Monoclonal Anitbody For Spike Protein Of Middle East Respiratory Syndrome Coronavirus And Use Thereof

Abstract

The present invention relates to monoclonal antibodies for a spike protein of the Middle East respiratory syndrome coronavirus (MERS-CoV), and a use thereof. Particularly, monoclonal antibodies 77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6, 90-F1 and 90-F2 according to the present invention have excellent attachment force with respect to a full-length spike protein of MERS-CoV and the Si domain of the protein, and, of the monoclonal antibodies, the monoclonal antibodies 90-F1, 90-E5, 90-E6, 90-F2, 77-A5 and 77-A6 have excellent attachment force with respect to an RBD antigen of MERS-CoV. Also, the antibodies 77-A5, 77-A6, 90-E5, 90-E6, 90-F1 and 90-F2 exhibit neutralizing capacity with respect to a MERS pseudovirus and MERS-CoV, and the antibodies 90-B2 and 90-B7 exhibit neutralizing capacity only with respect to MERS-CoV. Further, the monoclonal antibodies have a particular monomeric form, and have excellent stability and thus may be useful for treating or diagnosing MERS.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

[0001] The present invention relates to monoclonal antibodies for a spike protein of Middle East respiratory syndrome coronavirus and a use thereof.

2. Description of the Related Art

[0002] Middle East respiratory syndrome (MERS) was first identified in the Middle East region in September 2012. According to the report made by WHO, MERS spread to 26 countries as of Jun. 5, 2017, resulting in 1,980 confirmed patients and 693 deaths worldwide. It is a high-risk disease with a mortality rate of 35%.

[0003] Middle East respiratory syndrome coronavirus (MERS-CoV), the pathogen of MERS, is a newly discovered beta-coronavirus (YCoV), which was first found in 2012. This virus is a 30-kb (+)-sense single-stranded RNA virus, and is similar to severe acute respiratory syndrome (SARS) virus. MERS-CoV penetrates into cells as being conjugated with a human DPP4 (dipeptidyl peptidase 4) receptor by using a spike protein (S protein) (Wang N, Shi X, Jiang L J, Zhang S, Wang D, Tong P, Guo D, et al., Cell Research. 2013:23:986). MERS-CoV has a latency of about one week and causes severe respiratory symptoms such as high fever, coughing, and difficulty in breathing, etc.

[0004] For the treatment of MERS, the immunomodulator interferon and the anti-viral agent ribarvirin or lopinavir are used (Public Health England, ISARIC, 2015. Sep. 5. ver 3.0; Chung Y P, Song J Y, Seo Y B, et al., Infection & Chemotherapy. 2015). Interferon is an immune protein released when a virus or germ enters in a human body, inducing the surrounding cells to emit antiviral cytokines, inhibiting virus proliferation, and inviting immune cells to remove the virus-infected cells. However, the use of interferon can cause side effects such as bone marrow suppression, anemia, decrease of white cell number, or decrease of platelet number.

[0005] Ribarvirin, the antiviral agent, inhibits the proliferation of various types of viruses by interrupting the RNA synthesis thereof in the form of a nucleoside analogue. However, ribarvirin also induces side effects such as toxicity, carcinogenesis, or hemolytic anemia. The administration of interferon and ribarvirin for the treatment of MERS has not been supported by the clinical tests. In the rhesus monkey animal model, the co-administration of interferon and ribarvirin was effective in treating MERS (Falzarano D, Wit E, Rasmussen A L., et al., Nature Medicine, 2013, 19, 10, p 1313-1318). However, when interferon and ribarvirin were co-administered to 5 MERS patients with severe conditions in Middle East, the treatment effect was hardly observed (Al-Tawfiq J A, Momattin H., Dib J., et al., International Journal of Infectious Diseases 2014, 20, p 42-46). Therefore, it is urgently requested to develop a biologically safe therapeutic antibody that can be used for severe MERS patients, immunocompromised patients, and underlying disease patients with suppressing virus proliferation effectively.

[0006] In relation to the above, Korean Patent No 10-1593641 describes a monoclonal antibody binding specifically to MERS-CoV nucleocapsid and a composition for diagnosing MERS-CoV comprising the same.

[0007] Thus, the present inventors tried to develop therapeutic antibodies for MERS-CoV. As a result, the inventors succeeded in preparation of 24 kinds of monoclonal antibodies specifically binding to the spike protein of MERS-CoV. The inventors further confirmed that 11 antibodies (77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6, 90-F1, and 90-F2) among them had excellent adhesion to MERS-CoV full-length spike and S1 antigen, and 90-F1, 90-E5, 90-E6, 90-F2, 77-A5, and 77-A6 antibodies demonstrated excellent adhesion to MERS-CoV RBD antigen. Also, 77-A5, 77-A6, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies showed neutralizing ability against MERS pseudovirus and MERS-CoV, 90-B2 and 90-B7 antibodies showed neutralizing ability only against MERS-CoV. In addition, the present inventors further confirmed that the antibodies of the present invention are in the form of a certain monomer and have excellent physical stability, leading to the completion of the present invention.

SUMMARY OF THE INVENTION

[0008] It is an object of the present invention to provide a monoclonal antibody binding specifically to the spike protein of Middle East respiratory syndrome coronavirus.

[0009] It is another object of the present invention to provide a gene encoding the monoclonal antibody of the invention, a recombinant vector comprising thereof, and a host cell introduced with the gene or the recombinant vector above.

[0010] It is also an object of the present invention to provide a use of the prevention, treatment or diagnosis of Middle East respiratory syndrome comprising the monoclonal antibody of the invention.

[0011] It is further an object of the present invention to provide a kit for the detection or quantification of an antigen of Middle East respiratory syndrome comprising the monoclonal antibody of the invention.

[0012] To achieve the above objects, the present invention provides a heavy chain variable region of the monoclonal antibody specifically binding to the MERS-CoV spike protein whose complementarity determining region (CDR) is composed of the amino acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or NO: 21.

[0013] The present invention also provides a gene encoding the heavy chain variable region of the monoclonal antibody of the invention.

[0014] The present invention also provides a recombinant vector comprising the gene encoding the heavy chain variable region of the monoclonal antibody of the invention.

[0015] The present invention also provides a host cell introduced with the gene encoding the heavy chain variable region of the monoclonal antibody of the invention or the recombinant vector comprising the gene above.

[0016] The present invention also provides a composition for the prevention, treatment or diagnosis of Middle East respiratory syndrome comprising the heavy chain variable region of the monoclonal antibody of the invention.

[0017] The present invention also provides a kit for the detection or quantification of an antigen of Middle East respiratory syndrome comprising the heavy chain variable region of the monoclonal antibody of the invention.

[0018] The present invention also provides a light chain variable region of the monoclonal antibody specifically binding to the MERS-CoV spike protein whose complementarity determining region (CDR) is composed of the amino acid sequence represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or NO: 22.

[0019] The present invention also provides a gene encoding the light chain variable region of the monoclonal antibody of the invention.

[0020] The present invention also provides a recombinant vector comprising the gene encoding the light chain variable region of the monoclonal antibody of the invention.

[0021] The present invention also provides a host cell introduced with the gene encoding the light chain variable region of the monoclonal antibody of the invention or the recombinant vector comprising the gene above.

[0022] The present invention also provides a composition for the prevention, treatment or diagnosis of Middle East respiratory syndrome comprising the light chain variable region of the monoclonal antibody of the invention.

[0023] The present invention also provides a kit for the detection or quantification of an antigen of Middle East respiratory syndrome comprising the light chain variable region of the monoclonal antibody of the invention.

[0024] The present invention also provides a monoclonal antibody specifically binding to the MERS-CoV spike protein containing a heavy chain variable region whose complementarity determining region is composed of the amino acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or NO: 21, and a light chain variable region whose complementarity determining region is composed of the amino acid sequence represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or NO: 22.

[0025] The present invention also provides a gene encoding the monoclonal antibody of the invention.

[0026] The present invention also provides a recombinant vector containing the gene encoding the monoclonal antibody of the invention.

[0027] The present invention also provides a host cell introduced with the gene encoding the monoclonal antibody of the invention or the recombinant vector comprising the gene above.

[0028] The present invention also provides a composition for the prevention, treatment or diagnosis of Middle East respiratory syndrome comprising the monoclonal antibody of the invention.

[0029] The present invention provides a kit for the detection or quantification of an antigen of Middle East respiratory syndrome comprising the monoclonal antibody of the invention.

[0030] The present invention also provides a method for preventing, treating or diagnosing Middle East respiratory syndrome comprising a step of administering the heavy chain variable region of the monoclonal antibody of the invention to a subject.

[0031] The present invention also provides a use of the heavy chain variable region of the monoclonal antibody of the invention for the manufacture of a medicament for preventing, treating or diagnosing Middle East respiratory syndrome.

[0032] The present invention also provides a method for preventing, treating or diagnosing Middle East respiratory syndrome comprising a step of administering the light chain variable region of the monoclonal antibody of the invention to a subject.

[0033] The present invention also provides a use of the light chain variable region of the monoclonal antibody of the invention for the manufacture of a medicament for preventing, treating or diagnosing Middle East respiratory syndrome.

[0034] The present invention also provides a method for preventing, treating or diagnosing Middle East respiratory syndrome comprising a step of administering the monoclonal antibody of the invention to a subject.

[0035] In addition, the present invention provides a use of the monoclonal antibody of the invention for the manufacture of a medicament for preventing, treating or diagnosing Middle East respiratory syndrome.

Advantageous Effect

[0036] Monoclonal antibodies 77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6, 90-F1 and 90-F2 according to the present invention have excellent attachment force with respect to a full-length spike protein of MERS-CoV and the S1 domain of the protein, and, of the monoclonal antibodies, the monoclonal antibodies 90-F1, 90-E5, 90-E6, 90-F2, 77-A5 and 77-A6 have excellent attachment force with respect to an RBD antigen of MERS-CoV. Also, the antibodies 77-A5, 77-A6, 90-E5, 90-E6, 90-F1 and 90-F2 exhibit neutralizing capacity with respect to a MERS pseudovirus and MERS-CoV, and the antibodies 90-B2 and 90-B7 exhibit neutralizing capacity only with respect to MERS-CoV. Further, the monoclonal antibodies have a particular monomeric form, and have excellent stability and thus may be useful for treating or diagnosing MERS.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

[0038] FIG. 1 is a graph illustrating the adhesion of 77-A1, 77-A4, 77-A5, 77-A6, 77-A9, 77-A10, 77-A11, 77-A12, 77-B3, 77-B4, 77-B12, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-C5, 90-D6, 90-E1, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies to the full-length spike trimer antigen of MERS-CoV. Herein, C2, A2, A10, G2, and G4 antibodies developed by NIH, USA, were used as the positive controls.

[0039] FIG. 2 is a graph illustrating the adhesion of the antibodies above to the MERS-CoV S1 antigen composed of the amino acids 1 to 757 of the spike protein.

[0040] FIG. 3 is a graph illustrating the adhesion of the antibodies above to the MERS-CoV RBD antigen composed of the amino acids 377 to 588 of the spike protein.

[0041] FIG. 4 is a graph illustrating the adhesion of the antibodies above to the MERS-CoV S2 antigen composed of the amino acids 757 to 1275 of the spike protein.

[0042] FIG. 5a is a set of graphs illustrating the neutralizing ability of 77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies against pseudovirus using the spike protein of MERS-CoV Erasmus strain.

[0043] FIG. 5b is a set of graphs illustrating the neutralizing ability of 77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies against pseudovirus using the spike protein of MERS-CoV Bisha 1 strain.

[0044] FIG. 6a is a set of graphs illustrating the neutralizing ability of 77-A5, 77-A6, 90-A3, 90-A9, 90-B2 and 90-B7 antibodies against MERS-CoV.

[0045] FIG. 6b is a set of graphs illustrating the neutralizing ability of 90-C4, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies against MERS-CoV.

[0046] FIG. 7 is a graph illustrating the morphology of the antibodies analyzed by SEC-HPLC.

[0047] FIG. 8a is a set graphs illustrating the antigen-antibody binding affinity investigated by SPR (Surface Plasmon Resonance) assay using S1 antigen.

[0048] FIG. 8b is a set graphs illustrating the antigen-antibody binding affinity investigated by SPR (Surface Plasmon Resonance) assay using RBD antigen.

