Anti-ACE2 monoclonal antibody

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

[Technical Field] 【0001】 This invention relates to a monoclonal antibody that binds to angiotensin-converting enzyme 2 (ACE2) and its use. [Background technology] 【0002】 The novel coronavirus infection (COVID-19), which originated in Wuhan, China in December 2019, spread rapidly throughout the world, not only causing many deaths but also inflicting serious damage on the global economy. SARS-CoV-2, the virus that causes COVID-19, is classified as a beta-coronavirus and has a single-stranded RNA genome. The surface of the virus particle has spike proteins, which are protruding proteins. These spike proteins interact with angiotensin-converting enzyme 2 (ACE2), which is expressed on the surface of human cells, allowing direct contact between the virus particle and human cells (Non-Patent Literature 1). COVID-19 can cause severe respiratory distress, and although the number of deaths worldwide exceeded 1.9 million by the end of 2020, there is still no effective treatment that can be considered a miracle cure. [Prior art documents] [Non-patent literature] 【0003】 [Non-Patent Document 1] Wrapp et al., Science 367, 1260-1263 (2020) [Overview of the Initiative] [Problems that the invention aims to solve] 【0004】 In this situation, the development of novel molecules that neutralize the binding between coronavirus and ACE2 is desirable. [Means for solving the problem] 【0005】 As a result of diligent research to solve the above problems, the inventors of this invention have succeeded in developing a novel anti-ACE2 monoclonal antibody that can neutralize the binding of coronavirus to ACE2, thereby completing the present invention. In other words, the present invention is as follows: 【0006】 [1] A monoclonal antibody or its antigen-binding fragment that binds to ACE2 (Angiotensin Converting Enzyme 2). [2] A monoclonal antibody or antigen-binding fragment thereof, as described in [1] above, which can neutralize the binding of coronavirus to ACE2. [3] A monoclonal antibody or antigen-binding fragment thereof as described in [2] above, wherein the coronavirus is a coronavirus that infects cells via ACE2. [4] A monoclonal antibody or antigen-binding fragment thereof according to any one of the above [1] to [3], wherein the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody. [5] (a) comprising 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 also comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 60, CDR-L2 containing the amino acid sequence of SEQ ID NO: 62, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 64, (b) A CDR-H1 containing the amino acid sequence of SEQ ID NO: 30, a CDR-H2 containing the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 containing the amino acid sequence of SEQ ID NO: 34, and a CDR-L1 containing the amino acid sequence of SEQ ID NO: 36, a CDR-L2 containing the amino acid sequence of SEQ ID NO: 38, and a CDR-L3 containing the amino acid sequence of SEQ ID NO: 40, (c) A CDR-H1 containing the amino acid sequence of SEQ ID NO: 18, a CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and a CDR-H3 containing the amino acid sequence of SEQ ID NO: 22, and a CDR-L1 containing the amino acid sequence of SEQ ID NO: 24, a CDR-L2 containing the amino acid sequence of SEQ ID NO: 26, and a CDR-L3 containing the amino acid sequence of SEQ ID NO: 28, (d) A CDR-H1 containing the amino acid sequence of SEQ ID NO: 6, a CDR-H2 containing the amino acid sequence of SEQ ID NO: 8, and a CDR-H3 containing the amino acid sequence of SEQ ID NO: 10, and a CDR-L1 containing the amino acid sequence of SEQ ID NO: 12, a CDR-L2 containing the amino acid sequence of SEQ ID NO: 14, and a CDR-L3 containing the amino acid sequence of SEQ ID NO: 16, (e) A CDR-H1 containing the amino acid sequence of SEQ ID NO: 42, a CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and a CDR-H3 containing the amino acid sequence of SEQ ID NO: 46, and a CDR-L1 containing the amino acid sequence of SEQ ID NO: 48, a CDR-L2 containing the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 containing the amino acid sequence of SEQ ID NO: 52, or (f) A CDR-H1 containing the amino acid sequence of SEQ ID NO: 66, a CDR-H2 containing the amino acid sequence of SEQ ID NO: 68, and a CDR-H3 containing the amino acid sequence of SEQ ID NO: 70, and a CDR-L1 containing the amino acid sequence of SEQ ID NO: 72, a CDR-L2 containing the amino acid sequence of SEQ ID NO: 74, and a CDR-L3 containing the amino acid sequence of SEQ ID NO: 76, A monoclonal antibody or its antigen-binding fragment as described in any of the above [1] to [4]. [6] (a) It includes a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 94 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 96, (b) comprising a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 86 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 88, (c) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 82 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 84, (d) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 78 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 80, (e) comprising a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 90 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 92, or (f) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 98 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 100, A monoclonal antibody or its antigen-binding fragment as described in any of the above [1] to [5]. [7] A monoclonal antibody or its antigen-binding fragment that can neutralize the binding of coronavirus to ACE2 and competes with the antibodies described in [5] or [6] above for binding to ACE2. [8] A pharmaceutical composition for treating or preventing coronavirus infection, comprising a monoclonal antibody or an antigen-binding fragment thereof as described in any of the above [1] to [7]. [9] The pharmaceutical composition according to [8] above, wherein coronavirus infection is a disease or condition caused by infection with a coronavirus that infects cells via ACE2.

[10] The pharmaceutical composition according to [9] above, wherein 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 symptoms, speech disorder, cognitive impairment, and cardiovascular symptoms.

[11] A method for treating or preventing coronavirus infection, comprising the step of administering a therapeutically effective amount of a monoclonal antibody or an antigen-binding fragment thereof, or a pharmaceutical composition containing the same, as described in any of the above [1] to [7].

[12] The method according to

[11] above, wherein coronavirus infection is a disease or condition caused by infection with a coronavirus that infects cells via ACE2.

[13] The method according to

[12] above, wherein the disease or symptom is at least one selected from the group consisting of respiratory diseases, fever, fatigue, chilliness, pain, taste or olfactory disorders, rash, digestive symptoms, language disorders, cognitive disorders, and circulatory symptoms.

[14] The monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [7] above for use in the treatment or prevention of coronavirus infection.

[15] The monoclonal antibody or antigen-binding fragment thereof according to

[14] above, wherein the coronavirus infection is a disease or symptom caused by the infection of a coronavirus that infects cells via ACE2.

[16] The monoclonal antibody or antigen-binding fragment thereof according to

[15] above, wherein the disease or symptom is at least one selected from the group consisting of respiratory diseases, fever, fatigue, chilliness, pain, taste or olfactory disorders, rash, digestive symptoms, language disorders, cognitive disorders, and circulatory symptoms.

