Antibodies specific to coronavirus spike protein and uses thereof

Abstract

The present invention relates to antibodies specific to coronavirus spike (S) protein and uses thereof.

Description

[Technical field]

[0001] The present invention relates to an antibody specific to a coronavirus spike (S) protein and uses thereof. [Background technology]

[0002] Coronaviruses were first discovered in chickens in 1937, and then in other animals such as dogs, pigs, and birds, before being discovered in humans in 1965. They were named after the corona phenomenon, which is the white light that appears around the sun's photosphere when it is hidden by the moon during a total solar eclipse.

[0003] Coronaviruses are known to cause mainly pneumonia and enteritis in humans and animals, and are also known to occasionally cause nervous system infections and hepatitis. Coronaviruses belong to the Coronaviridae family and are positive sense RNA viruses with a spherical outer membrane, measuring approximately 100 to 120 nm in size. Coronaviruses consist of a total of five structural proteins, including the spike protein (S) on the outermost shell, the hemagglutinin-esterase (HE) protein, the transmembrane (M) protein, the small membrane (E) protein, and the nucleocapsid (N) protein. Of these, the spike protein acts as a ligand that binds to cell receptors and induces fusion between the host cell and the virus, and is known to be the protein that mutates most frequently.

[0004] Until now, coronaviruses have been recognized as pathogens that rarely infect humans, but mainly infect animals such as dogs, pigs, and cows. Even if they infect humans, they are one of many viruses that cause respiratory symptoms, and only cause a simple cold or intestinal diseases such as diarrhea, which are not very dangerous for children. However, as it became known that the causative bacteria of Severe Acute Respiratory Syndrome (SARS), which has caused hundreds of deaths and thousands of patients worldwide, Middle East Respiratory Syndrome (MERS), and coronavirus disease 2019 (COVID-19) are new (variant) coronaviruses, they have gradually attracted attention.

[0005] COVID-19, a disease that was first identified in Wuhan, the capital of Hubei province in China, at the end of 2019 and has since spread and progressed to become a pandemic worldwide, is an infectious disease caused by SARS-CoV-2. As of May 7, 2020, over 3.75 million cases have been reported in 187 countries, with the death toll reaching 263 thousand and 1.24 million recoveries. Universal symptoms are fever, cough, fatigue, shortness of breath and loss of smell and taste. In most cases, symptoms are mild, but they can progress to viral pneumonia, multi-organ failure and cytokine storm. The time from onset of symptoms to onset is generally around 5 days, ranging from 2 to 14 days. The virus is transmitted between humans mainly by droplets generated during close contact and sometimes during coughing, sneezing and talking. It is most contagious during the first 3 days after symptoms appear, but can also be transmitted before symptoms appear and during the late stages of the disease. The standard method for diagnosing this is by real-time reverse transcription polymerase chain reaction (rRT-PCR) from a nasopharyngeal swab.

[0006] SARS-coronavirus 2 is known as a variant coronavirus belonging to the Betacoronavirus genus and is thought to have originated from bats, which are the primary reservoir of various coronaviruses (Non-Patent Document 1), but its exact route of infection has not yet been fully elucidated.

[0007] On the other hand, the antiviral drugs developed so far have severe side effects, so they must be applied with great care. These drugs are not effective and side effects have been reported. In addition, there is still no new coronavirus treatment that can prevent and treat SARS-coronavirus 2, has excellent infection suppression effects, and is less toxic, and there is an increasing need to develop one. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Antiviral Res., 101:45-56 [Non-Patent Document 2] Lai and Homes, 2001. Fields Virology [Non-Patent Document 3] Cascella, M., Rajnik, M., Cuomo, A., Dulebohn, SC, & Di Napoli, R. (2020). Features, Evaluation and Treatment Coronavirus (COVID-19). In StatPearls. Treasure Island (FL) [Non-Patent Document 4] Koeher and Milstein, 1976, Nature, 256:495 Summary of the Invention [Problem to be solved by the invention]

[0009] As a result of efforts to develop a novel SARS-CoV-2 therapeutic agent, the inventors confirmed that a novel antibody specific to the Receptor Binding Domain (RBD) of the SARS-CoV-2 S protein has a therapeutic effect against coronaviruses, particularly SARS-CoV-2, and thus completed the present invention. [Means for solving the problem]

[0010] The present invention aims to provide an antibody or antigen-binding fragment thereof that specifically binds to a coronavirus spike protein.

[0011] Another object of the present invention is to provide a polynucleotide encoding the antibody or antigen-binding fragment thereof, an expression vector comprising the polynucleotide, and a transformant comprising the expression vector.

[0012] A further object of the present invention is to provide a method for producing the antibody or antigen-binding fragment thereof.

[0013] Furthermore, the present invention aims to provide a composition for diagnosing coronavirus infection, which contains the antibody or an antigen-binding fragment thereof, and a kit containing the same.

[0014] Furthermore, the present invention aims to provide a method for providing information for diagnosing coronavirus infection, comprising a step of detecting coronavirus present in a biological sample using the antibody or an antigen-binding fragment thereof.

[0015] Furthermore, the present invention aims to provide a pharmaceutical composition for preventing or treating coronavirus, comprising the antibody or an antigen-binding fragment thereof.

[0016] A further object of the present invention is to provide a method for treating coronavirus using the antibody or antigen-binding fragment thereof. Effect of the Invention

[0017] The antibody of the present invention is not only useful for detecting and diagnosing coronaviruses, but also useful for preventing and treating coronavirus infectious diseases since it has the ability to neutralize coronaviruses. [Brief description of the drawings]

[0018] [Figure 1] This is a diagram showing the size and purity of the product obtained by purifying the antigen and confirming the size and purity of the product by SDS PAGE. [Diagram 2] This figure shows the size and purity of the product confirmed by SDS PAGE after expression and purification of the anti-SARS-CoV-2 antibody obtained by library screening. Specifically, the size of the entire antibody (~150 kDa) was confirmed by non-reduced PAGE, and the sizes of the antibody heavy chain (~50 kDa) and light chain (~25 kDa) were confirmed by reduced PAGE. [Diagram 3] This figure shows the results of sandwich ELISA performed to select antigen-specific anti-COVID-19 antibodies, which selected antibodies that exhibit specific binding to the antigen. [Figure 4] This figure shows the results of an experiment to determine whether the selected anti-SARS-CoV2 antibodies also bind to the S (S1S2) proteins of other betacoronaviruses, SARS-CoV and MERS-CoV. [Diagram 5] This figure shows the results of IgG expression and purification of humanized clones confirmed by SDS-PAGE. Specifically, the size of the whole antibody (~150 kDa) was confirmed by non-reduced PAGE, and the sizes of the antibody heavy chain (~50 kDa) and light chain (~25 kDa) were confirmed by reduced PAGE. [Figure 6] This figure shows the results of sandwich ELISA to select antigen-specific anti-COVID-19 humanized antibodies. Specifically, it was confirmed that all humanized clones maintained the same binding ability as conventional antibodies. [Figure 7]This figure shows the results of comparing the binding strength of humanized antibodies to SARS-CoV-2 mutant antigens. Specifically, it was confirmed that both clones M4-4 and M5-4 maintained binding strength to mutant antigens at or above the wild type level. [Figure 8a] This figure shows the kinetics of the binding properties of antibodies to anti-SARS-CoV-2 antigens. Specifically, the dissociation constant of the humanized antibody M4-4 was measured to be 10 times higher than that of the conventional antibody M4. [Figure 8b] This figure shows the results of evaluating the binding strength of antibodies against anti-SARS-CoV-2 antigens to Alpha, Beta, and Gamma mutations. Specifically, it was confirmed that M4-4 showed very strong binding strength to all mutations. [Figure 9a] FIG. 1 shows the results of observing ACE2 expression in ACE2-HEK293 cells using a flow cytometer. [Figure 9b] This figure shows the results of measuring the SARS-CoV-2 pseudovirus neutralizing ability of candidate antibodies using SARS-CoV-2 pseudovirus and ACE2-HEK293 cells. [Figure 10a] FIG. 1 shows the results of observing Spike protein expression in HT1080-S cells using a flow cytometer. [Figure 10b] FIG. 1 shows the results of measuring the ADCC effect of candidate antibodies using ADCC effector cells and HT1080-S cells. [Figure 10c] FIG. 1 shows the results of measuring the ADCP effect of candidate antibodies using FcγRIIa-H effector cells and HT1080-S cells. [Figure 11] This figure shows the neutralizing activity of anti-SARS-CoV-2 antibodies against authentic, wild type antigens, and alpha, beta, and gamma mutant antigens. Specifically, the M4-4 antibody in all cases showed a neutralizing effect equal to or greater than that of the comparative antibody. [Figure 12]1 shows the results of epitope binning of the M4-4 antibody, specifically, the results suggest that M4-4 binds to a wide range of epitopes that compete 100% with REGN10987, S309, and ACE2 receptors. [Figure 13] 1 shows the results of predicting the epitope of the M4-4 antibody by simulation docking. Specifically, it was confirmed that the epitope coincided with the predicted data. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] These will be described in detail below. Each description and embodiment disclosed in this application is also applicable to other descriptions and embodiments. That is, all combinations of various elements disclosed in the present invention are included in this application. In addition, the present invention is not limited to the following specific description. Furthermore, many papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosure contents of the cited papers and patent documents are incorporated in their entirety into this specification by reference, thereby more clearly explaining the state of the art to which the present invention belongs and the contents of the present invention.

