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

Anti-ACE2 monoclonal antibody

[0001] The present invention relates to monoclonal antibodies that bind to angiotensin-converting enzyme 2 (ACE2) and uses thereof.

[0002] The novel coronavirus disease (COVID-19) that emerged in Wuhan, China in December 2019, rapidly spread worldwide, causing numerous deaths and severe damage to the global economy. SARS-CoV-2, the virus that causes COVID-19, is classified as a betacoronavirus and has a single-stranded RNA genome. The surface of the virus particle contains spike proteins, which interact with angiotensin-converting enzyme 2 (ACE2) expressed on the surface of human cells, resulting in direct contact between the virus particle and the human cell (Non-Patent Document 1). COVID-19 can cause severe respiratory distress, and the global death toll exceeded 1.9 million by the end of 2020. However, no effective treatment has yet been found to be effective.

[0003] Wrapp et al., Science 367, 1260-1263 (2020)

[0004] In this situation, it is desirable to develop novel molecules that neutralize the binding of coronaviruses to ACE2.

[0005] As a result of intensive research to solve the above problems, the present inventors have succeeded in developing a novel anti-ACE2 monoclonal antibody that can neutralize the binding of coronavirus to ACE2, thereby completing the present invention.

[0006] [1] A monoclonal antibody or antigen-binding fragment thereof that binds to ACE2 (Angiotensin Converting Enzyme 2). [2] The monoclonal antibody or antigen-binding fragment thereof according to [1] above, which is capable of neutralizing the binding of coronavirus to ACE2. [3] The monoclonal antibody or antigen-binding fragment thereof according to [2] above, wherein the coronavirus infects cells via ACE2. [4] The monoclonal antibody or antigen-binding fragment thereof according to any of [1] to [3] above, wherein the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.[5] (a) A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 54, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 56, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 58, and also comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 60, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 62, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64; (b) A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 30, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 34, and also comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40; or (c) (d) A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 6, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; or (e) A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 6, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16. (f) the monoclonal antibody or antigen-binding fragment thereof according to any of [1] to [4] above, comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 42, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and also comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 48, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 52; or[6] (a) comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 94 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 96; (b) comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 86 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 88; (c) comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 82 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 84; (d) comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 78 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 80; (e) comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 90 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 92; or (f) comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 98 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 100.

[0016] The monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [5] above. [7] A monoclonal antibody or antigen-binding fragment thereof that can neutralize the binding of coronavirus to ACE2 and competes with the antibody according to [5] or [6] above for binding to ACE2. [8] A pharmaceutical composition for treating or preventing coronavirus infection, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [7] above. [9] The pharmaceutical composition according to [8] above, wherein the coronavirus infection is a disease or symptom 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, fatigue, chills, pain, taste or smell disorders, rash, gastrointestinal symptoms, speech disorders, cognitive disorders, and cardiovascular symptoms.

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

[12] The method according to

[11] above, wherein the coronavirus infection is a disease or symptom 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 disease, fever, fatigue, chills, pain, taste or smell disorders, rash, gastrointestinal symptoms, speech disorders, cognitive disorders, and cardiovascular symptoms.

[14] The monoclonal antibody or antigen-binding fragment thereof according to any of [1] to [7] above, for use in treating or preventing coronavirus infection.

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

[14] above, wherein the coronavirus infection is a disease or symptom caused by infection with 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 disease, fever, fatigue, chills, pain, taste or smell disorders, rash, gastrointestinal symptoms, speech disorders, cognitive disorders, and cardiovascular 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 treating or preventing coronavirus infection.

[18] A reagent or kit comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [7] above.

[0007] The present invention makes it possible to neutralize the binding of coronaviruses to ACE2. Because the antibodies of the present invention bind to ACE2, a coronavirus receptor, they can inhibit the binding of coronaviruses to ACE2 even if the coronavirus mutates, as long as the mutant virus infects via ACE2.

[0008]

[0033] Figure 1 shows the results of a binding inhibition test between the receptor binding domain (RBD) of SARS-CoV-2 spike protein and ACE2 using antibodies of the present invention.

[0034] Figure 2 shows a schematic diagram of a competitive inhibition test using antibodies of the present invention.

[0035] Figure 3 shows the results of a competitive inhibition test using antibodies of the present invention.

[0036] Figure 4 shows the results of confirming the reactivity of antibodies of the present invention with human ACE2, mouse ACE2, and mouse / human fusion ACE2.

[0037] Figure 5 shows the results of confirming the reactivity of antibodies of the present invention with human ACE2-expressing cells.

[0038] A: Shows the reactivity of mouse antibodies of the present invention with human ACE2-expressing cells.

[0039] B: Shows the reactivity of chimeric antibodies of the present invention with human ACE2-expressing cells.

[0039] C: Shows the reactivity of chimeric antibodies of the present invention with 4T1 cells that do not express human ACE2.

[0039] Figure 1 shows the results of a binding inhibition test between the receptor binding domain (RBD) of wild-type or mutant SARS-CoV-2 spike protein and ACE2 using antibodies of the present invention.

[0009] The present invention will be described in detail below. The following embodiments are examples for explaining the present invention and are not intended to limit the present invention to these embodiments. The present invention can be implemented in various forms without departing from the gist of the invention. This specification also includes the contents described in the specification and drawings of the Japanese Patent Application (Patent Application No. 2021-036928) filed on March 9, 2021, which is the basis for claiming priority to this application.

[0010] 1. Overview Although the global death toll from COVID-19 exceeded 1.9 million by the end of 2020, no therapeutic drug has yet demonstrated a promising therapeutic effect. While compounds such as favipiravir and remdesivir are currently being considered as potential treatments for COVID-19, these drugs were originally developed to treat infectious diseases such as influenza and Ebola hemorrhagic fever, and it is unclear whether they are optimal for COVID-19. Antibody drugs such as tocilizumab and ravulizumab are also considered potential treatments. However, they do not directly recognize the virus; rather, they target IL-6R and complement (C5), which are biological factors involved in the immune system, and therefore act indirectly. Furthermore, as the global pandemic continues, reports of SARS-CoV-2 mutants with altered spike proteins and altered amino acid sequences have been emerging, and more are expected to emerge. In this situation, the development of new therapeutic drugs that can be used to treat or prevent coronavirus infections, even in the event of a mutation, is desired. The present inventors focused on ACE2, the receptor that coronaviruses bind to when infecting human cells, and as a result of extensive research into antibodies against ACE2, succeeded in developing a novel monoclonal antibody that can neutralize the binding of coronaviruses to ACE2. Because the antibody of the present invention recognizes ACE2, it is extremely useful in that it can inhibit the binding of coronaviruses to ACE2, even if the coronavirus mutates, as long as the mutant virus infects via ACE2. Furthermore, because the antibody of the present invention can neutralize the binding of wild-type and mutant strains of SARS-CoV-2 to and infecting human cells, it is extremely effective in treating or preventing coronavirus infection.

[0011] 2. Angiotensin-Converting Enzyme The antibodies of the present invention bind 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. ACE2 also 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 are able to infect human cells by binding their spike proteins (S proteins) to ACE2.

[0012] ACE2 is mainly expressed in cells of the lungs, digestive system, heart, blood vessels, eyes, kidneys, cerebral cortex, amygdala, brainstem, medulla oblongata, etc. 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 infection, such as respiratory diseases.

[0013] The ACE2 of the present invention may be derived from any mammal. Examples of such mammals include mice, rats, rabbits, cats, dogs, goats, monkeys, and humans, and preferably, but not limited to, mice, rats, cats, dogs, and humans. Examples of the nucleotide and amino acid sequences of these ACE2s are shown in SEQ ID NOs: 1 and 2, respectively, but the nucleotide and amino acid sequences of the ACE2 of the present invention are not limited to these. The nucleotide and amino acid sequences of human ACE2 are registered in the GenBank database under the following accession numbers:

[0014] Base sequence of DNA encoding human ACE2: NM_001371415 (SEQ ID NO: 1) Amino acid sequence of human ACE2: NP_001358344.1 (SEQ ID NO: 2)

[0015] ACE2 in the present invention includes the following proteins (a) to (c): (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2; (b) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 in which one or several amino acids are deleted, substituted, or added, and having the activity of binding to the receptor binding domain (RBD) of the coronavirus spike protein; and (c) a protein comprising an amino acid sequence having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2, and having the activity of binding to the RBD of the coronavirus spike protein.

[0016] In the present invention, a "protein comprising the amino acid sequence shown in SEQ ID NO: 2" includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 2. Furthermore, examples of the "amino acid sequence shown in SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted, or added" include: (i) an amino acid sequence in which 1 to 10 (e.g., 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1) amino acids have been deleted from the amino acid sequence shown in SEQ ID NO: 2, (ii) an amino acid sequence in which 1 to 10 (e.g., 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1) amino acids have been substituted with other amino acids from the amino acid sequence shown in SEQ ID NO: 2, (iii) an amino acid sequence in which 1 to 10 (e.g., 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1) amino acids have been added to the amino acid sequence shown in SEQ ID NO: 2, and (iv) an amino acid sequence mutated by a combination of the above (i) to (iii).