[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 the treatment or prevention of 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. [Effects of the Invention] 【0007】 According to the present invention, the binding between coronavirus and ACE2 can be neutralized. Since the antibody of the present invention binds to ACE2, which is a receptor of the coronavirus, even if the coronavirus mutates, as long as the mutant virus infects via ACE2, the binding between the mutant virus and ACE2 can be inhibited. [Brief Description of the Drawings] 【0008】 [Figure 1]This figure shows the results of a binding inhibition test between the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and ACE2 using the antibody of the present invention. [Figure 2] This is a schematic diagram of a competitive inhibition test using the antibody of the present invention. [Figure 3] This figure shows the results of a competitive inhibition test using the antibody of the present invention. [Figure 4] This figure shows the results of confirming the reactivity of the antibody of the present invention with human ACE2, mouse ACE2, and mouse / human fusion ACE2, respectively. [Figure 5] This figure shows the results of confirming the reactivity of the antibody of the present invention with human ACE2-expressing cells. A: Shows the reactivity of the mouse antibody of the present invention with human ACE2-expressing cells. B: Shows the reactivity of the chimeric antibody of the present invention with human ACE2-expressing cells. C: Shows the reactivity of the chimeric antibody of the present invention with 4T1 cells that do not express human ACE2. [Figure 6] This figure shows the results of a binding inhibition test between the receptor-binding domain (RBD) of the spike protein of wild-type or mutant SARS-CoV-2 and ACE2, using the antibody of the present invention. [Modes for carrying out the invention] 【0009】 The present invention will now be described in detail. The following embodiments are illustrative for illustrating the present invention and are not intended to limit the present invention to these embodiments only. The present invention can be implemented in various forms without departing from its spirit. Furthermore, this specification includes the contents described in the specification and drawings of the Japanese Patent Application No. 2021-036928 filed on March 9, 2021, which forms the basis of this application's priority claim. 【0010】 1. overview Although the number of deaths worldwide from COVID-19 exceeded 1.9 million by the end of 2020, there is still no treatment that can be considered a miracle cure. Compounds such as favipiravir and remdesivir have been mentioned as potential treatments for COVID-19, but these were originally used to treat infectious diseases such as influenza and Ebola hemorrhagic fever, and it is unclear whether they are the optimal treatments for COVID-19. Antibody drugs such as tocilizumab and ravulizumab are also being considered as treatment candidates, but these are not antibodies that directly recognize the virus, but rather target endogenous factors involved in the immune system, such as IL-6R and complement (C5), so their effects are indirect. Furthermore, amidst the prolonged global pandemic, there have been numerous reports of mutant SARS-CoV-2 cases in which the spike protein has mutated, altering the amino acid sequence, and this trend is expected to continue. In this situation, there is a need for the development of new therapeutic drugs that can be used to treat or prevent coronavirus infections, even if the coronavirus mutates. The inventors focused on ACE2, a receptor to which coronaviruses bind when infecting human cells, and after diligently studying antibodies against ACE2, they succeeded in developing a novel monoclonal antibody that can neutralize the binding of coronaviruses to ACE2. The antibody of the present invention is extremely useful because, since it recognizes ACE2, it can inhibit the binding of the mutated virus to ACE2, even if the coronavirus mutates, as long as the mutated virus infects via ACE2. Furthermore, the antibodies of the present invention can neutralize the binding and infection of human cells by wild-type and mutant strains of SARS-CoV-2, making them extremely effective in treating or preventing coronavirus infections. 【0011】 2. Angiotensin-converting enzyme The antibody of the present invention binds to angiotensin-converting enzyme 2 (ACE2). ACE2 is a membrane protein present in the cell 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 for the coronaviruses SARS-CoV-1 and SARS-CoV-2 when they infect human cells. Specifically, SARS-CoV-1 and SARS-CoV-2 can infect human cells when their spike protein (S protein) binds to ACE2. 【0012】 ACE2 is primarily expressed in cells of the lungs, digestive system, heart, blood vessels, eyes, kidneys, cerebral cortex, amygdala, brainstem, and medulla oblongata. Therefore, by inhibiting coronavirus infection of cells in these organs, the present invention can contribute to the treatment or prevention of various diseases and symptoms associated with coronavirus infections, such as respiratory diseases. 【0013】 The ACE2 in this invention may be derived from any mammal. Examples of such mammals include mice, rats, rabbits, cats, dogs, goats, monkeys, and humans, and preferably mice, rats, cats, dogs, and humans, but is not limited to these. As examples of the nucleotide and amino acid sequences of these ACE2s, the nucleotide and amino acid sequences of human ACE2 are shown as Sequence IDs 1 and 2, respectively, but the nucleotide and amino acid sequences of the ACE2 in this invention are not limited to these. The nucleotide and amino acid sequences of human ACE2 are registered in the GenBank database with the following predetermined accession numbers. 【0014】 DNA sequence encoding human ACE2: NM_001371415 (Sequence ID 1) Amino acid sequence of human ACE2: NP_001358344.1 (SEQ ID NO: 2) 【0015】 The ACE2 in this invention includes the following proteins (a) to (c). (a) Protein containing the amino acid sequence shown in Sequence ID No. 2 (b) A protein having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 2, and which has the activity to bind to the receptor-binding domain (RBD) of the coronavirus spike protein. (c) A protein that contains an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in Sequence ID No. 2, and that has the activity to bind to the RBD of the coronavirus spike protein. 【0016】 In the present invention, "proteins containing the amino acid sequence shown in SEQ ID NO: 2" includes proteins consisting of the amino acid sequence shown in SEQ ID NO: 2. Furthermore, "an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 2" includes, for example, (i) an amino acid sequence in which 1 to 10 amino acids (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, even more preferably 1) are deleted from the amino acid sequence shown in SEQ ID NO: 2 (ii) An amino acid sequence in which 1 to 10 amino acids (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, even more preferably 1) in the amino acid sequence shown in Sequence ID No. 2 are replaced with other amino acids. (iii) An amino acid sequence in which 1 to 10 amino acids (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, even more preferably 1) are added to the amino acid sequence shown in Sequence ID No. 2, (iv) Amino acid sequences mutated by the combination of (i) to (iii) above These are some examples. 【0017】 In the present invention, the presence or absence of "activity to bind coronavirus spike protein to RBD" can be measured by known methods, such as immunological techniques such as immunoprecipitation, Western blotting, EIA (enzyme immunoassay), ELISA (enzyme-linked immunosorbent assay) (e.g., polypeptide-based ELISA, cell ELISA, etc.), or pull-down assays. Furthermore, "activity to bind coronavirus spike protein to RBD" means having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and preferably 90% or more of activity compared to the activity of the protein consisting of the amino acid sequence shown in Sequence ID No. 2, which is set to 100. 【0018】 Furthermore, the ACE2 in the present invention includes a protein that contains 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 has the activity to bind to the RBD of the coronavirus spike protein. Such proteins also include those that contain an amino acid sequence having approximately 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and have the activity to bind to the RBD of the coronavirus spike protein. Sequence identity can be checked using homology search sites via the internet, such as the DNA Data Bank of Japan (DDBJ), where homology searches such as FASTA, BLAST, and PSI-BLAST can be used. In addition, searches using BLAST can be performed at the National Center for Biotechnology Information (NCBI). 【0019】 To prepare a protein having the above mutation, a mutagenesis kit utilizing site-directed mutagenesis methods such as the Kunkel method or the Gapped duplex method is used to introduce the mutation into the DNA encoding the protein, such as QuikChange. TM Site-Directed Mutagenesis Kit (manufactured by Stratagene), GeneTailor TM This can be performed using a Site-Directed Mutagenesis System (Invitrogen), a TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: Takara Bio), or similar systems. Alternatively, methods such as site-directed mutagenesis described in "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)) can be used. 【0020】 In ACE2, the amino acid sequence of the region to which the antibody of the present invention binds (the region where the epitope to which the antibody of the present invention binds exists) is not limited, and examples include the extracellular region of ACE2 or a part thereof. The amino acid sequence of the extracellular region of ACE2 is not limited, and in the case of human ACE2, examples include the amino acid sequence consisting of amino acid residues 18 to 740 (the amino acid sequence of SEQ ID NO: 4), counting from the N-terminal amino acid residue of the full-length amino acid sequence of ACE2 (SEQ ID NO: 2). The base sequence of the DNA encoding the amino acid sequence consisting of amino acid residues 18 to 740 of human ACE2 is shown in SEQ ID NO: 3. Furthermore, the amino acid sequence of the extracellular region of ACE2 is not limited to this, and includes, for example, the amino acid sequence of the coronavirus binding region of ACE2 or a part thereof, or the amino acid sequence of the region of ACE2 other than the coronavirus binding region or a part thereof. Moreover, the amino acid sequence of the coronavirus binding region of ACE2 or a part thereof is not limited to this, and for example, in the case of human ACE2, it includes an amino acid sequence that includes at least one amino acid residue selected from 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), counting from the N-terminal amino acid residue of the full-length amino acid sequence of ACE2. 【0021】 Furthermore, examples of amino acid sequences of a portion of the extracellular region of ACE2 include, but are not limited to, the amino acid sequence consisting of amino acid residues 1 to 120 (SEQ ID NO: 132) and the amino acid sequence consisting of amino acid residues 121 to 740 (SEQ ID NO: 134) in the case of human ACE2, counting from the N-terminal amino acid residue of the full-length amino acid sequence of ACE2. The base sequences of the DNA encoding the amino acid sequence consisting of amino acid residues 1 to 120 and the DNA encoding the amino acid sequence consisting of amino acid residues 121 to 740 are shown in SEQ ID NOs: 131 and 133, respectively. 【0022】 Furthermore, the amino acid sequence of the region to which the antibody of the present invention binds (the region where the epitope to which the antibody of the present invention binds exists) 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 NOs: 4, 132, or 134, (b) an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NOs: 4, 132 or 134, or an amino acid sequence consisting of such a sequence (c) an amino acid sequence having approximately 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence shown in SEQ ID NOs: 4, 132, or 134, or an amino acid sequence consisting of such a sequence Those skilled in the art can easily confirm whether the antibodies of the present invention bind to these amino acid sequences using known immunological methods, such as immunoprecipitation, Western blotting, EIA, and ELISA (e.g., polypeptide-based ELISA, cell ELISA, etc.). 【0023】 The epitopes to which the antibodies of the present invention bind and the regions in which such epitopes exist are not limited, as long as the antibodies of the present invention can neutralize the binding of coronavirus to ACE2. Furthermore, those skilled in the art can determine whether the test antibody can neutralize the binding of coronavirus to ACE2 without identifying the epitopes of the test antibody, based on the description herein (for example, the description in Example 3). In other words, according to the description herein, those skilled in the art do not need to identify the epitopes of the test antibody to determine whether the test antibody can neutralize the binding of coronavirus to ACE2. 【0024】 3. The 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 interactions, electrostatic forces, van der Waals forces, etc. In the present invention, "binding" includes "specifically binding." In the present invention, "specifically binding" means recognizing an epitope in a target protein or target polypeptide and binding selectively or preferentially to a protein or polypeptide that has that epitope compared to a protein or polypeptide that does not have that epitope. In the present invention, examples of "antigen-binding fragments" include, but are not limited to, scFv (single chain Fv), sc(Fv)2, Fab, Fab', diabody (dsFv), F(ab')2, and multispecific antibodies. The above antibody-binding fragments can be obtained by genetic engineering techniques or by known methods such as papain digestion and pepsin digestion. In one aspect of the present invention, the antibody of the present invention is an isolated antibody. 【0025】 The antibody of the present invention can neutralize the binding of coronavirus to ACE2. In the present invention, "coronavirus" refers to a coronavirus that infects cells via ACE2. Examples of such coronaviruses include SARS-CoV-1, SARS-CoV-2, and their variants, with SARS-CoV-2 and its variants being preferred. These coronaviruses infect cells when the receptor-binding domain (RBD) of their spike protein binds to ACE2. In the present invention, "neutralizing" means inhibiting the interaction (e.g., binding) between the coronavirus RBD and ACE2, and inhibiting the binding or infection (e.g., entry) of the coronavirus into cells. Whether the test antibody inhibits the interaction between the coronavirus RBD and ACE2 can be investigated, for example, by detecting the ACE2-bound RBD using a fluorescently labeled antibody capable of binding to RBD, as described in Example 3 of this specification. Specifically, if, after reacting the test antibody with ACE2, RBD is further added, and no ACE2-bound RBD is detected, it can be determined that the test antibody inhibits the interaction between RBD and ACE2. Furthermore, whether the test antibody inhibits (neutralizes) the binding or infection (e.g., entry) of coronavirus to cells can be confirmed, for example, by bringing the test antibody and coronavirus into contact with cultured cells and observing whether or not there is a morphological change (cytopathic effect: CPE) in the cultured cells. 