative control.
  • Anti-CD63 antibody (Clone 8A12, manufactured by Cosmo Bio) and Europium-labeled streptavidin (manufactured by PerkinElmer) were mixed, stirred, and reacted at room temperature for 2 hours.
  • Example 2-3 Construction of sandwich ELISA
  • the anti-peplomer monoclonal antibody identified in Example 2-2 Using clone A2-5A12, a sandwich ELISA measurement system for quantifying Exo-S of Example 2-1 was constructed. bottom. Specifically, a 96-well microplate solid-phased with an anti-peplomer monoclonal antibody was prepared. The antibody solution adjusted to 5 ⁇ g / mL with Tris-hydrochloric acid buffer was added to a 96-well microplate at a rate of 100 ⁇ L / well and reacted overnight.
  • TMB solution 3,3', 5,5'-tetramethylbenzidine solution ( manufactured by Moss), which is a color-developing substrate, was added and reacted at room temperature for 20 minutes.
  • 50 ⁇ L of 2N sulfuric acid was added thereto to stop the reaction, and the absorbance of each well at 450 nm was measured.
  • concentration-dependent measured values were obtained for Exo-S, and no significant measured values were obtained for negative control exosomes.
  • a sandwich ELISA measurement system for quantifying Exo-S was constructed by the anti-peplomer monoclonal antibody: clone A2-5A12 shown in FIG.
  • Example 3 Evaluation of inhibitory activity of spike protein and its receptor ACE2 in a monoclonal antibody against the spike protein of SARS-CoV-2 3-1 Preparation of ACE2 extracellular domain protein cDNA encoding the extracellular domain of human ACE2 protein Is a primer for 5'-AAAGCTAGCCATGTCAAGCTCTTCCTGGC-3'(SEQ ID NO: 9) and 5'-TGTGCGGCCGCAGGGGGCTGGTTAGGAG-3' (SEQ ID NO: 10) using the plasmid pcDNA3.1 + / C (K) DYK-ACE2 manufactured by Genscript Co., Ltd. as a template.
  • the RBD region of the SARS-CoV-2 spike protein prepared in Example 1-1 was biotinylated to the biotin-labeled protein used for detection. Specifically, the RBD region protein was reacted with a 20-fold molar amount of Sulfo-NHS-LC-Biotin (Thermo Fisher) for 2 hours at room temperature and gel-filtered on a Zeba desalting column (Thermo Fisher). Prepared.
  • a monoclonal antibody (catalog number 40592-MM57) against the spike protein RBD region of SARS-CoV-2 marketed by Synobiological Co., Ltd.
  • both antibodies inhibited the binding of the spike protein RBD region to ACE2, but the IC50 values of the cloned A2-5A12 monoclonal antibody and the commercially available antibody were 23.6 ng / mL and 1270 ng / mL, respectively, and the clone A2-5A12. It was revealed that the inhibitory activity of the monoclonal antibody was 53.8 times stronger than that of the commercially available antibody (Fig. 5).
  • Example 3-2 Similar to Example 3-2, as the antibody to be compared, a monoclonal antibody against the spike protein of SARS-CoV-2 marketed by Synobiological Co., Ltd. was used. After the reaction with each antibody, the cells were washed 3 times with a washing solution, 100 ⁇ L of 1 ⁇ g / mL HRP-labeled anti-human CD9 antibody (manufactured by Cosmo Bio Co., Ltd.) was added, and the reaction was carried out at room temperature for 2 hours. After washing three times, 100 ⁇ L / well of TMB solution (manufactured by Moss) was added, and the mixture was reacted at room temperature for 20 minutes. 50 ⁇ L / well of 2N sulfuric acid was added thereto to stop the reaction, and the absorbance of each well at 450 nm was measured.
  • 1 ⁇ g / mL HRP-labeled anti-human CD9 antibody manufactured by Cosmo Bio Co., Ltd.
  • TMB solution manufactured by Moss
  • both antibodies inhibited the binding of Exo-S to ACE2, but the IC50 values of the cloned A2-5A12 monoclonal antibody and the commercially available antibody were 27.7 ng / mL and 1590 ng / mL, respectively, and the cloned A2-5A12 monoclonal antibody. It was revealed that the inhibitory activity of the antibody was 57.4 times stronger than that of the commercially available antibody (Fig. 6).
  • Example 4 Structural analysis of anti-peplomer monoclonal antibody A2-5A12 Structural analysis of H-chain variable region and L-chain variable region of anti-peplomer monoclonal antibody A2-5A12 was performed by the bioinformatics analysis system of Repertoire Genesis Co., Ltd. Specifically, cDNA was synthesized using messenger RNA extracted from a hybridoma that produces a monoclonal antibody of clone A2-5A12 as a template, and the gene sequences of the H-chain variable region and the L-chain variable region were determined by a next-generation sequencer, respectively. The sequence of was replaced with an amino acid sequence.
  • the H chain variable region of the monoclonal antibody of clone A2-5A12 is shown in FIG. 7 (amino acid sequence: SEQ ID NO: 12 and base sequence: SEQ ID NO: 13), and the L chain variable region is shown in FIG. 8 (amino acid sequence: SEQ ID NO: 20 and Nucleotide sequence: shown in SEQ ID NO: 21).
  • Example 5 Preparation of Monoclonal Antibodies to Spike Protein of SARS-CoV-2 (2) Using the methods described in Example 1, Example 2-1 and Example 2-2, immunity and cell fusion, and hybridoma screening were performed to obtain a monoclonal antibody showing reactivity with Exo-S. Six more clones: A1-1C7, A1-1F6, A1-5C3, A2-2B6, A2-7G6 and A2-9D2 were obtained. These antibodies were purified with Protein G Sepharose (manufactured by Citiva), and the antibody titers were measured together with the clones A2-5A12 obtained in Example 2-2.
  • Protein G Sepharose manufactured by Citiva
  • purified antibody was added to a 96-well microplate solid-phased with goat anti-mouse IgG antibody so that the final concentration was 0.1, 1, 10, 100, 1000 ng / mL, and further biotin-labeled rabbit Fc fusion RBD.