[0017] In the present invention, the presence or absence of "activity to bind to the RBD of coronavirus spike protein" can be measured using known methods, such as immunological techniques such as immunoprecipitation, Western blotting, enzyme immunoassay (EIA), and enzyme-linked immunosorbent assay (ELISA) (e.g., ELISA using polypeptides, cell ELISA, etc.), or pull-down assays. Furthermore, "activity to bind to the RBD of coronavirus spike protein" refers to having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and preferably 90% of the activity of a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, defined as 100.

[0018] In addition to the amino acid sequence set forth in SEQ ID NO: 2, the ACE2 of the present invention also includes proteins that contain an amino acid sequence with 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and have binding activity to the RBD of the coronavirus spike protein. Such proteins also include those that contain an amino acid sequence with 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 to the amino acid sequence set forth in SEQ ID NO: 2 and have binding activity to the RBD of the coronavirus spike protein. Sequence identity can be determined using online homology search sites, such as the DNA Data Bank of Japan (DDBJ), using FASTA, BLAST, PSI-BLAST, and other similar searches. BLAST searches can also be performed at the National Center for Biotechnology Information (NCBI).

[0019] To prepare a protein having the above mutation, a mutation can be introduced into DNA encoding the protein using a site-directed mutagenesis method such as the Kunkel method or the Gapped duplex method, using a mutation introduction kit such as QuikChange TMSite-Directed Mutagenesis Kit (Stratagene), GeneTailor TM This can be performed using the Site-Directed Mutagenesis System (Invitrogen), the Takara Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: Takara Bio Inc.), etc. 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] The amino acid sequence of the region of ACE2 to which the antibodies of the present invention bind (the region containing the epitope to which the antibodies of the present invention bind) is not limited, and examples thereof include the amino acid sequence of the extracellular region of ACE2 or a portion thereof. The amino acid sequence of the extracellular region of ACE2 is not limited, and in the case of human ACE2, an amino acid sequence containing the amino acid sequence of amino acid residues 18 to 740 (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) is exemplified. The nucleotide sequence of DNA encoding the amino acid sequence of amino acid residues 18 to 740 of human ACE2 is shown in SEQ ID NO: 3. Furthermore, examples of the amino acid sequence of a portion of the amino acid sequence of the extracellular region of ACE2 include, but are not limited to, the amino acid sequence of the coronavirus-binding region of ACE2 or a portion thereof, and the amino acid sequence of a region of ACE2 other than the coronavirus-binding region or a portion thereof. Furthermore, the amino acid sequence of the coronavirus-binding region or a portion thereof in ACE2 is not limited to, and examples thereof include, in the case of human ACE2, amino acid sequences containing 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 partial amino acid sequences of the amino acid sequence of the extracellular domain 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), counting from the N-terminal amino acid residue of the full-length amino acid sequence of ACE2. The nucleotide sequences of DNA encoding the amino acid sequence consisting of amino acid residues 1 to 120 of human ACE2 and the nucleotide sequences of 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 containing the epitope to which the antibody of the present invention binds) includes the following amino acid sequences (a) to (c): (a) an amino acid sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO: 4, 132, or 134, (b) an amino acid sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO: 4, 132, or 134 in which one or several amino acids have been deleted, substituted, or added, and (c) an amino acid sequence comprising or consisting of an amino acid sequence having about 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 to the amino acid sequence shown in SEQ ID NO: 4, 132, or 134. Those skilled in the art can easily confirm whether the antibody of the present invention binds to these amino acid sequences using known immunological techniques, such as immunoprecipitation, Western blotting, EIA, ELISA (e.g., ELISA using polypeptides, cell ELISA, etc.), etc.

[0023] The epitope to which the antibody of the present invention binds and the region in which the epitope is located are not limited, as long as the antibody of the present invention can neutralize the binding of coronavirus to ACE2. Furthermore, based on the description herein (e.g., the description in Example 3), a person skilled in the art can determine whether a test antibody can neutralize the binding of coronavirus to ACE2 without identifying the epitope of the test antibody. In other words, based on the description herein, a person skilled in the art does not need to identify the epitope of the test antibody to determine whether the test antibody can neutralize the binding of coronavirus to ACE2.

[0024] 3. Antibodies of the Present Invention The antibodies of the present invention are monoclonal antibodies that bind to ACE2 (hereinafter also referred to as "anti-ACE2 monoclonal antibodies") or antigen-binding fragments thereof. In the present invention, "binding to ACE2" means forming a reversible non-covalent bond with ACE2 through hydrogen bonds, hydrophobic interactions, electrostatic forces, van der Waals forces, etc. In the present invention, "binding" includes "specific binding." In the present invention, "specific binding" refers to recognizing an epitope in a target protein or target polypeptide and selectively or preferentially binding to a protein or polypeptide containing that epitope compared to proteins or polypeptides that do not contain that epitope. Examples of "antigen-binding fragments" in the present invention include, but are not limited to, single-chain Fvs (scFvs), sc(Fv)2, Fab, Fab', diabodies (dsFvs), F(ab')2, and multispecific antibodies. The antibody-binding fragments can be obtained by known methods, such as genetic engineering, papain digestion, or pepsin digestion. In one aspect of the invention, the antibody of the invention is an isolated antibody.

[0025] The antibodies of the present invention can neutralize the binding of coronaviruses to ACE2. In the present invention, coronavirus refers to coronaviruses that infect 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 through the binding of the receptor-binding domain (RBD) of their spike protein to ACE2. In the present invention, "neutralizing" refers to inhibiting the interaction (e.g., binding) between the coronavirus RBD and ACE2, or inhibiting the coronavirus's binding to or infection (e.g., entry) of cells. Whether a test antibody inhibits the interaction between the coronavirus RBD and ACE2 can be determined by detecting RBD bound to ACE2 using a fluorescently labeled antibody capable of binding to RBD, as described in Example 3 of the present specification. Specifically, if RBD is added after reacting the test antibody with ACE2 and no RBD bound to ACE2 is detected, the test antibody can be determined to inhibit the interaction between RBD and ACE2. Furthermore, whether a test antibody inhibits (neutralizes) coronavirus binding to or infection (e.g., invasion) of cells can be confirmed, for example, by contacting the test antibody and coronavirus with cultured cells and observing the presence or absence of morphological changes in the cultured cells (cytopathic effect: CPE). If no morphological changes are 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 is described below. (1) Preparation of Antigen: As immunogens for producing the antibody of the present invention, (i) ACE2 and (ii) ACE2-expressing cells (mammalian cells in which ACE2 (e.g., human ACE2) is forcibly expressed on the cell surface) can be used. When ACE2 is used as an immunogen, the full-length ACE2 polypeptide (SEQ ID NO: 2) or a partial polypeptide thereof can be used. The partial ACE2 polypeptide used as an antigen or immunogen is not limited, and examples thereof include polypeptides containing all or a portion of the amino acid sequence of the extracellular domain of ACE2. The full-length or partial amino acid sequence of the extracellular domain of ACE2 is not limited, and examples thereof include, in the case of human ACE2, amino acid sequences containing all or a portion of the amino acid sequence consisting of amino acid residues 18 to 740, counting from the N-terminal amino acid residue, of the full-length ACE2 amino acid sequence (SEQ ID NO: 4). Polypeptides comprising a portion of the amino acid sequence of the extracellular domain of ACE2 include, but are not limited to, polypeptides comprising the amino acid sequence of the coronavirus-binding domain of ACE2 or a portion thereof, and polypeptides comprising the amino acid sequence of a domain other than the coronavirus-binding domain of ACE2 or a portion thereof. For example, in the case of human ACE2, such polypeptides 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), counting from the N-terminus of the full-length amino acid sequence of ACE2.

[0027] When ACE2-expressing cells are used as immunogens, they can be prepared, for example, using the following method. First, DNA encoding the full-length human ACE2 is incorporated into a lentiviral vector, and then transfected into mammalian cells (e.g., 293T cells) together 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), allowing cells to be obtained that stably express human ACE2 on the cell membrane. However, the method for preparing ACE2-expressing cells is not limited to this method.

[0028] The ACE2 used as an antigen or immunogen can be native ACE2 purified from tissues or cells of mice, rats, rabbits, cats, dogs, goats, monkeys, humans, etc., or genetically engineered ACE2. For example, a biological sample containing ACE2 expression can be fractionated into soluble and insoluble fractions using various detergents, such as Triton-X or Sarkosyl. ACE2 can be isolated by dissolving the insoluble fraction in urea or guanidine hydrochloride and binding it to various columns, such as a heparin column or a binding resin. 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 synthesizers. The synthesized peptide can be conjugated 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 ACE2 or a partial polypeptide prepared as described above is administered alone or together with a carrier or diluent to non-human mammals, such as mice, rats, rabbits, cats, dogs, goats, and monkeys, for immunization. The antigen dose 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 typically carried out by intravenous, subcutaneous, or intraperitoneal injection. The interval between immunizations is not particularly limited; immunizations are typically performed 2 to 10 times, preferably 2 to 5 times, at intervals of several days to several weeks, preferably 1 to 2 weeks. Those skilled in the art can determine the interval between immunizations based on the antibody titer obtained. It is preferable to perform a blood sample after 3 to 4 subcutaneous immunizations to measure the antibody titer. Measurement of antibody titers in serum can be performed by ELISA, EIA, radioimmunoassay (RIA), etc. After confirming that the antibody titer has risen sufficiently, individuals in which an increase in antibody titer has been observed are selected and antibody-producing cells are collected. Examples of antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, with spleen cells or lymph node cells being preferred.