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 (e.g., Example 10). 【0026】 The method for producing the antibody of the present invention will be described below. (1) Antigen preparation As immunogens for producing the antibodies 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) can be used. When using ACE2 as an immunogen, the full-length polypeptide of ACE2 (SEQ ID NO: 2) or a partial polypeptide thereof can be used. The partial polypeptide of ACE2 used as an antigen or immunogen is not limited to, but includes, for example, a polypeptide containing all or part of the amino acid sequence of the extracellular region of ACE2. The amino acid sequence of all or part of the extracellular region of ACE2 is not limited to this, but for example, in the case of human ACE2, it includes an amino acid sequence that includes all or part of the amino acid sequence (SEQ ID NO: 4) consisting of amino acid residues 18 to 740, counting from the N-terminal amino acid residue of the full-length amino acid sequence of ACE2. Polypeptides containing a portion of the amino acid sequence of the extracellular region of ACE2 are not limited to these, and include, for example, polypeptides containing the coronavirus binding region of ACE2 or a portion thereof, and polypeptides containing the region of ACE2 other than the coronavirus binding region or a portion thereof. Examples of such polypeptides in the case of human ACE2 include, but are not limited to, amino acid sequences consisting of amino acid residues 1 to 120 (SEQ ID NO: 132) and amino acid sequences consisting of amino acid residues 121 to 740 (SEQ ID NO: 134), counting from the N-terminal amino acid residue of the full-length amino acid sequence of ACE2. 【0027】 When using ACE2-expressing cells as an immunogen, these cells can be produced, for example, by the following method. First, the full-length DNA encoding human ACE2 is incorporated into a lentiviral vector and introduced into mammalian cells (e.g., 293T cells) along with a packaging plasmid and an envelope plasmid. The recombinant lentivirus produced in the culture supernatant of these cells is then used to infect mammalian cells (e.g., breast cancer cell line 4T1 cells) to obtain cells that stably express human ACE2 on their cell membranes. However, the method for producing ACE2-expressing cells is not limited to this method. 【0028】 The ACE2 used as an antigen or immunogen may be naturally occurring ACE2 purified from tissues or cells of mice, rats, rabbits, cats, dogs, goats, monkeys, humans, etc., or it may be genetically engineered ACE2. For example, a biological sample showing ACE2 expression can be fractionated into a soluble fraction and an insoluble fraction using various surfactants, such as Triton-X or Sarkosyl. The insoluble fraction can then be dissolved in urea or guanidine hydrochloride and bound to various columns, such as heparin columns or binding resins, to obtain ACE2. The ACE2 used as an antigen can also be synthesized by specifying its amino acid sequence using known protein synthesis methods such as solid-phase synthesis or commercially available protein synthesis equipment. The synthesized peptide can be bound to a carrier protein such as Keyhole Limpet Hemocyanin (KLH) or Thyroglobulin and used as an immunogen. 【0029】 (2) Production of monoclonal antibodies (i) Collection of antibody-producing cells The ACE2 or partial polypeptide prepared as described above is administered either by itself or together with a carrier and diluent to non-human mammals such as mice, rats, rabbits, cats, dogs, goats, and monkeys to induce immunization. The dose of antigen per animal is 0.1 to 10 mg when an adjuvant is used. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is mainly performed by intravenous, subcutaneous, or intraperitoneal injection. The interval between immunizations is not particularly limited, and 2 to 10 immunizations, preferably 2 to 5, are performed at intervals of several days to several weeks, preferably 1 to 2 weeks. The interval between immunizations can be set by a person skilled in the art, taking into account the antibody titer obtained. It is preferable to collect test blood after 3 to 4 subcutaneous immunizations and measure the antibody titer. Antibody titers in serum can be measured by ELISA, EIA, radioimmunoassay (RIA), etc. After confirming that the antibody titer has risen sufficiently, individuals with elevated antibody titers are selected and antibody-producing cells are collected. Examples of antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, but spleen cells or lymph node cells are preferred. 【0030】 (ii) cell fusion To obtain hybridomas, cell fusion is performed between antibody-producing cells and myeloma cells. The fusion procedure can be carried out according to known methods, such as the method of Kohler et al. As the myeloma cells to be fused with antibody-producing cells, commonly available cell lines from animals such as mice can be used. Preferred cell lines are those that exhibit drug selectivity, cannot survive in HAT selective medium (containing hypoxanthine, aminopterin, and thymidine) in an unfused state, and can only survive when fused with antibody-producing cells. 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. 【0031】 Cell fusion between the above myeloma cells and antibody-producing cells was performed in animal cell culture media such as serum-free DMEM or RPMI-1640 medium, at a rate of 1 × 10⁶ 8 ~5×10 8 A single antibody-producing cell and 2 × 10⁶ 7 ~10×10 7 A mixture of antibody-producing cells and myeloma cells (with a cell ratio of 10:1 to 1:1) is used to initiate the fusion reaction in the presence of a cell fusion promoter. Polyethylene glycol or Sendai virus with an average molecular weight of 1000 to 6000 daltons can be used as the cell fusion promoter. Alternatively, the antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device that utilizes electrical stimulation (e.g., electroporation). 【0032】 (iii) Selection and cloning of hybridomas The desired hybridomas are selected from cells after cell fusion processing. This is done by appropriately diluting the cell suspension with, for example, RPMI-1640 medium containing 10-20% fetal bovine serum, then seeding approximately 0.3 cells / well onto a microtiter plate using the limiting dilution method. A selective medium such as HAT medium is added to each well, and the cells are cultured thereafter, with the selective medium being replaced as needed. As a result, cells that begin to grow approximately 10 days after the start of culture in the selective medium can be obtained as hybridomas. 【0033】 Next, the developed hybridomas are further screened. Screening of hybridomas can be done using standard methods and is not particularly limited. For example, a portion of the culture supernatant contained in the wells in which hybridomas are cultured can be collected and screened using ELISA, EIA, radioimmunoassay, etc. Specifically, after adsorbing the antigen onto a 96-well plate, it is blocked with calf serum. The culture supernatant of hybridoma cells is reacted with the immobilized antigen at 37°C for 1 hour, then reacted with peroxidase-labeled anti-mouse IgG at 37°C for 1 hour, and color development is induced using orthophenylenediamine as a substrate. After stopping the reaction with acid, screening can be performed by measuring the absorbance at a wavelength of 490 nm. Hybridomas that produce monoclonal antibodies that show a positive result using the above measurement method are cloned using the limiting dilution method, etc. Finally, hybridomas that produce monoclonal antibodies that bind to ACE2 are established. 【0034】 (iv) Collection of monoclonal antibodies As a method for collecting monoclonal antibodies from established hybridomas, conventional cell culture methods or ascites formation methods can be employed. In the cell culture method, hybridomas are cultured for 7 to 14 days under normal culture conditions (e.g., 37°C, 5% CO2 concentration) in animal cell culture media such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium, and antibodies are obtained from the culture supernatant. In the case of ascites formation, hybridomas are introduced into the peritoneal cavity of mammalian allogenes derived from myeloma cells, such as mice (ICR, BALB / c), at a rate of approximately 5 × 10⁶. 6 ~2×10 7 Individual doses are administered to induce massive proliferation of hybridomas. Then, ascites fluid is collected 1 to 2 weeks later. If antibody purification is required in the above antibody collection method, it can be purified by appropriately selecting or combining known methods such as ammonium sulfate salting-out, ion exchange chromatography, gel filtration, and affinity chromatography. 【0035】 Examples of antibodies of the present invention include, but are not limited to, the following monoclonal antibodies or their antigen-binding fragments. (a) The heavy chain variable region (VH) includes a heavy chain complementarity determining region (CDR) 1 (CDR-H1) containing or consisting of the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR2 (CDR-H2) containing or consisting of the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 (CDR-H3) containing or consisting of the amino acid sequence of SEQ ID NO: 10, and / or the light chain variable region (VL) includes a light chain CDR1 (CDR-L1) containing or consisting of the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 (CDR-L2) containing or consisting of the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 (CDR-L3) containing or consisting of the amino acid sequence of SEQ ID NO: 16, (b) The heavy chain variable region (VH) includes the amino acid sequence of SEQ ID NO: 18 or CDR-H1 consisting of said amino acid sequence, the amino acid sequence of SEQ ID NO: 20 or CDR-H2 consisting of said amino acid sequence, and the amino acid sequence of SEQ ID NO: 22 or CDR-H3 consisting of said amino acid sequence, and / or the light chain variable region (VL) includes the amino acid sequence of SEQ ID NO: 24 or CDR-L1 consisting of said amino acid sequence, the amino acid sequence of SEQ ID NO: 26 or CDR-L2 consisting of said amino acid sequence, and the amino acid sequence of SEQ ID NO: 28 or CDR-L3 consisting of said amino acid sequence, (c) The heavy chain variable region (VH) includes the amino acid sequence of SEQ ID NO: 30 or CDR-H1 consisting of said amino acid sequence, the amino acid sequence of SEQ ID NO: 32 or CDR-H2 consisting of said amino acid sequence, and the amino acid sequence of SEQ ID NO: 34 or CDR-H3 consisting of said amino acid sequence, and / or the light chain variable region (VL) includes the amino acid sequence of SEQ ID NO: 36 or CDR-L1 consisting of said amino acid sequence, the amino acid sequence of SEQ ID NO: 38 or CDR-L2 consisting of said amino acid sequence, and the amino acid sequence of SEQ ID NO: 40 or CDR-L3 consisting of said amino acid sequence, (d) The heavy chain variable region (VH) includes the amino acid sequence of SEQ ID NO: 42 or CDR-H1 consisting of said amino acid sequence, the amino acid sequence of SEQ ID NO: 44 or CDR-H2 consisting of said amino acid sequence, and the amino acid sequence of SEQ ID NO: 46 or CDR-H3 consisting of said amino acid sequence, and / or the light chain variable region (VL) includes the amino acid sequence of SEQ ID NO: 48 or CDR-L1 consisting of said amino acid sequence, the amino acid sequence of SEQ ID NO: 50 or CDR-L2 consisting of said amino acid sequence, and the amino acid sequence of SEQ ID NO: 52 or CDR-L3 consisting of said amino acid sequence, (e) The heavy chain variable region (VH) includes the amino acid sequence of SEQ ID NO: 54 or CDR-H1 consisting of said amino acid sequence, the amino acid sequence of SEQ ID NO: 56 or CDR-H2 consisting of said amino acid sequence, and the amino acid sequence of SEQ ID NO: 58 or CDR-H3 consisting of said amino acid sequence, and / or the light chain variable region (VL) includes the amino acid sequence of SEQ ID NO: 60 or CDR-L1 consisting of said amino acid sequence, the amino acid sequence of SEQ ID NO: 62 or CDR-L2 consisting of said amino acid sequence, and the amino acid sequence of SEQ ID NO: 64 or CDR-L3 consisting of said amino acid sequence, or (f) The heavy chain variable region (VH) includes CDR-H1 which contains or consists of the amino acid sequence of SEQ ID NO: 66, CDR-H2 which contains or consists of the amino acid sequence of SEQ ID NO: 68, and CDR-H3 which contains or consists of the amino acid sequence of SEQ ID NO: 70, and / or the light chain variable region (VL) includes CDR-L1 which contains or consists of the amino acid sequence of SEQ ID NO: 72, CDR-L2 which contains or consists of the amino acid sequence of SEQ ID NO: 74, and CDR-L3 which contains or consists of the amino acid sequence of SEQ ID NO: 76. A monoclonal antibody or its antigen-binding fragment. 【0036】 In the present invention, the base sequence of the DNA encoding CDR-H1 includes, but is not limited to, the base sequence shown in SEQ ID NOs. 5, 17, 29, 41, 53, or 65. In the present invention, the base sequence of the DNA encoding CDR-H2 includes, but is not limited to, the base sequence shown in SEQ ID NOs: 7, 19, 31, 43, 55, or 67. In the present invention, the base sequence of the DNA encoding CDR-H3 includes, but is not limited to, the base sequence shown in SEQ ID NOs: 9, 21, 33, 45, 57, or 69. In the present invention, the base sequence of the DNA encoding CDR-L1 includes, but is not limited to, the base sequence shown in SEQ ID NOs: 11, 23, 35, 47, 59, or 71, or consists of such a base sequence. In the present invention, the base sequence of the DNA encoding CDR-L2 includes, but is not limited to, the base sequence shown in SEQ ID NOs: 13, 25, 37, 49, 61, or 73. In the present invention, the base sequence of the DNA encoding CDR-L3 includes, but is not limited to, the base sequence shown in SEQ ID NOs. 15, 27, 39, 51, 63, or 75, or consists of such a base sequence. 【0037】 In another embodiment, the antibodies of the present invention include, but are not limited to, the following monoclonal antibodies or their antigen-binding fragments. (a) A heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 78 or said amino acid sequence, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 80 or said amino acid sequence, (b) A heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 82 or said amino acid sequence, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 84, (c) A heavy chain variable region (VH) containing or consisting of the amino acid sequence of SEQ ID NO: 86 and a light chain variable region (VL) containing or consisting of the amino acid sequence of SEQ ID NO: 88, (d) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 90 or consisting thereof, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 92 or consisting thereof, (e) A heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 94 or said amino acid sequence and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 96 or said amino acid sequence, or (f) A heavy chain variable region (VH) containing or consisting of the amino acid sequence of SEQ ID NO: 98 and a light chain variable region (VL) containing or consisting of the amino acid sequence of SEQ ID NO: 100 A monoclonal antibody or its antigen-binding fragment. 