CA3152245A1

CA3152245A1 - Anti-mers-cov antibody and use thereof

Info

Publication number: CA3152245A1
Application number: CA3152245A
Authority: CA
Inventors: Junho Chung; Sang Il Kim; Myoung-don OH; Wan Beom PARK; Seungtaek KIM; Ji-Young Min; Jinhee Kim; So Young Chang; Jung Min Shim; Sujeong Kim
Original assignee: Seoul National University R&DB Foundation; Institut Pasteur Korea
Current assignee: SNU R&DB Foundation; Institut Pasteur Korea
Priority date: 2019-09-23
Filing date: 2020-09-23
Publication date: 2021-04-01
Also published as: WO2021060837A1; KR20220070262A

Abstract

Provided is an anti-MERS-CoV antibody or an antigen-binding fragment thereof, and medical uses thereof.

Description

Description Title of Invention: ANTI-MERS-COV ANTIBODY AND USE
THEREOF
Technical Field [1] Provided is an anti-MERS-CoV antibody or an antigen-binding fragment thereof, and medical uses thereof.

[2]
Background Art [31 Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was first identified in Saudi Arabia in 2012 from a patient who suffered acute pneumonia and subsequent renal failure. Since then, the World Health Organization has reported 2,254 laboratory-confirmed cases of MERS-CoV infections in 27 different countries around the world, and South Korea has recorded the highest number of cases outside of the Middle East.
Despite resilient efforts throughout the scientific and medical communities, no vaccine or antiviral agent for MERS-CoV is currently available.
[4] MERS-CoV is a large (30 kb), enveloped, single-stranded, positive-sense RNA virus.
The viral genome encodes four major structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. The S glycoprotein is a major envelope protein and interacts with the cellular receptor dipeptidyl peptidase 4 (DPP4) for entry into the host cell. This protein consists of the 51 and S2 subunits. The receptor-binding domain (RBD) within the 51 subunit mediates receptor binding, whereas the S2 subunit facilitates membrane fusion. DPP4 is expressed on a variety of human cells, including fibroblasts, intestinal epithelial cells, and hepatocytes, as well as in the lung parenchyma and interstitium. MERS-CoV is detected in respiratory secretions and the lower respiratory tract of the infected patients. In the most severe cases of MERS-CoV
infection, aggravating respiratory failure ultimately results in mechanical ventilation.
These observations suggest that the MERS-CoV virus primarily infects the human res-piratory tract and replicates within the human airway epithelium.
[51 Antibodies play a crucial role in the prevention and treatment of viral infection.
Polysera taken from recovered patients and vaccinated donors have been used as pro-phylactic agents for hepatitis B, rabies, and other viral diseases.
Palivizumab (Synagis, Medimmune, Gaithersburg, MD, USA) was approved for the prophylaxis of RSV
(Respiratory Syndrome Virus) in 1998, and ibalizumab-uiyk (Trogarzo, TailMed Biologics, Taiwan) became clinically available in 2018 for the treatment of human im-munodeficiency virus type 1 (HIV-1) infection in treatment-experienced adults with multi-drug-resistant HIV-1 and failure to respond to the current antiretroviral regimen.

[6] In response to the ongoing epidemic, several groups have developed anti-MERS-CoV neutralizing monoclonal or polyclonal antibodies that target RBD.
These antibodies were generated from B cells derived from convalescent patients, nonimmune human antibody phage-display libraries, fully humanized mice, tran-schromosomic bovines, or hybridomas from mice that were immunized with MERS-CoV S. These antibodies potently inhibit RBD binding to the DPP4 receptor. Fur-thermore, therapeutic effects of RBD-specific neutralizing antibodies were evaluated in several animal models, including Ad5/hDPP4-trasduced mice, humanized DPP4 mice, and hDPP4-transgenic mice as well as hDPP4 knockin mice, rabbits, and rhesus monkeys.
171 All MERS-CoV neutralizing antibodies were developed for intravenous (i. v.) delivery; however, recent reports indicate that the amount of antibody delivered to lung tissue is often quite limited following systemic delivery (Hart TK, Cook RM, Zia-Amirhosseini P, Minthorn E, Sellers TS, Maleeff BE, et al. Preclinical efficacy and safety of mepolizumab (SB-240563), a humanized monoclonal antibody to IL-5, in cynomolgus monkeys. J Allergy Clin Immunol. 2001;108(2):250-257; Koleba T, Ensom MH. Pharmacokinetics of intravenous immunoglobulin: a systematic review.

Pharmacotherapy. 2006;26(6):813-827). In cynomolgus monkeys, bronchoalveolar lavage fluid contained dose-proportional concentrations of systemically administrated antibody, and these concentrations were approximately 500-fold less than those in plasma. Therefore, delivery of therapeutic antibody to lung tissues via inhalation has garnered considerable interest. Following delivery via the airway, cetuximab, an anti-epidermal growth factor receptor (EGFR) antibody, accumulated in normal and cancerous tissues in the lung at a concentration that was twice that achieved after i. v.
delivery. In addition, recent studies showed that Fc fusion proteins and nanobodies are also efficiently delivered via the pulmonary route (Bitonti AJ, Dumont JA.
Pulmonary administration of therapeutic proteins using an immunoglobulin transport pathway.
Adv Drug Deliv Rev. 2006;58(9-10):1106-1118). Therefore, MERS-CoV neutralizing antibody may also accumulate at higher concentrations following delivery via a pulmonary route, guaranteeing higher efficacy. In order for this pulmonary delivery to be successful, the antibody must be sufficiently stable to resist denaturation during the process of nebulization.
[81 Disclosure of Invention Technical Problem [91 Provided an antibody against a coronavirus, MERS-CoV, or an antigen-binding fragment thereof, having improved stability and efficacy, and uses thereof.

3 [10] An embodiment provides an anti-MERS-CoV antibody or an antigen-binding fragment thereof comprising:
[11] a VL-CDR1(complementarity determining region 1) comprising an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7;
[12] a VL-CDR2 comprising an amino acid sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, or 71;
[13] a VL-CDR3 comprising an amino acid sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, or 72;
[14] a VH-CDR1 comprising an amino acid sequence of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, or 73;
[15] a VH-CDR2 comprising an amino acid sequence of SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, or 74; and [16] a VH-CDR3 comprising an amino acid sequence of SEQ ID NO: 38, 39, 40, 41, 42, 43, or 75.
[17] Another embodiment provides an anti-MERS-CoV antibody or an antigen-binding fragment thereof comprising:
[18] a light chain variable region comprising a VL-CDR1 comprising an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, a VL-CDR2 comprising an amino acid sequence of SEQ ID NO: 8,9, 10, 11, 12, 13, 14, or 71, and a VL-CDR3 comprising an amino acid sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, or 72; and [19] a heavy chain variable region comprising a VH-CDR1 comprising an amino acid sequence of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, or 73, a VH-CDR2 comprising an amino acid sequence of SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, or 74, and a VH-comprising an amino acid sequence of SEQ ID NO: 38, 39, 40, 41, 42, 43, or 75.
[20] Another embodiment provides an anti-MERS-CoV antibody or an antigen-binding fragment thereof comprising:
[21] a light chain variable region comprising or consisting essentially of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 76; and [22] a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, or 77.
[23] Another embodiment provides a pharmaceutical composition comprising the anti-MERS-CoV antibody or an antigen-binding fragment thereof. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The pharma-ceutical composition may be used for preventing and/or treating MERS-CoV
infection and/or a disease associated with MERS-CoV infection.
[24] Another embodiment provides a method of treating and/or preventing MERS-CoV
infection and/or a disease associated with MERS-CoV infection, comprising admin-istering a pharmaceutically effective amount of the anti-MERS-CoV antibody or an

4 antigen-binding fragment thereof or the pharmaceutical composition to a subject in need of treating and/or preventing the MERS-CoV infection and/or the disease.
The method may further comprise a step of identifying the subject in need of treating and/
or preventing MERS-CoV infection and/or a disease associated with MERS-CoV
infection, prior to the administering step.
[25] Another embodiment provides a use of the anti-MERS-CoV antibody or an antigen-binding fragment thereof or the pharmaceutical composition in treating and/or preventing MERS-CoV infection and/or a disease associated with MERS-CoV
infection. Another embodiment provides a use of the anti-MERS-CoV antibody or an antigen-binding fragment thereof in preparing a pharmaceutical composition for treating and/or preventing MERS-CoV infection and/or a disease associated with MERS-CoV infection.
[26] An embodiment provides a polynucleotide encoding the anti-MERS-CoV
antibody or an antigen-binding fragment thereof. In particular, an embodiment provides a first polynucleotide encoding a combination of VL-CDR1, VL-CDR2, and VL-CDR3, a heavy chain variable region, or a heavy chain of the anti-MERS-CoV antibody or an antigen-binding fragment thereof. Another embodiment provides a second polynu-cleotide encoding a combination of VH-CDR1, VH-CDR2, and VH-CDR3, a light chain variable region, or a light chain of the anti-MERS-CoV antibody or an antigen-binding fragment thereof.
[27] An embodiment provides a recombinant vector comprising the first polynucleotide, the second polynucleotide, or a combination thereof. The recombinant vector may be used as an expression vector of the polynucleotide. Another embodiment provides a re-combinant cell transfected with the recombinant vector.
[28] Another embodiment provides a method of preparing the anti-MERS-CoV
antibody or an antigen-binding fragment thereof, comprising expressing the first polynucleotide and the second polynucleotide in a cell. The step of expressing the polynucleotides may be conducted by culturing the cell comprising the polynucleotides (for example, each of the polynucleotides is carried by each recombinant vector, or both of the polynucleotides are carried by one recombinant vector) under a condition allowing the expression of the polynucleotides. The method may further comprise isolating and/or purifying the anti-MERS-CoV antibody or an antigen-binding fragment thereof from the cell culture, after the step of expressing or culturing.
[29]
Solution to Problem [30] In this disclosure, an Ab-phage library was constructed using as a source peripheral blood mononuclear cell (PMBC) isolated from biological specimen of recovered