  • the region protein and europium-labeled streptavidin (manufactured by PerkinElmer) were added and reacted at room temperature for 2 hours.
  • Example 6 Relative evaluation of the reactivity of 7 cloned monoclonal antibodies with spike protein or Exo-S 6-1: Confirmation of reactivity with RBD region protein Clone A2-5A12 and Example 5 identified in Example 2-2.
  • the sandwich ELISA for quantifying the RBD region protein using the monoclonal antibody of a total of 7 clones of 6 clones obtained in the above the detection sensitivity by the combination of all 7 clones as a solid phase antibody and a labeled antibody was examined.
  • clones of the antibody solid phased on the 96-well microplate were cloned into A2-5A12, A1-1C7, A1-1F6, A1-5C3, A2-2B6, A2-7G6.
  • 10 ng of purified RBD region protein was added per well, and the clones of the labeled antibody were cloned into A2-5A12, A1-1C7, A1-1F6, A1-5C3, A2-2B6, A2.
  • the absorbance at 450 nm when the reaction was carried out as -7G6 and A2-9D2 was measured (Fig. 10).
  • Example 6-1 Confirmation of reactivity with Exo-S
  • a strong signal was detected in the combination of antibodies that are thought to recognize different epitopes.
  • the strongest reactivity was confirmed when A1-1C7 was used as the solid phase antibody and A2-2B6 or A2-7G6 was used as the labeled antibody.
  • Example 7 Relative evaluation of the inhibitory activity of the 7-clone monoclonal antibody against the binding of ACE2 to ACE2 or peplomer protein 7-1: Inhibition of binding of ACE2 to peplomer by the same method as described in Example 3-2. , 7 monoclonal antibodies were investigated for their inhibitory activity against the binding of the ACE2 protein immobilized on a 96-well microplate to the biotin-labeled RBD region protein (Fig. 12). The IC50 values shown by each antibody were 13.7 for clones A2-5A12, 10.5 for A1-1C7, 18.5 for A1-1F6, 712.4 for A1-5C3, 12.9 for A2-2B6, 11.4 for A2-7G6, and A2-9D2. Was 40.7 (both units are ng / mL), and clone A1-1C7 showed the strongest inhibitory activity.
  • Example 8 Structural Analysis of Anti-Spiked Protein Monoclonal Antibodies A1-1C7, A1-5C3
  • the seven anti-spiked protein monoclonal antibodies are classified into three groups according to the difference in reaction specificity to the epitope. I was able to. That is, the group consisting of clones A2-5A12, A1-1F6, A2-2B6 and A2-7G6, the group consisting of clones A1-1C7 and A2-9D2, and the clone A1-5C3.
  • the structure of clones A2-5A12 is described in Example 4.
  • H-chain variable region and the L-chain variable region of these antibodies was performed by the bioinformatics analysis system of Repertoire Genesis Co., Ltd.
  • CDNA was synthesized using messenger RNA extracted from hybridomas producing each antibody as a template, gene sequences of H-chain variable region and L-chain variable region were determined by a next-generation sequencer, and each sequence was replaced with an amino acid sequence.
  • the H chain variable region is shown in FIG. 14 (amino acid sequence: SEQ ID NO: 29 and base sequence: SEQ ID NO: 30), and the L chain variable region is shown in FIG. 15 (amino acid sequence: SEQ ID NO: 37 and base sequence: It is shown in SEQ ID NO: 38).
  • the H chain variable region is shown in FIG. 16 (amino acid sequence: SEQ ID NO: 45 and base sequence: SEQ ID NO: 46), and the L chain variable region is shown in FIG. 17 (amino acid sequence: SEQ ID NO: 53 and base sequence: It is shown in SEQ ID NO: 54).
  • Example 9 Reactivity of 7 clones of monoclonal antibody to peaplomer of SARS-CoV-2 mutant 9-1 Preparation of peaplomer derived from mutant
  • VOC-202012 / 01 UK type
  • 501Y.V2 Common to the three mutant strains of South African type
  • 501Y.V3 Brazilian type
  • N501Y in which the 501st amino acid asparagine is mutated to tyrosine, and there are two types, South African type and Brazilian type.
  • a common mutation is E484K, in which the 484th glutamic acid is mutated to lysine.
  • the 417th lysine is mutated to asparagine in the South African type and to tyrosine in the Brazilian type.
  • RBD region proteins with N501Y mutations, as well as proteins with N501Y and E484K mutations were prepared. Examined.
  • PCR was performed using the primers of No. 62) or sequence: 5'-GGCTTCCAACCAACCTATGGAGTGGGCTACCAAC-3'(SEQ ID NO: 63) and sequence: 5'-ACTACCGGTGGTGCTCTTCTTTGGTCC-3' (SEQ ID NO: 64) to obtain two types of mutant PCR fragments. Made.
  • PCR was performed using these PCR fragments as templates using the primers of SEQ ID NO: 61 and SEQ ID NO: 64 to prepare RBD region fragments having the N501Y mutation.
  • This pD603-CoV-2SB (N501Y) -rFc was introduced into Expi293 cells and transiently expressed, then purified using HisMag Sepharose excel (manufactured by Citiva), and an RBD region protein having an N501Y mutation. And said.
  • sequence: 5'-GGAGTGAAGGGCTTCAACTGTTAC-3'(SEQ ID NO: 65) and sequence: 5'-GAAGCCCTTCACTCCATTACATGG-3' PCR was performed using the primer of (SEQ ID NO: 66), and the restriction enzyme DpnI was added to the reaction solution after the reaction to perform fragmentation treatment of the methylation template plasmid to transform Escherichia coli.
  • the cloned plasmid was sequenced to obtain an expression vector pD603-CoV-2SB (E484K, N501Y) -rFc with a mutation of E484K.
  • This plasmid was also introduced into Expi293 cells and transiently expressed, and then purified using His Mag Sepharose excel (manufactured by Citiva) to obtain an RBD region protein having an E484K mutation and an N501Y mutation.
  • Example 9-2 A1-1C7 was not affected by the N501Y mutation, but since it loses reactivity with the E484K and N501Y mutations, A1-1C7 loses its binding ability to the RBD region protein due to the E484K mutation. It was suggested to do.