【0038】 In the present invention, the base sequence of DNA encoding the heavy chain variable region includes, but is not limited to, the base sequence shown by SEQ ID NOs. 77, 81, 85, 89, 93, or 97. Furthermore, in the present invention, the base sequence of DNA encoding the light chain variable region includes, but is not limited to, the base sequence shown by SEQ ID NOs. 79, 83, 87, 91, 95, or 99. 【0039】 (3) Production of genetically modified antibodies One preferred embodiment of the antibody of the present invention is a genetically modified antibody. Examples of genetically modified antibodies are not limited to, but include chimeric antibodies, canine-type antibodies, human-type antibodies, and fully human antibodies. 【0040】 A chimeric antibody is an antibody produced by linking immunoglobulin gene fragments from different animal species. In this invention, examples of chimeric antibodies include human-type chimeric antibodies, but the types of animals from which the variable and constant regions of the chimeric antibody originate are not limited. A human-type chimeric antibody is, for example, an antibody in which the variable region of a mouse-derived antibody is linked (conjugated) to the constant region of a human-derived antibody (see Proc. Natl. Acad. Sci. USA 81, 6851-6855, (1984), etc.). Examples of subclasses of such human-derived antibodies include, but are not limited to, IgG1, IgG2, and IgG4. Chimeric antibodies can be easily constructed by genetic recombination technology. 【0041】 In the present invention, when producing humanized antibodies, a method known as CDR grafting (CDR transplantation) can be employed. CDR grafting is a method of producing a reconstituted variable region in which the complementarity-determining region (CDR) from the variable region of a mouse antibody is transplanted into the human variable region, and the framework region (FR) is derived from humans and the CDR is derived from mice. Next, these humanized reconstituted human variable regions are ligated to the human constant region. Such methods for producing humanized antibodies are well known in this field (see 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); Japanese Patent Publication No. 2828340, etc.). 【0042】 In the present invention, fully human antibodies can be produced, for example, using mammals capable of producing human antibodies, in accordance with known methods (e.g., WO96 / 9634096, WO98 / 24893). 【0043】 In the present invention, the human heavy chain constant region that can be used in chimeric antibodies and humanized antibodies includes, but is not limited to, a human heavy chain constant region that includes an amino acid sequence derived from the human IgG4 heavy chain constant region, for example, a human heavy chain constant region that includes or consists of the amino acid sequence shown in SEQ ID NO: 102. Furthermore, the human light chain constant region that can be used in chimeric antibodies and humanized antibodies includes, but is not limited to, a human light chain constant region that includes an amino acid sequence derived from the human IgG4 light chain constant region, for example, a human light chain constant region that includes or consists of the amino acid sequence shown in SEQ ID NO: 104. Furthermore, the DNA encoding the human heavy chain constant region includes, for example, DNA that includes or consists of the base sequence shown in SEQ ID NO: 101, and the DNA encoding the human light chain constant region includes, for example, DNA that includes or consists of the base sequence shown in SEQ ID NO: 103. 【0044】 In the present invention, chimeric antibodies and humanized antibodies can be produced using hybridomas or DNA or RNA extracted from said hybridomas as raw materials, in accordance with the known methods described above. Furthermore, the antibody-fused protein of the present invention can be produced by fusing the variable region of the antibody with other proteins using known genetic recombination methods. Alternatively, the fusion protein can be produced by crosslinking a monoclonal antibody with other proteins using a crosslinker. 【0045】 (4) Preparation of antibody-binding fragments The antigen-binding fragment of the antibody of the present invention binds to ACE2. The antigen-binding fragment of the antibody refers to a polypeptide containing a portion of the antigen-binding portion of the antibody of the present invention. Examples of antigen-binding fragments include, but are not limited to, scFv (single chain Fv), sc(Fv)2, Fab, Fab', diabody (dsFv), F(ab')2, and multispecific antibodies. The above antibody-binding fragments can be obtained by genetic engineering techniques or by known methods such as papain digestion and pepsin digestion. 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. Furthermore, Fab' can be obtained by cleaving the disulfide bond in the hinge region of F(ab')2. In the case of scFv, cDNA encoding the H chain V region and L chain V region of the antibody is obtained, and DNA encoding scFv is constructed. This DNA is inserted into an expression vector, and scFv can be produced by introducing this expression vector into a host organism and expressing it. In the case of diabody, cDNA encoding the V region of the antibody's heavy chain and light chain is obtained, and DNA encoding scFv is constructed such that the length of the peptide linker amino acid sequence is 8 residues or less. By inserting this DNA into an expression vector and introducing the expression vector into a host organism for expression, diabody can be produced. In the case of dsFv, cDNA encoding the H chain V region and L chain V region of the antibody is obtained, and DNA encoding dsFv is constructed. This DNA is inserted into an expression vector, and dsFv can be produced by introducing this expression vector into a host organism and expressing it. Multispecific antibodies, such as bispecific antibodies, bind to two or more antigens or epitopes and can be produced by various known methods (e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321). 【0046】 Antigen-binding fragments (peptides) containing CDRs are composed of at least one region of the VH or VL CDRs (CDR1-3). Antigen-binding fragments containing multiple CDRs can be conjugated directly or via a suitable peptide linker. Antigen-binding fragments containing CDRs can be produced by constructing DNA encoding the VH and VL CDRs of an antibody, inserting this DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryote to induce expression. Alternatively, 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). 【0047】 (5) binding affinity The affinity of an antibody for an antigen can generally be expressed by the equilibrium dissociation constant (KD), and a lower KD value indicates that the antibody has a higher affinity. Antibody affinity can be measured using known instruments and methods (e.g., Biacore®-3000 (GE Healthcare), ProteON XPR36 (Bio-Rad), etc.). 【0048】 (6) Antibodies that compete with the reference antibody The present invention provides a monoclonal antibody or its antigen-binding fragment that can neutralize the binding of coronavirus to ACE2 and competes with any of the following reference antibodies or their antigen-binding fragments for binding to ACE2. In the present invention, the following monoclonal antibodies can be used as reference antibodies. (a) A monoclonal antibody in which the heavy chain variable region (VH) contains or comprises the amino acid sequence of SEQ ID NO: 6, CDR-H1, CDR-H2, and CDR-H3, and / or the light chain variable region (VL) contains or comprises the amino acid sequence of SEQ ID NO: 12, CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of SEQ ID NO: 16. (b) A monoclonal antibody in which the heavy chain variable region (VH) comprises 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 the light chain variable region (VL) comprises CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 24, 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) A monoclonal antibody in which the heavy chain variable region (VH) includes CDR-H1 containing or consisting of the amino acid sequence of SEQ ID NO: 30, CDR-H2 containing or consisting of the amino acid sequence of SEQ ID NO: 32, and CDR-H3 containing or consisting of the amino acid sequence of SEQ ID NO: 34, and / or the light chain variable region (VL) includes CDR-L1 containing or consisting of the amino acid sequence of SEQ ID NO: 36, CDR-L2 containing or consisting of the amino acid sequence of SEQ ID NO: 38, and CDR-L3 containing or consisting of the amino acid sequence of SEQ ID NO: 40. (d) A monoclonal antibody in which the heavy chain variable region (VH) includes or comprises the amino acid sequence of SEQ ID NO: 42, CDR-H1, CDR-H2, and CDR-H3, and / or the light chain variable region (VL) includes or comprises the amino acid sequence of SEQ ID NO: 48, CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of SEQ ID NO: 52. (e) A monoclonal antibody in which the heavy chain variable region (VH) contains or comprises the amino acid sequence of SEQ ID NO: 54, CDR-H1 containing or comprising the amino acid sequence of SEQ ID NO: 56, CDR-H2 containing or comprising the amino acid sequence of SEQ ID NO: 58, and / or the light chain variable region (VL) contains or comprises the amino acid sequence of SEQ ID NO: 60, CDR-L1 containing or comprising the amino acid sequence of SEQ ID NO: 62, CDR-L2 containing or comprising the amino acid sequence of SEQ ID NO: 64, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 64. (f) A monoclonal antibody in which the heavy chain variable region (VH) comprises CDR-H1 containing or consisting of the amino acid sequence of SEQ ID NO: 66, CDR-H2 containing or consisting of the amino acid sequence of SEQ ID NO: 68, and CDR-H3 containing or consisting of the amino acid sequence of SEQ ID NO: 70, and / or the light chain variable region (VL) comprises CDR-L1 containing or consisting of the amino acid sequence of SEQ ID NO: 72, CDR-L2 containing or consisting of the amino acid sequence of SEQ ID NO: 74, and CDR-L3 containing or consisting of the amino acid sequence of SEQ ID NO: 76. (g) A monoclonal antibody comprising a heavy chain variable region (VH) containing or consisting of the amino acid sequence of SEQ ID NO: 78 and a light chain variable region (VL) containing or consisting of the amino acid sequence of SEQ ID NO: 80, (h) A monoclonal antibody comprising a heavy chain variable region (VH) containing or consisting of the amino acid sequence of SEQ ID NO: 82 and a light chain variable region (VL) containing or consisting of the amino acid sequence of SEQ ID NO: 84, (i) A monoclonal antibody comprising a heavy chain variable region (VH) containing or consisting of the amino acid sequence of SEQ ID NO: 86 and a light chain variable region (VL) containing or consisting of the amino acid sequence of SEQ ID NO: 88, (j) A monoclonal antibody comprising a heavy chain variable region (VH) containing or consisting of the amino acid sequence of SEQ ID NO: 90 and a light chain variable region (VL) containing or consisting of the amino acid sequence of SEQ ID NO: 92, (j) A monoclonal antibody comprising the amino acid sequence of SEQ ID NO: 94 or a heavy chain variable region (VH) consisting of said amino acid sequence and the amino acid sequence of SEQ ID NO: 96 or a light chain variable region (VL) consisting of said amino acid sequence, or (k) A monoclonal antibody comprising a heavy chain variable region (VH) containing or consisting of the amino acid sequence of SEQ ID NO: 98 and a light chain variable region (VL) containing or consisting of the amino acid sequence of SEQ ID NO: 100. 【0049】 In the present invention, "competing" with respect to binding to ACE2 means that the antibody of the present invention or its antigen-binding fragment inhibits (e.g., suppresses) the binding of the reference antibody or its antigen-binding fragment to ACE2, or that the binding of the antibody of the present invention or its antigen-binding fragment to ACE2 is inhibited (e.g., suppressed) by the reference antibody or its antigen-binding fragment. Whether the antibody of the present invention or its antigen-binding fragment competes with either the reference antibody or its antigen-binding fragment for binding to ACE2 can be investigated using known methods, such as competitive RIA, competitive EIA, competitive ELISA (competitive ELISA using polypeptides, competitive cell ELISA), epitope binning, etc. Antibody-based competition testing is an established technique in the field of antibody technology to determine whether a test antibody or its antigen-binding fragment competes with a reference antibody for binding to the same or adjacent epitopes 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 term "adjacent epitopes" refers to epitopes that are so close to the test antibody or its antigen-binding fragment and the reference antibody that steric interference occurs in their binding. For example, as shown in Example 6 of this specification, the four monoclonal antibodies OMRad004G05, OMRad028D08, OMRad031H08, and OMRad045G03 are competing antibodies for binding to ACE2, and furthermore, as shown in Example 10 of this specification, these competing antibodies have equivalent neutralizing activity against coronavirus binding to or infection of cells. On the other hand, OMRad004G05 and OMRad028D08 and OMRad031H08 and OMRad045G03 have different reactivity to mouse ACE2 (Example 7), and therefore may bind to different epitopes. 【0050】 As shown in Example 6 of this specification, in order to investigate whether the antibody or its antigen-binding fragment of the present invention competes with the above-mentioned reference antibody or its antigen-binding fragment for binding to ACE2, it is not necessary to have information about the structure of the antibody or the structure of the epitope (for example, what amino acid sequence the CDR and variable region have, whether it is linear or three-dimensional, etc.). Furthermore, by performing a series of steps, including producing an anti-ACE monoclonal antibody based on a conventional method and selecting an antibody that competes with the reference antibody described herein for binding to ACE2, it is possible to obtain antibodies that compete with the reference antibody described herein for binding to ACE2, in addition to the antibodies whose specific structures are identified in the examples herein. In other words, a monoclonal antibody or antigen-binding fragment of the present invention that competes with either the above-mentioned reference antibody or its antigen-binding fragment for binding to ACE2 can be obtained by a person skilled in the art without excessive experimentation by performing a competition test using the above-mentioned reference antibody. 