WO2022191162A1 - Anti-ace2 monoclonal antibody - Google Patents

Anti-ace2 monoclonal antibody Download PDF

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WO2022191162A1
WO2022191162A1 PCT/JP2022/009860 JP2022009860W WO2022191162A1 WO 2022191162 A1 WO2022191162 A1 WO 2022191162A1 JP 2022009860 W JP2022009860 W JP 2022009860W WO 2022191162 A1 WO2022191162 A1 WO 2022191162A1
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amino acid
acid sequence
seq
cdr
ace2
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PCT/JP2022/009860
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French (fr)
Japanese (ja)
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脩一郎 柏葉
弘匡 秋山
英治 岡
洋子 岡部
聖之 福岡
康文 村上
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株式会社オーダーメードメディカルリサーチ
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Priority to JP2023505563A priority Critical patent/JPWO2022191162A1/ja
Publication of WO2022191162A1 publication Critical patent/WO2022191162A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the present invention relates to a monoclonal antibody that binds to angiotensin converting enzyme (ACE2: Angiotensin Converting Enzyme 2) and uses thereof.
  • ACE2 Angiotensin Converting Enzyme 2
  • COVID-19 The novel coronavirus disease (COVID-19), which broke out in Wuhan, China in December 2019, quickly spread around the world, causing not only many deaths but also a serious blow to the global economy.
  • SARS-CoV-2 the virus that causes COVID-19, is classified as a betacoronavirus and has a single-stranded RNA genome.
  • Virus particles have protruding proteins called spike proteins on their surface, and these spike proteins interact with Angiotensin Converting Enzyme 2 (ACE2) expressed on the surface of human cells to form virus particles. and human cells are directly contacted (Non-Patent Document 1).
  • COVID-19 can cause severe respiratory distress, and although the global death toll exceeded 1.9 million by the end of 2020, there is still no therapeutic agent that can be called a silver bullet.
  • the present inventors succeeded in developing a novel anti-ACE2 monoclonal antibody that can neutralize the binding between coronavirus and ACE2, and have completed the present invention. Completed. That is, the present invention is as follows.
  • ACE2 Angiotensin Converting Enzyme 2
  • the coronavirus is a coronavirus that infects cells via ACE2.
  • a monoclonal antibody or an antigen-binding fragment thereof which is capable of neutralizing the binding of coronavirus to ACE2 and competes with the antibody of [5] or [6] above for binding to ACE2.
  • a pharmaceutical composition for treating or preventing coronavirus infection comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [7] above.
  • coronavirus infection is a disease or condition caused by infection with a coronavirus that infects cells via ACE2.
  • the disease or symptom is at least one selected from the group consisting of respiratory disease, fever, malaise, chills, pain, taste or smell disorder, rash, gastrointestinal symptom, speech disorder, cognitive disorder and cardiovascular symptom.
  • coronavirus infection is a disease or condition caused by infection with a coronavirus that infects cells via ACE2.
  • the disease or symptom is at least one selected from the group consisting of respiratory disease, fever, malaise, chills, pain, taste or smell disorder, rash, gastrointestinal symptom, speech disorder, cognitive disorder and cardiovascular symptom.
  • the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [7] above for use in treating or preventing coronavirus infection.
  • the disease or symptom is at least one selected from the group consisting of respiratory disease, fever, malaise, chills, pain, taste or smell disorder, rash, gastrointestinal symptom, speech disorder, cognitive disorder and cardiovascular symptom.
  • a reagent or kit comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [7] above.
  • the present invention can neutralize the binding between coronavirus and ACE2. Since the antibody of the present invention binds to ACE2, which is a receptor for coronaviruses, even if the coronavirus mutates, as long as the mutant virus infects via ACE2, the binding between the mutant virus and ACE2 is suppressed. can be inhibited.
  • FIG. 2 shows the results of a binding inhibition test between the receptor binding domain (RBD) of SARS-CoV-2 spike protein and ACE2 using the antibody of the present invention.
  • RBD receptor binding domain
  • 1 is a schematic diagram of a competitive inhibition test using the antibodies of the present invention.
  • FIG. 2 shows the results of a competitive inhibition test using the antibodies of the present invention.
  • FIG. 3 shows the results of confirming the reactivity of the antibody of the present invention with human ACE2, mouse ACE2, and mouse/human fusion ACE2.
  • FIG. 2 shows the results of confirming the reactivity between the antibody of the present invention and human ACE2-expressing cells.
  • A Shows the reactivity of the mouse antibody of the present invention to human ACE2-expressing cells.
  • FIG. 2 shows the results of a binding inhibition test between wild-type or mutant SARS-CoV-2 spike protein receptor-binding domain (RBD) and ACE2 using the antibody of the present invention.
  • the antibodies of the present invention can neutralize the binding and infection of wild type and mutant strains of SARS-CoV-2 to human cells, and are thus highly effective in treating or preventing coronavirus infections.
  • Angiotensin Converting Enzyme The antibody of the present invention binds to angiotensin converting enzyme (ACE2: Angiotensin Converting Enzyme 2).
  • ACE2 is a membrane protein present in the plasma membrane of human cells and plays an important role as a regulator of the renin-angiotensin system.
  • ACE2 functions as a receptor when the coronaviruses SARS-CoV-1 and SARS-CoV-2 infect human cells. Specifically, SARS-CoV-1 and SARS-CoV-2 can infect human cells by binding their spike protein (S protein) to ACE2.
  • ACE2 is mainly expressed in cells such as the lung, digestive system, heart, blood vessels, eyes, kidney, cerebral cortex, amygdala, brain stem, and medulla oblongata. Therefore, the present invention can contribute to the treatment or prevention of various diseases and symptoms associated with coronavirus infections, such as respiratory diseases, by inhibiting the infection of coronaviruses to cells of these organs.
  • ACE2 in the present invention may be derived from any mammal. Examples of such mammals include, but are not limited to, mice, rats, rabbits, cats, dogs, goats, monkeys, and humans, preferably mice, rats, cats, dogs, and humans. .
  • these ACE2 nucleotide and amino acid sequences the human ACE2 nucleotide and amino acid sequences are shown in SEQ ID NOS: 1 and 2, respectively, but the ACE2 nucleotide and amino acid sequences of the present invention are not limited to these.
  • the nucleotide sequence and amino acid sequence of human ACE2 are registered in the GenBank database under the given Accession No. below.
  • ACE2 in the present invention includes the following proteins (a) to (c).
  • (b) contains an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and binds to the receptor binding domain (RBD) of the coronavirus spike protein
  • active protein a protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and having activity to bind to the RBD of the coronavirus spike protein;
  • a protein comprising the amino acid sequence shown by SEQ ID NO:2 includes proteins consisting of the amino acid sequence shown by SEQ ID NO:2.
  • amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 2 include: (i) 1 to 10 (eg, 1 to 5, preferably 1 to 3, more preferably 1 to 2, still more preferably 1) amino acids in the amino acid sequence represented by SEQ ID NO: 2 are deleted; missing amino acid sequence, (ii) 1 to 10 (eg, 1 to 5, preferably 1 to 3, more preferably 1 to 2, still more preferably 1) amino acids in the amino acid sequence represented by SEQ ID NO: 2 are other an amino acid sequence substituted with an amino acid of (iii) 1 to 10 (eg, 1 to 5, preferably 1 to 3, more preferably 1 to 2, still more preferably 1) amino acids added to the amino acid sequence shown in SEQ ID NO: 2 amino acid sequence, (iv) Amino acid sequences mutated by combinations
  • the presence or absence of "coronavirus spike protein RBD-binding activity" can be determined by known methods such as immunoprecipitation, Western blotting, EIA (enzyme immunoassay), ELISA (enzyme-linked immunosorbent assay) (e.g., It can be measured by using an immunological technique such as ELISA using a polypeptide, cell ELISA, etc., or a method such as a pull-down assay.
  • the activity of binding to the RBD of the coronavirus spike protein is defined as at least 10% or more, 20% or more, 30% or more when the activity of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is set to 100. % or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, preferably 90% or higher.
  • ACE2 in the present invention includes an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 in addition to the amino acid sequence shown in SEQ ID NO: 2, and the RBD of the coronavirus spike protein.
  • Proteins with binding activity are included. Such proteins include about 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% of the amino acid sequence shown in SEQ ID NO: 2. Also included are those that contain an amino acid sequence with a % or more sequence identity and that have the activity of binding to the RBD of the coronavirus spike protein.
  • homology searches such as FASTA, BLAST, and PSI-BLAST can be used on internet homology search sites such as the DNA Data Bank of Japan (DDBJ). You can also search using BLAST at the National Center for Biotechnology Information (NCBI).
  • NCBI National Center for Biotechnology Information
  • a mutagenesis kit using a site-directed mutagenesis method such as the Kunkel method or the gapped duplex method, such as QuikChange TM Site-Directed Mutagenesis Kit (Stratagene), GeneTailor TM Site-Directed Mutagenesis System (Invitrogen), TakaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: Takara Bio) etc.
  • a site-directed mutagenesis method such as the Kunkel method or the gapped duplex method, such as QuikChange TM Site-Directed Mutagenesis Kit (Stratagene), GeneTailor TM Site-Directed Mutagenesis System (Invitrogen), TakaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: Takara Bio) etc.
  • methods such as site-directed mutagenesis described in "Molecular Cloning, A Laboratory Manual (4th edition)” (Cold Spring
  • the amino acid sequence of the region to which the antibody of the present invention binds (the region in which the epitope to which the antibody of the present invention binds) is not limited. Amino acid sequences are included.
  • the amino acid sequence of the extracellular region of ACE2 is not limited, and in the case of human ACE2, the 18th to 740th amino acid residues counted from the N-terminal amino acid residue of the full-length amino acid sequence of ACE2 (SEQ ID NO: 2). (amino acid sequence of SEQ ID NO: 4).
  • the base sequence of DNA encoding the amino acid sequence consisting of the 18th to 740th amino acid residues of human ACE2 is shown in SEQ ID NO:3.
  • the partial amino acid sequence of the amino acid sequence of the extracellular region of ACE2 is not limited. A region other than the binding region or a partial amino acid sequence thereof can be mentioned. Furthermore, the amino acid sequence of the coronavirus-binding region of ACE2 or a portion thereof is not limited.
  • the 24th glutamine residue (Q24), the 30th aspartic acid residue (D30), the 34th histidine residue (H34), the 41st tyrosine residue (Y41), the 42nd glutamine residue (Q42 ), the 82nd methionine residue (M82), the 353rd lysine residue (K353) and the 357th arginine residue (R357) is not limited.
  • the partial amino acid sequence of the amino acid sequence of the extracellular region of ACE2 includes, for example, in the case of human ACE2, the 1st to 120th amino acids counted from the N-terminal amino acid residue of the full-length amino acid sequence of ACE2.
  • Examples include, but are not limited to, an amino acid sequence consisting of residues (SEQ ID NO: 132), an amino acid sequence consisting of amino acid residues 121 to 740 (SEQ ID NO: 134), and the like.
  • the base sequence of the DNA encoding the amino acid sequence consisting of the 1st to 120th amino acid residues of human ACE2 and the base sequence of the DNA encoding the amino acid sequence consisting of the 121st to 740th amino acid residues of human ACE2 are SEQ ID NO: 131, respectively. and 133.
  • the amino acid sequence of the region to which the antibody of the present invention binds includes the following amino acid sequences (a) to (c).
  • An amino acid sequence comprising or consisting of an amino acid sequence having the above sequence identity A person skilled in the art can determine whether the antibody of the present invention binds to these amino acid sequences by known immunological techniques such as immunization. It can be easily confirmed
  • the epitope to which the antibody of the present invention binds and the region in which the epitope is present are not limited as long as the antibody of the present invention can neutralize the binding between coronavirus and ACE2.
  • a person skilled in the art can, based on the description of the present specification (e.g., the description of Example 3), neutralize the binding of the subject antibody to ACE2 without specifying the epitope of the subject antibody. You can check if you can. That is, according to the description herein, one skilled in the art can identify the epitope of the subject antibody in order to confirm whether the subject antibody can neutralize the binding of the coronavirus to ACE2. do not have to.
  • the antibody of the present invention is a monoclonal antibody that binds to ACE2 (hereinafter also referred to as “anti-ACE2 monoclonal antibody”) or an antigen-binding fragment thereof.
  • binding to ACE2 means forming a reversible non-covalent bond with ACE2 through hydrogen bonding, hydrophobic interaction, electrostatic force, van der Waals force, or the like.
  • bind includes “specifically bind”.
  • “specifically binds” means recognizing an epitope in a target protein or target polypeptide and selectively or preferentially binding to that epitope compared to a protein or polypeptide that does not have that epitope.
  • the "antigen-binding fragment” includes, for example, scFv (single chain Fv), sc(Fv) 2 , Fab, Fab', diabody (dsFv), F(ab') 2 , multispecific Examples include, but are not limited to, antibodies and the like.
  • the above antibody-binding fragments can be obtained by known methods such as genetic engineering techniques, papain digestion, pepsin digestion, and the like.
  • the antibody of the invention is an isolated antibody.
  • a coronavirus means a coronavirus that infects cells via ACE2.
  • Such coronaviruses include, for example, SARS-CoV-1, SARS-CoV-2 and variants thereof, with SARS-CoV-2 and variants thereof being preferred.
  • These coronaviruses infect cells by binding the receptor-binding domain (RBD) of their spike protein to ACE2.
  • RBD receptor-binding domain
  • neutralize means inhibiting the interaction (e.g., binding) between RBD and ACE2 of coronaviruses, and inhibiting binding or infection (e.g., entry) of coronaviruses into cells. .
  • Whether the subject antibody inhibits the interaction of the RBD of the coronavirus with ACE2 can be determined by binding the RBD bound to ACE2 to the RBD, e.g., as described in Example 3 herein. can be examined by detection using a fluorescently labeled antibody. Specifically, after reacting the antibody to be tested with ACE2, when RBD is further added to this, if RBD bound to ACE2 is not detected, the antibody to be tested is the RBD and ACE2 It can be determined that the interaction is inhibited.
  • the antibody to be tested inhibits (neutralizes) the binding or infection (e.g., entry) of the coronavirus to cells can be determined, for example, by contacting the antibody to be tested and the coronavirus with cultured cells, It can be confirmed by observing the presence or absence of morphological changes (cytopathic effect: CPE). If no morphological change is observed in the cultured cells, it can be confirmed that the test antibody has neutralizing activity against coronavirus at the antibody concentration used in the test (eg, Example 10).
  • CPE cytopathic effect
  • ACE2 and ACE2-expressing cells are used as immunogens for preparing the antibody of the present invention.
  • ACE2 e.g., human ACE2
  • ACE2 e.g., human ACE2
  • ACE2 e.g., human ACE2
  • ACE2 the full-length ACE2 polypeptide (SEQ ID NO: 2) or a partial polypeptide thereof can be used.
  • Partial polypeptides of ACE2 used as antigens or immunogens are not limited, and include, for example, polypeptides containing all or part of the amino acid sequence of the extracellular domain of ACE2.
  • amino acid sequence of all or part of the amino acid sequence of the extracellular region of ACE2 is not limited.
  • An amino acid sequence including all or part of the amino acid sequence (SEQ ID NO: 4) consisting of the 18th to 740th amino acid residues is included.
  • Polypeptides containing a partial amino acid sequence of the amino acid sequence of the extracellular region of ACE2 are not limited. Examples thereof include polypeptides containing a region other than the coronavirus-binding region of ACE2 or a partial amino acid sequence thereof.
  • polypeptides examples include, in the case of human ACE2, an amino acid sequence consisting of the 1st to 120th amino acid residues counted from the N-terminal amino acid residue of the full-length amino acid sequence of ACE2 (SEQ ID NO: 132). , an amino acid sequence consisting of 121st to 740th amino acid residues (SEQ ID NO: 134), etc., but not limited thereto.
  • ACE2-expressing cells can be produced, for example, using the following method.
  • a DNA encoding full-length human ACE2 is incorporated into a lentiviral vector, and the vector is introduced into mammalian cells (eg, 293T cells) together with a packaging plasmid and an envelope plasmid.
  • mammalian cells eg, breast cancer cell line 4T1 cells
  • the method for producing ACE2-expressing cells is not limited to this method.
  • ACE2 used as an antigen or immunogen may be natural ACE2 purified from tissues or cells of mice, rats, rabbits, cats, dogs, goats, monkeys, humans, etc., or genetically engineered ACE2. good.
  • a biological sample in which ACE2 expression is observed is fractionated into a soluble fraction and an insoluble fraction using various surfactants such as Triton-X and Sarkosyl.
  • ACE2 can be obtained by dissolving the insoluble fraction in urea, guanidine hydrochloride or the like and binding it to various columns such as a heparin column or a binding resin.
  • ACE2 used as an antigen can also be synthesized using a known protein synthesis method such as a solid-phase method or a commercially available protein synthesizer by specifying its amino acid sequence.
  • the synthesized peptide can be bound to a carrier protein such as Keyhole Limpet Hemocyanin (KLH) or Thyroglobulin and used as an immunogen.
  • KLH Keyhole Limpet Hemocyanin
  • Thyroglobulin used as an immunogen.
  • the immunization interval is not particularly limited, and immunization is performed 2 to 10 times, preferably 2 to 5 times, at intervals of several days to several weeks, preferably 1 to 2 weeks.
  • the interval between immunizations can be set by those skilled in the art in consideration of the obtained antibody titer.
  • Antibody titers in serum can be measured by ELISA, EIA, radioimmunoassay (RIA), or the like. After confirming that the antibody titer has sufficiently increased, the individual in which the antibody titer has increased is selected and the antibody-producing cells are collected.
  • Antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells and the like, and spleen cells or lymph node cells are preferred.
  • (ii) Cell fusion To obtain cell fusion hybridomas, antibody-producing cells and myeloma cells are fused. Fusion manipulations can be performed according to known methods, such as the method of Kohler et al. As myeloma cells to be fused with antibody-producing cells, commonly available cell lines of animals such as mice can be used. The cell line used has drug selectivity and cannot survive in a HAT selection medium (containing hypoxanthine, aminopterin, and thymidine) in an unfused state, and can survive only in a state fused with antibody-producing cells. is preferred.
  • HAT selection medium containing hypoxanthine, aminopterin, and thymidine
  • myeloma cells include mouse myeloma cell lines such as P3X63Ag8.653, P3X63Ag8U.1, SP2/O-Ag14, PAI, P3U1, NSI/1-Ag4-1 and NSO/1.
  • Cell fusion between the myeloma cells and the antibody-producing cells was carried out in a serum-free DMEM, RPMI-1640 medium, or other animal cell culture medium containing 1 ⁇ 10 8 to 5 ⁇ 10 8 antibody-producing cells and 2 ⁇ 10 8 antibody-producing cells.
  • 10 7 to 10 ⁇ 10 7 myeloma cells are mixed (the cell ratio of antibody-producing cells to myeloma cells is 10:1 to 1:1), and a fusion reaction is performed in the presence of a cell fusion promoter.
  • a cell fusion accelerator polyethylene glycol having an average molecular weight of 1000 to 6000 daltons, Sendai virus, or the like can be used.
  • antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device that utilizes electrical stimulation (eg, electroporation).
  • Hybridoma selection and cloning Target hybridomas are selected from the cells after the cell fusion treatment.
  • the cell suspension is diluted appropriately with, for example, RPMI-1640 medium containing 10-20% fetal bovine serum, and then seeded on a microtiter plate at a calculation of 0.3 cells/well by the limiting dilution method.
  • a selection medium such as HAT medium is added to the wells, and thereafter the selection medium is appropriately exchanged for culturing.
  • hybridoma cells can be obtained from around 10 days after initiation of culture in the selective medium.
  • hybridomas are further screened. Screening of hybridomas may be performed according to conventional methods, and is not particularly limited. For example, a portion of the culture supernatant contained in wells in which hybridomas were cultured can be collected and screened by ELISA, EIA, radioimmunoassay, or the like. Specifically, antigens are adsorbed on a 96-well plate and then blocked with calf serum. The hybridoma cell culture supernatant is reacted with the immobilized antigen at 37°C for 1 hour, then peroxidase-labeled anti-mouse IgG is reacted at 37°C for 1 hour, and color is developed using orthophenylenediamine as a substrate.
  • Screening can be performed by measuring absorbance at a wavelength of 490 nm after quenching the reaction with acid.
  • Hybridomas producing monoclonal antibodies that are positive in the above assay are cloned by limiting dilution or the like.
  • a hybridoma which is a cell that produces a monoclonal antibody that binds to ACE2, is established.
  • (iv) Collection of monoclonal antibodies As a method for collecting monoclonal antibodies from established hybridomas, a conventional cell culture method, ascites formation method, or the like can be employed. In the cell culture method, hybridomas are cultured in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium under normal culture conditions (e.g., 37°C, 5% CO 2 concentration). After culturing for 7 to 14 days, antibodies are obtained from the culture supernatant.
  • an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium under normal culture conditions (e.g., 37°C, 5% CO 2 concentration). After culturing for 7 to 14 days, antibodies are obtained from the culture supernatant.
  • hybridomas derived from myeloma cells are administered intraperitoneally to mammals and allogeneic animals, such as mice (ICR, BALB/c), and large amounts of hybridomas are administered. grow to After 1 to 2 weeks, ascites fluid is collected.
  • known methods such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, and affinity chromatography are appropriately selected, or by combining these can be refined.
  • Antibodies of the present invention include, but are not limited to, the following monoclonal antibodies or antigen-binding fragments thereof.
  • the heavy chain variable region (VH) comprises or consists of the amino acid sequence of SEQ ID NO: 6, the heavy chain complementarity determining region (CDR) 1 (CDR-H1), the amino acid sequence of SEQ ID NO: 8; a heavy chain CDR2 (CDR-H2) comprising or consisting of the amino acid sequence of SEQ ID NO: 10 and a heavy chain CDR3 (CDR-H3) comprising or consisting of the amino acid sequence of SEQ ID NO: 10, and/or a light chain variable region (VL) comprises a light chain CDR1 (CDR-L1) comprising or consisting of the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 14 (CDR- L2) and a light chain CDR3 (CDR-L3) comprising or consisting of the amino acid sequence of SEQ
  • the nucleotide sequence of DNA encoding CDR-H1 includes, for example, those containing or consisting of the nucleotide sequence represented by SEQ ID NO: 5, 17, 29, 41, 53 or 65. but not limited to these.
  • the nucleotide sequence of the DNA encoding CDR-H2 includes, for example, those containing or consisting of the nucleotide sequence represented by SEQ ID NO: 7, 19, 31, 43, 55 or 67. but not limited to these.
  • the nucleotide sequence of the DNA encoding CDR-H3 includes, for example, those containing or consisting of the nucleotide sequence represented by SEQ ID NO: 9, 21, 33, 45, 57 or 69.
  • the nucleotide sequence of the DNA encoding CDR-L1 includes, for example, those containing or consisting of the nucleotide sequence represented by SEQ ID NO: 11, 23, 35, 47, 59 or 71. but not limited to these.
  • the nucleotide sequence of DNA encoding CDR-L2 includes, for example, those comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 13, 25, 37, 49, 61 or 73. but not limited to these.
  • the nucleotide sequence of the DNA encoding CDR-L3 includes, for example, those containing or consisting of the nucleotide sequence represented by SEQ ID NO: 15, 27, 39, 51, 63 or 75. but not limited to these.
  • antibodies of the present invention include, but are not limited to, the following monoclonal antibodies or antigen-binding fragments thereof.
  • a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 78 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 80; mosquito,
  • a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:82 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:84; mosquito
  • a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:86 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:88 mosquito
  • a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:90 and a light chain variable region
  • the nucleotide sequence of the DNA encoding the heavy chain variable region includes, for example, those containing or consisting of the nucleotide sequence represented by SEQ ID NO: 77, 81, 85, 89, 93 or 97. include but are not limited to:
  • the nucleotide sequence of the DNA encoding the light chain variable region includes, for example, the nucleotide sequence represented by SEQ ID NO: 79, 83, 87, 91, 95 or 99, or consists of the nucleotide sequence. include, but are not limited to.
  • One of preferred embodiments of the antibody of the present invention is a genetically engineered antibody.
  • genetically modified antibodies include, but are not limited to, chimeric antibodies, canine antibodies, humanized antibodies, and fully human antibodies.
  • a chimeric antibody refers to an antibody produced by linking immunoglobulin gene fragments of a different animal.
  • chimeric antibodies include, for example, human chimeric antibodies, but the types of animals from which the variable regions and constant regions of chimeric antibodies are derived are not limited.
  • Human chimeric antibodies are, for example, antibodies in which the variable region of a mouse-derived antibody is linked (joined) to the constant region of a human-derived antibody (see Proc. Natl. Acad. Sci. U.S.A. 81, 6851-6855, (1984), etc.) ).
  • subclasses of the human-derived antibody include, but are not limited to, IgG1, IgG2, IgG4, and the like.
  • a chimeric antibody can be easily constructed by gene recombination technology.
  • CDR transplantation when producing a humanized antibody, a technique called so-called CDR grafting (CDR transplantation) can be adopted.
  • CDR grafting is the grafting of complementarity determining regions (CDRs) from mouse antibody variable regions to human variable regions, with framework regions (FRs) of human origin and CDRs of mouse origin. It is a method of making a constructed variable region. These humanized reshaped human variable regions are then ligated to human constant regions.
  • Methods for producing such humanized antibodies are well known in the art (Nature, 321, 522-525 (1986); J. Mol. Biol., 196, 901-917 (1987); Queen C et al. ., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989); see Japanese Patent No. 2828340, etc.).
  • Fully human antibodies can be produced, for example, using mammals capable of producing human antibodies according to known techniques (WO96/9634096, WO98/24893, etc.).
  • the human heavy chain constant region that can be used for chimeric antibodies and humanized antibodies includes those containing an amino acid sequence derived from a human IgG4 heavy chain constant region, for example, the amino acid sequence shown in SEQ ID NO: 102. It includes, but is not limited to, a human heavy chain constant region comprising or consisting of said amino acid sequence.
  • the human light chain constant region that can be used for chimeric antibodies and humanized antibodies includes those containing an amino acid sequence derived from a human IgG4 light chain constant region, for example, the amino acid sequence shown in SEQ ID NO: 104. Alternatively, it may be a human light chain constant region consisting of said amino acid sequence, but is not limited thereto.
  • DNAs encoding human heavy chain constant regions include DNAs containing or consisting of the nucleotide sequence shown in SEQ ID NO: 101.
  • DNAs encoding human light chain constant regions include: Examples include, but are not limited to, DNA containing or consisting of the nucleotide sequence shown in SEQ ID NO:103.
  • chimeric antibodies and humanized antibodies can be produced from hybridomas or DNA or RNA extracted from such hybridomas as raw materials according to the above-described known methods.
  • a protein fused with an antibody of the present invention can be produced by fusing an antibody variable region with another protein using a known genetic recombination method.
  • the fusion protein can be produced by cross-linking a monoclonal antibody and another protein using a cross-linker.
  • the antigen-binding fragment of the antibody of the present invention binds to ACE2.
  • An antigen-binding fragment of an antibody refers to a polypeptide comprising a portion of the antigen-binding portion of the antibody of the invention.
  • antigen-binding fragments include scFv (single chain Fv), sc(Fv) 2 , Fab, Fab', diabody (dsFv), F(ab') 2 , multispecific antibodies and the like. , but not limited to.
  • the above antibody-binding fragments can be obtained by known methods such as genetic engineering techniques, papain digestion, pepsin digestion, and the like.
  • Fab can be obtained by treating an antibody molecule with papain, and F(ab') 2 can be obtained by treating an antibody molecule with pepsin.
  • Fab' can also be obtained by cleaving the disulfide bond in the hinge region of F(ab') 2 above.
  • scFv cDNAs encoding the antibody H-chain V region and L-chain V region are obtained to construct scFv-encoding DNA.
  • scFv can be produced by inserting this DNA into an expression vector, introducing the expression vector into a host organism, and expressing the scFv.
  • diabody In the case of diabody, cDNAs encoding the H chain V region and L chain V region of the antibody are obtained, and scFv-encoding DNA is constructed so that the amino acid sequence length of the peptide linker is 8 residues or less.
  • a diabody can be produced by inserting this DNA into an expression vector, introducing the expression vector into a host organism, and expressing the DNA.
  • dsFv cDNAs encoding the H chain V region and L chain V region of the antibody are obtained, and DNA encoding the dsFv is constructed.
  • a dsFv can be produced by inserting this DNA into an expression vector, introducing the expression vector into a host organism, and expressing the dsFv.
  • Multispecific antibodies such as bispecific antibodies, target and bind to two or more antigens or epitopes, and can be produced by various known techniques (Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321).
  • Antigen-binding fragments (peptides) containing CDRs are composed of at least one region or more of CDRs (CDR1-3) of VH or VL. Antigen-binding fragments containing multiple CDRs can be conjugated directly or via suitable peptide linkers. Antigen-binding fragments containing CDRs are obtained by constructing DNAs encoding the VH and VL CDRs of an antibody, inserting the DNAs into prokaryotic or eukaryotic expression vectors, and converting the expression vectors into prokaryotic or eukaryotic expression vectors. It can be expressed and produced by introduction into eukaryotes. Peptides containing CDRs can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
  • Binding Affinity The affinity of an antibody for an antigen can generally be expressed by the equilibrium dissociation constant (KD), with lower values of KD indicating higher affinity of the antibody.
  • KD equilibrium dissociation constant
  • Antibody affinity can be measured using known instruments and methods (eg, Biacore (registered trademark)-3000 (GE Healthcare), ProteON XPR36 (Bio-Rad), etc.).
  • the present invention is a monoclonal antibody capable of neutralizing the binding of a coronavirus to ACE2 and that competes with any of the reference antibodies or antigen-binding fragments thereof for binding to ACE2. Antibodies or antigen-binding fragments thereof are provided.
  • reference antibodies include the following monoclonal antibodies.
  • VH heavy chain variable region
  • VH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8
  • CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 10 and/or a CDR-H3 in which the light chain variable region (VL) comprises or consists of the amino acid sequence of SEQ ID NO: 12 a monoclonal antibody comprising L1, a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 14 and a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 16;
  • VH heavy chain variable region
  • VH CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 18, CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 20, and CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 22, and
  • “competing” for binding to ACE2 means that the antibody or antigen-binding fragment thereof of the present invention inhibits (for example, suppresses) the binding of the above-mentioned reference antibody or antigen-binding fragment thereof to ACE2; Alternatively, it means that the binding of the antibody of the present invention or antigen-binding fragment thereof to ACE2 is inhibited (eg, suppressed) by the reference antibody or antigen-binding fragment thereof.
  • Whether the antibody or antigen-binding fragment thereof of the present invention competes with any of the reference antibodies or antigen-binding fragments thereof for binding to ACE2 can be determined by known methods such as competitive RIA, competitive EIA, competitive ELISA (poly It can be examined using competitive ELISA using peptides, competitive cell ELISA), epitope binning, and the like.
  • a competition test using an antibody is a technique for examining whether an antibody to be tested or an antigen-binding fragment thereof and a reference antibody compete for binding to the same epitope or a neighboring epitope on an antigen.
  • adjacent epitope refers to an epitope in which the antibody or antigen-binding fragment thereof to be tested and the reference antibody are in close proximity to the extent that binding to the epitope is sterically hindered.
  • OMRad004G05 For example, as shown in Example 6 herein, four monoclonal antibodies, OMRad004G05, OMRad028D08, OMRad031H08, and OMRad045G03, are antibodies that compete with each other for binding to ACE2, and these competing antibodies are , as shown in Example 10 herein, have comparable neutralizing activity against coronavirus cell binding or infection.
  • two types, OMRad004G05 and OMRad028D08, and two types, OMRad031H08 and OMRad045G03 have different reactivities to mouse ACE2 (Example 7), and thus may bind to different epitopes.
  • Example 6 to determine whether the antibody or antigen-binding fragment thereof of the present invention competes with the reference antibody or antigen-binding fragment thereof for binding to ACE2, What kind of structures and epitope structures are (for example, what kind of amino acid sequences the CDRs and variable regions are, whether they are linear or three-dimensional (steric) structures, etc.) does not require information about
  • the practice of the present invention is then performed by following a series of steps of generating anti-ACE monoclonal antibodies based on routine methods and selecting antibodies that compete with the reference antibodies described herein for binding to ACE2.
  • antibodies can be obtained that compete with the reference antibodies described herein for binding to ACE2.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention which competes with either the reference antibody or the antigen-binding fragment thereof, can be tested by a person skilled in the art using the reference antibody. can be obtained without undue experimentation.
  • the pharmaceutical composition of the present invention is a pharmaceutical composition for treating or preventing coronavirus infection, which contains the antibody or antigen-binding fragment thereof described in "3. Antibody of the present invention" as an active ingredient.
  • a coronavirus infection that is the target of the pharmaceutical composition of the present invention means a disease or symptom caused by infection with a coronavirus.
  • Such coronavirus infections include, for example, diseases or conditions caused by infection with coronaviruses that infect cells via ACE2.
  • the "coronavirus that infects cells via ACE2" is not limited, and includes, for example, SARS-CoV-1 and SARS-CoV-2, preferably SARS-CoV-2.
  • Examples of "diseases or symptoms caused by infection with a coronavirus that infects cells via ACE2" include organs or tissues in which ACE2 is mainly expressed, such as lungs, digestive system, heart, blood vessels, eyes, and kidneys. , cerebral cortex, amygdala, brain stem, medulla oblongata, etc., diseases or symptoms based on immune response, and the like. Such diseases or symptoms include, for example, respiratory diseases, fever, malaise, chills, pain, taste or smell disorders, rashes, gastrointestinal symptoms, speech disorders, cognitive disorders, cardiovascular symptoms, It is not limited to these. Pharmaceutical compositions containing the antibodies of the present invention are effective in treating or preventing various diseases or symptoms associated with coronavirus infection by inhibiting coronavirus infection that infects cells via ACE2.
  • the term “treatment” refers to the use of the antibody of the present invention or an antigen-binding fragment thereof, or a pharmaceutical composition comprising the same (hereinafter also referred to as “the antibody and pharmaceutical composition of the present invention”) after the onset of a disease. It means that by contacting (e.g., administering) the subject, the symptoms of the disease are alleviated compared to when the subject is not contacted, but does not necessarily mean that the symptoms of the disease are completely suppressed.
  • “onset of disease” includes not only symptoms of the disease appearing in the body, but also a positive result in a test for coronavirus infection (such as a PCR test) even if no symptoms appear.
  • treatment of coronavirus infection includes suppression or inhibition of aggravation of coronavirus infection.
  • prevention means suppression or inhibition of the onset of coronavirus infection, suppression or inhibition of aggravation, but does not necessarily mean complete suppression of the onset.
  • prevention refers to contacting (e.g., administering) the pharmaceutical composition etc. of the present invention with a subject/subject before the onset of the disease, compared to the case of no contact, after the onset of the disease. This includes relieving symptoms.
  • the pharmaceutical composition of the present invention can contain a monoclonal antibody that binds to ACE2 or an antigen-binding fragment thereof, as well as a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” means any carrier suitable for pharmaceutical compositions (liposomes, lipid vesicles, micelles, etc.), diluents, excipients, wetting agents, buffers, suspending agents, lubricants, Refers to adjuvants, emulsifiers, disintegrants, absorbents, preservatives, surfactants, colorants, flavorants or sweeteners.
  • Antibodies and pharmaceutical compositions of the present invention include injections, freeze-dried products, tablets, hard capsules, soft capsules, granules, powders, pills, syrups, suppositories, poultices, ointments, creams, Dosage forms such as eye drops can be taken.
  • Liquid formulations such as injections may be in the form of powders (for example, freeze-dried powders) for preparation before use, which are dissolved in physiological saline or the like before use.
  • the antibodies and pharmaceutical compositions of the present invention can be administered locally or systemically by any means known to those skilled in the art.
  • an administration route of the pharmaceutical composition of the present invention both oral administration and parenteral administration are possible. etc.), intradermal administration, topical administration (such as transdermal administration), or transrectal administration.
  • the pharmaceutical composition of the present invention can be administered in dosage forms suitable for these administration routes.
  • the dosage of the antibody and pharmaceutical composition of the present invention varies depending on factors such as age, body weight, health condition, sex, symptoms, animal species, administration route, administration frequency, and dosage form of the subject/subject. Suitable administration procedures can be set by those skilled in the art.
  • the dosage of the antibody of the present invention for the treatment of coronavirus infection is, for example, 0.1 mg to 100 mg/day, preferably 1 mg to 15 mg/day, more preferably 2-12 mg per kg body weight of the subject/subject. / day, but not limited to.
  • As for the administration frequency it can be administered 1 to 5 times a day.
  • the timing of administration of the antibody and pharmaceutical composition of the present invention can be appropriately determined according to symptoms, and multiple doses can be administered simultaneously or separately at intervals. Also, the pharmaceutical composition of the present invention may be administered to a subject/subject prior to the onset of the disease or after the onset of the disease.
  • the pharmaceutical composition of the present invention can be administered to mammals as subjects/subjects.
  • Mammals include, for example, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, goats, pigs, sheep, cows, horses, monkeys, humans and the like.
  • coronavirus infection is treated or prevented by administering a monoclonal antibody that binds to ACE2, an antigen-binding fragment thereof, or a pharmaceutical composition containing the same to a subject. can do. That is, the present invention provides a method of treating or preventing coronavirus infection, comprising the step of administering to a subject/subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention, or a pharmaceutical composition comprising the same. .
  • the therapeutically effective amount of the antibody or antigen-binding fragment thereof of the present invention, or a pharmaceutical composition comprising the same depends on factors such as subject/subject's age, body weight, health condition, sex, symptoms, route of administration, frequency of administration, and dosage form. Varies depending on A person of ordinary skill in the art can readily determine the therapeutically effective amount required to treat or prevent coronavirus infection.
  • subject or “subject” includes a subject/subject in need of treatment or prevention of coronavirus infection.
  • mammals to be treated or prevented as “subjects” or “subjects” are as described above.
  • coronavirus infection Treatment
  • prevention in the method for treating or preventing coronavirus infection of the present invention are as described above.
  • the dosage forms, administration routes, dosages, administration periods, etc. of the antibody or antigen-binding fragment thereof of the present invention, or the pharmaceutical composition containing the same are also described above.
  • binding inhibitor of coronavirus and ACE2 Since the antibody of the present invention neutralizes (inhibits) the binding between coronavirus and ACE2, it can inhibit coronavirus infection due to this binding. That is, the present invention provides an inhibitor of binding between coronavirus and ACE2, which comprises the antibody or antigen-binding fragment thereof described in "3. Antibodies of the present invention" as an active ingredient.
  • the inhibitor of the present invention can be used as a test reagent or for the treatment of mammals, and its dosage form, additives, administration route, administration subject, dosage, etc. are as described in "4. Pharmaceutical composition” above. It can be appropriately selected according to the description. However, the inhibitor of the present invention may contain only the antibody of the present invention or an antigen-binding fragment thereof.
  • Antibodies or antigen-binding fragments thereof of the present invention can be used in methods of treating or preventing coronavirus infections or in the manufacture of medicaments for treating or preventing coronavirus infections.
  • the present invention provides antibodies or antigen-binding fragments thereof of the present invention for use in methods of treating or preventing coronavirus infection.
  • the present invention also provides the antibody or antigen-binding fragment thereof of the present invention for use in manufacturing a medicament for treating or preventing coronavirus infection.
  • the present invention provides the antibody or antigen-binding fragment thereof of the present invention for use in producing a binding inhibitor between coronavirus and ACE2.
  • compositions of the present invention can be used for co-administration with at least one other therapeutic agent.
  • Other therapeutic agents for use in the present invention include, for example, remdesivir, dexamethasone, favipiravir, nafamostat, camostat, ivermectin, tricizumab, baricitinib, and the like.
  • the combined administration of the pharmaceutical composition of the present invention and at least one other therapeutic agent is expected to provide even better effects than using each agent alone.
  • the excellent effect includes the effect of reducing side effects more than before while maintaining the therapeutic effect.
  • “combination” means administering the pharmaceutical composition of the present invention and at least one of the other therapeutic agents simultaneously or separately.
  • Simultaneously means administered at the same timing in one administration schedule, and the timing of administration need not be exactly the same.
  • “Separately” means administered at different times in one administration schedule.
  • the dosage form, administration route, and administration target of the pharmaceutical composition and other therapeutic agents used in the combination therapy of the present invention are not particularly limited, and can be appropriately selected according to the description in "4. Pharmaceutical Compositions" above.
  • the dosage forms or dosages of drugs to be used in combination may differ from each other, and can be appropriately adjusted depending on the combination to be used in combination.
  • the dosage can be reduced accordingly.
  • an effective amount of a pharmaceutical composition of the invention and an effective amount of another therapeutic agent (i) an effective amount of the pharmaceutical composition of the invention and an ineffective amount of the other therapeutic agent; (iii) an ineffective amount of a pharmaceutical composition of the invention and an effective amount of another therapeutic agent; and (iv) Combinations of ineffective amounts of the pharmaceutical compositions of the invention and ineffective amounts of other therapeutic agents can be employed. Even if one or both of the pharmaceutical composition and the other therapeutic agent are used in an ineffective amount, if the combined use can exhibit a pharmacological effect, the combined administration can be performed in such a manner.
  • Reagents, Kits Antibodies or antigen-binding fragments thereof of the present invention can be included in reagents or kits.
  • the invention provides reagents and kits comprising the antibodies or antigen-binding fragments thereof of the invention.
  • the reagents and kits of the present invention can be used, for example, as test reagents or kits for coronaviruses.
  • the antibody or antigen-binding fragment thereof of the present invention can be used, for example, after making it easy to handle by a method such as freezing, or it can be mixed with known pharmaceutical agents such as excipients, extenders, binders and lubricants.
  • kit of the present invention can also contain the antibody of the present invention or an antigen-binding fragment thereof, as well as a buffer solution, an enzyme solution, a secondary antibody, a diluent solution, instructions for use, and the like.
  • a DNA encoding full-length human ACE2 was incorporated into a lentiviral vector, and the vector was introduced into 293T cells together with a packaging plasmid and an envelope plasmid. 4T1 cells were infected with the recombinant lentivirus produced in the culture supernatant of the cells to prepare cells stably expressing human ACE2 on the cell membrane.
  • the partial polypeptide of (ii) above was prepared by introducing a vector containing the DNA (SEQ ID NO: 105) encoding the partial polypeptide into E. coli, expressing it in E. coli, and then purifying it.
  • mice or BALB/c mice both from Sankyo Labo Service Co., Ltd.
  • the mouse-derived cells (i) above are administered into the peritoneal cavity of BALB/c mice, and a mixture of the mouse-derived cells (i) above and the partial polypeptide and adjuvant (ii) above is administered into the peritoneal cavity of ICR mice. It was carried out by administering
  • OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, OMRad045G03, OMRad052B05 show the nucleotide sequences and amino acid sequences of the variable regions in the table below.
  • OMRad004G05 and OMRad028D08 were produced from hybridomas obtained by immunizing BALB/c mice with the mouse-derived cells described in (i) above.
  • the results of this example demonstrated that the antibody of the present invention can bind to ACE2 and neutralize the binding between ACE2 and RBD.
  • a chimeric antibody was prepared by replacing the constant region with the amino acid sequence of human IgG4.
  • the amino acid sequence of the heavy chain constant region and the amino acid sequence of the light chain constant region used in this example are shown in SEQ ID NO: 102 and SEQ ID NO: 104, respectively.
  • a chimeric antibody was prepared by the following method. First, from the anti-ACE2 antibody-producing hybridoma produced in Example 1, the heavy chain variable region gene and light chain variable region gene of the anti-ACE2 antibody were cloned.
  • these genes were ligated to the base sequences of the heavy chain constant region gene (SEQ ID NO: 101) or the light chain ( ⁇ chain) constant region gene (SEQ ID NO: 103) of human IgG4, respectively.
  • a primer (SEQ ID NO: 109) having the 5'-terminal nucleotide sequence of the light chain variable region gene and the restriction enzyme EcoRI sequence, and the complementary sequence of the 3'-terminal nucleotide sequence and the restriction enzyme were used.
  • PCR was performed using an antisense primer (SEQ ID NO: 110) with the BsiWI sequence.
  • the base sequences of the primers used in the above PCR are shown in the table below.
  • the obtained amplification product was treated with restriction enzymes EcoRI and NheI or EcoRI and BsiWI, and the EcoRI-NheI site of the human IgG4 heavy chain constant region expression plasmid (pFUSEss-CHIg-hG4; InvivoGen) or the light chain of human Ig. ( ⁇ chain) was integrated into the EcoRI-BsiWI site of a constant region expression plasmid (pFUSE2ss-CLIg-hk; InvivoGen).
  • pFUSEss-CHIg-hG4 was loaded with the heavy chain constant region gene of human IgG4, and pFUSE-CLIg-hk was loaded with the light chain ( ⁇ chain) constant region gene of human Ig.
  • mice heavy chain variable region and the human heavy chain constant region were linked by the restriction enzyme NheI sequence, and the mouse light chain variable region and the human light chain constant region were linked by the restriction enzyme BsiWI sequence.
  • a chimeric antibody based on the antibody (OMRad052B05) prepared in Example 1 could be prepared.
  • chimeric antibodies were prepared by replacing the constant region with the amino acid sequence of human IgG4 for OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, and OMRad045G03. did.
  • the primers listed in the table below were used in PCR for amplifying the variable regions.
  • each anti-ACE2 mouse antibody was used as a reference antibody (Reference Antibody), and each chimeric antibody was used as a test antibody (antibody to be detected (Detection Antibody)).
  • 4T1 cells (4T1 -hACE2 cells) were reacted with one type of reference antibody, and then reacted with all the test antibodies in round robin.
  • FIG. A schematic diagram of this test is shown in FIG. Specifically, 4T1-hACE2 cells were seeded into each well of a 96-well cell culture plate at 1 x 104 cells/well and cultured for 2 days.
  • a solution containing 30 ⁇ g/mL of each anti-human ACE2 mouse monoclonal antibody (reference antibody) was prepared using a blocking buffer (PBS(-) containing 3% BSA and 0.2% bovine serum).
  • the culture medium was removed from the 96-well plate, a reference antibody solution diluted at 50 ⁇ L/well was added, and the plate was allowed to stand at 4 ° C. for 1 hour. After the wells were washed three times with 250 ⁇ L of washing buffer (PBS(-) containing 0.1% BSA), 2.5% formalin was added at 50 ⁇ L/well and allowed to stand at room temperature for 10 minutes to fix the cells.
  • each anti-human ACE2 mouse/human chimeric monoclonal antibody (test antibody (antibody to be detected)) diluted to 3 ⁇ g/mL with blocking buffer was added to 50 ⁇ L/well. was added to each well and allowed to stand at room temperature for 1 hour. After washing three times with 250 ⁇ L of washing buffer, 50 ⁇ L/well of PBS(-) containing 3% hydrogen peroxide was added and allowed to stand at room temperature for 5 minutes to inactivate endogenous peroxidase. .
  • fusion ACE2 gene (polynucleotide) in which residues 1 to 120 from the N-terminus of human ACE2 were replaced with mouse ACE2, and generated mouse/human fusion-type ACE2-expressing cells expressing this by the following procedure. made.
  • An ACE2 gene construct was generated using NEBuilder Hi-Fi DNA Assembly (New England Biolabs) and incorporated into a lentiviral vector.
  • This lentiviral construct was transfected into 293T cells together with the packaging plasmid and envelope plasmid, and mouse 4T1 cells were infected with the recombinant lentivirus produced in the culture supernatant.
  • 4T1 cells expressing human mouse/human fusion ACE2 on the cell membrane (mouse/human fusion ACE2-expressing 4T1 cells) were obtained.
  • Mouse/human fusion ACE2 has the amino acid sequence of mouse ACE2 in residues 1 to 120 from the N-terminus, and the amino acid sequence of human ACE2 in residues 121 and beyond.
  • Immunocytostaining 4T1 cells, human ACE2-expressing 4T1 cells, mouse ACE2-expressing 4T1 cells, and mouse/human fusion ACE2-expressing 4T1 cells were plated at 3.2 x 10 3 cells/well in glass-bottom 96-well plates for fluorescence observation. , cultured for 2 days. After the plate was placed on ice to remove the medium, 200 mL of RPMI1640 medium containing 20 mg/mL anti-ACE2 mouse monoclonal antibody was added, and the plate was allowed to stand on ice for 1 hour.
  • RPMI1640 medium supplemented with Alexa Fluor 488-labeled anti-mouse IgG antibody (Thermo Fisher Scientific, A11001) was added and allowed to react on ice for 30 minutes. After washing, 100 mL of 4% paraformaldehyde was added and allowed to stand on ice for 10 minutes. After thoroughly washing with PBS, they were observed under a fluorescence microscope.
  • OMRad044E11 and OMRad052B05 bound to human ACE2, but did not bind to residues 1-120 of mouse ACE2 and residues 121-740 of human ACE2. From this, it was shown that these antibodies mainly bind to the region containing residues 1 to 120 from the N-terminus of human ACE2.
  • OMRad004G05 and OMRad028D08 bound to full-length human ACE2 and residues 121-740 of human ACE2, but did not bind to mouse ACE2. From this, these antibodies are mainly the extracellular region after the 121st (up to the 740th serine) of human ACE2, and the position or region that can distinguish the difference between human ACE2 and mouse ACE2 (e.g. , regions with low homology between human ACE2 and mouse ACE2, positions with different amino acids between the two, etc.).
  • OMRad031H08 and OMRad045G03 reacted to all of human ACE2, mouse ACE2, and human/mouse fusion ACE2, so it was not possible to narrow down the region to which these antibodies bind in this test.
  • OMRad031H08 and OMRad045G03 competed with OMRad004G05 and OMRad028D08 belonging to the same Group 1, and considering the results of this test, these antibodies are mainly 121-740 in human ACE2.
  • a region containing the second amino acid residue that does not discriminate between human ACE2 and mouse ACE2 e.g., a region with high homology between human ACE2 and mouse ACE2, the same amino acid in both It was suggested that it binds to a position having
  • OMRad004G05 and OMRad028D08, OMRad031H08 and OMRad045G03, and OMRad044E11 and OMRad052B05 showed similar reactivity to ACE2 is consistent with the fact that the CDR amino acid sequences of these antibodies are similar to each other.
  • Group 1 antibodies (OMRad004G05, OMRad028D08, OMRad031H08, OMRad045G03) shown in Example 6 are mainly in the region containing amino acid residues 121-740, similar to other antibodies belonging to the same Group.
  • Group 2 antibodies (OMRad044E11, OMRad052B05) were shown to bind mainly to a region containing amino acid residues 1-120 from the N-terminus, similar to the other antibodies in the same group. .
  • Binding affinity evaluation test 1 Among the antibodies obtained in Method Example 1, six types of anti-ACE2 mouse antibodies (OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, OMRad045G03, OMRad052B05) were evaluated for their affinity to human ACE2 by cell-based ELISA.
  • cell-based ELISA was performed using the six types of mouse antibodies described above and the chimeric antibodies prepared in Examples 4 and 5 based on these antibodies to determine the Kd value. Specifically, the test was conducted according to the following procedures.
  • 4T1 cells and 4T1 cells stably expressing human ACE2 (hereinafter referred to as "4T1-hACE2") were seeded in each well of a 96-well cell culture plate at 1 x 104 cells/well. , cultured for 2 days.
  • blocking buffer PBS(-) containing 3% BSA and 0.2% bovine serum
  • a dilution series of was prepared.
  • the culture medium was removed from the 96-well plate, an antibody solution diluted at 50 ⁇ L/well was added, and the plate was allowed to stand at 4°C for 1 hour.
  • washing buffer PBS(-) containing 0.05% Tween20
  • 10% formalin was added at 50 ⁇ L/well and allowed to stand at room temperature for 10 minutes to fix the cells.
  • PBS(-) containing 3% hydrogen peroxide was added at 50 ⁇ L/well and allowed to stand at room temperature for 5 minutes to inactivate endogenous peroxidase.
  • HRP-labeled anti-mouse Kappa chain antibody (Bethyl, A90-119P) or HPR-labeled anti-human IgG-Fc antibody (Bethyl, A80-304P) diluted 10,000-fold with blocking buffer ) was added at 50 ⁇ L/well and allowed to stand at room temperature for 1 hour.
  • OPD substrate solution was added at 100 ⁇ L/well and allowed to react at room temperature for about 10 minutes. The reaction was terminated by adding 100 ⁇ L of 1.5 M sulfuric acid solution and stirring, and the absorbance at 490 nm was measured using an absorptiometer. The obtained data were analyzed by GraphPad Prism to determine the Kd value.
  • the Kd values in this test ranged from 1.97 nM to 5.81 nM for the mouse antibody and from 0.86 nM to 2.62 nM for the chimeric antibody, as shown in the table below. Since the secondary antibodies used are different, the Kd value of the mouse antibody and the Kd value of the chimeric antibody cannot be directly compared.
  • mutant spike protein RBD Binding inhibition test between mutant spike protein RBD and ACE2
  • the six chimeric antibodies described above were used to analyze the extent to which these antibodies inhibited binding between mutant RBD and human ACE2-expressing cells. did. 1.
  • mutant spike protein RBD In this example, wild type (WT (Wuhan type)), alpha type, beta type, delta type, epsilon type, eta type, theta type, and kappa type are used as mutant spike protein RBDs.
  • WT Wild type
  • beta type beta type
  • delta type epsilon type
  • eta type theta type
  • kappa type An RBD with the same amino acid sequence as that of SARS-CoV2 was generated.
  • a 6 x histidine tag was added to the 3' end of the region corresponding to the RBD (319th lysine to 541st phenylalanine counting from the N-terminus).
  • a construct to which a base sequence was added was prepared, and the construct was incorporated into an expression vector for mammalian cells. Mutations shown in the table below were introduced into the resulting plasmid by OE-PCR method.
  • the resulting plasmid DNA was introduced into CHO cells, and the mutant recombinant RBD was purified from the culture supernatant using Ni-NTA agarose.
  • Binding inhibition test between wild-type and mutant spike protein RBD and human ACE2 100 ⁇ L of cell suspension of 4T1-hACE2 cells was seeded into each well of a 96-well plate for cell culture at 10,000 cells per well. , and cultured at 37°C in the presence of 5% CO 2 for 2 days. Wild-type and each mutant RBD were added to Blocking Buffer (PBS containing 3% BSA, 0.2% bovine serum) to a final concentration of 5 nM, and anti-human ACE2 was added with Blocking Buffer containing wild-type and each mutant RBD. Chimeric antibodies were diluted.
  • Blocking Buffer PBS containing 3% BSA, 0.2% bovine serum
  • the medium of the 96-well plate was removed by decantation, 30 ⁇ L of the prepared anti-human ACE2 chimeric antibody solution was added to each well, and the plate was allowed to stand at 4°C for 1 hour.
  • the anti-human ACE2 chimeric antibody solution in the 96-well plate was removed by decantation, and each well was washed twice with 250 ⁇ L of PBS(-).
  • 30 ⁇ L of S28 antibody solution in which mouse anti-SARS-CoV-2 Spike antibody (clone S28) was diluted with blocking buffer to 1 ⁇ g/mL, was added to each well and allowed to stand at 4° C. for 1 hour.
  • the S28 antibody solution was removed by decantation, and each well was washed twice with 250 ⁇ L of PBS(-). 50 ⁇ L of a 10% formalin solution was added to each well of a 96-well plate, and the plate was allowed to stand at room temperature for 10 minutes to immobilize the cells. The 10% formalin solution was removed by decantation, and each well was washed twice with 250 ⁇ L of washing buffer (PBS(-) containing 0.05% Tween20). 50 ⁇ L of a 3% hydrogen peroxide solution was added to each well of a 96-well plate and allowed to stand at room temperature for 5 minutes to deactivate endogenous peroxidase.
  • PBS(-) washing buffer
  • the 3% hydrogen peroxide solution was removed by decantation, and each well was washed three times with 250 ⁇ L of washing buffer (PBS(-) containing 0.05% Tween20).
  • PBS(-) washing buffer
  • 50 ⁇ L of secondary antibody (Mouse IgG-Fc Fragment cross-adsorbed Antibody HRP Conjugated, Bethyl, A90-231P) diluted 10,000 times with Blocking Buffer to each well of a 96-well plate, and leave to stand at room temperature in the dark for 1 hour. placed.
  • the secondary antibody solution was removed by decantation, and each well was washed three times with 250 ⁇ L of washing buffer (PBS(-) containing 0.05% Tween20).
  • OMRad004G05 OMRad028D08, OMRad031H08, and OMRad045G03 in particular exhibited similar inhibitory effects on the binding of any mutant RBD to ACE2.
  • these four chimeric antibodies exhibited higher inhibitory effects (lower IC50) than OMRad044E11 and OMRad052B05.
  • the neutralizing antibody titer of the chimeric antibody was measured.
  • the antibody diluent is a cell maintenance medium [Dulbecco's Modified Eagle Medium (Nacalai Tesque, Inc.) containing 2% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 ⁇ g/mL), geneticin (G418, 1 mg/mL) added].
  • a cell maintenance medium Dulbecco's Modified Eagle Medium (Nacalai Tesque, Inc.) containing 2% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 ⁇ g/mL), geneticin (G418, 1 mg/mL) added.
  • live virus refers to a virus that can infect and proliferate in living cells.
  • virus strains were used as the live virus of SARS-CoV-2.
  • SARS-CoV-2 wild type strain JPY/TY/WK-521)
  • SARS-CoV-2 alpha variant strain QHN001 VOC-202012/01 strain
  • SARS-CoV-2 beta variant TY8-612 B.1.351 lineage
  • SARS-CoV-2 delta variant TY11-927 (B.1.617.2 strain)
  • SARS-CoV-2 Omicron variant TY38-873 (BA.1 strain)
  • VeroE6/TMPRSS2 cells JCRB1819 were used in this example.
  • the above SARS-CoV-2 virus suspension was prepared as follows. (i) Cell growth medium [10% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 ⁇ g/mL), geneticin (G418) (1 mg /mL)] was used to culture VeroE6/TMPRSS2 cells in monolayers in tissue culture flasks.
  • the antibodies of the present invention neutralize binding and infection of SARS-CoV-2 wild type and mutant strains to human cells. That is, the antibodies of the present invention were shown to be extremely effective in treating or preventing coronavirus infections caused by SARS-CoV-2 wild strains or mutant strains.
  • the antibodies belonging to Group 1 shown in Example 6 are equivalent to other antibodies belonging to the same Group for binding and infection of SARS-CoV-2 wild type and mutant strains to human cells. It was shown to have synergistic activity.
  • OMRad031H08 and OMRa045G03 differed by one amino acid residue in their framework amino acid sequences, both showed the same neutralizing activity as shown in the table above. From this, in the antibody of the present invention, when the CDR amino acid sequences are the same or similar between the two antibodies, even if the framework amino acid sequences do not completely match between the two, equivalent effects (medium activity, etc.).
  • the present invention can provide a novel monoclonal antibody that can bind to ACE2.
  • SEQ ID NOs: 3 to 134 synthetic DNA or synthetic peptide