[0020] One aspect of the invention provides an antibody, or antigen-binding fragment thereof, that specifically binds to a coronavirus spike (S) protein.

[0021] In the present invention, the term "coronavirus" refers to a positive-sense single-stranded RNA virus (ssRNA) that belongs to the Coronaviridae family, has a spherical outer membrane, and is about 80 to 220 nm in size. Coronaviruses are known to contain five structural proteins, including the spike (S) protein on the outermost shell, the hemagglutinin esterase (HE) protein, the membrane (M) protein, the envelope (E) protein, and the nucleocapsid (NP) protein (Non-Patent Document 2).

[0022] In one embodiment, the coronavirus is SARS-CoV, SARS-CoV-2, or MERS-CoV, specifically, but not limited to, SARS-CoV-2. The coronavirus spike (S) protein is spike 1 (S1) protein, which is a form in which a partial domain of the spike (S) protein of SARS-CoV-2 is truncated, but may be any coronavirus to which the antibody or antigen-binding fragment thereof of the present invention specifically binds.

[0023] In the present invention, "SARS-CoV-2" refers to the virus that causes the respiratory disease novel coronavirus disease (COVID-19).

[0024] SARS-CoV-2 is a single-stranded positive-stranded RNA virus. Taxonomically, SARS-CoV-2 is a SARS-CoV strain with zoonotic origins and is considered to have close genetic similarity to bat coronaviruses. SARS-CoV-2 is primarily transmitted by close contact between humans and / or through droplets generated during coughing and sneezing, and is known to enter human cells mainly by binding the spike (S) protein of the viral envelope to angiotensin-converting enzyme 2 (ACE2), a receptor present on the surface of human cells.

[0025] SARS-CoV-2 contains RNA-dependent RNA polymerase, spike (S), membrane (M), envelope (E) and nucleocapsid (NP) proteins and / or genes encoding said proteins. Among the SARS-CoV-2 structural proteins, the spike (S) protein is present on the surface of the virus particle and is involved in host cell invasion, and is divided into S1 and S2. The S1 protein interacts with a host cell receptor, and the receptor-binding domain (RBD) present in the S1 protein is known as the core part that binds to the ACE2 receptor present on the host cell surface. The S2 protein is involved in fusion with the host cell membrane. The contents of the above structure are described in detail in Non-Patent Document 3.

[0026] Specifically, the antibody or antigen-binding fragment thereof provided in the present invention may specifically bind to spike 1 (S1) protein, which is a truncated form of a partial domain of the spike (S) protein of SARS-CoV-2. Specifically, the antibody specifically binding to the coronavirus spike protein may be a mouse antibody, a chimeric antibody, or a humanized antibody.

[0027] For example, the antigenic determinant (epitope) recognized by the antibody or antigen-binding fragment thereof provided in the present invention may be a part of the binding domain (CD) present in the spike 1 (S1) protein of SARS-CoV-2.

[0028] In the present invention, the term "antibody" refers to a protein molecule that includes an immunoglobulin molecule that is immunologically reactive to a specific antigen and that plays the role of a ligand that specifically recognizes the antigen, and includes polyclonal antibodies, monoclonal antibodies, whole antibodies, and antibody fragments. The term also includes chimeric antibodies (e.g., humanized mouse antibodies), bivalent or bispecific molecules (e.g., bispecific antibodies), diabodies, tribodies, and tetrabodies. The term further includes single-chain antibodies, scAbs, derivatives of antibody constant regions, and artificial antibodies based on protein scaffolds that have a binding function to FcRn (neonatal Fc receptor). A whole antibody is a structure having two full-length light chains (LC) and two full-length heavy chains (HC), and each light chain is linked to a heavy chain by a disulfide bond. The whole antibodies include IgA, IgD, IgE, IgM and IgG, and IgG includes subtypes of IgG1, IgG2, IgG3 and IgG4. In one embodiment, the antibody provided in the present invention may be an IgG antibody.

[0029] The terms "fragment", "polypeptide binding fragment" and "antibody fragment" refer to any fragment of the antibody of the present invention that has the antigen-binding activity of the antibody, and are used interchangeably. Examples of antibody fragments include, but are not limited to, single chain antibodies, Fd, Fab, Fab', F(ab')2, dsFv or scFv. The term Fd refers to the heavy chain portion contained in the Fab fragment. The Fab has a structure having light and heavy chain variable regions, a light chain constant region (framework region, FR), and a first constant region (CH1 domain) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing at least one cysteine ​​residue at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is generated by disulfide bonding of the cysteine ​​residues in the hinge region of Fab'. The term Fv (variable fragment) refers to the minimum antibody fragment having only a heavy chain variable region and a light chain variable region. In disulfide-stabilized Fv (dsFv), the heavy chain variable region and the light chain variable region are linked by a disulfide bond, and in single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are generally linked by a covalent bond via a peptide linker. Such antibody fragments can be obtained by using a protease (for example, Fab is obtained by restrictive cleavage of the whole antibody with papain, and F(ab')2 fragments are obtained by cleavage with pepsin), and can be produced, for example, by genetic recombination techniques.

[0030] In the present invention, the term "antigenic determinant" is also called "epitope", and refers to the region or site of an antigen to which an antibody or an antibody fragment specifically binds.

[0031] Furthermore, the antibody or antigen-binding fragment thereof competitively binds to an epitope derived from the coronavirus spike to which casirivimab (REGN10933), imdevimab (REGN10987) and / or S309 binds, but is not limited to these.

[0032] In one embodiment, the antibody provided in the present invention may be a monoclonal antibody.

[0033] By "monoclonal antibody" in this invention is meant an antibody molecule of single molecular composition obtained from a population of substantially homogeneous antibodies, which monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0034] Typically, immunoglobulins have heavy and light chains, each of which contains a constant region and a variable region (these regions are also known as domains). The variable regions of the light and heavy chains contain three hypervariable regions called complementarity-determining regions (hereinafter referred to as "CDRs") and four framework regions. The CDRs mainly play a role in binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 in order from the N-terminus, and are also identified by the chain in which a particular CDR is located.

[0035] In general, immunoglobulins have a heavy chain and a light chain, each of which contains a constant region and a variable region. The variable regions of the light and heavy chains contain three hypervariable regions called complementarity-determining regions (hereinafter referred to as "CDRs") and four framework regions (hereinafter referred to as "FRs"). The CDRs of each chain mainly play a role in binding to an epitope of an antigen, and are typically called CDR1, CDR2, and CDR3 from the N-terminus. The FRs of each chain are called FR1, FR2, FR3, and FR4 from the N-terminus.

[0036] In the present invention, the variable region of the heavy chain is referred to as "V H The light chain variable region is called "V L" and the heavy chain CDRs are called "V H -CDR1", "V H -CDR2", "V H -CDR3" and the light chain CDRs are called "V L -CDR1", "V L -CDR2", "V L -CDR3" and the heavy chain FR is "V H -FR1", "V H -FR2", "V H The light chain FR is called "VH-FR3" and "VH-FR4". L -FR1", "V L -FR2", "V L -FR3", "V L -FR4".