Abstract

L'invention concerne un procédé de détection et/ou de quantification efficace du SARS-CoV-2 et/ou d'un variant du SARS-CoV-2.　La présente invention concerne une molécule d'anticorps monoclonal capable de reconnaître spécifiquement une protéine de spicule du SARS-CoV-2 ou un dérivé de l'anticorps monoclonal, une composition pharmaceutique contenant la molécule d'anticorps monoclonal ou le dérivé, un procédé de détection et/ou de quantification du SARS-CoV-2 à l'aide de la molécule d'anticorps monoclonal ou du dérivé, et un procédé de détection et/ou de quantification d'un variant du SARS-CoV-2 à l'aide de la molécule d'anticorps monoclonal ou du dérivé, chacun d'entre eux étant basé sur la molécule d'anticorps ou le dérivé.
PCT/JP2021/041726 2020-11-13 2021-11-12 Anticorps visant la protéine de spicule du sars-cov-2 WO2022102744A1 (fr)

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CN111875701A (zh) * 2020-08-14 2020-11-03 江苏中慧元通生物科技有限公司 一种SARS-CoV-2病毒的单链抗体及用途
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CN112505325A (zh) * 2021-01-22 2021-03-16 山东康华生物医疗科技股份有限公司 一种针对SARS-CoV-2野生株与突变株抗原检测试纸条、检测卡及试剂盒

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JP7235365B1 (ja) 2022-07-22 2023-03-08 株式会社mAbProtein ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質とそのヒト受容体アンジオテンシン変換酵素２との結合阻害を評価する方法及びキット
JP2024014412A (ja) * 2022-07-22 2024-02-01 株式会社mAbProtein ＳＡＲＳ－ＣｏＶ－２スパイクタンパク質とそのヒト受容体アンジオテンシン変換酵素２との結合阻害を評価する方法及びキット

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