阅读说明：本技术 针对中东呼吸综合征-冠状病毒具有中和活性的结合分子 (Binding molecules having neutralizing activity against middle east respiratory syndrome-coronavirus ) 是由 李修泳 李癸淑 金哲珉 宋京珉 裵延珍 金宇柱 郑熙真 宋俊暎 朴晚成 鲁芝允 于 2018-11-30 设计创作，主要内容包括：本发明涉及具有针对中东呼吸综合征-冠状病毒(MERS-CoV)的中和活性的结合分子。更特别地,本发明涉及具有结合至MERS-CoV的S蛋白的强大能力和针对MERS-CoV的中和活性的结合分子,因此在MERS-CoV感染的预防、治疗或诊断中非常有用。(The present invention relates to binding molecules having neutralizing activity against middle east respiratory syndrome-coronavirus (MERS-CoV). More particularly, the present invention relates to a binding molecule having a strong ability to bind to the S protein of MERS-CoV and a neutralizing activity against MERS-CoV, and thus is very useful in the prevention, treatment or diagnosis of MERS-CoV infection.)

1. A neutralizing binding molecule that binds to the spike protein (S protein) on the surface of middle east respiratory syndrome-coronavirus (MERS-CoV).

2. The binding molecule according to claim 1, wherein the binding molecule is any one selected from the group consisting of the following binding molecules i) to vi):

i) a binding molecule comprising a) a heavy chain variable region comprising the CDR1 region of SEQ ID No. 1, the CDR2 region of SEQ ID No. 2 and the CDR3 region of SEQ ID No. 3, and b) a light chain variable region comprising the CDR1 region of SEQ ID No. 4, the CDR2 region of SEQ ID No. 5 and the CDR3 region of SEQ ID No. 6;

ii) a binding molecule comprising a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO 7, the CDR2 region of SEQ ID NO 8 and the CDR3 region of SEQ ID NO 9 and b) a light chain variable region comprising the CDR1 region of SEQ ID NO 10, the CDR2 region of SEQ ID NO 11 and the CDR3 region of SEQ ID NO 12;

iii) a binding molecule comprising a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO 13, the CDR2 region of SEQ ID NO 14 and the CDR3 region of SEQ ID NO 15, and b) a light chain variable region comprising the CDR1 region of SEQ ID NO 16, the CDR2 region of SEQ ID NO 17 and the CDR3 region of SEQ ID NO 18;

iv) a binding molecule comprising a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO 19, the CDR2 region of SEQ ID NO 20 and the CDR3 region of SEQ ID NO 21 and b) a light chain variable region comprising the CDR1 region of SEQ ID NO 22, the CDR2 region of SEQ ID NO 23 and the CDR3 region of SEQ ID NO 24;

v) a binding molecule comprising a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO. 25, the CDR2 region of SEQ ID NO. 26 and the CDR3 region of SEQ ID NO. 27, and b) a light chain variable region comprising the CDR1 region of SEQ ID NO. 28, the CDR2 region of SEQ ID NO. 29 and the CDR3 region of SEQ ID NO. 30; and

vi) a binding molecule comprising a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO:31, the CDR2 region of SEQ ID NO:32 and the CDR3 region of SEQ ID NO:33, and b) a light chain variable region comprising the CDR1 region of SEQ ID NO:34, the CDR2 region of SEQ ID NO:35 and the CDR3 region of SEQ ID NO: 36.

3. The binding molecule according to claim 1, wherein the binding molecule is any one selected from the group consisting of the following binding molecules i) to vi):

i) a binding molecule comprising a) a heavy chain variable region having 95% or more sequence identity to the heavy chain variable region of the polypeptide sequence of SEQ ID NO:37, and b) a light chain variable region having 95% or more sequence identity to the light chain variable region of the polypeptide sequence of SEQ ID NO: 38;

ii) a binding molecule comprising a) a heavy chain variable region having 95% or more sequence identity to the heavy chain variable region of the polypeptide sequence of SEQ ID NO:39, and b) a light chain variable region having 95% or more sequence identity to the light chain variable region of the polypeptide sequence of SEQ ID NO: 40;