[0037] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to the coronavirus spike protein of the present invention may comprise at least one heavy chain variable region selected from the group consisting of any of the sequences of SEQ ID NOs: 5 to 45, 87 to 91, and 97 to 115, and at least one light chain variable region selected from the group consisting of any of the sequences of SEQ ID NOs: 46 to 86, 92 to 96, and 116 to 131. Specifically, the heavy chain variable region of SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 46, the heavy chain variable region of SEQ ID NO: 6 and the light chain variable region of SEQ ID NO: 47, the heavy chain variable region of SEQ ID NO: 7 and the light chain variable region of SEQ ID NO: 48, the heavy chain variable region of SEQ ID NO: 8 and the light chain variable region of SEQ ID NO: 49, the heavy chain variable region of SEQ ID NO: 9 and the light chain variable region of SEQ ID NO: 50, the heavy chain variable region of SEQ ID NO: 10 and the light chain variable region of SEQ ID NO: 51, the heavy chain variable region of SEQ ID NO: 11 and the light chain variable region of SEQ ID NO: 52, the heavy chain variable region of SEQ ID NO: 12 and the light chain variable region of SEQ ID NO: 53, the heavy chain variable region of SEQ ID NO: 13 and the light chain variable region of SEQ ID NO: 54, the heavy chain variable region of SEQ ID NO: 14 and the light chain variable region of SEQ ID NO: 55, the heavy chain variable region of SEQ ID NO: 15 and the light chain variable region of SEQ ID NO: 56, the heavy chain variable region of SEQ ID NO: 16 and the light chain variable region of SEQ ID NO: 57, the heavy chain variable region of SEQ ID NO: 17 and the light chain variable region of SEQ ID NO: 58, the heavy chain variable region of SEQ ID NO: 18, and the light chain variable region of SEQ ID NO:59, the heavy chain variable region of SEQ ID NO:19 and the light chain variable region of SEQ ID NO:60, the heavy chain variable region of SEQ ID NO:20 and the light chain variable region of SEQ ID NO:61, the heavy chain variable region of SEQ ID NO:21 and the light chain variable region of SEQ ID NO:62, the heavy chain variable region of SEQ ID NO:22 and the light chain variable region of SEQ ID NO:63, the heavy chain variable region of SEQ ID NO:23 and the light chain variable region of SEQ ID NO:64, the heavy chain variable region of SEQ ID NO:24 and the light chain variable region of SEQ ID NO:65, the heavy chain variable region of SEQ ID NO:25 and the light chain variable region of SEQ ID NO:66, the heavy chain variable region of SEQ ID NO:26 and the light chain variable region of SEQ ID NO:67, the heavy chain variable region of SEQ ID NO:27 and the light chain variable region of SEQ ID NO:68, the heavy chain variable region of SEQ ID NO:28 and the light chain variable region of SEQ ID NO:69, the heavy chain variable region of SEQ ID NO:29 and the light chain variable region of SEQ ID NO:70, the heavy chain variable region of SEQ ID NO:30 and the light chain variable region of SEQ ID NO:71, the heavy chain variable region of SEQ ID NO:31 and the light chain variable region of SEQ ID NO:72,A heavy chain variable region of SEQ ID NO: 32 and a light chain variable region of SEQ ID NO: 73, a heavy chain variable region of SEQ ID NO: 33 and a light chain variable region of SEQ ID NO: 74, a heavy chain variable region of SEQ ID NO: 34 and a light chain variable region of SEQ ID NO: 75, a heavy chain variable region of SEQ ID NO: 35 and a light chain variable region of SEQ ID NO: 76, a heavy chain variable region of SEQ ID NO: 36 and a light chain variable region of SEQ ID NO: 77, a heavy chain variable region of SEQ ID NO: 37 and a light chain variable region of SEQ ID NO: 78, a heavy chain variable region of SEQ ID NO: 38 and a light chain variable region of SEQ ID NO: 79, a heavy chain variable region of SEQ ID NO: 39 and a light chain variable region of SEQ ID NO: 80, a heavy chain variable region of SEQ ID NO: 40, and the light chain variable region of SEQ ID NO: 81, the heavy chain variable region of SEQ ID NO: 41 and the light chain variable region of SEQ ID NO: 82, the heavy chain variable region of SEQ ID NO: 42 and the light chain variable region of SEQ ID NO: 83, the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 84, the heavy chain variable region of SEQ ID NO: 44 and the light chain variable region of SEQ ID NO: 85, the heavy chain variable region of SEQ ID NO: 45 and the light chain variable region of SEQ ID NO: 86, the heavy chain variable region of SEQ ID NO: 87 and the light chain variable region of SEQ ID NO: 92, the heavy chain variable region of SEQ ID NO: 88 and the light chain variable region of SEQ ID NO: 93, the heavy chain variable region of SEQ ID NO: 89 and the light chain variable region of SEQ ID NO: 94 , a heavy chain variable region of SEQ ID NO: 90 and a light chain variable region of SEQ ID NO: 95, a heavy chain variable region of SEQ ID NO: 91 and a light chain variable region of SEQ ID NO: 96, a heavy chain variable region of SEQ ID NO: 100 and a light chain variable region of SEQ ID NO: 116, a heavy chain variable region of SEQ ID NO: 101 and a light chain variable region of SEQ ID NO: 117, a heavy chain variable region of SEQ ID NO: 102 and a light chain variable region of SEQ ID NO: 118, a heavy chain variable region of SEQ ID NO: 103 and a light chain variable region of SEQ ID NO: 119, a heavy chain variable region of SEQ ID NO: 104 and a light chain variable region of SEQ ID NO: 120, a heavy chain variable region of SEQ ID NO: 105 and a light chain variable region of SEQ ID NO: 121, A heavy chain variable region of sequence number 106 and a light chain variable region of sequence number 122, a heavy chain variable region of sequence number 107 and a light chain variable region of sequence number 123, a heavy chain variable region of sequence number 108 and a light chain variable region of sequence number 124, a heavy chain variable region of sequence number 109 and a light chain variable region of sequence number 125, a heavy chain variable region of sequence number 110 and a light chain variable region of sequence number 126, a heavy chain variable region of sequence number 111 and a light chain variable region of sequence number 127, a heavy chain variable region of sequence number 112 and a light chain variable region of sequence number 128, a heavy chain variable region of sequence number 113 and a light chain variable region of sequence number 129,The heavy chain variable region of SEQ ID NO: 114 and the light chain variable region of SEQ ID NO: 130, the heavy chain variable region of SEQ ID NO: 115 and the light chain variable region of SEQ ID NO: 131, but is not limited thereto.

[0038] For purposes of the present invention, the antibody or antigen-binding fragment thereof may be cross-reactive with two or more coronaviruses, including but not limited to SARS-CoV, SARS-CoV-2, or MERS-CoV, as long as the antibody or antigen-binding fragment thereof is cross-reactive with two or more coronaviruses.

[0039] For example, the antibody or antigen-binding fragment thereof that cross-reacts with two or more types of coronaviruses comprises at least one heavy chain variable region selected from the group consisting of any of the sequences of SEQ ID NOs: 6 to 12, 14 to 22, 24, 25, 32, 37, 45, 87 to 91, and 97 to 99, and at least one light chain variable region selected from the group consisting of any of the sequences of SEQ ID NOs: 47 to 53, 55 to 63, 65, 66, 73, 78, 86, and 92 to 96, but is not limited thereto.

[0040] Specifically, the heavy chain variable region of SEQ ID NO: 6 and the light chain variable region of SEQ ID NO: 47, the heavy chain variable region of SEQ ID NO: 7 and the light chain variable region of SEQ ID NO: 48, the heavy chain variable region of SEQ ID NO: 8 and the light chain variable region of SEQ ID NO: 49, the heavy chain variable region of SEQ ID NO: 9 and the light chain variable region of SEQ ID NO: 50, the heavy chain variable region of SEQ ID NO: 10 and the light chain variable region of SEQ ID NO: 51, the heavy chain variable region of SEQ ID NO: 11 and the light chain variable region of SEQ ID NO: 52, the heavy chain variable region of SEQ ID NO: 12 and the light chain variable region of SEQ ID NO: 53, the heavy chain variable region of SEQ ID NO: 14 and the light chain variable region of SEQ ID NO: 55, the heavy chain variable region of SEQ ID NO: 15 and the light chain variable region of SEQ ID NO: 56, the heavy chain variable region of SEQ ID NO: 16 and the light chain variable region of SEQ ID NO: 57, the heavy chain variable region of SEQ ID NO: 17 and the light chain variable region of SEQ ID NO: 58, the heavy chain variable region of SEQ ID NO: 18 and the light chain variable region of SEQ ID NO: 59, the heavy chain variable region of SEQ ID NO: 19 and the light chain variable region of SEQ ID NO: 60, the heavy chain variable region of SEQ ID NO: 20 The heavy chain variable region of SEQ ID NO: 45 and the light chain variable region of SEQ ID NO: 86, the heavy chain variable region of SEQ ID NO: 87 and the light chain variable region of SEQ ID NO: 92, the heavy chain variable region of SEQ ID NO: 88 and the light chain variable region of SEQ ID NO: 93, the heavy chain variable region of SEQ ID NO: 89 and the light chain variable region of SEQ ID NO: 94, the heavy chain variable region of SEQ ID NO: 90 and the light chain variable region of SEQ ID NO: 95, the heavy chain variable region of SEQ ID NO: 91 and the light chain variable region of SEQ ID NO: 96.