【0051】 4. Pharmaceutical composition The pharmaceutical composition of the present invention contains the antibody or its antigen-binding fragment described in "3. The Antibody of the Present Invention" above as an active ingredient, and is a pharmaceutical composition for treating or preventing coronavirus infection. The coronavirus infections targeted by the pharmaceutical compositions of the present invention refer to diseases or symptoms caused by coronavirus infection. Such coronavirus infections include, for example, diseases or symptoms caused by coronavirus infection that infects cells via ACE2. "Coronavirus that infects cells via ACE2" is not limited to, but includes, for example, SARS-CoV-1 and SARS-CoV-2, with SARS-CoV-2 being preferred. "Diseases or symptoms caused by infection with coronaviruses that infect cells via ACE2" include, for example, diseases or symptoms related to organs or tissues where ACE2 is primarily expressed, such as the lungs, digestive system, heart, blood vessels, eyes, kidneys, cerebral cortex, amygdala, brainstem, and medulla oblongata, as well as diseases or symptoms based on immune responses. Such diseases or symptoms include, but are not limited to, respiratory diseases, fever, malaise, chills, pain, taste or smell disorders, rashes, gastrointestinal symptoms, speech disorders, cognitive impairment, and cardiovascular symptoms. The pharmaceutical composition containing the antibody of the present invention is effective in treating or preventing various diseases or symptoms associated with coronavirus infection by inhibiting the infection of coronaviruses that infect cells via ACE2. 【0052】 In the present invention, "treatment" means reducing the symptoms of a disease by exposing a subject to (e.g., administering) the antibody or its antigen-binding fragment or a pharmaceutical composition containing the same (hereinafter also referred to as "the antibody and pharmaceutical composition of the present invention") after the onset of the disease, compared to not exposing the subject to the treatment. It does not necessarily mean completely suppressing the symptoms of the disease. In the present invention, "onset of disease" includes not only the appearance of symptoms of the disease on the body, but also a positive result in a test for coronavirus infection (such as a PCR test) even if no symptoms are present. Furthermore, in the present invention, "treatment" of coronavirus infection includes suppression or inhibition of the worsening of coronavirus infection. In the present invention, "prevention" means suppressing or inhibiting the onset of coronavirus infection, or suppressing or inhibiting its severity, but does not necessarily mean completely suppressing the onset of the disease. In the present invention, "prevention" includes reducing the symptoms after the onset of the disease by exposing the subject / target to the pharmaceutical composition of the present invention (e.g., administering it) before the onset of the disease, compared to not being exposed. 【0053】 The pharmaceutical composition of the present invention may contain a monoclonal antibody bound to ACE2 or an antigen-binding fragment thereof, as well as a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to any carrier suitable for the pharmaceutical composition (liposomes, lipid vesicles, micelles, etc.), diluents, excipients, wetting agents, buffers, suspending agents, lubricants, adjuvants, emulsifiers, disintegrants, absorbents, preservatives, surfactants, colorants, flavorings, or sweeteners. 【0054】 The antibodies and pharmaceutical compositions of the present invention can take the form of injections, lyophilized products, tablets, hard capsules, soft capsules, granules, powders, pills, syrups, suppositories, poultices, ointments, creams, eye drops, and other dosage forms. Liquid formulations such as injections may also be in the form of a powder (e.g., lyophilized powder) that is dissolved in physiological saline or the like before use. 【0055】 The antibodies and pharmaceutical compositions of the present invention can be administered topically or systemically by any means known to those skilled in the art. The pharmaceutical compositions of the present invention can be administered orally or parenterally. Parenteral administration can include intratissue administration (subcutaneous, intraperitoneal, intramuscular, intravenous, etc.), intradermal administration, topical administration (transdermal, etc.), or rectal administration. The pharmaceutical compositions of the present invention can be administered in a form suitable for these administration routes. 【0056】 The dosage of the antibody and pharmaceutical composition of the present invention varies depending on factors such as the subject's age, weight, health status, sex, symptoms, animal species, route of administration, number of administrations, and dosage form, and specific administration procedures can be determined by those skilled in the art. For the treatment of coronavirus infection, the dosage of the antibody of the present invention is, for example, 0.1 mg to 100 mg per kg of body weight per day, preferably 1 mg to 15 mg per day, more preferably 2 to 12 mg per day, but is not limited thereto. The dosage can be 1 to 5 times per day. 【0057】 The timing of administration of the antibody and pharmaceutical composition of the present invention can be appropriately determined according to the symptoms, and multiple doses can be administered simultaneously or separately at intervals. Furthermore, the pharmaceutical composition of the present invention may be administered to subjects / targets before the onset of the disease, or after the onset of the disease. 【0058】 The pharmaceutical composition of the present invention can be administered to mammals as subjects / targets. Examples of mammals include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, goats, pigs, sheep, cattle, horses, monkeys, and humans. 【0059】 5. Treatment or prevention methods for coronavirus infection In the present invention, coronavirus infection can be treated or prevented by administering a monoclonal antibody that binds to ACE2, its antigen-binding fragment, or a pharmaceutical composition containing the same to a subject / target. That is, the present invention provides a method for treating or preventing coronavirus infection, comprising the step of administering a therapeutically effective amount of the antibody or its antigen-binding fragment, or a pharmaceutical composition containing the same, to a subject / target. The therapeutically effective amount of the antibody or its antigen-binding fragment, or a pharmaceutical composition containing the same, varies depending on factors such as the subject / target's age, weight, health status, sex, symptoms, route of administration, number of administrations, and dosage form. Those skilled in the art can easily determine the therapeutically effective amount necessary for treating or preventing coronavirus infection. In the present invention, "subject" or "target" includes subjects / targets who require treatment or prevention of coronavirus infection. Furthermore, the mammals targeted for treatment or prevention as "subjects" or "targets" are as described above. In the present invention's method for treating or preventing coronavirus infection, the terms "coronavirus infection," "treatment," and "prevention" are described above. Furthermore, the dosage form, route of administration, dosage, and timing of administration of the antibody or its antigen-binding fragment or the pharmaceutical composition containing it are also described above. 【0060】 6. Inhibitors that bind coronavirus to ACE2 The antibody of the present invention neutralizes (inhibits) the binding of coronavirus to ACE2, and therefore can inhibit coronavirus infection caused by such binding. In other words, the present invention provides a coronavirus-ACE2 binding inhibitor containing the antibody described in "3. The Antibody of the Present Invention" above or its antigen-binding fragment 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 administration form, additives, route of administration, target of administration, dosage, etc., can be appropriately selected in accordance with the description in "4. Pharmaceutical Compositions" above. However, the inhibitor of the present invention may contain only the antibody of the present invention or its antigen-binding fragment. 【0061】 7. Use of antibodies The antibody or antigen-binding fragment thereof of the present invention can be used in methods for treating or preventing coronavirus infection, or in the manufacture of pharmaceuticals for treating or preventing coronavirus infection. That is, the present invention provides the antibody or antigen-binding fragment thereof for use in methods for treating or preventing coronavirus infection. The present invention also provides the antibody or antigen-binding fragment thereof for use in the manufacture of pharmaceuticals for treating or preventing coronavirus infection. Furthermore, the present invention provides the antibody or antigen-binding fragment thereof for use in the manufacture of inhibitors for binding between coronavirus and ACE2. 【0062】 8. Combination therapy The pharmaceutical composition of the present invention can be used for combination administration with at least one other therapeutic agent. Examples of other therapeutic agents used in the present invention include remdesivir, dexamethasone, favipiravir, nafamostat, camostat, ivermectin, tricizumab, and baricitinib. 【0063】 By administering the pharmaceutical composition of the present invention in combination with at least one other therapeutic agent, a more superior effect can be expected compared to using each agent alone. This superior effect includes maintaining therapeutic efficacy while reducing side effects compared to conventional methods. In the present invention, "combined administration" means administering the pharmaceutical composition of the present invention and at least one of the other therapeutic agents simultaneously or separately. "Simultaneous administration" means administering at the same time in a single administration schedule, but the administration times do not need to be exactly the same. "Separate administration" means administering at different times in a single administration schedule. The administration form, route of administration, and 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 in accordance with the description in "4. Pharmaceutical Composition" above. Furthermore, the administration forms or dosages of the drugs used in combination may differ from each other and can be appropriately adjusted depending on the combination used. When the pharmaceutical composition of the present invention is used in combination with other therapeutic agents, the dosage can be appropriately reduced. Therefore, in combinations of the pharmaceutical composition of the present invention and other therapeutic agents, (i) an effective amount of the pharmaceutical composition of the present invention and an effective amount of another therapeutic agent, (ii) an effective amount of the pharmaceutical composition of the present invention and an ineffective amount of another therapeutic agent, (iii) an ineffective amount of the pharmaceutical composition of the present invention and an effective amount of another therapeutic agent, (iv) Ineffective amount of the pharmaceutical composition of the present invention and ineffective amount of other therapeutic agents A combination of these can be adopted. Even if one or both of the pharmaceutical composition and other therapeutic agents are used in an ineffective amount, if they can exert a pharmacological effect when used together, they may be administered in such a manner. 【0064】 9. Reagents, kits The antibody or antigen-binding fragment thereof of the present invention can be included in a reagent or kit. That is, the present invention provides reagents and kits comprising the antibody or antigen-binding fragment thereof of the present invention. The reagents and kits of the present invention can be used, for example, as reagents or kits for testing against coronavirus. In the reagents and kits of the present invention, the antibody or antigen-binding fragment of the present invention may be made easier to handle by methods such as freezing, and then used as is or mixed with known pharmaceutically acceptable carriers such as excipients, bulking agents, binders, lubricants, and known additives (including buffers, isotonic agents, chelating agents, colorants, preservatives, fragrances, flavoring agents, sweeteners, etc.). The kit of the present invention may also include, in addition to the antibody of the present invention or its antigen-binding fragment, a buffer solution, an enzyme solution, a secondary antibody, a diluent, instructions for use, and the like. 【0065】 The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Examples] 【0066】 Production of monoclonal antibodies (1) immunogen In this embodiment, either (i) mouse-derived cells stably overexpressing human ACE2, or (ii) a partial polypeptide of ACE2 containing an amino acid sequence (SEQ ID NO: 106) consisting of amino acid residues from the 19th to the 85th amino acid residues counting from the N-terminus of human ACE2 (SEQ ID NO: 2), was used as the immunogen. Mouse-derived cells (4T1 cells) that stably overexpressed human ACE2 as described in (i) above were produced by the following method. Specifically, first, the full-length DNA encoding human ACE2 was incorporated into a lentiviral vector and introduced into 293T cells along with a packaging plasmid and an envelope plasmid. Recombinant lentivirus produced in the culture supernatant of these cells was used to infect 4T1 cells, thereby producing cells that stably express human ACE2 on the cell membrane. The partial polypeptide described in (ii) above was prepared by introducing a vector containing the DNA encoding the partial polypeptide (SEQ ID NO: 105) into E. coli, expressing it in E. coli, and then purifying it. 【0067】 (2) immunity ICR mice or BALB / c mice (both from Sankyo Lab Services Co., Ltd.) were used as immunized animals. Immunization was performed by administering the mouse-derived cells described in (i) above into the peritoneal cavity of BALB / c mice, and by administering a mixture of the mouse-derived cells described in (i) above, the partial polypeptide described in (ii) above, and the adjuvant into the peritoneal cavity of ICR mice. 【0068】 (3) Hybridoma fabrication The immune response was monitored by cell ELISA using the mouse-derived cells (4T1 cells) described in (i) above, or by ELISA using the partial polypeptide described in (ii) above. Once the increase in the immune response stagnated (i.e., the antibody titer had risen sufficiently), spleen cells were harvested from the immunized mice and fused with mouse myeloma cells P3X63Ag8.653 (ATCC) to create a hybridoma cell line. 