iii) a binding molecule comprising a) a heavy chain variable region having 95% or more sequence identity to the heavy chain variable region of the polypeptide sequence of SEQ ID NO:41 and b) a light chain variable region having 95% or more sequence identity to the light chain variable region of the polypeptide sequence of SEQ ID NO: 42;

iv) a binding molecule comprising a) a heavy chain variable region having 95% or more sequence identity to the heavy chain variable region of the polypeptide sequence of SEQ ID NO 43, and b) a light chain variable region having 95% or more sequence identity to the light chain variable region of the polypeptide sequence of SEQ ID NO 44;

v) a binding molecule comprising a) a heavy chain variable region having 95% or more sequence identity to the heavy chain variable region of the polypeptide sequence of SEQ ID NO:45, and b) a light chain variable region having 95% or more sequence identity to the light chain variable region of the polypeptide sequence of SEQ ID NO: 46; and

vi) a binding molecule comprising a) a heavy chain variable region having 95% or more sequence identity to the heavy chain variable region of the polypeptide sequence of SEQ ID NO:47, and b) a light chain variable region having 95% or more sequence identity to the light chain variable region of the polypeptide sequence of SEQ ID NO: 48.

4. The binding molecule of any one of claims 1 to 3, wherein the binding molecule is a Fab fragment, Fv fragment, diabody, chimeric antibody, humanized antibody, or human antibody.

5. An immunoconjugate, wherein at least one tag is additionally bound to the binding molecule of any one of claims 1 to 3.

6. A nucleic acid molecule encoding the binding molecule of any one of claims 1 to 3.

7. An expression vector into which the nucleic acid molecule of claim 6 is inserted.

8. A cell line wherein the expression vector of claim 7 is transformed into a host cell to produce a binding molecule that binds to MERS-CoV and thus has neutralizing activity.

9. The cell line of claim 8, wherein the host cell is any one selected from the group consisting of CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, HT1080 cells, A549 cells, HEK293 cells, and HEK293T cells.

10. A composition for preventing or treating MERS-CoV infection comprising the binding molecule of any one of claims 1 to 3.

11. The composition of claim 10, wherein the composition is a sterile injectable solution, a lyophilized formulation, a pre-filled syringe solution, an oral formulation, a topical formulation, or a suppository.

12. A method of diagnosing, preventing, or treating a disease caused by MERS-CoV infection, comprising administering a therapeutically effective amount of the composition of claim 10 or 11 to a subject having a disease caused by MERS-CoV infection.

13. A kit for diagnosing MERS-CoV comprising the binding molecule of any one of claims 1 to 3.

Technical Field

The present invention relates to binding molecules having neutralizing activity against middle east respiratory syndrome-coronavirus (MERS-CoV). More particularly, the present invention relates to a binding molecule having a strong ability to bind to a spike protein (S protein) on the surface of MERS-CoV and a neutralizing activity against MERS-CoV, and thus is very useful in the prevention, treatment or diagnosis of MERS-CoV infection.

Background

Middle east respiratory syndrome-coronavirus (MERS-CoV) is an infectious disease caused by a coronavirus belonging to the genus b coronavirus (Betacoronavirus), and the first disease virus was found in middle east unexplained pneumonia patients in 2012. MERS-CoV is considered to originate from bats and is hitherto known to be introduced into humans through camels. Although its route of transmission has not been fully characterized, it appears that the virus has been repeatedly transmitted from camels to humans in the middle east, and limited and non-sustained interpersonal transmission has occurred.

To date MERS-CoV has occurred in 27 countries, with 2,078 patients from 9 months 2012 to 9 months 2017 on day 29. Of these patients, 730 died, indicating 35.1% lethality (WHO). There is no specific therapeutic or prophylactic agent, but the effect of antiviral drugs has not been clearly confirmed. In view of the high mortality and morbidity of MERS-CoV, active treatment with antiviral drugs, such as combination therapy with ribavirin (ribavirin), interferon alpha-2 alpha and lopinavir (lopinavir)/ritonavir (ritonavir), is recommended in the early stages of the disease, but is problematic due to its side effects.