5 patients from MERS-CoV; and from convalescent MERS-CoV infected patients, several potent human neutralizing antibodies that specifically bind to RBD in the Si domain of S glycoprotein of MERS-CoV were successfully identified and char-acterized. More specifically, a phage-display library was constructed from two con-valescent MERS-CoV-infected patients and nine MERS-CoV RBD-specific neu-tralizing monoclonal antibodies (mAbs) were successfully isolated therefrom.
After ad-ministration (e.g. nebulization), these antibodies showed significant aggregation and reduced reactivity to recombinant S glycoprotein. In this disclosure, the number of hy-drophobic residues was reduced and solubilizing mutations were introduced within the complementarity-determining regions (CDRs), thereby generating an antibody that is resistant to aggregation during administration (e.g. nebulization) and retains its MERS-CoV neutralizing activity.
[31] One embodiment provides a MERS-CoV neutralizing antibody that bind to RBD in the Si domain of S glycoprotein of MERS-CoV or an antigen-binding fragment thereof. In an embodiment, the antibody or antigen-binding fragment thereof may be for delivery via nebulization.
[32] Another embodiment provides medical uses of the anti- MERS-CoV
antibody or an antigen-binding fragment thereof for treating and/or preventing MERS-CoV
infection and/or a disease associated with MERS-CoV infection.
[33]
[34] Hereinafter, more detailed descriptions are provided.
[35]
[36] Definitions [37] As used herein, 'consisting of a sequence,' consisting essentially of a sequence,' or 'comprising a sequence' may refer to any case comprising the sequence, but it may not be intended to exclude a case comprising further sequence other than the sequence.
[38] As used herein, the term 'a protein or polypeptide comprising or consisting of an amino acid sequence identified by SEQ ID NO' and 'a gene or polynucleotide comprising or consisting of a nucleic acid sequence identified by SEQ ID NO' may refer to a protein (or polypeptide) or gene (or polynucleotide), which consists es-sentially of the amino acid sequence or nucleic acid sequence, or which has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence or nucleic acid sequence with maintaining its inherent activity and/or function.
[39] As used herein, the term "antibody" may encompass various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will ap-preciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (7, [I, a,

6

7 PCT/KR2020/012887 6, E) with some subclasses among them (e.g., 71-74), and light chains are classified as either kappa or lambda (K, X). It is the nature of this chain that determines the "class"
of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to confer functional specialization.
[40] An intact antibody includes two full-length light chains and two full-length heavy chains, in which each light chain is linked to a heavy chain by disulfide bonds. The antibody has a heavy chain constant region and a light chain constant region.
The heavy chain constant region is of a gamma (7), mu (pc), alpha (a), delta (6), or epsilon (E) type, which may be further categorized as gamma 1 (71), gamma 2(72), gamma 3(73), gamma 4(74), alpha 1(a1), or alpha 2(a2). The light chain constant region is of either a kappa (cc) or lambda (X) type.
[41] The term "heavy chain" refers to a full-length heavy chain or a fragment thereof, including a variable region VH that includes amino acid sequences sufficient to provide specificity to antigens, and three constant regions, CHI, CH2, and CH3, and a hinge. The term "light chain" refers to a full-length light chain or a fragment thereof, including a variable region VL that includes amino acid sequences sufficient to provide specificity to antigens, and a constant region CL.
[42] The term "complementarity determining region (CDR)" refers to an amino acid sequence found in a hyper variable region of a heavy chain or a light chain of im-munoglobulin. The light and heavy chains may respectively include three CDRs (light chain: VL-CDR1, VL-CDR2, and VL-CDR3; heavy chain: HL-CDR1, HL-CDR2, and HL-CDR3). The CDR may provide residues that play an important role in the binding of antibodies to an antigens or epitope. The terms "specifically binding" or "specifically recognized" is well known to one of ordinary skill in the art, and indicates that an antibody and an antigen specifically interact with each other to lead to an im-munological activity.
[43] In this disclosure, the term "epitope" may refer to a site of an antigen where an antibody or an antigen-binding fragment binds, recognizes, targets, and/or interacts.
[44] In this disclosure, the antibody may include, but not be limited to, polyclonal or monoclonal; and/or human, humanized, animal (e.g., mouse, rabbit, etc.) derived antibody, or chimeric antibodies (e.g., mouse-human chimeric antibody, rabbit-human chimeric antibody, etc.).
[45] An animal-derived antibody which is produced by immunizing an animal with a desired antigen may generally trigger an immune rejection response when administered to humans for treatment purpose, and a chimeric antibody has been developed to suppress such immune rejection response. A chimeric antibody is formed by replacing the constant region of an animal-derived antibody, which is a cause of anti-isotype response, with the constant region of a human antibody using genetic engineering methods. The chimeric antibody has considerably improved anti-isotype response in comparison with animal-derived antibodies, but animal-derived amino acids are still present in its variable regions and thus it still contains potential side effects resulting from an anti-idiotypic response. It is a humanized antibody that has been developed to improve such side effects. This is manufactured by grafting CDR
(complementarity de-termining regions) which, of the variable regions of a chimeric antibody, has an important role in antigen binding into a human antibody framework.
[46] As used herein, the term "antigen binding fragment" refers to a fragment derived from a full immunoglobulin structure including a portion capable of binding to an antigen such as CDRs. For example, the antigen binding fragment may be scFv, (scFv) 2, Fab, Fab', or F(abt)2, but not be limited thereto. In the present disclosure, the antigen binding fragment may be a fragment derived from an antibody, including at least one complementarity determining region, for example, selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab' and F(ab')2.
[47] Of the antigen binding fragments, Fab is a structure having variable regions of a light chain and a heavy chain, a constant region of the light chain, and the first constant region (CHI) of the heavy chain, and it has one antigen binding site.
[48] Fab' is different from Fab in that it has a hinge region including one or more cysteine residues at the C-terminal of heavy chain CHI domain. An F(ab')2antibody is formed through disulfide bond of the cysteine residues at the hinge region of Fab'.
[49] Fv is a minimal antibody piece having only a heavy chain variable region and light chain variable region, and a recombinant technique for producing the Fv fragment is well known in the pertinent art. Two-chain Fv may have a structure in which the heavy chain variable region is linked to the light chain variable region by a non-covalent bond, and single-chain Fv (scFv) may generally have a dimer structure in which the variable region of a heavy chain and the variable region of a light chain are covalently linked via a peptide linker or they are directly (i.e., without a peptide linker) linked to each other. The peptide linker may comprise about 1 to about 100 amino acids, about 2 to about 50 amino acids, or about 5 to about 25 amino acids, and any kinds of amino acids may be included therein without any restrictions.
[50] The antigen binding fragments may be obtained using proteases (for example, a whole antibody is digested with papain to obtain Fab fragments, and is digested with pepsin to obtain F(abt)2 fragments), and may be prepared by a genetic recombinant technique.
[51] Immunoglobulin (e.g., a human immunoglobulin) or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, IgY, etc.), class (e.g., IgGl, IgG2, IgG3, IgG4, IgG5, IgAl, IgA2, etc.), or subclass of immunoglobulin

8 molecule.
[52] In the antibody or antibody fragment, portions(e.g., frameworks, constant regions, etc.) except the CDRs or variable regions may be derived from an immunoglobulin (e.g., a human immunoglobulin), and particularly, they may be derived from IgG, IgA, IgD, IgE, IgM, or IgY, for example, IgGl, IgG2, IgG 3, or IgG4.
[53] In this disclosure, the term "antibody" may include an antigen binding fragment thereof comprising any combination of 6 CDRs or VH (heavy chain variable region) and VL (light chain variable region) as well as a complete (full length) antibody (e.g., IgG), unless otherwise indicated or clearly contradicted by context.
[54] The antibody or antigen binding fragment may be chemically or recombinantly syn-thesized (not naturally occurring).
[55] The term "subject" may refer to any subject, particularly a mammalian subject, for whom diagnosis, prophylaxis, and/or therapy is desired. Mammalian subjects may include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.
[56]
[57] RBD and/or S protein of MERS-CoV as an epitope [58] One embodiment provides an anti-MERS-CoV antibody that binds to (and/or recognizes and/or targets) a receptor-binding domain (RBD) in the Si domain of S
protein (spike protein) of MERS-CoV or an antigen-binding fragment thereof.
[59] An epitope for an anti-MERS-CoV antibody or an antigen-binding fragment thereof provided herein may be positioned in RBD of S protein of MERS-CoV. The epitope may be positioned at least partially overlapping or adjacent to a receptor binding site of S protein of MERS-CoV, thereby allowing the antibody or an antigen-binding fragment thereof to inhibit the binding of S protein of MERS-CoV to a cell (hereinafter, "host cell") of a subject infected by MERS-CoV.
[60] The RBD or S protein of MERS-CoV which is targeted by an anti-MERS-CoV

antibody or an antigen-binding fragment thereof provided herein may be a wild-type and/or variants thereof.
[61] For example, the S protein of MERS-CoV may be represented by GenBank Accession No. AF588936.1 (SEQ ID NO: 70), wherein the region from E367 to Y606 amino acid residues corresponds to the RBD. For example, the variant of S
protein of MERS-CoV may have a polymorphism (mutation) from a wild type. In a specific em-bodiment, the variant of S protein may have mutation (e.g., amino acid substitution) at position 510, 529, or both thereof, in SEQ ID NO: 70. For example, the variant of S
protein may have one or both of mutation D5 10G (wherein the amino acid (D) corre-sponding to position 510 is substituted with G) and I529T(wherein the amino acid (I) corresponding to position 529 is substituted with T) in SEQ ID NO: 70.

[62] The receptor binding site of the S protein may be a binding site with a receptor on a host cell. In an embodiment, the receptor on a host cell may be dipeptidyl peptidase IV
(DPP4). For example, when the subject infected by MERS-CoV is a human being (i.e., the host cell is a human cell), and the receptor on a host cell is human DPP4 (e.g., NCBI Accession No. NP 001926.2 etc.), the receptor binding site of the S
protein with DPP4 may comprise at least one selected from amino acid residues corresponding to positions 452, 454, 460, 461, 462, 463, 466, 499, 501, 502, 504, 505, 506, 510, 511, 512, 513, 515, 535, 536, 537, 538, 539, 540, 541, 542, 553, 555, 557, 559, and 562 of SEQ ID NO: 70 (https://www.nature.com/articlesincomms9223?origin=ppub).
[63] The epitope may be at least partially overlapping the receptor binding site of S
protein (SEQ ID NO: 70) as described above. For example, an anti-MERS-CoV
antibody or an antigen-binding fragment thereof provided herein may bind to an epitope comprising at least one (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, or at least twenty, etc.) selected from amino acid residues corresponding to positions 498 to 554 of the S
protein (SEQ
ID NO: 70) or variants thereof.
[64] In a specific embodiment, the epitope for an anti-MERS-CoV antibody or an antigen-binding fragment thereof provided herein may be:
[65] (a) an epitope comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8,