[0030] (ii) Cell Fusion: To obtain hybridomas, antibody-producing cells are fused with myeloma cells. The fusion procedure can be performed by known methods, such as the method of Kohler et al. Publicly available established cell lines from animals such as mice can be used as myeloma cells to be fused with antibody-producing cells. The cell lines used are preferably drug-selective and cannot survive in HAT selection medium (containing hypoxanthine, aminopterin, and thymidine) in the unfused state, but can survive only 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] The cell fusion between the above myeloma cells and antibody-producing cells was carried out in an animal cell culture medium such as serum-free DMEM or RPMI-1640 medium at 1×10 8 ~5×10 8 antibody-producing cells and 2 × 10 7 ~10×10 7 The antibody-producing cells are mixed with myeloma cells (a cell ratio of antibody-producing cells to myeloma cells of 10:1 to 1:1), and a fusion reaction is carried out in the presence of a cell fusion accelerator. Examples of cell fusion accelerators that can be used include polyethylene glycol with an average molecular weight of 1,000 to 6,000 daltons and Sendai virus. Alternatively, 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) Hybridoma Selection and Cloning: The desired hybridomas are selected from the cells after cell fusion. To do this, the cell suspension is appropriately diluted, for example, in RPMI-1640 medium containing 10-20% fetal bovine serum, and seeded onto a microtiter plate at approximately 0.3 cells per well by limiting dilution. A selective medium such as HAT medium is added to each well, and the selective medium is then replaced as appropriate. As a result, hybridomas can be obtained from the cells that begin to grow around 10 days after the start of culture in the selective medium.

[0033] Next, the grown hybridomas are further screened. Hybridoma screening can be performed according to conventional methods and is not particularly limited. For example, a portion of the culture supernatant contained in the wells in which the hybridomas were cultured can be collected and screened by ELISA, EIA, radioimmunoassay, or the like. Specifically, antigen is adsorbed onto a 96-well plate and then blocked with calf serum. The hybridoma cell culture supernatant is reacted with the immobilized antigen at 37°C for 1 hour, followed by reaction with peroxidase-labeled anti-mouse IgG at 37°C for 1 hour, and color development is achieved using orthophenylenediamine as a substrate. After the reaction is stopped with acid, screening can be performed by measuring the absorbance at a wavelength of 490 nm. Hybridomas producing monoclonal antibodies that test positive in the above assay are cloned by limiting dilution or the like. Finally, hybridomas, which are cells producing monoclonal antibodies that bind to ACE2, are established.

[0034] (iv) Collection of Monoclonal Antibodies Monoclonal antibodies can be collected from established hybridomas by conventional cell culture or ascites formation methods. In the cell culture method, hybridomas are cultured in animal cell culture media such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium under conventional culture conditions (e.g., 37°C, 5% CO2 concentration) for 7 to 14 days, and antibodies are collected from the culture supernatant. In the ascites formation method, approximately 5 x 10 hybridomas are cultured in the abdominal cavity of an animal of the same species as the mammal from which the myeloma cells were derived, such as a mouse (ICR, BALB / c). 6 ~2×10 7 The hybridomas are then mass-produced by administering the antibody to the animals. After 1 to 2 weeks, ascites fluid is collected. If antibody purification is required in the above antibody collection method, the antibody can be purified by any known method, such as ammonium sulfate precipitation, ion exchange chromatography, gel filtration, or affinity chromatography, or by a combination of these methods.

[0035] Antibodies of the present invention include, but are not limited to, the following monoclonal antibodies or antigen-binding fragments thereof: (a) a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region (CDR) 1 (CDR-H1) comprising or consisting of the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR2 (CDR-H2) comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 (CDR-H3) comprising or consisting of the amino acid sequence of SEQ ID NO: 10, and / or a light chain variable region (VL) comprising a light chain CDR1 (CDR-L1) comprising or consisting of the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 (CDR-L2) comprising or consisting of the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 (CDR-L3) comprising or consisting of the amino acid sequence of SEQ ID NO: 16; (c) the heavy chain variable region (VH) comprises a CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 18, a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 20, and a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 22; and / or the light chain variable region (VL) comprises a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 24, a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 26, and a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 28; (d) the heavy chain variable region (VH) comprises a CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 30, a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 34; and / or the light chain variable region (VL) comprises a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 36, a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 38, and a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 40;(e) the heavy chain variable region (VH) comprises a CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 42, a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 44, and a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 46; and / or the light chain variable region (VL) comprises a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 48, a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 52; (f) the heavy chain variable region (VH) comprises a CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 54, a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 56, and a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 58; and / or the light chain variable region (VL) comprises a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 60, a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 62, and a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 64; A monoclonal antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region (VH) comprises CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 66, CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 68, and CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 70, and / or the light chain variable region (VL) comprises CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 72, CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 74, and CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 76.

[0036] In the present invention, examples of DNA nucleotide sequences encoding CDR-H1 include, but are not limited to, those comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 5, 17, 29, 41, 53, or 65. In the present invention, examples of DNA nucleotide sequences encoding CDR-H2 include, but are not limited to, those comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 7, 19, 31, 43, 55, or 67. In the present invention, examples of DNA nucleotide sequences encoding CDR-H3 include, but are not limited to, those comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 9, 21, 33, 45, 57, or 69. In the present invention, examples of DNA nucleotide sequences encoding CDR-L1 include, but are not limited to, those comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 11, 23, 35, 47, 59, or 71. In the present invention, examples of the nucleotide sequence of DNA encoding CDR-L2 include, but are not limited to, those comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 13, 25, 37, 49, 61, or 73. In the present invention, examples of the nucleotide sequence of DNA encoding CDR-L3 include, but are not limited to, those comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 15, 27, 39, 51, 63, or 75.

[0037] In yet another aspect, antibodies of the present invention include, but are not limited to, the following monoclonal antibodies or antigen-binding fragments thereof: (a) comprising a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 78 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 80, (b) comprising a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 82 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 84, (c) comprising a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 86 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 88, (d) comprising a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 90 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 92, or (e) (f) a monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 94 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 96; or (f) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 98 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 100.

[0038] In the present invention, examples of DNA nucleotide sequences encoding heavy chain variable regions include, but are not limited to, those comprising or consisting of the nucleotide sequences shown in SEQ ID NOs: 77, 81, 85, 89, 93, or 97. In addition, examples of DNA nucleotide sequences encoding light chain variable regions include, but are not limited to, those comprising or consisting of the nucleotide sequences shown in SEQ ID NOs: 79, 83, 87, 91, 95, or 99.

[0039] (3) Production of Recombinant Antibodies One preferred embodiment of the antibody of the present invention is a recombinant antibody. Examples of recombinant antibodies include, but are not limited to, chimeric antibodies, canine antibodies, humanized antibodies, and fully human antibodies.

[0040] A chimeric antibody is an antibody produced by linking immunoglobulin gene fragments from different animal species. In the present invention, chimeric antibodies include, for example, human chimeric antibodies, but the species of animal from which the variable and constant regions of the chimeric antibody are derived is not limited. A human 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, for example, Proc. Natl. Acad. Sci. USA 81, 6851-6855, (1984)). Examples of subclasses of such human-derived antibodies include, but are not limited to, IgG1, IgG2, and IgG4. Chimeric antibodies can be easily constructed using genetic recombination techniques.

[0041] In the present invention, a technique known as CDR grafting can be used to produce humanized antibodies. CDR grafting involves grafting complementarity-determining regions (CDRs) from the variable region of a mouse antibody onto a human variable region to produce a reshaped variable region in which the framework regions (FRs) are human and the CDRs are mouse-derived. These humanized reshaped human variable regions are then linked to human constant regions. Such methods for producing humanized antibodies are well known in the art (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 techniques (WO96 / 9634096, WO98 / 24893, etc.).

[0043] In the present invention, human heavy chain constant regions that can be used in chimeric antibodies and humanized antibodies include, but are not limited to, those containing an amino acid sequence derived from the human IgG4 heavy chain constant region, for example, a human heavy chain constant region containing or consisting of the amino acid sequence shown in SEQ ID NO: 102. Furthermore, human light chain constant regions that can be used in chimeric antibodies and humanized antibodies include, but are not limited to, those containing an amino acid sequence derived from the human IgG4 light chain constant region, for example, a human light chain constant region containing or consisting of the amino acid sequence shown in SEQ ID NO: 104. Furthermore, DNA encoding a human heavy chain constant region includes, for example, DNA containing or consisting of the nucleotide sequence shown in SEQ ID NO: 101, and DNA encoding a human light chain constant region includes, for example, DNA containing or consisting of the nucleotide sequence shown in SEQ ID NO: 103, but is not limited to these.

[0044] In the present invention, chimeric antibodies and humanized antibodies can be produced using hybridomas or DNA or RNA extracted from the hybridomas as raw materials in accordance with the above-mentioned known methods. Furthermore, proteins to which the antibodies of the present invention are fused can be produced by fusing the variable regions of the antibodies with other proteins using known genetic recombination methods. Furthermore, such fusion proteins can be produced by crosslinking the monoclonal antibody with other proteins using a crosslinker.