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Abstract

It is required to provide a novel molecule for neutralizing the binding between coronavirus and angiotensin converting enzyme 2 (ACE2). The present invention provides a monoclonal antibody binding to ACE2.

Description

抗ＡＣＥ２モノクローナル抗体Anti-ACE2 monoclonal antibody
　本発明は、アンジオテンシン変換酵素（ACE2：Angiotensin Converting Enzyme 2)に結合するモノクローナル抗体及びその使用に関する。 The present invention relates to a monoclonal antibody that binds to angiotensin converting enzyme (ACE2: Angiotensin Converting Enzyme 2) and uses thereof.
　2019年12月に中国武漢市で発生した新型コロナウイルス感染症(COVID-19)は瞬く間に世界中に拡がり、多くの死者を出すにとどまらず、世界経済に深刻な打撃を与えた。
　COVID-19の原因ウイルスであるSARS-CoV-2は、ベータコロナウイルスに分類され、一本鎖のRNAをゲノムとして有する。ウイルス粒子の表面にはスパイクタンパク質と呼ばれる突起状のタンパク質が存在しており、このスパイクタンパク質がヒト細胞表面に発現するアンジオテンシン変換酵素（ACE2：Angiotensin Converting Enzyme 2)と相互作用することで、ウイルス粒子とヒト細胞の直接的な接触が成立する(非特許文献1)。
　COVID-19は重篤な呼吸器困難を引き起こす場合があり、全世界における死者数は2020年末までに190万人を超えたものの、特効薬と言える治療効果を示す治療薬は未だない。 The novel coronavirus disease (COVID-19), which broke out in Wuhan, China in December 2019, quickly spread around the world, causing not only many deaths but also a serious blow to the global economy.
SARS-CoV-2, the virus that causes COVID-19, is classified as a betacoronavirus and has a single-stranded RNA genome. Virus particles have protruding proteins called spike proteins on their surface, and these spike proteins interact with Angiotensin Converting Enzyme 2 (ACE2) expressed on the surface of human cells to form virus particles. and human cells are directly contacted (Non-Patent Document 1).
COVID-19 can cause severe respiratory distress, and although the global death toll exceeded 1.9 million by the end of 2020, there is still no therapeutic agent that can be called a silver bullet.
　このような状況においては、コロナウイルスとACE2との結合を中和する新規分子の開発が望まれる。 Under these circumstances, it is desirable to develop novel molecules that neutralize the binding of coronaviruses to ACE2.
　本発明者らは、上記課題を解決するために鋭意研究を行った結果、コロナウイルスとACE2との結合を中和することができる、新規の抗ACE2モノクローナル抗体の開発に成功し、本発明を完成するに至った。
　すなわち、本発明は以下の通りである。 As a result of intensive research to solve the above problems, the present inventors succeeded in developing a novel anti-ACE2 monoclonal antibody that can neutralize the binding between coronavirus and ACE2, and have completed the present invention. Completed.
That is, the present invention is as follows.
［１］
　ACE2（Angiotensin Converting Enzyme 2）に結合するモノクローナル抗体又はその抗原結合断片。
［２］
　コロナウイルスとACE2との結合を中和することができる、上記［１］に記載のモノクローナル抗体又はその抗原結合断片。
［３］
　コロナウイルスが、ACE2を介して細胞に感染するコロナウイルスである、上記［２］に記載のモノクローナル抗体又はその抗原結合断片。
［４］
　前記抗体が、キメラ抗体、ヒト化抗体又は完全ヒト抗体である、上記［１］～［３］のいずれかに記載のモノクローナル抗体又はその抗原結合断片。
［５］
　(a) 配列番号５４のアミノ酸配列を含むCDR-H1、配列番号５６のアミノ酸配列を含むCDR-H2及び配列番号５８のアミノ酸配列を含むCDR-H3を含み、かつ、配列番号６０のアミノ酸配列を含むCDR-L1、配列番号６２のアミノ酸配列を含むCDR-L2及び配列番号６４のアミノ酸配列を含むCDR-L3を含むか、
　(b) 配列番号３０のアミノ酸配列を含むCDR-H1、配列番号３２のアミノ酸配列を含むCDR-H2及び配列番号３４のアミノ酸配列を含むCDR-H3を含み、かつ、配列番号３６のアミノ酸配列を含むCDR-L1、配列番号３８のアミノ酸配列を含むCDR-L2及び配列番号４０のアミノ酸配列を含むCDR-L3を含むか、
　(c) 配列番号１８のアミノ酸配列を含むCDR-H1、配列番号２０のアミノ酸配列を含むCDR-H2及び配列番号２２のアミノ酸配列を含むCDR-H3を含み、かつ、配列番号２４のアミノ酸配列を含むCDR-L1、配列番号２６のアミノ酸配列を含むCDR-L2及び配列番号２８のアミノ酸配列を含むCDR-L3を含むか、
　(d) 配列番号６のアミノ酸配列を含むCDR-H1、配列番号８のアミノ酸配列を含むCDR-H2及び配列番号１０のアミノ酸配列を含むCDR-H3を含み、かつ、配列番号１２のアミノ酸配列を含むCDR-L1、配列番号１４のアミノ酸配列を含むCDR-L2及び配列番号１６のアミノ酸配列を含むCDR-L3を含むか、
　(e) 配列番号４２のアミノ酸配列を含むCDR-H1、配列番号４４のアミノ酸配列を含むCDR-H2及び配列番号４６のアミノ酸配列を含むCDR-H3を含み、かつ、配列番号４８のアミノ酸配列を含むCDR-L1、配列番号５０のアミノ酸配列を含むCDR-L2及び配列番号５２のアミノ酸配列を含むCDR-L3を含むか、又は
　(f) 配列番号６６のアミノ酸配列を含むCDR-H1、配列番号６８のアミノ酸配列を含むCDR-H2及び配列番号７０のアミノ酸配列を含むCDR-H3を含み、かつ、配列番号７２のアミノ酸配列を含むCDR-L1、配列番号７４のアミノ酸配列を含むCDR-L2及び配列番号７６のアミノ酸配列を含むCDR-L3を含む、
上記［１］～［４］のいずれかに記載のモノクローナル抗体又はその抗原結合断片。
［６］
　(a) 配列番号９４のアミノ酸配列を含む重鎖可変領域（VH）及び配列番号９６のアミノ酸配列を含む軽鎖可変領域（VL）を含むか、
　(b) 配列番号８６のアミノ酸配列を含む重鎖可変領域（VH）及び配列番号８８のアミノ酸配列を含む軽鎖可変領域（VL）を含むか、
　(c) 配列番号８２のアミノ酸配列を含む重鎖可変領域（VH）及び配列番号８４のアミノ酸配列を含む軽鎖可変領域（VL）を含むか、
　(d) 配列番号７８のアミノ酸配列を含む重鎖可変領域（VH）及び配列番号８０のアミノ酸配列を含む軽鎖可変領域（VL）を含むか、
　(e) 配列番号９０のアミノ酸配列を含む重鎖可変領域（VH）及び配列番号９２のアミノ酸配列を含む軽鎖可変領域（VL）を含むか、又は
　(f) 配列番号９８のアミノ酸配列を含む重鎖可変領域（VH）及び配列番号１００のアミノ酸配列を含む軽鎖可変領域（VL）を含む、
上記［１］～［５］のいずれかに記載のモノクローナル抗体又はその抗原結合断片。
［７］
　コロナウイルスとACE2との結合を中和することができ、ACE2との結合について、上記［５］又は［６］に記載の抗体と競合する、モノクローナル抗体又はその抗原結合断片。
［８］
　上記［１］～［７］のいずれかに記載のモノクローナル抗体又はその抗原結合断片を含む、コロナウイルス感染症を治療又は予防するための医薬組成物。
［９］
　コロナウイルス感染症が、ACE2を介して細胞に感染するコロナウイルスの感染によって引き起こされる疾患又は症状である、上記［８］に記載の医薬組成物。
［１０］
　前記疾患又は症状が、呼吸器疾患、発熱、倦怠感、悪寒、疼痛、味覚若しくは嗅覚の障害、発疹、消化器症状、言語障害、認知障害及び循環器症状からなる群から選択される少なくとも一つのものである、上記［９］に記載の医薬組成物。
［１１］
　上記［１］～［７］のいずれかに記載のモノクローナル抗体若しくはその抗原結合断片又はそれを含む医薬組成物の治療上有効量を対象に投与する工程を含む、コロナウイルス感染症を治療又は予防する方法。
［１２］
　コロナウイルス感染症が、ACE2を介して細胞に感染するコロナウイルスの感染によって引き起こされる疾患又は症状である、上記［１１］に記載の方法。
［１３］
　前記疾患又は症状が、呼吸器疾患、発熱、倦怠感、悪寒、疼痛、味覚若しくは嗅覚の障害、発疹、消化器症状、言語障害、認知障害及び循環器症状からなる群から選択される少なくとも一つのものである、上記［１２］に記載の方法。
［１４］
　コロナウイルス感染症の治療又は予防において使用するための上記［１］～［７］のいずれかに記載のモノクローナル抗体又はその抗原結合断片。
［１５］
　コロナウイルス感染症が、ACE2を介して細胞に感染するコロナウイルスの感染によって引き起こされる疾患又は症状である、上記［１４］に記載のモノクローナル抗体又はその抗原結合断片。
［１６］
　前記疾患又は症状が、呼吸器疾患、発熱、倦怠感、悪寒、疼痛、味覚若しくは嗅覚の障害、発疹、消化器症状、言語障害、認知障害及び循環器症状からなる群から選択される少なくとも一つのものである、上記［１５］に記載のモノクローナル抗体又はその抗原結合断片。
［１７］
　コロナウイルス感染症の治療又は予防のための医薬の製造における、上記［１］～［７］のいずれかに記載のモノクローナル抗体又はその抗原結合断片の使用。
［１８］
　上記［１］～［７］のいずれかに記載のモノクローナル抗体又はその抗原結合断片を含む、試薬又はキット。 [1]
A monoclonal antibody or antigen-binding fragment thereof that binds to ACE2 (Angiotensin Converting Enzyme 2).
[2]
The monoclonal antibody or antigen-binding fragment thereof according to [1] above, which is capable of neutralizing binding between coronavirus and ACE2.
[3]
The monoclonal antibody or antigen-binding fragment thereof according to [2] above, wherein the coronavirus is a coronavirus that infects cells via ACE2.
[4]
The monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [3] above, wherein the antibody is a chimeric antibody, a humanized antibody or a fully human antibody.
[5]
(a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 54, CDR-H2 containing the amino acid sequence of SEQ ID NO: 56, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 58, and the amino acid sequence of SEQ ID NO: 60 CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 62 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64;
(b) CDR-H1 containing the amino acid sequence of SEQ ID NO: 30, CDR-H2 containing the amino acid sequence of SEQ ID NO: 32, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 34, and the amino acid sequence of SEQ ID NO: 36 CDR-L1 comprising the amino acid sequence of SEQ ID NO:38, CDR-L2 comprising the amino acid sequence of SEQ ID NO:38 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:40;
(c) CDR-H1 containing the amino acid sequence of SEQ ID NO: 18, CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 22, and the amino acid sequence of SEQ ID NO: 24 CDR-L1 comprising the amino acid sequence of SEQ ID NO: 26, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 28;
(d) CDR-H1 containing the amino acid sequence of SEQ ID NO: 6, CDR-H2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 10, and the amino acid sequence of SEQ ID NO: 12 CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16;
(e) CDR-H1 containing the amino acid sequence of SEQ ID NO: 42, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 46, and the amino acid sequence of SEQ ID NO: 48 CDR-L1 comprising the amino acid sequence of SEQ ID NO: 50 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 52, or (f) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 66, SEQ ID NO: CDR-H2 comprising the amino acid sequence of 68 and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 70, and CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74, and comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 76;
The monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [4] above.
[6]
(a) comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:94 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:96;
(b) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:86 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:88, or
(c) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:82 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:84;
(d) comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:78 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:80;
(e) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:90 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:92; or (f) comprising the amino acid sequence of SEQ ID NO:98. a heavy chain variable region (VH) and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 100;
The monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [5] above.
[7]
A monoclonal antibody or an antigen-binding fragment thereof, which is capable of neutralizing the binding of coronavirus to ACE2 and competes with the antibody of [5] or [6] above for binding to ACE2.
[8]
A pharmaceutical composition for treating or preventing coronavirus infection, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [7] above.
[9]
The pharmaceutical composition according to [8] above, wherein the coronavirus infection is a disease or condition caused by infection with a coronavirus that infects cells via ACE2.
[10]
The disease or symptom is at least one selected from the group consisting of respiratory disease, fever, malaise, chills, pain, taste or smell disorder, rash, gastrointestinal symptom, speech disorder, cognitive disorder and cardiovascular symptom. The pharmaceutical composition according to [9] above, which is
[11]
Treatment or prevention of coronavirus infection, comprising the step of administering to a subject a therapeutically effective amount of the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [7] above, or a pharmaceutical composition containing the same. how to.
[12]
The method according to [11] above, wherein the coronavirus infection is a disease or condition caused by infection with a coronavirus that infects cells via ACE2.
[13]
The disease or symptom is at least one selected from the group consisting of respiratory disease, fever, malaise, chills, pain, taste or smell disorder, rash, gastrointestinal symptom, speech disorder, cognitive disorder and cardiovascular symptom. The method according to [12] above.
[14]
The monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [7] above for use in treating or preventing coronavirus infection.
[15]
The monoclonal antibody or antigen-binding fragment thereof according to [14] above, wherein the coronavirus infection is a disease or condition caused by infection with a coronavirus that infects cells via ACE2.
[16]
The disease or symptom is at least one selected from the group consisting of respiratory disease, fever, malaise, chills, pain, taste or smell disorder, rash, gastrointestinal symptom, speech disorder, cognitive disorder and cardiovascular symptom. The monoclonal antibody or antigen-binding fragment thereof according to [15] above, which is a
[17]
Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [7] above in the manufacture of a medicament for treating or preventing coronavirus infection.
[18]
A reagent or kit comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [7] above.
　本発明により、コロナウイルスとACE2との結合を中和することができる。本発明の抗体は、コロナウイルスの受容体であるACE2に結合するため、コロナウイルスが変異したとしても、当該変異型ウイルスがACE2を介して感染する限り、当該変異型ウイルスとACE2との結合を阻害することができる。 The present invention can neutralize the binding between coronavirus and ACE2. Since the antibody of the present invention binds to ACE2, which is a receptor for coronaviruses, even if the coronavirus mutates, as long as the mutant virus infects via ACE2, the binding between the mutant virus and ACE2 is suppressed. can be inhibited.
本発明の抗体を用いた、SARS-CoV-2スパイクタンパク質の受容体結合ドメイン（RBD）とACE2との結合阻害試験の結果を示す図である。FIG. 2 shows the results of a binding inhibition test between the receptor binding domain (RBD) of SARS-CoV-2 spike protein and ACE2 using the antibody of the present invention. 本発明の抗体を用いた競合阻害試験の概略図である。1 is a schematic diagram of a competitive inhibition test using the antibodies of the present invention. FIG. 本発明の抗体を用いた競合阻害試験の結果を示す図である。FIG. 2 shows the results of a competitive inhibition test using the antibodies of the present invention. 本発明の抗体と、ヒトACE2、マウスACE2及びマウス/ヒト融合ACE2のそれぞれとの反応性を確認した結果を示す図である。FIG. 3 shows the results of confirming the reactivity of the antibody of the present invention with human ACE2, mouse ACE2, and mouse/human fusion ACE2. 本発明の抗体とヒトACE2発現細胞との反応性を確認した結果を示す図である。Ａ：本発明のマウス抗体のヒトACE2発現細胞に対する反応性を示す。Ｂ：本発明のキメラ抗体のヒトACE2発現細胞に対する反応性を示す。Ｃ：本発明のキメラ抗体の、ヒトACE2を発現していない4T1細胞に対する反応性を示す。FIG. 2 shows the results of confirming the reactivity between the antibody of the present invention and human ACE2-expressing cells. A: Shows the reactivity of the mouse antibody of the present invention to human ACE2-expressing cells. B: Shows the reactivity of the chimeric antibody of the present invention to human ACE2-expressing cells. C: Shows the reactivity of the chimeric antibody of the present invention to 4T1 cells that do not express human ACE2. 本発明の抗体を用いた、野生型又は変異型SARS-CoV-2のスパイクタンパク質の受容体結合ドメイン（RBD）とACE2との結合阻害試験の結果を示す図である。FIG. 2 shows the results of a binding inhibition test between wild-type or mutant SARS-CoV-2 spike protein receptor-binding domain (RBD) and ACE2 using the antibody of the present invention.
　以下、本発明を詳細に説明する。以下の実施の形態は、本発明を説明するための例示であり、本発明をこの実施の形態のみに限定する趣旨ではない。本発明は、その要旨を逸脱しない限り、様々な形態で実施をすることができる。また、本明細書は、本願優先権主張の基礎となる2021年3月9日に出願された日本国特許出願（特願2021-036928号）の明細書及び図面に記載の内容を包含する。 The present invention will be described in detail below. The following embodiments are examples for explaining the present invention, and are not meant to limit the present invention only to these embodiments. The present invention can be embodied in various forms without departing from the gist thereof. In addition, this specification includes the contents described in the specification and drawings of the Japanese patent application (Japanese Patent Application No. 2021-036928) filed on March 9, 2021, which is the basis for claiming priority of the present application.
１．概要
　新型コロナウイルス感染症（COVID-19）による全世界における死者数は2020年末までに190万人を超えたものの、特効薬と言える治療効果を示す治療薬は未だない。COVID-19の治療薬としては、ファビピラビルやレムデシビルといった化合物が挙げられるが、これらは元々インフルエンザやエボラ出血熱等を対象とした感染症の治療薬であり、COVID-19に最適な治療薬であるかについては不明である。また、トシリズマブやラブリズマブなどの抗体医薬も治療薬の候補とされているが、いずれもウイルスを直接認識する抗体ではなく、免疫系に関与する生体内因子であるIL-6R、補体(C5)を標的としていることから、その作用は間接的である。
　また、長引く世界的流行の中、スパイクタンパク質に変異が生じ、アミノ酸配列が変化した変異型SARS-CoV-2の報告が相次いでおり、今後も増えることが予想される。
　このような状況においては、コロナウイルスが変異した場合においても、コロナウイルスによる感染症の治療又は予防に使用することができる、新たな治療薬の開発が望まれる。
　本発明者は、コロナウイルスがヒト細胞に感染する際に結合する受容体であるACE2に着目し、ACE2に対する抗体について鋭意検討を行った結果、コロナウイルスとACE2との結合を中和することができる、新規モノクローナル抗体の開発に成功した。
　本発明の抗体は、ACE2を認識するため、たとえコロナウイルスが変異したとしても、当該変異型ウイルスがACE2を介して感染する限り、当該変異型ウイルスとACE2との結合を阻害することができる点で極めて有用である。
　さらに、本発明の抗体は、SARS-CoV-2の野生株及び変異株のヒト細胞への結合及び感染を中和することができるため、コロナウイルス感染症の治療又は予防に極めて有効である。 1. OverviewAlthough the number of deaths worldwide due to the new coronavirus infection (COVID-19) exceeded 1.9 million by the end of 2020, there is still no therapeutic drug that can be called a silver bullet. Compounds such as favipiravir and remdesivir are among the therapeutic drugs for COVID-19, but these are originally therapeutic drugs for infectious diseases such as influenza and Ebola hemorrhagic fever, so it is unclear whether they are the best therapeutic drugs for COVID-19. is unknown. In addition, antibody drugs such as tocilizumab and rablizumab are also considered as candidates for therapeutic drugs, but none of them are antibodies that directly recognize viruses, and are in vivo factors involved in the immune system, IL-6R and complement (C5). Its effects are indirect because it targets
In addition, amid the protracted global epidemic, there have been a series of reports of mutant SARS-CoV-2, in which the spike protein has mutated and the amino acid sequence has changed, and it is expected that the number will increase in the future.
Under such circumstances, it is desired to develop new therapeutic agents that can be used for treatment or prevention of infectious diseases caused by coronavirus even when the coronavirus mutates.
The present inventor focused on ACE2, a receptor that binds when coronavirus infects human cells, and conducted intensive studies on antibodies against ACE2. We succeeded in developing a novel monoclonal antibody that can
Since the antibody of the present invention recognizes ACE2, even if the coronavirus mutates, it can inhibit the binding of the mutant virus to ACE2 as long as the mutant virus infects via ACE2. very useful in
Furthermore, the antibodies of the present invention can neutralize the binding and infection of wild type and mutant strains of SARS-CoV-2 to human cells, and are thus highly effective in treating or preventing coronavirus infections.
２．アンジオテンシン変換酵素
　本発明の抗体は、アンジオテンシン変換酵素（ACE2：Angiotensin Converting Enzyme 2)に結合する。
　ACE2は、ヒト細胞の細胞膜に存在する膜タンパク質であり、レニン-アンジオテンシン系の調節因子として重要な役割を果たす。一方、ACE2は、コロナウイルスであるSARS-CoV-1及びSARS-CoV-2がヒト細胞に感染する際の受容体として機能する。具体的には、当該SARS-CoV-1及びSARS-CoV-2は、そのスパイクタンパク質（Sタンパク質）がACE2に結合することで、ヒト細胞に感染することができる。 2. Angiotensin Converting Enzyme The antibody of the present invention binds to angiotensin converting enzyme (ACE2: Angiotensin Converting Enzyme 2).
ACE2 is a membrane protein present in the plasma membrane of human cells and plays an important role as a regulator of the renin-angiotensin system. On the other hand, ACE2 functions as a receptor when the coronaviruses SARS-CoV-1 and SARS-CoV-2 infect human cells. Specifically, SARS-CoV-1 and SARS-CoV-2 can infect human cells by binding their spike protein (S protein) to ACE2.
　ACE2は、主に肺、消化器系、心臓、血管、眼、腎臓、大脳皮質、扁桃体、脳幹、延髄などの細胞で発現する。したがって、本発明は、コロナウイルスのこれらの器官の細胞への感染を阻害することにより、呼吸器疾患等のコロナウイルス感染症に伴う多様な疾患及び症状の治療又は予防に寄与することができる。 　ACE2 is mainly expressed in cells such as the lung, digestive system, heart, blood vessels, eyes, kidney, cerebral cortex, amygdala, brain stem, and medulla oblongata. Therefore, the present invention can contribute to the treatment or prevention of various diseases and symptoms associated with coronavirus infections, such as respiratory diseases, by inhibiting the infection of coronaviruses to cells of these organs.
　本発明におけるACE2は、任意の哺乳動物に由来するものであってよい。そのような哺乳動物としては、例えば、マウス、ラット、ウサギ、ネコ、イヌ、ヤギ、サル、ヒトなどが挙げられ、好ましくは、マウス、ラット、ネコ、イヌ、ヒトであるが、これらに限定されない。これらのACE2の塩基配列及びアミノ酸配列の一例として、ヒトACE2の塩基配列及びアミノ酸配列を、それぞれ、配列番号１及び２で示すが、本発明のACE2の塩基配列及びアミノ酸配列はこれらに限定されない。ヒトACE2の塩基配列及びアミノ酸配列は、それぞれGenBankデータベースにおいて、下記の所定のアクセッション番号（Accession No.）により登録されている。 ACE2 in the present invention may be derived from any mammal. Examples of such mammals include, but are not limited to, mice, rats, rabbits, cats, dogs, goats, monkeys, and humans, preferably mice, rats, cats, dogs, and humans. . As examples of these ACE2 nucleotide and amino acid sequences, the human ACE2 nucleotide and amino acid sequences are shown in SEQ ID NOS: 1 and 2, respectively, but the ACE2 nucleotide and amino acid sequences of the present invention are not limited to these. The nucleotide sequence and amino acid sequence of human ACE2 are registered in the GenBank database under the given Accession No. below.
　ヒトACE2をコードするDNAの塩基配列：NM_001371415（配列番号１）
　ヒトACE2のアミノ酸配列：NP_001358344.1（配列番号２） Nucleotide sequence of DNA encoding human ACE2: NM_001371415 (SEQ ID NO: 1)
Amino acid sequence of human ACE2: NP_001358344.1 (SEQ ID NO: 2)
　本発明におけるACE2には、以下の（a）～（c）のタンパク質が含まれる。
(a) 配列番号２で示されるアミノ酸配列を含むタンパク質
(b) 配列番号２で示されるアミノ酸配列において、１若しくは数個のアミノ酸が、欠失、置換若しくは付加されたアミノ酸配列を含み、かつコロナウイルススパイクタンパク質の受容体結合ドメイン（RBD）と結合する活性を有するタンパク質
(c) 配列番号２で示されるアミノ酸配列に対して80％以上の配列同一性を有するアミノ酸配列を含み、かつコロナウイルススパイクタンパク質のRBDと結合する活性を有するタンパク質 ACE2 in the present invention includes the following proteins (a) to (c).
(a) a protein comprising the amino acid sequence shown in SEQ ID NO:2
(b) contains an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and binds to the receptor binding domain (RBD) of the coronavirus spike protein; active protein
(c) a protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and having activity to bind to the RBD of the coronavirus spike protein;
　本発明において、「配列番号２で示されるアミノ酸配列を含むタンパク質」には、配列番号２で示されるアミノ酸配列からなるタンパク質が含まれる。
　また、「配列番号２で示されるアミノ酸配列において、１若しくは数個のアミノ酸が、欠失、置換若しくは付加されたアミノ酸配列」としては、例えば、
　(i) 配列番号２で示されるアミノ酸配列中の１～１０個（例えば、１～５個、好ましくは１～３個、より好ましくは１～２個、さらに好ましくは１個）のアミノ酸が欠失したアミノ酸配列、
　(ii) 配列番号２で示されるアミノ酸配列中の１～１０個（例えば、１～５個、好ましくは１～３個、より好ましくは１～２個、さらに好ましくは１個）のアミノ酸が他のアミノ酸で置換されたアミノ酸配列、
　(iii) 配列番号２で示されるアミノ酸配列に１～１０個（例えば、１～５個、好ましくは１～３個、より好ましくは１～２個、さらに好ましくは１個）のアミノ酸が付加したアミノ酸配列、
　(iv) 上記(i)～(iii) の組合せにより変異されたアミノ酸配列
　などが挙げられる。 In the present invention, "a protein comprising the amino acid sequence shown by SEQ ID NO:2" includes proteins consisting of the amino acid sequence shown by SEQ ID NO:2.
Examples of "amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 2" include:
(i) 1 to 10 (eg, 1 to 5, preferably 1 to 3, more preferably 1 to 2, still more preferably 1) amino acids in the amino acid sequence represented by SEQ ID NO: 2 are deleted; missing amino acid sequence,
(ii) 1 to 10 (eg, 1 to 5, preferably 1 to 3, more preferably 1 to 2, still more preferably 1) amino acids in the amino acid sequence represented by SEQ ID NO: 2 are other an amino acid sequence substituted with an amino acid of
(iii) 1 to 10 (eg, 1 to 5, preferably 1 to 3, more preferably 1 to 2, still more preferably 1) amino acids added to the amino acid sequence shown in SEQ ID NO: 2 amino acid sequence,
(iv) Amino acid sequences mutated by combinations of (i) to (iii) above.
　本発明において、「コロナウイルススパイクタンパク質のRBDと結合する活性」の有無については、公知の方法、例えば免疫沈降法、ウェスタンブロッティング、EIA（enzyme immunoassay）、ELISA(enzyme-linked immunosorbent assay)（例えば、ポリペプチドを用いたELISA、cell ELISA等）などの免疫学的手法やプルダウンアッセイ等の方法を用いることにより測定することができる。また、「コロナウイルススパイクタンパク質のRBDと結合する活性」とは、配列番号２で示されるアミノ酸配列からなるタンパク質の活性を100としたときと比較して、少なくとも10%以上、20％以上、30%以上、40%以上、50%以上、60%以上、70%以上、80%以上、好ましくは90%以上の活性を有することを意味する。 In the present invention, the presence or absence of "coronavirus spike protein RBD-binding activity" can be determined by known methods such as immunoprecipitation, Western blotting, EIA (enzyme immunoassay), ELISA (enzyme-linked immunosorbent assay) (e.g., It can be measured by using an immunological technique such as ELISA using a polypeptide, cell ELISA, etc., or a method such as a pull-down assay. In addition, "the activity of binding to the RBD of the coronavirus spike protein" is defined as at least 10% or more, 20% or more, 30% or more when the activity of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is set to 100. % or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, preferably 90% or higher.
　また、本発明におけるACE2には、配列番号２で示されるアミノ酸配列のほか配列番号２で示されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列を含み、かつコロナウイルススパイクタンパク質のRBDと結合する活性を有するタンパク質が挙げられる。このようなタンパク質としては、配列番号２で示されるアミノ酸配列に対して、約80％以上、85％以上、90％以上、95％以上、96％以上、97％以上、98％以上、又は99％以上の配列同一性を有するアミノ酸配列を含み、かつコロナウイルススパイクタンパク質のRBDと結合する活性を有するものも含まれる。配列同一性は、インターネットを利用したホモロジー検索サイト、例えば日本DNAデータバンク（DDBJ）において、FASTA、BLAST、PSI-BLAST等の相同性検索を利用できる。また、National Center for Biotechnology Information (NCBI) において、BLASTを用いた検索を行うこともできる。 In addition, ACE2 in the present invention includes an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 in addition to the amino acid sequence shown in SEQ ID NO: 2, and the RBD of the coronavirus spike protein. Proteins with binding activity are included. Such proteins include about 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% of the amino acid sequence shown in SEQ ID NO: 2. Also included are those that contain an amino acid sequence with a % or more sequence identity and that have the activity of binding to the RBD of the coronavirus spike protein. For sequence identity, homology searches such as FASTA, BLAST, and PSI-BLAST can be used on internet homology search sites such as the DNA Data Bank of Japan (DDBJ). You can also search using BLAST at the National Center for Biotechnology Information (NCBI).
　上記の変異を有するタンパク質を調製するために、該タンパク質をコードするDNAに変異を導入するには、Kunkel法やGapped duplex法等の部位特異的突然変異誘発法を利用した変異導入用キット、例えばQuikChangeTM Site-Directed Mutagenesis Kit（ストラタジーン社製）、GeneTailorTM Site-Directed Mutagenesis System（インビトロジェン社製）、TaKaRa Site-Directed Mutagenesis System（Mutan-K、Mutan-Super Express Km等：タカラバイオ社製）等を用いて行うことができる。また、「Molecular Cloning, A Laboratory Manual(4th edition)」（Cold Spring Harbor Laboratory Press (2012)）等に記載された部位特異的変異誘発法等の方法を用いることができる。 In order to prepare a protein having the mutation described above, to introduce a mutation into the DNA encoding the protein, a mutagenesis kit using a site-directed mutagenesis method such as the Kunkel method or the gapped duplex method, such as QuikChange ™ Site-Directed Mutagenesis Kit (Stratagene), GeneTailor ™ Site-Directed Mutagenesis System (Invitrogen), TakaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: Takara Bio) etc. can be used. In addition, methods such as site-directed mutagenesis described in "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)) and the like can be used.
　ACE2において、本発明の抗体が結合する領域（本発明の抗体が結合するエピトープが存在する領域）のアミノ酸配列としては、限定されるものではなく、例えば、ACE2の細胞外領域又はその一部のアミノ酸配列が挙げられる。ACE2の細胞外領域のアミノ酸配列としては、限定されるものではなく、ヒトACE2の場合、ACE2の全長のアミノ酸配列（配列番号２）のN末端のアミノ酸残基から数えて、18番目～740番目のアミノ酸残基からなるアミノ酸配列（配列番号４のアミノ酸配列）を含むアミノ酸配列が挙げられる。ヒトACE2の18番目～740番目のアミノ酸残基からなるアミノ酸配列をコードするDNAの塩基配列は、配列番号３で示される。
　また、ACE2の細胞外領域のアミノ酸配列の一部のアミノ酸配列としては、限定されるものではなく、例えば、ACE2におけるコロナウイルスとの結合領域又はその一部のアミノ酸配列、ACE2におけるコロナウイルスとの結合領域以外の領域又はその一部のアミノ酸配列が挙げられる。さらに、ACE2におけるコロナウイルスとの結合領域又はその一部のアミノ酸配列としては、限定されるものではなく、例えば、ヒトACE2の場合、ACE2の全長のアミノ酸配列のN末端のアミノ酸残基から数えて、24番目のグルタミン残基（Q24）、30番目のアスパラギン酸残基（D30）、34番目のヒスチジン残基（H34）、41番目のチロシン残基（Y41）、42番目のグルタミン残基（Q42）、82番目のメチオニン残基（M82）、353番目のリジン残基（K353）及び357番目のアルギニン残基（R357）から選ばれる少なくとも１つのアミノ酸残基を含むアミノ酸配列が挙げられる。 In ACE2, the amino acid sequence of the region to which the antibody of the present invention binds (the region in which the epitope to which the antibody of the present invention binds) is not limited. Amino acid sequences are included. The amino acid sequence of the extracellular region of ACE2 is not limited, and in the case of human ACE2, the 18th to 740th amino acid residues counted from the N-terminal amino acid residue of the full-length amino acid sequence of ACE2 (SEQ ID NO: 2). (amino acid sequence of SEQ ID NO: 4). The base sequence of DNA encoding the amino acid sequence consisting of the 18th to 740th amino acid residues of human ACE2 is shown in SEQ ID NO:3.