9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23) selected from amino acid residues corresponding to positions 498 to 520 (e.g., SYINKCSRLLSDDRTEVPQLVNA (SEQ ID NO: 67;
wild-type) or SYINKCSRLLSDGRTEVPQLVNA (SEQ ID NO: 68; variant (including mutation D5 10G)) of the S protein (SEQ ID NO: 70) or variants thereof;
[66] (b) an epitope comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) selected from amino acid residues corresponding to positions 540 to 554 (YYRKQLSPLEGGGWL (SEQ ID NO: 69)) of the S protein (SEQ ID NO: 70) or variants thereof; or [67] (c) both of (a) and (b).
[68] The epitope and receptor binding site positioned in S protein (SEQ ID
NO: 70) are il-lustrated below.
[69] 1 MIHSVFLLMF LLTPTESYVD VGPDSVKSAC IEVDIQQTFF DKTWPRPIDV
SKADGIIYPQ
[70] 61 GRTYSNITIT YQGLFPYQGD HGDMYVYSAG HATGITPQKL
FVANYSQDVK QFANGFVVRI
[71] 121 GAAANSTGTV IISPSTSATI RKIYPAFMLG SSVGNFSDGK
MGRFFNHTLV LLPDGCGTLL

10 [72] 181 RAFYCILEPR SGNHCPAGNS YTSFATYHTP ATDCSDGNYN
RNASLNSFKE YFNLRNCTFM
[73] 241 YTYNITEDEI LEWFGITQTA QGVHLFSSRY VDLYGGNMFQ
FATLPVYDTI KYYSIIPHSI
[74] 301 RSIQSDRKAW AAFYVYKLQP LTFLLDFSVD GYIRRAIDCG
FNDLSQLHCS YESFDVESGV
[75] 361 YSVSSFEAKP SGSVVEQAEG VECDFSPLLS GTPPQVYNFK
RLVFTNCNYN LTKLLSLFSV
[76] 421 NDFTCSQISP AAIASNCYSS LILDYFSYPL SMKSDLSVSS AGPISQFNYK
QSFSNPTCLI
[77] 481 LATVPHNLTT ITKPLKYSYI NKCSRLLSDD RTEVPQLVNA
NQYSPCVSIV PSTVWEDGDY
[78] 541 YRKQLSPLEG GGWLVASGST VAMTEQLQMG FGITVQYGTD
TNSVCPKLEF ANDTKIASQL
[79] 601 GNCVEYSLYG VSGRGVFQNC TAVGVRQQRF VYDAYQNLVG
YYSDDGNYYC LRACVSVPVS
[80] 661 VIYDKETKTH ATLFGSVACE HISSTMSQYS RSTRSMLKRR
DSTYGPLQTP VGCVLGLVNS
[81] 721 SLFVEDCKLP LGQSLCALPD TPSTLTPRSV RSVPGEMRLA
SIAFNHPIQV DQLNSSYFKL
[82] 781 SIPTNFSFGV TQEYIQTTIQ KVTVDCKQYV CNGFQKCEQL
LREYGQFCSK INQALHGANL
[83] 841 RQDDSVRNLF ASVKSSQSSP IIPGFGGDFN LTLLEPVSIS TGSRSARSAI
EDLLFDKVTI
[84] 901 ADPGYMQGYD DCMQQGPASA RDLICAQYVA GYKVLPPLMD VN-MEAAYTSS LLGSIAGVGW
[85] 961 TAGLSSFAAI PFAQSIFYRL NGVGITQQVL SENQKLIANK FN-QALGAMQT GFTTTNEAFR
[86] 1021 KVQDAVNNNA QALSKLASEL SNTFGAISAS IGDIIQRLDV
LEQDAQIDRL INGRLTTLNA
[87] 1081 FVAQQLVRSE SAALSAQLAK DKVNECVKAQ SKRSGFCGQG
THIVSFVVNA PNGLYFMHVG
[88] 1141 YYPSNHIEVV SAYGLCDAAN PTNCIAPVNG YFIKTNNTRI
VDEWSYTGSS FYAPEPITSL
[89] 1201 NTKYVAPQVT YQNISTNLPP PLLGNSTGID FQDELDEFFK
NVSTSIPNFG SLTQINTTLL
[90] 1261 DLTYEMLSLQ QVVKALNESY IDLKELGNYT YYNKWPWYIW
LGFIAGLVAL ALCVFFILCC

11 [91] 1321 TGCGTNCMGK LKCNRCCDRY EEYDLEPHKV HVH
[92] (the amino sequence of GenBank Accession No. AFS88936.1 (SEQ ID NO:
70), wherein the possible region where the epitope may position is indicated in bold and the receptor binding site is underlined) [93]
[94] Anti-MERS-CoV antibody or antigen-binding fragment thereof [95] Provided an antibody against a coronavirus, MERS-CoV, or an antigen-binding fragment thereof, having improved stability and efficacy, and uses thereof.
The antibody may be a neutralizing antibody against MERS-CoV. The antibody or an antigen-binding fragment thereof may bind to an epitope positioned at least partially overlapping or adjacent to a receptor binding site of S protein of MERS-CoV, to competing with RBD and/or S protein) of MERS-CoV for binding a receptor on a host cell, thereby inhibiting the binding of S protein of MERS-CoV to the host cell. The S
protein of MERS-CoV may be a wild type and/or a variant (e.g., including D510G, I529T, or both thereof).
[96] An embodiment provides an anti-MERS-CoV antibody or an antigen-binding fragment thereof, which binds to an epitope positioned in a RBD in the 51 domain of S
protein of MERS-CoV or an antigen-binding fragment thereof.
[97] In a specific embodiment, an anti-MERS-CoV antibody or an antigen-binding fragment thereof provided herein may bind to [98] (a) an epitope comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23) selected from amino acid residues corresponding to positions 498 to 520 (e.g., SYINKCSRLLSDDRTEVPQLVNA (SEQ ID NO: 67;
wild-type) or SYINKCSRLLSDGRTEVPQLVNA (SEQ ID NO: 68; variant (including D5 10G)) of the S protein (SEQ ID NO: 70) or variants thereof;
[99] (b) an epitope comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) selected from amino acid residues corresponding to positions 540 to 554 (YYRKQLSPLEGGGWL (SEQ ID NO: 69)) of the S protein (SEQ ID NO: 70) or variants thereof; or [100] (c) both of (a) and (b).
[101]
[102] Another embodiment provides an anti-MERS-CoV antibody or an antigen-binding fragment thereof comprising:
[103] a VL-CDR1 comprising an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7;
[104] a VL-CDR2 comprising an amino acid sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14 or 71;
[105] a VL-CDR3 comprising an amino acid sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, or 72;

12 [106] a VH-CDR1 comprising an amino acid sequence of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, or 73;
[107] a VH-CDR2 comprising an amino acid sequence of SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, or 74; and [108] a VH-CDR3 comprising an amino acid sequence of SEQ ID NO: 38, 39, 40, 41, 42, 43, or 75.
[109] Amino acid sequences of CDRs of the anti-MERS-CoV antibody or an antigen-binding fragment thereof are illustrated in Table 1, wherein the CDRs are determined according to IMGT numbering:

13 [110] [Table 11 CDR Amino Acid Sequence SEQ
ID NO

14 [111] Another embodiment provides an anti-MERS-CoV antibody or an antigen-binding fragment thereof comprising:a light chain variable region comprising a VL-CDR1 comprising an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, a VL-comprising an amino acid sequence of SEQ ID NO: 8,9, 10, 11, 12, 13, 14, or 71, and a VL-CDR3 comprising an amino acid sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, or 72; and [112] a heavy chain variable region comprising a VH-CDR1 comprising an amino acid sequence of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, or 73, a VH-CDR2 comprising an amino acid sequence of SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, or 74, and a VH-

15 comprising an amino acid sequence of SEQ ID NO: 38, 39, 40, 41, 42, 43, or 75.
[113] Another embodiment provides an anti-MERS-CoV antibody or an antigen-binding fragment thereof comprising:
[114] a light chain variable region comprising or consisting essentially of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 76; and [115] a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, or 77.
[116] The amino acid sequences of the variable regions of the anti-MERS-CoV
antibody or an antigen-binding fragment are illustrated in Table 2:

16 [117] [Table 2]
SEQ ID Light chain variable region targeting MERS-CoV (e.g., RBD in Si NO domain of S glycoprotein of MERS-CoV) QSPQLLIYLGSNRASGVPDRFS GS GS GTDFTLKIGRVEAEDVGIYYC
MQALQTPLTFGGGTKVEIK

QSPQLLIYLGSNRASGVPDRFS GS GS GTDFTLKIGRVEAEDVGIYYC
MQAVQTPLTFGGGTKVEIK

QSPQLLIYEGSNRASGVPDRFS GS GS GTDFTLKIGRVEAEDVGIYYC
MQALQTPLTFGGGTKVEIK

LIYSNNQRPS GVPDRFS GS KS GT S ASLAIS GLRSEDEADYYCA TWD
DNLSGPVFGGGTKVTVLG

PGQPPKLLIYWASTRES GVPDRFS GS GSGTDFTLTIS SVQTEDVAVY
YCQQYYGSPYTFGQGTKLEIK

PGQPPKLLIS WA STRES GVPERFS GS GS GTDFTLTIS GLQAEDVAVY
YCQQYYSTPPTFGQGTKVDIK

KLLIYGNSNRPSGVPDRFS GS KS GTSASLAIS GLQSEDEGDYYCAA
WDDSLSGPVFGGGTELTVLG

LLIYSNNHRPS GVPDRFS GS KS GTSASLAIS GLRSEDEAVYYCAAW
DDSLSGVVFGGGTELTVLG

KLLIYSNSQRPSGVPDRFS GS KS GTSASLVIS GLRSEDEADYYCAA
WDDSLSGPVFGGGTQLTVLG

LIYSNNQRPS GVPDRFS GS KS GT S ASLAINGLQSED EADYYCAAWD
DSLNGPVFGGGTKLTVLG

PVLVIYQDSRRASGIPERFS GSNS GNTATLTIS GT QAMDEADYYCQ.