[0045] (4) Preparation of Antibody-Binding Fragments Antigen-binding fragments of the antibodies of the present invention bind to ACE2. An antigen-binding fragment of an 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, single-chain Fv (scFv), sc(Fv)2, Fab, Fab', diabody (dsFv), F(ab')2, and multispecific antibodies. The antibody-binding fragments can be obtained by known methods, such as genetic engineering, papain digestion, and pepsin digestion. For example, Fab can be obtained by treating antibody molecules with papain, and F(ab')2 can be obtained by treating antibody molecules with pepsin. Fab' can also be obtained by cleaving the disulfide bond in the hinge region of the F(ab')2. In the case of scFv, cDNA encoding the heavy chain variable region and light chain variable region of the antibody is obtained, and DNA encoding the scFv is constructed. This DNA is inserted into an expression vector, and the expression vector is introduced into a host organism for expression, thereby producing an scFv. In the case of diabodies, cDNA encoding the heavy and light chain V regions of an antibody is obtained, and a DNA encoding an scFv is constructed so that the amino acid sequence of the peptide linker is eight residues or less. This DNA is inserted into an expression vector, and the expression vector is introduced into a host organism for expression, thereby producing a diabody. In the case of dsFv, cDNA encoding the heavy and light chain V regions of an antibody is obtained, and a DNA encoding a dsFv is constructed. This DNA is inserted into an expression vector, and the expression vector is introduced into a host organism for expression, thereby producing a dsFv. Multispecific antibodies, such as bispecific antibodies, bind to two or more antigens or epitopes as targets and can be produced by various known techniques (e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321).

[0046] CDR-containing antigen-binding fragments (peptides) comprise at least one region of the VH or VL CDRs (CDRs 1 to 3). Antigen-binding fragments containing multiple CDRs can be linked directly or via an appropriate peptide linker. CDR-containing antigen-binding fragments can be produced by constructing DNA encoding the VH and VL CDRs of an antibody, inserting the DNA into a prokaryotic or eukaryotic expression vector, and expressing the vector by introducing it into a prokaryotic or eukaryotic organism. CDR-containing peptides can also be produced by chemical synthesis methods such as the Fmoc (fluorenylmethyloxycarbonyl) method and the tBoc (t-butyloxycarbonyl) method.

[0047] (5) Binding Affinity The affinity of an antibody for an antigen can generally be expressed as the equilibrium dissociation constant (KD), and a lower KD value indicates a higher affinity of the antibody. 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 reference antibodies The present invention provides monoclonal antibodies or antigen-binding fragments thereof that can neutralize the binding of coronavirus to ACE2 and compete with any of the following reference antibodies or antigen-binding fragments thereof for binding to ACE2. In the present invention, the reference antibodies include the following monoclonal antibodies: (a) a monoclonal antibody whose heavy chain variable region (VH) comprises a CDR-H1 that includes or consists of the amino acid sequence of SEQ ID NO: 6, a CDR-H2 that includes or consists of the amino acid sequence of SEQ ID NO: 8, and a CDR-H3 that includes or consists of the amino acid sequence of SEQ ID NO: 10, and / or whose light chain variable region (VL) comprises a CDR-L1 that includes or consists of the amino acid sequence of SEQ ID NO: 12, a CDR-L2 that includes or consists of the amino acid sequence of SEQ ID NO: 14, and a CDR-L3 that includes or consists of the amino acid sequence of SEQ ID NO: 16; (b) (c) a monoclonal antibody in which the heavy chain variable region (VH) comprises a CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 18, a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 20, and a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 22, and / or the light chain variable region (VL) comprises a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 24, a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 26, and a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 28;(d) a monoclonal antibody in which the heavy chain variable region (VH) comprises a CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 30, a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 34, and / or the light chain variable region (VL) comprises a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 36, a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 38, and a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 40; (e) a monoclonal antibody, the heavy chain variable region (VH) comprising CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 42, CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 44, and CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 46, and / or the light chain variable region (VL) comprising CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 48, CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 50, and CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 52; (f) 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: 54, CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 56, and CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 58, and / or the light chain variable region (VL) comprises CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 60, CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 62, and CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 64;(g) A monoclonal antibody comprising a heavy chain variable region (VH) comprising CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 66, CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 68, and CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 70, and / or a light chain variable region (VL) comprising CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 72, CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 74, and CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 76; (h) A monoclonal antibody comprising a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 78 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 80; (i) A monoclonal antibody comprising a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 82 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 84; (j) A monoclonal antibody comprising a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 90 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 92; (j) A monoclonal antibody comprising a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 94 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 96; or (k) A monoclonal antibody comprising a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 98 and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 100.

[0049] In the present invention, "competing" for binding to ACE2 means that an antibody or antigen-binding fragment thereof of the present invention inhibits (e.g., suppresses) the binding of the reference antibody or antigen-binding fragment thereof to ACE2, or that the binding of an antibody or antigen-binding fragment thereof to ACE2 is inhibited (e.g., suppressed) by the reference antibody or antigen-binding fragment thereof. Whether an antibody or antigen-binding fragment thereof of the present invention competes with either the reference antibody or antigen-binding fragment thereof for binding to ACE2 can be determined using known techniques, such as competitive RIA, competitive EIA, competitive ELISA (competitive ELISA using polypeptides, competitive cell ELISA), epitope binning, etc. Antibody competition tests are a method established in the antibody technical field for determining whether a test antibody or its antigen-binding fragment competes with a reference antibody for binding to the same epitope 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 sufficiently close to each other that steric hindrance occurs between the test antibody or its antigen-binding fragment and the reference antibody in binding to the epitope. For example, as shown in Example 6 herein, four monoclonal antibodies, OMRad004G05, OMRad028D08, OMRad031H08, and OMRad045G03, compete with each other for binding to ACE2, and furthermore, as shown in Example 10 herein, these competing antibodies have equivalent neutralizing activity against coronavirus binding to or infection of cells. On the other hand, OMRad004G05 and OMRad028D08 differ from OMRad031H08 and OMRad045G03 in their reactivity to mouse ACE2 (Example 7), and therefore, the epitopes they bind to may be different.

[0050] As shown in Example 6 herein, determining whether an antibody or antigen-binding fragment thereof of the present invention competes with the reference antibody or antigen-binding fragment thereof for binding to ACE2 does not require information about the structure of the antibody or epitope (e.g., the amino acid sequences of the CDRs and variable regions, whether the structure is linear or three-dimensional (stereoscopic)). Furthermore, by performing a series of procedures in which an anti-ACE monoclonal antibody is prepared according to conventional methods and an antibody that competes with the reference antibody described herein for binding to ACE2 is selected, antibodies that compete with the reference antibody described herein for binding to ACE2 can be obtained, in addition to the antibodies whose specific structures are identified in the Examples herein. That is, a monoclonal antibody or antigen-binding fragment thereof of the present invention that competes with either the reference antibody or its antigen-binding fragment for binding to ACE2 can be obtained by one of ordinary skill in the art without undue experimentation by performing a competition test using the reference antibody.

[0051] 4. Pharmaceutical Compositions The pharmaceutical compositions of the present invention contain as an active ingredient the antibodies or antigen-binding fragments thereof described in "3. Antibodies of the Present Invention" above, and are intended for treating or preventing coronavirus infection. Coronavirus infections that are the subject of the pharmaceutical compositions of the present invention refer to diseases or symptoms caused by coronavirus infection. Examples of such coronavirus infections include diseases or symptoms caused by infection with coronaviruses that infect cells via ACE2. Examples of "coronaviruses that infect cells via ACE2" include, but are not limited to, SARS-CoV-1 and SARS-CoV-2, with SARS-CoV-2 being preferred. Examples of "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 caused by immune responses. Such diseases or symptoms include, but are not limited to, respiratory diseases, fever, fatigue, chills, pain, taste or smell disorders, rash, gastrointestinal symptoms, speech disorders, cognitive disorders, and cardiovascular symptoms.The pharmaceutical composition containing the antibody of the present invention inhibits infection by coronaviruses that infect cells via ACE2, and is therefore effective in treating or preventing various diseases or symptoms associated with coronavirus infection.

[0052] In the present invention, "treatment" refers to contacting (e.g., administering) an antibody or antigen-binding fragment thereof, or a pharmaceutical composition containing the antibody or antigen-binding fragment thereof (hereinafter also referred to as "the antibody and pharmaceutical composition of the present invention") with a subject / object after the onset of the disease, thereby alleviating the symptoms of the disease compared to when the subject / object is not contacted, but does not necessarily mean completely suppressing the symptoms of the disease. In the present invention, "onset of the disease" includes not only the physical appearance of disease symptoms, but also a positive result in a test for coronavirus infection (e.g., PCR test) even when no symptoms are present. Furthermore, in the present invention, "treatment" of coronavirus infection includes suppressing or inhibiting the worsening of coronavirus infection. In the present invention, "prevention" refers to suppressing or inhibiting the onset or worsening of coronavirus infection, but does not necessarily mean completely suppressing the onset. In the present invention, "prevention" includes contacting (e.g., administering) a pharmaceutical composition or the like of the present invention with a subject / object before the onset of the disease, thereby alleviating symptoms after the onset of the disease compared to when the subject / object is not contacted.