Meanwhile, as a conventional technique for MERS-CoV binding antibodies, korean patent No. 10-1593641 discloses an antibody recognizing MERS-CoV nucleocapsid, a diagnostic composition comprising the same, a kit and a method for detecting MERS-CoV using the same. This document relates to the determination of MERS-CoV infection using antibodies that specifically bind to the nucleocapsid of MERS-CoV, but the neutralizing activity of antibodies against MERS-CoV is unknown, and thus there is a continuing need for antibodies with MERS-CoV therapeutic effects.

BRIEF SUMMARY OF THE PRESENT DISCLOSURE

Technical problem

Accordingly, the present inventors have developed a binding molecule having the ability to bind to the S protein of MERS-CoV in order to solve the problems encountered in the related art, and have determined that the binding molecule has a neutralizing efficacy against MERS-CoV, thereby completing the present invention.

It is an object of the present invention to provide binding molecules that bind to the S protein of MERS-CoV and thus have neutralizing activity against MERS-CoV.

It is another object of the invention to provide compositions comprising binding molecules for use in the prevention or treatment of MERS-CoV.

It is still another object of the present invention to provide a kit comprising a binding molecule for diagnosing MERS-CoV.

Technical scheme

To achieve the above object, embodiments of the present invention provide a neutralizing binding molecule that binds to spike protein (S protein) on the surface of MERS-CoV (middle east respiratory syndrome-coronavirus).

Another embodiment of the invention provides a composition comprising a binding molecule for use in the prevention or treatment of MERS-CoV.

Yet another embodiment of the invention provides a kit comprising a binding molecule for diagnosing MERS-CoV.

Hereinafter, a detailed description of the present invention will be given.

Embodiments of the invention relate to neutralizing binding molecules that bind to the S protein of MERS-CoV.

Embodiments of the present invention relate to a neutralizing binding molecule, which is any one selected from the group consisting of the following binding molecules i) to vi).

i) A binding molecule comprising a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO. 1, the CDR2 region of SEQ ID NO. 2 and the CDR3 region of SEQ ID NO. 3, and b) a light chain variable region comprising the CDR1 region of SEQ ID NO. 4, the CDR2 region of SEQ ID NO. 5 and the CDR3 region of SEQ ID NO. 6.

ii) a binding molecule comprising a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO 7, the CDR2 region of SEQ ID NO 8 and the CDR3 region of SEQ ID NO 9 and b) a light chain variable region comprising the CDR1 region of SEQ ID NO 10, the CDR2 region of SEQ ID NO 11 and the CDR3 region of SEQ ID NO 12.

iii) a binding molecule comprising a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO 13, the CDR2 region of SEQ ID NO 14 and the CDR3 region of SEQ ID NO 15 and b) a light chain variable region comprising the CDR1 region of SEQ ID NO 16, the CDR2 region of SEQ ID NO 17 and the CDR3 region of SEQ ID NO 18.

iv) a binding molecule comprising a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO 19, the CDR2 region of SEQ ID NO 20 and the CDR3 region of SEQ ID NO 21 and b) a light chain variable region comprising the CDR1 region of SEQ ID NO 22, the CDR2 region of SEQ ID NO 23 and the CDR3 region of SEQ ID NO 24.

v) a binding molecule comprising a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO. 25, the CDR2 region of SEQ ID NO. 26 and the CDR3 region of SEQ ID NO. 27, and b) a light chain variable region comprising the CDR1 region of SEQ ID NO. 28, the CDR2 region of SEQ ID NO. 29 and the CDR3 region of SEQ ID NO. 30.

vi) a binding molecule comprising a) a heavy chain variable region comprising the CDR1 region of SEQ ID NO 31, the CDR2 region of SEQ ID NO 32 and the CDR3 region of SEQ ID NO 33, and b) a light chain variable region comprising the CDR1 region of SEQ ID NO 34, the CDR2 region of SEQ ID NO 35 and the CDR3 region of SEQ ID NO 36.

In an embodiment of the invention, the binding molecules include antibodies 1 to 36, as shown in table 1 below.