[0049] FIG. 9a is a set of graphs illustrating the thermo-stability of 77-A5 and 77-A6 antibodies investigated by PTS (Protein Thermal Shift) assay.

[0050] FIG. 9b is a set of graphs illustrating the thermo-stability of 90-B2 and 90-B7 antibodies investigated by PTS (Protein Thermal Shift) assay.

[0051] FIG. 9c is a set of graphs illustrating the thermo-stability of 90-F1, and 90-F2 antibodies investigated by PTS (Protein Thermal Shift) assay.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Hereinafter, the present invention is described in detail.

[0053] The present invention provides a heavy chain variable region of the monoclonal antibody binding specifically to the spike protein of MERS-CoV whose complementarity determining region (CDR) is composed of the amino acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or NO: 21.

[0054] The spike protein above can be a polypeptide composed of any sequence known to those in the art. The polypeptide above can be a variant or a fragment of an amino acid sequence having a different sequence made by deletion, insertion, substitution or combination thereof as long as it does not affect the protein function. The exchange of amino acids in a protein or a peptide, without changing the overall molecular activity, has been well-informed to those in the art. The most common exchanges are between the amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly. In some cases, such modifications as phosphorylation, sulfation, acrylation, glycosylation, methylation, and farnesylation can be occurred. In an example of the present invention, the spike protein can be composed of the amino acid sequence represented by SEQ. ID. NO: 23.

[0055] The said heavy chain variable region can specifically bind to the 1.sup.st to 757.sup.th amino acid sites from the N-terminus of the spike protein of MERS-CoV. Among the heavy chain variable regions, the heavy chain variable region of the monoclonal antibody whose complementarity determining region is composed of the amino acid sequence represented by SEQ. ID. NO: 1, 3, 15, 17, 19, or NO: 21 can bind specifically to the 377.sup.th to 588.sup.th amino acid sites from the N-terminus of the spike protein of MERS-CoV.

[0056] In a preferred embodiment of the present invention, the present inventors prepared 24 kinds of monoclonal antibodies binding specifically to the spike protein of MERS-CoV. Among them, 77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies were confirmed to have excellent adhesion to the full-length spike protein and S1 antigen of MERS-CoV (see FIGS. 1 and 2). 90-F1, 90-E5, 90-E6, 90-F2, 77-A5, and 77-A6 antibodies were confirmed to have excellent adhesion to the RBD antigen of MERS-CoV (see FIG. 3).

[0057] The present invention also provides a gene encoding the heavy chain variable region of the monoclonal antibody; a recombinant vector containing the gene above; and a host cell introduced with the gene or the recombinant vector above.

[0058] The heavy chain variable region above can be characterized by the description above. For example, the heavy chain variable region can be the heavy chain variable region of the monoclonal antibody binding specifically to the spike protein of MERS-CoV whose complementarity determining region (CDR) is composed of the amino acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or NO: 21.

[0059] The gene above includes not only a sequence encoding the heavy chain variable region but also a polynucleotide having the same nucleotide sequence substantially identical to the gene and a fragment thereof. The polynucleotide having the same nucleotide sequence substantially identical to the gene can have at least 80%, preferably 90% s, and more preferably at least 95% homology with the polypeptide of the present invention. As described above, the polynucleotide of the present invention can include a variant in which one or more nucleotide sequences are substituted, deleted or inserted, as long as the variant encode a protein having the equivalent activity.

[0060] The said vector is a recombinant vector capable of expressing a target peptide in a desired host cell, which indicates a gene construct containing a necessary regulatory element operably linked thereto in order to express the gene insert. The vector includes such expression regulatory elements as initiation codon, termination codon, promoter, and operator, etc. At this time, the initiation codon and termination codon are generally considered as a part of the nucleotide encoding a polypeptide and they have to be in frame with the coding sequence so as to be functioning in a subject when the gene construct is inserted.

[0061] The term "operably linked" herein in this invention indicates a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked to perform a general function. For example, a promoter can be operably linked to a nucleic acid sequence encoding a protein or RNA, which affects the expression of the coding sequence. The operable linkage can be achieved by using the genetic recombination technique which is well-known to those in the art. The site-specific DNA cleavage and linkage can be accomplished by using the enzyme well known to those in the art.

[0062] The vector system of the present invention can be constructed through various methods known to those in the art, and a specific method for this is disclosed in "Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001)".

[0063] The vector of the present invention can be constructed as a typical cloning vector or an expression vector. Also, the vector of the present invention can be constructed by using a prokaryotic cell or a eukaryotic cell as a host cell. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host cell, it generally includes a strong promoter capable of promoting transcription (for example, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL promoter, pR promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. When E. coli (for example, HB101, BL21, DH5.alpha., etc) is used as a host cell, the promoter and operator region of E. coli tryptophan biosynthesis pathway (Yanofsky, C., J. Bacteriol., 158:1018-1024(1984)) and the pL promoter of phage (pL promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445(1980)) can be used as a regulatory region. When a Bacillus bacterium is used as a host cell, a promoter of a toxin protein gene of Bacillus thuringiensis (Appl. Environ. Microbiol. 64:3932-3938(1998); Mol. Gen. Genet. 250:734-741(1996)) or any promoter capable of expressing in Bacillus can be used as a regulatory region.

[0064] The vector usable in this invention can be constructed by manipulating plasmids (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pRSET, pBluescript, pGEX2T, pCR1, pMB9, RP4, and pUC19 etc.), phages (for example, gt4.B, -Charon, Ez1, and M13) or viruses (for example, SV40 etc.) frequently used in this field.

[0065] When the vector of the invention is an expression vector and the host cell is an eukaryotic cell, a promoter originated from a mammalian cell genome (for example, metallothionine promoter, .gamma.-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter originated from a mammalian virus (for example, bovine papulardermatitis virus promoter, adeno-associated virus promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, moloney virus promoter, Epstein-Barr virus (EBV) promoter, tobacco mosaic virus promoter, and roas sarcoma virus (RSV) promoter) can be used and a polyadenylation sequence is generally included therein as a transcription termination sequence.

[0066] The vectors of the present invention can be fused with other sequences to facilitate the purification of the antibodies expressed therefrom. The sequences adequate for the fusion are exemplified by glutathione S-transferase, maltose binding protein, FLAG, and 6.times. His, etc.

[0067] Since the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified through a protein A column without any additional sequence for the purification.

[0068] The vector above can also include a selection marker. The selection marker herein is a marker for the selection of a transformed microorganism or a recombinant vector, which can be used to give selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or surface protein expression. When a vector containing the selection marker is used, only those cells expressing the selection marker can survive in the selective medium, so that the transformed cells can be easily selected. Any selection marker that can be used in the conventional art can be used. For example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin or tetracycline can be used as a selection marker herein.

[0069] Various host cells can be used for the expression of the heavy chain variable region of the monoclonal antibody according to the present invention. For example, the host cells can be prokaryotic cells such as Escherichia sp., Bacillus sp., Streptomyces sp., Pseudomonas sp., Proteus sp., or Staphylococcus sp., fungi such as Aspergillus sp., yeasts such as Pichia sp., Saccharomyces sp., Schizosaccharomyces sp., or Neurospora sp., other lower eukaryotic cells, or eukaryotic sells such as animal cells and plant cells.

[0070] As a method of introducing the recombinant vector of the invention into a host cell and transforming it, the conventional gene manipulation method can be used. For example, as a physical method, microinjection, liposome dependent method, direct DNA uptake, receptor-mediated DNA transfer, Ca.sup.++-directed DNA transfer, or virus-mediated gene transfer can be used.

[0071] In a preferred embodiment of the present invention, the present inventors prepared monoclonal antibodies binding specifically to the spike protein of MERS-CoV. Among them, 77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies were confirmed to have excellent adhesion to the full-length spike protein and S1 antigen of MERS-CoV (see FIGS. 1 and 2). 90-F1, 90-E5, 90-E6, 90-F2, 77-A5, and 77-A6 antibodies were confirmed to have excellent adhesion to the RBD antigen of MERS-CoV (see FIG. 3).

[0072] The present invention also provides a composition for the prevention, treatment or diagnosis of Middle East respiratory syndrome comprising the heavy chain variable region of the monoclonal antibody of the invention.

[0073] The heavy chain variable region above can be characterized by the description above. For example, the heavy chain variable region can be the heavy chain variable region of the monoclonal antibody binding specifically to the spike protein of MERS-CoV whose complementarity determining region (CDR) is composed of the amino acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or NO: 21.

[0074] The composition for the treatment of MERS of the present invention can be administered as an independent drug or can be co-administered with other drugs. In the case of co-treatment, it can be administered simultaneously together with the conventional drug or stepwise.

[0075] The composition comprising the heavy chain variable region of the monoclonal antibody above can be used for the treatment of virus infection by introducing into a living body in the form of the antibody-drug conjugate. At this time, the drug can be a chemotherapeutic agent, a radionuclide, an immunotherapeutic agent, a cytokine, a chemokine, a toxin, a biological agent, or an enzyme inhibitor. An example of the method to bind an antibiotic to an antibody is explained in the following references: G. Gregoriadies, ed., Academic Press London, (1979); Arnon et al., Recent Results in Cancer Res., 75: 236 (1980); and Moolton et al., Immunolog. Res., 62:47 (1982).

[0076] The drug that is preferred for the coupling with the antibody of the present invention include antibiotics, antiparasitic, antifungal, and related agents such as, for example, sulfonamide, penicillin and cephalosporin, aminoglycoside, tetracycline, chloramphenicol, piperazine, chloroquine, diaminopyridine, metroniazide, isoniazid, rifampin, streptomycin, sulfone, erythromycin, polymyxin, nistatin, amphotericin, 5-fluorocytosine, 5-iodo-2'-deoxyuridine, 1-adamanthamine, adenine arabinoside, ammanidin, ribavirin or azatrimidine (AZT). The conditions appropriate and preferred for delivering a drug to a specific target are explained in Trouet et al., Plenum Press, New York and London, 19-30 (1982). The immunoregulator usable as a drug in the form of the antibody-drug conjugate can be a lymphokine or a cytokine, but not always limited thereto.

[0077] The composition for the prevention or treatment of MERS of the present invention can include the heavy chain variable region of the monoclonal antibody of the invention as an active ingredient at the concentration of 10-95 weight % by the total weight of the composition. The composition of the present invention can include, in addition to the active ingredient, one or more effective ingredients having the same or similar function to the active ingredient.

[0078] The composition for the prevention or treatment of MERS of the present invention can include any generally used carrier, diluent, excipient, or a combination of at least two of those. The pharmaceutically acceptable carrier can be any carrier that is able to deliver the composition of the present invention in a living body without limitation, which is exemplified by the compounds described in Merck Index, 13.sup.th ed., Merck & Co. Inc., such as saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and a mixture comprising one or more of those components. If necessary, a general additive such as antioxidant, buffer, and bacteriostatic agent can be additionally added.

[0079] The composition of the present invention can be prepared for oral or parenteral administration by mixing with generally used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactant. Solid formulations for oral administration are tablets, pills, powders, granules, capsules, and troches. These solid formulations are prepared by mixing the composition of the present invention with one or more suitable excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Lubricants such as magnesium stearate and talc can also be included therein. Liquid formulations for oral administrations are suspensions, solutions, emulsions and syrups, and the above-mentioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives.

[0080] Formulations for parenteral administration are sterilized aqueous solutions, water-insoluble excipients, suspensions, and emulsions.

[0081] Water insoluble excipients and suspensions can contain propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc.

[0082] The composition for the prevention or treatment of MERS of the present invention can be administered orally or parenterally and the parenteral administration includes intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, and intra-thoracic injection.

[0083] The composition for the prevention or treatment of MERS of the present invention is administered in a pharmaceutically effective dose. The effective dose can be determined by considering many factors such as the type of disease, severity of the disease, activity of the drug, sensitivity to the drug, administration frequency and pathway, excretion, term of treatment, co-treatment drug and other factors regarded as relevant in the medicinal field. In the case of co-treatment, the composition of the invention can be administered simultaneously together with the other drugs or stepwise.

[0084] For the preferred effect, the amount of the active ingredient included in the composition of the present invention can be 0.001.about.10,000 mg/kg, specifically 0.1.about.5 g/kg. The administration frequency can be once a day or a few times a day.

[0085] The diagnostic composition of the present invention indicates a major tool used for the diagnosis of a target disease, which can include the materials useful for diagnosing MERS-CoV according to the purpose of the invention. The diagnostic method can include the step of contacting an antibody or an antibody fragment with a sample.