In addition, the partial amino acid sequence of the amino acid sequence of the extracellular region of ACE2 is not limited. A region other than the binding region or a partial amino acid sequence thereof can be mentioned. Furthermore, the amino acid sequence of the coronavirus-binding region of ACE2 or a portion thereof is not limited. For example, in the case of human ACE2, , the 24th glutamine residue (Q24), the 30th aspartic acid residue (D30), the 34th histidine residue (H34), the 41st tyrosine residue (Y41), the 42nd glutamine residue (Q42 ), the 82nd methionine residue (M82), the 353rd lysine residue (K353) and the 357th arginine residue (R357).
　また、ACE2の細胞外領域のアミノ酸配列の一部のアミノ酸配列としては、例えば、ヒトACE2の場合、ACE2の全長のアミノ酸配列のN末端のアミノ酸残基から数えて、1番目～120番目のアミノ酸残基からなるアミノ酸配列（配列番号１３２）、121番目～740番目のアミノ酸残基からなるアミノ酸配列（配列番号１３４）などが挙げられるが、これらに限定されない。ヒトACE2の1番目～120番目のアミノ酸残基からなるアミノ酸配列をコードするDNAの塩基配列、121番目～740番目のアミノ酸残基からなるアミノ酸配列をコードするDNAの塩基配列は、それぞれ配列番号１３１及び１３３で示される。 In addition, the partial amino acid sequence of the amino acid sequence of the extracellular region of ACE2 includes, for example, in the case of human ACE2, the 1st to 120th amino acids counted from the N-terminal amino acid residue of the full-length amino acid sequence of ACE2. Examples include, but are not limited to, an amino acid sequence consisting of residues (SEQ ID NO: 132), an amino acid sequence consisting of amino acid residues 121 to 740 (SEQ ID NO: 134), and the like. The base sequence of the DNA encoding the amino acid sequence consisting of the 1st to 120th amino acid residues of human ACE2 and the base sequence of the DNA encoding the amino acid sequence consisting of the 121st to 740th amino acid residues of human ACE2 are SEQ ID NO: 131, respectively. and 133.
　さらに、本発明の抗体が結合する領域（本発明の抗体が結合するエピトープが存在する領域）のアミノ酸配列としては、以下の（a）～（c）のアミノ酸配列が含まれる。
(a) 配列番号４、１３２若しくは１３４で示されるアミノ酸配列を含む又は当該配列からなるアミノ酸配列、
(b) 配列番号４、１３２若しくは１３４で示されるアミノ酸配列において、１若しくは数個のアミノ酸が、欠失、置換若しくは付加されたアミノ酸配列を含む又は当該配列からなるアミノ酸配列
(c) 配列番号４、１３２若しくは１３４で示されるアミノ酸配列に対して約80％以上、85％以上、90％以上、95％以上、96％以上、97％以上、98％以上、又は99％以上の配列同一性を有するアミノ酸配列を含む又は当該配列からなるアミノ酸配列
　本発明の抗体がこれらのアミノ酸配列に結合するかどうかについては、当業者であれば、公知の免疫学的手法、例えば免疫沈降法、ウェスタンブロッティング、EIA、ELISA（例えば、ポリペプチドを用いたELISA、cell ELISA等）などを用いて容易に確認することができる。 Furthermore, the amino acid sequence of the region to which the antibody of the present invention binds (the region in which the epitope to which the antibody of the present invention binds) includes the following amino acid sequences (a) to (c).
(a) an amino acid sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO: 4, 132 or 134;
(b) an amino acid sequence comprising or consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, 132 or 134;
(c) About 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% of the amino acid sequence shown in SEQ ID NO: 4, 132 or 134 An amino acid sequence comprising or consisting of an amino acid sequence having the above sequence identity A person skilled in the art can determine whether the antibody of the present invention binds to these amino acid sequences by known immunological techniques such as immunization. It can be easily confirmed using a sedimentation method, Western blotting, EIA, ELISA (eg, ELISA using polypeptide, cell ELISA, etc.).
　本発明の抗体が結合するエピトープ及び当該エピトープが存在する領域は、本発明の抗体がコロナウイルスとACE2との結合を中和することができる限り、限定されない。また、当業者は、本明細書の記載（例えば実施例３の記載）に基づいて、被験対象の抗体のエピトープを特定することなく、被験対象の抗体がコロナウイルスとACE2との結合を中和することができるかどうかを確認することができる。すなわち、本明細書の記載によれば、当業者は、被験対象の抗体がコロナウイルスとACE2との結合を中和することができるかどうかを確認するために、被験対象の抗体のエピトープを特定する必要はない。 The epitope to which the antibody of the present invention binds and the region in which the epitope is present are not limited as long as the antibody of the present invention can neutralize the binding between coronavirus and ACE2. In addition, a person skilled in the art can, based on the description of the present specification (e.g., the description of Example 3), neutralize the binding of the subject antibody to ACE2 without specifying the epitope of the subject antibody. You can check if you can. That is, according to the description herein, one skilled in the art can identify the epitope of the subject antibody in order to confirm whether the subject antibody can neutralize the binding of the coronavirus to ACE2. do not have to.
３．本発明の抗体
　本発明の抗体は、ACE2に結合するモノクローナル抗体（以下「抗ACE2モノクローナル抗体」とも称する）又はその抗原結合断片である。
　本発明において、「ACE2に結合する」とは、水素結合、疎水性相互作用、静電力、ファン・デル・ワールス力等により、ACE2との可逆的な非共有結合を形成することを意味する。本発明において、「結合する」には、「特異的に結合する」ことが含まれる。本発明において、「特異的に結合する」とは、標的タンパク質又は標的ポリペプチドにおけるエピトープを認識して、そのエピトープを有さないタンパク質又はポリペプチドと比較して、選択的又は優先的にそのエピトープを有するタンパク質又はポリペプチドに結合することをいう。
　本発明において、「抗原結合断片」としては、例えば、scFv（single chain Fv）、sc(Fv)2、Fab、Fab'、ダイアボディ（diabody、dsFv）、F(ab')2、多重特異性抗体などが挙げられるが、これらに限定されない。上記抗体結合断片は、遺伝子工学的手法や、パパイン消化、ペプシン消化など、公知の方法により取得することができる。
　本発明の一態様において、本発明の抗体は、単離された抗体である。 3. Antibody of the Present Invention The antibody of the present invention is a monoclonal antibody that binds to ACE2 (hereinafter also referred to as "anti-ACE2 monoclonal antibody") or an antigen-binding fragment thereof.
In the present invention, "binding to ACE2" means forming a reversible non-covalent bond with ACE2 through hydrogen bonding, hydrophobic interaction, electrostatic force, van der Waals force, or the like. In the present invention, "bind" includes "specifically bind". In the present invention, "specifically binds" means recognizing an epitope in a target protein or target polypeptide and selectively or preferentially binding to that epitope compared to a protein or polypeptide that does not have that epitope. It refers to binding to a protein or polypeptide having
In the present invention, the "antigen-binding fragment" includes, for example, scFv (single chain Fv), sc(Fv) 2 , Fab, Fab', diabody (dsFv), F(ab') 2 , multispecific Examples include, but are not limited to, antibodies and the like. The above antibody-binding fragments can be obtained by known methods such as genetic engineering techniques, papain digestion, pepsin digestion, and the like.
In one aspect of the invention, the antibody of the invention is an isolated antibody.
　本発明の抗体は、コロナウイルスとACE2との結合を中和することができる。
　本発明において、コロナウイルスとは、ACE2を介して細胞に感染するコロナウイルスを意味する。そのようなコロナウイルスとしては、例えば、SARS-CoV-1、SARS-CoV-2及びそれらの変異型が挙げられ、SARS-CoV-2及びその変異型が好ましい。これらのコロナウイルスは、そのスパイクタンパク質の受容体結合ドメイン（RBD）がACE2に結合することにより、細胞に感染する。本発明において「中和する」とは、コロナウイルスのRBDとACE2との相互作用（例えば結合）を阻害すること、コロナウイルスの細胞への結合又は感染（例えば侵入）を阻害することを意味する。被験対象の抗体がコロナウイルスのRBDとACE2との相互作用を阻害するかどうかは、例えば、本明細書の実施例３に記載されているように、ACE2に結合したRBDを、RBDに結合可能な蛍光標識抗体を用いて検出することにより調べることができる。具体的には、被験対象の抗体とACE2とを反応させた後で、これにさらにRBDを添加した場合に、ACE2に結合したRBDが検出されなければ、被験対象の抗体がRBDとACE2との相互作用を阻害すると判断できる。また、被験対象の抗体がコロナウイルスの細胞への結合又は感染（例えば侵入）を阻害（中和）するかどうかは、例えば、被験対象の抗体とコロナウイルスを培養細胞に接触させ、当該培養細胞の形態変化（細胞変性効果：CPE）の有無を観察することで確認することができる。当該培養細胞の形態変化が認められなければ、当該被験抗体は、試験に用いた抗体濃度においてコロナウイルスに対する中和活性を有することを確認することができる（例えば実施例１０）。 The antibodies of the invention are capable of neutralizing the binding of coronaviruses to ACE2.
In the present invention, a coronavirus means a coronavirus that infects cells via ACE2. Such coronaviruses include, for example, SARS-CoV-1, SARS-CoV-2 and variants thereof, with SARS-CoV-2 and variants thereof being preferred. These coronaviruses infect cells by binding the receptor-binding domain (RBD) of their spike protein to ACE2. In the present invention, "neutralize" means inhibiting the interaction (e.g., binding) between RBD and ACE2 of coronaviruses, and inhibiting binding or infection (e.g., entry) of coronaviruses into cells. . Whether the subject antibody inhibits the interaction of the RBD of the coronavirus with ACE2 can be determined by binding the RBD bound to ACE2 to the RBD, e.g., as described in Example 3 herein. can be examined by detection using a fluorescently labeled antibody. Specifically, after reacting the antibody to be tested with ACE2, when RBD is further added to this, if RBD bound to ACE2 is not detected, the antibody to be tested is the RBD and ACE2 It can be determined that the interaction is inhibited. In addition, whether the antibody to be tested inhibits (neutralizes) the binding or infection (e.g., entry) of the coronavirus to cells can be determined, for example, by contacting the antibody to be tested and the coronavirus with cultured cells, It can be confirmed by observing the presence or absence of morphological changes (cytopathic effect: CPE). If no morphological change is observed in the cultured cells, it can be confirmed that the test antibody has neutralizing activity against coronavirus at the antibody concentration used in the test (eg, Example 10).
　以下、本発明の抗体の作製方法について説明する。
（１）抗原の調製
　本発明の抗体を作製するための免疫原としては、(i) ACE2及び(ii) ACE2発現細胞（ACE2（例えばヒトACE2）を細胞表面に強制発現させた哺乳類細胞）を使用することができる。
　免疫原としてACE2を用いる場合、ACE2の全長ポリペプチド（配列番号２）又はその部分ポリペプチドを使用することができる。抗原又は免疫原として用いられるACE2の部分ポリペプチドとしては、限定されるものではなく、例えば、ACE2の細胞外領域のアミノ酸配列の全部又は一部のアミノ酸配列を含むポリペプチドが挙げられる。
　ACE2の細胞外領域のアミノ酸配列の全部又は一部のアミノ酸配列としては、限定されるものではなく、例えば、ヒトACE2の場合、ACE2の全長のアミノ酸配列のN末端のアミノ酸残基から数えて、18番目～740番目のアミノ酸残基からなるアミノ酸配列（配列番号４）の全部又は一部のアミノ酸配列を含むアミノ酸配列が挙げられる。
　ACE2の細胞外領域のアミノ酸配列の一部のアミノ酸配列を含むポリペプチドとしては、限定されるものではなく、例えば、ACE2におけるコロナウイルスとの結合領域又はその一部のアミノ酸配列を含むポリペプチド、ACE2におけるコロナウイルスとの結合領域以外の領域又はその一部のアミノ酸配列を含むポリペプチドが挙げられる。このようなポリペプチドとしては、例えば、ヒトACE2の場合、ACE2の全長のアミノ酸配列のN末端のアミノ酸残基から数えて、1番目～120番目のアミノ酸残基からなるアミノ酸配列（配列番号１３２）、121番目～740番目のアミノ酸残基からなるアミノ酸配列（配列番号１３４）などが挙げられるが、これらに限定されない。 A method for producing the antibody of the present invention will be described below.
(1) Preparation of Antigen As immunogens for preparing the antibody of the present invention, (i) ACE2 and (ii) ACE2-expressing cells (mammalian cells in which ACE2 (e.g., human ACE2) is forcibly expressed on the cell surface) are used. can be used.
When ACE2 is used as an immunogen, the full-length ACE2 polypeptide (SEQ ID NO: 2) or a partial polypeptide thereof can be used. Partial polypeptides of ACE2 used as antigens or immunogens are not limited, and include, for example, polypeptides containing all or part of the amino acid sequence of the extracellular domain of ACE2.
The amino acid sequence of all or part of the amino acid sequence of the extracellular region of ACE2 is not limited. An amino acid sequence including all or part of the amino acid sequence (SEQ ID NO: 4) consisting of the 18th to 740th amino acid residues is included.
Polypeptides containing a partial amino acid sequence of the amino acid sequence of the extracellular region of ACE2 are not limited. Examples thereof include polypeptides containing a region other than the coronavirus-binding region of ACE2 or a partial amino acid sequence thereof. Examples of such a polypeptide include, in the case of human ACE2, an amino acid sequence consisting of the 1st to 120th amino acid residues counted from the N-terminal amino acid residue of the full-length amino acid sequence of ACE2 (SEQ ID NO: 132). , an amino acid sequence consisting of 121st to 740th amino acid residues (SEQ ID NO: 134), etc., but not limited thereto.
　免疫原としてACE2発現細胞を用いる場合、ACE2発現細胞は、例えば以下の方法を用いて作製することができる。まず、ヒトACE2の全長をコードするDNAをレンチウイルスベクターに組込み、パッケージングプラスミド、エンベローププラスミドと共に哺乳動物細胞（例えば293T細胞）に遺伝子導入する。当該細胞の培養上清中に産生された組換えレンチウイルスを哺乳動物細胞（例えば、乳がん細胞株4T1細胞）に感染させ、ヒトACE2を細胞膜上に安定的に発現する細胞を取得することができる。ただし、ACE2発現細胞の作製方法は、この方法に限定されない。 When using ACE2-expressing cells as an immunogen, ACE2-expressing cells can be produced, for example, using the following method. First, a DNA encoding full-length human ACE2 is incorporated into a lentiviral vector, and the vector is introduced into mammalian cells (eg, 293T cells) together with a packaging plasmid and an envelope plasmid. By infecting mammalian cells (eg, breast cancer cell line 4T1 cells) with the recombinant lentivirus produced in the culture supernatant of the cells, cells stably expressing human ACE2 on the cell membrane can be obtained. . However, the method for producing ACE2-expressing cells is not limited to this method.
　抗原又は免疫原として用いるACE2は、マウス、ラット、ウサギ、ネコ、イヌ、ヤギ、サル、ヒト等の組織や細胞から精製された天然型のACE2でもよいし、遺伝子工学的に生産されたACE2でもよい。例えば、ACE2の発現が認められる生体試料を各種界面活性剤、例えばTriton-X、Sarkosylなどを用い、可溶性画分と不溶性画分に分画する。さらに不溶性画分を尿素やグアニジン塩酸などに溶解し、各種カラム、例えばヘパリンカラムあるいは結合樹脂に結合させることによりACE2を得ることができる。抗原として用いるACE2は、そのアミノ酸配列を指定することにより、固相法などの公知のタンパク質合成法又は市販のタンパク質合成装置を用いて合成することもできる。合成したペプチドは、Keyhole Limpet Hemocyanin（KLH）又はThyroglobulinなどの担体タンパク質と結合させ、免疫原として用いることができる。 ACE2 used as an antigen or immunogen may be natural ACE2 purified from tissues or cells of mice, rats, rabbits, cats, dogs, goats, monkeys, humans, etc., or genetically engineered ACE2. good. For example, a biological sample in which ACE2 expression is observed is fractionated into a soluble fraction and an insoluble fraction using various surfactants such as Triton-X and Sarkosyl. Furthermore, ACE2 can be obtained by dissolving the insoluble fraction in urea, guanidine hydrochloride or the like and binding it to various columns such as a heparin column or a binding resin. ACE2 used as an antigen can also be synthesized using a known protein synthesis method such as a solid-phase method or a commercially available protein synthesizer by specifying its amino acid sequence. The synthesized peptide can be bound to a carrier protein such as Keyhole Limpet Hemocyanin (KLH) or Thyroglobulin and used as an immunogen.
（２）モノクローナル抗体の作製
　(i) 抗体産生細胞の採取
　前記のようにして作製したACE2又は部分ポリペプチドをそれ自体で、あるいは担体、希釈剤と共に非ヒト哺乳動物、例えばマウス、ラット、ウサギ、ネコ、イヌ、ヤギ、サル等に投与することにより免疫する。抗原の動物１匹当たりの投与量は、アジュバントを用いるときは0.1～10 mgである。アジュバントとしては、フロイント完全アジュバント（FCA）、フロイント不完全アジュバント（FIA）、水酸化アルミニウムアジュバント等が挙げられる。免疫は、主として静脈内、皮下又は腹腔内等に注入することにより行われる。また、免疫の間隔は特に限定されず、数日から数週間間隔、好ましくは1～2週間間隔で、2～10回、好ましくは2～5回免疫を行う。免疫の間隔は、当業者であれば得られる抗体価を勘案して設定することができる。3～4回皮下免疫を行った時点で試採血を行い、抗体価を測定することが好ましい。血清中の抗体価の測定は、ELISA、EIA、放射性免疫測定法（RIA; radioimmuno assay）等によって行うことができる。抗体価が十分上昇したことを確認した後、抗体価の上昇が認められた個体を選択し抗体産生細胞を採集する。抗体産生細胞としては、脾臓細胞、リンパ節細胞、末梢血細胞等が挙げられるが、脾臓細胞又はリンパ節細胞が好ましい。 (2) Preparation of monoclonal antibody (i) Collection of antibody-producing cells ACE2 or partial polypeptide prepared as described above is prepared by itself or together with a carrier or diluent to a non-human mammal such as mouse, rat, rabbit, Cats, dogs, goats, monkeys, etc. are immunized by administration. The dose of antigen per animal is 0.1-10 mg when an adjuvant is used. Adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), aluminum hydroxide adjuvant and the like. Immunization is mainly performed by intravenous, subcutaneous or intraperitoneal injection. The immunization interval is not particularly limited, and immunization is performed 2 to 10 times, preferably 2 to 5 times, at intervals of several days to several weeks, preferably 1 to 2 weeks. The interval between immunizations can be set by those skilled in the art in consideration of the obtained antibody titer. After subcutaneous immunization 3 to 4 times, it is preferable to sample the blood and measure the antibody titer. Antibody titers in serum can be measured by ELISA, EIA, radioimmunoassay (RIA), or the like. After confirming that the antibody titer has sufficiently increased, the individual in which the antibody titer has increased is selected and the antibody-producing cells are collected. Antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells and the like, and spleen cells or lymph node cells are preferred.
　(ii) 細胞融合
　ハイブリドーマを得るため、抗体産生細胞とミエローマ細胞との細胞融合を行う。融合操作は既知の方法、例えばKohlerらの方法に従い実施できる。抗体産生細胞と融合させるミエローマ細胞として、マウスなどの動物の一般に入手可能な株化細胞を使用することができる。使用する細胞株としては、薬剤選択性を有し、未融合の状態ではHAT選択培地（ヒポキサンチン、アミノプテリン、チミジンを含む）で生存できず、抗体産生細胞と融合した状態でのみ生存できる性質を有するものが好ましい。ミエローマ細胞としては、例えば、P3X63Ag8.653、P3X63Ag8U.1、SP2/O-Ag14、PAI、P3U1、NSI/1-Ag4-1、NSO/1などのマウスミエローマ細胞株などが挙げられる。 (ii) Cell fusion To obtain cell fusion hybridomas, antibody-producing cells and myeloma cells are fused. Fusion manipulations can be performed according to known methods, such as the method of Kohler et al. As myeloma cells to be fused with antibody-producing cells, commonly available cell lines of animals such as mice can be used. The cell line used has drug selectivity and cannot survive in a HAT selection medium (containing hypoxanthine, aminopterin, and thymidine) in an unfused state, and can survive only in a state fused with antibody-producing cells. is preferred. Examples of myeloma cells include mouse myeloma cell lines such as P3X63Ag8.653, P3X63Ag8U.1, SP2/O-Ag14, PAI, P3U1, NSI/1-Ag4-1 and NSO/1.
　上記ミエローマ細胞と抗体産生細胞との細胞融合は、血清を含まないDMEM、RPMI-1640培地などの動物細胞培養用培地中で、1×108～5×108個の抗体産生細胞と2×107～10×107個のミエローマ細胞とを混合し（抗体産生細胞とミエローマ細胞との細胞比10：1～1：1）、細胞融合促進剤の存在下で融合反応を行う。細胞融合促進剤として、平均分子量1000～6000ダルトンのポリエチレングリコール又はセンダイウイルス等を使用することができる。また、電気刺激（例えばエレクトロポレーション）を利用した市販の細胞融合装置を用いて抗体産生細胞とミエローマ細胞とを融合させることもできる。 Cell fusion between the myeloma cells and the antibody-producing cells was carried out in a serum-free DMEM, RPMI-1640 medium, or other animal cell culture medium containing 1×10 8 to 5×10 8 antibody-producing cells and 2×10 8 antibody-producing cells. 10 7 to 10×10 7 myeloma cells are mixed (the cell ratio of antibody-producing cells to myeloma cells is 10:1 to 1:1), and a fusion reaction is performed in the presence of a cell fusion promoter. As a cell fusion accelerator, polyethylene glycol having an average molecular weight of 1000 to 6000 daltons, Sendai virus, or the like can be used. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device that utilizes electrical stimulation (eg, electroporation).
 (iii) ハイブリドーマの選別及びクローニング
　細胞融合処理後の細胞から目的とするハイブリドーマを選別する。その方法として、細胞懸濁液を、例えば10～20%のウシ胎児血清含有RPMI-1640培地などで適当に希釈後、マイクロタイタープレート上に限界希釈法で計算上0.3 個／well程度まき、各ウェルにHAT培地などの選択培地を加え、以後適当に選択培地を交換して培養を行う。その結果、選択培地で培養開始後、10日前後から生育してくる細胞をハイブリドーマとして得ることができる。 (iii) Hybridoma selection and cloning Target hybridomas are selected from the cells after the cell fusion treatment. As a method, the cell suspension is diluted appropriately with, for example, RPMI-1640 medium containing 10-20% fetal bovine serum, and then seeded on a microtiter plate at a calculation of 0.3 cells/well by the limiting dilution method. A selection medium such as HAT medium is added to the wells, and thereafter the selection medium is appropriately exchanged for culturing. As a result, hybridoma cells can be obtained from around 10 days after initiation of culture in the selective medium.
　次に、生育してきたハイブリドーマをさらにスクリーニングする。ハイブリドーマのスクリーニングは、通常の方法に従えばよく、特に限定されるものではない。例えば、ハイブリドーマを培養したウェルに含まれる培養上清の一部を採集し、ELISA、EIA、放射性免疫測定法等によって、スクリーニングすることができる。具体的には、96ウェルプレートに抗原を吸着させた後、仔牛血清でブロッキングする。ハイブリドーマ細胞の培養上清を、固相化した抗原に37℃で1時間反応させた後、ペルオキシダーゼ標識した抗マウスIgGを37℃で1時間反応させ、オルトフェニレンジアミンを基質として用いて発色させる。酸で反応を停止させた後、490nmの波長における吸光度を測定することにより、スクリーニングすることができる。上記測定法により陽性を示したモノクローナル抗体を産生するハイブリドーマを、限界希釈法等によりクローニングする。そして、最終的に、ACE2に結合するモノクローナル抗体を産生する細胞であるハイブリドーマを樹立する。 Next, the grown hybridomas are further screened. Screening of hybridomas may be performed according to conventional methods, and is not particularly limited. For example, a portion of the culture supernatant contained in wells in which hybridomas were cultured can be collected and screened by ELISA, EIA, radioimmunoassay, or the like. Specifically, antigens are adsorbed on a 96-well plate and then blocked with calf serum. The hybridoma cell culture supernatant is reacted with the immobilized antigen at 37°C for 1 hour, then peroxidase-labeled anti-mouse IgG is reacted at 37°C for 1 hour, and color is developed using orthophenylenediamine as a substrate. Screening can be performed by measuring absorbance at a wavelength of 490 nm after quenching the reaction with acid. Hybridomas producing monoclonal antibodies that are positive in the above assay are cloned by limiting dilution or the like. Finally, a hybridoma, which is a cell that produces a monoclonal antibody that binds to ACE2, is established.
 (iv) モノクローナル抗体の採取
　樹立したハイブリドーマからモノクローナル抗体を採取する方法として、通常の細胞培養法又は腹水形成法等を採用することができる。細胞培養法においては、ハイブリドーマを10％ウシ胎児血清含有RPMI-1640培地、MEM培地又は無血清培地等の動物細胞培養培地中で、通常の培養条件（例えば37℃、5% CO2濃度）で7～14日間培養し、その培養上清から抗体を取得する。腹水形成法の場合は、ミエローマ細胞由来の哺乳動物と同種系動物、例えばマウス（ICR、BALB/c）の腹腔内にハイブリドーマを約5×106 ～2×107個投与し、ハイブリドーマを大量に増殖させる。そして、1～2週間後に腹水を採取する。上記抗体の採取方法において抗体の精製が必要とされる場合は、硫安塩析法、イオン交換クロマトグラフィー、ゲル濾過、アフィニティークロマトグラフィーなどの公知の方法を適宜選択して、又はこれらを組み合わせることにより精製することができる。 (iv) Collection of monoclonal antibodies As a method for collecting monoclonal antibodies from established hybridomas, a conventional cell culture method, ascites formation method, or the like can be employed. In the cell culture method, hybridomas are cultured in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium under normal culture conditions (e.g., 37°C, 5% CO 2 concentration). After culturing for 7 to 14 days, antibodies are obtained from the culture supernatant. In the case of the ascites formation method, approximately 5×10 6 to 2×10 7 hybridomas derived from myeloma cells are administered intraperitoneally to mammals and allogeneic animals, such as mice (ICR, BALB/c), and large amounts of hybridomas are administered. grow to After 1 to 2 weeks, ascites fluid is collected. When antibody purification is required in the antibody collection method, known methods such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, and affinity chromatography are appropriately selected, or by combining these can be refined.
　本発明の抗体としては、例えば、以下のモノクローナル抗体又はその抗原結合断片が挙げられるが、これらに限定されない。
　(a) 重鎖可変領域（VH）が、配列番号６のアミノ酸配列を含むか若しくは該アミノ酸配列からなる重鎖相補性決定領域（CDR）1（CDR-H1）、配列番号８のアミノ酸配列を含むか若しくは該アミノ酸配列からなる重鎖CDR2（CDR-H2）及び配列番号１０のアミノ酸配列を含むか若しくは該アミノ酸配列からなる重鎖CDR3（CDR-H3）を含み、かつ／又は軽鎖可変領域（VL）が、配列番号１２のアミノ酸配列を含むか若しくは該アミノ酸配列からなる軽鎖CDR1（CDR-L1）、配列番号１４のアミノ酸配列を含むか若しくは該アミノ酸配列からなる軽鎖CDR2（CDR-L2）及び配列番号１６のアミノ酸配列を含むか若しくは該アミノ酸配列からなる軽鎖CDR3（CDR-L3）を含むか、
　(b) 重鎖可変領域（VH）が、配列番号１８のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H1、配列番号２０のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H2及び配列番号２２のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H3を含み、かつ／又は軽鎖可変領域（VL）が、配列番号２４のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L1、配列番号２６のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L2及び配列番号２８のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L3を含むか、
　(c) 重鎖可変領域（VH）が、配列番号３０のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H1、配列番号３２のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H2及び配列番号３４のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H3を含み、かつ／又は軽鎖可変領域（VL）が、配列番号３６のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L1、配列番号３８のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L2及び配列番号４０のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L3を含むか、
　(d) 重鎖可変領域（VH）が、配列番号４２のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H1、配列番号４４のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H2及び配列番号４６のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H3を含み、かつ／又は軽鎖可変領域（VL）が、配列番号４８のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L1、配列番号５０のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L2及び配列番号５２のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L3を含むか、
　(e) 重鎖可変領域（VH）が、配列番号５４のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H1、配列番号５６のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H2及び配列番号５８のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H3を含み、かつ／又は軽鎖可変領域（VL）が、配列番号６０のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L1、配列番号６２のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L2及び配列番号６４のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L3を含むか、又は
　(f) 重鎖可変領域（VH）が、配列番号６６のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H1、配列番号６８のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H2及び配列番号７０のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H3を含み、かつ／又は軽鎖可変領域（VL）が、配列番号７２のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L1、配列番号７４のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L2及び配列番号７６のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L3を含む、
モノクローナル抗体又はその抗原結合断片。 Antibodies of the present invention include, but are not limited to, the following monoclonal antibodies or antigen-binding fragments thereof.
(a) the heavy chain variable region (VH) comprises or consists of the amino acid sequence of SEQ ID NO: 6, the heavy chain complementarity determining region (CDR) 1 (CDR-H1), the amino acid sequence of SEQ ID NO: 8; a heavy chain CDR2 (CDR-H2) comprising or consisting of the amino acid sequence of SEQ ID NO: 10 and a heavy chain CDR3 (CDR-H3) comprising or consisting of the amino acid sequence of SEQ ID NO: 10, and/or a light chain variable region (VL) comprises a light chain CDR1 (CDR-L1) comprising or consisting of the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 14 (CDR- L2) and a light chain CDR3 (CDR-L3) comprising or consisting of the amino acid sequence of SEQ ID NO: 16;
(b) heavy chain variable region (VH) CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 18, CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 20, and CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 22, and/or a CDR-H3 in which the light chain variable region (VL) comprises or consists of the amino acid sequence of SEQ ID NO: 24 L1, CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 26 and CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 28,
(c) heavy chain variable region (VH) CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 30, CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 32, and CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 34, and/or a CDR-H3 in which the light chain variable region (VL) comprises or consists of the amino acid sequence of SEQ ID NO: 36 L1, CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 38 and CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 40,