[0041] In the present invention, the term "human antibody" refers to a molecule derived from human immunoglobulin, in which all amino acid sequences constituting the antibody, including the complementarity determining region and the framework region, are composed of amino acid sequences of human immunoglobulin. Human antibodies are usually used to treat human diseases, and have at least three potential advantages. First, they interact better with the human immune system and can destroy target cells more efficiently, for example by complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Second, they have the advantage that the human immune system does not recognize the antibody as foreign. Third, they have the advantage that their half-life in the human circulation is similar to that of naturally occurring antibodies, even when smaller and less frequent doses of the drug are administered. Thus, the human antibody according to the present invention has a prophylactic or therapeutic effect against coronaviruses, particularly SARS-CoV-2, and is therefore useful for treating SARS-CoV-2.

[0042] When the human monoclonal antibody of the present invention contains a constant region, the constant region may be derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid of these.

[0043] In the present invention, the term "combination" refers to the binding of a polypeptide encoding a single-chain immunoglobulin constant region of the same origin to a single-chain polypeptide of a different origin when forming a dimer or multimer. For example, a dimer or multimer can be formed from at least two constant regions selected from the group consisting of IgG, IgA, IgD, IgE, and IgM constant regions.

[0044] In the present invention, the term "hybrid" means that a single-chain immunoglobulin heavy chain constant region contains sequences corresponding to immunoglobulin heavy chain constant regions of at least two different origins, and may be, for example, a hybrid of one to four domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG, IgA, IgD, IgE, and IgM.

[0045] Furthermore, in the present invention, the variable region and constant region of an antibody or antigen-binding fragment thereof may be of the same or different origins, and the CDRs, the variable region excluding the CDRs, and the constant region may be of the same or different origins.

[0046] For example, an antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 139, a heavy chain CDR2 of SEQ ID NO: 140, and a heavy chain CDR3 of SEQ ID NO: 141, and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 142 or 148, a light chain CDR2 of SEQ ID NO: 143 or 146, and a light chain CDR3 of SEQ ID NO: 144 or 147, but is not limited thereto.

[0047] Specifically, it includes a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 139, a heavy chain CDR2 of SEQ ID NO: 140, and a heavy chain CDR3 of SEQ ID NO: 141, and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 142, a light chain CDR2 of SEQ ID NO: 143, and a light chain CDR3 of SEQ ID NO: 144, or it includes a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 139, a heavy chain CDR2 of SEQ ID NO: 140, and a heavy chain CDR3 of SEQ ID NO: 141, and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 145, a light chain CDR2 of SEQ ID NO: 146, and a light chain CDR3 of SEQ ID NO: 147, but is not limited thereto.

[0048] Furthermore, in the present invention, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 103 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 119, but is not limited thereto.

[0049] In one embodiment of the present invention, an antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 103 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 119 was named "M4-4" or "MG1141A."

[0050] Furthermore, the antibodies of the present invention comprise a heavy chain variable region comprising a heavy chain Framework region 1 of SEQ ID NO: 148, 152 or 155, a heavy chain FR2 of SEQ ID NO: 149 or 156, a heavy chain FR3 of SEQ ID NO: 150, 153 or 157, and a heavy chain FR4 of SEQ ID NO: 151, 154 or 158, and a light chain variable region comprising a light chain FR1 of SEQ ID NO: 159, 163 or 166, a light chain FR2 of SEQ ID NO: 160 or 167, a light chain FR3 of SEQ ID NO: 161, 164 or 168, and a light chain FR4 of SEQ ID NO: 162, 165 or 169, but are not limited thereto.

[0051] Specifically, the antibody comprises a heavy chain variable region comprising a heavy chain FR (Framework region) 1 of SEQ ID NO: 148, a heavy chain FR2 of SEQ ID NO: 149, a heavy chain FR3 of SEQ ID NO: 150, and a heavy chain FR4 of SEQ ID NO: 151, and a light chain variable region comprising a light chain FR1 of SEQ ID NO: 159, a light chain FR2 of SEQ ID NO: 160, a light chain FR3 of SEQ ID NO: 161, and a light chain FR4 of SEQ ID NO: 162; a heavy chain FR (Framework region) 1 of SEQ ID NO: 152, a heavy chain FR2 of SEQ ID NO: 149, a heavy chain FR3 of SEQ ID NO: 153, and a heavy chain FR4 of SEQ ID NO: 154, and a light chain variable region comprising a light chain FR1 of SEQ ID NO: 163, a light chain FR2 of SEQ ID NO: 160, a light chain FR3 of SEQ ID NO: 164, and a light chain FR4 of SEQ ID NO: 165; or a heavy chain FR (Framework region) 1 of SEQ ID NO: 155. and a light chain variable region comprising a light chain FR1 of SEQ ID NO: 166, a light chain FR2 of SEQ ID NO: 167, a light chain FR3 of SEQ ID NO: 168, and a light chain FR4 of SEQ ID NO: 169, but is not limited to these.

[0052] Another aspect of the invention provides a method for producing the novel antibody or antigen-binding fragment thereof.

[0053] The novel antibody or antigen-binding fragment thereof of the present invention can be easily produced by known antibody production techniques, such as, but not limited to, producing hybridomas using B lymphocytes obtained from immunized animals (Non-Patent Document 4) or using phage display technology.

[0054] An antibody library using phage display technology is a method in which antibody genes are directly obtained from B lymphocytes and expressed on the surface of phages without producing hybridomas. Using phage display technology, it is possible to overcome many of the conventional difficulties involved in producing monoclonal antibodies by B cell immortalization. In general, phage display technology consists of the steps of: 1) inserting an oligonucleotide with a random sequence into a gene region corresponding to the N-terminus of the phage coat protein pIII (or pIV); 2) expressing a fusion protein of a part of the natural coat protein and a polypeptide encoded by the oligonucleotide with the random sequence; 3) treating with a receptor substance that binds to the polypeptide encoded by the oligonucleotide; 4) eluting the peptide-phage particles bound to the receptor using low pH or a molecule that has binding competitive power; 5) amplifying the phage eluted by panning in a host cell; 6) repeating the above method to obtain a desired amount; and 7) determining the sequence of an active peptide from the DNA sequence of the phage clone selected by panning.

[0055] Yet another aspect of the present invention provides a polynucleotide encoding the novel antibody, an expression vector containing the polynucleotide, and a transformant containing the expression vector.

[0056] Yet another aspect of the present invention provides an expression vector comprising an antibody or antigen-binding fragment thereof provided herein, and a host cell into which said vector has been introduced.

[0057] The antibody is as described above.

[0058] The expression vector comprising the polynucleotide encoding the antibody provided in the present invention is a vector that replicates and / or expresses the polynucleotide in eukaryotic or prokaryotic cells, including, but not limited to, mammalian cells (e.g., human, monkey, rabbit, rat, hamster, mouse cells, etc.), plant cells, yeast cells, insect cells, or bacterial cells (e.g., E. coli, etc.), and specifically, a vector that is operably linked to an appropriate promoter so that the nucleotide is expressed in a host cell and contains at least one selection marker. For example, the polynucleotide may be introduced into a phage, plasmid, cosmid, mini-chromosome, virus, retrovirus vector, etc.

[0059] The expression vector comprising a polynucleotide encoding the antibody may be an expression vector comprising a polynucleotide encoding the heavy chain or a polynucleotide encoding the light chain of the antibody, or may be an expression vector comprising both a polynucleotide encoding the heavy chain and a polynucleotide encoding the light chain.

[0060] The transformant introduced with the expression vector provided in the present invention includes, but is not limited to, bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc., fungal cells such as yeast cells, Pichia pastoris, etc., insect cells such as Drosophila and Spodoptera Sf9 cells, animal cells such as CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, bow melanoma cells, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), PERC.6 (human retinal cells), etc., or plant cells, all of which have been transformed with the expression vector.