[ Table 1]

Classification

Heavy chain CDR1

Heavy chain CDR2

Heavy chain CDR3

Light chain CDR1

Light chain CDR2

Light chain CDR3

Antibody 1

SEQ ID NO:1

SEQ ID NO:2

SEQ ID NO:3

SEQ ID NO:4

SEQ ID NO:5

SEQ ID NO:6

Antibody 2

SEQ ID NO:7

SEQ ID NO:8

SEQ ID NO:9

SEQ ID NO:10

SEQ ID NO:11

SEQ ID NO:12

Antibody 3

SEQ ID NO:13

SEQ ID NO:14

SEQ ID NO:15

SEQ ID NO:16

SEQ ID NO:17

SEQ ID NO:18

Antibody 4

SEQ ID NO:19

SEQ ID NO:20

SEQ ID NO:21

SEQ ID NO:22

SEQ ID NO:23

SEQ ID NO:24

Antibody 5

SEQ ID NO:25

SEQ ID NO:26

SEQ ID NO:27

SEQ ID NO:28

SEQ ID NO:29

SEQ ID NO:30

Antibody 6

SEQ ID NO:31

SEQ ID NO:32

SEQ ID NO:33

SEQ ID NO:34

SEQ ID NO:35

SEQ ID NO:36

In the present invention, CDRs of variable regions are determined by a typical method using a system designed by Kabat et al (Kabat et al, Sequences of Proteins of Immunological Interest (5 th edition), National Institutes of health, Bethesda, Md. (1991)). The CDR numbering used in the present invention is determined using the Kabat method, but the invention also encompasses binding molecules comprising CDRs determined by other methods such as the IMGT method, Chothia method, AbM method, etc.

Embodiments of the present invention relate to a neutralizing binding molecule, which is any one selected from the group consisting of the following binding molecules i) to vi).

i) A binding molecule comprising a) a heavy chain variable region having 95% or more sequence identity to the heavy chain variable region of the polypeptide sequence of SEQ ID NO:37, and b) a light chain variable region having 95% or more sequence identity to the light chain variable region of the polypeptide sequence of SEQ ID NO: 38.

ii) a binding molecule comprising a) a heavy chain variable region having 95% or more sequence identity to the heavy chain variable region of the polypeptide sequence of SEQ ID NO:39, and b) a light chain variable region having 95% or more sequence identity to the light chain variable region of the polypeptide sequence of SEQ ID NO:40

iii) a binding molecule comprising a) a heavy chain variable region having 95% or more sequence identity to the heavy chain variable region of the polypeptide sequence of SEQ ID NO:41 and b) a light chain variable region having 95% or more sequence identity to the light chain variable region of the polypeptide sequence of SEQ ID NO: 42.

iv) a binding molecule comprising a) a heavy chain variable region having 95% or more sequence identity to the heavy chain variable region of the polypeptide sequence of SEQ ID NO:43 and b) a light chain variable region having 95% or more sequence identity to the light chain variable region of the polypeptide sequence of SEQ ID NO: 44.

v) a binding molecule comprising a) a heavy chain variable region having 95% or more sequence identity to the heavy chain variable region of the polypeptide sequence of SEQ ID NO:45 and b) a light chain variable region having 95% or more sequence identity to the light chain variable region of the polypeptide sequence of SEQ ID NO: 46.

vi) a binding molecule comprising a) a heavy chain variable region having 95% or more sequence identity to the heavy chain variable region of the polypeptide sequence of SEQ ID NO:47, and b) a light chain variable region having 95% or more sequence identity to the light chain variable region of the polypeptide sequence of SEQ ID NO: 48.

In an embodiment of the invention, the binding molecules include antibodies 1 to 6, as shown in table 2 below.

[ Table 2]

In the embodiment of the present invention, the binding molecule may be a Fab fragment, Fv fragment, diabody, chimeric antibody, humanized antibody, or human antibody, but is not limited thereto. Embodiments of the invention provide fully human antibodies that bind to S protein. As used herein, the term "antibody" is used in its broadest possible sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from two or more intact antibodies, and antibody fragments that exhibit the desired biological activity. Antibodies are proteins produced by the immune system that are capable of recognizing and binding to specific antigens. Typically, antibodies are configured as Y-shaped proteins having four amino acid chains (two heavy chains and two light chains). Each antibody has two regions including a variable region and a constant region. The variable region at the end of the arm of the Y binds to and interacts with the target antigen. The variable region includes Complementarity Determining Regions (CDRs) that recognize and bind to specific binding sites on specific antigens. The constant region located at the tail of the Y is recognized by and interacts with the immune system. The target antigen has multiple binding sites, called epitopes, that are recognized by CDRs on the antibody. Corresponding antibodies that specifically bind to different epitopes have different structures. Thus, a single antigen may have at least one antibody corresponding thereto.