17 AWDSRRAVFGGGTELTVLG

AIYODTKRPS GIPERFS GS NS GNTATLTIS GTQPMDEADYYC QAWD
NNFYVFGTGTKLTVLG
SEQ ID Heavy chain variable region targeting MERS-CoV (e.g., RBD in Si NO domain of S glycoprotein of MERS-CoV) EWMGGIIPFFGTA NYAQKFQGRVTITADES TS TAYMELS S LRS EDT
AVYYCARDGRKDYYGSGSYLHYYGMDVWGQGTTVTVSS

LEWM GGIIPFFDKANYAQKFQGRVTITADES TS TAYMELS SLRS ED
TAVYYCARDGRKDYYGSGSYLHYYGMDVWGQGTTVTVSS

LEWM GGIIPFFDKANYAQKFQGRVTITADES TS TAYMELS SLRS ED
TAVYYCARDGRKDYYGSGSYLHYYGMDVWGQGTTVTVSS

LEWIGS IFYIGNTYYNPS LKS RVTIS VDT S KNQFS LRLS S VTAADTA
VYYCARQEGSSIIRFDPWGQGTLVTVSS

LEWM GRIIPILGIANYAQKFQGRVTITAD KS TS TAYMELS SLRS ED
TAVYYCASLFDSSGYYPYYFDYWGQGTLVTVSS

EWMGRIIPIFGIA NYAQKFQ GRVTITAD KST GTAYMELS SLRS EDT
AVYYCATHFGASGYDPYYFDYWGQGTLVTVSS

LEWIGS IFYIGNTYYNPS LKS RVTIS VDT S KNQFS LKLS S VTAADTA
VYYCARQEGSSIIRFDPWGQGTLVTVSS

LEWIGS IYYTGNTYYNPS LKS RLTIS VDT S KNQFS LKLS S VTAADT
AVYYCARQVADLGYFDYWGQGTLVTVSS

EWVS SI ST TGSYIFYAD S VKGRFTIS RDNAKNS LYLQMNTLRPEDT
ALYYCAKGTAFDGGLAFDIWGQGTIVTVSS

LEWVS GISWDSGSIAYADSVKGRFTISRDNAKNSLYLQMNSLRAE

18 DTAVYYCAREKQLVPYYYYGMDVWGQGTTVTVSS
[118] (CDR1, CDR2, and CDR3, which are determined by IMGT numbering, are un-derlined in order)In a specific embodiment, the anti-MERS-CoV antibody or an antigen-binding fragment thereof may comprise:
[119] a light chain variable region comprising or consisting essentially of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 76; and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO:
55;
[120] a light chain variable region comprising or consisting essentially of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 76; and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO:
56;
[121] a light chain variable region comprising or consisting essentially of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 76; and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO:
57;
[122] a light chain variable region comprising or consisting essentially of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 76; and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO:
58;
[123] a light chain variable region comprising or consisting essentially of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 76; and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO:
59;
[124] a light chain variable region comprising or consisting essentially of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 76; and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO:
60;
[125] a light chain variable region comprising or consisting essentially of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 76; and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO:
61;
[126] a light chain variable region comprising or consisting essentially of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 76; and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO:
62;
[127] a light chain variable region comprising or consisting essentially of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 76; and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO:
63; or [128] a light chain variable region comprising or consisting essentially of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 76; and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO:
77.
[129]
[130] For example, the anti-MERS-CoV antibody or an antigen-binding fragment thereof may comprise:

19 [131] (1) a VL-CDR1 of SEQ ID NO: 1, a VL-CDR2 of SEQ ID NO: 8, a VL-CDR3 of SEQ ID NO: 15, a VH-CDR1 of SEQ ID NO: 24, a VH-CDR2 of SEQ ID NO: 31, and a VH-CDR3 of SEQ ID NO: 38;
[132] (2) a VL-CDR1 of SEQ ID NO: 1, a VL-CDR2 of SEQ ID NO: 8, a VL-CDR3 of SEQ ID NO: 15, a VH-CDR1 of SEQ ID NO: 25, a VH-CDR2 of SEQ ID NO: 32, and a VH-CDR3 of SEQ ID NO: 38;
[133] (3) a VL-CDR1 of SEQ ID NO: 2, a VL-CDR2 of SEQ ID NO: 8, a VL-CDR3 of SEQ ID NO: 16, a VH-CDR1 of SEQ ID NO: 25, a VH-CDR2 of SEQ ID NO: 32, and a VH-CDR3 of SEQ ID NO: 38;
[134] (4) a VL-CDR1 of SEQ ID NO: 1, a VL-CDR2 of SEQ ID NO: 8, a VL-CDR3 of SEQ ID NO: 15, a VH-CDR1 of SEQ ID NO: 26, a VH-CDR2 of SEQ ID NO: 32, and a VH-CDR3 of SEQ ID NO: 38;
[135] (5) a VL-CDR1 of SEQ ID NO: 1, a VL-CDR2 of SEQ ID NO: 9, a VL-CDR3 of SEQ ID NO: 15, a VH-CDR1 of SEQ ID NO: 26, a VH-CDR2 of SEQ ID NO: 32, and a VH-CDR3 of SEQ ID NO: 38;
[136] (6) a VL-CDR1 of SEQ ID NO: 3, a VL-CDR2 of SEQ ID NO: 10, a VL-CDR3 of SEQ ID NO: 17, a VH-CDR1 of SEQ ID NO: 27, a VH-CDR2 of SEQ ID NO: 33, and a VH-CDR3 of SEQ ID NO: 39;
[137] (7) a VL-CDR1 of SEQ ID NO: 4, a VL-CDR2 of SEQ ID NO: 11, a VL-CDR3 of SEQ ID NO: 18, a VH-CDR1 of SEQ ID NO: 28, a VH-CDR2 of SEQ ID NO: 34, and a VH-CDR3 of SEQ ID NO: 40;
[138] (8) a VL-CDR1 of SEQ ID NO: 4, a VL-CDR2 of SEQ ID NO: 11, a VL-CDR3 of SEQ ID NO: 19, a VH-CDR1 of SEQ ID NO: 29, a VH-CDR2 of SEQ ID NO: 35, and a VH-CDR3 of SEQ ID NO: 41;
[139] (9) a VL-CDR1 of SEQ ID NO: 5, a VL-CDR2 of SEQ ID NO: 12, a VL-CDR3 of SEQ ID NO: 20, a VH-CDR1 of SEQ ID NO: 27, a VH-CDR2 of SEQ ID NO: 33, and a VH-CDR3 of SEQ ID NO: 39;
[140] (10) a VL-CDR1 of SEQ ID NO: 3, a VL-CDR2 of SEQ ID NO: 10, a VL-CDR3 of SEQ ID NO: 21, a VH-CDR1 of SEQ ID NO: 27, a VH-CDR2 of SEQ ID NO: 36, and a VH-CDR3 of SEQ ID NO: 42;
[141] (11) a VL-CDR1 of SEQ ID NO: 6, a VL-CDR2 of SEQ ID NO: 13, a VL-CDR3 of SEQ ID NO: 20, a VH-CDR1 of SEQ ID NO: 27, a VH-CDR2 of SEQ ID NO: 36, and a VH-CDR3 of SEQ ID NO: 42;
[142] (12) a VL-CDR1 of SEQ ID NO: 3, a VL-CDR2 of SEQ ID NO: 10, a VL-CDR3 of SEQ ID NO: 22, a VH-CDR1 of SEQ ID NO: 27, a VH-CDR2 of SEQ ID NO: 36, and a VH-CDR3 of SEQ ID NO: 42;
[143] (13) a VL-CDR1 of SEQ ID NO: 7, a VL-CDR2 of SEQ ID NO: 14, a VL-CDR3 of SEQ ID NO: 23, a VH-CDR1 of SEQ ID NO: 30, a VH-CDR2 of SEQ ID NO: 37, and

20 a VH-CDR3 of SEQ ID NO: 43; or [144] (14) a VL-CDR1 of SEQ ID NO: 7, a VL-CDR2 of SEQ ID NO: 71, a VL-CDR3 of SEQ ID NO: 72, a VH-CDR1 of SEQ ID NO: 73, a VH-CDR2 of SEQ ID NO: 74, and a VH-CDR3 of SEQ ID NO: 75.
[145] For example, the anti-MERS-CoV antibody or an antigen-binding fragment thereof may comprise:
[146] (1) a light chain variable region comprising or consisting essentially of SEQ ID NO:
44 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 55;
[147] (2) a light chain variable region comprising or consisting essentially of SEQ ID NO:
44 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 56;
[148] (3) a light chain variable region comprising or consisting essentially of SEQ ID NO:
45 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 56;
[149] (4) a light chain variable region comprising or consisting essentially of SEQ ID NO:
44 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 57;
[150] (5) a light chain variable region comprising or consisting essentially of SEQ ID NO:
46 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 55;
[151] (6) a light chain variable region comprising or consisting essentially of SEQ ID NO:
47 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 58;
[152] (7) a light chain variable region comprising or consisting essentially of SEQ ID NO:
48 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 59;
[153] (8) a light chain variable region comprising or consisting essentially of SEQ ID NO:
49 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 60;
[154] (9) a light chain variable region comprising or consisting essentially of SEQ ID NO:
50 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 61;
[155] (10) a light chain variable region comprising or consisting essentially of SEQ ID NO:
51 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 62;
[156] (11) a light chain variable region comprising or consisting essentially of SEQ ID NO:
52 and a heavy chain variable region comprising or consisting essentially of an amino

21 acid sequence of SEQ ID NO: 62;
[157] (12) a light chain variable region comprising or consisting essentially of SEQ ID NO:
53 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 62;
[158] (13) a light chain variable region comprising or consisting essentially of SEQ ID NO:
54 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 63; or [159] (14) a light chain variable region comprising or consisting essentially of SEQ ID NO:
76 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 77.
[160]
[161] The antibody provided herein may include an antigen binding fragment thereof comprising any combination of VH (heavy chain variable region) and VL (light chain variable region), such as a scFv or a scFv linked with Fc and/or hinge region, as well as a complete (full length) antibody (e.g., IgGl, IgG2, IgG3, IgG4, etc.).
[162] In an embodiment, the anti-MERS-CoV antibody or antigen-binding fragment thereof may be in a full-length form of IgGl, IgG2, IgG3, or IgG4, comprising:
[163] a light chain comprising a light chain variable region comprising or consisting es-sentially of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 76; and [164] a heavy chain comprising a heavy chain variable region comprising or consisting es-sentially of an amino acid sequence of SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, or 77.
[165] In another embodiment, anti-MERS-CoV antibody or antigen-binding fragment thereof may be in a scFv (single chain variable fragment) form, comprising:
[166] a light chain variable region comprising a VL-CDR1 comprising an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, a VL-CDR2 comprising an amino acid sequence of SEQ ID NO: 8,9, 10, 11, 12, 13, 14, or 71, and a VL-CDR3 comprising an amino acid sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, or 72; and [167] a heavy chain variable region comprising a VH-CDR1 comprising an amino acid sequence of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, or 73, a VH-CDR2 comprising an amino acid sequence of SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, or 74, and a VH-comprising an amino acid sequence of SEQ ID NO: 38, 39, 40, 41, 42, 43, or 75.
[168] Another embodiment provides an anti-MERS-CoV antibody or an antigen-binding fragment thereof in a scFv form, comprising:
[169] a light chain variable region comprising or consisting essentially of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 76; and [170] a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, or 77.