[0053] The pharmaceutical compositions of the present invention can contain a pharmaceutically acceptable carrier in addition to a monoclonal antibody that binds to ACE2 or an antigen-binding fragment thereof. The term "pharmaceutically acceptable carrier" refers to any carrier (e.g., liposome, lipid vesicle, micelle), diluent, excipient, wetting agent, buffer, suspending agent, lubricant, adjuvant, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavoring agent, or sweetener that is suitable for a pharmaceutical composition.

[0054] The antibodies and pharmaceutical compositions of the present invention can be in the form of injections, lyophilized products, tablets, hard capsules, soft capsules, granules, powders, pills, syrups, suppositories, patches, ointments, creams, eye drops, etc. Liquid preparations such as injections may also be in the form of powders (e.g., lyophilized powders) to be dissolved in physiological saline or the like before use to be reconstituted immediately.

[0055] The antibodies and pharmaceutical compositions of the present invention can be administered locally or systemically by any means known to those skilled in the art. The pharmaceutical compositions of the present invention can be administered either orally or parenterally. In the case of parenteral administration, administration can be intratissue (subcutaneous, intraperitoneal, intramuscular, intravenous, etc.), intradermal, topical (transdermal, etc.), or rectally. The pharmaceutical compositions of the present invention can be administered in dosage forms suitable for these administration routes.

[0056] The dosage of the antibodies and pharmaceutical compositions of the present invention varies depending on factors such as the age, weight, health condition, sex, symptoms, animal species, administration route, frequency of administration, and dosage form of the subject / subject, and specific administration procedures can be determined by those skilled in the art. The dosage of the antibodies of the present invention for treating coronavirus infection is, for example, but not limited to, 0.1 mg to 100 mg per kg of subject / subject body weight per day, preferably 1 mg to 15 mg per kg, and more preferably 2-12 mg per kg. The dosage can be one to five times per day.

[0057] The timing of administration of the antibody and pharmaceutical composition of the present invention can be determined appropriately depending on the symptoms, and multiple doses can be administered simultaneously or separately at intervals. Furthermore, the pharmaceutical composition of the present invention can be administered to a subject / subject before or after the onset of a disease.

[0058] The pharmaceutical composition of the present invention can be administered to mammals as test subjects / subjects, including, for example, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, goats, pigs, sheep, cows, horses, monkeys, and humans.

[0059] 5. Method for Treating or Preventing Coronavirus Infection: In the present invention, coronavirus infection can be treated or prevented by administering to a subject / subject a monoclonal antibody that binds to ACE2, or an antigen-binding fragment thereof, or a pharmaceutical composition containing the same. Specifically, the present invention provides a method for treating or preventing coronavirus infection, comprising administering to a subject / subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of the present invention, or a pharmaceutical composition containing the antibody or antigen-binding fragment thereof. The therapeutically effective amount of the antibody or antigen-binding fragment thereof of the present invention or a pharmaceutical composition containing the antibody or antigen-binding fragment thereof will vary depending on factors such as the subject / subject's age, weight, health condition, sex, symptoms, route of administration, frequency of administration, and dosage form. Those skilled in the art can easily determine the therapeutically effective amount required for treating or preventing coronavirus infection. In the present invention, the term "subject" or "subject" includes subjects / subjects in need of treatment or prevention of coronavirus infection. Furthermore, the mammals targeted for treatment or prevention as "subjects" or "subjects" are as described above. In the method for treating or preventing coronavirus infection of the present invention, the terms "coronavirus infection," "treatment," and "prevention" are as described above. Furthermore, the dosage form, administration route, dosage, administration time, etc. of the antibody or antigen-binding fragment thereof of the present invention, or the pharmaceutical composition containing same are also as described above.

[0060] 6. Coronavirus ACE2 Binding Inhibitors The antibodies of the present invention neutralize (inhibit) the binding of coronavirus to ACE2, thereby inhibiting coronavirus infection caused by such binding. Specifically, the present invention provides coronavirus ACE2 binding inhibitors that contain, as an active ingredient, the antibodies or antigen-binding fragments thereof described in "3. Antibodies of the Present Invention" above. The inhibitors of the present invention can be used as test reagents or for treating mammals, and their dosage forms, additives, administration routes, target subjects, and dosages can be appropriately selected in accordance with the descriptions in "4. Pharmaceutical Compositions" above. However, the inhibitors of the present invention may contain only the antibodies of the present invention or their antigen-binding fragments.

[0061] 7. Uses of Antibodies The antibodies or antigen-binding fragments thereof of the present invention can be used in methods for treating or preventing coronavirus infections or in the manufacture of medicaments for treating or preventing coronavirus infections. That is, the present invention provides the antibodies or antigen-binding fragments thereof of the present invention for use in methods for treating or preventing coronavirus infections. The present invention also provides the antibodies or antigen-binding fragments thereof of the present invention for use in the manufacture of medicaments for treating or preventing coronavirus infections. Furthermore, the present invention provides the antibodies or antigen-binding fragments thereof of the present invention for use in the manufacture of inhibitors of 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, such as remdesivir, dexamethasone, favipiravir, nafamostat, camostat, ivermectin, tricizumab, or baricitinib.

[0063] The combined administration of the pharmaceutical composition of the present invention and at least one other therapeutic agent is expected to produce more effective results than when each agent is used alone. The more effective results include maintaining therapeutic efficacy while reducing side effects compared to conventional methods. In the present invention, "combined administration" refers to simultaneous or separate administration of the pharmaceutical composition of the present invention and at least one other therapeutic agent. "Simultaneous" refers to administration at the same timing within a single administration schedule, and the administration times do not necessarily have to be exactly the same. "Separately" refers to administration at different timings within a single administration schedule. The dosage forms, administration routes, and administration targets 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 descriptions in "4. Pharmaceutical Compositions" above. Furthermore, the dosage forms or dosage amounts of the drugs used in combination may differ from each other and can be appropriately adjusted depending on the combination. When the pharmaceutical composition of the present invention is used in combination with other therapeutic agents, the dosage can also be appropriately reduced. Therefore, in the combination of the pharmaceutical composition of the present invention and the other therapeutic agent, the following combinations can be used: (i) an effective amount of the pharmaceutical composition of the present invention and an effective amount of the other therapeutic agent, (ii) an effective amount of the pharmaceutical composition of the present invention and an ineffective amount of the other therapeutic agent, (iii) an ineffective amount of the pharmaceutical composition of the present invention and an effective amount of the other therapeutic agent, and (iv) an ineffective amount of the pharmaceutical composition of the present invention and an ineffective amount of the other therapeutic agent. Even if one or both of the pharmaceutical composition and the other therapeutic agent are used in an ineffective amount, they can be administered in combination in such a manner as long as the combination can exert a pharmacological effect.

[0064] 9. Reagents and Kits The antibodies or antigen-binding fragments thereof of the present invention can be included in reagents or kits. That is, the present invention provides reagents and kits comprising the antibodies or antigen-binding fragments thereof of the present invention. The reagents and kits of the present invention can be used, for example, as reagents or kits for testing coronaviruses. In the reagents and kits of the present invention, the antibodies or antigen-binding fragments thereof of the present invention can 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, and lubricants, known additives (including buffers, isotonic agents, chelating agents, colorants, preservatives, fragrances, flavoring agents, sweeteners, etc.). In addition to the antibodies or antigen-binding fragments thereof of the present invention, the kits of the present invention can also include buffer solutions, enzyme solutions, secondary antibodies, diluents, instructions for use, etc.

[0065] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0066] Preparation of Monoclonal Antibodies (1) Immunogen In this example, the immunogens used were (i) mouse-derived cells stably expressing human ACE2, or (ii) a partial polypeptide of ACE2 containing the amino acid sequence (SEQ ID NO: 106) consisting of the 19th to 85th amino acid residues counting from the N-terminus of human ACE2 (SEQ ID NO: 2). The mouse-derived cells (4T1 cells) stably expressing human ACE2 (i) above were prepared as follows. Specifically, DNA encoding the full-length human ACE2 was first incorporated into a lentiviral vector, which was then transfected into 293T cells together with a packaging plasmid and an envelope plasmid. The recombinant lentivirus produced in the culture supernatant of these cells was then infected into 4T1 cells to generate cells stably expressing human ACE2 on the cell membrane. The partial polypeptide (ii) above was prepared by introducing a vector containing DNA (SEQ ID NO: 105) encoding the partial polypeptide into Escherichia coli, expressing the polypeptide in E. coli, and then purifying it.

[0067] (2) Immunization ICR mice or BALB / c mice (both from Sankyo Laboservice Co., Ltd.) were used as immunized animals. Immunization was performed by intraperitoneally administering the mouse-derived cells described in (i) above to BALB / c mice, and by intraperitoneally administering a mixture of the mouse-derived cells described in (i) above with the partial polypeptide and adjuvant described in (ii) above to ICR mice.

[0068] (3) Hybridoma Generation Immune responses were monitored by cell ELISA using mouse-derived cells (4T1 cells) described in (i) above, or by ELISA using the partial polypeptide described in (ii) above. When the immune response reached a plateau (i.e., when antibody titers had risen sufficiently), spleen cells were collected from the immunized mice and fused with mouse myeloma cells P3X63Ag8.653 (ATCC) to generate hybridoma cell lines.