(d) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 42, CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 44, CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 44; CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 46, and/or a CDR-H3 in which the light chain variable region (VL) comprises or consists of the amino acid sequence of SEQ ID NO: 48 L1, CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 50 and CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 52,
(e) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:54 CDR-H1, comprising or consisting of the amino acid sequence of SEQ ID NO:56 CDR-H2 and CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 58 and/or the light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 60 (f) heavy chain variable CDR-H1 region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 66, CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 68, and the amino acid sequence of SEQ ID NO: 70 and/or the light chain variable region (VL) comprises or consists of the amino acid sequence of SEQ ID NO: 72, CDR-L1, SEQ ID NO: 74 a CDR-L2 comprising or consisting of the amino acid sequence and a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 76;
A monoclonal antibody or antigen-binding fragment thereof.
　本発明において、CDR-H1をコードするDNAの塩基配列としては、例えば、配列番号５、１７、２９、４１、５３又は６５で示される塩基配列を含むか又は該塩基配列からなるものが挙げられるが、これらに限定されない。
　本発明において、CDR-H2をコードするDNAの塩基配列としては、例えば、配列番号７、１９、３１、４３、５５又は６７で示される塩基配列を含むか又は該塩基配列からなるものが挙げられるが、これらに限定されない。
　本発明において、CDR-H3をコードするDNAの塩基配列としては、例えば、配列番号９、２１、３３、４５、５７又は６９で示される塩基配列を含むか又は該塩基配列からなるものが挙げられるが、これらに限定されない。
　本発明において、CDR-L1をコードするDNAの塩基配列としては、例えば、配列番号１１、２３、３５、４７、５９又は７１で示される塩基配列を含むか又は該塩基配列からなるものが挙げられるが、これらに限定されない。
　本発明において、CDR-L2をコードするDNAの塩基配列としては、例えば、配列番号１３、２５、３７、４９、６１又は７３で示される塩基配列を含むか又は該塩基配列からなるものが挙げられるが、これらに限定されない。
　本発明において、CDR-L3をコードするDNAの塩基配列としては、例えば、配列番号１５、２７、３９、５１、６３又は７５で示される塩基配列を含むか又は該塩基配列からなるものが挙げられるが、これらに限定されない。 In the present invention, the nucleotide sequence of DNA encoding CDR-H1 includes, for example, those containing or consisting of the nucleotide sequence represented by SEQ ID NO: 5, 17, 29, 41, 53 or 65. but not limited to these.
In the present invention, the nucleotide sequence of the DNA encoding CDR-H2 includes, for example, those containing or consisting of the nucleotide sequence represented by SEQ ID NO: 7, 19, 31, 43, 55 or 67. but not limited to these.
In the present invention, the nucleotide sequence of the DNA encoding CDR-H3 includes, for example, those containing or consisting of the nucleotide sequence represented by SEQ ID NO: 9, 21, 33, 45, 57 or 69. but not limited to these.
In the present invention, the nucleotide sequence of the DNA encoding CDR-L1 includes, for example, those containing or consisting of the nucleotide sequence represented by SEQ ID NO: 11, 23, 35, 47, 59 or 71. but not limited to these.
In the present invention, the nucleotide sequence of DNA encoding CDR-L2 includes, for example, those comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 13, 25, 37, 49, 61 or 73. but not limited to these.
In the present invention, the nucleotide sequence of the DNA encoding CDR-L3 includes, for example, those containing or consisting of the nucleotide sequence represented by SEQ ID NO: 15, 27, 39, 51, 63 or 75. but not limited to these.
　また別の態様において、本発明の抗体としては、例えば、以下のモノクローナル抗体又はその抗原結合断片が挙げられるが、これらに限定されない。
　(a) 配列番号７８のアミノ酸配列を含むか若しくは該アミノ酸配列からなる重鎖可変領域（VH）及び配列番号８０のアミノ酸配列を含むか若しくは該アミノ酸配列からなる軽鎖可変領域（VL）を含むか、
　(b) 配列番号８２のアミノ酸配列を含むか若しくは該アミノ酸配列からなる重鎖可変領域（VH）及び配列番号８４のアミノ酸配列を含むか若しくは該アミノ酸配列からなる軽鎖可変領域（VL）を含むか、
　(c) 配列番号８６のアミノ酸配列を含むか若しくは該アミノ酸配列からなる重鎖可変領域（VH）及び配列番号８８のアミノ酸配列を含むか若しくは該アミノ酸配列からなる軽鎖可変領域（VL）を含むか、
　(d) 配列番号９０のアミノ酸配列を含むか若しくは該アミノ酸配列からなる重鎖可変領域（VH）及び配列番号９２のアミノ酸配列を含むか若しくは該アミノ酸配列からなる軽鎖可変領域（VL）を含むか、
　(e) 配列番号９４のアミノ酸配列を含むか若しくは該アミノ酸配列からなる重鎖可変領域（VH）及び配列番号９６のアミノ酸配列を含むか若しくは該アミノ酸配列からなる軽鎖可変領域（VL）を含むか、又は
　(f) 配列番号９８のアミノ酸配列を含むか若しくは該アミノ酸配列からなる重鎖可変領域（VH）及び配列番号１００のアミノ酸配列を含むか若しくは該アミノ酸配列からなる軽鎖可変領域（VL）を含む、
モノクローナル抗体又はその抗原結合断片。 In another aspect, antibodies of the present invention include, but are not limited to, the following monoclonal antibodies or antigen-binding fragments thereof.
(a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 78 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 80; mosquito,
(b) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:82 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:84; mosquito,
(c) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:86 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:88 mosquito,
(d) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:90 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:92; mosquito,
(e) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:94 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:96; or (f) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:98 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:100 )including,
A monoclonal antibody or antigen-binding fragment thereof.
　本発明において、重鎖可変領域をコードするDNAの塩基配列としては、例えば、配列番号７７、８１、８５、８９、９３又は９７で示される塩基配列を含むか又は該塩基配列からなるものが挙げられるが、これらに限定されない。また、本発明において、軽鎖可変領域をコードするDNAの塩基配列としては、例えば、配列番号７９、８３、８７、９１、９５又は９９で示される塩基配列を含むか又は該塩基配列からなるものが挙げられるが、これらに限定されない。 In the present invention, the nucleotide sequence of the DNA encoding the heavy chain variable region includes, for example, those containing or consisting of the nucleotide sequence represented by SEQ ID NO: 77, 81, 85, 89, 93 or 97. include but are not limited to: In the present invention, the nucleotide sequence of the DNA encoding the light chain variable region includes, for example, the nucleotide sequence represented by SEQ ID NO: 79, 83, 87, 91, 95 or 99, or consists of the nucleotide sequence. include, but are not limited to.
（３）遺伝子組換え抗体の作製
　本発明の抗体の好ましい態様の一つとして、遺伝子組換え抗体が挙げられる。遺伝子組換え抗体としては、限定されるものではなく、例えば、キメラ抗体、イヌ型化抗体、ヒト型化抗体、完全ヒト抗体が挙げられる。 (3) Preparation of genetically engineered antibody One of preferred embodiments of the antibody of the present invention is a genetically engineered antibody. Examples of genetically modified antibodies include, but are not limited to, chimeric antibodies, canine antibodies, humanized antibodies, and fully human antibodies.
　キメラ抗体は、異種の動物の免疫グロブリン遺伝子断片を連結して作製された抗体をいう。本発明において、キメラ抗体としては、例えば、ヒト型キメラ抗体が挙げられるが、キメラ抗体の可変領域及び定常領域が由来する動物の種類は限定されない。ヒト型キメラ抗体は、例えば、マウス由来抗体の可変領域をヒト由来抗体の定常領域に連結（接合）した抗体（Proc. Natl. Acad. Sci. U.S.A. 81, 6851-6855, (1984) 等を参照）である。当該ヒト由来抗体のサブクラスとしては、例えば、IgG1、IgG2、IgG4などが挙げられるが、これらに限定されない。キメラ抗体は、遺伝子組換え技術によって容易に構築できる。 A chimeric antibody refers to an antibody produced by linking immunoglobulin gene fragments of a different animal. In the present invention, chimeric antibodies include, for example, human chimeric antibodies, but the types of animals from which the variable regions and constant regions of chimeric antibodies are derived are not limited. Human chimeric antibodies are, for example, antibodies in which the variable region of a mouse-derived antibody is linked (joined) to the constant region of a human-derived antibody (see Proc. Natl. Acad. Sci. U.S.A. 81, 6851-6855, (1984), etc.) ). Examples of subclasses of the human-derived antibody include, but are not limited to, IgG1, IgG2, IgG4, and the like. A chimeric antibody can be easily constructed by gene recombination technology.
　本発明において、ヒト型化抗体を作製する場合は、いわゆるCDRグラフティング（CDR移植）と呼ばれる手法を採用することができる。CDRグラフティングとは、マウス抗体の可変領域から相補性決定領域（CDR）をヒト可変領域に移植して、フレームワーク領域（FR）はヒト由来のものでCDRはマウス由来のものからなる、再構成した可変領域を作製する方法である。次に、これらのヒト型化された再構成ヒト可変領域をヒト定常領域に連結する。このようなヒト型化抗体の作製法は、当分野において周知である（Nature, 321, 522-525 (1986)；J. Mol. Biol., 196, 901-917 (1987)；Queen C et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989)；特許第2828340号公報等を参照）。 In the present invention, when producing a humanized antibody, a technique called so-called CDR grafting (CDR transplantation) can be adopted. CDR grafting is the grafting of complementarity determining regions (CDRs) from mouse antibody variable regions to human variable regions, with framework regions (FRs) of human origin and CDRs of mouse origin. It is a method of making a constructed variable region. These humanized reshaped human variable regions are then ligated to human constant regions. Methods for producing such humanized antibodies are well known in the art (Nature, 321, 522-525 (1986); J. Mol. Biol., 196, 901-917 (1987); Queen C et al. ., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989); see Japanese Patent No. 2828340, etc.).
　本発明において、完全ヒト抗体は、例えば、ヒト抗体を産生することができる哺乳動物を用いて、公知の手法に準じて作製することができる（WO96／9634096、WO98/24893など）。 In the present invention, fully human antibodies can be produced, for example, using mammals capable of producing human antibodies according to known techniques (WO96/9634096, WO98/24893, etc.).
　本発明において、キメラ抗体及びヒト型化抗体に用いることができるヒト重鎖定常領域としては、ヒトIgG4重鎖定常領域に由来するアミノ酸配列を含むもの、例えば、配列番号１０２で示されるアミノ酸配列を含むか若しくは該アミノ酸配列からなるヒト重鎖定常領域が挙げられるが、これに限定されない。また、キメラ抗体及びヒト型化抗体に用いることができるヒト軽鎖定常領域としては、ヒトIgG4軽鎖定常領域に由来するアミノ酸配列を含むもの、例えば、配列番号１０４で示されるアミノ酸配列を含むか若しくは該アミノ酸配列からなるヒト軽鎖定常領域が挙げられるが、これに限定されない。また、ヒト重鎖定常領域をコードするDNAとしては、例えば、配列番号１０１で示される塩基配列を含むか若しくは該塩基配列からなるDNAが挙げられ、ヒト軽鎖定常領域をコードするDNAとしては、例えば、配列番号１０３で示される塩基配列を含むか若しくは該塩基配列からなるDNAが挙げられるが、これに限定されない。 In the present invention, the human heavy chain constant region that can be used for chimeric antibodies and humanized antibodies includes those containing an amino acid sequence derived from a human IgG4 heavy chain constant region, for example, the amino acid sequence shown in SEQ ID NO: 102. It includes, but is not limited to, a human heavy chain constant region comprising or consisting of said amino acid sequence. In addition, the human light chain constant region that can be used for chimeric antibodies and humanized antibodies includes those containing an amino acid sequence derived from a human IgG4 light chain constant region, for example, the amino acid sequence shown in SEQ ID NO: 104. Alternatively, it may be a human light chain constant region consisting of said amino acid sequence, but is not limited thereto. Examples of DNAs encoding human heavy chain constant regions include DNAs containing or consisting of the nucleotide sequence shown in SEQ ID NO: 101. DNAs encoding human light chain constant regions include: Examples include, but are not limited to, DNA containing or consisting of the nucleotide sequence shown in SEQ ID NO:103.
　本発明においては、ハイブリドーマ又は当該ハイブリドーマから抽出したDNA若しくはRNAなどを原料として、上述した公知の方法に準じてキメラ抗体及びヒト型化抗体を作製することができる。
　さらに、本発明の抗体が融合したタンパク質は、抗体の可変領域とその他のタンパク質を公知の遺伝子組換え方法を用いて融合することにより作製することができる。また、当該融合タンパク質は、モノクローナル抗体と他のタンパク質とをクロスリンカーを用いて架橋することにより作製することができる。 In the present invention, chimeric antibodies and humanized antibodies can be produced from hybridomas or DNA or RNA extracted from such hybridomas as raw materials according to the above-described known methods.
Furthermore, a protein fused with an antibody of the present invention can be produced by fusing an antibody variable region with another protein using a known genetic recombination method. Alternatively, the fusion protein can be produced by cross-linking a monoclonal antibody and another protein using a cross-linker.
（４）抗体結合断片の作製
　本発明の抗体の抗原結合断片は、ACE2に結合する。
　抗体の抗原結合断片は、本発明の抗体における抗原結合部分の一部を含むポリペプチドを意味する。抗原結合断片としては、例えば、scFv（single chain Fv）、sc(Fv)2、Fab、Fab'、ダイアボディ（diabody、dsFv）、F(ab')2、多重特異性抗体などが挙げられるが、これらに限定されない。上記抗体結合断片は、遺伝子工学的手法や、パパイン消化、ペプシン消化など、公知の方法により取得することができる。
  例えば、Fabは、抗体分子をパパインで処理することにより、F(ab')2は、抗体分子をペプシンで処理することによりそれぞれ得ることができる。また、Fab'は、上記F(ab')2のヒンジ領域のジスルフィド結合を切断することで得ることができる。
  scFvの場合は、抗体のH鎖V領域及びL鎖V領域をコードするcDNAを取得し、scFvをコードするDNAを構築する。このDNAを発現ベクターに挿入し、当該発現ベクターを宿主生物に導入して発現させることにより、scFvを製造することができる。
　diabodyの場合は、抗体のH鎖V領域及びL鎖V領域をコードするcDNAを取得し、ペプチドリンカーのアミノ酸配列の長さが８残基以下となるようにscFvをコードするDNAを構築する。このDNAを発現ベクターに挿入し、当該発現ベクターを宿主生物に導入して発現させることにより、diabodyを製造することができる。
  dsFvの場合は、抗体のH鎖V領域及びL鎖V領域をコードするcDNAを取得し、dsFvをコードするDNAを構築する。このDNAを発現ベクターに挿入し、当該発現ベクターを宿主生物に導入して発現させることにより、dsFvを製造することができる。
　多重特異性抗体、例えば二重特異性抗体は、二以上の抗原又はエピトープを標的として結合するものであり、様々な公知の手法により作製することができる（Songsivilai and Lachmann, 1990, Clin.Exp.Immunol.79:315-321など）。 (4) Preparation of antibody-binding fragment The antigen-binding fragment of the antibody of the present invention binds to ACE2.
An antigen-binding fragment of an antibody refers to a polypeptide comprising a portion of the antigen-binding portion of the antibody of the invention. Examples of antigen-binding fragments include scFv (single chain Fv), sc(Fv) 2 , Fab, Fab', diabody (dsFv), F(ab') 2 , multispecific antibodies and the like. , but not limited to. The above antibody-binding fragments can be obtained by known methods such as genetic engineering techniques, papain digestion, pepsin digestion, and the like.
For example, Fab can be obtained by treating an antibody molecule with papain, and F(ab') 2 can be obtained by treating an antibody molecule with pepsin. Fab' can also be obtained by cleaving the disulfide bond in the hinge region of F(ab') 2 above.
In the case of scFv, cDNAs encoding the antibody H-chain V region and L-chain V region are obtained to construct scFv-encoding DNA. scFv can be produced by inserting this DNA into an expression vector, introducing the expression vector into a host organism, and expressing the scFv.
In the case of diabody, cDNAs encoding the H chain V region and L chain V region of the antibody are obtained, and scFv-encoding DNA is constructed so that the amino acid sequence length of the peptide linker is 8 residues or less. A diabody can be produced by inserting this DNA into an expression vector, introducing the expression vector into a host organism, and expressing the DNA.
In the case of dsFv, cDNAs encoding the H chain V region and L chain V region of the antibody are obtained, and DNA encoding the dsFv is constructed. A dsFv can be produced by inserting this DNA into an expression vector, introducing the expression vector into a host organism, and expressing the dsFv.
Multispecific antibodies, such as bispecific antibodies, target and bind to two or more antigens or epitopes, and can be produced by various known techniques (Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321).
　CDRを含む抗原結合断片（ペプチド）は、VH又はVLのCDR（CDR1～3）の少なくとも１領域以上を含んで構成される。複数のCDRを含む抗原結合断片は、直接又は適当なペプチドリンカーを介して結合させることができる。CDRを含む抗原結合断片は、抗体のVH及びVLのCDRをコードするDNAを構築し、該DNAを原核生物用発現ベクター又は真核生物用発現ベクターに挿入して、該発現ベクターを原核生物又は真核生物へ導入することにより発現させて、製造することができる。また、CDRを含むペプチドは、Fmoc法（フルオレニルメチルオキシカルボニル法）及びtBoc法（ｔ－ブチルオキシカルボニル法）等の化学合成法によって製造することもできる。 Antigen-binding fragments (peptides) containing CDRs are composed of at least one region or more of CDRs (CDR1-3) of VH or VL. Antigen-binding fragments containing multiple CDRs can be conjugated directly or via suitable peptide linkers. Antigen-binding fragments containing CDRs are obtained by constructing DNAs encoding the VH and VL CDRs of an antibody, inserting the DNAs into prokaryotic or eukaryotic expression vectors, and converting the expression vectors into prokaryotic or eukaryotic expression vectors. It can be expressed and produced by introduction into eukaryotes. Peptides containing CDRs can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
（５）結合親和性
  抗体の抗原に対する親和性は、一般に、平衡解離定数（KD）で表すことができ、KDの値が低いほど、抗体が高い親和性を有することを示す。
　抗体の親和性は、公知の機器及び方法を用いて測定することができる（例えばBiacore（登録商標）-3000 (GE Healthcare社）、ProteON XPR36 (Bio-Rad社)など）。 (5) Binding Affinity The affinity of an antibody for an antigen can generally be expressed by the equilibrium dissociation constant (KD), with lower values of KD indicating higher affinity of the antibody.
Antibody affinity can be measured using known instruments and methods (eg, Biacore (registered trademark)-3000 (GE Healthcare), ProteON XPR36 (Bio-Rad), etc.).
（６）参照抗体と競合する抗体
　本発明は、コロナウイルスとACE2との結合を中和することができ、ACE2との結合について、下記の参照抗体又はその抗原結合断片のいずれかと競合する、モノクローナル抗体又はその抗原結合断片を提供する。
　本発明において、参照抗体としては、以下のモノクローナル抗体が挙げられる。
　(a) 重鎖可変領域（VH）が、配列番号６のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H1、配列番号８のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H2及び配列番号１０のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H3を含み、かつ／又は軽鎖可変領域（VL）が、配列番号１２のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L1、配列番号１４のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L2及び配列番号１６のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L3を含む、モノクローナル抗体、
　(b) 重鎖可変領域（VH）が、配列番号１８のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H1、配列番号２０のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H2及び配列番号２２のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H3を含み、かつ／又は軽鎖可変領域（VL）が、配列番号２４のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L1、配列番号２６のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L2及び配列番号２８のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L3を含む、モノクローナル抗体、
　(c) 重鎖可変領域（VH）が、配列番号３０のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H1、配列番号３２のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H2及び配列番号３４のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H3を含み、かつ／又は軽鎖可変領域（VL）が、配列番号３６のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L1、配列番号３８のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L2及び配列番号４０のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L3を含む、モノクローナル抗体、
　(d) 重鎖可変領域（VH）が、配列番号４２のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H1、配列番号４４のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H2及び配列番号４６のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H3を含み、かつ／又は軽鎖可変領域（VL）が、配列番号４８のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L1、配列番号５０のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L2及び配列番号５２のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L3を含む、モノクローナル抗体、
　(e) 重鎖可変領域（VH）が、配列番号５４のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H1、配列番号５６のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H2及び配列番号５８のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H3を含み、かつ／又は軽鎖可変領域（VL）が、配列番号６０のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L1、配列番号６２のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L2及び配列番号６４のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L3を含む、モノクローナル抗体、
　(f) 重鎖可変領域（VH）が、配列番号６６のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H1、配列番号６８のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H2及び配列番号７０のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-H3を含み、かつ／又は軽鎖可変領域（VL）が、配列番号７２のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L1、配列番号７４のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L2及び配列番号７６のアミノ酸配列を含むか若しくは該アミノ酸配列からなるCDR-L3を含む、モノクローナル抗体、
　(g) 配列番号７８のアミノ酸配列を含むか若しくは該アミノ酸配列からなる重鎖可変領域（VH）及び配列番号８０のアミノ酸配列を含むか若しくは該アミノ酸配列からなる軽鎖可変領域（VL）を含む、モノクローナル抗体、
　(h) 配列番号８２のアミノ酸配列を含むか若しくは該アミノ酸配列からなる重鎖可変領域（VH）及び配列番号８４のアミノ酸配列を含むか若しくは該アミノ酸配列からなる軽鎖可変領域（VL）を含む、モノクローナル抗体、
　(i) 配列番号８６のアミノ酸配列を含むか若しくは該アミノ酸配列からなる重鎖可変領域（VH）及び配列番号８８のアミノ酸配列を含むか若しくは該アミノ酸配列からなる軽鎖可変領域（VL）を含む、モノクローナル抗体、
　(j) 配列番号９０のアミノ酸配列を含むか若しくは該アミノ酸配列からなる重鎖可変領域（VH）及び配列番号９２のアミノ酸配列を含むか若しくは該アミノ酸配列からなる軽鎖可変領域（VL）を含む、モノクローナル抗体、
　(j) 配列番号９４のアミノ酸配列を含むか若しくは該アミノ酸配列からなる重鎖可変領域（VH）及び配列番号９６のアミノ酸配列を含むか若しくは該アミノ酸配列からなる軽鎖可変領域（VL）を含む、モノクローナル抗体、又は
　(k) 配列番号９８のアミノ酸配列を含むか若しくは該アミノ酸配列からなる重鎖可変領域（VH）及び配列番号１００のアミノ酸配列を含むか若しくは該アミノ酸配列からなる軽鎖可変領域（VL）を含む、モノクローナル抗体。 (6) An antibody that competes with a reference antibody The present invention is a monoclonal antibody capable of neutralizing the binding of a coronavirus to ACE2 and that competes with any of the reference antibodies or antigen-binding fragments thereof for binding to ACE2. Antibodies or antigen-binding fragments thereof are provided.
In the present invention, reference antibodies include the following monoclonal antibodies.
(a) heavy chain variable region (VH) CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 6, CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 10 and/or a CDR-H3 in which the light chain variable region (VL) comprises or consists of the amino acid sequence of SEQ ID NO: 12 a monoclonal antibody comprising L1, a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 14 and a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 16;
(b) heavy chain variable region (VH) CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 18, CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 20, and CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 22, and/or a CDR-H3 in which the light chain variable region (VL) comprises or consists of the amino acid sequence of SEQ ID NO: 24 a monoclonal antibody comprising L1, a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 26 and a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 28;
(c) heavy chain variable region (VH) CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 30, CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 32, and CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 34, and/or a CDR-H3 in which the light chain variable region (VL) comprises or consists of the amino acid sequence of SEQ ID NO: 36 a monoclonal antibody comprising L1, a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO:38 and a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO:40;
(d) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 42, CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 44, CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 44; CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 46, and/or a CDR-H3 in which the light chain variable region (VL) comprises or consists of the amino acid sequence of SEQ ID NO: 48 a monoclonal antibody comprising L1, a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 50 and a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 52;
(e) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:54 CDR-H1, comprising or consisting of the amino acid sequence of SEQ ID NO:56 CDR-H2 and CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 58 and/or the light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 60 a monoclonal antibody comprising L1, a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 62 and a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 64;
(f) heavy chain variable region (VH) CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 66, CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 68, and CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 70 and/or the light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 72 a monoclonal antibody comprising L1, a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 74, and a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 76;
(g) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:78 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:80; , monoclonal antibodies,
(h) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:82 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:84 , monoclonal antibodies,
(i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:86 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:88; , monoclonal antibodies,
(j) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:90 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:92; , monoclonal antibodies,
(j) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:94 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:96; , a monoclonal antibody, or (k) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 98 and a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 100 Monoclonal antibodies, including (VL).
　本発明において、ACE2との結合について「競合する」とは、本発明の抗体又はその抗原結合断片が、上記参照抗体又はその抗原結合断片のACE2への結合を阻害する（例えば抑制する）こと、あるいは、本発明の抗体又はその抗原結合断片のACE2への結合が、上記参照抗体又はその抗原結合断片により阻害される（例えば抑制される）ことを意味する。本発明の抗体又はその抗原結合断片が、ACE2への結合について、上記参照抗体又はその抗原結合断片のいずれかと競合するかどうかは、公知の手法、例えば、競合RIA、競合EIA、競合ELISA（ポリペプチドを用いた競合ELISA、競合cell ELISA）、エピトープビニング（epitope binning）などを用いて調べることができる。抗体を用いた競合試験は、被験対象の抗体又はその抗原結合断片と参照抗体とが、抗原における同じエピトープ又は近接するエピトープへの結合について、競合するかどうかを調べるための手法として抗体の技術分野において確立された手法である（例えば、Ju-Won Kwak et al., Journal of Immunological Methods 191 (1996) 49-54、Sindy Liao-Chan, et al. J Immunol Methods. 2014 Mar;405:1-14等）。上記「近接するエピトープ」とは、被験対象の抗体又はその抗原結合断片と参照抗体とが、エピトープへの結合について立体的妨害が生じる程度に近接するエピトープをいう。例えば、本明細書の実施例６に示されるとおり、OMRad004G05、OMRad028D08、OMRad031H08、OMRad045G03の4種類のモノクローナル抗体は、ACE2への結合について、互いに競合する抗体であり、さらに、これらの競合する抗体は、本明細書の実施例１０に示されるとおり、コロナウイルスの細胞への結合又は感染に対して同等の中和活性を有する。一方、OMRad004G05及びOMRad028D08の2種類とOMRad031H08及びOMRad045G03の2種類とは、マウスACE2に対する反応性が異なるため（実施例７）、結合するエピトープは異なる可能性がある。 In the present invention, "competing" for binding to ACE2 means that the antibody or antigen-binding fragment thereof of the present invention inhibits (for example, suppresses) the binding of the above-mentioned reference antibody or antigen-binding fragment thereof to ACE2; Alternatively, it means that the binding of the antibody of the present invention or antigen-binding fragment thereof to ACE2 is inhibited (eg, suppressed) by the reference antibody or antigen-binding fragment thereof. Whether the antibody or antigen-binding fragment thereof of the present invention competes with any of the reference antibodies or antigen-binding fragments thereof for binding to ACE2 can be determined by known methods such as competitive RIA, competitive EIA, competitive ELISA (poly It can be examined using competitive ELISA using peptides, competitive cell ELISA), epitope binning, and the like. A competition test using an antibody is a technique for examining whether an antibody to be tested or an antigen-binding fragment thereof and a reference antibody compete for binding to the same epitope or a neighboring epitope on an antigen. (e.g., Ju-Won Kwak et al., Journal of Immunological Methods 191 (1996) 49-54, Sindy Liao-Chan, et al. J Immunol Methods. 2014 Mar;405:1-14 etc). The above-mentioned "adjacent epitope" refers to an epitope in which the antibody or antigen-binding fragment thereof to be tested and the reference antibody are in close proximity to the extent that binding to the epitope is sterically hindered. For example, as shown in Example 6 herein, four monoclonal antibodies, OMRad004G05, OMRad028D08, OMRad031H08, and OMRad045G03, are antibodies that compete with each other for binding to ACE2, and these competing antibodies are , as shown in Example 10 herein, have comparable neutralizing activity against coronavirus cell binding or infection. On the other hand, two types, OMRad004G05 and OMRad028D08, and two types, OMRad031H08 and OMRad045G03, have different reactivities to mouse ACE2 (Example 7), and thus may bind to different epitopes.