[0061] In the present invention, "introduction" refers to a method of incorporating a vector containing a polynucleotide encoding the antibody into a host cell. The introduction can be performed by various methods known in the art, such as calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electron bombardment, microinjection, liposome fusion, lipofectamine, and protoplast fusion. Transduction refers to the delivery of a target substance into a cell by infection using virus particles. Furthermore, a vector can be introduced into a host cell by gene bombardment or the like. In the present invention, introduction is used interchangeably with transformation.

[0062] Yet another aspect of the present invention provides a composition for diagnosing coronavirus infection, comprising an antibody or antigen-binding fragment thereof provided herein.

[0063] Yet another aspect of the present invention provides a kit for diagnosing coronavirus infection, comprising an antibody or antigen-binding fragment thereof provided herein.

[0064] Yet another aspect of the present invention provides a method for diagnosing coronavirus infection or a method for providing information for diagnosis, comprising the step of detecting coronavirus present in a biological sample isolated from an individual suspected of coronavirus infection using an antibody or an antigen-binding fragment thereof provided in the present invention, a composition for diagnosing infection comprising the same, or a kit comprising the composition.

[0065] "Diagnosis" in the present invention includes determining an individual's susceptibility to a particular disease or disorder, determining whether an individual currently has a particular disease or disorder, or monitoring an individual's condition to provide information regarding the effectiveness of a treatment.

[0066] For the purposes of the present invention, the diagnosis may be to confirm whether or not a coronavirus infection disease has developed. The term "coronavirus infection disease" refers to a disease caused by infection with a coronavirus that invades the body of a host organism. Specifically, the coronavirus may be SARS-CoV-2. For example, the coronavirus infection disease may be COVID-19.

[0067] The coronavirus infection diagnostic composition of the present invention may contain an antibody or an antigen-binding fragment thereof other than the antibody or antigen-binding fragment thereof of the present invention. In addition, the composition may further contain a substance, such as a reagent, necessary for the binding reaction between the antibody or antigen-binding fragment thereof of the present invention and an antigen. Furthermore, the composition may further contain a substance necessary for detecting the binding, a substance for stabilizing the antibody or antigen-binding fragment thereof of the present invention, and the like.

[0068] For example, the antibody or antigen-binding fragment thereof contained in the diagnostic composition of the present invention may be labeled for detection. A variety of methods for molecular labeling are well known to those skilled in the art and are within the scope of the present invention.

[0069] Examples of the types of labels that can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds. Commonly used labels include fluorescent substances (e.g., fluorescein, rhodamine, Texas Red, etc.), enzymes (e.g., horseradish peroxidase, β-galactosidase, alkaline phosphatase), radioisotopes (e.g., 32P or 125I), biotin, digoxigenin, colloidal metals, chemiluminescent or bioluminescent compounds (e.g., dioxetane, luminol, or acridinium). Labeling methods such as covalent binding of enzymes or biotinyl groups, iodination, phosphorylation, and biotinylation are also well known.

[0070] The kit of the present invention may further comprise reagents, instruments, etc. necessary for detecting the binding between the antibody of the present invention or an antigen-binding fragment thereof and an antigen.

[0071] For example, a kit container of the invention may include a solid support to which an antibody of the invention is attached, which may be porous or non-porous, planar or non-planar.

[0072] For example, the kit includes a solid carrier, a sample pad that contains a sample to be analyzed and has a buffer input section and a sample input section, a conjugate pad having an antibody that specifically binds to coronavirus contained in the sample flowing in from the sample pad, a signal detection pad having a signal detection section that detects whether or not coronavirus is present in the sample and a control section that checks whether or not the sample has moved to the absorbent pad regardless of the presence or absence of an analyte, and an absorbent pad that absorbs the sample after the signal detection reaction has completed, but is not limited to this.

[0073] For example, the kit of the present invention may be in the form of an ELISA (Enzyme-linked immunosorbent assay) kit. In the present invention, "ELISA" is also referred to as an enzyme immunoassay, which is a method in which an enzyme is bound to an antibody to form an antigen-antibody complex, and the enzyme reacts with a substrate to perform quantification using absorbance. The ELISA includes direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in an antibody complex that recognizes the antigen attached to a solid support, direct sandwich ELISA using another labeled antibody that recognizes an antigen in an antibody-antigen complex attached to a solid support, and indirect sandwich ELISA using a labeled secondary antibody that reacts with another antibody that recognizes the antigen in an antibody-antigen complex attached to a solid support and then recognizes that antibody.

[0074] The antibody or antigen-binding fragment thereof of the present invention may be used to detect the presence or absence of coronavirus in a biological sample by contacting the sample with the antibody or antigen-binding fragment thereof and observing the reaction.

[0075] The biological sample may be any one selected from the group consisting of sputum, saliva, blood, sweat, lung cells, mucus from lung tissue, respiratory tissue, and saliva from a subject, but is not limited thereto, and may be prepared by any conventional method known to those skilled in the art.

[0076] For example, the method of providing information for diagnosing coronavirus infection of the present invention may include the steps of (a) contacting the antibody or antigen-binding fragment thereof provided in the present invention with a biological sample, and (b) detecting a complex of the antibody or antigen-binding fragment thereof of the present invention and the coronavirus spike protein formed by the contact as described above. Specifically, in the step (b), the complex of the antibody or antigen-binding fragment thereof and the coronavirus spike protein may be a conjugate of the binding domain (CD) site of the SARS-CoV-2 spike protein.

[0077] In the present invention, the term "antigen-antibody complex" refers to a complex of a protein antigen in a sample and an antibody that recognizes the antigen. The antigen-antibody complex may be detected by a method known in the art, for example, a spectroscopic, photochemical, biochemical, immunochemical, electrical, absorbance, chemical, or other method. Specifically, the antigen-antibody complex may be detected by any method selected from the group consisting of a colorimetric method, an electrochemical method, a fluorimetric method, a luminometry method, a particle counting method, a visual assessment, and a scintillation counting method. Examples of the method include western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence-activated cell sorting (FACS), protein chip, and the like. Methods such as, but not limited to, the use of a holographic chip can be used.

[0078] Yet another aspect of the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection, comprising the antibody or antigen-binding fragment thereof. Specifically, the coronavirus may be SARS-CoV-2. For example, the composition may be for preventing or treating COVID-19.

[0079] In the present invention, "COVID-19" refers to a disease caused by infection with SARS-CoV-2. The antibody of the present invention has the ability to neutralize SARS-CoV-2 and inhibit infection, and can therefore be used to prevent or treat COVID-19.

[0080] In the present invention, "prevention" refers to any action of suppressing or delaying the onset of coronavirus infection by administering the composition, and "treatment" refers to any action of improving or favorably altering symptoms of coronavirus infection by administering the composition. The pharmaceutical composition may further comprise a pharma- ceutical acceptable carrier.

[0081] In the present invention, the term "pharmaceutical acceptable carrier" refers to a carrier or diluent that does not stimulate the organism and does not impair the biological activity and properties of the administered compound. The pharmaceutical carrier acceptable for the composition formulated as a liquid solution is one that is sterilized and compatible with the living body, and can be used by mixing at least one of saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, glucose solution, maltodextrin solution, glycerin, ethanol, and components thereof, and other common additives such as antioxidants, buffers, bacteriostatic agents, etc., can be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants can be further added to formulate the composition into an injectable dosage form such as an aqueous solution, suspension, or emulsion, or into pills, capsules, granules, or tablets.

[0082] The pharmaceutical composition may be in various oral or parenteral dosage forms. When formulated, it is prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, or surfactant. Oral solid preparations include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing at least one compound with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to the usual excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, internal liquids, emulsions, and syrups, and in addition to the usual diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives are used. Parenteral preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of non-aqueous solvents and suspension solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. Examples of suppository bases include witepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin, etc.

[0083] The pharmaceutical composition may have any dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories.

[0084] The compositions of the present invention are administered in a pharma- ceutically effective amount.

[0085] In the present invention, the term "pharmacologically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level is determined by factors including the type and severity of the individual, age, sex, type of cancer, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors known in the medical field. The composition of the present invention may be administered alone or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be administered singly or multiple times. It is important to administer an amount that provides maximum effect with minimal amount without side effects, taking into account all of the above factors, and this can be easily determined by those skilled in the art.

[0086] Yet another aspect of the invention provides a method of preventing or treating a coronavirus infection using the antibody.