【0069】 (4) screening Using the culture supernatant of hybridomas, screening was performed using cell ELISA with mouse-derived cells (4T1 cells) as described in (i) above, or ELISA with the partial polypeptide described in (ii) above, to select hybridoma cell lines that produce monoclonal antibodies that bind to human ACE2. This allowed us to produce a monoclonal antibody that binds to human ACE2. [Examples] 【0070】 Sequencing of anti-ACE2 monoclonal antibodies From the anti-ACE2 monoclonal antibodies obtained in Example 1, several hybridoma cell lines that produced monoclonal antibodies with high reactivity in cell ELISA or ELISA using partial polypeptides were selected. For the selected hybridoma cell lines, the antibody gene was cloned, and the amino acid sequence of the antibody was determined by analyzing its gene sequence. The nucleotide and amino acid sequences of the variable regions of representative antibodies (OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, OMRad045G03, OMRad052B05) obtained from the numerous anti-ACE2 monoclonal antibodies obtained in Example 1 are shown in the table below. Of these antibodies, OMRad004G05 and OMRad028D08 were produced from hybridomas obtained by immunizing BALB / c mice with the mouse-derived cells described in (i) above, while OMRad031H08, OMRad044E11, OMRad045G03, and OMRad052B05 were produced from hybridomas obtained by immunizing ICR mice with a mixture of the mouse-derived cells described in (i) above and the partial polypeptide described in (ii) above. The CDR of each antibody was identified according to the Kabat numbering system. Furthermore, by performing the tests and evaluations shown in the following examples on antibodies other than the six types of antibodies mentioned above, it is possible to obtain antibodies with equivalent or superior activity or effects to these six types of antibodies. 【0071】 [Table 1A] [Table 1B] 【0072】 [Table 2A] [Table 2B] 【0073】 [Table 3A] [Table 3B] 【0074】 [Table 4A] [Table 4B] 【0075】 [Table 5A] [Table 5B] 【0076】 [Table 6A] [Table 6B] [Examples] 【0077】 Inhibition of the binding of spike protein RBD to ACE2 using the anti-ACE2 monoclonal antibody of the present invention Mouse 4T1 cells stably overexpressing human ACE2 were seeded at 10,000 cells / well in each well of a collagen-coated 96-well glass-bottom plate (Greiner Bio-One). The plate was incubated at 37°C and 5% CO2 for 2 days. After removing the culture medium, 200 μL / well of RPMI1640 medium containing 4 μg / mL of the anti-ACE2 monoclonal antibody prepared in Example 1 was added, and the plate was incubated on ice for 1 hour. After removing the antibody-containing medium and washing twice with RPMI1640 medium, 200 μL / well of RPMI 1640 medium containing SARS-CoV-2 spike protein RBD expressed in mammalian cells (CHO cells) at a concentration of 0.5 μg / mL was added, and the mixture was 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. 【0078】 After washing twice with RPMI1640 medium, 200 μL / well of fluorescently labeled anti-His tag antibody (MBL) diluted to 1 μg / mL in RPMI 1640 medium was added, and the cells were incubated in the dark at room temperature for 30 minutes. 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. 【0079】 The results are shown in Figure 1. In the sample to which the anti-ACE2 monoclonal antibody was not added, a fluorescence signal was detected (the "Antidoby(-)" panel in Figure 1). This indicates that in this sample, ACE2 on the cell membrane bound to RBD, and the fluorescently labeled anti-His tag antibody bound to the His tag on RBD, resulting in the detection of the fluorescence signal. 【0080】 In contrast, no fluorescence signal was detected in the samples to which the anti-ACE2 monoclonal antibody prepared in Example 1 was added (in Figure 1, "OMRad052B05", "OMRad004G05", "OMRad028D08", "OMRad031H08", "OMRad044E11", and "OMRad045G03"). This indicates that in these samples, the anti-ACE2 monoclonal antibody prepared in Example 1 bound to ACE2 on the cell membrane, thereby inhibiting (neutralizing) the binding of ACE2 to RBD. As a result, the fluorescently labeled anti-His tag antibody could not bind to RBD, and therefore the fluorescence signal of the anti-His tag antibody was not detected. 【0081】 In other words, the results of this embodiment demonstrate that the antibody of the present invention can bind to ACE2 and neutralize the binding between ACE2 and RBD. [Examples] 【0082】 Production of chimeric antibodies Of the antibodies obtained in Example 1, a chimeric antibody was created for OMRad052B05 by replacing the constant region with the amino acid sequence of human IgG4. The amino acid sequences of the heavy chain constant region and the light chain constant region used in this example are shown in SEQ ID NOs: 102 and 104, respectively. Specifically, chimeric antibodies were produced using the following method. First, the heavy chain variable region gene and light chain variable region gene of the anti-ACE2 antibody were cloned from the hybridoma producing the anti-ACE2 antibody prepared in Example 1. Next, these genes (DNA) were ligated to the nucleotide sequences of the human IgG4 heavy chain constant region gene (SEQ ID NO: 101) or the light chain (κ chain) constant region gene (SEQ ID NO: 103), respectively. To amplify the heavy chain variable region gene, PCR was performed using a primer (SEQ ID NO: 107) containing the 5' terminal sequence of the heavy chain variable region, the Kozak sequence, and the restriction enzyme EcoRI sequence, and an antisense primer (SEQ ID NO: 108) containing the complementary sequence of the 3' terminal sequence and the restriction enzyme NheI sequence. Furthermore, to amplify the light chain variable region gene, PCR was performed using a primer (SEQ ID NO: 109) containing the 5' terminal sequence of the light chain variable region gene and the restriction enzyme EcoRI sequence, and an antisense primer (SEQ ID NO: 110) containing the complementary sequence of the 3' terminal sequence and the restriction enzyme BsiWI sequence. The base sequences of the primers used in the above PCR are shown in the table below. 【0083】 [Table 7] 【0084】 The resulting amplification products were treated with restriction enzymes EcoRI and NheI, or EcoRI and BsiWI, and incorporated into the EcoRI-NheI site of a human IgG4 heavy chain constant region expression plasmid (pFUSEss-CHIg-hG4; InvivoGen) or the EcoRI-BsiWI site of a human Ig light chain (κ chain) constant region expression plasmid (pFUSE2ss-CLIg-hk; InvivoGen). pFUSEss-CHIg-hG4 contained the human IgG4 heavy chain constant region gene, and pFUSE-CLIg-hk contained the human Ig light chain (κ chain) constant region gene. The NheI restriction enzyme sequence ligated the mouse heavy chain variable region and the human heavy chain constant region, and the BsiWI restriction enzyme sequence ligated the mouse light chain variable region and the human light chain constant region. This example demonstrated the ability to produce a chimeric antibody based on the antibody (OMRad052B05) prepared in Example 1. [Examples] 【0085】 Production of chimeric antibodies Following the same procedure as in Example 4, chimeric antibodies were prepared by replacing the constant region of OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, and OMRad045G03, obtained in Example 1, with the amino acid sequence of human IgG4. However, the primers listed in the table below were used for PCR to amplify the variable region. 【0086】 [Table 8] In this example, we were able to produce chimeric antibodies based on the mouse antibodies OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, and OMRad045G03, which were obtained in Example 1. [Examples] 【0087】 Competitive Inhibition Test In this example, among the antibodies obtained in Example 1, six types of anti-ACE2 mouse antibodies (OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, OMRad045G03, OMRad052B05) selected as representative examples, and chimeric antibodies prepared in Examples 4 and 5 based on these antibodies were used to test whether competing antibodies could be obtained among these antibodies for binding to ACE2. This competitive inhibition test was performed by cell-based ELISA referring to a known method (Sindy Liao-Chan, et al. J Immunol Methods. 2014 Mar;405:1-14, PMID: 24380699). 【0088】 In this example, each of the above anti-ACE2 mouse antibodies was used as a reference antibody, and each of the above chimeric antibodies was used as a test antibody (detection antibody for the target antibody). First, a certain type of reference antibody was reacted with 4T1 cells expressing human ACE2 (4T1-hACE2 cells), and then all the test antibodies were reacted one by one. A schematic diagram of this test is shown in Figure 2. Specifically, 4T1-hACE2 cells were seeded at 1 x 10 4 cells / well in each well of a 96-well cell culture plate and cultured for 2 days. Using a blocking buffer (PBS(-) containing 3% BSA and 0.2% bovine serum), a solution containing each anti-human ACE2 mouse monoclonal antibody (reference antibody) at 30 μg / mL was prepared. The culture medium in the 96-well plate was removed, and the diluted reference antibody solution at 50 μL / well was added, and 4 oThe cells were allowed to stand in 1°C for 1 hour. After washing the wells three times with 250 μL of washing buffer (PBS(-) containing 0.1% BSA), 50 μL / well of 2.5% formalin was added, and the cells were fixed by standing at room temperature for 10 minutes. After washing three times with 250 μL of washing buffer, 50 μL / well of 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 each well, and the cells were 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 the cells were allowed to stand at room temperature for 5 minutes to inactivate endogenous peroxidase. After washing three times with 250 μL of washing buffer, 50 μL / well of HRP-labeled anti-human IgG-Fc antibody (Bethyl, A80-304P), diluted 10,000-fold with blocking buffer, was added and allowed to stand at room temperature for 1 hour. After washing three times with 250 μL of blocking buffer, 100 μL / well of OPD substrate solution was added and the mixture was allowed to react at room temperature for about 10 minutes. The reaction was stopped by adding 100 μL of 1.5 M sulfuric acid solution and stirring, and the absorbance at 490 nm was measured using a spectrophotometer. The obtained absorbance was corrected by setting the value of the well using only each chimeric antibody prepared as a control to 100. 【0089】 The results are shown in Figure 3. Four chimeric antibodies, OMRad004G05, OMRad028D08, OMRad031H08, and OMRad045G03, showed inhibitory effects comparable to the relative values ​​indicating competition between a reference antibody (anti-ACE2 mouse antibody) and a test antibody (chimeric antibody) based on the same clone (for example, 22.8 when both the reference antibody and the test antibody are OMRad004G05) (Group 1). Additionally, two other antibodies, OMRad044E11 and OMRad052B05, showed inhibitory effects comparable to the relative values ​​indicating competition between a reference antibody and a test antibody based on the same clone (Group 2). Antibodies in Group 1 and Group 2 competed slightly with each other. 【0090】 This demonstrated that antibodies belonging to Group 1 and Group 2 compete with other antibodies belonging to the same group. In this embodiment, the above-mentioned anti-ACE2 mouse antibody was used as the reference antibody and each of the above-mentioned chimeric antibodies was used as the test antibody. However, it is also possible to use each of the above-mentioned chimeric antibodies as the reference antibody and screen for anti-ACE2 mouse antibodies that compete with it. In summary, this example demonstrates that an antibody can be obtained that competes with the antibody obtained in Example 1 and the chimeric antibody produced based thereon in terms of binding to ACE. 【0091】 In the following examples, the function and activity of each antibody were evaluated, and it was also assessed whether antibodies belonging to Group 1 and Group 2 had equivalent activity to other antibodies belonging to the same group. [Examples] 【0092】 Epitope identification testing 1. Generation of mouse / human fusion ACE2-expressing cells Previous reports have indicated that the SARS-CoV-2 spike protein interacts with the N-terminus of human ACE2, and that SARS-CoV-2 cannot recognize mouse ACE2. Therefore, we investigated whether six types of anti-ACE2 mouse antibodies (OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, OMRad045G03, OMRad052B05) obtained in Example 1 could bind to the N-terminal region of human ACE2. Specifically, we created a fusion ACE2 gene (polynucleotide) in which 1-120 residues from the N-terminus of human ACE2 were replaced with those of mouse ACE2, and mouse / human fusion ACE2-expressing cells expressing this gene were created using the following procedure. First, a fusion ACE2 gene construct was created using NEBuilder Hi-Fi DNA Assembly (New England Biolabs) by combining a polynucleotide containing the 1st to 360th bases from the 5' end of the mouse ACE2 encoding sequence with a polynucleotide containing the 361st to 2418th bases from the 5' end of the human ACE2 encoding sequence. This construct was then incorporated into a lentiviral vector. This lentiviral construct, along with a packaging plasmid and an envelope plasmid, was introduced into 293T cells, and the recombinant lentivirus produced in the culture supernatant was used to infect mouse 4T1 cells. In this way, 4T1 cells expressing human-mouse / human fusion ACE2 on the cell membrane (mouse / human fusion ACE2-expressing 4T1 cells) were obtained. In mouse / human fusion ACE2, the amino acid sequence of mouse ACE2 is from residues 1-120 from the N-terminus, and the amino acid sequence of human ACE2 is from residue 121 onwards. 【0093】 2. Immune cell staining 4T1 cells, human ACE2-expressing 4T1 cells, mouse ACE2-expressing 4T1 cells, and mouse / human fusion ACE2-expressing 4T1 cells were placed in a glass-bottom 96-well plate for fluorescence observation in a 3.2 x 10⁶ arrangement. 3 Cells were seeded in each well and cultured for 2 days. After removing the culture medium by placing the plate on ice, 200 mL of RPMI1640 medium containing 20 mg / mL of anti-ACE2 mouse monoclonal antibody was added and 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 thorough washing with PBS, the samples were observed under a fluorescence microscope. 【0094】 3. result The results are shown in Figure 4. All six types of anti-ACE2 monoclonal antibodies bound to human ACE2. Two types of mice, OMRad031H08 and OMRad045G03, bound to mouse ACE2, but OMRad004G05, OMRad028D08, OMRad044E11, and OMRad052B05 did not bind to mouse ACE2. OMRad004G05, OMRad028D08, OMRad031H08, and OMRad045G03 bound to mouse / human fusion ACE2, but OMRad044E11 and OMRad052B05 did not bind to mouse / human fusion ACE2 (Figure 4). Furthermore, none of the antibodies bound to 4T1 cells that did not express ACE2. The results are summarized in the table below (in the table, "Y" indicates binding, "N" indicates non-binding, "Total Length" indicates the total length of the extracellular region, and "aa" indicates the amino acid position). 【0095】 [Table 9] 【0096】 As shown in the table above, OMRad044E11 and OMRad052B05 bound to human ACE2, but did not bind to residues 1-120 of mouse ACE2 or residues 121-740 of human ACE2. This indicates that these antibodies primarily bind to the region of human ACE2 containing residues 1-120 from the N-terminus. 