[0086] The sample herein can be sputum, cells or a tissues taken from nasal cavity, sinus cavity, salivary gland, lung, liver, pancreas, kidney, ear, eye, placenta, digestive tract, heart, ovary, pituitary, adrenal, thyroid, brain or skin, urine, whole blood, serum, plasma, feces, cell culture supernatant, or ruptured eukaryotic cells.

[0087] The formation of the sample-antibody conjugate can be detected by colormetric method, electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment, or scintillation counting method.

[0088] The detection is to detect the sample-antibody conjugate, for which various markers can be used. As the marker, an enzyme, a fluorescent material, a ligand, a luminescent material, a microparticle or a radioactive isotope can be used.

[0089] The enzyme usable as a detection marker is exemplified by acetylcholinesterase, alkaline phosphatase, .gamma.-D-galactosidase, horseradish peroxidase, and .gamma.-lactamase. The fluorescent material usable as a detection marker can be fluorescein, Eu.sup.3+, Eu.sup.3+ chelate, or cryptate. As the ligand, a biotin derivative can be used. As the luminescent material, acridinium ester and an isoluminol derivative can be used. Colloidal gold and colored latex can be used as the microparticle, and .sup.57Co, .sup.3H, .sup.125I, and .sup.125I-Bonton Hunter reagent can be used as the radioactive isotope.

[0090] In a preferred embodiment of the present invention, the present inventors prepared monoclonal antibodies binding specifically to the spike protein of MERS-CoV. Among them, 77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies were confirmed to have excellent adhesion to the full-length spike protein and S1 antigen of MERS-CoV (see FIGS. 1 and 2). 90-F1, 90-E5, 90-E6, 90-F2, 77-A5, and 77-A6 antibodies were confirmed to have excellent adhesion to the RBD antigen of MERS-CoV (see FIG. 3). 77-A5, 77-A6, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies exhibited neutralizing ability against MERS pseudovirus and MERS-CoV, and 90-B2 and 90-B7 antibodies showed neutralizing ability only against MERS-CoV (see FIGS. 5a, FIG. 5b, FIG. 6a and FIG. 6b). The monoclonal antibodies above were confirmed to be in the form of a certain monomer (see FIG. 7) and have excellent stability (see FIG. 9a to 9c).

[0091] The present invention also provides a kit for the detection or quantification of an antigen of Middle East respiratory syndrome by using the antibody and protein comprising the heavy chain variable region of the monoclonal antibody of the invention.

[0092] The heavy chain variable region above can be characterized by the description above. For example, the heavy chain variable region can be the heavy chain variable region of the monoclonal antibody binding specifically to the spike protein of MERS-CoV whose complementarity determining region (CDR) is composed of the amino acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or NO: 21.

[0093] The said kit can be prepared by the conventional method well known to those in the art. The kit can additionally include a buffer, a stabilizer, and an inactive protein. The kit can be used in the following method: fluorescence method performed by detecting fluorescence of the fluorescent material attached as a detection marker to investigate the amount of the detection reagent, high throughput screening (HTS) system through radiation method performed by detecting radiation of the radioactive isotope attached as a detection marker, surface plasmon resonance (SPR) method to measure the surface plasmon resonance change in real time without labeling a detection marker, or surface plasmon resonance imaging (SPRI) method for imaging and verifying SPR system.

[0094] In a preferred embodiment of the present invention, the present inventors prepared monoclonal antibodies binding specifically to the spike protein of MERS-CoV. Among them, 77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies were confirmed to have excellent adhesion to the full-length spike protein and S1 antigen of MERS-CoV (see FIGS. 1 and 2). 90-F1, 90-E5, 90-E6, 90-F2, 77-A5, and 77-A6 antibodies were confirmed to have excellent adhesion to the RBD antigen of MERS-CoV (see FIG. 3). 77-A5, 77-A6, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies exhibited neutralizing ability against MERS pseudovirus and MERS-CoV, and 90-B2 and 90-B7 antibodies showed neutralizing ability only against MERS-CoV (see FIGS. 5a, FIG. 5b, FIG. 6a and FIG. 6b). The monoclonal antibodies above were confirmed to be in the form of a certain monomer (see FIG. 7) and have excellent stability (see FIG. 9a to FIG. 9c).

[0095] The present invention also provides a method for preventing, treating or diagnosing Middle East respiratory syndrome comprising a step of administering the heavy chain variable region of the monoclonal antibody of the invention to a subject.

[0096] The heavy chain variable region above can be characterized by the description above. For example, the heavy chain variable region can be the heavy chain variable region of the monoclonal antibody binding specifically to the spike protein of MERS-CoV whose complementarity determining region (CDR) is composed of the amino acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or NO: 21.

[0097] In addition, the subject can be a mammal, specifically a human.

[0098] The heavy chain variable region of the present invention can be administered orally or parenterally and the parenteral administration includes intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, and intra-thoracic injection.

[0099] The heavy chain variable region of the present invention is administered in a pharmaceutically effective dose. The effective dose can be determined by considering many factors such as the type of disease, severity of the disease, activity of the drug, sensitivity to the drug, administration frequency and pathway, excretion, term of treatment, co-treatment drug and other factors regarded as relevant in the medicinal field. In the case of co-treatment, the heavy chain variable region of the invention can be administered simultaneously together with the other drugs or stepwise.

[0100] For the preferred effect, the amount of the active ingredient included in the heavy chain variable region of the present invention can be 0.001-10,000 mg/kg, specifically 0.1-5 g/kg. The administration frequency can be once a day or a few times a day.

[0101] The heavy chain variable region of the present invention indicates a major tool used for the diagnosis of a target disease, which can include the materials useful for diagnosing MERS-CoV according to the purpose of the invention. The diagnostic method can include the step of contacting an antibody or an antibody fragment with a sample.

[0102] The sample herein can be sputum, cells or a tissues taken from nasal cavity, sinus cavity, salivary gland, lung, liver, pancreas, kidney, ear, eye, placenta, digestive tract, heart, ovary, pituitary, adrenal, thyroid, brain or skin, urine, whole blood, serum, plasma, feces, cell culture supernatant, or ruptured eukaryotic cells.

[0103] The formation of the sample-antibody conjugate can be detected by colormetric method, electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment, or scintillation counting method.

[0104] The detection is to detect the sample-antibody conjugate, for which various markers can be used. As the marker, an enzyme, a fluorescent material, a ligand, a luminescent material, a microparticle or a radioactive isotope can be used.

[0105] The enzyme usable as a detection marker is exemplified by acetylcholinesterase, alkaline phosphatase, .gamma.-D-galactosidase, horseradish peroxidase, and .gamma.-lactamase. The fluorescent material usable as a detection marker can be fluorescein, Eu3+, Eu3+ chelate, or cryptate. As the ligand, a biotin derivative can be used. As the luminescent material, acridinium ester and an isoluminol derivative can be used. Colloidal gold and colored latex can be used as the microparticle, and 57Co, 3H, 125I, and 125I-Bonton Hunter reagent can be used as the radioactive isotope.

[0106] The present invention also provides a use of the heavy chain variable region of the monoclonal antibody of the invention for the manufacture of a medicament for preventing, treating or diagnosing Middle East respiratory syndrome.

[0107] The heavy chain variable region above can be characterized by the description above.

[0108] The present invention also provides a light chain variable region of the monoclonal antibody specifically binding to the spike protein of MERS-CoV whose complementarity determining region (CDR) is composed of the amino acid sequence represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or NO: 22.

[0109] The spike protein above can be characterized by the description above. In a preferred embodiment of the present invention, the spike protein can be composed of the amino acid sequence represented by SEQ. ID. NO: 23.

[0110] The said light chain variable region can specifically bind to the 1.sup.st to 757.sup.th amino acid sites from the N-terminus of the spike protein of MERS-CoV. Among the light chain variable regions, the light chain variable region of the monoclonal antibody whose complementarity determining region is composed of the amino acid sequence represented by SEQ. ID. NO: 2, 4, 16, 18, 20, or NO: 22 can bind specifically to the 377.sup.th to 588.sup.th amino acid sites from the N-terminus of the spike protein of MERS-CoV.

[0111] The present invention also provides a gene encoding the light chain variable region of the monoclonal antibody of the invention; a recombinant vector containing the gene above; and a host cell introduced with the gene or the recombinant vector above.

[0112] The light chain variable region above can be characterized by the description above. For example, the light chain variable region can be the light chain variable region of the monoclonal antibody binding specifically to the spike protein of MERS-CoV whose complementarity determining region (CDR) is composed of the amino acid sequence represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or NO: 22.

[0113] The gene above is characterized by the description above.

[0114] The said vector is a recombinant vector capable of expressing a target peptide in a desired host cell, which indicates a gene construct containing a necessary regulatory element operably linked thereto in order to express the gene insert.

[0115] Various host cells can be used in order to express the light chain variable region of the monoclonal antibody according to the present invention. The usable host cells are characterized by the description above.

[0116] As a method of introducing the recombinant vector of the invention into a host cell and transforming it, the conventional gene manipulation method can be used. For example, as a physical method, microinjection, liposome dependent method, direct DNA uptake, receptor-mediated DNA transfer, Ca.sup.++-directed DNA transfer, or virus-mediated gene transfer can be used.

[0117] The present invention also provides a composition for the prevention, treatment or diagnosis of Middle East respiratory syndrome comprising the light chain variable region of the monoclonal antibody of the invention.

[0118] The light chain variable region above can be characterized by the description above. For example, the light chain variable region can be the light chain variable region of the monoclonal antibody binding specifically to the spike protein of MERS-CoV whose complementarity determining region (CDR) is composed of the amino acid sequence represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or NO: 22.

[0119] The composition for the prevention or treatment of the invention is characterized by the description above.

[0120] The diagnostic composition of the present invention indicates a major tool used for the diagnosis of a target disease, which can include the materials useful for diagnosing MERS-CoV according to the purpose of the invention. The diagnostic method can include the step of contacting an antibody or an antibody fragment with a sample. The sample herein can be sputum, cells or a tissues taken from nasal cavity, sinus cavity, salivary gland, lung, liver, pancreas, kidney, ear, eye, placenta, digestive tract, heart, ovary, pituitary, adrenal, thyroid, brain or skin, urine, whole blood, serum, plasma, feces, cell culture supernatant, or ruptured eukaryotic cells.

[0121] The present invention also provides a kit for the detection or quantification of an antigen of Middle East respiratory syndrome by using the antibody and protein comprising the light chain variable region of the monoclonal antibody of the invention.

[0122] The light chain variable region above can be characterized by the description above. For example, the light chain variable region can be the light chain variable region of the monoclonal antibody binding specifically to the spike protein of MERS-CoV whose complementarity determining region (CDR) is composed of the amino acid sequence represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or NO: 22.

[0123] The kit above is characterized by the description above. For example, it can be prepared by the conventional method known to those in the art, and can additionally include a buffer, a stabilizer, and an inactive protein, etc. The kit can be used in the following method: fluorescence method performed by detecting fluorescence of the fluorescent material attached as a detection marker to investigate the amount of the detection reagent, high throughput screening (HTS) system through radiation method performed by detecting radiation of the radioactive isotope attached as a detection marker, surface plasmon resonance (SPR) method to measure the surface plasmon resonance change in real time without labeling a detection marker, or surface plasmon resonance imaging (SPRI) method for imaging and verifying SPR system.

[0124] The present invention also provides a method for preventing, treating or diagnosing Middle East respiratory syndrome comprising a step of administering the light chain variable region of the monoclonal antibody of the invention to a subject.

[0125] The light chain variable region above can be characterized by the description above. For example, the light chain variable region can be the light chain variable region of the monoclonal antibody binding specifically to the spike protein of MERS-CoV whose complementarity determining region (CDR) is composed of the amino acid sequence represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or NO: 22.

[0126] In addition, the subject can be a mammal, specifically a human.

[0127] The light chain variable region of the present invention can be administered orally or parenterally and the parenteral administration includes intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, and intra-thoracic injection.

[0128] The light chain variable region of the present invention is administered in a pharmaceutically effective dose. The effective dose can be determined by considering many factors such as the type of disease, severity of the disease, activity of the drug, sensitivity to the drug, administration frequency and pathway, excretion, term of treatment, co-treatment drug and other factors regarded as relevant in the medicinal field. In the case of co-treatment, the light chain variable region of the invention can be administered simultaneously together with the other drugs or stepwise.