[0069] (4) Screening: Hybridoma cell lines producing monoclonal antibodies that bind to human ACE2 were selected 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 using the culture supernatant of the hybridoma. This enabled the production of monoclonal antibodies that bind to human ACE2.

[0070] Sequencing of Anti-ACE2 Monoclonal Antibodies Among the anti-ACE2 monoclonal antibodies obtained in Example 1, several hybridoma cell lines producing monoclonal antibodies with high reactivity in cell ELISA or ELISA using partial polypeptides were selected. For the selected hybridoma cell lines, the antibody genes were cloned and the gene sequences were analyzed to determine the amino acid sequences of the antibodies. The nucleotide and amino acid sequences of the variable regions of representative antibodies (OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, OMRad045G03, OMRad052B05) 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 CDRs of each antibody were identified according to the Kabat numbering system. Among the numerous anti-ACE2 monoclonal antibodies obtained in Example 1, antibodies other than the six antibodies described above may be tested and evaluated as described in the following examples to obtain antibodies with actions or effects equivalent to or superior to those of these six antibodies.

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] Test for inhibition of binding between the RBD of spike protein and ACE2 using the anti-ACE2 monoclonal antibody of the present invention. Mouse 4T1 cells stably expressing human ACE2 were seeded at 10,000 cells / well into 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 medium, 200 μL / well of RPMI 1640 medium containing the anti-ACE2 monoclonal antibody prepared in Example 1 at 4 μg / mL was added and incubated on ice for 1 hour. After removing the antibody-containing medium and washing twice with RPMI 1640 medium, 200 μL / well of RPMI 1640 medium containing the RBD of the SARS-CoV-2 spike protein expressed in mammalian cells (CHO cells) at a concentration of 0.5 μg / mL was added and incubated on ice for 1 hour. This RBD contains a His tag, which allows it to be detected using a fluorescently labeled anti-His tag antibody.

[0078] After washing twice with RPMI 1640 medium, 200 μL / well of fluorescently labeled anti-His tag antibody (MBL) diluted to 1 μg / mL in RPMI 1640 medium was added and incubated for 30 minutes at room temperature in the dark. After fixation with 4% paraformaldehyde and nuclear staining with DAPI, the RBD of the SARS-CoV-2 spike protein bound to ACE2 on the cell membrane was detected using a fluorescence microscope.

[0079] The results are shown in Figure 1. A fluorescent signal was detected in the sample to which no anti-ACE2 monoclonal antibody was added (the "Antidoby(-)" panel in Figure 1). This indicates that in this sample, ACE2 on the cell membrane binds to the RBD, and the fluorescently labeled anti-His tag antibody binds to the His tag on the RBD, resulting in the detection of the fluorescent signal.

[0080] ​In contrast, no fluorescent signal was detected in the samples to which the anti-ACE2 monoclonal antibody prepared in Example 1 was added ("OMRad052B05," "OMRad004G05," "OMRad028D08," "OMRad031H08," "OMRad044E11," and "OMRad045G03" in Figure 1). 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 between ACE2 and RBD, and therefore the fluorescently labeled anti-His tag antibody was unable to bind to RBD, and therefore the fluorescent signal of the anti-His tag antibody was not detected.

[0081] That is, the results of this example demonstrated that the antibodies of the present invention can bind to ACE2 and neutralize the binding between ACE2 and RBD.

[0082] Preparation of Chimeric Antibodies Of the antibodies obtained in Example 1, a chimeric antibody was prepared 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 light chain constant region used in this example are shown in SEQ ID NO: 102 and SEQ ID NO: 104, respectively. Specifically, the chimeric antibody was prepared using the following method. First, the heavy chain variable region genes and light chain variable region genes of the anti-ACE2 antibody prepared in Example 1 were cloned from the hybridoma producing the anti-ACE2 antibody. Next, these genes (DNA) were linked to the base sequence of the heavy chain constant region gene (SEQ ID NO: 101) or light chain (κ chain) constant region gene (SEQ ID NO: 103) of human IgG4, respectively. To amplify the heavy chain variable region gene, PCR was performed using a primer (SEQ ID NO: 107) containing the 5'-terminal nucleotide sequence of the heavy chain variable region, a Kozak sequence, and the restriction enzyme EcoRI sequence, and an antisense primer (SEQ ID NO: 108) containing the complementary sequence of the 3'-terminal nucleotide sequence and the restriction enzyme NheI sequence. To amplify the light chain variable region gene, PCR was performed using a primer (SEQ ID NO: 109) containing the 5'-terminal nucleotide 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 nucleotide sequence and the restriction enzyme BsiWI sequence. The nucleotide sequences of the primers used in the PCR are shown in the table below.

[0083]

[0084] The resulting amplified product was digested with the restriction enzymes EcoRI and NheI or EcoRI and BsiWI and inserted 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 mouse heavy chain variable region and human heavy chain constant region were linked via the NheI restriction enzyme sequence, and the mouse light chain variable region and human light chain constant region were linked via the BsiWI restriction enzyme sequence. This example enabled the production of a chimeric antibody based on the antibody (OMRad052B05) prepared in Example 1.

[0085] Preparation of chimeric antibodies Chimeric antibodies were prepared by replacing the constant regions with the amino acid sequence of human IgG4 for OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, and OMRad045G03, among the antibodies obtained in Example 1, using the same procedure as in Example 4. However, the primers listed in the table below were used for PCR to amplify the variable regions.

[0086] In this example, chimeric antibodies based on the mouse antibodies OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, and OMRad045G03 obtained in Example 1 were successfully produced.

[0087] Competitive Inhibition Test In this example, six anti-ACE2 mouse antibodies (OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, OMRad045G03, and OMRad052B05) selected as representative examples from the antibodies obtained in Example 1, along with the chimeric antibodies prepared in Examples 4 and 5 based on these antibodies, were used to test whether antibodies that compete with these antibodies for binding to ACE2 could be obtained. This competitive inhibition test was performed by cell-based ELISA using 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 anti-ACE2 mouse antibodies was used as a reference antibody, and each of the chimeric antibodies was used as a test antibody (detection antibody). First, a certain reference antibody was reacted with 4T1 cells expressing human ACE2 (4T1-hACE2 cells), and then all test antibodies were reacted in a round-robin manner. A schematic diagram of this test is shown in Figure 2. Specifically, 1 x 10 4T1-hACE2 cells were cultured. 4 Cells / well were seeded into each well of a 96-well cell culture plate and cultured for 2 days. A solution containing each anti-human ACE2 mouse monoclonal antibody (reference antibody) at 30 μg / mL was prepared using blocking buffer (PBS(-) containing 3% BSA and 0.2% bovine serum). The culture medium was removed from the 96-well plate, and 50 μL / well of diluted reference antibody solution was added. oThe wells were then incubated at RT for 1 hour. After washing three times with 250 μL of wash buffer (PBS(-) containing 0.1% BSA), 50 μL of 2.5% formalin was added per well and the wells were incubated at room temperature for 10 minutes to fix the cells. After washing three times with 250 μL of wash buffer, 50 μL of each anti-human ACE2 mouse / human chimeric monoclonal antibody (test antibody (antibody to be detected)) diluted to 3 μg / mL in blocking buffer was added to each well and the wells were incubated at room temperature for 1 hour. After washing three times with 250 μL of wash buffer, 50 μL of 3% hydrogen peroxide in PBS(-) was added per well and the wells were incubated at room temperature for 5 minutes to inactivate endogenous peroxidase. After washing three times with 250 μL of wash buffer, 50 μL / well of HRP-labeled anti-human IgG-Fc antibody (Bethyl, A80-304P) diluted 1:10,000 in blocking buffer was added and incubated 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 incubated at room temperature for approximately 10 minutes. The reaction was stopped by adding 100 μL of 1.5 M sulfuric acid solution and vortexing, and the absorbance at 490 nm was measured using an absorption spectrophotometer. The absorbance was normalized to 100 for control wells containing only each chimeric antibody.

[0089] The results are shown in Figure 3. Four chimeric antibodies, OMRad004G05, OMRad028D08, OMRad031H08, and OMRad045G03, exhibited 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 (e.g., 22.8 when the reference antibody and test antibody were OMRad004G05) (Group 1). Furthermore, two antibodies, OMRad044E11 and OMRad052B05, exhibited 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 showed weak competition.

[0090] This demonstrates that antibodies belonging to Group 1 and Group 2 compete with other antibodies belonging to the same Group. In this example, the anti-ACE2 mouse antibody was used as the reference antibody and each of the chimeric antibodies was used as the test antibody; however, it is also possible to use each of the chimeric antibodies as the reference antibody and screen for anti-ACE2 mouse antibodies that compete with it. From the above, this example demonstrates that antibodies can be obtained that compete with the antibody obtained in Example 1 and the chimeric antibody prepared based on it for binding to ACE.

[0091] In the following Examples, the function and activity of each antibody was evaluated, and antibodies belonging to Group 1 and Group 2 were also evaluated to determine whether they had activity equivalent to that of other antibodies belonging to the same group.