Furthermore, the invention includes functional variants of the binding molecules. Such binding molecules are considered to be functional variants of the binding molecules of the invention, as long as the variants are capable of competing with the binding molecules of the invention for specific binding to MERS-CoV or its S protein, and also have neutralizing activity against MERS-CoV. Such functional variants include, but are not limited to, derivatives (whose primary conformational sequences are substantially similar, and examples of which include in vitro or in vivo modifications), chemical and/or biochemical substances, and which are not found in the parent monoclonal antibodies of the invention. Examples of such modifications may include acetylation, acylation, covalent binding of nucleotides or nucleotide derivatives, co-binding of lipids or lipid derivatives, cross-linking, disulfide bonding, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolysis, and phosphorylation. The functional variant may alternatively be an antibody comprising an amino acid sequence obtained by substitution, insertion, deletion or combination thereof of at least one amino acid as compared to the amino acid sequence of the parent antibody. In addition, functional variants may include truncated forms of the amino acid sequence in one or both of the amino-terminus and the carboxy-terminus. The functional variants of the invention may have the same or different binding affinity as, i.e. greater or less than, the binding affinity of the parent antibody of the invention, but may still bind to MERS-CoV or its S protein. For example, the amino acid sequence of the variable region may be modified, including but not limited to, the framework structure or hypervariable regions, particularly the CDRs (complementarity determining regions) of the light or heavy chain. Typically, the light or heavy chain region includes three hypervariable regions comprising three CDRs and more conserved regions, Framework Regions (FRs). Hypervariable regions comprise amino acid residues from the CDRs and amino acid residues from the hypervariable loops. Functional variants falling within the scope of the invention may have about 50% to 99%, about 60% to 99%, about 80% to 99%, about 90% to 99%, about 95% to 99%, about 97% to 99% amino acid sequence homology with a parent antibody of the invention. For optimal alignment of the amino acid sequences to be compared, and also for defining similar or identical amino acid residues, Gap or Best-fit known to those skilled in the art can be used in computer algorithms. Functional variants may be obtained by subjecting the parent antibody or part thereof to known molecular biological processes (including PCR or mutagenesis/partial mutagenesis using oligonucleotides) or organic synthetic processes, although the invention is not limited thereto.

Furthermore, the drug may additionally be attached to a binding molecule. In particular, the binding molecules according to the invention may be used in the form of antibody-drug conjugates comprising a drug conjugated thereto. When antibody-drug conjugates (ADCs), i.e. immunoconjugates, are used to deliver drugs locally, targeted delivery of the drug moiety to the infected cell becomes possible. When administered without a conjugated pharmaceutical agent, unacceptable levels of toxicity to normal cells can result. By increasing not only drug conjugation and drug release, but also the selectivity of polyclonal and monoclonal antibodies (mabs), the maximum efficacy and minimum toxicity of ADCs can be achieved.

Typical means for attaching a drug moiety to an antibody, such as the use of covalent binding, can result in the production of a heterogeneous mixture of molecules in which the drug moiety is attached to a number of sites on the antibody. For example, cytotoxic drugs are conjugated to antibodies through many lysine residues of the antibodies, thereby creating a heterogeneous antibody-drug conjugate mixture. Depending on the reaction conditions, such heterogeneous mixtures typically have a distribution whereby the number of antibodies attached to the drug moiety ranges from 0 to about 8 or more. In addition, each subgroup of conjugates comprising a drug moiety and an antibody in a specific integer ratio is a potentially heterogeneous mixture in which the drug moiety is attached to various sites on the antibody. Antibodies are large, complex and structurally diverse biomolecules, and typically have many reactive functional groups. The reactivity of the linker reagent and drug linker intermediate depends on factors such as pH, concentration, salt concentration, and co-solvent.

Furthermore, embodiments of the present invention provide nucleic acid molecules encoding binding molecules.