[0087] Specifically, the coronavirus may be SARS-CoV-2.

[0088] For example, the method may include administering the antibody to an individual suspected of SARS-CoV-2 infection.

[0089] The method may include administering to an individual infected with coronavirus a pharmaceutical composition further comprising the antibody and a pharma- ceutically acceptable carrier, wherein the pharma- ceutically acceptable carrier is as described above.

[0090] The individual may be any individual, including mammals such as cows, pigs, sheep, chickens, dogs, and humans, birds, etc., as long as the coronavirus infection is treated by administration of the composition of the present invention. Here, the antibody may be administered singly or multiple times in a pharma- ceutically effective amount. Here, the antibody may be administered in the form of a liquid, powder, aerosol, capsule, enteric coating, capsule, or suppository. The route of administration may include, but is not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary, and rectal administration.

[0091] The method may also be used to prevent or treat coronavirus infections, particularly SARS-CoV-2 infections, including, but not limited to, COVID-19.

[0092] Yet another aspect of the present invention provides a use of the antibody in the manufacture of a medicament for the prevention or treatment of coronavirus infection. The medicament is for use in the prevention or treatment of SARS-CoV-2 infection, such as, but not limited to, COVID-19. The antibody and the prevention or treatment of SARS-CoV-2 infection are as described above. EXAMPLES

[0093] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative of the present invention and are not intended to limit the present invention.

[0094] Experimental Example 1. Expression and purification of recombinant proteins Genes encoding the ectodomain (spike-ECD) and RBD (RBD-his) of the SARS-CoV-2 spike glycoprotein were synthesized and cloned into the pCIW mammalian expression vector with a C-terminal 6x histidine tag. Genes encoding RBD-Fc and human ACE2-Fc were cloned into the pCIW vector. The pCIW mammalian vector was cloned into Expi293FTM The recombinant proteins were transformed into 100% ribosomal RNA (Cat. no. A1435101) cells. All steps to analyze the expression were performed according to the manufacturer's guidelines. After 5–6 days of cell culture, the cells were harvested by centrifugation, and the supernatant was filtered through a 0.22 μm filter to remove cell debris. For His-tag purification of the recombinant proteins, Ni-NTA resin was added to the supernatant. MabSelect Xtra (Cat. no. 17-5269-02, GE Healthcare) was used as the resin for protein-A purification of Fc fusion proteins. All purification steps were performed according to the manufacturer's guidelines. The buffer of the eluted proteins was exchanged into phosphate-buffered saline (PBS) using Zeba Spin Desalting Columns. Protein concentrations were quantified using a Nanodrop 2000C spectrophotometer (Thermo Scientific) (Figure 1).

[0095] Experimental example 2. Production of antibodies against SARS-CoV2 spike protein A common antibody production technique was used to produce antibodies against the SARS-CoV2 spkie protein. Specifically, a human B cell-derived library, a mouse immune library, and a human domain antibody synthetic library were constructed and screened. A commonly used monoclonal ELISA method was used for screening. The monoclonal ELISA screening method is a method in which a spike protein is mixed with an antibody expressed monoclonally to confirm whether the antibody is specific to the spike protein. Through screening, 41 clones (clones 1 to 41) specific to the spike protein were obtained in the human B cell-derived library, 5 clones (clones M1 to M5) in the mouse immune library, and 3 clones (clones 5E9, 6D9, and 6H10) in the human domain antibody synthetic library. The heavy and light chain sequences of the obtained antibodies were confirmed by sequence analysis (Figure 3, Table 1, Table 2, Table 3). However, the clones obtained from the human domain antibody synthetic library only contain heavy chain sequence information because the synthetic library itself is a library consisting of only heavy chain sequences.

[0096] [Table 1] JPEG0007599186000002.jpg180170JPEG0007599186000003.jpg178170JPEG0007599186000004.jpg181170JPEG0007599186000005.jpg18317 0JPEG0007599186000006.jpg179170JPEG0007599186000007.jpg182170JPEG0007599186000008.jpg180170JPEG0007599186000009.jpg96170

[0097] [Table 2]

[0098] [Table 3]

[0099] Experimental Example 3. Antibody expression and purification The anti-SARS-CoV-2 scFv was converted to human IgG1 format by subcloning into pCIW. TM The cells were incubated in 30 mL of Expi293 TM 2.5 × 10 in expression medium 6The cells were prepared at a concentration of 1000 cells / mL (37°C, 8% CO2, 125 rpm, viability ≥ 95%). These cells were transformed with a DNA mixture consisting of 30 μg of DNA (pCLW-anti-SARS-CoV-2 heavy chain: 15 μg, pcIw-anti-SARS-CoV-2 light chain: 15 μg), OptiProTM SEM medium, and ExpiFectamineTM 293 reagent. 0.1 mL of protein A resin washed with PBS was added to the expressed supernatant, and then incubated overnight at 4°C. The cells were transferred to a purification column and then washed 20 times more than the resin with IgG binding buffer. The bound IgG was eluted using elution buffer. The concentration was measured using 280 nm wavelength and summarized (Figure 2).

[0100] The preservative was filtered by centrifugation at 1000g for 2 minutes in a desalting column. PBS buffer was added to the column to match its volume, and then centrifugation was performed at 1000g for 2 minutes. Column washing was repeated three times. The sample was added, and then centrifugation was performed at 1000g for 2 minutes. The concentration was measured again using 280 nm wavelength.

[0101] Experimental Example 4. Confirmation of IgG cross-reactivity by ELISA ELISA was performed to measure cross-reactivity for 29 clones that were successfully converted to IgG1 form. 50 μl of 3 μg / ml antigen was coated on an ELISA plate overnight at 4°C. The antigens used were Recombinant SARS (1-1190) (Beta Lifescience, BLIT-0210), SARS-CoV-2 (2019-nCoV) Spike Protein (S1 + S2 ECD, His tag) (Sino Biological., 40589-V08B1), and MERS-CoV Spike Protein (ECD, aa 1-1297, His Tag) (Sino Biological., 40069-V08B). The next day, the antigen was removed from the ELISA plate and washed three times with PBS + Tween 20 0.05%. Each well coated with the antigen was blocked with 200 μl of PBS + BSA 5% at room temperature for 1 hour. After 1 hour, the plate was washed three times with PBS+tween 20 0.05%. 35 kinds of antibodies were diluted to 300nM and measured. 50μl of the diluted antibodies were added to each well and binding was allowed to occur at room temperature for 1 hour. Similarly, the plate was washed three times with PBS+tween 20 0.05%. 50μl of anti-human Fc-HRP (3000:1) was added to each well and incubated at room temperature for 1 hour. The plate was washed three times with PBS+tween 20 0.05%. 50μl of TMB solution was added to each well and incubated for 1-15 minutes. 50μl of stop solution was added to each well to stop the reaction. The degree of color development was measured using an ELISA reader.

[0102] Cross-reactivity experiments of selected IgG against SARS-CoV-2, SARS-CoV, and MERS-CoV confirmed clones with cross-reactivity to at least two coronaviruses (Table 4, Figure 4).

[0103] [Table 4]

[0104] Specifically, cross-reactivity tests using ELISA confirmed that 2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 28, 33, 41, and M3 (21 types) antibodies also bind to SARS-CoV1 (cut-off OD 0.5 or higher). In addition, 6H10 and M3 antibodies (2 types) were confirmed to also bind to MERS-CoV (cut-off OD 0.5 or higher). From these results, it is possible to select clones from among anti-SARS-CoV2 antibodies that show cross-reactivity to SARS-CoV or MERS-CoV2 (Figure 4).

[0105] Experimental Example 5. Preparation of chimeric antibodies against M4 and M5 Among the mouse immune clones, humanization was performed on the M4 and M5 clones. The framework region sequences of the M4 and M5 antibodies were identified to the mouse germline sequences IGHV5-6 and IGKV6-32, respectively. In the M4 and M5 clones, the HCDR sequences are identical, but the LCDRs are different. For humanization, a common method, CDR-grafting, was used. An Igblast search was performed to search for a human framework with the highest homology to M4 and M5. As a result of the search, it was confirmed that the highest homology was with IGHV3-21 and IGKV1-39, and the CDRs of M4 and M5 were grafted to the human antibody frameworks IGHV3-21 and IGKV1-39, respectively. After grafting, in order to minimize the decrease in stability and affinity due to the change in the framework, core residues that play an important role in determining the stability of the antibody structure and the canonical structure of the CDR were confirmed, and then further mutations were introduced to design variants (Table 5).