　本明細書の実施例６に示されるとおり、本発明の抗体又はその抗原結合断片が、ACE2への結合について、上記参照抗体又はその抗原結合断片と競合するかどうかを調べるためには、抗体の構造やエピトープの構造がどのような構造であるか（例えば、CDR及び可変領域がどのようなアミノ酸配列であるか、線状であるか三次元的（立体的）な構造であるか、など）についての情報を必要としない。
　そして、通常の方法に基づいて抗ACEモノクローナル抗体を作製し、ACE2との結合について本明細書に記載の参照抗体と競合する抗体を選択するという一連の手順を行うことにより、本明細書の実施例において具体的な構造が特定された抗体以外にも、ACE2との結合について、本明細書に記載の参照抗体と競合する抗体を取得し得る。
　すなわち、ACE2との結合について、上記参照抗体又はその抗原結合断片のいずれかと競合する、本発明のモノクローナル抗体又はその抗原結合断片は、当業者であれば、上記参照抗体を用いて競合試験を行うことにより、過度の実験を伴うことなく得ることができる。 As shown in Example 6 herein, to determine whether the antibody or antigen-binding fragment thereof of the present invention competes with the reference antibody or antigen-binding fragment thereof for binding to ACE2, What kind of structures and epitope structures are (for example, what kind of amino acid sequences the CDRs and variable regions are, whether they are linear or three-dimensional (steric) structures, etc.) does not require information about
The practice of the present invention is then performed by following a series of steps of generating anti-ACE monoclonal antibodies based on routine methods and selecting antibodies that compete with the reference antibodies described herein for binding to ACE2. In addition to the antibodies whose specific structures are specified in the examples, antibodies can be obtained that compete with the reference antibodies described herein for binding to ACE2.
That is, for binding to ACE2, the monoclonal antibody or antigen-binding fragment thereof of the present invention, which competes with either the reference antibody or the antigen-binding fragment thereof, can be tested by a person skilled in the art using the reference antibody. can be obtained without undue experimentation.
４．医薬組成物
　本発明の医薬組成物は、上記「３．本発明の抗体」に記載した抗体又はその抗原結合断片を有効成分として含有し、コロナウイルス感染症を治療又は予防するための医薬組成物である。
　本発明の医薬組成物の対象となるコロナウイルス感染症としては、コロナウイルスの感染によって引き起こされる疾患又は症状を意味する。このようなコロナウイルス感染症としては、例えば、ACE2を介して細胞に感染するコロナウイルスの感染によって引き起こされる疾患又は症状が挙げられる。「ACE2を介して細胞に感染するコロナウイルス」としては、限定されるものではなく、例えば、SARS-CoV-1、SARS-CoV-2が挙げられ、SARS-CoV-2が好ましい。
「ACE2を介して細胞に感染するコロナウイルスの感染によって引き起こされる疾患又は症状」としては、例えば、ACE2が主に発現する器官又は組織、例えば、肺、消化器系、心臓、血管、眼、腎臓、大脳皮質、扁桃体、脳幹、延髄などに関連する疾患又は症状、免疫応答に基づく疾患又は症状などが挙げられる。このような疾患又は症状としては、例えば、呼吸器疾患、発熱、倦怠感、悪寒、疼痛、味覚若しくは嗅覚の障害、発疹、消化器症状、言語障害、認知障害、循環器症状が挙げられるが、これらに限定されない。
　本発明の抗体を含む医薬組成物は、ACE2を介して細胞に感染するコロナウイルスの感染を阻害することにより、コロナウイルス感染症に伴う多様な疾患又は症状の治療又は予防に有効である。 4. Pharmaceutical composition The pharmaceutical composition of the present invention is a pharmaceutical composition for treating or preventing coronavirus infection, which contains the antibody or antigen-binding fragment thereof described in "3. Antibody of the present invention" as an active ingredient. is.
A coronavirus infection that is the target of the pharmaceutical composition of the present invention means a disease or symptom caused by infection with a coronavirus. Such coronavirus infections include, for example, diseases or conditions caused by infection with coronaviruses that infect cells via ACE2. The "coronavirus that infects cells via ACE2" is not limited, and includes, for example, SARS-CoV-1 and SARS-CoV-2, preferably SARS-CoV-2.
Examples of "diseases or symptoms caused by infection with a coronavirus that infects cells via ACE2" include organs or tissues in which ACE2 is mainly expressed, such as lungs, digestive system, heart, blood vessels, eyes, and kidneys. , cerebral cortex, amygdala, brain stem, medulla oblongata, etc., diseases or symptoms based on immune response, and the like. Such diseases or symptoms include, for example, respiratory diseases, fever, malaise, chills, pain, taste or smell disorders, rashes, gastrointestinal symptoms, speech disorders, cognitive disorders, cardiovascular symptoms, It is not limited to these.
Pharmaceutical compositions containing the antibodies of the present invention are effective in treating or preventing various diseases or symptoms associated with coronavirus infection by inhibiting coronavirus infection that infects cells via ACE2.
　本発明において、「治療」とは、疾患の発症後に本発明の抗体若しくはその抗原結合断片又はそれを含む医薬組成物（以下「本発明の抗体及び医薬組成物」とも称する）を被験者／対象（subject）に接触させる（例えば、投与する）ことにより、接触させない場合に比べて、当該疾患の症状を軽減することを意味し、必ずしも疾患の症状を完全に抑制することを意味するものではない。本発明における「疾患の発症」には、疾患の症状が身体に現れることに加えて、症状が現れていなくてもコロナウイルス感染に関する検査（PCR検査など）において陽性であることも含まれる。また、本発明において、コロナウイルス感染症の「治療」には、コロナウイルス感染症の重篤化の抑制又は阻害が含まれる。
　本発明において、「予防」とは、コロナウイルス感染症の発症の抑制又は阻害、重篤化の抑制又は阻害を意味するが、必ずしも発症を完全に抑制することを意味するものではない。本発明において、「予防」には、疾患の発症前に本発明の医薬組成物等を被験者／対象に接触させる（例えば、投与する）ことにより、接触させない場合に比べて、疾患の発症後の症状を軽減することが含まれる。 In the present invention, the term "treatment" refers to the use of the antibody of the present invention or an antigen-binding fragment thereof, or a pharmaceutical composition comprising the same (hereinafter also referred to as "the antibody and pharmaceutical composition of the present invention") after the onset of a disease. It means that by contacting (e.g., administering) the subject, the symptoms of the disease are alleviated compared to when the subject is not contacted, but does not necessarily mean that the symptoms of the disease are completely suppressed. In the present invention, "onset of disease" includes not only symptoms of the disease appearing in the body, but also a positive result in a test for coronavirus infection (such as a PCR test) even if no symptoms appear. In addition, in the present invention, “treatment” of coronavirus infection includes suppression or inhibition of aggravation of coronavirus infection.
In the present invention, "prevention" means suppression or inhibition of the onset of coronavirus infection, suppression or inhibition of aggravation, but does not necessarily mean complete suppression of the onset. In the present invention, "prevention" refers to contacting (e.g., administering) the pharmaceutical composition etc. of the present invention with a subject/subject before the onset of the disease, compared to the case of no contact, after the onset of the disease. This includes relieving symptoms.
　本発明の医薬組成物は、ACE2に結合するモノクローナル抗体又はその抗原結合断片のほか、薬学的に許容できる担体を含むことができる。「薬学的に許容できる担体」とは、医薬組成物に適する任意の担体（リポソーム、脂質小胞体、ミセル等）、希釈剤、賦形剤、湿潤剤、緩衝剤、懸濁剤、潤滑剤、アジュバント、乳化剤、崩壊剤、吸収剤、保存料、界面活性剤、着色料、着香料又は甘味料を指す。 The pharmaceutical composition of the present invention can contain a monoclonal antibody that binds to ACE2 or an antigen-binding fragment thereof, as well as a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" means any carrier suitable for pharmaceutical compositions (liposomes, lipid vesicles, micelles, etc.), diluents, excipients, wetting agents, buffers, suspending agents, lubricants, Refers to adjuvants, emulsifiers, disintegrants, absorbents, preservatives, surfactants, colorants, flavorants or sweeteners.
　本発明の抗体及び医薬組成物は、注射剤、凍結乾燥品、錠剤、硬カプセル剤、軟カプセル剤、顆粒剤、散剤、丸剤、シロップ剤、坐剤、バップ剤、軟膏剤、クリーム剤、点眼剤等の剤型をとることができる。注射剤などの液体製剤は、使用前に生理食塩水等で溶解する用時調製用粉末（例えば凍結乾燥粉末）の形態であってもよい。 Antibodies and pharmaceutical compositions of the present invention include injections, freeze-dried products, tablets, hard capsules, soft capsules, granules, powders, pills, syrups, suppositories, poultices, ointments, creams, Dosage forms such as eye drops can be taken. Liquid formulations such as injections may be in the form of powders (for example, freeze-dried powders) for preparation before use, which are dissolved in physiological saline or the like before use.
　本発明の抗体及び医薬組成物は、当業者に既知である任意の手段によって局所的又は全身に投与することができる。本発明の医薬組成物の投与経路としては、経口投与及び非経口投与のいずれも可能であり、非経口投与の場合は、組織内投与（皮下投与、腹腔内投与、筋肉内投与、静脈内投与など）、皮内投与、局所投与（経皮投与など）又は経直腸的に投与することができる。本発明の医薬組成物は、これらの投与経路に適した投与形態で投与することができる。 The antibodies and pharmaceutical compositions of the present invention can be administered locally or systemically by any means known to those skilled in the art. As an administration route of the pharmaceutical composition of the present invention, both oral administration and parenteral administration are possible. etc.), intradermal administration, topical administration (such as transdermal administration), or transrectal administration. The pharmaceutical composition of the present invention can be administered in dosage forms suitable for these administration routes.
　本発明の抗体及び医薬組成物の投与量は、被験者／対象の年齢、体重、健康状態、性別、症状、動物種、投与経路、投与回数、剤型などの要因に応じて変化し、具体的な投与手順は当業者により設定することができる。コロナウイルス感染症の治療のための本発明の抗体の投与量としては、被験者／対象の体重1 kgあたり、例えば0.1mg～100mg/日、好ましくは1mg～15mg/日、より好ましくは2-12mg/日であるが、これに限定されない。投与回数としては、一日に１～５回投与することができる。 The dosage of the antibody and pharmaceutical composition of the present invention varies depending on factors such as age, body weight, health condition, sex, symptoms, animal species, administration route, administration frequency, and dosage form of the subject/subject. Suitable administration procedures can be set by those skilled in the art. The dosage of the antibody of the present invention for the treatment of coronavirus infection is, for example, 0.1 mg to 100 mg/day, preferably 1 mg to 15 mg/day, more preferably 2-12 mg per kg body weight of the subject/subject. / day, but not limited to. As for the administration frequency, it can be administered 1 to 5 times a day.
　本発明の抗体及び医薬組成物の投与時期は、症状に応じて適宜定めることができ、複数回分を同時に又は時間をおいて別々に投与することができる。また、本発明の医薬組成物は、疾患の発症前に被験者／対象に投与してもよいし、疾患の発症後に投与してもよい。 The timing of administration of the antibody and pharmaceutical composition of the present invention can be appropriately determined according to symptoms, and multiple doses can be administered simultaneously or separately at intervals. Also, the pharmaceutical composition of the present invention may be administered to a subject/subject prior to the onset of the disease or after the onset of the disease.
　本発明の医薬組成物は、哺乳動物を被験者／対象として投与することができる。哺乳動物としては、例えば、マウス、ラット、ハムスター、モルモット、ウサギ、ネコ、イヌ、ヤギ、ブタ、ヒツジ、ウシ、ウマ、サル、ヒトなどが挙げられる。 The pharmaceutical composition of the present invention can be administered to mammals as subjects/subjects. Mammals include, for example, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, goats, pigs, sheep, cows, horses, monkeys, humans and the like.
５．コロナウイルス感染症の治療又は予防方法
　本発明においては、ACE2に結合するモノクローナル抗体若しくはその抗原結合断片又はこれを含む医薬組成物を被験者／対象に投与することにより、コロナウイルス感染症を治療又は予防することができる。すなわち、本発明は、治療上有効量の本発明の抗体若しくはその抗原結合断片又はそれを含む医薬組成物を被験者／対象に投与する工程を含む、コロナウイルス感染症の治療又は予防方法を提供する。本発明の抗体若しくはその抗原結合断片又はそれを含む医薬組成物の治療上の有効量は、被験者／対象の年齢、体重、健康状態、性別、症状、投与経路、投与回数、剤型などの要因に応じて変化する。当業者であれば、コロナウイルス感染症の治療又は予防に必要な治療上の有効量を容易に決定することができる。本発明において、「被験者」又は「対象」には、コロナウイルス感染症の治療又は予防を必要とする被験者／対象が含まれる。また、「被験者」又は「対象」として治療又は予防の対象となる哺乳動物は、上記の通りである。
　本発明のコロナウイルス感染症の治療又は予防方法において、「コロナウイルス感染症」、「治療」及び「予防」についての説明は上記の通りである。また、本発明の抗体若しくはその抗原結合断片又はそれを含む医薬組成物の剤形、投与経路、投与量、投与時期などの説明についても上記の通りである。 5. Method for treating or preventing coronavirus infection In the present invention, coronavirus infection is treated or prevented by administering a monoclonal antibody that binds to ACE2, an antigen-binding fragment thereof, or a pharmaceutical composition containing the same to a subject. can do. That is, the present invention provides a method of treating or preventing coronavirus infection, comprising the step of administering to a subject/subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention, or a pharmaceutical composition comprising the same. . The therapeutically effective amount of the antibody or antigen-binding fragment thereof of the present invention, or a pharmaceutical composition comprising the same, depends on factors such as subject/subject's age, body weight, health condition, sex, symptoms, route of administration, frequency of administration, and dosage form. Varies depending on A person of ordinary skill in the art can readily determine the therapeutically effective amount required to treat or prevent coronavirus infection. As used herein, "subject" or "subject" includes a subject/subject in need of treatment or prevention of coronavirus infection. In addition, mammals to be treated or prevented as "subjects" or "subjects" are as described above.
"Coronavirus infection", "treatment" and "prevention" in the method for treating or preventing coronavirus infection of the present invention are as described above. The dosage forms, administration routes, dosages, administration periods, etc. of the antibody or antigen-binding fragment thereof of the present invention, or the pharmaceutical composition containing the same are also described above.
６．コロナウイルスとACE2との結合阻害剤 
　本発明の抗体は、コロナウイルスとACE2との結合を中和（阻害）するため、当該結合によるコロナウイルス感染を阻害することができる。すなわち、本発明は、上記「３．本発明の抗体」に記載された抗体又はその抗原結合断片を有効成分として含む、コロナウイルスとACE2との結合阻害剤を提供する。
　本発明の阻害剤は、試験用試薬として、あるいは哺乳動物の治療に用いることができ、その投与形態、添加物、投与経路、投与対象、投与量などは、上記「４．医薬組成物」の記載に準じて適宜選択することができる。ただし、本発明の阻害剤は、本発明の抗体又はその抗原結合断片のみを含有するものであってもよい。 6. Binding inhibitor of coronavirus and ACE2
Since the antibody of the present invention neutralizes (inhibits) the binding between coronavirus and ACE2, it can inhibit coronavirus infection due to this binding. That is, the present invention provides an inhibitor of binding between coronavirus and ACE2, which comprises the antibody or antigen-binding fragment thereof described in "3. Antibodies of the present invention" as an active ingredient.
The inhibitor of the present invention can be used as a test reagent or for the treatment of mammals, and its dosage form, additives, administration route, administration subject, dosage, etc. are as described in "4. Pharmaceutical composition" above. It can be appropriately selected according to the description. However, the inhibitor of the present invention may contain only the antibody of the present invention or an antigen-binding fragment thereof.
７．抗体の使用
　本発明の抗体又はその抗原結合断片は、コロナウイルス感染症の治療又は予防方法において、あるいはコロナウイルス感染症を治療又は予防するための医薬の製造において、使用することができる。すなわち、本発明は、コロナウイルス感染症の治療又は予防方法において使用するための、本発明の抗体又はその抗原結合断片を提供する。また、本発明は、コロナウイルス感染症を治療又は予防するための医薬の製造において使用するための、本発明の抗体又はその抗原結合断片を提供する。さらに、本発明は、コロナウイルスとACE2との結合阻害剤の製造において使用するための、本発明の抗体又はその抗原結合断片を提供する。 7. Use of Antibodies Antibodies or antigen-binding fragments thereof of the present invention can be used in methods of treating or preventing coronavirus infections or in the manufacture of medicaments for treating or preventing coronavirus infections. Thus, the present invention provides antibodies or antigen-binding fragments thereof of the present invention for use in methods of treating or preventing coronavirus infection. The present invention also provides the antibody or antigen-binding fragment thereof of the present invention for use in manufacturing a medicament for treating or preventing coronavirus infection. Furthermore, the present invention provides the antibody or antigen-binding fragment thereof of the present invention for use in producing a binding inhibitor between coronavirus and ACE2.
８．併用療法
　本発明の医薬組成物は、他の治療剤の少なくとも１種と併用投与するために用いることができる。本発明に使用される他の治療剤としては、例えば、レムデシビル、デキサメタゾン、ファビピラビル、ナファモスタット、カモスタット、イベルメクチン、トリシズマブ、バリシチニブなどが挙げられる。 8. Combination Therapy The pharmaceutical compositions of the present invention can be used for co-administration with at least one other therapeutic agent. Other therapeutic agents for use in the present invention include, for example, remdesivir, dexamethasone, favipiravir, nafamostat, camostat, ivermectin, tricizumab, baricitinib, and the like.
　本発明の医薬組成物と他の治療剤の少なくとも1種とを併用投与することにより、それぞれ単独で用いるよりもさらに優れた効果が期待される。優れた効果には、治療効果を維持しつつ従来よりも副作用を軽減するという効果が含まれる。
　本発明において「併用」とは、本発明の医薬組成物と前記他の治療剤の少なくとも1種とを、同時又は別々に投与することを意味する。「同時」とは、一つの投与スケジュールにおいて同一のタイミングで投与されることを意味し、投与の時分が完全に同一である必要はない。「別々」とは、一つの投与スケジュールにおいて異なるタイミングで投与されることを意味する。
　本発明の併用療法に用いる医薬組成物及び他の治療剤の投与形態、投与経路、投与対象は特に限定されず、上記「４．医薬組成物」の記載に準じて適宜選択することができる。また、併用する薬剤の投与形態又は投与量が互いに異なっていてもよく、その併用する組み合わせにより、適宜調整することができる。
　本発明の医薬組成物を他の治療剤と併用する場合は、投与量を適宜減らすことも可能である。従って、本発明の医薬組成物と他の治療剤との組合せにおいては、
(i) 本発明の医薬組成物の有効量と、他の治療剤の有効量、
(ii) 本発明の医薬組成物の有効量と、他の治療剤の非有効量、
(iii) 本発明の医薬組成物の非有効量と、他の治療剤の有効量、及び
(iv) 本発明の医薬組成物の非有効量と、他の治療剤の非有効量
の組合せを採用することができる。
　医薬組成物及び他の治療剤の一方又は両者が非有効量の使用態様であっても、併用により薬理効果を発揮することができる場合は、そのような態様により併用投与することができる。 The combined administration of the pharmaceutical composition of the present invention and at least one other therapeutic agent is expected to provide even better effects than using each agent alone. The excellent effect includes the effect of reducing side effects more than before while maintaining the therapeutic effect.
In the present invention, "combination" means administering the pharmaceutical composition of the present invention and at least one of the other therapeutic agents simultaneously or separately. "Simultaneously" means administered at the same timing in one administration schedule, and the timing of administration need not be exactly the same. "Separately" means administered at different times in one administration schedule.
The dosage form, administration route, and administration target of the pharmaceutical composition and other therapeutic agents used in the combination therapy of the present invention are not particularly limited, and can be appropriately selected according to the description in "4. Pharmaceutical Compositions" above. In addition, the dosage forms or dosages of drugs to be used in combination may differ from each other, and can be appropriately adjusted depending on the combination to be used in combination.
When the pharmaceutical composition of the present invention is used in combination with other therapeutic agents, the dosage can be reduced accordingly. Accordingly, in combination of the pharmaceutical composition of the present invention with other therapeutic agents,
(i) an effective amount of a pharmaceutical composition of the invention and an effective amount of another therapeutic agent;
(ii) an effective amount of the pharmaceutical composition of the invention and an ineffective amount of the other therapeutic agent;
(iii) an ineffective amount of a pharmaceutical composition of the invention and an effective amount of another therapeutic agent; and
(iv) Combinations of ineffective amounts of the pharmaceutical compositions of the invention and ineffective amounts of other therapeutic agents can be employed.
Even if one or both of the pharmaceutical composition and the other therapeutic agent are used in an ineffective amount, if the combined use can exhibit a pharmacological effect, the combined administration can be performed in such a manner.
９．試薬、キット
　本発明の抗体又はその抗原結合断片は、試薬又はキットに含めることができる。すなわち、本発明は、本発明の抗体又はその抗原結合断片を含む試薬及びキットを提供する。本発明の試薬及びキットは、例えば、コロナウイルスに関する試験用試薬又はキットとして使用することができる。
　本発明の試薬及びキットにおいて、本発明の抗体又はその抗原結合断片は、例えば凍結などの方法により扱いやすくした後、そのまま若しくは賦形剤、増量剤、結合剤、滑沢剤等公知の薬学的に許容される担体、公知の添加剤（緩衝剤、等張化剤、キレート剤、着色剤、保存剤、香料、風味剤、甘味剤等が含まれる）などと混合してもよい。
　本発明のキットには、本発明の抗体又はその抗原結合断片のほか、緩衝液、酵素液、二次抗体、希釈用溶液、使用説明書などを含めることもできる。 9. Reagents, Kits Antibodies or antigen-binding fragments thereof of the present invention can be included in reagents or kits. Thus, the invention provides reagents and kits comprising the antibodies or antigen-binding fragments thereof of the invention. The reagents and kits of the present invention can be used, for example, as test reagents or kits for coronaviruses.
In the reagents and kits of the present invention, the antibody or antigen-binding fragment thereof of the present invention can be used, for example, after making it easy to handle by a method such as freezing, or it can be mixed with known pharmaceutical agents such as excipients, extenders, binders and lubricants. It may be mixed with a carrier acceptable for the above, known additives (including buffering agents, tonicity agents, chelating agents, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, etc.) and the like.
The kit of the present invention can also contain the antibody of the present invention or an antigen-binding fragment thereof, as well as a buffer solution, an enzyme solution, a secondary antibody, a diluent solution, instructions for use, and the like.
  以下、実施例により本発明を詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.
モノクローナル抗体の作製
（１）免疫原
　本実施例においては、免疫原として、(i) ヒトACE2を安定的に強制発現させたマウス由来細胞、又は(ii)ヒトACE2（配列番号２）のN末端から数えて19番目～85番目のアミノ酸残基からなるアミノ酸配列（配列番号１０６）を含む、ACE2の部分ポリペプチドを用いた。
　上記(i)のヒトACE2を安定的に強制発現させたマウス由来細胞（4T1細胞）は、以下の方法により作製した。具体的には、まず、ヒトACE2の全長をコードするDNAをレンチウイルスベクターに組込み、パッケージングプラスミド、エンベローププラスミドと共に293T細胞に遺伝子導入した。当該細胞の培養上清中に産生された組換えレンチウイルスを4T1細胞に感染させ、ヒトACE2を細胞膜上に安定的に発現する細胞を作製した。
　上記(ii)の部分ポリペプチドは、当該部分ポリペプチドをコードするDNA（配列番号１０５）を含むベクターを大腸菌に導入し、大腸菌で発現させ、その後精製することにより調製した。 Preparation of Monoclonal Antibodies (1) Immunogen In this example, (i) mouse-derived cells in which human ACE2 was stably overexpressed, or (ii) the N-terminus of human ACE2 (SEQ ID NO: 2) was used as the immunogen. A partial polypeptide of ACE2 containing an amino acid sequence (SEQ ID NO: 106) consisting of the 19th to 85th amino acid residues counted from was used.
Mouse-derived cells (4T1 cells) in which human ACE2 was stably expressed in (i) above were prepared by the following method. Specifically, first, a DNA encoding full-length human ACE2 was incorporated into a lentiviral vector, and the vector was introduced into 293T cells together with a packaging plasmid and an envelope plasmid. 4T1 cells were infected with the recombinant lentivirus produced in the culture supernatant of the cells to prepare cells stably expressing human ACE2 on the cell membrane.
The partial polypeptide of (ii) above was prepared by introducing a vector containing the DNA (SEQ ID NO: 105) encoding the partial polypeptide into E. coli, expressing it in E. coli, and then purifying it.
（２）免疫
　免疫動物としては、ICRマウス又はBALB/cマウス（いずれも三共ラボサービス株式会社）を用いた。
　免疫は、上記(i)のマウス由来細胞をBALB/cマウスの腹腔に投与し、上記(i)のマウス由来細胞と上記(ii)の部分ポリペプチド及びアジュバントとの混合物をICRマウスの腹腔に投与することにより行った。 (2) Immunization As animals for immunization, ICR mice or BALB/c mice (both from Sankyo Labo Service Co., Ltd.) were used.
For immunization, the mouse-derived cells (i) above are administered into the peritoneal cavity of BALB/c mice, and a mixture of the mouse-derived cells (i) above and the partial polypeptide and adjuvant (ii) above is administered into the peritoneal cavity of ICR mice. It was carried out by administering
（３）ハイブリドーマの作製
　上記(i)のマウス由来細胞（4T1細胞）を用いたcell ELISA、又は上記(ii)の部分ポリペプチドを用いたELISAにより、免疫応答をモニターした。
　免疫応答の増大が停滞したところ（抗体価が十分に上昇したところ）で、免疫したマウスから脾臓細胞を回収し、マウスミエローマ細胞P3X63Ag8.653（ATCC）と融合させてハイブリドーマ細胞株を作製した。 (3) Preparation of hybridoma Immune responses were monitored by cell ELISA using the mouse-derived cells (4T1 cells) of (i) above or ELISA using the partial polypeptide of (ii) above.
When the immune response stopped increasing (antibody titer increased sufficiently), spleen cells were collected from the immunized mice and fused with mouse myeloma cells P3X63Ag8.653 (ATCC) to produce a hybridoma cell line.
（４）スクリーニング
　ハイブリドーマの培養上清を用いて、上記(i)のマウス由来細胞（4T1細胞）を用いたcell ELISA又は上記(ii)の部分ポリペプチドを用いたELISAによるスクリーニングを行い、ヒトACE2に結合するモノクローナル抗体を産生するハイブリドーマ細胞株を選択した。
　これにより、ヒトACE2に結合するモノクローナル抗体を作製することができた。 (4) Screening Using the hybridoma culture supernatant, screening is performed by cell ELISA using the mouse-derived cells (4T1 cells) of (i) or ELISA using the partial polypeptide of (ii) above, and human ACE2 A hybridoma cell line producing a monoclonal antibody that binds to was selected.
As a result, we were able to produce a monoclonal antibody that binds to human ACE2.
抗ACE2モノクローナル抗体の配列決定
　実施例１で得られた抗ACE2モノクローナル抗体のうち、cell ELISA又は部分ポリペプチドを用いたELISAの反応性が高かったモノクローナル抗体を産生するハイブリドーマ細胞株をいくつか選択した。選択したハイブリドーマ細胞株について、抗体遺伝子をクローニングし、その遺伝子配列を解析することで抗体のアミノ酸配列を決定した。
　実施例１で得られた多数の抗ACE2モノクローナル抗体のうち、代表例の抗体（OMRad004G05、OMRad028D08、OMRad031H08、OMRad044E11、OMRad045G03、OMRad052B05）の可変領域の塩基配列及びアミノ酸配列を下記の表に示す。これらの抗体のうち、OMRad004G05及びOMRad028D08は、上記(i)のマウス由来細胞をBALB/cマウスに免疫して得られたハイブリドーマから産生されたものであり、OMRad031H08、OMRad044E11、OMRad045G03及びOMRad052B05は、上記(i)のマウス由来細胞と上記(ii)の部分ポリペプチドとの混合物をICRマウスに免疫して得られたハイブリドーマから産生されたものである。各抗体のCDRは、Kabatのナンバリングシステムに従って特定した。
　なお、実施例１で得られた多数の抗ACE2モノクローナル抗体のうち、上記6種類の抗体以外の抗体について下記の実施例に示す試験及び評価を行えば、これら6種類の抗体と同等又はそれ以上の作用若しくは効果を有する抗体を取得し得る。
  Sequencing of Anti-ACE2 Monoclonal Antibodies Among the anti-ACE2 monoclonal antibodies obtained in Example 1, some hybridoma cell lines producing monoclonal antibodies exhibiting high reactivity in cell ELISA or ELISA using partial polypeptides were selected. . For the selected hybridoma cell line, the antibody gene was cloned and the gene sequence was analyzed to determine the amino acid sequence of the antibody.
Among the numerous anti-ACE2 monoclonal antibodies obtained in Example 1, representative examples of the antibodies (OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, OMRad045G03, OMRad052B05) show the nucleotide sequences and amino acid sequences of the variable regions in the table below. Among these antibodies, OMRad004G05 and OMRad028D08 were produced from hybridomas obtained by immunizing BALB/c mice with the mouse-derived cells described in (i) above. It is produced from a hybridoma obtained by immunizing an ICR mouse with a mixture of the mouse-derived cells of (i) and the partial polypeptide of (ii). The CDRs of each antibody were identified according to the Kabat numbering system.
Of the many anti-ACE2 monoclonal antibodies obtained in Example 1, antibodies other than the above 6 types of antibodies were tested and evaluated in the following Examples, and were found to be equivalent to or higher than these 6 types of antibodies. An antibody having the action or effect of
Figure JPOXMLDOC01-appb-T000001
  