22 [171] More specific description of the light chain variable and the heavy chain variable region is the same as above.
[172] In the scFv, the light chain variable region and the heavy chain variable region may be linked in any order. For example, the scFv may comprise the light chain variable region and the heavy chain variable region in a direction of N- to C-terminus (i.e., the C-terminus of the light chain variable region and the N-terminus of the heavy chain variable region are linked; N-VL-VH-C). Alternatively, the scFv may comprise the heavy chain variable region and the light chain variable region in a direction of N- to C-terminus (i.e., the C-terminus of the heavy chain variable region and the N-terminus of the light chain variable region are linked; N-VH-VL-C).
[173] In the scFv, the light chain variable region and the heavy chain variable region may be linked via a peptide linker (e.g., N-VL-linker-VH-C or N-VH-linker-VL-C) or directly (without a peptide linker). The peptide linker may be an oligopeptide including 1 to 100 amino acids or 5 to 25 amino acids, each of which may be any kind of amino acids without any restrictions. Any conventional peptide linker may be used with or without an appropriate modification to comply with specific purposes. In a specific embodiment, the peptide linker may comprise, for example, at least one Gly, at least one Ser, and/or at least one Arg residues, in any order. The amino acid sequences suitable for the peptide linker may be known in the relevant art. The length of the peptide linker can be properly determined within such a limit that the functions of the polypeptide and/or scFv will not be affected. For instance, the peptide linker may be formed by including a total of about 1 to about 100 amino acids, about 2 to about 50 amino acids, about 5 to about 30 amino acids, or about 5 to about 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acids, each of which is independently selected from the group consisting of Gly, Ser, Arg, and the like, in any order. In one embodiment, the peptide linker may be represented as "GGSSRSSSSGGGGSGGGG" (SEQ IF NO: 65), or (GmS1)õ (m, 1, and n are the number of "G", "S", and "(GmS1)", respectively, and independently selected from integers of about 1 to about 10, particularly, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). For example, the peptide linker can be amino acids of SEQ ID NO: 65, (GGGGS)2, (GGGGS)3, (GGGGS)4, or (GS)9, but not be limited thereto.
[174] In an embodiment, the anti-MERS-CoV antibody or antigen-binding fragment thereof may be in a scFv-Fc form, wherein a scFv and Fc region are linked to each other via a peptide linker or directly (without a peptide linker). In the scFv-Fc, the scFv and the peptide linker are described as above; the Fc region may be derived from IgG
(e.g., IgGl, IgG2, IgG3, or IgG4), for example, represented by SEQ ID NO: 66;
and the Fc region may be linked to the N-terminus or C-terminus of the scFv in direction of N- to C-terminus or C- to N-terminus (i.e., the N-terminus of the scFv and the N-

23 terminus of the Fc region are linked, the N-terminus of the scFv and the C-terminus of the Fc region are linked, the C-terminus of the scFv and the N-terminus of the Fc region are linked, or the C-terminus of the scFv and the C-terminus of the Fc region are linked).
[175] In an embodiment, the scFv and/or the scFv-Fc antibody may further comprise a hinge region. In this case, the scFv and/or scFv-Fc are described as above;
the hinge region may be derived from IgG (e.g., IgGl, IgG2, IgG3, or IgG4), for example, rep-resented by SEQ ID NO: 65; and the hinge region may be linked to the N-terminus or C-terminus (e.g., N-terminus) of the Fc region in the scFv and/or the scFv-Fc.
[176] As used herein, the immunoglobulin such as IgG (e.g., IgGl, IgG2, IgG3, or IgG4) may be a human or murine immunoglobulin, for example, a human immunoglobulin (hIgG; e.g., hIgGl, hIgG2, hIgG3, or hIgG4).
[177]
[178] The binding affinity of an antibody or its antigen-binding fragment provided in this disclosure to RBD and/or S protein of MERS-CoV can be assessed using one or more techniques well established in the art. For example, the binding affinity may be assessed by a binding assay such as ELISA assays using a recombinant RBD
and/or S
protein of MERS-CoV, but not be limited thereto. For example, the binding affinity of an antibody or its antigen-binding fragment provided in this disclosure to RBD
and/or S protein of MERS-CoV may be KD of 1 x 106 M or less, 1 x 10 7 M or less, or 1 x 10 M or less, for example, 1.01 x 10 9 M or less, but not be limited thereto.
[179] Without limitation, the anti-MERS-CoV antibody or fragment thereof is a chimeric antibody, a humanized antibody, or a fully human antibody. In one aspect, antibody or fragment thereof is not naturally occurring, or chemically or recombinantly syn-thesized.
[180] Given that each of antibodies can bind to MERS-CoV, for example, receptor-binding domain (RBD) of MERS-CoV, the CDR sequences, or VL (heavy chain variable region) and VL (light chain variable region) sequences as disclosed herein can be "mixed and matched" to create other Anti-MERS-CoV binding molecules.
[181]
[182] Medical use [183] Provided is a medical use of an anti-MERS-CoV antibody or an antigen-binding fragment thereof provided herein for treating and/or preventing MERS-CoV
infection and/or a disease associated with MERS-CoV infection. Such medical use of the antibody or antigen-binding fragment may be due to the capability to compete with MERS-CoV (RBD and or S protein) for binding to a receptor on a host cell, thereby in-hibiting the binding of MERS-CoV to a receptor on a host cell and inhibiting infection of MERS-CoV.

24 [184] More specifically, an embodiment provides a pharmaceutical composition comprising the anti-MERS-CoV antibody or an antigen-binding fragment thereof as an active ingredient. The pharmaceutical composition may further comprise a pharma-ceutically acceptable carrier. The pharmaceutical composition may be used for preventing and/or treating MERS-CoV infection and/or a disease associated with MERS-CoV infection.
[185] Another embodiment provides a method of treating and/or preventing MERS-CoV
infection and/or a disease associated with MERS-CoV infection, comprising admin-istering a pharmaceutically effective amount of the anti-MERS-CoV antibody or an antigen-binding fragment thereof or the pharmaceutical composition to a subject in need of treating and/or preventing the MERS-CoV infection and/or the disease.
The method may further comprise a step of identifying the subject in need of treating and/
or preventing MERS-CoV infection and/or a disease associated with MERS-CoV
infection, prior to the administering step.
[186] Another embodiment provides a use of the anti-MERS-CoV antibody or an antigen-binding fragment thereof or the pharmaceutical composition in treating and/or preventing MERS-CoV infection and/or a disease associated with MERS-CoV
infection. Another embodiment provides a use of the anti-MERS-CoV antibody or an antigen-binding fragment thereof in preparing a pharmaceutical composition for treating and/or preventing MERS-CoV infection and/or a disease associated with MERS-CoV infection.
[187] The disease associated with MERS-CoV infection may be selected from any res-piratory syndrome caused by MERS-CoV infection (e.g., pneumonia, acute upper res-piratory infection, etc.) and/or complications thereof (e.g., respiratory failure, septic shock, etc.) and/or symptoms (e.g., cough, fever, shortness of breath, headache, chill, sore throat, rhinorrhea, muscular pain, inappetence, nausea, stomachache, emesis, diarrhea, etc.) associated with the respiratory syndrome.
[188] The pharmaceutical composition may further comprise a pharmaceutical acceptable carrier, solubilizer, diluent, emulsifier, preservative, excipient, and/or adjuvant, in addition to the active ingredient. For example, the pharmaceutically acceptable carrier may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. In certain embodiments, the phar-maceutical composition may further comprise formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or pen-

25 etration of the composition. In such embodiments, suitable formulation materials may be at least one selected from the group consisting of, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials;
an-tioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite);
buffers (such as borate, bicarbonate, Tris-HC1, citrates, phosphates or other organic acids);
bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin);
fillers;
monosaccharides; disaccharides; and other carbohydrates (such as glucose, sucrose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins);
coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol);
suspending agents;
surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; and excipients and/or pharmaceutical adjuvants.
[189] The antibody or antigen-binding fragment, or the pharmaceutical composition may be administered to a subject orally or parenterally. The parenteral administration may be intranasal administration, intrapulmonary administration, intravenous injection, sub-cutaneous injection, muscular injection, intraperitoneal injection, endothelial admin-istration, or local administration. Since oral administration leads to digestion of proteins or peptides, an active ingredient in the compositions for oral administration must be coated or formulated to prevent digestion in stomach. In addition, the com-positions may be administered using an optional device that enables the active in-gredient to be delivered to target cells or organ.
[190] For example, the antibody or antigen-binding fragment may be administered by in-tranasal or intrapulmonary route, such as nasal spray, inhalation, or nebulization.
[191] As used herein, the term "the pharmaceutically effective amount" may refer to an amount at which the active ingredient (the antibody or antigen-binding fragment) can exert pharmaceutically meaningful effects in preventing or treating MERS-CoV
infection and/or a disease associated with MERS-CoV infection. The pharmaceutically effective amount of the active ingredient, or a suitable dosage of the pharmaceutical composition indicated by an amount of the active ingredient, may be prescribed in a

26 variety of ways, depending on various factors, such as age, body weight, gender, pathologic conditions, diets, excretion speed, and/or reaction sensitivity of a patient, formulation types, administration time, administration route, administration manner, and the like. For example, the pharmaceutically effective amount of the active in-gredient, or a suitable dosage of the pharmaceutical composition, may be in the range from about 0.001 to about 1000 mg(amount of the antibody or antigen-binding fragment)/kg(body weight), about 0.01 to about 100 mg/kg, or 0.1 to 50 mg/kg per day for an adult.
[192] The subject to which the antibody or antigen-binding fragment or the pharmaceutical composition is administered may be one selected from mammals, for example, humans, monkeys, rats, mice, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on, or a cell or tissue obtained therefrom, but are not limited thereto. The subject may be one having a risk of MERS-CoV infection or suffering from MERS-CoV infection or a disease associated with MERS-CoV infection.
[193] The pharmaceutical composition may be formulated with a pharmaceutically ac-ceptable carrier and/or excipient into a unit or a multiple dosage form by a method easily carried out by a skilled person in the pertinent art. The dosage form may be a solution in oil or an aqueous medium, a suspension, syrup, an emulsifying solution, an extract, powder, granules, a tablet, or a capsule, and may further include a dispersing or a stabilizing agent. For example, the pharmaceutical composition may be formulated for intranasal or intrapulmonary delivery of the antibody or antigen-binding fragment, such as nasal spray, inhalation, or nebulization formulation.
[194]
[195] Polynucleotide, recombinant vector, and preparation of antibody [196] An embodiment provides a polynucleotide encoding the anti-MERS-CoV
antibody or an antigen-binding fragment thereof. In particular, an embodiment provides a first polynucleotide encoding a combination of VL-CDR1, VL-CDR2, and VL-CDR3, a heavy chain variable region, or a heavy chain of the anti-MERS-CoV antibody or an antigen-binding fragment thereof. Another embodiment provides a second polynu-cleotide encoding a combination of VH-CDR1, VH-CDR2, and VH-CDR3, a light chain variable region, or a light chain of the anti-MERS-CoV antibody or an antigen-binding fragment thereof.
[197] An embodiment provides a recombinant vector comprising the first polynucleotide, the second polynucleotide, or a combination thereof. The recombinant vector may be used as an expression vector of the polynucleotide. Another embodiment provides a re-combinant cell transfected with the recombinant vector.
[198] Another embodiment provides a method of preparing the anti-MERS-CoV
antibody or an antigen-binding fragment thereof, comprising expressing the first polynucleotide