[0092] Epitope Identification Test 1. Generation of Mouse / Human Fusion ACE2-Expressing Cells Previous reports have shown that the SARS-CoV-2 spike protein interacts with the N-terminus of human ACE2, while others have shown that SARS-CoV-2 cannot recognize mouse ACE2. Therefore, we investigated whether six anti-ACE2 mouse antibodies (OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, OMRad045G03, and OMRad052B05) from the antibodies obtained in Example 1 bind to the N-terminal region of human ACE2. Specifically, we generated a fusion ACE2 gene (polynucleotide) in which residues 1-120 from the N-terminus of human ACE2 were replaced with mouse ACE2, and then generated mouse / human fusion ACE2-expressing cells expressing this gene using the following procedure. First, a fusion ACE2 gene construct was created using NEBuilder Hi-Fi DNA Assembly (New England Biolabs), consisting of a polynucleotide containing bases 1-360 from the 5' end of the mouse ACE2 coding sequence and a polynucleotide containing bases 361-2418 from the 5' end of the human ACE2 coding sequence. This construct was then incorporated into a lentiviral vector. This lentiviral construct, along with a packaging and envelope plasmid, was transfected into 293T cells, and the recombinant lentivirus produced in the culture supernatant was then 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. The amino acid sequence of mouse ACE2 is the 1-120 amino acid sequence from the N-terminus, and the amino acid sequence of human ACE2 is the 121 amino acid sequence from the N-terminus.

[0093] 2. Immunocytostaining 4T1 cells, human ACE2-expressing 4T1 cells, mouse ACE2-expressing 4T1 cells, and mouse / human fusion ACE2-expressing 4T1 cells were plated at 3.2 x 10 in a glass-bottom 96-well plate for fluorescence observation. 3Cells were seeded at 1000 cells / well and cultured for 2 days. The plate was placed on ice, the medium was removed, and 200 mL of RPMI 1640 medium containing 20 mg / mL anti-ACE2 mouse monoclonal antibody was added and incubated on ice for 1 hour. After washing with 150 mL of ice-cold RPMI 1640 medium, RPMI 1640 medium containing Alexa Fluor 488-labeled anti-mouse IgG antibody (Thermo Fisher Scientific, A11001) was added and incubated on ice for 30 minutes. After washing, 100 mL of 4% paraformaldehyde was added and incubated on ice for 10 minutes. After thorough washing with PBS, the plates were observed under a fluorescence microscope.

[0094] 3. Results The results are shown in Figure 4. All six anti-ACE2 monoclonal antibodies bound to human ACE2. Two, OMRad031H08 and OMRad045G03, bound to mouse ACE2, whereas OMRad004G05, OMRad028D08, OMRad044E11, and OMRad052B05 did not. OMRad004G05, OMRad028D08, OMRad031H08, and OMRad045G03 bound to mouse / human fusion ACE2, whereas OMRad044E11 and OMRad052B05 did not (Figure 4). None of the antibodies bound to 4T1 cells, which do not express ACE2. The results are summarized in the table below (in the table, "Y" indicates binding, and "N" indicates no binding. "Full length" indicates the full length of the extracellular domain, and "aa" indicates the amino acid position).

[0095]

[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, indicating that these antibodies mainly bind to the region of human ACE2 that includes residues 1-120 from the N-terminus.

[0097] Furthermore, OMRad004G05 and OMRad028D08 bound to full-length human ACE2 and residues 121-740 of human ACE2, but not to mouse ACE2. This suggests that these antibodies bind to the extracellular region of human ACE2, mainly from residue 121 onwards (up to serine at residue 740), at positions or regions that allow for the distinction between human ACE2 and mouse ACE2 (e.g., regions with low homology between human ACE2 and mouse ACE2, or positions with different amino acids between the two).

[0098] On the other hand, because OMRad031H08 and OMRad045G03 reacted with all of human ACE2, mouse ACE2, and human / mouse fusion ACE2, this test did not allow us to narrow down the region to which these antibodies bind. However, taking into account the results of this test and the fact that OMRad031H08 and OMRad045G03 competed with OMRad004G05 and OMRad028D08, which also belong to Group 1 in Example 6, these antibodies are suggested to bind to a region of human ACE2 mainly containing amino acid residues 121-740, a position or region that does not distinguish between human ACE2 and mouse ACE2 (e.g., a region highly homologous between human ACE2 and mouse ACE2, a position with identical amino acids between both).

[0099] The fact that OMRad004G05 and OMRad028D08, OMRad031H08 and OMRad045G03, and OMRad044E11 and OMRad052B05 showed similar results in terms of reactivity to ACE2 is consistent with the similarity of the amino acid sequences of the CDRs of these antibodies.

[0100] Furthermore, the antibodies in Group 1 shown in Example 6 (OMRad004G05, OMRad028D08, OMRad031H08, OMRad045G03), like other antibodies in the same Group, were shown to bind mainly to a region containing amino acid residues 121-740, while the antibodies in Group 2 (OMRad044E11, OMRad052B05), like other antibodies in the same Group, were shown to bind mainly to a region containing amino acid residues 1-120 from the N-terminus.

[0101] Binding Affinity Evaluation Test 1. Method The affinity of six anti-ACE2 mouse antibodies (OMRad004G05, OMRad028D08, OMRad031H08, OMRad044E11, OMRad045G03, and OMRad052B05) from the antibodies obtained in Example 1 for human ACE2 was evaluated by cell-based ELISA. In this example, cell-based ELISA was performed using the six mouse antibodies and the chimeric antibodies prepared in Examples 4 and 5 based on these antibodies to determine the Kd values. Specifically, the test was performed according to the following procedure. First, 4T1 cells and 4T1 cells stably expressing human ACE2 (hereinafter referred to as "4T1-hACE2") were cultured at 1 x 10 4Cells / well were seeded into each well of a 96-well cell culture plate and cultured for two days. Solutions containing the six mouse and chimeric antibodies listed above were prepared at 100 μg / mL in blocking buffer (PBS(-) containing 3% BSA and 0.2% bovine serum). A two-fold dilution series was then prepared using blocking buffer. The culture medium was removed from the 96-well plate, and 50 μL of diluted antibody solution was added per well. The plate was then incubated at 4°C for 1 hour. After washing the wells three times with 250 μL of wash buffer (PBS(-) containing 0.05% Tween 20), 50 μL of 10% formalin was added per well and incubated at room temperature for 10 minutes to fix the cells. After washing twice with wash buffer, 50 μL of 3% hydrogen peroxide in PBS(-) was added per well and incubated at room temperature for 5 minutes to inactivate endogenous peroxidase. After washing three times with 250 μL of wash buffer, 50 μL / well of HRP-labeled anti-mouse kappa chain antibody (Bethyl, A90-119P) or HPR-labeled anti-human IgG-Fc antibody (Bethyl, A80-304P), diluted 1:10,000 in blocking buffer, was added and incubated 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 incubated at room temperature for approximately 10 minutes. The reaction was stopped by adding 100 μL of 1.5 M sulfuric acid solution and vortexing, and the absorbance at 490 nm was measured using an absorption spectrophotometer. The data obtained was analyzed using GraphPad Prism to determine the Kd value.

[0102] 2. Results: In cell-based ELISA, all six mouse and chimeric antibodies showed a concentration-dependent increase in absorbance against 4T1-hACE2 cells expressing human ACE2 (Figures 5A and 5B). On the other hand, when each chimeric antibody was reacted with 4T1 cells not expressing human ACE2 as a control, no concentration-dependent increase in absorbance was observed (Figure 5C). These results demonstrate that all six mouse 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 the mouse antibodies and from 0.86 nM to 2.62 nM for the chimeric antibodies, as shown in the table below. Note that the Kd values ​​of the mouse and chimeric antibodies cannot be directly compared due to the different secondary antibodies used.

[0103]

[0104] Inhibition of Binding of Mutant Spike Protein RBD to ACE2: In this example, the six chimeric antibodies described above were used to analyze the extent to which these antibodies inhibited the binding of mutant RBD to human ACE2-expressing cells. 1. Construction of Mutant Spike Protein RBD: In this example, mutant spike protein RBDs were constructed with the same amino acid sequence as the wild-type (WT (Wuhan)), alpha, beta, delta, epsilon, eta, theta, and kappa SARS-CoV-2 RBDs. Specifically, a 6x histidine tag was added to the 3' end of the SARS-CoV-2 spike protein cDNA region corresponding to the RBD (from lysine 319 to phenylalanine 541, counting from the N-terminus). This construct was then inserted into a mammalian cell expression vector. The mutations listed in the table below were introduced into the resulting plasmid using OE-PCR.

[0105]

[0106] The resulting plasmid DNA was transfected into CHO cells, and the mutant recombinant RBD was purified from the culture supernatant using Ni-NTA agarose.