【0097】 Furthermore, OMRad004G05 and OMRad028D08 bound to the entire human ACE2 molecule and residues 121-740 of human ACE2, but did not bind to mouse ACE2. This suggests that these antibodies primarily bind to the extracellular region of human ACE2 from residue 121 onward (up to serine residue 740) at locations or regions that can distinguish between human ACE2 and mouse ACE2 (for example, regions with low homology between human ACE2 and mouse ACE2, or locations with different amino acids in the two). 【0098】 On the other hand, since OMRad031H08 and OMRad045G03 reacted with human ACE2, mouse ACE2, and human / mouse fusion ACE2, this study could not narrow down the region to which these antibodies bind. However, considering that OMRad031H08 and OMRad045G03 competed with OMRad004G05 and OMRad028D08, which belong to the same Group 1, in Example 6, along with the results of this study, it is suggested that these antibodies bind to a region of human ACE2, mainly containing amino acid residues 121-740, at a position or region that does not distinguish between human ACE2 and mouse ACE2 (for example, a region with high homology between human ACE2 and mouse ACE2, or a position where both have the same amino acid). 【0099】 The similar results observed between OMRad004G05 and OMRad028D08, OMRad031H08 and OMRad045G03, and OMRad044E11 and OMRad052B05 regarding their reactivity to ACE2 are consistent with the similarity of the amino acid sequences of the CDRs of these antibodies. 【0100】 Furthermore, it was shown that the Group 1 antibodies (OMRad004G05, OMRad028D08, OMRad031H08, OMRad045G03) shown in Example 6 bind mainly to the region containing amino acid residues 121-740, similar to other antibodies belonging to the same group, while the Group 2 antibodies (OMRad044E11, OMRad052B05) bind mainly to the region containing amino acid residues 1-120 from the N-terminus, similar to the other antibody in the same group. [Examples] 【0101】 Binding affinity evaluation test 1. method The affinity of six anti-ACE2 mouse antibodies (OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, OMRad045G03, OMRad052B05) obtained in Example 1 for human ACE2 was evaluated 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 values. Specifically, the tests were conducted according to the following procedure. First, 1 x 10⁶ 4T1 cells and 4T1 cells that stably express human ACE2 (hereinafter referred to as "4T1-hACE2") were prepared. 4Cells were seeded in each well of a 96-well cell culture plate at a rate of cells / well and cultured for 2 days. Using blocking buffer (PBS(-) containing 3% BSA and 0.2% bovine serum), solutions containing the six types of mouse antibodies and chimeric antibodies at 100 μg / mL were prepared, and a 2-fold dilution series was created using the blocking buffer. The culture medium was removed from the 96-well plate, and antibody solutions diluted to 50 μL / well were added and allowed to stand at 4°C for 1 hour. The wells were washed three times with 250 μL of washing buffer (PBS(-) containing 0.05% Tween20), and then 10% formalin was added at 50 μL / well. Cells were fixed by allowing to stand at room temperature for 10 minutes. Endogenous peroxidase was inactivated by washing twice with washing buffer, and then adding PBS(-) containing 3% hydrogen peroxide at 50 μL / well. After washing three times with 250 μL of washing buffer, 50 μL / well of either 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 and allowed to stand at room temperature for 1 hour. After washing three times with 250 μL of blocking buffer, 100 μL / well of OPD substrate solution was added and the mixture was reacted at room temperature for approximately 10 minutes. The reaction was stopped by adding 100 μL of 1.5 M sulfuric acid solution and stirring, and the absorbance at 490 nm was measured using a spectrophotometer. The obtained data was analyzed using GraphPad Prism to determine the Kd value. 【0102】 2. result As a result, in cell-based ELISA, all six types of mouse antibodies and chimeric antibodies showed a concentration-dependent increase in absorbance in 4T1-hACE2 cells expressing human ACE2 (Figures 5A and 5B). On the other hand, as a control, each chimeric antibody was reacted with 4T1 cells that do not express human ACE2, but no concentration-dependent increase in absorbance was observed (Figure 5C). These results demonstrate that all six types of mouse antibodies and chimeric antibodies bind with high specificity to native human ACE2 expressed on cells. Furthermore, the Kd values ​​in this test ranged from 1.97 nM to 5.81 nM for mouse antibodies and from 0.86 nM to 2.62 nM for chimeric antibodies, as shown in the table below. Furthermore, because different secondary antibodies are used, it is not possible to directly compare the Kd values ​​of the mouse antibody and the chimeric antibody. 【0103】 [Table 10] [Examples] 【0104】 Inhibition study on the binding of mutant spike protein RBD to ACE2 In this example, we analyzed the extent to which the six types of chimeric antibodies described above inhibited the binding of mutant RBD to human ACE2-expressing cells. 1. Production of mutant spike protein RBD In this example, mutant spike protein RBDs were created that had the same amino acid sequences as the wild-type (WT (Wuhan type)), alpha, beta, delta, epsilon, eta, theta, and kappa RBDs of SARS-CoV-2. Specifically, a construct was created in which a 6x histidine tag sequence was added to the 3' end of the region corresponding to the RBD (from lysine position 319 to phenylalanine position 541 from the N-terminus) of the SARS-CoV-2 spike protein cDNA, and this construct was incorporated into an expression vector for mammalian cells. The mutations shown in the table below were introduced into the resulting plasmid by OE-PCR. 【0105】 [Table 11] 【0106】 The obtained plasmid DNA was introduced into CHO cells, and the mutant recombinant RBD was purified from the culture supernatant using Ni-NTA agarose. 【0107】 2. Inhibition study of binding of wild-type and mutant spike protein RBD to human ACE2 A cell suspension of 4T1-hACE2 cells was seeded at a rate of 100 μL per well in each well of a 96-well cell culture plate, resulting in 10,000 cells per well. The cells were cultured at 37°C in the presence of 5% CO2 for 2 days. Wild-type and each mutant RBD were added to Blocking Buffer (PBS containing 3% BSA and 0.2% bovine serum) to a final concentration of 5 nM, and anti-human ACE2 chimeric antibodies were diluted with the Blocking Buffer containing wild-type and each mutant RBD. The culture medium was removed from the 96-well plate by decantation, and 30 μL of the prepared anti-human ACE2 chimeric antibody solution was added to each well. The mixture was then allowed to stand at 4°C for 1 hour. The anti-human ACE2 chimeric antibody solution was removed from the 96-well plate by decantation, and each well was washed twice with 250 μL of PBS(-). 30 μL of S28 antibody solution, prepared by diluting mouse anti-SARS-CoV-2 Spike antibody (clone S28) to 1 μg / mL in Blocking Buffer, 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 10% formalin solution was added to each well of a 96-well plate, and the cells were fixed by standing at room temperature for 10 minutes. 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 3% hydrogen peroxide solution was added to each well of a 96-well plate, and the endogenous peroxidase was inactivated by standing at room temperature for 5 minutes. 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). 50 μL of secondary antibody (Mouse IgG-Fc Fragment cross-adsorbed Antibody HRP Conjugated, Bethyl, A90-231P) solution, diluted 10,000-fold in Blocking Buffer, was added to each well of the 96-well plate, and the mixture was allowed to stand at room temperature in the dark for 1 hour. 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 OPD solution was added to each well of the 96-well plate, and the mixture was allowed to stand at room temperature while observing the color development. 100 μL of 1.5 M sulfuric acid solution was then added to each well to stop the color reaction. The absorbance at 490 nm was measured using a spectrophotometer to detect RBD bound to human ACE2 on the cell membrane. The inhibition rates of binding between wild-type and each mutant RBD and human ACE2 by anti-human ACE2 chimeric antibodies were calculated using wells containing only each RBD as positive controls and wells containing only anti-human ACE2 chimeric antibodies as negative controls. 【0108】 3.Results As a result, all six chimeric antibodies inhibited the binding of recombinant RBD to 4T1-hACE2 cells in a concentration-dependent manner (Figure 6). The IC50 values ​​for binding of each mutant RBD to 4T1-hACE2 cells were 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), and 5.5–15.4 μg / mL (kappa), respectively. Of the six chimeric antibodies, OMRad004G05, OMRad028D08, OMRad031H08, and OMRad045G03 showed similar levels of inhibitory effect against the binding of all mutant RBDs to ACE2. Furthermore, these four chimeric antibodies exhibited higher inhibitory effects (lower IC50) compared to OMRad044E11 and OMRad052B05. These results are shown in the table below. 【0109】 [Table 12] 【0110】 The results of this embodiment demonstrate that all antibodies of the present invention neutralize the binding of wild-type and all mutant recombinant RBDs to human ACE2. Furthermore, the antibodies belonging to Group 1 (OMRad004G05, OMRad028D08, OMRad031H08, OMRad045G03) and the antibodies belonging to Group 2 (OMRad044E11, OMRad052B05) shown in Example 6 were demonstrated to have activity equivalent to other antibodies belonging to the same group. [Examples] 【0111】 3. Neutralization test against live viruses (1) Summary of antibodies used and tests In this neutralization test, four of the six chimeric antibodies used in Example 9 (OMRad004G05, OMRad028D08, OMRad031H08, and OMRad045G03) that showed particularly high inhibitory effects were used. The neutralization test method was carried out with some modifications to the method described in the National Institute of Infectious Diseases' "COVID-19 Serological Testing Manual." Specifically, after contacting cells with the dilutions of the above chimeric antibodies (1.5-fold dilution series from 10 μg / mL (except for the Omicron strain) or 2.5 μg / mL (Omicron strain) for each antibody), 100 TCID was performed. 50 Equal volumes of wild-type and various mutant SARS-CoV-2 suspensions, adjusted to 50 μL concentration, were added and incubated for 3 days in a carbon dioxide incubator (CO2 concentration: 5%) at 37°C ± 1°C. The neutralizing antibody titers of the chimeric antibodies were then measured. Antibody dilutions were prepared using cell maintenance medium [Dulbecc's modified Eagle medium (Nacalai Tesque Co., Ltd.) with 2% fetal bovine serum, penicillin (100 U / mL), streptomycin (100 μg / mL), and geneticin (G418, 1 mg / mL)]. 【0112】 (2) The viruses and cells used In this embodiment, "live virus" refers to a virus that can infect and multiply in living cells. In this example, the following virus strain was used as the live SARS-CoV-2 virus. (i) SARS-CoV-2 wild type strain (JPY / TY / WK-521) (ii) SARS-CoV-2 alpha mutant strain QHN001 (VOC-202012 / 01 strain) (iii) SARS-CoV-2 beta mutant strain TY8-612 (B.1.351 lineage strain) (iv) SARS-CoV-2 delta variant strain (TY11-927 (B.1.617.2 strain)) (v) SARS-CoV-2 omicron mutant (TY38-873 (BA.1 strain)) Furthermore, in this example, VeroE6 / TMPRSS2 cells (JCRB1819) were used. 【0113】 (3) Preparation of virus suspension The above SARS-CoV-2 virus suspension was prepared as follows: (i) VeroE6 / TMPRSS2 cells were cultured in a monolayer in a tissue culture flask using cell proliferation medium [Dulbecc's modified Eagle medium (Nacalai Tesque Co., Ltd.) supplemented with 10% fetal bovine serum, penicillin (100 U / mL), streptomycin (100 μg / mL), and Geneticin (G418) (1 mg / mL)]. (ii) After cell culture, the cell growth medium was removed from the flask, various SARS-CoV-2 strains were inoculated, and cell maintenance medium [Dulbecc's modified Eagle medium (Nacalai Tesque Co., Ltd.) with 2% fetal bovine serum, penicillin (100 U / mL), streptomycin (100 μg / mL), and Geneticin (G418) (1 mg / mL) added] was added and the cells were incubated in a carbon dioxide incubator (CO2 concentration: 5%) at 37°C ± 1°C 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) had 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. 【0114】 (4) Measurement of neutralizing antibody titer VeroE6 / TMPRSS2 cells were cultured in a monolayer in a tissue culture microplate (flat-bottom, 96 wells) using cell growth medium. After removing the cell growth medium, the cells were washed with cell maintenance medium, and the cell maintenance medium was removed. Next, 50 μL of the above chimeric antibody dilution, serially diluted 1.5 times, was added to each of the four wells, followed by inoculation with 50 μL of the above virus suspension. The wells were then incubated in a carbon dioxide incubator (CO2 concentration: 5%) at 37°C ± 1°C for 3 days. After culturing, the presence or absence of morphological changes in the cells (cytopathic effect: CPE) was observed using an inverted phase-contrast microscope, the supernatant was removed, and the cells were fixed and washed with water. After staining and washing the fixed cells, the highest dilution factor at which CPE (cytotoxicity-induced hyperplasia) was suppressed in two or more wells was calculated. The antibody concentration required for neutralization was then calculated from this highest dilution factor and the amount of antibody, and this was defined as the neutralizing antibody titer. This test was repeated twice. As a result, all four antibodies used in the test showed neutralizing activity against the infection of cells by all live virus strains, both wild-type and mutant. These results are shown in the table below. 【0115】 [Table 13] 【0116】 These results demonstrate that the antibodies of the present invention neutralize the binding of SARS-CoV-2 wild-type and mutant strains to and infect human cells. In other words, the antibodies of the present invention have been shown to be extremely effective in treating or preventing coronavirus infections caused by wild-type or mutant strains of SARS-CoV-2. Furthermore, the antibodies belonging to Group 1 shown in Example 6 were demonstrated to have equivalent neutralizing activity against the binding and infection of human cells by SARS-CoV-2 wild-type and mutant strains to other antibodies belonging to the same Group. Furthermore, despite the fact that OMRad031H08 and OMRa045G03 differ by one amino acid residue in their framework amino acid sequences, they both exhibited the same neutralizing activity, as shown in the table above. This demonstrates that, in the antibodies of the present invention, if the CDR amino acid sequences of two antibodies are identical or similar, they can have equivalent effects (neutralizing activity, etc.) even if the framework amino acid sequences are not completely identical. [Industrial applicability] 【0117】 The present invention provides a novel monoclonal antibody capable of binding to ACE2. [Sequence Listing Free Text] 【0118】 Sequence IDs 3-134: Synthetic DNA or synthetic peptides