The nucleic acid molecules of the invention include any nucleic acid molecule in which the amino acid sequence of an antibody provided by the invention is converted into a polynucleotide sequence known to those skilled in the art. Thus, various polynucleotide sequences can be prepared using ORFs (open reading frames) and can also be incorporated into the nucleic acid molecules of the invention.

In addition, embodiments of the present invention provide expression vectors into which nucleic acid molecules are inserted.

The expression vector may include, but is not limited to, one selected from the group consisting of expression vectors obtained from celltron such as MarEx vector (korean patent No. 10-1076602), and commercially widely used pCDNA vector, F, R1, RP1, Co1, pBR322, ToL, and Ti vector; sticking particles; bacteriophages such as λ, λ class (lambdoid), M13, Mu, P1, P22, Q μ, T-even, T2, T3, T7, and the like; and a plant virus; any expression vector known to those skilled in the art may be used in the present invention, and the expression vector may be selected according to the nature of the host cell of interest. Introduction of the vector into the host cell may be performed by calcium phosphate transfection, viral infection, DEAE-dextran mediated transfection, lipofection, or electroporation, but the present invention is not limited thereto, and those skilled in the art may employ introduction procedures suitable for the expression vector and the host cell. For example, the expression vector may comprise at least one selection marker, but is not limited thereto, and the selection may depend on whether the product can be obtained using a vector that does not comprise a selection marker. The selection of the selection marker depends on the host cell of interest and is performed using any method known to those skilled in the art, and the present invention is therefore not limited in this regard.

To facilitate purification of the binding molecules of the invention, the tag sequence may be inserted into and thus fused to an expression vector. The tag may include, but is not limited to, a six histidine tag, a hemagglutinin tag, a myc tag, or a tag, and any tag may be used in the present invention as long as it facilitates purification, as known to those skilled in the art.

In addition, embodiments of the invention provide cell lines in which the expression vector is transformed into a host cell to produce a binding molecule that binds to MERS-CoV and thus has neutralizing activity.

In the present invention, cell lines may include, but are not limited to, cells of mammalian, plant, insect, fungal or cellular origin. Any one selected from the group consisting of mammalian cells such as CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, HT1080 cells, a549 cells, HEK293 cells, and HEK293T cells may be used as the host cell, but the present invention is not limited thereto, and any cells may be used as long as they can be used as host cells for mammals, as known to those skilled in the art.

In addition, embodiments of the invention relate to compositions comprising binding molecules for use in the prevention or treatment of MERS-CoV infection. In addition to the binding molecule, the compositions of the invention may include a pharmaceutically acceptable excipient. Such pharmaceutically acceptable excipients are well known to those skilled in the art.

The compositions of the present invention may further comprise at least one other therapeutic or diagnostic agent. For example, in addition to binding molecules, the compositions of the invention may further comprise an interferon, an anti-S protein monoclonal antibody, an anti-S protein polyclonal antibody, a nucleoside analog, a DNA polymerase inhibitor, an siRNA preparation, or a therapeutic vaccine as an antiviral drug.

The composition of the present invention including the binding molecule may be provided in the form of a preparation such as a sterile injectable solution, a lyophilized preparation, a pre-filled syringe solution, an oral preparation, an external preparation or a suppository by respective typical processes, but the present invention is not limited thereto.

In addition, the compositions of the invention comprising the binding molecules may be administered orally or parenterally. For example, the route of administration can be intravenous administration, but is not limited thereto.

The compositions of the present invention are administered to mammals, including humans, to thereby prevent or treat MERS-CoV infection and diseases caused by MERS-CoV infection. Here, the amount of binding molecule (e.g., antibody) administered depends on the subject being treated, the severity of the disease or condition, the rate of administration, and the prescription of the physician.