27 and the second polynucleotide in a cell. The step of expressing the polynucleotides may be conducted by culturing the cell comprising the polynucleotides (for example, each of the polynucleotides is carried by each recombinant vector, or both of the polynucleotides are carried by one recombinant vector) under a condition allowing the expression of the polynucleotides. The method may further comprise isolating and/or purifying the anti-MERS-CoV antibody or an antigen-binding fragment thereof from the cell culture, after the step of expressing or culturing.
[199] The term "vector" refers to a means for expressing a target gene in a host cell, as ex-emplified by a plasmid vector, a cosmid vector, and a viral vector such as a bacte-riophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. The recombinant vector may be constructed from plasmids frequently used in the art (for example, pComb3XSS vector, pSC101, pGV1106, pACYC177, ColEL pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, p1161, pLAFR1, pHV14, pGEX series, pET series, and pUC19), phages (for example, Xgt4XB, X-Charon, XAzl, and M13) or by manipulating viruses (for example, SV40, etc.).
[200] In the recombinant vector, the polynucleotide may be operatively linked to a promoter. The term "operatively linked" is intended to pertain to a functional linkage between a nucleotide sequence of interest and an expression regulatory sequence (for example, a promoter sequence). When being "operatively linked", the regulatory element can control the transcription and/or translation of the nucleotide of interest.
[201] The recombinant vector may be constructed typically as a cloning vector or an ex-pression vector. For recombinant expression vectors, a vector generally available in the relevant art for expressing a foreign protein in plant, animal, or microbial cells may be employed. Various methods well known in the art may be used for the construction of recombinant vectors.
[202] For use in hosts, such as prokaryotic or eukaryotic cells, the recombinant vector may be constructed accordingly. For example, when a vector is constructed as an expression vector for use in a prokaryotic host, the vector typically includes a strong promoter for transcription (e.g., a pLia promoter, a CMV promoter, a trp promoter, a lac promoter, a tac promoter, a T7 promoter, etc.), a ribosomal binding site for initiating translation, and transcriptional/translational termination sequences. On the other hand, an ex-pression vector for use in a eukaryotic host includes an origin of replication operable in a eukaryotic cell, such as an fl origin of replication, an SV40 origin of replication, a pMB1 origin of replication, an adeno origin of replication, an AAV origin of replication, and a BBV origin of replication, but is not limited thereto. In addition, the expression vector typically includes a promoter derived from genomes of mammalian cells (for example, metallothionein promoter) or from mammalian viruses (for example, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cy-

28 tomegalovirus promoter, and tk promoter of HSV), and a polyadenylation sequence as a transcription termination sequence.
[203] The recombinant cell may be prepared by introducing the recombinant vector into a suitable host cell. As long as it allows the sequential cloning and expression of the re-combinant vector in a stable manner, any host cell known in the art may be employed in the present disclosure. Examples of the prokaryotic host cell available for the present disclosure may be selected from E. coli, Bacillus spp. such as Bacillussubtilis and Bacillus thuringiensis, and enterobacteriaceae strains such as Salmonella ty-phimurium, Serratia marcescens and various Pseudomonas species. Eukaryotic host cells that may be used for transformation may selected from, but are not limited to, Saccharomyce cerevisiae, insect cells, and animal cells, such as Sp2/0, CHO
(Chinese hamster ovary) Kl, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK.
[204] The polynucleotide or a recombinant vector carrying the same may be introduced (transfected) into a host cell using a method well known in the relevant art.
For example, this transfection may be carried out using a CaCl2 or electroporation method when the host cell is prokaryotic. For eukaryotic host cells, the genetic introduction may be achieved using, but not limited to, microinjection, calcium phosphate pre-cipitation, electroporation, liposome-mediated transfection, or particle bombardment.
[205] To select a transformed host cell, advantage may be taken of a phenotype associated with a selection marker according to methods well known in the art. For example, when the selection marker is a gene conferring resistance to a certain antibiotic, the host cells may be grown in the presence of the antibiotic in a medium to select a transformant of interest.
[206] Another embodiment provides a method for production of the antibody or antigen-binding fragment, the method comprising a step of expressing the polynucleotide or the recombinant vector in a host cell. In one embodiment, the production method may comprise culturing a recombinant cell harboring the polynucleotide or the recombinant vector thereat, and optionally isolating and/or purifying the antibody from the culture medium.
[207]
Advantageous Effects of Invention [208] Middle East respiratory syndrome coronavirus (MERS-CoV) induces severe ag-gravating respiratory failure in infected patients, frequently resulting in mechanical ventilation. As limited therapeutic antibody is accumulated in lung tissue following systemic administration, inhalation is newly recognized as an alternative, possibly better, route of therapeutic antibody for pulmonary diseases. The nebulization process,

29 however, generates diverse physiological stresses, and thus, the therapeutic antibody must be resistant to these stresses, remain stable, and form minimal aggregates. The disclosure provides antibodies against MERS-CoV with significantly greater stability, reactivity and neutralizing activity following nebulization.
[209]
Brief Description of Drawings [210] Figures la and lb are graphs showing reactivity of scFv clones before and after neb-ulization.
[211] Figure 2 (A-D) shows results of flow cytometry analysis of the inhibition of re-combinant S glycoprotein binding to hDPP4-expressing cells.
[212] Figure 3 (A-B) schematically shows sequential randomization of CDR
residues of the C-8 clone.
[213] Figure 4 (A-D) shows graphs showing reactivity of C-8, C-8-2, and C-8-2-4B clones in scFv-Fc fusion protein form or IgG1 (full-length) form before and after nebu-lization.
[214] Figure 5 (A-C) shows graphs showing Reactivity of anti-MERS-CoV IgG1 an-tibodies before and after nebulization.
[215] Figure 6 shows graphs showing DLS analysis results.
[216] Figures 7a-7c are graphs showing relative infection (%), indicating neutralization of MERS-CoV by pre- and post-nebulized IgGl.
[217] Figure 8 shows graphs showing reactivity of anti-MERS-CoV IgG1 antibodies against recombinant MERS-CoV S RBD mutants.
[218] Figures 9a and 9b are graphs showing results of flow cytometry analysis of the in-hibition of recombinant mutant MERS-CoV RBD protein binding to hDPP4-expressing cells.
[219] Figures 10a-10c show HDX profiles of free- and C-8 IgGi-bound MERS-CoV RBD, wherein 10a shows three-dimensional structure of MERS-CoV RBD, 10b shows graphs corresponding to (A) region (5er498-Ala520) indicated in 10a (upper graph:
deuterium uptake of 5498-L506; lower graph: deuterium uptake of RBD L507-A520), and 10c shows a graph corresponding to (B) region (Tyr540-Leu554) indicated in 10a, wherein "Single" indicates MERS-CoV RBD only (C-8 IgG1 antibody does not bind to MERS-CoV RBD), and "complex" indicates a complex of MERS-CoV RBD and C-8 IgG1 antibody.
[220] Figure 11 illustrates mapping of the C-8 epitope and DPP4 binding site on MERS-CoV RBD sequence.
[221]
Mode for the Invention

30 [222] Hereafter, the present invention will be described in detail by examples. The following examples are intended merely to illustrate the invention and are not construed to restrict the invention.
[223]
[224] Example 1. Construction of a human scFv phage-display library and three ran-domization libraries [225] PBMCs were isolated from two MERS-CoV-infected convalescent patients using a Ficoll-Paque density gradient medium (GE Healthcare, Pittsburgh, PA, USA) as described previously (Kanof ME, Smith PD, Zola H. Isolation of whole mononuclear cells from peripheral blood and cord blood. Curr Protoc Immunol. 2001;Chapter 7:Unit 7 1). The PBMCs were subjected to total RNA isolation using the TRI Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The RNA
was used to synthesize cDNA using Superscript III First-Strand Synthesis system (Invitrogen) with oligo(dT) primers according to the manufacturer's instructions.
Using the cDNA as a template, the genes encoding the variable regions of heavy and light chains (VH and Vic/V),) were amplified and used for the construction of a human scFv phage-display libraries as described previously (Phage Display: A
Laboratory Manual. Carlos F. Barbas III, Dennis R. Burton, Jamie K. Scott, Gregg J.
Silverman.
2001;76(4):487-488; Andris-Widhopf J, Steinberger P, Fuller R, Rader C, Barbas CF, 3rd. Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences. Cold Spring Harb Protoc.
2011;2011(9)).
[226] For the construction of the first randomization library, a set of degenerate Ultramer DNA oligonucleotides (Integrated DNA Technologies, Coralville, IA, USA) encoding residues from H1 to H65 of clone C-8 (VH,H) was chemically synthesized to contain either a codon encoding the wild-type amino acid or a GAK degenerate codon at the H29, H32, H51, H52, H53, and H54 residues (Table 3). Then, the gene fragment (VHc) encoding residues from H58 to H113 of clone C-8 was amplified by PCR in a T100 Thermal Cycler (Bio-Rad, Carlsbad, CA, USA). The PCR conditions were as follows:
preliminary denaturation at 94 C for 5 min, followed by 25 cycles of 15 s at 94 C, 15 s at 56 C and 90 s at 72 C. A final extension was then conducted for 10 min at 72 C.
[227] After electrophoresis on a 1% agarose gel, the PCR products were purified using QIAquick gel extraction kit (Qiagen Inc., Valencia, CA, USA) according to the manu-facturer's instructions. The purified VHNT1 and Vfic gene fragments were mixed at a con-centration of 100 ng and subjected to linker PCR in a T100 Thermal Cycler to yield the VH1 fragment. The PCR conditions were as follows: preliminary denaturation at for 5 min, followed by 25 cycles of 15 s at 94 C, 15 s at 56 C and 120 s at 72 C. The reaction was ended with an extension step for 10 min at 72 C. The gene fragment encoding VL (VIA) of clone C-8 was amplified by PCR with the same PCR
conditions