[0106] The gene sequences of the designed M4 and M5 antibodies were synthesized and used as inserts (SEQ ID NOs: 132 to 138). The synthesized genes were cloned by the infusion cloning method, and the chimeric antibodies were cultured and purified as described above (FIG. 5).

[0107] [Table 5] JPEG0007599186000014.jpg171170JPEG0007599186000015.jpg171170JPEG0007599186000016.jpg97170

[0108] Experimental Example 6. Evaluation of the binding ability of humanized antibodies to spike proteins ELISA was performed to confirm whether each humanized antibody specifically binds to the SARS-CoV2 spike protein. Spike protein 3μg / ml, 100μl per well, was coated on an ELISA plate overnight at 4℃. The next day, spike protein was removed from the ELISA plate, and each well was blocked with 200μl of PBS+BSA 2% at 37℃ for 1 hour. The plate was washed three times with PBS+tween 20 0.05%. M4, M5 and each humanized antibody were diluted 11 times from the initial concentration (100nM) by 3-fold with PBS, and the final concentration was set to 0.5pM. 100μl of the diluted antibody solution was added to each well and binding was performed at 37℃ for 1 hour. Similarly, the plate was washed three times with PBS+tween 20 0.05%. 50μl of HRP conjugated anti-Human IgG-Fc antibody (5000:1) was added to each well and incubated at 37℃ for 1 hour. The wells were washed four times with PBS + Tween 20 0.05%. 50 μl of TMB solution was added to each well and incubated for 5 minutes. 50 μl of Stop solution was added to each well to stop the reaction. The binding strength of the humanized clones to the Spike protein was evaluated, and it was confirmed that all of them maintained the same binding strength as the conventional M4 and M5 clones even after humanization (Figure 6).

[0109] Experimental Example 7. Evaluation of antibody binding to RBD and spike protein mutants ELISA was performed to confirm whether the humanized antibody M4-4 of M4 and the humanizaed antibody M5-4 of M5 bind to various mutants of RBD and spike protein in addition to the wild type. The nine mutants shown in Table 6 were used.

[0110] [Table 6]

[0111] ELISA plates were coated with 100 μl of RBD WT and mutant at 3 μg / ml (0.1 μM) at 4°C overnight. Spike protein was coated with 100 μl of WT and mutant at 14 ug / ml (0.1 μM) at 4°C overnight. The next day, WT and mutant were removed from the ELISA plate, and each well was blocked with 200 μl of PBS+BSA 2% at 37°C for 1 hour. The plates were washed three times with PBS+tween 20 0.05%. Conventional antibodies (S309,REGN 10933+10987 combination), M4, M5 and each humanized antibody were adjusted to 1 nM, and 100 μl was added to each well and bound at 37°C for 1 hour. Similarly, the plates were washed three times with PBS+tween 20 0.05%. 50 μl of HRP conjugated anti-Human IgG-Fc antibody (5000:1) was added to each well and incubated at 37°C for 1 hour. The wells were washed four times with PBS + Tween 20 0.05%. 50 μl of TMB solution was added to each well and incubated for 5 minutes. 50 μl of Stop solution was added to each well to stop the reaction. The binding ability of M4-4 and M5-4 to the mutant was evaluated, and both M4-4 and M5-4 were confirmed to have binding ability to the mutant at or above the WT RBD level. It was confirmed that the binding ability of REGN10933 and REGN10987, which were used as comparison antibodies, to the Y453F and N439K mutants, respectively, was reduced by more than 50% when treated alone (Figure 7).

[0112] Experimental Example 8. Binding characteristics of antibodies against S protein and its mutants Antigen-antibody binding characteristics were examined to determine whether anti-SARS-CoV-2 spike protein antibodies exhibit different binding characteristics for spike protein and its variants, alpha (UK), beta (South Africa), and gamma (Brazil). The kinetics of the binding characteristics of anti-S protein antibodies were determined using a Biacore T-200 biosensor (Cytiva). The antibody was diluted with HBS-EP buffer to adjust the concentration to 10 μg / mL and immobilized on the SA chip at a flow rate of 10 μL / min until Rmax reached 200Ru. S protein diluted to a concentration of 0.3125-20 nM in HBS-EP buffer was flowed through the SA chip with the antibody immobilized at a flow rate of 30 μL / min for 3 minutes for binding and 30 minutes for dissociation. Next, 10 mM glycine-HCl (pH 1.5) was reacted for 30 seconds to wash the S protein bound to the antibody. The sensorgrams of the antibodies were analyzed using Biacore evaluation software. The antigen-antibody binding kinetics showed that the dissociation constant of the humanized antibody M4-4 was 10-fold higher than that of the mouse antibody M4, and was approximately 10-fold higher than that of the comparative antibodies REGN10933 and REGN10987 (FIG. 8a).

[0113] We next assessed the binding ability of antibodies against the RBD of alpha (UK), beta (South Africa) and gamma (Brazil) variants. In this experiment, we used M4, M4-4 and control antibodies (REGN10933, REGN10987 and REGN mix (REGN10933+10987, 1:1 mix)). The REGN mix was used to compare whether the epitope against RBM shows synergistic effects against variants when used in a mixture with the other two antibodies.

[0114] REGN10933, an RBM-targeting neutralizing antibody, showed similar binding affinity to the alpha mutant as well as the conventional RBD, but binding of REGN10933 was significantly reduced to the beta and gamma mutants. In contrast, M4 and M4-4 showed similar binding affinity to the conventional RBD and all mutants (Figure 8b).

[0115] This suggests that the antibodies of the present invention maintain their ability to bind to the newly emerged SARS-CoV-2 mutants and have the ability to bind to and neutralize various mutant viruses.

[0116] Experimental Example 9. Production of Pseudovirus Pseudoviruses carrying the full-length spike protein of SARS-CoV-2 and mutants carrying a firefly luciferase reporter gene were produced from Lenti-X 293T cells (Takara). Namely, 10 μg of psPAX2 (Addgene), 10 μg of pLVX-Luciferase, and 10 μg of SARS-CoV-2 S (codon-optimized) expression plasmid were transformed into polyethylenimine (mass ratio 1:2) Lenti-X 293T cells, and after 72 hours, the supernatant was centrifuged at 600 × g for 15 min, then filtered through a 0.45 μm filter and stored at -70 °C for further use. The titer of the pseudovirus was determined by measuring the relative luciferase units.

[0117] Experimental Example 10. Neutralization analysis using SARS-CoV-2 pseudovirus The ACE2-HEK293 stable cell line was maintained in DMEM medium containing 10% fetal bovine serum and an antibiotic-antimycotic cocktail. 96-well flat-bottom plates were coated with 10 μg / mL type I collagen and incubated at 37 °C in an atmosphere containing 5% CO2 for 4 h. ACE2-HEK293 cells were suspended in DMEM at 1 × 10 51000 cells / well were dispensed into collagen-coated 96-well flat-bottom plates and incubated for 24 hours. Antibodies were serially diluted 3-fold starting at 10 μg / mL (66.67 nM) in DMEM containing 2% FBS and antibiotic-antimycotic cocktail. Antibody dilutions were mixed 1:1 with pseudovirus and incubated for 1 hour at 37°C in 5% CO2 atmosphere. The supernatant was removed from ACE2-HEK293 cells and replaced with 50 μL of antibody-pseudovirus mixture. The cells were then incubated for 1 hour at 37°C in 5% CO2 atmosphere. 50 μL of infection medium was added to the cells and the cells were incubated for an additional 48 hours at 37°C in 5% CO2 atmosphere. The cells were lysed in 5× reporter lysis buffer (Promega) and relative luciferase activity was determined using a luciferase assay system and luminescence system. The relative luciferase units were converted to percent neutralization and fitted to a nonlinear regression curve using Prism Software (GraphPad, Prism 8.0).The results confirmed that M4 and M5 chimeric antibodies, as well as the humanized candidate antibodies M4-4, 5, 7, 8 and M5-4, 5, 7, 8, showed similar levels of neutralization activity to REGN10933 (Figure 9a, Figure 9b).

[0118] Example 11. Generation of cell lines expressing the spike protein of SARS-CoV-2 To generate a stable cell line expressing the spike protein of SARS-CoV-2 (HT1080-S), the cells were transfected with SARS-CoV-2 pcDNA3.1-spike cDNA. Cell lines expressing SARS-CoV-2 S protein were selected with 1.5 mg / mL Geneticin. Expression of SARS-CoV-2 S protein was confirmed by flow cytometry (FACS LSR Fortessa, BD Biosciences) using the anti-SARS-CoV-2 antibody REGN10933 (Figure 10a).