[0129] For the preferred effect, the amount of the active ingredient included in the light chain variable region of the present invention can be 0.001-10,000 mg/kg, specifically 0.1-5 g/kg. The administration frequency can be once a day or a few times a day.

[0130] The light chain variable region of the present invention indicates a major tool used for the diagnosis of a target disease, which can include the materials useful for diagnosing MERS-CoV according to the purpose of the invention. The diagnostic method can include the step of contacting an antibody or an antibody fragment with a sample.

[0131] The sample herein can be sputum, cells or a tissues taken from nasal cavity, sinus cavity, salivary gland, lung, liver, pancreas, kidney, ear, eye, placenta, digestive tract, heart, ovary, pituitary, adrenal, thyroid, brain or skin, urine, whole blood, serum, plasma, feces, cell culture supernatant, or ruptured eukaryotic cells.

[0132] The formation of the sample-antibody conjugate can be detected by colormetric method, electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment, or scintillation counting method.

[0133] The detection is to detect the sample-antibody conjugate, for which various markers can be used. As the marker, an enzyme, a fluorescent material, a ligand, a luminescent material, a microparticle or a radioactive isotope can be used.

[0134] The enzyme usable as a detection marker is exemplified by acetylcholinesterase, alkaline phosphatase, .gamma.-D-galactosidase, horseradish peroxidase, and .gamma.-lactamase. The fluorescent material usable as a detection marker can be fluorescein, Eu3+, Eu3+ chelate, or cryptate. As the ligand, a biotin derivative can be used. As the luminescent material, acridinium ester and an isoluminol derivative can be used. Colloidal gold and colored latex can be used as the microparticle, and 57Co, 3H, 125I, and 125I-Bonton Hunter reagent can be used as the radioactive isotope.

[0135] The present invention also provides a use of the light chain variable region of the monoclonal antibody of the invention for the manufacture of a medicament for preventing, treating or diagnosing Middle East respiratory syndrome.

[0136] The light chain variable region above can be characterized by the description above.

[0137] The present invention also provides a monoclonal antibody specifically binding to the spike protein of MERS-CoV containing a heavy chain variable region whose complementarity determining region is composed of the amino acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or NO: 21, and a light chain variable region whose complementarity determining region is composed of the amino acid sequence represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or NO: 22.

[0138] The spike protein above can be characterized by the description above. In a preferred embodiment of the present invention, the spike protein can be composed of the amino acid sequence represented by SEQ. ID. NO: 23. The light chain variable region and the heavy chain variable region can be characterized by the description above.

[0139] The monoclonal antibody is an antibody produced by a single antibody-forming cell, which has a uniform primary structure (amino acid sequence). The monoclonal antibody recognizes only one antigenic determinant and is generally produced by culturing the hybridoma cells prepared by the fusion of cancer cells and antibody producing cells. This antibody also can be produced by using other recombinant protein expressing host cells using the obtained antibody gene sequence.

[0140] The present invention also provides a gene encoding the monoclonal antibody of the invention; a recombinant vector containing the gene above; and a host cell introduced with the gene or the recombinant vector above.

[0141] The monoclonal antibody above can be characterized by the description above. For example, the monoclonal antibody can be the antibody binding specifically to the spike protein of MERS-CoV containing a heavy chain variable region whose complementarity determining region is composed of the amino acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or NO: 21, and a light chain variable region whose complementarity determining region is composed of the amino acid sequence represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or NO: 22.

[0142] The gene above is characterized by the description above.

[0143] The said vector is a recombinant vector capable of expressing a target peptide in a desired host cell, which indicates a gene construct containing a necessary regulatory element operably linked thereto in order to express the gene insert.

[0144] Various host cells can be used in order to express the monoclonal antibody according to the present invention. The usable host cells are characterized by the description above.

[0145] As a method of introducing the recombinant vector of the invention into a host cell and transforming it, the conventional gene manipulation method can be used. For example, as a physical method, microinjection, liposome dependent method, direct DNA uptake, receptor-mediated DNA transfer, Ca.sup.++-directed DNA transfer, or virus-mediated gene transfer can be used.

[0146] The present invention also provides a composition for the prevention, treatment or diagnosis of Middle East respiratory syndrome comprising the monoclonal antibody of the invention.

[0147] The monoclonal antibody above can be characterized by the description above. For example, the monoclonal antibody can be the antibody binding specifically to the spike protein of MERS-CoV containing a heavy chain variable region whose complementarity determining region is composed of the amino acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or NO: 21, and a light chain variable region whose complementarity determining region is composed of the amino acid sequence represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or NO: 22.

[0148] The composition for the prevention or treatment of the invention is characterized by the description above.

[0149] The diagnostic composition of the present invention indicates a major tool used for the diagnosis of a target disease, which can include the materials useful for diagnosing MERS-CoV according to the purpose of the invention. The diagnostic method can include the step of contacting an antibody or an antibody fragment with a sample. The sample herein can be sputum, cells or a tissues taken from nasal cavity, sinus cavity, salivary gland, lung, liver, pancreas, kidney, ear, eye, placenta, digestive tract, heart, ovary, pituitary, adrenal, thyroid, brain or skin, urine, whole blood, serum, plasma, feces, cell culture supernatant, or ruptured eukaryotic cells.

[0150] The present invention also provides a kit for the detection or quantification of an antigen of Middle East respiratory syndrome comprising the monoclonal antibody of the invention.

[0151] The monoclonal antibody above can be characterized by the description above. For example, the monoclonal antibody can be the antibody binding specifically to the spike protein of MERS-CoV containing a heavy chain variable region whose complementarity determining region is composed of the amino acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or NO: 21, and a light chain variable region whose complementarity determining region is composed of the amino acid sequence represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or NO: 22.

[0152] The kit above is characterized by the description above. For example, it can be prepared by the conventional method known to those in the art, and can additionally include a buffer, a stabilizer, and an inactive protein, etc. The kit can be used in the following method: fluorescence method performed by detecting fluorescence of the fluorescent material attached as a detection marker to investigate the amount of the detection reagent, high throughput screening (HTS) system through radiation method performed by detecting radiation of the radioactive isotope attached as a detection marker, surface plasmon resonance (SPR) method to measure the surface plasmon resonance change in real time without labeling a detection marker, or surface plasmon resonance imaging (SPRI) method for imaging and verifying SPR system.

[0153] The present invention also provides a method for preventing, treating or diagnosing Middle East respiratory syndrome comprising a step of administering the monoclonal antibody of the invention to a subject.

[0154] The monoclonal antibody above can be characterized by the description above. For example, the monoclonal antibody can be the antibody binding specifically to the spike protein of MERS-CoV containing a heavy chain variable region whose complementarity determining region is composed of the amino acid sequence represented by SEQ. ID. NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or NO: 21, and a light chain variable region whose complementarity determining region is composed of the amino acid sequence represented by SEQ. ID. NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or NO: 22.

[0155] In addition, the subject can be a mammal, specifically a human.

[0156] The monoclonal antibody of the present invention can be administered orally or parenterally and the parenteral administration includes intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, and intra-thoracic injection.

[0157] The monoclonal antibody of the present invention is administered in a pharmaceutically effective dose. The effective dose can be determined by considering many factors such as the type of disease, severity of the disease, activity of the drug, sensitivity to the drug, administration frequency and pathway, excretion, term of treatment, co-treatment drug and other factors regarded as relevant in the medicinal field. In the case of co-treatment, The monoclonal antibody of the invention can be administered simultaneously together with the other drugs or stepwise.

[0158] For the preferred effect, the amount of the active ingredient included in the monoclonal antibody of the present invention can be 0.001.about.10,000 mg/kg, specifically 0.1.about.5 g/kg. The administration frequency can be once a day or a few times a day.

[0159] The monoclonal antibody of the present invention indicates a major tool used for the diagnosis of a target disease, which can include the materials useful for diagnosing MERS-CoV according to the purpose of the invention. The diagnostic method can include the step of contacting an antibody or an antibody fragment with a sample.

[0160] The sample herein can be sputum, cells or a tissues taken from nasal cavity, sinus cavity, salivary gland, lung, liver, pancreas, kidney, ear, eye, placenta, digestive tract, heart, ovary, pituitary, adrenal, thyroid, brain or skin, urine, whole blood, serum, plasma, feces, cell culture supernatant, or ruptured eukaryotic cells.

[0161] The formation of the sample-antibody conjugate can be detected by colormetric method, electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment, or scintillation counting method.

[0162] The detection is to detect the sample-antibody conjugate, for which various markers can be used. As the marker, an enzyme, a fluorescent material, a ligand, a luminescent material, a microparticle or a radioactive isotope can be used.

[0163] The enzyme usable as a detection marker is exemplified by acetylcholinesterase, alkaline phosphatase, .gamma.-D-galactosidase, horseradish peroxidase, and .gamma.-lactamase. The fluorescent material usable as a detection marker can be fluorescein, Eu3+, Eu3+ chelate, or cryptate. As the ligand, a biotin derivative can be used. As the luminescent material, acridinium ester and an isoluminol derivative can be used. Colloidal gold and colored latex can be used as the microparticle, and 57Co, 3H, 125I, and 125I-Bonton Hunter reagent can be used as the radioactive isotope.

[0164] The present invention also provides a use of the monoclonal antibody of the invention for the manufacture of a medicament for preventing, treating or diagnosing Middle East respiratory syndrome.

[0165] The monoclonal antibody above can be characterized by the description above.

[0166] Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

[0167] However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Preparation of Monoclonal Antibody Binding Specifically to MERS-CoV Spike Protein

[0168] In the period between May and July 2015 when MERS occurred in Korea, blood samples were taken from patients with early stage MERS. Peripheral blood mononuclear cells (PBMCs) were isolated from those blood samples by the conventional method, which were cryopreserved. B cells having MERS antibody were separated as single cells (single cell sorting) from the PBMCs isolated by FACS using the MERS-CoV spike trimer protein probe (PE fluorescent dye), anti-human IgG-FITC and anti-human CD20 IgG-Cy55PerCP, provided by NIH/NIAID/VRC, USA. Unnecessary immune cells except B cells were negatively selected by attaching CD3, CD4, CD8 and CD14 markers and removed by FACS. CDR regions of the heavy chain, kappa light chain and lambda light chain of the antibody gene separated from the B cells obtained from patients with early phase MERS were amplified by PCR, and the sequences were confirmed. The nucleotide sequence appropriate for the immunoglobulin antibody expression was examined by using a web-based IMGT (International Immunogenetics Information systems, http://www.imgt.org) program. CDR regions of the heavy chain, kappa light chain and lambda light chain of the antibody were cloned in pVRC vector comprising mouse IgM signal peptide and IgG constant region provided by NIH/NIAID/VRC, USA. Then, Expi293F cells were transfected with the prepared antibody plasmid. 5 days later, the cell culture fluid was obtained, from which the antibody was prepared by purifying with protein G agarose column.

Experimental Example 1: Investigation of Antibody Adhesion to Antigen

<1-1> Investigation of Antibody Adhesion to MERS-CoV Full-Length Spike Protein Trimer Antigen

[0169] The binding affinity of 24 kinds of the antibodies prepared in Example 1 to the full-length spike trimer antigen of MERS-CoV, which is composed of amino acids 1 to 1275 from the N-terminus of the spike protein and is lack of the transmembrane region, was confirmed. The adhesion was compared among the monoclonal antibodies prepared in Example 1, the human anti-S1 monoclonal antibodies A2 and A10, the human anti-RBD monoclonal antibody C2, the mouse anti-S2 monoclonal antibody G4, and the mouse anti-S1 monoclonal antibody G2, developed by NIH, USA.

[0170] Particularly, MERS-CoV full spike trimer antigen was loaded in MaxiSorp 96-well plate (Nunc) at the concentration of 0.2 .mu.g/100 .mu. PBS/well, followed by reaction at 4.degree. C. for a day. Upon completion of the reaction, the reaction solution was eliminated, followed by blocking with a blocking solution containing 5% skim milk and 2% BSA in PBST at room temperature for 1 hour. Next, the antibody prepared in Example 1 was diluted in the blocking solution at the concentration of 10, 2.5, 0.625, 0.15625, 0.039, 0.009, 0.002, 0.0006, or 0.0001 .mu.g/m, which was loaded in the plate coated with the full spike trimer antigen above, followed by reaction at room temperature for 1 hour. The plate was washed with PBST 6 times, to which rabbit anti-human IgG H&L-HRP (horse radish peroxidase) secondary antibody was added, followed by reaction for 1 hour. Anti-mouse IgG-HRP secondary antibody was added to the well containing G2 and G4 antibodies, followed by reaction for 1 hour. After washing with PBST 6 times, 100 .mu. of TMB solution was added to each well, followed by reaction at room temperature for 30 minutes with blocking light. Upon completion of the reaction, 100 .mu. of stop solution (Enzygost) was added to each well. Then, OD.sub.450 was measured to investigate the antibody adhesion to antigen.