31 described above for amplification of VHc. Then, the VH1 and VII fragments were subjected to electrophoresis on a 1% agarose gel, and excised bands were purified using the QIAquick gel extraction kit. The purified VH1 and VII fragments were used for the synthesis of the scFv gene (scFv) using PCR as described previously (Phage Display: A Laboratory Manual. Carlos F. Barbas III, Dennis R. Burton, Jamie K.
Scott, Gregg J. Silverman. 2001;76(4):487-488). The amplified scFv i fragment was purified and cloned into the phagemid vector as described (Phage Display: A Laboratory Manual. Carlos F. Barbas III, Dennis R. Burton, Jamie K. Scott, Gregg J.
Silverman.
2001;76(4):487-488; Andris-Widhopf J, Steinberger P, Fuller R, Rader C, Barbas CF, 3rd. Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences. Cold Spring Harb Protoc.
2011;2011(9)).
[228] For the construction of the second randomization library, a set of degenerate Ultramer DNA oligonucleotides encoding residues from H1 to H65 of clone C-8-2 (V
HN2) was chemically synthesized to contain either a codon encoding the wild-type amino acid or a GAK degenerate codon at the H26 to H33 (HCDR1) and H51 to H57 (HCDR2) residues (Table 3), excluding the previously randomized residues. The and VHc gene fragments were mixed at equal ratios at 100 ng and subjected to linker PCR in a T100 Thermal Cycler to yield the VH2 gene fragment as described above. The VH2 gene fragment was purified as described above and subjected to linker PCR
with V
Li fragments to yield the scFv2 gene fragment, which was cloned into the phagemid vector as described above.
[229] For the construction of the third randomization library, two sets of degenerate Ultramer DNA oligonucleotides with a length of 200 nucleotides were chemically syn-thesized. One set encoded from Li to L61 residues of clone C-8 (VLN), while the other one encoded from L56 to L107 of clone C-8 (Vir). These degenerate oligonucleotides contained either a codon encoding the wild-type amino acid or a GAK degenerate codon at L27B, L27C, L30, L32, L50, L89, L92, and L96 residues (Table 3). The VLN
and Vir gene fragments (100 ng each) were subjected to a linker PCR in a T100 Thermal Cycler to produce the VL2 gene fragment using the same PCR conditions as described above for the amplification of the VH1 gene fragment. The gene fragment encoding VH of C-8-2-4B (VH3) was amplified by PCR using the same PCR
conditions used for the amplification of the Vfic gene fragment as described above. After pu-rification, VL2 and VH3 gene fragments were used to produce the scFv3 gene fragment, which was cloned into the phagemid vector as described above.

32 [230] [Table 31 Degenerate ccidoiic in7ed in the randomized libraries The fir.: H:

flu -r acid De-Jen KW
erate KAK RVVK RVVK KWK KWK
cidon acids encode D,E, bill I 7'1 N F.L Y FLY
he .
F DE
,LV ,V.b \e õ ,b, ! ly"
DE DE
de-Jene ra7:e.
codon The second library KaI.r.a H52 H26 H27 H28 H29 H30 H31 nu i-ieisr A
GGTESSEA I I P F F GT A
Dn acid era:e GRK GRF lb RRK RRK GRK i:YAK
aciciD
P R, G.D G.D, T KH A.D G. .D
K,A .
!he E E E
D.E
degene E E
ra,.e en The third library Kai:a4, L2 L2 L2 L2 L2 L2 L2 L2 L3 L3 L31 L8 L9 L9 L9 L9 L9 L9 L9 L9 nu rie'sr 7 Th7B70707E 8 9 0 1 2 9 0 1 2 3 4 5 6 7 A
LL HS NGYN Y L GS MDALIDTPLI
acid erx.e. K K
K K K
cocion K
Eno acids LF L,F d LF M.1 L.F L,F
.
encoe ,HõH, , .H, Y, Y, H
d 00. D N. 0. 0, D
V, V , , V, V, E , ID, 0, E ,D
D. 0, 0, EE
codon [231]
[232] Example 2. Biopanning [233] The human scFv phage-display libraries were subjected to four rounds of biopanning against recombinant MERS-CoV S RBD protein (Sino Biological Inc., Beijing, China)

33 as described previously (Lee Y, Kim H, Chung J. An antibody reactive to the Gly63-Lys68 epitope of NT-proBNP exhibits 0-glycosylation-independent binding.

Exp Mol Med. 2014;46:e114). Briefly, the scFv phage-display libraries (-10"
phage) were added to 3[1g of the recombinant MERS-CoV S RBD protein conjugated to 5.0 x 106 magnetic beads (Dynabeads M-270 epoxy, Invitrogen) and incubated with rotation for 2 h at 37 C. The beads were washed once with 500 [IL of 0.05% (v/v) Tween-(Sigma-Aldrich, St. Louis, MO, USA) in PBS (PBST) during the first round of biopanning. The number of washes was increased to three for the other rounds.
Phages bound to beads were eluted, neutralized, allowed to infect E. coli ER2738 (New England Biolabs, Ipswich, MA, USA), and rescued as described previously (Lee Y, Kim H, Chung J. An antibody reactive to the Gly63-Lys68 epitope of NT-proBNP
exhibits 0-glycosylation-independent binding. Exp Mol Med. 2014;46:e114).
[234] The first randomized scFv library was subjected to two rounds of biopanning against recombinant MERS-CoV S RBD protein. The scFv phage-display library (-10"
phage) was added to 1.5[1g of the recombinant MERS-CoV S RBD protein conjugated to 2.5 x 106 magnetic beads and incubated with rotation for 2 h at 37 C. The beads were washed once with 500 [AL of 0.5% PBST and three times with 500 [IL of 0.5%
PBST during the first and second rounds of biopanning, respectively. After each round of washing, bound phages were eluted and rescued as described above.
[235] For first round of biopanning for the second and third randomized scFv libraries, the scFv phage-display libraries (-10" phage) were added to 1.5[1g of the recombinant MERS-CoV S RBD protein conjugated to 2.5 x 106 magnetic beads and incubated with rotation for 2 h at 37 C. After washing three times with 500 [IL of 0.5% PBST, bound phages were eluted and rescued as described above.
[236] Before the second round of biopanning of the second and third randomized scFv libraries, 10 [tg of recombinant MERS-CoV S RBD protein was conjugated to 200 [tg of non-magnetic beads (Nacalai, San Diego, CA, USA) following the manufacturer's instructions. Then, the scFv phage-display libraries (-10" phage) were added to 1.5[1g of recombinant MERS-CoV S RBD protein conjugated to 2.5 x 106 magnetic beads and incubated on a rotator for 2 h at 37 C. After washing three times with 500 [IL of 0.5% PBST, magnetic beads were resuspended in 100 [AL of PBS and transferred to a microtube (microTUBE AFA Fiber Pre-Slit Snap-Cap, 520045, Covaris, Woburn, MA, USA) along with the recombinant MERS-COV S RBD protein-conjugated non-magnetic beads resuspended in 30 [IL of PBS at a concentration of 0.33 [tg/mL.
Then, these bead mixtures were subjected to an ultrasound washing step using an ultra-sonicator (M220, Covaris) with the following conditions: duty factor (DF) 20%, peak incident power (PIP) 12.5 W, cycles/burst 50, 20 min, and 24 C. After ultrasonication, magnetic beads were transferred to 1.5-mL microcentrifuge tube and washed three

34 times with 0.5% PBST. Then, the bound phages were eluted and rescued as described above.
[237]
[238] Example 3. High-throughput retrieval of scFv clones and phage ELISA
[239] After the fourth round of biopanning of human scFv phage-display libraries, the plasmid DNA was obtained from overnight cultures of E. coli cells and subjected to high-throughput retrieval of scFv clones by TrueRepertoire analysis as described previously (Celemics, Seoul, Republic of Korea) (Noh J, Kim 0, Jung Y, Han H, Kim JE, Kim S, et al. High-throughput retrieval of physical DNA for NGS-identifiable clones in phage display library. MAbs. 2019;11(3):532-545).
[240] To select reactive clones to recombinant MERS-CoV S RBD protein, the scFv genes obtained from TrueRepertoire were cloned into the pComb3XSS vector (Phage Display: A Laboratory Manual. Carlos F. Barbas III, Dennis R. Burton, Jamie K.
Scott, Gregg J. Silverman. 2001;76(4):487-488) and used to transform E. coli ER2738 cells.
After overnight culture, the phages were rescued from individual colonies using the M13K07 helper phage and subjected to phage ELISA as described previously (Phage Display: A Laboratory Manual. Carlos F. Barbas III, Dennis R. Burton, Jamie K.
Scott, Gregg J. Silverman. 2001;76(4):487-488). Microtiter plates (Costar, Cambridge, MA, USA) were coated with 100 ng of recombinant MERS-CoV S RBD protein in coating buffer (0.1 M sodium bicarbonate, pH 8.6) at 4 C overnight. The wells were blocked with 3% (w/v) bovine serum albumin (BSA; Thermo Scientific, Waltham, MA, USA) dissolved in PBS for 1 h at 37 C, and culture supernatant containing scFv-displayed phages that were rescued from individual colonies were added into each well.
After in-cubation for 2 h at 37 C, the microtiter plates were washed three times with 0.05%
PBST. Then, horseradish peroxidase (HRP)-conjugated anti-M13 monoclonal antibody (GE Healthcare) in 3% BSA/PBS was added into wells, and the plate was incubated for 1 h at 37 C. After washing three times with PBST, 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid solution (Thermo Scientific) was used as the substrate for HRP. Absorbance was measured at 405 nm with a Multiskan Ascent microplate reader (Labsystems, Helsinki, Finland).
[241] To select reactive clones from the randomized libraries, phage ELISA
was performed as described previously (Phage Display: A Laboratory Manual. Carlos F. Barbas III, Dennis R. Burton, Jamie K. Scott, Gregg J. Silverman. 2001;76(4):487-488) using re-combinant MERS-CoV S RBD protein-coated microtiter plates. The nucleotide sequences of positive scFv clones were determined by Sanger sequencing (Cosmogenetech, Seoul, Republic of Korea).
[242]
[243] Example 4. Expression of scFv-hFc and IgG1