[0119] Experimental Example 12. Measurement of Fc-mediated effects To confirm whether anti-SARS-CoV-2 antibodies exhibit Fc-mediated antibody-dependent cellular phagocytosis, ADCC and ADCP effects were measured. In the ADCC analysis, HT1080-S cells were used as target cells and Jurkat-NFAT-Luc / FcγRIIIa was used as effector cells. HT1080-S cells were cultured in the appropriate medium for 18 hours, and ADCC assay buffer was added. The antibody was then diluted and added and reacted for 15 minutes. Effector cells were added and cultured for 18 hours, after which Bio-Glo luciferase assay reagent was added and luminescence was measured according to the manufacturer's instructions. A nonlinear regression curve was drawn from the measured luminescence values ​​using Prism software (Graphpad), and the EC50 value was obtained. In the ADCP analysis, measurements were performed in the same manner as above. As a result of the measurement, it was confirmed that M4, M5 chimeric antibody, and humanized candidate antibody M4-4, 5, 7, 8 and M5-4, 5, 7, 8 showed the same level of ADCC effect as the reference antibody (REGN10987, REGN mix) (Figure 10b). In addition to the ADCC effect, it was further confirmed that M4-4 induced ADCP activity comparable to a single Regeneron antibody or a cocktail of Regeneron antibodies (Figure 10c). In summary, the data in Figure 10 indicates that M4-4 may induce ADCC and ADCP, which are Fc-mediated antibody effect functions, to mediate maximum therapeutic effect (Figures 10b, 10c).

[0120] Experimental Example 13. Measurement of neutralizing activity against SARS-CoV-2 spike protein mutants To evaluate the neutralizing effect of M4-4 against SARS-CoV-2 spike variants, alpha (UK), beta (South Africa) and gamma (Brazil), pseudovirus neutralization assays were performed using lentiviral systems expressing each SARS-CoV-2 spike variant. M4 and M4-4 were confirmed to be able to neutralize the three mutant viruses with neutralizing effects comparable to REGN10987 or the Regeneron cocktail. REGN10933 was unable to mediate 100% neutralization of beta or gamma mutant pseudovirus infection at high cell concentrations (Figure 11).

[0121] In summary, we confirmed that the humanized SARS-CoV-2 antibody M4-4 was able to neutralize WT and selected spike mutant SARS-CoV-2 pseudovirus particles with a neutralizing effect comparable to that of the Regeneron cocktail (Figure 11).

[0122] Experimental Example 14. Epitope binning Epitope binning was performed in three steps using a BLI system (Octet Qke). Step 1 involved antigen immobilization, step 2 included primary antibody binding, and step 3 included secondary antibody binding. A 60-second reference step was set between each step to confirm the baseline signal. In step 1, recombinant SARS-CoV-2 spike protein or RBD His purified to 20 μg / mL was immobilized on a pre-hydrated antipenta-His biosensor at 1000 rpm for 200 seconds. In step 2, 37.5 μg / mL of different primary binding Abs were bound for 300 seconds. In step 3, one secondary antibody (18.75 μg / mL) was bound for 150 seconds to confirm the degree of self-binding and competition. The degree of competition was defined as a percentage. The binding of only the secondary antibody to the immobilized antigen was defined as 100%. A binding level of less than 33% was defined as complete competition, a level between 33% and 66% was defined as intermediate competition, and a level of 66% or more was defined as non-competition. Binning confirmed that M4-4 competed 100% with the epitopes of REG10987 and S309, and also partially competed with the epitope of REGN10933. In contrast, REGN10987 and S309 were observed to have non-competing epitopes with REGN10933 (Figure 12).

[0123] This suggests that M4-4 binds to a broader region of epitope than REGN10933, REGN10987, and S309.

[0124] We also performed a similar experiment to confirm competitive binding of the ACE2 receptor. The difference is that the ratio of the binding affinity of the recombinant human ACE2 receptor to the recombinant SARS-CoV-2 spike protein RBD and the affinity of the mAb to the RBD is about 10-100. Therefore, the ACE2 receptor was only used in step 3 for secondary antibody binding. M4-4 was also observed to compete 100% with the ACE2 receptor. Therefore, it was confirmed that M4-4 inhibits 100% of the antigen binding to the ACE2 receptor (Figure 12).

[0125] Experimental example 15.Simulation docking BioLuminate in the Schrodinger Suite was used for scFv homology modeling of M4-4 using the structures of anti-PD1 (PDB code: 6JJP) and anti-SIRPα antibody (PDB code: 6NMR) as heavy and light chain templates, respectively. Docking of M4-4 scFv structure to RBD (PDB code: 6M0J) was performed using PIPER in BioLuminate56. The distance between Lys440 and Thr500 in RBD and CDR loops was restricted to 2 Å to 10 Å based on the results of epitope binning. BioLuminate proposed 30 best complexes with 70,000 possible protein-protein structures. The final complex of M4-4 was selected based on the cluster size and agreement with the epitope binning results, followed by energy minimization. The binding site of M4-4 against RBD predicted by the simulation docking results was a position that inhibited all of the binding of REGN10933, REGN10987, S309 and ACE2 receptors. This is consistent with the binning results, and the epitope of M4-4 can be predicted (Figure 13).

[0126] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be interpreted as including all modifications and alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. It specifically binds to the coronavirus spike protein, a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 139, a heavy chain CDR2 of SEQ ID NO: 140, and a heavy chain CDR3 of SEQ ID NO: 141; A light chain variable region according to any one of (i) to (iii): (i) a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 142, a light chain CDR2 of SEQ ID NO: 143, and a light chain CDR3 of SEQ ID NO: 144; (ii) a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 145, a light chain CDR2 of SEQ ID NO: 146, and a light chain CDR3 of SEQ ID NO: 147; or (iii) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 117, 120, or 123. and

2. The antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 139, a heavy chain CDR2 of SEQ ID NO: 140, and a heavy chain CDR3 of SEQ ID NO: 141; A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 142, a light chain CDR2 of SEQ ID NO: 143, and a light chain CDR3 of SEQ ID NO:

144. The antibody or antigen-binding fragment thereof of claim 1 .

3. The antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 139, a heavy chain CDR2 of SEQ ID NO: 140, and a heavy chain CDR3 of SEQ ID NO: 141; A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 145, a light chain CDR2 of SEQ ID NO: 146, and a light chain CDR3 of SEQ ID NO:

147. The antibody or antigen-binding fragment thereof of claim 1 .

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the coronavirus is SARS-CoV, SARS-CoV-2 or MERS-CoV.

5. The antibody or antigen-binding fragment thereof of claim 1 , which has cross-reactivity with two or more types of coronaviruses.

6. The antibody or antigen-binding fragment thereof comprises: A heavy chain variable region consisting of any one of the sequences selected from the group consisting of SEQ ID NOs: 90, 103, 104, 106 and 107, and / or A light chain variable region consisting of any one sequence selected from the group consisting of SEQ ID NOs: 95, 119, 120, 122 and 123. The antibody or antigen-binding fragment thereof of claim 1 .

7. The antibody or antigen-binding fragment thereof comprises: A heavy chain variable region consisting of any one of the sequences selected from the group consisting of SEQ ID NOs: 91, 111, 112, 114 and 115, and / or A light chain variable region consisting of any one sequence selected from the group consisting of SEQ ID NOs: 96, 127, 128, 130 and 131. The antibody or antigen-binding fragment thereof of claim 1 .

8. the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 103; The antibody or antigen-binding fragment thereof of claim 1, wherein the light chain variable region consists of the amino acid sequence of SEQ ID NO:

119.

9. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. An expression vector comprising the polynucleotide of claim 9.

11. A transformant incorporating the expression vector according to claim 10.

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

13. A method for preventing or treating a coronavirus infection, comprising the step of administering to a non-human individual the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

14. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and a drug bound thereto.

15. A composition for diagnosing coronavirus infection, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

16. A kit for diagnosing coronavirus infection comprising the composition of claim 15.

17. A method for providing information for diagnosing coronavirus infection, comprising a step of detecting coronavirus present in a biological sample isolated from an individual suspected of coronavirus infection, using the antibody or antigen-binding fragment thereof described in any one of claims 1 to 8, a composition for diagnosing infection containing the same, or a kit containing the composition.

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