[0107] 2. Binding Inhibition Test of Wild-Type and Mutant Spike Protein RBD with Human ACE2. 100 μL of a cell suspension of 4T1-hACE2 cells was seeded into each well of a 96-well cell culture plate at 10,000 cells per well and cultured at 37°C for 2 days in the presence of 5% CO2. Wild-type and mutant RBDs were added to Blocking Buffer (PBS containing 3% BSA and 0.2% bovine serum) to a final concentration of 5 nM. Anti-human ACE2 chimeric antibodies were diluted in Blocking Buffer containing wild-type and mutant RBDs. The 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 and incubated at 4°C for 1 hour. The anti-human ACE2 chimeric antibody solution was then decanted from the 96-well plate, and each well was washed twice with 250 μL of PBS(-). Mouse anti-SARS-CoV-2 Spike antibody (clone S28) was diluted to 1 μg / mL in blocking buffer, and 30 μL of the S28 antibody solution was added to each well and incubated at 4°C for 1 hour. The S28 antibody solution was then decanted, and each well was washed twice with 250 μL of PBS(-). 50 μL of 10% formalin solution was added to each well of the 96-well plate and incubated at room temperature for 10 minutes to fix the cells. The 10% formalin solution was then decanted, and each well was washed twice with 250 μL of wash buffer (PBS(-) containing 0.05% Tween 20). 50 μL of 3% hydrogen peroxide solution was added to each well of the 96-well plate and incubated at room temperature for 5 minutes to inactivate endogenous peroxidase. The 3% hydrogen peroxide solution was removed by decantation, and each well was washed three times with 250 μL of washing buffer (PBS(-) containing 0.05% Tween 20). 50 μL of secondary antibody (Mouse IgG-Fc Fragment cross-adsorbed antibody HRP conjugated, Bethyl, A90-231P) solution diluted 10,000-fold with blocking buffer was added to each well of the 96-well plate, and the plate was left 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 wash buffer (PBS(-) containing 0.05% Tween 20). 100 μL of OPD solution was added to each well of a 96-well plate, and the plate was left to stand at room temperature while monitoring the color development. 100 μL of 1.5 M sulfuric acid solution was added to each well to stop the color reaction. Absorbance at 490 nm was measured using an absorption spectrophotometer to detect RBD bound to human ACE2 on the cell membrane. The inhibition rate of the binding of wild-type and each mutant RBD to human ACE2 by anti-human ACE2 chimeric antibodies was calculated using wells containing only each RBD as a positive control and wells containing only anti-human ACE2 chimeric antibodies as a negative control.

[0108] 3. Results All six chimeric antibodies inhibited the binding of recombinant RBD to 4T1-hACE2 cells in a concentration-dependent manner (Fig. 6). The IC50 values ​​for the mutant RBD binding 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. Among the six chimeric antibodies, OMRad004G05, OMRad028D08, OMRad031H08, and OMRad045G03 showed comparable inhibitory effects against the binding of all mutant RBDs to ACE2. Furthermore, these four chimeric antibodies showed higher inhibitory effects (lower IC50) than OMRad044E11 and OMRad052B05. The results are shown in the table below.

[0109]

[0110] The results of this example demonstrated that all antibodies of the present invention neutralized 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 Group 2 (OMRad044E11, OMRad052B05) shown in Example 6 were shown to have activity equivalent to that of other antibodies in the same group.

[0111] 3. Neutralization Test Against Live Virus (1) Antibodies Used and Test Overview In this neutralization test, four of the six chimeric antibodies used in Example 9 above (OMRad004G05, OMRad028D08, OMRad031H08, and OMRad045G03) that showed particularly high inhibitory effects were used. The neutralization test was performed using a modified version of the method described in the National Institute of Infectious Diseases' "COVID-19 Serological Testing Manual." Specifically, dilutions of the chimeric antibodies (all antibodies were diluted 1.5-fold from 10 μg / mL (except for the Omicron strain) or 2.5 μg / mL (Omicron strain)) were contacted with cells, and then 100 TCID 50 Equal volumes of wild-type and various mutant SARS-CoV-2 suspensions adjusted to a concentration of 50 μL per 50 μL were added and cultured for 3 days in a carbon dioxide incubator (CO2 concentration: 5%) at 37°C ± 1°C, and the neutralizing antibody titers of the chimeric antibodies were measured. Antibody dilutions were prepared using cell maintenance medium [Dulbecco's modified Eagle's medium (Nacalai Tesque, Inc.) supplemented with 2% fetal bovine serum, penicillin (100 U / mL), streptomycin (100 μg / mL), and geneticin (G418, 1 mg / mL)].

[0112] (2) Viruses and Cells Used In this example, "live virus" refers to a virus that can infect and replicate in living cells. In this example, the following virus strains were used as live SARS-CoV-2 viruses: (i) SARS-CoV-2 wild-type strain (JPY / TY / WK-521); (ii) SARS-CoV-2 alpha variant QHN001 (VOC-202012 / 01 lineage); (iii) SARS-CoV-2 beta variant TY8-612 (B.1.351 lineage); (iv) SARS-CoV-2 delta variant TY11-927 (B.1.617.2 lineage); and (v) SARS-CoV-2 omicron variant TY38-873 (BA.1 lineage). VeroE6 / TMPRSS2 cells (JCRB1819) were used in this example.

[0113] (3) Preparation of virus suspension The SARS-CoV-2 virus suspension was prepared as follows: (i) VeroE6 / TMPRSS2 cells were cultured in monolayer in tissue culture flasks using cell growth medium [Dulbecco's modified Eagle's medium (Nacalai Tesque) 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 flasks, and various SARS-CoV-2 strains were inoculated. Cell maintenance medium [Dulbecco's Modified Eagle's Medium (Nacalai Tesque, Inc.) supplemented with 2% fetal bovine serum, penicillin (100 U / mL), streptomycin (100 μg / mL), and geneticin (G418) (1 mg / mL)] was added and the cells were cultured for 3 days in a carbon dioxide incubator (CO2 concentration: 5%) at 37°C ± 1°C. (iii) After 3 days of culture, cell morphology was observed using an inverted phase-contrast microscope to confirm the occurrence of morphological changes (cytopathic effect: CPE). The culture medium was then 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 grown in monolayer in tissue culture microplates (96-well flat-bottom) using cell growth medium. The cell growth medium was then removed, washed with cell maintenance medium, and the cell maintenance medium was then removed. Next, 50 μL of the above-mentioned chimeric antibody dilutions, serially diluted 1.5 times, were added to four wells, followed by inoculation with 50 μL of the above-mentioned virus suspension. The cells were then cultured for 3 days in a carbon dioxide incubator (CO2 concentration: 5%) at 37°C ± 1°C. After incubation, the presence or absence of cytopathic effect (CPE) was observed using an inverted phase-contrast microscope. The supernatant was removed, and the cells were fixed and washed with water. The fixed cells were stained and washed, and the highest dilution ratio at which CPE was suppressed in at least two wells was calculated. The antibody concentration required for neutralization was calculated from the highest dilution ratio and the antibody amount, and this was used as the neutralizing antibody titer. This test was repeated twice. As a result, all four antibodies used in the test showed neutralizing activity against cell infection by all live viruses, both wild-type and mutant strains. The results are shown in the table below.

[0115]

[0116] These results demonstrated that the antibodies of the present invention neutralize the binding and infection of wild-type and mutant SARS-CoV-2 strains to human cells. This indicates that the antibodies of the present invention are highly effective in treating or preventing coronavirus infection caused by wild-type or mutant SARS-CoV-2 strains. Furthermore, the antibodies belonging to Group 1 shown in Example 6 were shown to have neutralizing activity against the binding and infection of wild-type and mutant SARS-CoV-2 strains to human cells equivalent to that of other antibodies belonging to the same Group. Furthermore, as shown in the table above, OMRad031H08 and OMRa045G03 exhibited the same level of neutralizing activity, despite differing by one amino acid residue in their framework amino acid sequences. These results demonstrate that the antibodies of the present invention, when the amino acid sequences of the CDRs of two antibodies are identical or similar, have equivalent effects (e.g., neutralizing activity) even if the framework amino acid sequences are not completely identical.

[0117] The present invention provides a novel monoclonal antibody capable of binding to ACE2.

[0118] SEQ ID NOs: 3 to 134: synthetic DNA or synthetic peptide

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof that binds to ACE2 (Angiotensin Converting Enzyme 2).

2. The monoclonal antibody or its antigen-binding fragment described in claim 1, which is capable of neutralizing the binding of coronavirus to ACE2.

3. The monoclonal antibody or its antigen-binding fragment according to claim 2, wherein the coronavirus is a coronavirus that infects cells via ACE2.

4. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is a chimeric antibody, a humanized antibody or a fully human antibody.

5. (a) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO:54, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:56, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:58, and comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:60, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:62, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:64; or (b) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 30, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 34, and comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40; or (c) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 22, and comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 24, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 28; or (d) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10, and comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; or (e) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 42, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 48, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 52; or (f) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO:66, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:68, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:70, and comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:72, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:74, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:76; A monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4.

6. (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 94 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 96; or (b) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 86 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 88; or (c) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 82 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 84; or (d) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 78 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 80; (e) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 90 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 92; or (f) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 98 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 100; A monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5.

7. A monoclonal antibody or an antigen-binding fragment thereof capable of neutralizing the binding of coronavirus to ACE2 and competing with the antibody described in claim 5 or 6 for binding to ACE2.

8. A pharmaceutical composition for treating or preventing a coronavirus infection, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. The pharmaceutical composition according to claim 8, wherein the coronavirus infection is a disease or symptom caused by infection with a coronavirus that infects cells via ACE2.

10. The pharmaceutical composition according to claim 9, wherein the disease or symptom is at least one selected from the group consisting of respiratory diseases, fever, fatigue, chills, pain, taste or smell disorders, rash, gastrointestinal symptoms, speech disorders, cognitive disorders and cardiovascular symptoms.

11. 8. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 in the manufacture of a medicament for the treatment or prevention of a coronavirus infection.

12. A reagent or kit comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.