In addition, embodiments of the present invention relate to diagnostic kits comprising the binding molecules. The binding molecules of the invention used in the diagnostic kit may be detectably labeled. Various methods that can be used to label biomolecules are well known to those skilled in the art and are considered to fall within the scope of the present invention. Examples of labels useful in the present invention may include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds. Common labels include fluorescent substances (e.g., fluorescein, rhodamine, texas red, etc.), enzymes (e.g., horseradish peroxidase, beta-galactosidase, or alkaline phosphatase), radioisotopes (e.g., 32P or 125I), biotin, digoxigenin, colloidal metals, or chemiluminescent or bioluminescent compounds (e.g., dioxane, luminol, or acridine). Methods for labeling enzymatic or biotin groups such as covalent binding, iodination, phosphorylation, biotinylation, etc. are well known in the art. Detection methods include, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, and the like. Commonly used detection assays are radioisotopic or non-radioisotopic. Particularly useful are immunoblots, overlay assays, RIA (radioimmunoassay), IRMA (immunoradioimmunoassay), EIA (enzyme immunoassay), ELISA (enzyme-linked immunosorbent assay), FIA (fluorescence immunoassay) and CLIA (chemiluminescent immunoassay).

The diagnostic kit of the present invention can be used to detect the presence or absence of MERS-CoV by contacting the sample with a binding molecule and observing the reaction.

The sample may be, but is not limited to, any one selected from the group consisting of sputum, saliva, blood, sweat, lung cells, lung tissue mucus, respiratory tissue, and saliva of the subject, and the sample may be prepared using a process generally known to those skilled in the art.

In addition, an embodiment of the present invention provides a kit for diagnosing, preventing or treating a disease caused by MERS-CoV, comprising:

a) a binding molecule; and

b) a container.

In the kit for diagnosis, prevention or treatment according to the present invention, the solid carrier may be included in a container thereof. The antibodies of the invention may be attached to a solid support, and the solid support may be porous or non-porous, or may be flat or non-flat.

In addition, the present invention provides a method of diagnosing, preventing or treating a disease caused by MERS-CoV infection, the method comprising administering a therapeutically effective amount of the above composition to a subject having a disease caused by MERS-CoV infection.

In embodiments of the invention, the diagnostic, prophylactic or therapeutic method may further comprise administering an antiviral drug, a viral entry inhibitor or a viral adhesion inhibitor.

The terms used in the present invention are defined as follows.

As used herein, the term "binding molecule" refers to a whole immunoglobulin, which includes monoclonal antibodies such as chimeric, humanized or human monoclonal antibodies; or an antigen-binding fragment which is an immunoglobulin that binds to an antigen. For example, it indicates a variable region, enzyme, receptor or protein comprising an immunoglobulin fragment that competes with an intact immunoglobulin for binding to the spike protein of MERS-CoV. Regardless of structure, an antigen-binding fragment binds to the same antigen recognized by an intact immunoglobulin. An antigen-binding fragment may comprise a peptide or polypeptide comprising an antibody amino acid sequence consisting of: 2 or more consecutive amino acid residues, 20 or more consecutive amino acid residues, 25 or more consecutive amino acid residues, 30 or more consecutive amino acid residues, 35 or more consecutive amino acid residues, 40 or more consecutive amino acid residues, 50 or more consecutive amino acid residues, 60 or more consecutive amino acid residues, 70 or more consecutive amino acid residues, 80 or more consecutive amino acid residues, 90 or more consecutive amino acid residues, 100 or more consecutive amino acid residues, 125 or more consecutive amino acid residues, 150 or more consecutive amino acid residues, 175 or more consecutive amino acid residues, 200 or more consecutive amino acid residues, or 250 or more consecutive amino acid residues.

As used herein, the term "antigen-binding fragment" indicates Fab, F (ab') 2, Fv, dAb, Fd, Complementarity Determining Region (CDR) fragments, single chain antibodies (scFv), bivalent single chain antibodies, single chain phage antibodies, diabodies, triabodies, tetrabodies, polypeptides, and the like, which comprise at least one fragment of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide. The above fragments may be produced synthetically or by enzymatic or chemical cleavage of intact immunoglobulins, or they may be genetically engineered by recombinant DNA techniques. Such production methods are well known in the art.

As used herein, the term "pharmaceutically acceptable excipient" means any inert substance that is combined with an active molecule, such as a drug, agent or antibody, to make an acceptable or convenient dosage form. A pharmaceutically acceptable excipient is one that is non-toxic, or at least has reduced toxicity, to a recipient at typical dosages and concentrations employed, and which is compatible with the other ingredients of the formulation, including the drug, agent or binding molecule.

As used herein, the term "therapeutically effective amount" refers to an amount of a binding molecule of the invention that is prophylactically or therapeutically effective before or after exposure to MERS-CoV.