TABLE-US-00001 TABLE 1 CDR amino acid sequences to 11 MERS antibodies CDR sequence of heavy CDR sequence of kappa CDR sequence of lambda Antiboby chain light chain light chain 77-A5 TGVHSQVQLVQSGAEVKKPGS TGSWAQSALTQPPSASGTPGQ SVKVSCKASGGTFRSHAISWV RVTISCSGSSSNIGSNTVNWY RQAPGQGLEWMGGIIPIFASA QQLPGTAPKLLIYSNNQRPSG NYAQKFQGRVTITADESTSTA VPDRFSGSKSGTSASLAISGL YMDLSSLRSDDTAVYYCAKNV QSEDEADYYCAAWDDSLSGHY SPKSYSGRYSISYFYGVDVWG VFGTGTKVTVLGQPKANPTVT QGTTVTVSSA LFPPS (SEQ.ID. NO: 1) (SEQ. ID. NO: 2) 77-A6 TGVHSEVQLLESGGGLVQPGG TGSWAQSALTQPPSVSAAPGQ SLRLSCADSGLTFSSYAMSWV KVTISCSGSSSNIGNNYVSWY RQAPGKGLEWVSAISVSGGST QHLPGTAPKLLIYDNIMRPSG YYSDSVKGRFTISRDNSKNTL IPDRFSGSKSGTSATLGITGL SLQMNSLRAEDTAVYYCVKAR QTGDEADYYCGTWDTSLSAW SIVGPFDYWGQGTLVTVSSAS FGGGTKLTVLGQPKAAPSVTL (SEQ. ID. NO: 3) FPPS (SEQ. ID. NO: 4) 90-A3 TGVHSQVQLVQSGAEVKKPGA TGSNSQAVVTQPPSVSAAPGQ SVKVSCMTSGYTFTSYGISWV KVTISCSGSSSNIGNNYVSWY RQAPGQGLEWMGWISAYNGNT QQLPGTAPKLLIYDNNKRPSG NYAQKLQGRVTMTTDTSTSTA IPDRFSGSKSGTSATLGITGL YMELSSLRSDDTAVYYCARDR QTGDEADYYCGTWDSSLSAW GAYWDCGGDCYLSAFDYWGQG FGGGTKLTVLGQPKAAPSVTL TLVTVSSAS FPPS (SEQ. ID. NO: 5) (SEQ. ID. NO: 6) 90-A9 TGVHSQVQLVQSGAEVKKPGS TGVHSEIVLTQSPGTLSL SVKVSCKASGGTFSSFPISWV SPGERATLSCRASQSVAS RQAPGQGLEWMGGIIPIFGAA SYLAWYQQKPGQAPRLLI NYAQKFQGRVTITADESTSTA YGTSSRATGIPDRFSGSG YMELSSLRSEDTAVYYCAENY SGADFTLTISRLEPEDFA EEIWIPAIMNFGYWGQGTLVT VYYCQQYGTSPLTFGGGT VSSAS KVEIK (SEQ. ID. NO: 7) (SEQ. ID. NO: 8) 90-B2 TGVHSQVQLQESGPGLVKPSE TGVHSEIVLTQSPATLSL TLSLTCTVSGGSISSSSYYWG SPGGRATLSCRASQSVSR WIRQPPGKGLEWIGSIYYSGN YLAWYQQKPGRAPRLLIY TYYNPSLKSRVTISVDTSKNK DASNRAPGIPARFSGSGS FSLRLSSVTAADTAVYFCARS GTDFTLTISSLEPEDFAV LPHYDSTGYLLYWGQGTLVTV YYCQQRSNWPQTTFGPGT SSAS KVDIKRT (SEQ. ID. NO: 9) (SEQ. ID. NO: 10) 90-B7 TGVHSEVQLLESGGGLVKPGG TGSNSQAVVTQPPSVSAAPGQ SLRLSCAASGFTFGSYSMTWV KVTISCSGSSSNIGNNYVSWY RQAPGKGLEWVSSISSSSSYI QQLPGTAPKLLIYDNNKRPSG YYADSVKGRFTISRDNAKNSL IPDRFSGSKSGTSATLGITGL FLQMNSLRAEDTAVYYCARGN QTGDEADYYCGTWDSSLSAFV GYCSHNSCYKIGVWFDPWGQG FGTGTKVTVLGQPKANPTVTL TLVTVSSAS FPPS (SEQ. ID. NO: 11) (SEQ. ID. NO: 12) 90-C4 TGVHSQVQLQESGPGLVKPSG TGSWAQSALTQPPSVSGAPGQ TLSLTCAVSGGSINSSNWWSW RVTISCTGSSSSIGAGYDVHW VRQPPGKGLEWIGEIYYSGST YQQLPGTAPKLLIYGNSNRPS NYNPSLKSRVTTSVDNSKNQF GVPDRFSGSKSGTSASLAITG SLKLSSVTAADTAVYYCATFD LQAEDEADYYCQSYDSSLSGY SGGYNPNWFDPWGQGTLVTVS VFGTGTKVTVLGQPKANPTVT SAS(SEQ. ID. NO: 13) LFPPS (SEQ. ID. NO: 14) 90-E5 TGVHSQVQLVQSGAEVKKPGS TGSWAQSVLTQPPSVSGAPGQ SVKVSCKASGGTFSSYTINWV RVTISCTGSSSNIGAGYDVHW RQAPGQGLEWMGGIIPIFGTA YQQLPGTAPKVLIYGNSNRPS NYAQKFQGRVTITADASTSTA GVPDRFSGSKSDTSASLAITG YMELSSLRSEDTAVYYCARVL LQAEDEADYYCQSYDSSLSW LRSSSWFSSNWFDPWGQGTLV FGGGTKLTVLGQPKAAPSVTL TVSSAS FPPS (SEQ. ID. NO: 15) (SEQ. ID. NO: 16) 90-E6 TGVHSEVQLVESGGGLVQPGR TGSWAQSVLTQPPSVSAAPGQ SLRLSCAASGFTFDDHAMHWV KVTISCSGSRSNIGNNYVSWY RQAPGKGLEWVSGFSWNSGSI QQLPGTAPKLLIYDNNKRPSG GYADSVKGRFTISRDNAKNSL IPDRFSGSKSGTSATLGITGL YLQMNSLRAEDTALYYCAKDR QTGDEADYYCGTWDSSLNAGV RSDYYFYGMDVWGQGTTVTVS FGGGTKLTVLGQPKAAPSVTL SAS FPPS (SEQ. ID. NO: 17) (SEQ. ID. NO: 18) 90-F1 TGVHSQVQLVQSGAEVKRPGS TGVHSEIVLTQSPATLSL SVKVSCKTSGGTFNNNAINWV SPGERATLSCGASQSVSS RQAPGQGLEWMGGIIPFFGIA SYLAWYQQKPGLAPRLLI KYAQKFQGRVTITADESTSTA YDASSRATGIPDRFSGSG YMELSSLRSEDTAVYYCARDL SGTDFTLTISRLEREDFA PRESSYGSGSYYTHYYAMDVW VYYCQQYGSSPLTFGGGT GQGTTVTVSSAS KVEIKRT (SEQ. ID. NO: 19) (SEQ. ID. NO: 20) 90-F2 TGVHSQVQLVQSGAEVKKPGA TGVHSDIQMTQSPSTLSA SVKVSCKASGYTFITYYMHWV SVGDRVTITCRASQTIST RQAPGQGLEWMGIINPSGGST WLAWYQQKPGKAPKLLIY SYAQKFQGRVTMTRDTSTSTV KASSLESGVPSRFSGSGS YMELSSLRSEDTAVYYCARGA GTEFTLTISSLQPDDFAT VWILDYWGQGTLVTVSSAS YYCQQYNSYSYTFGQGTK (SEQ. ID. NO: 21) LEIKRT (SEQ. ID. NO: 22)

[0171] As a result, as shown in FIG. 1, among the 24 antibodies, 77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies demonstrated excellent adhesion to MERS-CoV full spike trimer antigen, which was greater than that of A2, A10, and G4 antibodies (FIG. 1). CDR sequences of 11 kinds of the antibodies demonstrating excellent adhesion to the full spike trimer antigen are shown in Table 1.

<1-2> Investigation of Antibody Adhesion to MERS-CoV S1 Antigen

[0172] The binding affinity of 24 kinds of the antibodies prepared in Example 1 was investigated by the same manner as described in Experimental Example 1-1 except that the MERS-CoV S1 domain composed of amino acids 1 to 757 from the N-terminus of the spike protein was used as an antigen.

[0173] As a result, as shown in FIG. 2, among the 24 antibodies, 77-A5, 77-A6, 90-A3, 90-A9, 90-B2, 90-B7, 90-C4, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies demonstrated excellent adhesion to the S1 antigen, which was greater than that of the A2 and A10 antibodies developed by NIH, USA. In particular, 90-F1, 90-B7, 90-E6, 77-A4, 77-A5, 90-F2, and 77-A6 antibodies demonstrated higher adhesion than that of C2 and G2 antibodies (FIG. 2).

<1-3> Investigation of Antibody Adhesion to MERS-CoV RBD Antigen

[0174] The binding affinity of 24 kinds of the antibodies prepared in Example 1 was investigated by the same manner as described in Experimental Example 1-1 except that the MERS-CoV RBD (receptor binding domain) composed of amino acids 377 to 588 from the N-terminus of the spike protein was used as an antigen.

[0175] As a result, as shown in FIG. 3, among those 24 antibodies, 90-F1, 90-E5, 90-E6, 90-F2, 77-A5, and 77-A6 antibodies showed higher adhesion to the RBD antigen than that of C2 antibody. The rest of those antibodies did not show adhesion to the RBD antigen (FIG. 3).

<1-4> Investigation of Antibody Adhesion to MERS-CoV S2 Antigen

[0176] The binding affinity of 24 kinds of the antibodies prepared in Example 1 was investigated by the same manner as described in Experimental Example 1-1 except that the MERS-CoV S2 domain composed of amino acids 757 to 1275 from the N-terminus of the spike protein was used as an antigen.

[0177] As a result, as shown in FIG. 4, all of those 24 antibodies prepared in Example 1 showed very low adhesion to the S2 antigen (FIG. 4).

Experimental Example 2: Investigation of Neutralizing Capacity of Antibody Against MERS Pseudovirus

[0178] Huh7.5 cell line was treated with trypsin and suspended. The cells were diluted with DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS, which were distributed in a 96-well tissue culture late at the density of 10.sup.4 cells/100 .mu./well, followed by culture for a day. In the meantime, 90 .mu. of the supernatant of recombinant lentivirus (recombinant MRES pseudovirus expressing luciferase, NIH/NIAID/VRC, USA) containing MERS-CoV spike protein (Erasmus strain or Bisha 1 strain) and 90 .mu. of the antibody of Example 1 diluted in cell culture medium at the concentration of 10, 1, 0.1, 0.01, or 0,001 .mu.s/m were mixed with DMEM containing 10% FBS, followed by reaction at 37.degree. C. for 45 minutes, resulting in the preparation of a reaction mixture. The mixture was added to the culture plate (50 .mu./well), to which DMEM containing 10% FBS was added (100 .mu./well), followed by culture in a 37.degree. C., 5% CO.sub.2 incubator for 3 days. After discarding the culture fluid, 1.times. lysis buffer (Promega, E153A) was added therein at the concentration of 25 .mu./well, followed by stirring for 15 minutes to lyse the cells. The lysed cells were added with luciferase substrate reagent (Promega, E151A) (50 .mu./well). Fluorescence was measured with a luminometer and neutralizing capacity was calculated by the following mathematical formula 1.