Figure JPOXMLDOC01-appb-T000002
  
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Figure JPOXMLDOC01-appb-T000003
  
Figure JPOXMLDOC01-appb-T000004
  
Figure JPOXMLDOC01-appb-T000003
  
Figure JPOXMLDOC01-appb-T000004
  
Figure JPOXMLDOC01-appb-T000005
  
Figure JPOXMLDOC01-appb-T000006
  
Figure JPOXMLDOC01-appb-T000005
  
Figure JPOXMLDOC01-appb-T000006
  
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Figure JPOXMLDOC01-appb-T000007
  
Figure JPOXMLDOC01-appb-T000008
  
Figure JPOXMLDOC01-appb-T000009
  
Figure JPOXMLDOC01-appb-T000010
  
Figure JPOXMLDOC01-appb-T000009
  
Figure JPOXMLDOC01-appb-T000010
  
Figure JPOXMLDOC01-appb-T000011
  
Figure JPOXMLDOC01-appb-T000012
 　
Figure JPOXMLDOC01-appb-T000011
  
Figure JPOXMLDOC01-appb-T000012
 　
本発明の抗ACE2モノクローナル抗体を用いた、スパイクタンパク質のRBDとACE2との結合阻害試験
　ヒトACE2を安定的に強制発現させたマウス4T1細胞を、コラーゲンでコートした96ウェルガラスボトムプレート（Greiner Bio-One）の各ウェルに、10000細胞/ウェルで播種した。プレートを、37℃、5% CO2で2日間インキュベーションした。培地を除いた後、実施例１で作製した抗ACE2モノクローナル抗体を4 μg/mLで含むRPMI1640培地を200 μL/ウェル添加し、氷上で1時間インキュベーションした。
　抗体含有培地を除き、RPMI1640培地で2回洗浄後、哺乳動物細胞（CHO細胞）で発現させたSARS-CoV-2スパイクタンパク質のRBDを0.5 μg/mLの濃度で含むRPMI 1640培地を、200 μL/ウェルで添加して、氷上で1時間インキュベーションした。このRBDは、Hisタグを有するため、蛍光標識された抗Hisタグ抗体を用いて検出することができる。 Inhibition test of spike protein RBD binding to ACE2 using the anti-ACE2 monoclonal antibody of the present invention. One) were seeded at 10000 cells/well in each well. Plates were incubated at 37° C., 5% CO 2 for 2 days. After removing the medium, 200 μL/well of RPMI1640 medium containing 4 μg/mL anti-ACE2 monoclonal antibody prepared in Example 1 was added and incubated on ice for 1 hour.
Remove the antibody-containing medium, wash twice with RPMI1640 medium, and add 200 μL of RPMI 1640 medium containing the RBD of SARS-CoV-2 spike protein expressed in mammalian cells (CHO cells) at a concentration of 0.5 μg/mL. /well and incubated on ice for 1 hour. Since this RBD has a His tag, it can be detected using a fluorescently labeled anti-His tag antibody.
　RPMI1640培地で2回洗浄後、RPMI 1640培地で1 μg/mLに希釈した蛍光標識された抗Hisタグ抗体(MBL社)を200 μL/ウェルで添加し、遮光、室温で30分間インキュベーションした。4% パラホルムアルデヒドによる固定、DAPIによる核染色を行った後、蛍光顕微鏡により、細胞膜上のACE2と結合したSARS-CoV-2スパイクタンパク質のRBDを検出した。 After washing twice with RPMI1640 medium, 200 μL/well of a fluorescence-labeled anti-His tag antibody (MBL) diluted to 1 μg/mL with RPMI 1640 medium was added and incubated for 30 minutes at room temperature in the dark. After fixation with 4% paraformaldehyde and nuclear staining with DAPI, the RBD of SARS-CoV-2 spike protein bound to ACE2 on the cell membrane was detected by fluorescence microscopy.
　その結果を図１に示す。抗ACE2モノクローナル抗体を添加しなかったサンプルでは、蛍光シグナルが検出された（図１中「Antidoby(-)」のパネル）。これは、当該サンプルでは、細胞膜上のACE2とRBDとが結合し、RBDが有するHisタグに、蛍光標識された抗Hisタグ抗体が結合するため、その蛍光シグナルが検出されたことを示す。 The results are shown in Figure 1. Fluorescent signals were detected in the sample to which no anti-ACE2 monoclonal antibody was added (“Antidoby(-)” panel in FIG. 1). This indicates that in the sample, ACE2 on the cell membrane binds to the RBD, and the fluorescently labeled anti-His tag antibody binds to the His tag of the RBD, so that the fluorescence signal was detected.
　これに対し、実施例１で作製した抗ACE2モノクローナル抗体を添加したサンプルでは、蛍光シグナルが検出されなかった（図１中、「OMRad052B05」、「OMRad004G05」、「OMRad028D08」、「OMRad031H08」、「OMRad044E11」及び「OMRad045G03」）。これは、当該サンプルでは、実施例１で作製した抗ACE2モノクローナル抗体が細胞膜上のACE2に結合し、これによりACE2とRBDとの結合を阻害（中和）したため、蛍光標識された抗Hisタグ抗体はRBDに結合することができず、当該抗Hisタグ抗体の蛍光シグナルが検出されなかったことを示す。 In contrast, no fluorescent signal was detected in the sample to which the anti-ACE2 monoclonal antibody prepared in Example 1 was added (in FIG. ” and “OMRad045G03”). This is because, in the sample, the anti-ACE2 monoclonal antibody prepared in Example 1 bound to ACE2 on the cell membrane, thereby inhibiting (neutralizing) the binding between ACE2 and RBD. was unable to bind to the RBD, indicating that the fluorescence signal of the anti-His tag antibody was not detected.
　すなわち、本実施例の結果により、本発明の抗体が、ACE2に結合し、ACE2とRBDとの結合を中和することができることが示された。 That is, the results of this example demonstrated that the antibody of the present invention can bind to ACE2 and neutralize the binding between ACE2 and RBD.
キメラ抗体の作製
　実施例１で得られた抗体のうち、OMRad052B05については、定常領域をヒトIgG4のアミノ酸配列に置き換えたキメラ抗体を作製した。本実施例で用いた重鎖定常領域のアミノ酸配列及び軽鎖定常領域のアミノ酸配列は、それぞれ、配列番号１０２及び配列番号１０４で示される。
　具体的には、以下のような方法でキメラ抗体を作製した。
　まず、実施例１で作製した抗ACE2抗体を産生するハイブリドーマから、抗ACE2抗体の重鎖可変領域遺伝子及び軽鎖可変領域遺伝子をクローニングした。次に、これら遺伝子（DNA）を、それぞれヒトIgG4の重鎖定常領域遺伝子（配列番号１０１）、あるいは軽鎖（κ鎖）定常領域遺伝子（配列番号１０３）の塩基配列に連結した。
　重鎖可変領域遺伝子を増幅するために、重鎖可変領域の5’末端塩基配列とコザック配列と制限酵素EcoRI配列を有するプライマー（配列番号１０７）、及び3’末端塩基配列の相補配列と制限酵素NheI配列を有するアンチセンスプライマー（配列番号１０８）を用いてPCRを実施した。また、軽鎖可変領域遺伝子を増幅するために、軽鎖可変領域遺伝子の5’末端塩基配列と制限酵素EcoRI配列を有するプライマー（配列番号１０９）、及び3’末端塩基配列の相補配列と制限酵素BsiWI配列を有するアンチセンスプライマー（配列番号１１０）を用いてPCRを実施した。
　上記PCRに用いたプライマーの塩基配列を下記の表に示す。 Preparation of Chimeric Antibody Among the antibodies obtained in Example 1, for OMRad052B05, a chimeric antibody was prepared by replacing the constant region with the amino acid sequence of human IgG4. The amino acid sequence of the heavy chain constant region and the amino acid sequence of the light chain constant region used in this example are shown in SEQ ID NO: 102 and SEQ ID NO: 104, respectively.
Specifically, a chimeric antibody was prepared by the following method.
First, from the anti-ACE2 antibody-producing hybridoma produced in Example 1, the heavy chain variable region gene and light chain variable region gene of the anti-ACE2 antibody were cloned. Next, these genes (DNA) were ligated to the base sequences of the heavy chain constant region gene (SEQ ID NO: 101) or the light chain (κ chain) constant region gene (SEQ ID NO: 103) of human IgG4, respectively.
A primer (SEQ ID NO: 107) having a heavy chain variable region 5′-terminal nucleotide sequence, a Kozak sequence, and a restriction enzyme EcoRI sequence, and a complementary sequence of the 3′-terminal nucleotide sequence and a restriction enzyme to amplify the heavy chain variable region gene PCR was performed using an antisense primer (SEQ ID NO: 108) with NheI sequence. In order to amplify the light chain variable region gene, a primer (SEQ ID NO: 109) having the 5'-terminal nucleotide sequence of the light chain variable region gene and the restriction enzyme EcoRI sequence, and the complementary sequence of the 3'-terminal nucleotide sequence and the restriction enzyme were used. PCR was performed using an antisense primer (SEQ ID NO: 110) with the BsiWI sequence.
The base sequences of the primers used in the above PCR are shown in the table below.
Figure JPOXMLDOC01-appb-T000013
  