Claims

[Claim 1] A monoclonal antibody or its antigen-binding fragment that binds to ACE2 (Angiotensin Converting Enzyme 2), (a) comprising 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 also comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 60, CDR-L2 containing the amino acid sequence of SEQ ID NO: 62, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 64, (b) A CDR-H1 containing the amino acid sequence of SEQ ID NO: 30, a CDR-H2 containing the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 containing the amino acid sequence of SEQ ID NO: 34, and a CDR-L1 containing the amino acid sequence of SEQ ID NO: 36, a CDR-L2 containing the amino acid sequence of SEQ ID NO: 38, and a CDR-L3 containing the amino acid sequence of SEQ ID NO: 40, (c) A CDR-H1 containing the amino acid sequence of SEQ ID NO: 18, a CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and a CDR-H3 containing the amino acid sequence of SEQ ID NO: 22, and a CDR-L1 containing the amino acid sequence of SEQ ID NO: 24, a CDR-L2 containing the amino acid sequence of SEQ ID NO: 26, and a CDR-L3 containing the amino acid sequence of SEQ ID NO: 28, or (d) A CDR-H1 containing the amino acid sequence of SEQ ID NO: 6, a CDR-H2 containing the amino acid sequence of SEQ ID NO: 8, and a CDR-H3 containing the amino acid sequence of SEQ ID NO: 10, and a CDR-L1 containing the amino acid sequence of SEQ ID NO: 12, a CDR-L2 containing the amino acid sequence of SEQ ID NO: 14, and a CDR-L3 containing the amino acid sequence of SEQ ID NO: 16, The monoclonal antibody or its antigen-binding fragment. [Claim 2] A monoclonal antibody or antigen-binding fragment thereof according to claim 1, which can neutralize the binding of coronavirus to ACE2. [Claim 3] The monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein the coronavirus is a coronavirus that infects cells via ACE2. [Claim 4] The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is a chimeric antibody or a humanized antibody. [Claim 5] (a) comprising a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 94 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 96, (b) comprising a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 86 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 88, (c) comprising a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 82 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 84, or (d) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 78 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 80, A monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4. [Claim 6] A pharmaceutical composition for treating or preventing coronavirus infection, comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5. [Claim 7] The pharmaceutical composition according to claim 6, wherein coronavirus infection is a disease or symptom caused by infection with a coronavirus that infects cells via ACE2. [Claim 8] The pharmaceutical composition according to claim 7, wherein 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, digestive symptoms, speech disorder, cognitive impairment, and cardiovascular symptoms. [Claim 9] Use of a monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 in the manufacture of a pharmaceutical product for the treatment or prevention of coronavirus infection. [Claim 10] A reagent or kit comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5.

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