Neutralizing Capacity (%)=100-(luciferase signal mean value of antibody-treated pseudovirus/background signal of antibody-non-treated pseudovirus.times.100) [Mathematical Formula 1]

TABLE-US-00002 TABLE 2 Neutralizing capacity of 11 antibodies Antibody Neutralizing Capacity (%) Conc. (.mu.g/ml) 77-A5 77-A6 90-A3 90-A9 90-B2 10 95.93 99.04 -29.25 -9.72 -152.78 2.5 92.87 99.53 5.01 31.92 -71.75 0.625 67.05 97.96 22.00 18.73 -59.75 0.15625 64.10 92.04 28.44 15.01 -59.65 0.039063 3.16 56.75 2.43 -6.41 -40.74 0.009766 28.89 41.59 -15.18 -1.78 -44.11 0.002441 -3.78 3.81 6.57 -18.34 -25.61 Virus alone 0.00 0.00 0.00 0.00 Antibody Neutralizing Capacity (%) Conc. (.mu.g/ml) 90-B7 90-C4 90-E5 90-E6 90-F1 90-F2 10 -49.92 20.88 91.00 58.10 99.83 98.17 2.5 -9.19 28.48 79.40 66.30 99.65 94.39 0.625 -32.26 -3.31 61.12 44.77 98.55 86.01 0.15625 -30.39 17.46 46.94 41.31 98.56 59.63 0.039063 -21.07 2.43 38.62 41.83 88.45 48.10 0.009766 0.10 -23.37 37.07 10.61 69.87 21.76 0.002441 -9.71 -7.53 38.20 36.45 54.92 48.40 Virus alone 0.00 0.00 15.11 26.03 0.00 0.00

[0179] As a result, as shown in FIG. 5a, FIG. 5b and Table 2, 77-A5, 77-A6, 90-E5, 90-E6, 90-F1, and 90-F2 antibodies demonstrated neutralizing capacity against MERS pseudovirus Erasmus strain. Among them, 77-A5, 77-A6, 90-F1, and 90-F2 antibodies showed neutralizing capacity against MERS pseudovirus Bisha 1 strain as well. 90-F1 antibody displayed the highest neutralizing capacity against MERS pseudovirus Erasmus strain and Bisha 1 strain (FIG. 5a, FIG. 5b and Table 2). Therefore, it was confirmed that 77-A5, 77-A6, 90-F1, and 90-F2 antibodies had a wide range of neutralizing capacity.

Experimental Example 3: Investigation of Neutralizing Capacity of Antibody Against MERS-CoV

[0180] To investigate neutralizing capacity of the antibodies prepared in Example 1, plaque reduction neutralizing test (PRNT) was performed with MERS-CoV (Erasmus strain and KNIH strain). First, Vero cells were distributed in a 24-well plate at the density of 10.sup.5 cells/well, followed by culture. The culture medium was discarded therefrom. The antibodies of Example 1 diluted in cell culture medium at the concentration of 10, 2.5, 0.625, 0.156, 0.039, 0.009, or 0.002 ng/m were mixed with the MERS-CoV (EMC, KNIH-002, KNIH-016, and KNIH-042 strains) diluted in 50 PFU/well, resulting in the preparation of a mixed solution. The mixed solution was added to the culture plate at the concentration of 100 .mu./well, followed by reaction at 37.degree. C. for 1 hour. Then, DMEM containing 1.5% CMC (carboxymethyl cellulose) was added to the plate at the concentration of 1 m/well, followed by culture for 3 days. Upon completion of the culture, plaques were observed by crystal violet staining. The number of plaques in each well was calculated, based on which neutralizing capacity was calculated by the following mathematical formula 2. ND.sub.50 was also calculated by the following mathematical formula 3 to calculate the neutralizing capacity. In the mathematical formula 3, m indicates a log.sub.10 value of the highest rate of dilution, .DELTA. indicates a constant interval between the rates of dilution expressed by a log.sub.10 value, and .SIGMA.p indicates the average number of plaques for sum of all ratios of plaques/virus control.

Neutralizing Capacity (%)=100-(plaque number of antibody-treated MERS virus/plaque number of antibody-non-treated MERS virus.times.100) [Mathematical Formula 2]

log.sub.10.sup.ND.sup.50=m-.DELTA.(.SIGMA.p-0.5) [Mathematical Formula 3]

TABLE-US-00003 TABLE 3 Neutralizing capacity of antibodies analyzed by PRNT Antibody ND.sub.50 ND.sub.50 antibody conc. (.mu.g/ml) 77-A5 35.60 0.28 77-A6 95.73 0.10 90-A3 0.63 15.81 90-A9 0.58 17.30 90-B2 39.52 0.25 90-B7 14.93 0.67 90-C4 0.78 12.76 90-E5 6.60 1.51 90-E6 0.68 14.78 90-F1 956.46 0.01 90-F2 53.96 0.19 Negative Control 1.00 10.04

[0181] As a result, as shown in FIG. 6a, FIG. 6b and Table 3, 77-A5, 77-A6, 90-B2, 90-B7, 90-F1, 90-F2, and 90-E5 antibodies demonstrated neutralizing capacity against MERS-CoV. Among them, 90-F1 antibody showed the highest neutralizing capacity (FIG. 6a, FIG. 6b and Table 3). The results were the same for all MERS-CoV strains (EMC, KNIH-002, KNIH-016, and KNIH-042 strains).

Experimental Example 4: Physicochemical Characterization of Antibodies

[0182] The physicochemical characteristics of the antibodies 77-A5, 77-A6, 90-B2, 90-B7, 90-F1, and 90-F2, demonstrating excellent neutralizing capacity in Experimental Examples 2 and 3, were analyzed by SEC-HPLC (size exclusion chromatography-HPLC), SPR assay (surface plasmon resonance assay) and PTS assay (protein thermal shift assay).

<4-1> Analysis of Antibody Morphology by SEC-HPLC

[0183] The antibody morphology was analyzed by using water SEC-HPLC system. Agilent Bio SEC3 was used as the HPLC column and ultraviolet absorption spectrophotometer (280 nm) was used as the detector. At this time, 10 .mu. of each antibody was loaded as the sample at the concentration of 1 mg/m. The flow rate was 0.3 m/min. 1.times.PBS was used as a moving phase for isocratic elution.

[0184] As a result, as shown in FIG. 7, 6 kinds of antibodies were in the form of a certain monomer, and coagulation was not observed (FIG. 7).

<4-2> Analysis of Antibody Avidity by SPR Assay

[0185] The avidity of the antibodies was analyzed by SPR assay using S1 antigen or RBD antigen. Particularly, S1 antigen or RBD antigen was attached on the surface of CM5 chip, to which the antibody was loaded. The association time was 3 minutes and the dissociation time was as listed in Table 4. HBS-EP was used as a running buffer, and regeneration solution was as shown in Table 4 for SPR assay.

TABLE-US-00004 TABLE 4 SPR assay conditions Dissociation Antibody Time Regeneration Solution 77-A5 30 min. 10 mM NaOH, 1M NaCl 77-A6 6 min. 10 mM NaOH 90-B2 30 min. 10 mM NaOH, 1M NaCl 90-B7 10 min. 10 mM NaOH, 250 mM NaCl 90-F1 10 min. 10 mM glycine(pH 2.0), 250 mM NaCl 90-F2 6 min. 10 mM NaOH, 500 mM NaCl

TABLE-US-00005 TABLE 5 Analysis of antibody avidity by SPR assay Characteristic 77-A5 77-A6 90-B2 90-B7 90-F1 90-F2 Avidity to S1 antigen K.sub.a(1/Ms) 1.33 .times. 10.sup.5 6.38 .times. 10.sup.4 1.31 .times. 10.sup.5 6.81 .times. 10.sup.5 6.48 .times. 10.sup.5 1.34 .times. 10.sup.5 K.sub.d(1/s) .ltoreq.1.00 .times. 10.sup.-5 4.43 .times. 10.sup.-5 .ltoreq.1.00 .times. 10.sup.-5 1.04 .times. 10.sup.-4 9.88 .times. 10.sup.-5 8.82 .times. 10.sup.-4 K.sub.D(M) .ltoreq.7.51 .times. 10.sup.-11 .sup. 6.95 .times. 10.sup.-10 .ltoreq.7.51 .times. 10.sup.-11 .sup. 1.53 .times. 10.sup.-10 .sup. 1.53 .times. 10.sup.-10 6.60 .times. 10.sup.-9 Avidity to RBD antigen K.sub.a(1/Ms) 1.30 .times. 10.sup.5 4.67 .times. 10.sup.4 -- -- 3.76 .times. 10.sup.5 2.38 .times. 10.sup.5 K.sub.d(1/s) .sup. 2.08 .times. 10.sup.-5 3.15 .times. 10.sup.-5 -- -- 2.71 .times. 10.sup.-5 1.40 .times. 10.sup.-5 K.sub.D(M) .sup. 1.61 .times. 10.sup.-10 .sup. 6.73 .times. 10.sup.-10 -- -- .sup. 7.20 .times. 10.sup.-11 5.86 .times. 10.sup.-9

[0186] As a result, as shown in FIG. 8a, FIG. 8b and Table 5, the antibodies 77-A5 and 90-B2 attached with S1 antigen showed a high avidity to the S1 antigen, confirmed by the low Kr, value of up to 75 pM. The antibody 90-F1 attached with RBD antigen showed a high avidity to the RBD antigen, confirmed by the low K.sub.D value of up to 72 pM. In particular, 90-B2 and 90-B7 antibodies did not bind to the RBD antigen, indicating that the 90-B2 and 90-B7 antibodies were specific to the S1 antigen (FIG. 8a, FIG. 8b and Table 5).

<4-3> Investigation of Antibody Stability by PTS Assay

[0187] The antibody stability was investigated in PBS (phosphate buffered saline, pH 7.4) which included neither calcium nor magnesium. Particularly, 0.75 .mu.g of each antibody prepared in Example 1 was used. PTS assay was performed by using Protein Thermal Shift.TM. Dye Kit (Catalog No. 4466038, Life technologies) according to the manufacturer's protocol.

[0188] As a result, as shown in FIG. 9a to FIG. 9c, the melting points of 77-A5, 77-A6, 90-B2, 90-B7, 90-F1, and 90-F2 antibodies were respectively 67.52.degree. C., 68.44.degree. C. and 75.44.degree. C., 72.46.degree. C., 68.21.degree. C. and 81.19.degree. C., 69.21.degree. C. and 79.81.degree. C., and 68.89.degree. C. and 75.10.degree. C. Therefore, all of the six antibodies were confirmed to have a stable structure because the protein hydrophobicity was exposed at the temperature of 67.52.degree. C. or higher (FIG. 9a to FIG. 9c).