Figure JPOXMLDOC01-appb-T000013
  
　得られた増幅産物を制限酵素EcoRI及びNheI、又はEcoRI及びBsiWIで処理し、ヒトIgG4重鎖定常領域発現プラスミド(pFUSEss-CHIg-hG4; InvivoGen社)のEcoRI-NheIサイト、あるいはヒトIgの軽鎖（κ鎖）定常領域発現プラスミド(pFUSE2ss-CLIg-hk; InvivoGen社)のEcoRI-BsiWIサイトに組み込んだ。pFUSEss-CHIg-hG4にはヒトIgG4の重鎖定常領域遺伝子が、またpFUSE-CLIg-hkにはヒトIgの軽鎖（κ鎖）定常領域遺伝子が搭載されていた。制限酵素NheI配列によりマウス重鎖可変領域とヒト重鎖定常領域が連結され、制限酵素BsiWI配列によりマウス軽鎖可変領域とヒト軽鎖定常領域が連結された。
　本実施例により、実施例１で作製した抗体（OMRad052B05）に基づくキメラ抗体を作製することができた。 The obtained amplification product was treated with restriction enzymes EcoRI and NheI or EcoRI and BsiWI, and the EcoRI-NheI site of the human IgG4 heavy chain constant region expression plasmid (pFUSEss-CHIg-hG4; InvivoGen) or the light chain of human Ig. (κ chain) was integrated into the EcoRI-BsiWI site of a constant region expression plasmid (pFUSE2ss-CLIg-hk; InvivoGen). pFUSEss-CHIg-hG4 was loaded with the heavy chain constant region gene of human IgG4, and pFUSE-CLIg-hk was loaded with the light chain (κ chain) constant region gene of human Ig. The mouse heavy chain variable region and the human heavy chain constant region were linked by the restriction enzyme NheI sequence, and the mouse light chain variable region and the human light chain constant region were linked by the restriction enzyme BsiWI sequence.
According to this example, a chimeric antibody based on the antibody (OMRad052B05) prepared in Example 1 could be prepared.
キメラ抗体の作製
　実施例４と同様の手順で、実施例１で得られた抗体のうち、OMRad004G05、OMRad028D08、OMRad031H08、OMRad044E11、OMRad045G03について、定常領域をヒトIgG4のアミノ酸配列に置き換えたキメラ抗体を作製した。ただし、可変領域を増幅するためのPCRには、下記の表に記載のプライマーを用いた。 Preparation of Chimeric Antibody Using the same procedure as in Example 4, among the antibodies obtained in Example 1, chimeric antibodies were prepared by replacing the constant region with the amino acid sequence of human IgG4 for OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, and OMRad045G03. did. However, the primers listed in the table below were used in PCR for amplifying the variable regions.
Figure JPOXMLDOC01-appb-T000014
  
　本実施例により、実施例１で得られた抗体のうち、OMRad004G05、OMRad028D08、OMRad031H08、OMRad044E11、OMRad045G03について、これらのマウス抗体に基づくキメラ抗体を作製することができた。
Figure JPOXMLDOC01-appb-T000014
According to this example, among the antibodies obtained in Example 1, for OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, and OMRad045G03, chimeric antibodies based on these mouse antibodies could be produced.
競合阻害試験
　本実施例では、実施例１で得られた抗体のうち、代表例として選択した6種類の抗ACE2マウス抗体（OMRad004G05、OMRad028D08、OMRad031H08、OMRad044E11、OMRad045G03、OMRad052B05）と、これらの抗体をベースに実施例４及び５で作製したキメラ抗体を用いて、ACE2との結合についてこれらの抗体の中で競合する抗体が得られるか試験した。本競合阻害試験は、公知の方法（Sindy Liao-Chan, et al. J Immunol Methods. 2014 Mar;405:1-14, PMID: 24380699）を参考にしてセルベースELISAにより実施した。 Competitive inhibition test In this example, six types of anti-ACE2 mouse antibodies (OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, OMRad045G03, OMRad052B05) selected as representative examples from among the antibodies obtained in Example 1, and these antibodies Using the chimeric antibodies produced in Examples 4 and 5 as a base, it was tested whether an antibody that competes among these antibodies for binding to ACE2 could be obtained. This competitive inhibition test was performed by cell-based ELISA with reference to a known method (Sindy Liao-Chan, et al. J Immunol Methods. 2014 Mar;405:1-14, PMID: 24380699).
　本実施例では、上記各抗ACE2マウス抗体を参照抗体（Reference Antibody）とし、上記各キメラ抗体を被験抗体（検出対象の抗体(Detection Antibody)）として、まず、ヒトACE2を発現する4T1細胞（4T1-hACE2細胞）に、ある一種類の参照抗体を反応させ、その後、全ての被験抗体を総当たりで反応させた。本試験の概略図を図２に示す。
　具体的には、4T1-hACE2細胞を1 x 104 cells/wellで96-wellの細胞培養プレートの各ウェルに播種し、2日間培養した。ブロッキングバッファー(3% BSA, 0.2% ウシ血清を含むPBS(-))を用いて、各抗ヒトACE2マウスモノクローナル抗体(参照抗体)を、30 μg/mLで含む溶液を調製した。96-wellプレートの培養液を除去し、50 μL/wellで希釈した参照抗体溶液を添加し、4oCで1時間静置した。ウェルを250 μLの洗浄バッファー(0.1% BSAを含むPBS(-))で3回洗浄後、2.5% ホルマリンを50 μL/wellで添加し、室温で10分間静置することで細胞を固定した。250 μLの洗浄バッファーで3回洗浄後、ブロッキングバッファーで3 μg/mLになるように希釈した各抗ヒトACE2マウス/ヒトキメラモノクローナル抗体(被験抗体（検出対象の抗体）)を、50 μL/wellで各ウェルに添加し、室温で1時間静置した。250 μLの洗浄バッファーで3回洗浄後、3%の過酸化水素を含むPBS(-)を50 μL/wellで添加し、室温で5分間静置することで、内在性のペルオキシダーゼを不活化した。250 μLの洗浄バッファーで3回洗浄後、ブロッキングバッファーで10,000倍に希釈した、HRP標識抗ヒトIgG-Fc抗体(Bethyl, A80-304P)を50 μL/wellで添加し、室温で1時間静置した。250 μLのブロッキングバッファーで3回洗浄後、OPD基質溶液を100 μL/wellで添加し、室温で10分間程度反応させた。1.5 M硫酸溶液を100 μL添加して攪拌することで反応を停止し、吸光光度計を用いて490 nmの吸光度を測定した。得られた吸光度を、コントロールとして用意した各キメラ抗体のみを使用したウェルの値を100として補正した。 In this example, each anti-ACE2 mouse antibody was used as a reference antibody (Reference Antibody), and each chimeric antibody was used as a test antibody (antibody to be detected (Detection Antibody)). First, 4T1 cells (4T1 -hACE2 cells) were reacted with one type of reference antibody, and then reacted with all the test antibodies in round robin. A schematic diagram of this test is shown in FIG.
Specifically, 4T1-hACE2 cells were seeded into each well of a 96-well cell culture plate at 1 x 104 cells/well and cultured for 2 days. A solution containing 30 μg/mL of each anti-human ACE2 mouse monoclonal antibody (reference antibody) was prepared using a blocking buffer (PBS(-) containing 3% BSA and 0.2% bovine serum). The culture medium was removed from the 96-well plate, a reference antibody solution diluted at 50 μL/well was added, and the plate was allowed to stand at 4 ° C. for 1 hour. After the wells were washed three times with 250 μL of washing buffer (PBS(-) containing 0.1% BSA), 2.5% formalin was added at 50 μL/well and allowed to stand at room temperature for 10 minutes to fix the cells. After washing 3 times with 250 μL of washing buffer, each anti-human ACE2 mouse/human chimeric monoclonal antibody (test antibody (antibody to be detected)) diluted to 3 μg/mL with blocking buffer was added to 50 μL/well. was added to each well and allowed to stand at room temperature for 1 hour. After washing three times with 250 µL of washing buffer, 50 µL/well of PBS(-) containing 3% hydrogen peroxide was added and allowed to stand at room temperature for 5 minutes to inactivate endogenous peroxidase. . After washing three times with 250 μL of washing buffer, add 50 μL/well of HRP-labeled anti-human IgG-Fc antibody (Bethyl, A80-304P) diluted 10,000-fold with blocking buffer and leave at room temperature for 1 hour. did. After washing three times with 250 μL of blocking buffer, OPD substrate solution was added at 100 μL/well and allowed to react at room temperature for about 10 minutes. The reaction was terminated by adding 100 μL of 1.5 M sulfuric acid solution and stirring, and the absorbance at 490 nm was measured using an absorptiometer. The obtained absorbance was corrected by setting the value of wells using only each chimeric antibody prepared as a control to 100.
　その結果を図３に示す。
　OMRad004G05、OMRad028D08、OMRad031H08、OMRad045G03の4種類のキメラ抗体が、互いに、同一クローンに基づく参照抗体（抗ACE2マウス抗体）と被験抗体（キメラ抗体）との競合を示す相対値（例えば、参照抗体及び被験抗体がOMRad004G05である場合は22.8）と同程度の阻害効果を示した(Group 1)。また、OMRad044E11、及びOMRad052B05の2種類が互いに、同一クローンに基づく参照抗体と被験抗体との競合を示す相対値と同程度の阻害効果を示した(Group 2)。Group 1の抗体とGroup 2の抗体は、わずかに競合した。 The results are shown in FIG.
Relative values (e.g., reference antibody and test When the antibody was OMRad004G05, it showed an inhibitory effect similar to 22.8) (Group 1). In addition, two types of OMRad044E11 and OMRad052B05 each showed an inhibitory effect comparable to the relative value showing competition between the reference antibody and the test antibody based on the same clone (Group 2). Group 1 and Group 2 antibodies competed slightly.
　これにより、Group 1及びGroup 2に属する抗体は、それぞれ同じGroup内に属する他の抗体と互いに競合することが示された。
　本実施例では、上記抗ACE2マウス抗体を参照抗体とし、上記各キメラ抗体を被験抗体としたが、上記各キメラ抗体を参照抗体とし、これと競合する抗ACE2マウス抗体をスクリーニングすることも可能である。
　以上より、本実施例により、ACEとの結合について、実施例１で得られた抗体及びこれに基づいて作製されたキメラ抗体と競合する抗体が得られることが示された。 This indicated that antibodies belonging to Group 1 and Group 2 compete with each other with other antibodies belonging to the same Group.
In this example, the above anti-ACE2 mouse antibody was used as a reference antibody, and each of the above chimeric antibodies was used as a test antibody. be.
From the above, it was shown that this example yields an antibody that competes with the antibody obtained in Example 1 and the chimeric antibody prepared based thereon for binding to ACE.
　以下の実施例では、各抗体の機能や活性を評価するとともに、Group 1及びGroup 2に属する抗体が、それぞれ同じGroup内に属する他の抗体と同等の活性を有するかについても評価した。 In the examples below, in addition to evaluating the functions and activities of each antibody, we also evaluated whether antibodies belonging to Group 1 and Group 2 have activities equivalent to those of other antibodies belonging to the same Group.
エピトープの特定試験
１．マウス/ヒト融合型ACE2発現細胞の作製
　過去の報告には、SARS-CoV-2スパイクタンパク質はヒトACE2のN末端側と相互作用するとの報告や、SARS-CoV-2はマウスACE2を認識できないという報告がある。
　そこで、実施例１で得られた抗体のうち、6種類の抗ACE2マウス抗体（OMRad004G05、OMRad028D08、OMRad031H08、OMRad044E11、OMRad045G03、OMRad052B05）が、ヒトACE2のN末端側領域に結合するかどうかを調べた。具体的には、ヒトACE2のN末端から1-120残基をマウスACE2に置き換えた融合ACE2遺伝子（ポリヌクレオチド）を作製し、これを発現するマウス/ヒト融合型ACE2発現細胞を以下の手順で作製した。
　まず、マウスACE2をコードする塩基配列の5'末端から1～360番目の塩基を含むポリヌクレオチドとヒトACE2をコードする塩基配列の5'末端から361～2418番目の塩基を含むポリヌクレオチドとの融合ACE2遺伝子コンストラクトを、NEBuilder Hi-Fi DNA Assembly (New England Biolabs社)を用いて作製し、このコンストラクトをレンチウイルスベクターに組込んだ。このレンチウイルスコンストラクトを、パッケージングプラスミド、エンベローププラスミドと共に293T細胞に遺伝子導入し、培養上清中に産生された組換えレンチウイルスを、マウス4T1細胞に感染させた。
　このようにして、ヒトマウス/ヒト融合ACE2を細胞膜上に発現する4T1細胞（マウス/ヒト融合ACE2発現4T1細胞）を得た。マウス/ヒト融合ACE2は、N末端から1-120残基がマウスACE2のアミノ酸配列であり、121残基以降がヒトACE2のアミノ酸配列である。 Epitope identification test 1. Generation of mouse/human fusion-type ACE2-expressing cells Previous reports have suggested that the SARS-CoV-2 spike protein interacts with the N-terminal side of human ACE2, and that SARS-CoV-2 cannot recognize mouse ACE2. I have a report.
Therefore, among the antibodies obtained in Example 1, six types of anti-ACE2 mouse antibodies (OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, OMRad045G03, and OMRad052B05) were examined to determine whether they bind to the N-terminal region of human ACE2. . Specifically, we prepared a fusion ACE2 gene (polynucleotide) in which residues 1 to 120 from the N-terminus of human ACE2 were replaced with mouse ACE2, and generated mouse/human fusion-type ACE2-expressing cells expressing this by the following procedure. made.
First, fusion of a polynucleotide containing bases 1 to 360 from the 5' end of the nucleotide sequence encoding mouse ACE2 with a polynucleotide containing bases 361 to 2418 from the 5' end of the nucleotide sequence encoding human ACE2. An ACE2 gene construct was generated using NEBuilder Hi-Fi DNA Assembly (New England Biolabs) and incorporated into a lentiviral vector. This lentiviral construct was transfected into 293T cells together with the packaging plasmid and envelope plasmid, and mouse 4T1 cells were infected with the recombinant lentivirus produced in the culture supernatant.
Thus, 4T1 cells expressing human mouse/human fusion ACE2 on the cell membrane (mouse/human fusion ACE2-expressing 4T1 cells) were obtained. Mouse/human fusion ACE2 has the amino acid sequence of mouse ACE2 in residues 1 to 120 from the N-terminus, and the amino acid sequence of human ACE2 in residues 121 and beyond.
２．免疫細胞染色
　4T1細胞、ヒトACE2発現4T1細胞、マウスACE2発現4T1細胞及びマウス/ヒト融合ACE2発現4T1細胞を、蛍光観察用のガラスボトム96-wellプレートに3.2 x 103 cells/wellでそれぞれ播種し、2日間培養した。プレートを氷上に置いて培地を除いた後、20 mg/mLの抗ACE2マウスモノクローナル抗体を含むRPMI1640培地を200 mL添加して氷上で1時間静置した。150 mLの氷冷したRPMI1640培地で洗浄後、Alexa Fluor 488 標識抗マウスIgG抗体(Thermo Fisher Scientific, A11001)を添加したRPMI1640培地を添加し、氷上で30分間反応させた。洗浄後、100 mLの4% パラホルムアルデヒドを添加し、氷上で10分間静置した。PBSで十分に洗浄した後、蛍光顕微鏡で観察した。 2. Immunocytostaining 4T1 cells, human ACE2-expressing 4T1 cells, mouse ACE2-expressing 4T1 cells, and mouse/human fusion ACE2-expressing 4T1 cells were plated at 3.2 x 10 3 cells/well in glass-bottom 96-well plates for fluorescence observation. , cultured for 2 days. After the plate was placed on ice to remove the medium, 200 mL of RPMI1640 medium containing 20 mg/mL anti-ACE2 mouse monoclonal antibody was added, and the plate was allowed to stand on ice for 1 hour. After washing with 150 mL of ice-cold RPMI1640 medium, RPMI1640 medium supplemented with Alexa Fluor 488-labeled anti-mouse IgG antibody (Thermo Fisher Scientific, A11001) was added and allowed to react on ice for 30 minutes. After washing, 100 mL of 4% paraformaldehyde was added and allowed to stand on ice for 10 minutes. After thoroughly washing with PBS, they were observed under a fluorescence microscope.
３．結果
　その結果を図４に示す。
　6種類の抗ACE2モノクローナル抗体は全て、ヒトACE2に結合した。
　OMRad031H08とOMRad045G03の2種類についてはマウスACE2と結合したが、OMRad004G05、OMRad028D08、OMRad044E11、OMRad052B05はマウスACE2と結合しなかった。
　OMRad004G05、OMRad028D08、OMRad031H08、OMRad045G03はマウス/ヒト融合ACE2に結合したが、OMRad044E11、OMRad052B05はマウス/ヒト融合ACE2に結合しなかった(図４)。
　なお、ACE2を発現していない4T1細胞にはいずれの抗体も結合しなかった。
　この結果を下記の表にまとめた（表中、「Y」は結合したことを示し、「N」は結合しなかったことを示す。「全長」は細胞外領域の全長を示し、「aa」はアミノ酸位置を示す）。 3. Results The results are shown in FIG.
All six anti-ACE2 monoclonal antibodies bound to human ACE2.
Two of them, OMRad031H08 and OMRad045G03, bound to mouse ACE2, but OMRad004G05, OMRad028D08, OMRad044E11, and OMRad052B05 did not bind to mouse ACE2.
OMRad004G05, OMRad028D08, OMRad031H08, OMRad045G03 bound to mouse/human fusion ACE2, but OMRad044E11, OMRad052B05 did not bind to mouse/human fusion ACE2 (Fig. 4).
None of the antibodies bound to 4T1 cells that do not express ACE2.
The results are summarized in the table below (where "Y" indicates binding, "N" indicates no binding, "full length" indicates the full length of the extracellular region, and "aa" indicates the amino acid position).
Figure JPOXMLDOC01-appb-T000015
  
Figure JPOXMLDOC01-appb-T000015
  
　上記表に示すとおり、OMRad044E11及びOMRad052B05の2種類は、ヒトACE2に結合し、マウスACE2の1-120番目の残基及びヒトACE2の121-740番目の残基に結合しなかった。このことから、これらの抗体は、ヒトACE2のうち、主にN末端から1-120番目の残基を含む領域に結合することが示された。 As shown in the table above, OMRad044E11 and OMRad052B05 bound to human ACE2, but did not bind to residues 1-120 of mouse ACE2 and residues 121-740 of human ACE2. From this, it was shown that these antibodies mainly bind to the region containing residues 1 to 120 from the N-terminus of human ACE2.
　また、OMRad004G05及びOMRad028D08については、ヒトACE2全長及びヒトACE2の121-740番目の残基に結合し、マウスACE2に結合しなかった。このことから、これらの抗体は、ヒトACE2のうち、主に121番目以降の細胞外領域(740番目のセリンまで)であって、ヒトACE2とマウスACE2との違いを識別できる位置又は領域（例えば、ヒトACE2とマウスACE2との間で相同性が低い領域、両者で異なるアミノ酸を有する位置など）に結合することが示唆された。 In addition, OMRad004G05 and OMRad028D08 bound to full-length human ACE2 and residues 121-740 of human ACE2, but did not bind to mouse ACE2. From this, these antibodies are mainly the extracellular region after the 121st (up to the 740th serine) of human ACE2, and the position or region that can distinguish the difference between human ACE2 and mouse ACE2 (e.g. , regions with low homology between human ACE2 and mouse ACE2, positions with different amino acids between the two, etc.).
　他方、OMRad031H08及びOMRad045G03は、ヒトACE2、マウスACE2、及びヒト/マウス融合ACE2のいずれにも反応したことから、本試験ではこれらの抗体が結合する領域を絞り込むことはできなかった。しかし、実施例６において、OMRad031H08及びOMRad045G03は、同じGroup 1に属する OMRad004G05及びOMRad028D08と競合したことと本試験の結果とを考え合わせると、これらの抗体は、ヒトACE2のうち、主に121-740番目のアミノ酸残基を含む領域であって、ヒトACE2とマウスACE2との違いを識別しない位置又は領域（例えば、ヒトACE2とマウスACE2との間で相同性が高い領域、両者で同一のアミノ酸を有する位置など）に結合することが示唆された。 On the other hand, OMRad031H08 and OMRad045G03 reacted to all of human ACE2, mouse ACE2, and human/mouse fusion ACE2, so it was not possible to narrow down the region to which these antibodies bind in this test. However, in Example 6, OMRad031H08 and OMRad045G03 competed with OMRad004G05 and OMRad028D08 belonging to the same Group 1, and considering the results of this test, these antibodies are mainly 121-740 in human ACE2. A region containing the second amino acid residue that does not discriminate between human ACE2 and mouse ACE2 (e.g., a region with high homology between human ACE2 and mouse ACE2, the same amino acid in both It was suggested that it binds to a position having
　OMRad004G05とOMRad028D08、OMRad031H08とOMRad045G03、OMRad044E11とOMRad052B05がACE2への反応性について同様の結果を示したことは、これらの抗体のCDRのアミノ酸配列が互いに類似することとも合致する。 The fact that OMRad004G05 and OMRad028D08, OMRad031H08 and OMRad045G03, and OMRad044E11 and OMRad052B05 showed similar reactivity to ACE2 is consistent with the fact that the CDR amino acid sequences of these antibodies are similar to each other.
　また、実施例６で示されたGroup 1の抗体（OMRad004G05、OMRad028D08、OMRad031H08、OMRad045G03）は、同じGroup内に属する他の抗体と同様に、主に121-740番目のアミノ酸残基を含む領域に結合し、Group 2の抗体（OMRad044E11、OMRad052B05）は、同じGroup内のもう一方の抗体と同様に、主にN末端から1-120番目のアミノ酸残基を含む領域に結合することが示された。 In addition, the Group 1 antibodies (OMRad004G05, OMRad028D08, OMRad031H08, OMRad045G03) shown in Example 6 are mainly in the region containing amino acid residues 121-740, similar to other antibodies belonging to the same Group. Group 2 antibodies (OMRad044E11, OMRad052B05) were shown to bind mainly to a region containing amino acid residues 1-120 from the N-terminus, similar to the other antibodies in the same group. .
結合親和性評価試験
１．方法
　実施例１で得られた抗体のうち、6種類の抗ACE2マウス抗体（OMRad004G05、OMRad028D08、OMRad031H08、OMRad044E11、OMRad045G03、OMRad052B05）のヒトACE2に対する親和性をセルベースELISA(cell ELISA)で評価した。
　本実施例では、上記6種類のマウス抗体と、これらの抗体をベースに実施例４及び５で作製したキメラ抗体を用いて、セルベースELISAを行い、Kd値を求めた。具体的には、次の手順にしたがって試験を行った。
　まず、4T1細胞、及びヒトACE2を安定的に発現する4T1細胞(以下、「4T1-hACE2」という)をそれぞれ、1 x 104 cells/wellで96-wellの細胞培養プレートの各ウェルに播種し、2日間培養した。ブロッキングバッファー(3% BSA, 0.2% ウシ血清を含むPBS(-))を用いて、上記6種類のマウス抗体及びキメラ抗体を100 μg/mLで含む溶液を調製し、ブロッキングバッファーを用いて2倍の希釈系列を作製した。96-wellプレートの培養液を除去し、50 μL/wellで希釈した抗体溶液を添加し、4℃で1時間静置した。ウェルを250 μLの洗浄バッファー(0.05% Tween20を含むPBS(-))で3回洗浄後、10% ホルマリンを50 μL/wellで添加し、室温で10分間静置することで細胞を固定した。洗浄バッファーで2回洗浄後、3%の過酸化水素を含むPBS(-)を50 μL/wellで添加し、室温で5分間静置することで、内在性のペルオキシダーゼを不活化した。250 μLの洗浄バッファーで3回洗浄後、ブロッキングバッファーで10,000倍に希釈した、HRP標識抗マウスKappa鎖抗体(Bethyl, A90-119P)、あるいはHPR標識抗ヒトIgG-Fc抗体(Bethyl, A80-304P)を50 μL/wellで添加し、室温で1時間静置した。250 μLのブロッキングバッファーで3回洗浄後、OPD基質溶液を100 μL/wellで添加し、室温で10分間程度反応させた。1.5 M硫酸溶液を100 μL添加して攪拌することで反応を停止し、吸光光度計を用いて490 nmの吸光度を測定した。得られたデータをGraphPad Prismにより解析し、Kd値を求めた。 Binding affinity evaluation test 1. Among the antibodies obtained in Method Example 1, six types of anti-ACE2 mouse antibodies (OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, OMRad045G03, OMRad052B05) were evaluated for their affinity to human ACE2 by cell-based ELISA.
In this example, cell-based ELISA was performed using the six types of mouse antibodies described above and the chimeric antibodies prepared in Examples 4 and 5 based on these antibodies to determine the Kd value. Specifically, the test was conducted according to the following procedures.
First, 4T1 cells and 4T1 cells stably expressing human ACE2 (hereinafter referred to as "4T1-hACE2") were seeded in each well of a 96-well cell culture plate at 1 x 104 cells/well. , cultured for 2 days. Using blocking buffer (PBS(-) containing 3% BSA and 0.2% bovine serum), prepare a solution containing 100 μg/mL of the above 6 types of mouse antibodies and chimeric antibodies, and double with blocking buffer. A dilution series of was prepared. The culture medium was removed from the 96-well plate, an antibody solution diluted at 50 µL/well was added, and the plate was allowed to stand at 4°C for 1 hour. After the wells were washed three times with 250 μL of washing buffer (PBS(-) containing 0.05% Tween20), 10% formalin was added at 50 μL/well and allowed to stand at room temperature for 10 minutes to fix the cells. After washing twice with a washing buffer, PBS(-) containing 3% hydrogen peroxide was added at 50 μL/well and allowed to stand at room temperature for 5 minutes to inactivate endogenous peroxidase. After washing three times with 250 μL of washing buffer, HRP-labeled anti-mouse Kappa chain antibody (Bethyl, A90-119P) or HPR-labeled anti-human IgG-Fc antibody (Bethyl, A80-304P) diluted 10,000-fold with blocking buffer ) was added at 50 μL/well and allowed to stand at room temperature for 1 hour. After washing three times with 250 μL of blocking buffer, OPD substrate solution was added at 100 μL/well and allowed to react at room temperature for about 10 minutes. The reaction was terminated by adding 100 μL of 1.5 M sulfuric acid solution and stirring, and the absorbance at 490 nm was measured using an absorptiometer. The obtained data were analyzed by GraphPad Prism to determine the Kd value.
２．結果
　その結果、セルベースELISAにおいて、上記6種類のマウス抗体及びキメラ抗体はいずれも、ヒトACE2を発現する4T1-hACE2細胞に対し、濃度依存的な吸光度の増大を示した(図５Ａ及び図５Ｂ)。他方、コントロールとして、ヒトACE2を発現していない4T1細胞に各キメラ抗体を反応させたが、濃度依存的な吸光度の増大は観察されなかった(図５Ｃ)。
　この結果から、上記6種類のマウス抗体及びキメラ抗体はいずれも、細胞上に発現したネイティブなヒトACE2に対して、高い特異性で結合することが示された。
　また、この試験におけるKd値は、下記の表に示すとおり、マウス抗体で1.97 nMから5.81 nM、キメラ抗体で0.86 nMから2.62 nMであった。
　なお、用いている二次抗体が異なるため、マウス抗体のKd値とキメラ抗体のKd値とを直接比較することはできない。 2. Results As a result, in cell-based ELISA, all of the above six mouse antibodies and chimeric antibodies showed a concentration-dependent increase in absorbance against 4T1-hACE2 cells expressing human ACE2 (Figs. 5A and 5B). ). On the other hand, as a control, each chimeric antibody was reacted with 4T1 cells not expressing human ACE2, but no concentration-dependent increase in absorbance was observed (Fig. 5C).
These results indicated that all of the above six types of mouse antibodies and chimeric antibodies bind with high specificity to native human ACE2 expressed on cells.
Also, the Kd values in this test ranged from 1.97 nM to 5.81 nM for the mouse antibody and from 0.86 nM to 2.62 nM for the chimeric antibody, as shown in the table below.
Since the secondary antibodies used are different, the Kd value of the mouse antibody and the Kd value of the chimeric antibody cannot be directly compared.
Figure JPOXMLDOC01-appb-T000016
  