Advantageous effects

According to the present invention, the binding molecule has a strong ability to bind to the S protein of MERS-CoV, thereby exhibiting neutralizing activity, and thus is very useful in the prevention, treatment, or diagnosis of MERS-CoV infection.

Brief description of the drawings

FIG. 1 shows the neutralizing activity against Korean isolate MERS-CoV (MERS-CoV/Korea/KNIH/002-05-2015) depending on the antibody concentration by plaque assay with two finally selected antibodies;

FIG. 2 shows virus titers determined by plaque after tissue culture by infecting human lung tissue with Korean isolate MERS-CoV and antibody in order to evaluate the neutralizing activity of the antibody using a human lung tissue infection model (ex vivo);

figure 3a shows the quantitative PCR results for animal treatment efficacy assessment using an animal model capable of MERS-CoV infection and proliferation (hpdp 4 (human dipeptidylpeptidase 4) receptor-overexpressed mice) and antibodies that bind to the virus;

figure 3b shows the results of plaque assays using animal models capable of MERS-CoV infection and proliferation (hpdp 4 (human dipeptidyl peptidase 4) receptor-overexpressed mice) and antibodies that bind to the virus for the assessment of the efficacy of animal treatment;

figure 4a shows the quantitative PCR results for the assessment of the prophylactic efficacy of antibodies against MERS-CoV using an animal model capable of MERS-CoV infection and proliferation (hDPP4 (human dipeptidylpeptidase 4) receptor-overexpressed mice) (. p <0.05,. p <0.01,. p < 0.001);

figure 4b shows the results of plaque assays using animal models capable of MERS-CoV infection and proliferation (hpdp 4 (human dipeptidylpeptidase 4) receptor-overexpressed mice) for the assessment of the prophylactic efficacy of antibodies against MERS-CoV (/ p <0.05,/p <0.01,/p < 0.001);

figure 5 shows tissue changes in the mouse lung assessed for prophylactic efficacy of antibodies against MERS-CoV using an animal model capable of MERS-CoV infection and proliferation (hpdp 4 (human dipeptidyl peptidase 4) receptor-overexpressed mice);

figure 6a shows mouse weight loss using an animal model capable of MERS-CoV infection and proliferation (hDPP4 (human dipeptidylpeptidase 4) receptor-overexpressed mice) for assessment of therapeutic efficacy of antibodies against MERS-CoV (/ p <0.05,/p <0.01,/p < 0.001);

figure 6b shows mouse survival (/ p <0.05,/p <0.01,/p <0.001) assessed using an animal model capable of MERS-CoV infection and proliferation (dpp 4 (human dipeptidylpeptidase 4) receptor-overexpressed mice) for therapeutic efficacy of antibodies against MERS-CoV;

figure 6c shows the quantitative PCR results for therapeutic efficacy assessment of antibodies against MERS-CoV using an animal model capable of MERS-CoV infection and proliferation (hDPP4 (human dipeptidylpeptidase 4) receptor-overexpressed mice) (. p <0.05,. p <0.01,. p < 0.001); and

figure 6d shows the results of plaque assays using animal models capable of MERS-CoV infection and proliferation (hpdp 4 (human dipeptidylpeptidase 4) receptor-overexpressed mice) for the assessment of therapeutic efficacy of antibodies against MERS-CoV (/ p <0.05,/p <0.01,/p < 0.001).

Modes of the invention

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed to limit the scope of the present invention. The documents cited herein are incorporated by reference into this application.

Example 1: PBMC isolation from blood of MERS-CoV rehabilitated patients

Blood donors are those confirmed to have been infected with MERS-CoV in 2015 and no longer have virus for treatment, and the donor selection and blood collection procedure was performed under approval of the Institutional Review Board (IRB). After donor selection, approximately 30ml of whole blood was collected and Ficoll-Paque was used^TMThe PLUS (GE healthcare) method separates PBMC (peripheral blood mononuclear cells). Isolated PBMC were washed twice with phosphate buffered saline and then 1 × 10 in cryo-medium (RPMI: FBS: DMSO ═ 5:4:1)⁷The concentration of individual cells/ml was stored in a liquid nitrogen tank.