[0189] Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Sequence CWU 1

1

231136PRTHomo sapiens 1Thr Gly Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val1 5 10 15Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly 20 25 30Thr Phe Arg Ser His Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln 35 40 45Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Ala Ser Ala Asn 50 55 60Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser65 70 75 80Thr Ser Thr Ala Tyr Met Asp Leu Ser Ser Leu Arg Ser Asp Asp Thr 85 90 95Ala Val Tyr Tyr Cys Ala Lys Asn Val Ser Pro Lys Ser Tyr Ser Gly 100 105 110Arg Tyr Ser Ile Ser Tyr Phe Tyr Gly Val Asp Val Trp Gly Gln Gly 115 120 125Thr Thr Val Thr Val Ser Ser Ala 130 1352131PRTHomo sapiens 2Thr Gly Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser1 5 10 15Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser 20 25 30Asn Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr 35 40 45Ala Pro Lys Leu Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala65 70 75 80Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala 85 90 95Trp Asp Asp Ser Leu Ser Gly His Tyr Val Phe Gly Thr Gly Thr Lys 100 105 110Val Thr Val Leu Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe 115 120 125Pro Pro Ser 1303126PRTHomo sapiens 3Thr Gly Val His Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu1 5 10 15Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Asp Ser Gly Leu 20 25 30Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys 35 40 45Gly Leu Glu Trp Val Ser Ala Ile Ser Val Ser Gly Gly Ser Thr Tyr 50 55 60Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser65 70 75 80Lys Asn Thr Leu Ser Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 85 90 95Ala Val Tyr Tyr Cys Val Lys Ala Arg Ser Ile Val Gly Pro Phe Asp 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 115 120 1254130PRTHomo sapiens 4Thr Gly Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser1 5 10 15Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser 20 25 30Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln His Leu Pro Gly Thr 35 40 45Ala Pro Lys Leu Leu Ile Tyr Asp Asn Ile Met Arg Pro Ser Gly Ile 50 55 60Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly65 70 75 80Ile Thr Gly Leu Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr 85 90 95Trp Asp Thr Ser Leu Ser Ala Val Val Phe Gly Gly Gly Thr Lys Leu 100 105 110Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro 115 120 125Pro Ser 1305135PRTHomo sapiens 5Thr Gly Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val1 5 10 15Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Met Thr Ser Gly Tyr 20 25 30Thr Phe Thr Ser Tyr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln 35 40 45Gly Leu Glu Trp Met Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn 50 55 60Tyr Ala Gln Lys Leu Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser65 70 75 80Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr 85 90 95Ala Val Tyr Tyr Cys Ala Arg Asp Arg Gly Ala Tyr Trp Asp Cys Gly 100 105 110Gly Asp Cys Tyr Leu Ser Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu 115 120 125Val Thr Val Ser Ser Ala Ser 130 1356130PRTHomo sapiens 6Thr Gly Ser Asn Ser Gln Ala Val Val Thr Gln Pro Pro Ser Val Ser1 5 10 15Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser 20 25 30Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr 35 40 45Ala Pro Lys Leu Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile 50 55 60Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly65 70 75 80Ile Thr Gly Leu Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr 85 90 95Trp Asp Ser Ser Leu Ser Ala Val Val Phe Gly Gly Gly Thr Lys Leu 100 105 110Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro 115 120 125Pro Ser 1307132PRTHomo sapiens 7Thr Gly Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val1 5 10 15Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly 20 25 30Thr Phe Ser Ser Phe Pro Ile Ser Trp Val Arg Gln Ala Pro Gly Gln 35 40 45Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Ala Ala Asn 50 55 60Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser65 70 75 80Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 85 90 95Ala Val Tyr Tyr Cys Ala Arg Asn Tyr Glu Glu Ile Val Val Ile Pro 100 105 110Ala Ile Met Asn Phe Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 115 120 125Ser Ser Ala Ser 1308113PRTHomo sapiens 8Thr Gly Val His Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu1 5 10 15Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln 20 25 30Ser Val Ala Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Ala Pro Arg Leu Leu Ile Tyr Gly Thr Ser Ser Arg Ala Thr Gly Ile 50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Thr65 70 75 80Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 85 90 95Tyr Gly Thr Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110Lys9130PRTHomo sapiens 9Thr Gly Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu1 5 10 15Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly 20 25 30Ser Ile Ser Ser Ser Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro 35 40 45Gly Lys Gly Leu Glu Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Asn Thr 50 55 60Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr65 70 75 80Ser Lys Asn His Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp 85 90 95Thr Ala Val Tyr Phe Cys Ala Arg Ser Leu Pro His Tyr Asp Ser Thr 100 105 110Gly Tyr Leu Leu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125Ala Ser 13010115PRTHomo sapiens 10Thr Gly Val His Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu1 5 10 15Ser Leu Ser Pro Gly Gly Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln 20 25 30Ser Val Ser Arg Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Arg Ala 35 40 45Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Pro Gly Ile Pro 50 55 60Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile65 70 75 80Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg 85 90 95Ser Asn Trp Pro Gln Thr Thr Phe Gly Pro Gly Thr Lys Val Asp Ile 100 105 110Lys Arg Thr 11511135PRTHomo sapiens 11Thr Gly Val His Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu1 5 10 15Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 20 25 30Thr Phe Gly Ser Tyr Ser Met Thr Trp Val Arg Gln Ala Pro Gly Lys 35 40 45Gly Leu Glu Trp Val Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr 50 55 60Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala65 70 75 80Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 85 90 95Ala Val Tyr Tyr Cys Ala Arg Gly Asn Gly Tyr Cys Ser His Asn Ser 100 105 110Cys Tyr Lys Ile Gly Val Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu 115 120 125Val Thr Val Ser Ser Ala Ser 130 13512130PRTHomo sapiens 12Thr Gly Ser Asn Ser Gln Ala Val Val Thr Gln Pro Pro Ser Val Ser1 5 10 15Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser 20 25 30Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr 35 40 45Ala Pro Lys Leu Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile 50 55 60Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly65 70 75 80Ile Thr Gly Leu Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr 85 90 95Trp Asp Ser Ser Leu Ser Ala Phe Val Phe Gly Thr Gly Thr Lys Val 100 105 110Thr Val Leu Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro 115 120 125Pro Ser 13013129PRTHomo sapiens 13Thr Gly Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu1 5 10 15Val Lys Pro Ser Gly Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly 20 25 30Ser Ile Asn Ser Ser Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly 35 40 45Lys Gly Leu Glu Trp Ile Gly Glu Ile Tyr Tyr Ser Gly Ser Thr Asn 50 55 60Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Thr Ser Val Asp Asn Ser65 70 75 80Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr 85 90 95Ala Val Tyr Tyr Cys Ala Thr Phe Asp Ser Gly Gly Tyr Asn Pro Asn 100 105 110Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 125Ser14131PRTHomo sapiens 14Thr Gly Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser1 5 10 15Gly Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser 20 25 30Ser Ile Gly Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly 35 40 45Thr Ala Pro Lys Leu Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly 50 55 60Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu65 70 75 80Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln 85 90 95Ser Tyr Asp Ser Ser Leu Ser Gly Tyr Val Phe Gly Thr Gly Thr Lys 100 105 110Val Thr Val Leu Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe 115 120 125Pro Pro Ser 13015132PRTHomo sapiens 15Thr Gly Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val1 5 10 15Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly 20 25 30Thr Phe Ser Ser Tyr Thr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln 35 40 45Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn 50 55 60Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Ala Ser65 70 75 80Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 85 90 95Ala Val Tyr Tyr Cys Ala Arg Val Leu Leu Arg Ser Ser Ser Trp Phe 100 105 110Ser Ser Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val 115 120 125Ser Ser Ala Ser 13016130PRTHomo sapiens 16Thr Gly Ser Trp Ala Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser1 5 10 15Gly Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser 20 25 30Asn Ile Gly Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly 35 40 45Thr Ala Pro Lys Val Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly 50 55 60Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Asp Thr Ser Ala Ser Leu65 70 75 80Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln 85 90 95Ser Tyr Asp Ser Ser Leu Ser Val Val Phe Gly Gly Gly Thr Lys Leu 100 105 110Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro 115 120 125Pro Ser 13017129PRTHomo sapiens 17Thr Gly Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu1 5 10 15Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 20 25 30Thr Phe Asp Asp His Ala Met His Trp Val Arg Gln Ala Pro Gly Lys 35 40 45Gly Leu Glu Trp Val Ser Gly Phe Ser Trp Asn Ser Gly Ser Ile Gly 50 55 60Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala65 70 75 80Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 85 90 95Ala Leu Tyr Tyr Cys Ala Lys Asp Arg Arg Ser Asp Tyr Tyr Phe Tyr 100 105 110Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 115 120 125Ser18130PRTHomo sapiens 18Thr Gly Ser Trp Ala Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser1 5 10 15Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Arg Ser 20 25 30Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr 35 40 45Ala Pro Lys Leu Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile 50 55 60Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly65 70 75 80Ile Thr Gly Leu Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr 85 90 95Trp Asp Ser Ser Leu Asn Ala Gly Val Phe Gly Gly Gly Thr Lys Leu 100 105 110Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro 115 120 125Pro Ser 13019138PRTHomo sapiens 19Thr Gly Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val1 5 10 15Lys Arg Pro Gly Ser Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly 20 25 30Thr Phe Asn Asn Asn Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln 35 40 45Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Phe Phe Gly Ile Ala Lys 50 55 60Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser65 70 75 80Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 85 90 95Ala Val Tyr Tyr Cys Ala Arg Asp Leu Pro Arg Glu Ser Ser Tyr Gly 100 105