 
Figure JPOXMLDOC01-appb-T000016
  
 
変異型スパイクタンパク質RBDとACE2との結合阻害試験
　本実施例では、上記６種類のキメラ抗体を用いて、これらの抗体が変異型RBDとヒトACE2発現細胞との結合をどの程度阻害するかを解析した。
１．変異型スパイクタンパク質RBDの作製
　本実施例では、変異型スパイクタンパク質RBDとして、野生型（WT(武漢型)）、alpha型、beta型、delta型、epsilon型、eta型、theta型、kappa型のSARS-CoV2のRBDと同じアミノ酸配列を有するRBDを作製した。具体的には、SARS-CoV-2のスパイクタンパク質のcDNAのうち、RBD（N末端から数えて319番目のリジンから541番目のフェニルアラニン）に該当する領域の3'末端に、6 x ヒスチジンタグの塩基配列を付加したコンストラクトを作製し、当該コンストラクトを哺乳類細胞用の発現ベクターに組込んだ。得られたプラスミドに、下記表で示す各変異をOE-PCR法により導入した。 Binding inhibition test between mutant spike protein RBD and ACE2 In this example, the six chimeric antibodies described above were used to analyze the extent to which these antibodies inhibited binding between mutant RBD and human ACE2-expressing cells. did.
1. Preparation of mutant spike protein RBD In this example, wild type (WT (Wuhan type)), alpha type, beta type, delta type, epsilon type, eta type, theta type, and kappa type are used as mutant spike protein RBDs. An RBD with the same amino acid sequence as that of SARS-CoV2 was generated. Specifically, in the SARS-CoV-2 spike protein cDNA, a 6 x histidine tag was added to the 3' end of the region corresponding to the RBD (319th lysine to 541st phenylalanine counting from the N-terminus). A construct to which a base sequence was added was prepared, and the construct was incorporated into an expression vector for mammalian cells. Mutations shown in the table below were introduced into the resulting plasmid by OE-PCR method.
Figure JPOXMLDOC01-appb-T000017
  
Figure JPOXMLDOC01-appb-T000017
  
　得られたプラスミドDNAをCHO細胞に導入し、その培養上清から変異型の組換えRBDをNi-NTAアガロースを用いて精製した。 The resulting plasmid DNA was introduced into CHO cells, and the mutant recombinant RBD was purified from the culture supernatant using Ni-NTA agarose.
２．野生型及び変異型スパイクタンパク質RBDとヒトACE2との結合阻害試験
　4T1-hACE2細胞の細胞懸濁液を、細胞培養用96ウェルプレートの各ウェルに1ウェル当たり10,000細胞になるように100 μL播種し、37℃、5% CO2存在下で2日間培養した。終濃度5 nMになるように野生型及び各変異型RBDをBlocking Buffer (3% BSA、0.2% ウシ血清を含むPBS)に添加し、野生型及び各変異型RBDを含むBlocking Bufferで抗ヒトACE2キメラ抗体を希釈した。
　96ウェルプレートの培地をデカンテーションで除去し、調製した抗ヒトACE2キメラ抗体溶液を各ウェルに30 μL添加し、4℃で１時間静置した。96ウェルプレートの抗ヒトACE2キメラ抗体溶液をデカンテーションで除去し、各ウェルを250 μLのPBS(-)で2回洗浄した。
　Blocking Bufferで1 μg/mLになるようにマウス抗SARS-CoV-2 Spike抗体 (クローンS28)を希釈したS28抗体溶液を各ウェルに30 μL添加し、4℃で1時間静置した。S28抗体溶液をデカンテーションで除去し、各ウェルを250 μLのPBS(-)で2回洗浄した。96ウェルプレートの各ウェルに10% ホルマリン溶液を50 μL添加し、室温で10分間静置し細胞を固定化した。10% ホルマリン溶液をデカンテーションで除去し、各ウェルを250 μLの洗浄Buffer (0.05% Tween20を含むPBS(-))で2回洗浄した。96ウェルプレートの各ウェルに3% 過酸化水素溶液を50 μL添加し、室温で5分間静置し内在性ペルオキシダーゼを失活させた。3% 過酸化水素溶液をデカンテーションで除去し、各ウェルを250 μLの洗浄Buffer (0.05% Tween20を含むPBS(-))で3回洗浄した。96ウェルプレートの各ウェルにBlocking Bufferで10,000倍希釈した二次抗体 (Mouse IgG-Fc Fragment cross-adsorbed Antibody HRP Conjugated, Bethyl, A90-231P)溶液を50 μL添加し、室温暗所で1時間静置した。二次抗体溶液をデカンテーションで除去し、各ウェルを250 μLの洗浄Buffer (0.05% Tween20を含むPBS(-))で3回洗浄した。96ウェルプレートの各ウェルにOPD溶液を100 μL添加し、発色を見ながら室温で静置し、各ウェルに1.5 M 硫酸溶液を100 μL添加し発色反応を停止させた。吸光度計を用い490 nmの吸光度を測定し、細胞膜上のヒトACE2と結合したRBDを検出した。
　野生型及び各変異型RBDとヒトACE2との結合に対する抗ヒトACE2キメラ抗体による阻害率は、各RBDのみを添加したウェルを陽性対照とし、抗ヒトACE2キメラ抗体のみを添加したウェルを陰性対照として用いて算出した。 2. Binding inhibition test between wild-type and mutant spike protein RBD and human ACE2 100 μL of cell suspension of 4T1-hACE2 cells was seeded into each well of a 96-well plate for cell culture at 10,000 cells per well. , and cultured at 37°C in the presence of 5% CO 2 for 2 days. Wild-type and each mutant RBD were added to Blocking Buffer (PBS containing 3% BSA, 0.2% bovine serum) to a final concentration of 5 nM, and anti-human ACE2 was added with Blocking Buffer containing wild-type and each mutant RBD. Chimeric antibodies were diluted.
The medium of the 96-well plate was removed by decantation, 30 µL of the prepared anti-human ACE2 chimeric antibody solution was added to each well, and the plate was allowed to stand at 4°C for 1 hour. The anti-human ACE2 chimeric antibody solution in the 96-well plate was removed by decantation, and each well was washed twice with 250 µL of PBS(-).
30 μL of S28 antibody solution, in which mouse anti-SARS-CoV-2 Spike antibody (clone S28) was diluted with blocking buffer to 1 μg/mL, was added to each well and allowed to stand at 4° C. for 1 hour. The S28 antibody solution was removed by decantation, and each well was washed twice with 250 µL of PBS(-). 50 μL of a 10% formalin solution was added to each well of a 96-well plate, and the plate was allowed to stand at room temperature for 10 minutes to immobilize the cells. The 10% formalin solution was removed by decantation, and each well was washed twice with 250 µL of washing buffer (PBS(-) containing 0.05% Tween20). 50 µL of a 3% hydrogen peroxide solution was added to each well of a 96-well plate and allowed to stand at room temperature for 5 minutes to deactivate endogenous peroxidase. The 3% hydrogen peroxide solution was removed by decantation, and each well was washed three times with 250 µL of washing buffer (PBS(-) containing 0.05% Tween20). Add 50 μL of secondary antibody (Mouse IgG-Fc Fragment cross-adsorbed Antibody HRP Conjugated, Bethyl, A90-231P) diluted 10,000 times with Blocking Buffer to each well of a 96-well plate, and leave to stand at room temperature in the dark for 1 hour. placed. The secondary antibody solution was removed by decantation, and each well was washed three times with 250 µL of washing buffer (PBS(-) containing 0.05% Tween20). 100 µL of the OPD solution was added to each well of the 96-well plate, and the plate was allowed to stand at room temperature while observing the color development. Absorbance at 490 nm was measured using an absorptiometer to detect RBD bound to human ACE2 on the cell membrane.
The rate of inhibition by the anti-human ACE2 chimeric antibody against the binding of wild-type and each mutant RBD to human ACE2 was determined by using the wells to which only each RBD was added as a positive control and the wells to which only the anti-human ACE2 chimeric antibody was added as a negative control. calculated using
３．結果
　その結果、６種類のキメラ抗体はいずれも、濃度依存的に4T1-hACE2細胞と組換えRBDの結合を阻害した(図６)。各変異型RBDの4T1-hACE2細胞への結合に対するIC50はそれぞれ、5.8～20.7 μg/mL (WT)、11.4～38.0 μg/mL (alpha)、4.8～20.0 μg/mL (beta)、6.0～17.6 μg/mL (delta)、6.1～17.7 μg/mL (epsilon)、6.0～16.8 μg/mL (eta)、11.6～23.3 μg/mL (theta)、5.5～15.4 μg/mL (kappa)であった。
　６種類のキメラ抗体のうち、特に、OMRad004G05、OMRad028D08、OMRad031H08、OMRad045G03については、いずれの変異型RBDのACE2への結合に対しても同程度の阻害効果を示した。また、これら4種類のキメラ抗体は、OMRad044E11及びOMRad052B05と比較して高い阻害効果（低いIC50）を示した。これらの結果を下記表に示す。 3. Results As a result, all six chimeric antibodies inhibited the binding of 4T1-hACE2 cells to recombinant RBD in a concentration-dependent manner (Fig. 6). The IC50 for binding of each mutant RBD to 4T1-hACE2 cells is 5.8-20.7 μg/mL (WT), 11.4-38.0 μg/mL (alpha), 4.8-20.0 μg/mL (beta), 6.0-17.6, respectively. μg/mL (delta), 6.1-17.7 μg/mL (epsilon), 6.0-16.8 μg/mL (eta), 11.6-23.3 μg/mL (theta), 5.5-15.4 μg/mL (kappa).
Of the six chimeric antibodies, OMRad004G05, OMRad028D08, OMRad031H08, and OMRad045G03 in particular exhibited similar inhibitory effects on the binding of any mutant RBD to ACE2. In addition, these four chimeric antibodies exhibited higher inhibitory effects (lower IC50) than OMRad044E11 and OMRad052B05. These results are shown in the table below.
Figure JPOXMLDOC01-appb-T000018
  
Figure JPOXMLDOC01-appb-T000018
  
　本実施例の結果により、本発明の抗体はいずれも、野生型及び全ての変異型組換えRBDとヒトACE2との結合を中和することが示された。
　また、実施例６で示されたGroup 1に属する抗体（OMRad004G05、OMRad028D08、OMRad031H08、OMRad045G03）とGroup 2に属する抗体（OMRad044E11、OMRad052B05）は、それぞれ同じGroup内に属する他の抗体と同等の活性を有することが示された。 The results of this example demonstrate that all antibodies of the invention neutralize the binding of wild-type and all mutant recombinant RBDs to human ACE2.
In addition, the antibodies belonging to Group 1 (OMRad004G05, OMRad028D08, OMRad031H08, OMRad045G03) and the antibodies belonging to Group 2 (OMRad044E11, OMRad052B05) shown in Example 6 exhibit activities equivalent to those of other antibodies belonging to the same Group. shown to have
３．生ウイルスに対する中和試験
（１）使用した抗体及び試験の概要
　本中和試験においては、上記実施例９で用いた６種類のキメラ抗体のうち、特に阻害効果が高かった４種類（OMRad004G05、OMRad028D08、OMRad031H08、OMRad045G03）を用いた。
　本中和試験の試験方法は、国立感染症研究所「COVID-19 血清学的検査マニュアル」に記載の方法を一部改変して実施した。具体的には、上記キメラ抗体の希釈液（いずれの抗体も10 μg/mL (オミクロン株以外)又は 2.5 μg/mL (オミクロン株)からの1.5倍の希釈系列）を細胞に接触させた後、100TCID50/50 μLに濃度調整した野生型及び各種変異型SARS-CoV-2浮遊液を等量添加し、37℃±1℃の炭酸ガスインキュベーター(CO2濃度：５%)内で3日間培養し、上記キメラ抗体の中和抗体価を測定した。
　抗体希釈液は、細胞維持培地［ダルベッコ変法イーグル培地(ナカライテスク株式会社)に2%量の牛胎児血清 、ペニシリン(100 U/mL)、ストレプトマイシン(100 μg/ mL )、ジェネティシン(G418, 1 mg/mL)を添加］を用いてそれぞれ作製した。 3. Neutralization test against live virus (1) Antibodies used and outline of test , OMRad031H08, OMRad045G03) were used.
The test method of this neutralization test was carried out by partially modifying the method described in the National Institute of Infectious Diseases "COVID-19 Serological Test Manual". Specifically, after contacting the diluent of the chimeric antibody (10 μg/mL (other than Omicron strain) or 2.5 μg/mL (Omicron strain) for any antibody in a 1.5-fold dilution series) to the cells, Equal amounts of wild-type and various mutant SARS-CoV-2 suspensions adjusted to 100TCID 50/50 μL were added, and cultured for 3 days in a carbon dioxide incubator (CO2 concentration: 5%) at 37°C ± 1°C. , the neutralizing antibody titer of the chimeric antibody was measured.
The antibody diluent is a cell maintenance medium [Dulbecco's Modified Eagle Medium (Nacalai Tesque, Inc.) containing 2% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 μg/mL), geneticin (G418, 1 mg/mL) added].
（２）使用したウイルス及び細胞
　本実施例において、「生ウイルス」（live virus）とは、生きた細胞に感染し増殖することができるウイルスのことをいう。
　本実施例において、SARS-CoV-2の生ウイルスとしては、次のウイルス株を用いた。
　(i) SARS-CoV-2　野生型株 (JPY/TY/WK-521)
　(ii) SARS-CoV-2　アルファ型変異株 QHN001 (VOC-202012/01系統株)
　(iii) SARS-CoV-2　ベータ型変異株 TY8-612 (B.1.351系統株)
　(iv) SARS-CoV-2　デルタ型変異株 (TY11-927 (B.1.617.2系統株))
　(v) SARS-CoV-2　オミクロン型変異株 (TY38-873 (BA.1系統株)
　また、本実施例においては、VeroE6/TMPRSS2細胞（JCRB1819）を用いた。 (2) Viruses and Cells Used In this example, the term "live virus" refers to a virus that can infect and proliferate in living cells.
In this example, the following virus strains were used as the live virus of SARS-CoV-2.
(i) SARS-CoV-2 wild type strain (JPY/TY/WK-521)
(ii) SARS-CoV-2 alpha variant strain QHN001 (VOC-202012/01 strain)
(iii) SARS-CoV-2 beta variant TY8-612 (B.1.351 lineage)
(iv) SARS-CoV-2 delta variant (TY11-927 (B.1.617.2 strain))
(v) SARS-CoV-2 Omicron variant (TY38-873 (BA.1 strain)
Moreover, VeroE6/TMPRSS2 cells (JCRB1819) were used in this example.
（３）ウイルス浮遊液の調製
　上記SARS-CoV-2のウイルス浮遊液は、次のようにして調製した。
　(i) 細胞増殖培地［ダルベッコ変法イーグル培地（ナカライテスク株式会社）に10％量の牛胎児血清、ペニシリン（100 U/mL）、ストレプトマイシン（100 μg/mL）、ジェネティシン（G418）（1 mg/mL）を添加］を用い、VeroE6/TMPRSS2細胞を組織培養フラスコ内に単層培養した。
　(ii) 細胞培養後にフラスコ内から細胞増殖培地を除き、各種SARS-CoV-2を接種し、細胞維持培地［ダルベッコ変法イーグル培地（ナカライテスク株式会社）に2％量の牛胎児血清、ペニシリン（100 U/mL）、ストレプトマイシン（100 μg/mL）、ジェネティシン（G418）（1 mg/mL）を添加］を加えて37℃±１℃の炭酸ガスインキュベーター（CO2濃度：5％）内で3日間培養した。
　(iii) 3日間培養後、倒立位相差顕微鏡を用いて細胞の形態を観察し、細胞に形態変化（細胞変性効果：CPE）が起こっていることを確認した。次に、培養液を遠心分離（3500 rpm/min、10 min）し、得られた上清を試験ウイルス浮遊原液とした。 (3) Preparation of virus suspension The above SARS-CoV-2 virus suspension was prepared as follows.
(i) Cell growth medium [10% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 μg/mL), geneticin (G418) (1 mg /mL)] was used to culture VeroE6/TMPRSS2 cells in monolayers in tissue culture flasks.
(ii) After cell culture, remove the cell growth medium from the flask, inoculate with various SARS-CoV-2, and add 2% fetal bovine serum, penicillin to Dulbecco's Modified Eagle Medium (Nacalai Tesque) (100 U/mL), streptomycin (100 μg/mL), and geneticin (G418) (1 mg/mL)] were added and placed in a carbon dioxide gas incubator (CO 2 concentration: 5%) at 37°C ± 1°C. Cultured for 3 days.
(iii) After culturing for 3 days, the morphology of the cells was observed using an inverted phase-contrast microscope to confirm that morphological changes (cytopathic effect: CPE) occurred in the cells. Next, the culture medium was centrifuged (3500 rpm/min, 10 min), and the resulting supernatant was used as the test virus suspension stock solution.
（４）中和抗体価の測定
　細胞増殖培地を用い、VeroE6/TMPRSS2細胞を組織培養用マイクロプレート（平底96穴）内で単層培養した後、細胞増殖培地を除き、細胞維持培地で洗浄し、細胞維持培地を取り除いた。
　次に、1.5倍で段階希釈した上記キメラ抗体の希釈液50 μLをそれぞれ4穴に添加後、上記ウイルス浮遊液50 μLを接種し、37℃±1℃の炭酸ガスインキュベーター（CO2濃度：5％）内で3日間培養した。
　培養後、倒立位相差顕微鏡を用いて細胞の形態変化（細胞変性効果：CPE）の有無を観察し、上清を除去し細胞を固定し水洗した。
　固定した細胞を染色・水洗後、2穴以上でCPE出現が抑制された最高希釈倍数を算出し、当該最高希釈倍数と抗体量から中和に必要な抗体濃度を算出し、これを中和抗体価とした。この試験を2回繰り返した。
　その結果、試験に用いた4種類の抗体はいずれも、野生型及び変異型株の全ての生ウイルスの細胞への感染に対して中和活性を示した。この結果を下記の表に示す。 (4) Measurement of Neutralizing Antibody Titer After monolayer culture of VeroE6/TMPRSS2 cells in tissue culture microplates (flat-bottom 96 wells) using cell growth medium, remove cell growth medium and wash with cell maintenance medium. , the cell maintenance medium was removed.
Next, after adding 50 μL of the chimeric antibody serially diluted 1.5-fold to each of the 4 wells, 50 μL of the virus suspension was inoculated and placed in a carbon dioxide gas incubator at 37°C ± 1°C (CO 2 concentration: 5°C). %) for 3 days.
After culturing, the cells were observed for morphological changes (cytopathic effect: CPE) using an inverted phase-contrast microscope, and the supernatant was removed, and the cells were fixed and washed with water.
After staining and washing the fixed cells, calculate the highest dilution ratio that suppresses the appearance of CPE in 2 or more wells, calculate the antibody concentration required for neutralization from the highest dilution ratio and the amount of antibody, and use this as the neutralizing antibody. valued. This test was repeated twice.
As a result, all of the 4 types of antibodies used in the test showed neutralizing activity against the infection of cells by all live viruses of wild-type and mutant strains. The results are shown in the table below.
Figure JPOXMLDOC01-appb-T000019
  
Figure JPOXMLDOC01-appb-T000019
  
　この結果から、本発明の抗体は、SARS-CoV-2野生株及び変異株のヒト細胞への結合及び感染を中和することが示された。
　すなわち、本発明の抗体は、SARS-CoV-2の野生株又は変異株によるコロナウイルス感染症の治療又は予防に極めて有効であることが示された。
　また、実施例６で示されたGroup 1に属する抗体は、SARS-CoV-2野生株及び変異株のヒト細胞への結合及び感染に対し、それぞれ同じGroup内に属する他の抗体と同等の中和活性を有することが示された。
　さらに、OMRad031H08とOMRa045G03とは、そのフレームワークのアミノ酸配列において1アミノ酸残基が異なるにもかかわらず、上記表に示されるとおり、両者は同じ値の中和活性を示した。このことから、本発明の抗体においては、２つの抗体間でCDRのアミノ酸配列が同一又は類似する場合は、フレームワークのアミノ酸配列が両者間で完全に一致しなくても、同等の効果（中和活性等）を有することが示された。 The results indicated that the antibodies of the present invention neutralize binding and infection of SARS-CoV-2 wild type and mutant strains to human cells.
That is, the antibodies of the present invention were shown to be extremely effective in treating or preventing coronavirus infections caused by SARS-CoV-2 wild strains or mutant strains.
In addition, the antibodies belonging to Group 1 shown in Example 6 are equivalent to other antibodies belonging to the same Group for binding and infection of SARS-CoV-2 wild type and mutant strains to human cells. It was shown to have synergistic activity.
Furthermore, although OMRad031H08 and OMRa045G03 differed by one amino acid residue in their framework amino acid sequences, both showed the same neutralizing activity as shown in the table above. From this, in the antibody of the present invention, when the CDR amino acid sequences are the same or similar between the two antibodies, even if the framework amino acid sequences do not completely match between the two, equivalent effects (medium activity, etc.).
　本発明により、ACE2に結合することができる新規モノクローナル抗体を提供することができる。 The present invention can provide a novel monoclonal antibody that can bind to ACE2.
　配列番号３～１３４：合成DNA又は合成ペプチド 　SEQ ID NOs: 3 to 134: synthetic DNA or synthetic peptide

Claims (18)

  1. 　ACE2（Angiotensin Converting Enzyme 2）に結合するモノクローナル抗体又はその抗原結合断片。 A monoclonal antibody or antigen-binding fragment thereof that binds to ACE2 (Angiotensin Converting Enzyme 2).
  2. 　コロナウイルスとACE2との結合を中和することができる、請求項１に記載のモノクローナル抗体又はその抗原結合断片。 The monoclonal antibody or antigen-binding fragment thereof according to claim 1, which is capable of neutralizing binding between coronavirus and ACE2.
  3. 　コロナウイルスが、ACE2を介して細胞に感染するコロナウイルスである、請求項２に記載のモノクローナル抗体又はその抗原結合断片。 The monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein the coronavirus is a coronavirus that infects cells via ACE2.
  4. 　前記抗体が、キメラ抗体、ヒト化抗体又は完全ヒト抗体である、請求項１～３のいずれか一項に記載のモノクローナル抗体又はその抗原結合断片。 The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is a chimeric antibody, a humanized antibody or a fully human antibody.
  5. 　(a) 配列番号５４のアミノ酸配列を含むCDR-H1、配列番号５６のアミノ酸配列を含むCDR-H2及び配列番号５８のアミノ酸配列を含むCDR-H3を含み、かつ、配列番号６０のアミノ酸配列を含むCDR-L1、配列番号６２のアミノ酸配列を含むCDR-L2及び配列番号６４のアミノ酸配列を含むCDR-L3を含むか、
    　(b) 配列番号３０のアミノ酸配列を含むCDR-H1、配列番号３２のアミノ酸配列を含むCDR-H2及び配列番号３４のアミノ酸配列を含むCDR-H3を含み、かつ、配列番号３６のアミノ酸配列を含むCDR-L1、配列番号３８のアミノ酸配列を含むCDR-L2及び配列番号４０のアミノ酸配列を含むCDR-L3を含むか、
    　(c) 配列番号１８のアミノ酸配列を含むCDR-H1、配列番号２０のアミノ酸配列を含むCDR-H2及び配列番号２２のアミノ酸配列を含むCDR-H3を含み、かつ、配列番号２４のアミノ酸配列を含むCDR-L1、配列番号２６のアミノ酸配列を含むCDR-L2及び配列番号２８のアミノ酸配列を含むCDR-L3を含むか、
    　(d) 配列番号６のアミノ酸配列を含むCDR-H1、配列番号８のアミノ酸配列を含むCDR-H2及び配列番号１０のアミノ酸配列を含むCDR-H3を含み、かつ、配列番号１２のアミノ酸配列を含むCDR-L1、配列番号１４のアミノ酸配列を含むCDR-L2及び配列番号１６のアミノ酸配列を含むCDR-L3を含むか、
    　(e) 配列番号４２のアミノ酸配列を含むCDR-H1、配列番号４４のアミノ酸配列を含むCDR-H2及び配列番号４６のアミノ酸配列を含むCDR-H3を含み、かつ、配列番号４８のアミノ酸配列を含むCDR-L1、配列番号５０のアミノ酸配列を含むCDR-L2及び配列番号５２のアミノ酸配列を含むCDR-L3を含むか、又は
    　(f) 配列番号６６のアミノ酸配列を含むCDR-H1、配列番号６８のアミノ酸配列を含むCDR-H2及び配列番号７０のアミノ酸配列を含むCDR-H3を含み、かつ、配列番号７２のアミノ酸配列を含むCDR-L1、配列番号７４のアミノ酸配列を含むCDR-L2及び配列番号７６のアミノ酸配列を含むCDR-L3を含む、
    請求項１～４のいずれか一項に記載のモノクローナル抗体又はその抗原結合断片。     (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 54, CDR-H2 containing the amino acid sequence of SEQ ID NO: 56, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 58, and the amino acid sequence of SEQ ID NO: 60 CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 62 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64;
    (b) CDR-H1 containing the amino acid sequence of SEQ ID NO: 30, CDR-H2 containing the amino acid sequence of SEQ ID NO: 32, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 34, and the amino acid sequence of SEQ ID NO: 36 CDR-L1 comprising the amino acid sequence of SEQ ID NO:38, CDR-L2 comprising the amino acid sequence of SEQ ID NO:38 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:40;
    (c) CDR-H1 containing the amino acid sequence of SEQ ID NO: 18, CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 22, and the amino acid sequence of SEQ ID NO: 24 CDR-L1 comprising the amino acid sequence of SEQ ID NO: 26, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 28;
    (d) CDR-H1 containing the amino acid sequence of SEQ ID NO: 6, CDR-H2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 10, and the amino acid sequence of SEQ ID NO: 12 CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16;
    (e) CDR-H1 containing the amino acid sequence of SEQ ID NO: 42, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 46, and the amino acid sequence of SEQ ID NO: 48 CDR-L1 comprising the amino acid sequence of SEQ ID NO: 50 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 52, or (f) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 66, SEQ ID NO: CDR-H2 comprising the amino acid sequence of 68 and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 70, and CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74, and comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 76;
    The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4.
  6. 　(a) 配列番号９４のアミノ酸配列を含む重鎖可変領域（VH）及び配列番号９６のアミノ酸配列を含む軽鎖可変領域（VL）を含むか、
    　(b) 配列番号８６のアミノ酸配列を含む重鎖可変領域（VH）及び配列番号８８のアミノ酸配列を含む軽鎖可変領域（VL）を含むか、
    　(c) 配列番号８２のアミノ酸配列を含む重鎖可変領域（VH）及び配列番号８４のアミノ酸配列を含む軽鎖可変領域（VL）を含むか、
    　(d) 配列番号７８のアミノ酸配列を含む重鎖可変領域（VH）及び配列番号８０のアミノ酸配列を含む軽鎖可変領域（VL）を含むか、
    　(e) 配列番号９０のアミノ酸配列を含む重鎖可変領域（VH）及び配列番号９２のアミノ酸配列を含む軽鎖可変領域（VL）を含むか、又は
    　(f) 配列番号９８のアミノ酸配列を含む重鎖可変領域（VH）及び配列番号１００のアミノ酸配列を含む軽鎖可変領域（VL）を含む、
    請求項１～５のいずれか一項に記載のモノクローナル抗体又はその抗原結合断片。     (a) comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:94 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:96;
    (b) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:86 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:88, or
    (c) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:82 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:84;
    (d) comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:78 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:80;
    (e) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:90 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:92; or (f) comprising the amino acid sequence of SEQ ID NO:98. a heavy chain variable region (VH) and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 100;
    The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-5.
  7. 　コロナウイルスとACE2との結合を中和することができ、ACE2との結合について、請求項５又は６に記載の抗体と競合する、モノクローナル抗体又はその抗原結合断片。 A monoclonal antibody or an antigen-binding fragment thereof, which is capable of neutralizing the binding of coronavirus to ACE2 and competes with the antibody according to claim 5 or 6 for binding to ACE2.
  8. 　請求項１～７のいずれか一項に記載のモノクローナル抗体又はその抗原結合断片を含む、コロナウイルス感染症を治療又は予防するための医薬組成物。 A pharmaceutical composition for treating or preventing coronavirus infection, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.
  9. 　コロナウイルス感染症が、ACE2を介して細胞に感染するコロナウイルスの感染によって引き起こされる疾患又は症状である、請求項８に記載の医薬組成物。 The pharmaceutical composition according to claim 8, wherein the coronavirus infection is a disease or condition caused by infection with a coronavirus that infects cells via ACE2.
  10. 　前記疾患又は症状が、呼吸器疾患、発熱、倦怠感、悪寒、疼痛、味覚若しくは嗅覚の障害、発疹、消化器症状、言語障害、認知障害及び循環器症状からなる群から選択される少なくとも一つのものである、請求項９に記載の医薬組成物。 The disease or symptom is at least one selected from the group consisting of respiratory disease, fever, malaise, chills, pain, taste or smell disorder, rash, gastrointestinal symptom, speech disorder, cognitive disorder and cardiovascular symptom. 10. The pharmaceutical composition of claim 9, which is
  11. 　請求項１～７のいずれか一項に記載のモノクローナル抗体若しくはその抗原結合断片又はそれを含む医薬組成物の治療上有効量を対象に投与する工程を含む、コロナウイルス感染症を治療又は予防する方法。 Treating or preventing coronavirus infection, comprising administering to a subject a therapeutically effective amount of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or a pharmaceutical composition comprising the same Method.
  12. 　コロナウイルス感染症が、ACE2を介して細胞に感染するコロナウイルスの感染によって引き起こされる疾患又は症状である、請求項１１に記載の方法。 The method according to claim 11, wherein the coronavirus infection is a disease or condition caused by infection with a coronavirus that infects cells via ACE2.
  13. 　前記疾患又は症状が、呼吸器疾患、発熱、倦怠感、悪寒、疼痛、味覚若しくは嗅覚の障害、発疹、消化器症状、言語障害、認知障害及び循環器症状からなる群から選択される少なくとも一つのものである、請求項１２に記載の方法。 The disease or symptom is at least one selected from the group consisting of respiratory disease, fever, malaise, chills, pain, taste or smell disorder, rash, gastrointestinal symptom, speech disorder, cognitive disorder and cardiovascular symptom. 13. The method of claim 12, which is a
  14. 　コロナウイルス感染症の治療又は予防において使用するための、請求項１～７のいずれか一項に記載のモノクローナル抗体又はその抗原結合断片。 The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, for use in treating or preventing coronavirus infection.
  15. 　コロナウイルス感染症が、ACE2を介して細胞に感染するコロナウイルスの感染によって引き起こされる疾患又は症状である、請求項１４に記載のモノクローナル抗体又はその抗原結合断片。 The monoclonal antibody or antigen-binding fragment thereof according to claim 14, wherein the coronavirus infection is a disease or condition caused by infection with a coronavirus that infects cells via ACE2.
  16. 　前記疾患又は症状が、呼吸器疾患、発熱、倦怠感、悪寒、疼痛、味覚若しくは嗅覚の障害、発疹、消化器症状、言語障害、認知障害及び循環器症状からなる群から選択される少なくとも一つのものである、請求項１５に記載のモノクローナル抗体又はその抗原結合断片。 The disease or symptom is at least one selected from the group consisting of respiratory disease, fever, malaise, chills, pain, taste or smell disorder, rash, gastrointestinal symptom, speech disorder, cognitive disorder and cardiovascular symptom. 16. The monoclonal antibody or antigen-binding fragment thereof according to claim 15, which is
  17. 　コロナウイルス感染症の治療又は予防のための医薬の製造における、請求項１～７のいずれか一項に記載のモノクローナル抗体又はその抗原結合断片の使用。 Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 in the manufacture of a medicament for treating or preventing coronavirus infection.
  18. 　請求項１～７のいずれか一項に記載のモノクローナル抗体又はその抗原結合断片を含む、試薬又はキット。 A reagent or kit comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.
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