110Ser Gly Ser Tyr Tyr Thr His Tyr Tyr Ala Met Asp Val Trp Gly Gln 115 120 125Gly Thr Thr Val Thr Val Ser Ser Ala Ser 130 13520115PRTHomo sapiens 20Thr Gly Val His Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu1 5 10 15Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Gly Ala Ser Gln 20 25 30Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Leu 35 40 45Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Ile 50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 85 90 95Tyr Gly Ser Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110Lys Arg Thr 11521125PRTHomo sapiens 21Thr Gly Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val1 5 10 15Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 20 25 30Thr Phe Thr Thr Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln 35 40 45Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser 50 55 60Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser65 70 75 80Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 85 90 95Ala Val Tyr Tyr Cys Ala Arg Gly Ala Val Val Val Ile Leu Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 115 120 12522114PRTHomo sapiens 22Thr Gly Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu1 5 10 15Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 20 25 30Thr Ile Ser Thr Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45Pro Lys Leu Leu Ile Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro 50 55 60Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile65 70 75 80Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr 85 90 95Asn Ser Tyr Ser Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg Thr231354PRTUnknownmiddle east respiratory syndrome coronavirus spike proteinmisc_feature(1354)..(1354)Xaa can be any naturally occurring amino acid 23Met Ile His Ser Val Phe Leu Leu Met Phe Leu Leu Thr Pro Thr Glu1 5 10 15Ser Tyr Val Asp Val Gly Pro Asp Ser Val Lys Ser Ala Cys Ile Glu 20 25 30Val Asp Ile Gln Gln Thr Phe Phe Asp Lys Thr Trp Pro Arg Pro Ile 35 40 45Asp Val Ser Lys Ala Asp Gly Ile Ile Tyr Pro Gln Gly Arg Thr Tyr 50 55 60Ser Asn Ile Thr Ile Thr Tyr Gln Gly Leu Phe Pro Tyr Gln Gly Asp65 70 75 80His Gly Asp Met Tyr Val Tyr Ser Ala Gly His Ala Thr Gly Thr Thr 85 90 95Pro Gln Lys Leu Phe Val Ala Asn Tyr Ser Gln Asp Val Lys Gln Phe 100 105 110Ala Asn Gly Phe Val Val Arg Ile Gly Ala Ala Ala Asn Ser Thr Gly 115 120 125Thr Val Ile Ile Ser Pro Ser Thr Ser Ala Thr Ile Arg Lys Ile Tyr 130 135 140Pro Ala Phe Met Leu Gly Ser Ser Val Gly Asn Phe Ser Asp Gly Lys145 150 155 160Met Gly Arg Phe Phe Asn His Thr Leu Val Leu Leu Pro Asp Gly Cys 165 170 175Gly Thr Leu Leu Arg Ala Phe Tyr Cys Ile Leu Glu Pro Arg Ser Gly 180 185 190Asn His Cys Pro Ala Gly Asn Ser Tyr Thr Ser Phe Ala Thr Tyr His 195 200 205Thr Pro Ala Thr Asp Cys Ser Asp Gly Asn Tyr Asn Arg Asn Ala Ser 210 215 220Leu Asn Ser Phe Lys Glu Tyr Phe Asn Leu Arg Asn Cys Thr Phe Met225 230 235 240Tyr Thr Tyr Asn Ile Thr Glu Asp Glu Ile Leu Glu Trp Phe Gly Ile 245 250 255Thr Gln Thr Ala Gln Gly Val His Leu Phe Ser Ser Arg Tyr Val Asp 260 265 270Leu Tyr Gly Gly Asn Met Phe Gln Phe Ala Thr Leu Pro Val Tyr Asp 275 280 285Thr Ile Lys Tyr Tyr Ser Ile Ile Pro His Ser Ile Arg Ser Ile Gln 290 295 300Ser Asp Arg Lys Ala Trp Ala Ala Phe Tyr Val Tyr Lys Leu Gln Pro305 310 315 320Leu Thr Phe Leu Leu Asp Phe Ser Val Asp Gly Tyr Ile Arg Arg Ala 325 330 335Ile Asp Cys Gly Phe Asn Asp Leu Ser Gln Leu His Cys Ser Tyr Glu 340 345 350Ser Phe Asp Val Glu Ser Gly Val Tyr Ser Val Ser Ser Phe Glu Ala 355 360 365Lys Pro Ser Gly Ser Val Val Glu Gln Ala Glu Gly Val Glu Cys Asp 370 375 380Phe Ser Pro Leu Leu Ser Gly Thr Pro Pro Gln Val Tyr Asn Phe Lys385 390 395 400Arg Leu Val Phe Thr Asn Cys Asn Tyr Asn Leu Thr Lys Leu Leu Ser 405 410 415Leu Phe Ser Val Asn Asp Phe Thr Cys Ser Gln Ile Ser Pro Ala Ala 420 425 430Ile Ala Ser Asn Cys Tyr Ser Ser Leu Ile Leu Asp Tyr Phe Ser Tyr 435 440 445Pro Leu Ser Met Lys Ser Asp Leu Ser Val Ser Ser Ala Gly Pro Ile 450 455 460Ser Gln Phe Asn Tyr Lys Gln Ser Phe Ser Asn Pro Thr Cys Leu Ile465 470 475 480Leu Ala Thr Val Pro His Asn Leu Thr Thr Ile Thr Lys Pro Leu Lys 485 490 495Tyr Ser Tyr Ile Asn Lys Cys Ser Arg Leu Leu Ser Asp Asp Arg Thr 500 505 510Glu Val Pro Gln Leu Val Asn Ala Asn Gln Tyr Ser Pro Cys Val Ser 515 520 525Ile Val Pro Ser Thr Val Trp Glu Asp Gly Asp Tyr Tyr Arg Lys Gln 530 535 540Leu Ser Pro Leu Glu Gly Gly Gly Trp Leu Val Ala Ser Gly Ser Thr545 550 555 560Val Ala Met Thr Glu Gln Leu Gln Met Gly Phe Gly Ile Thr Val Gln 565 570 575Tyr Gly Thr Asp Thr Asn Ser Val Cys Pro Lys Leu Glu Phe Ala Asn 580 585 590Asp Thr Lys Ile Ala Ser Gln Leu Gly Asn Cys Val Glu Tyr Ser Leu 595 600 605Tyr Gly Val Ser Gly Arg Gly Val Phe Gln Asn Cys Thr Ala Val Gly 610 615 620Val Arg Gln Gln Arg Phe Val Tyr Asp Ala Tyr Gln Asn Leu Val Gly625 630 635 640Tyr Tyr Ser Asp Asp Gly Asn Tyr Tyr Cys Leu Arg Ala Cys Val Ser 645 650 655Val Pro Val Ser Val Ile Tyr Asp Lys Glu Thr Lys Thr His Ala Thr 660 665 670Leu Phe Gly Ser Val Ala Cys Glu His Ile Ser Ser Thr Met Ser Gln 675 680 685Tyr Ser Arg Ser Thr Arg Ser Met Leu Lys Arg Arg Asp Ser Thr Tyr 690 695 700Gly Pro Leu Gln Thr Pro Val Gly Cys Val Leu Gly Leu Val Asn Ser705 710 715 720Ser Leu Phe Val Glu Asp Cys Lys Leu Pro Leu Gly Gln Ser Leu Cys 725 730 735Ala Leu Pro Asp Thr Pro Ser Thr Leu Thr Pro Arg Ser Val Arg Ser 740 745 750Val Pro Gly Glu Met Arg Leu Ala Ser Ile Ala Phe Asn His Pro Ile 755 760 765Gln Val Asp Gln Leu Asn Ser Ser Tyr Phe Lys Leu Ser Ile Pro Thr 770 775 780Asn Phe Ser Phe Gly Val Thr Gln Glu Tyr Ile Gln Thr Thr Ile Gln785 790 795 800Lys Val Thr Val Asp Cys Lys Gln Tyr Val Cys Asn Gly Phe Gln Lys 805 810 815Cys Glu Gln Leu Leu Arg Glu Tyr Gly Gln Phe Cys Ser Lys Ile Asn 820 825 830Gln Ala Leu His Gly Ala Asn Leu Arg Gln Asp Asp Ser Val Arg Asn 835 840 845Leu Phe Ala Ser Val Lys Ser Ser Gln Ser Ser Pro Ile Ile Pro Gly 850 855 860Phe Gly Gly Asp Phe Asn Leu Thr Leu Leu Glu Pro Val Ser Ile Ser865 870 875 880Thr Gly Ser Arg Ser Ala Arg Ser Ala Ile Glu Asp Leu Leu Phe Asp 885 890 895Lys Val Thr Ile Ala Asp Pro Gly Tyr Met Gln Gly Tyr Asp Asp Cys 900 905 910Met Gln Gln Gly Pro Ala Ser Ala Arg Asp Leu Ile Cys Ala Gln Tyr 915 920 925Val Ala Gly Tyr Lys Val Leu Pro Pro Leu Met Asp Val Asn Met Glu 930 935 940Ala Ala Tyr Thr Ser Ser Leu Leu Gly Ser Ile Ala Gly Val Gly Trp945 950 955 960Thr Ala Gly Leu Ser Ser Phe Ala Ala Ile Pro Phe Ala Gln Ser Ile 965 970 975Phe Tyr Arg Leu Asn Gly Val Gly Ile Thr Gln Gln Val Leu Ser Glu 980 985 990Asn Gln Lys Leu Ile Ala Asn Lys Phe Asn Gln Ala Leu Gly Ala Met 995 1000 1005Gln Thr Gly Phe Thr Thr Thr Asn Glu Ala Phe Gln Lys Val Gln 1010 1015 1020Asp Ala Val Asn Asn Asn Ala Gln Ala Leu Ser Lys Leu Ala Ser 1025 1030 1035Glu Leu Ser Asn Thr Phe Gly Ala Ile Ser Ala Ser Ile Gly Asp 1040 1045 1050Ile Ile Gln Arg Leu Asp Val Leu Glu Gln Asp Ala Gln Ile Asp 1055 1060 1065Arg Leu Ile Asn Gly Arg Leu Thr Thr Leu Asn Ala Phe Val Ala 1070 1075 1080Gln Gln Leu Val Arg Ser Glu Ser Ala Ala Leu Ser Ala Gln Leu 1085 1090 1095Ala Lys Asp Lys Val Asn Glu Cys Val Lys Ala Gln Ser Lys Arg 1100 1105 1110Ser Gly Phe Cys Gly Gln Gly Thr His Ile Val Ser Phe Val Val 1115 1120 1125Asn Ala Pro Asn Gly Leu Tyr Phe Met His Val Gly Tyr Tyr Pro 1130 1135 1140Ser Asn His Ile Glu Val Val Ser Ala Tyr Gly Leu Cys Asp Ala 1145 1150 1155Ala Asn Pro Thr Asn Cys Ile Ala Pro Val Asn Gly Tyr Phe Ile 1160 1165 1170Lys Thr Asn Asn Thr Arg Ile Val Asp Glu Trp Ser Tyr Thr Gly 1175 1180 1185Ser Ser Phe Tyr Ala Pro Glu Pro Ile Thr Ser Leu Asn Thr Lys 1190 1195 1200Tyr Val Ala Pro Gln Val Thr Tyr Gln Asn Ile Ser Thr Asn Leu 1205 1210 1215Pro Pro Pro Leu Leu Gly Asn Ser Thr Gly Ile Asp Phe Gln Asp 1220 1225 1230Glu Leu Asp Glu Phe Phe Lys Asn Val Ser Thr Ser Ile Pro Asn 1235 1240 1245Phe Gly Ser Leu Thr Gln Ile Asn Thr Thr Leu Leu Asp Leu Thr 1250 1255 1260Tyr Glu Met Leu Ser Leu Gln Gln Val Val Lys Ala Leu Asn Glu 1265 1270 1275Ser Tyr Ile Asp Leu Lys Glu Leu Gly Asn Tyr Thr Tyr Tyr Asn 1280 1285 1290Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Val 1295 1300 1305Ala Leu Ala Leu Cys Val Phe Phe Ile Leu Cys Cys Thr Gly Cys 1310 1315 1320Gly Thr Asn Cys Met Gly Lys Leu Lys Cys Asn Arg Cys Cys Asp 1325 1330 1335Arg Tyr Glu Glu Tyr Asp Leu Glu Pro His Lys Val His Val His 1340 1345 1350Xaa

Claims

1. A heavy chain variable region of a monoclonal antibody having binding specificity to Middle East Respiratory Syndrome coronavirus (MERS-CoV) spike protein, wherein the complementarity determining region (CDR) of said heavy chain variable region comprises an amino acid sequence represented by SEQ. ID. NO: 1, SEQ. ID. NO: 3, SEQ. ID. NO: 5, SEQ. ID. NO: 7, SEQ. ID. NO: 9, SEQ. ID. NO: 11, SEQ. ID. NO: 13, SEQ. ID. NO: 15, SEQ. ID. NO: 17, SEQ. ID. NO: 19, or SEQ. ID. NO: 21.

2. The heavy chain variable region according to claim 1, wherein the MERS-CoV spike protein comprises an amino acid sequence represented by SEQ. ID. NO: 23.

3. The heavy chain variable region according to claim 1, wherein the heavy chain variable region has binding specificity to a region of the MERS-CoV spike protein comprising the 1.sup.st to 757.sup.th amino acids from the N-terminus of the MERS-CoV spike protein.

4. A nucleic acid encoding the heavy chain variable region of claim 1.

5. A recombinant vector comprising the nucleic acid of claim 4.

6. A host cell having the nucleic acid of claim 4 introduced therein.

7. A host cell having the recombinant vector of claim 5 introduced therein.

8. A kit for the detection or quantification of an antigen of Middle East respiratory syndrome (MERS), the kit comprising the heavy chain variable region of claim 1.

9. A light chain variable region of a monoclonal antibody having binding specificity to Middle East Respiratory Syndrome coronavirus (MERS-CoV) spike protein, wherein the complementarity determining region (CDR) of said light chain variable region comprises an amino acid sequence represented by SEQ. ID. NO: 2, SEQ. ID. NO: 4, SEQ. ID. NO: 6, SEQ. ID. NO: 8, SEQ. ID. NO: 10, SEQ. ID. NO: 12, SEQ. ID. NO: 14, SEQ. ID. NO: 16, SEQ. ID. NO: 18, SEQ. ID. NO: 20, or SEQ. ID. NO: 22.

10. The light chain variable region according to claim 9, wherein the MERS-CoV spike protein comprises an amino acid sequence represented by SEQ. ID. NO: 23.

11. The light chain variable region according to claim 9, wherein the light chain variable region has binding specificity to a region of the MERS-CoV spike protein comprising the 1.sup.st to 757.sup.th amino acids from the N-terminus of the MERS-CoV spike protein.

12. A nucleic acid encoding the light chain variable region of claim 9.

13. A recombinant vector comprising the nucleic acid of claim 12.

14. A host cell having the nucleic acid of claim 12 introduced therein.

15. A host cell having the recombinant vector of claim 13 introduced therein.

16. A kit for the detection or quantification of an antigen of Middle East respiratory syndrome (MERS), the kit comprising the light chain variable region of claim 9.

17. A monoclonal antibody having binding specificity to Middle East Respiratory Syndrome coronavirus (MERS-CoV) spike protein, the monoclonal antibody comprising a heavy chain variable region comprising a first complementarity determining region comprising an amino acid sequence represented by SEQ. ID. NO: 1, SEQ. ID. NO: 3, SEQ. ID. NO: 5, SEQ. ID. NO: 7, SEQ. ID. NO: 9, SEQ. ID. NO: 11, SEQ. ID. NO: 13, SEQ. ID. NO: 15, SEQ. ID. NO: 17, SEQ. ID. NO: 19, or SEQ. ID. NO: 21, and a light chain variable region comprising a second complementarity determining region comprising an amino acid sequence represented by SEQ. ID. NO: 2, SEQ. ID. NO: 4, SEQ. ID. NO: 6, SEQ. ID. NO: 8, SEQ. ID. NO: 10, SEQ. ID. NO: 12, SEQ. ID. NO: 14, SEQ. ID. NO: 16, SEQ. ID. NO: 18, SEQ. ID. NO: 20, or SEQ. ID. NO: 22.

18. A nucleic acid encoding the monoclonal antibody of claim 17.

19. A recombinant vector comprising the nucleic acid of claim 18.

20. A host cell having the nucleic acid of claim 18 introduced therein.

21. A host cell having the recombinant vector of claim 19 introduced therein.

22. A kit for the detection or quantification of an antigen of Middle East respiratory syndrome (MERS), the kit comprising the monoclonal antibody of claim 17.

23. A method for preventing, treating or diagnosing Middle East respiratory syndrome (MERS), the method comprising a step of administering the heavy chain variable region of claim 1 to a subject.

24. (canceled)

25. A method for preventing, treating or diagnosing Middle East respiratory syndrome comprising a step of administering the light chain variable region of claim 9 to a subject.

26. (canceled)

27. A method for preventing, treating or diagnosing Middle East respiratory syndrome comprising a step of administering the monoclonal antibody of claim 17 to a subject.

28. (canceled)