35 [244] The genes encoding the selected scFv clones were cloned into a modified mammalian expression vector containing the hIgGi Fc regions (hFc) at the C-terminus as described previously (Lee S, Yoon IH, Yoon A, Cook-Mills JM, Park CG, Chung J.
An antibody to the sixth Ig-like domain of VCAM-1 inhibits leukocyte transendothelial migration without affecting adhesion. J. Immunol.
2012;189(9):4592-4601). The expression vectors were transfected into HEK293F
cells (Invitrogen), and the fusion proteins were purified by Protein A affinity chro-matography as described previously (Lee S, Yoon IH, Yoon A, Cook-Mills JM, Park CG, Chung J. An antibody to the sixth Ig-like domain of VCAM-1 inhibits leukocyte transendothelial migration without affecting adhesion. J. Immunol.
2012;189(9):4592-4601).
[245] For the expression of IgGI (with kappa light chain), genes encoding VH and V, were amplified from the phage clones, cloned into a mammalian expression vector, and transfected into HEK293F cells. Then, IgGI was purified by Protein A affinity chro-matography as described previously (Jin J, Park G, Park JB, Kim S, Kim H, Chung J.
An anti-EGFR x cotinine bispecific antibody complexed with cotinine-conjugated duo-carmycin inhibits growth of EGFR-positive cancer cells with KRAS mutations.
Exp Mol Med. 2018;50(5):67). Then the eluate containing IgGI was subjected to gel filtration chromatography. A total of 4 mg of IgGI was injected at a flow rate of 1 mL/
min and purified by gel filtration using a XK16/100 column packed with Superdex 200 pg at pH 7.4 (AKTA pure, GE Healthcare). The chromatogram was recorded at a UV

absorbance of 280nm. The fractions containing IgGI were pooled by collection criteria and concentrated.
[246]
[247] Example 5. Experimental methods [248] 5.1. ELISA
[249] Microtiter plates (Costar) were coated with 100 ng of recombinant S
glycoprotein (GenBank Accession No. AF588936.1; wild type) in coating buffer at 4 C
overnight.
The wells were blocked with 3% BSA/PBS for 1 h at 37 C. Both nebulized and non-nebulized scFv-hFc or IgGI were serially diluted (5-fold, 12 dilutions starting from 500 nM for scFv-hFc fusion protein or 1,000 nM for IgGI) in blocking buffer and added into individual wells. After incubation for 1 h at 37 C, the microtiter plates were washed three times with 0.05% PBST. Then, HRP-conjugated rabbit anti-human IgG

antibody (Invitrogen) in blocking buffer (1:5,000) was added into wells, and the plate was incubated for 1 h at 37 C. After washing three times with PBST, 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid solution (Thermo Scientific) was used as the substrate. Absorbance was measured at 405 nm using a microplate spec-trophotometer (Multiskan GO; Thermo Scientific).

36

37 PCT/KR2020/012887 [250]
[251] 5.2. Nebulization [252] A nebulizer (Aerogen Pro, Aerogen, Galway, Ireland) was used for all experiments following the manufacturer's instructions. The nebulizer containing 1 mL of scFv-hFc fusion proteins or IgGI antibodies was placed on top of a 50-mL conical tube (SPL Life Sciences, Pocheon, Republic of Korea) and nebulized at a concentration of either 0.1, 0.3, or 1 mg/mL in phosphate-buffered saline (PBS).
[253]
[254] 5.3. Microneutralization assay [255] The virus (MERS-CoV/KOR/KNIH/002 05 2015, accession number KT029139.1) was obtained from the Korea National Institute of Health (kindly provided by Dr. Sung Soon Kim) and propagated in Vero cells (ATCC CCL-81) in Dulbecco's Modified Eagle's Medium (DMEM, Welgene, Gyeongsan, Republic of Korea) in the presence of 2% fetal bovine serum (Gibco). The cells were grown in T-75 flasks, inoculated with MERS-CoV, and incubated at 37 C in a 5% CO2 environment. Then 3 days after in-oculation, the viruses were harvested and stored at -80 C. The virus titer was de-termined via a TCID50 assay (Reed LJ, Muench H. Lancaster Press, In-corporated:1938).
[256] A neutralization assay was performed as previously described (Jiang L, Wang N, Zuo T, Shi X, Poon KM, Wu Y, et al. Potent neutralization of MERS-CoV by human neu-tralizing monoclonal antibodies to the viral spike glycoprotein. Sci Transl Med.
2014;6(234):234ra259). Briefly, Vero cells were seeded in 96-well plates (1 x cells/well) in Opti-PRO SFM (Thermo Scientific) supplemented with 4 mM L-glutamine and lx Antibiotics-Antimycotic (Thermo Scientific) and grown for 24 h at 37 C in a 5% CO2 environment. Two-fold serially diluted scFv-hFc fusion proteins were mixed with 100 TCID50 of MERS-CoV, and the mixture was incubated for 30 min at 37 C. Then, the mixture was added to the Vero cells in tetrad and incubated for 4 days at 37 C in a 5% CO2 environment. The cytopathic effect (CPE) in each well was visualized following crystal violet staining 4 days post-infection. The IC50 values were calculated using the dose-response inhibition equation of GraphPad Prism (GraphPad Software, La Jolla, CA, USA).
[257]
[258] 5.4. Flow cytometry [259] The scFv-hFc fusion proteins (2,000, 1000, 250, or 200 nM) were incubated either with 200 nM of the recombinant S glycoprotein fused with a polyhistidine tag at the C-terminus (Sino Biological Inc.) or without S protein in 50 [IL of 1% (w/v) BSA
in PBS
containing 0.02% (w/v) sodium azide (FACS buffer) at 37 C for 1 h. The m336 scFv-hFc and irrelevant scFv-hFc fusion proteins were used as positive and negative controls, respectively. Huh-7 cells (hDPP4+)(KCLB (Korean Cell Line Bank) Accession number KCLB #60104) were added into v-bottom 96-well plates (Corning, Corning, NY, USA) at a density of 3 x 105 cells per well, and then, the mixture was added to the wells. After incubation at 37 C for 1 h, cells were washed three times with FACS buffer and incubated with FITC-labeled rabbit anti-HIS Ab (Abcam, Cambridge, UK) at 37 C for 1 h. Then, the cells were washed three times with FACS
buffer, resuspended in 200 [AL of PBS, and subjected to analysis by flow cytometry using a FACS Canto II instrument (BD Bioscience, San Jose, CA, USA). For each sample, 10,000 cells were assessed, and the data were analyzed using the FlowJo software (TreeStar, Ashland, OR, USA).
[260]
[261] 5.5. SE-HPLC (size exclusion HPLC) [262] Non-nebulized and nebulized samples were analyzed using Waters e2695 HPLC
system (Waters Corporation, Milford, MA, USA) equipped with a BioSuite high-resolution size-exclusion chromatography column (250 A 7.5 mm x 300 mm). Each sample (10 [tg) was injected at a flow rate of 1 mL/min. The mobile phase was PBS
(pH 7.4), and UV detection was performed at 280 nm/220 nm. The sample tray and column holder were maintained at 4 and 30 C, respectively, throughout data ac-quisition. The molecular weights corresponding to the antibody peaks were calculated using the Empower software (Waters Corporation, USA).
[263]
[264] 5.6. DLS (Dynamic light scattering) assay [265] DLS experiments were performed using a Zetasizer Nano S (Malvern Panalytical Ltd, Malvern, UK) and a 633-nm/4-mW laser at a 173 detection angle as described previously (Van Heeke G, Allosery K, De Brabandere V, De Smedt T, Detalle L, de Fougerolles A. Nanobodies(R) as inhaled biotherapeutics for lung diseases.
Pharmacol Ther. 2017;169:47-56). Non-nebulized and nebulized samples were analyzed by performing three acquisitions per sample. PBS (pH 7.4) was used as the reference solvent. The results were evaluated with the Zetasizer software 7.02.
[266]
[267] 5.7. PRNT (plaque reduction neutralization test) assay [268] Vero cells were seeded in 12-well plates (3.5 x 105 cells/well) in Opti-PRO SFM
supplemented with 4 mM L-glutamine and lx Antibiotics-Antimycotic (Thermo Scientific) and grown for 24 h at 37 C in a 5% CO2 environment. IgGI
antibodies were serially diluted three-fold in Dulbecco's PBS (Welgene) and mixed with an equal volume of culture media containing MERS-CoV/KOR/KNIH/002 05 2015 (100 pfu).
After incubation for 1 h at 37 C in a 5% CO2 environment, the virus-antibody mixture was added to the cells and maintained for 1 h at room temperature. The mixture was

38 then removed, and the cells were overlaid with 1% agarose in DMEM. After in-cubation for 2 days at 37 C in a 5% CO2 environment, the cells were washed with PBS
and fixed for 24 h with 4% paraformaldehyde. The agarose overlay was removed, and the cell monolayer was gently washed with water to remove residual agarose.
The cells were stained with 0.5% crystal violet solution, and the plaques were counted manually.
The number of plaques was plotted as a function of IgGI antibodies, and the con-centration of IgGI at which the number of plaques was reduced by 50% compared to that in the absence of IgGI (PRNT50) was calculated using GraphPad Prism 6.
[269]
[270] Example 6. Generation of antibodies reactive to recombinant MERS-CoV
RBD
protein from patients [271] Human single-chain variable fragment (scFv) phage-display libraries were generated using peripheral blood mononuclear cells (PBMCs) isolated from two MERS-CoV-infected convalescent patients. One patient (P014) was considered to be the super spreader, and the other patient (P002) was the wife of the index patient. The complexity of the libraries exceeded 3.6 x 109 and 1.9 x 109 colony-forming units for patients P002 and P014, respectively. After the third and fourth rounds of biopanning against recombinant MERS-CoV S RBD protein, the scFv clones were retrieved in a high-throughput manner as described previously (Noh J, Kim 0, Jung Y, Han H, Kim JE, Kim S, et al. High-throughput retrieval of physical DNA for NGS-identifiable clones in phage display library. MAbs. 2019;11(3):532-545). Briefly, 1,800 micro-colonies formed on the TR chip, and of these, 542 clones with unique VH and Vic/V), were identified. In these clones, 44 unique hCDR3 sequences were identified.
We selected 44 clones encoding unique hCDR3 sequences and rescued phages for phage enzyme-linked immunosorbent assay (ELISA) analysis. A total of 36 unique scFv clones were highly reactive to recombinant MERS-CoV S RBD protein (data not shown). These clones were prepared as scFv fused with human Fc (scFv-hFc) using a eukaryotic expression vector and HEK293F cells. A human anti-MERS-CoV neu-tralizing mAb reported previously, m336 (https://www.nature.com/articlesincomms9223?origin=ppub), was also prepared in this same form for use as a positive control.
[272] Amino acid sequences of prepared antibodies (scFv or scFv-Fc) are illustrated in Tables 4 to 17:

39 [273] [Table 41 Clone/ Part Amino Acid Sequence (N¨>C) SEQ ID
name NO

(N-VL-V VL CDR2 LGS 8 H-C) VL

NGYNYLDWYLQRPGQSPQLLIYLGSNRASG
VPDRFSGSGSGTDFTLKIGRVEAEDVGIYYC
MQALQTPLTFGGGTKVEIK
VH

SYAISWVRQAPGQGLEWMGGIIPFFGTANY
AQKFQGRVTITADESTSTAYMELSSLRSEDT
AVYYCARDGRKDYYGSGSYLHYYGMDV
WGQGTTVTVSS

40 [274] [Table 51 Clone/ Part Amino Acid Sequence (N¨>C) SEQ ID
name NO

(N-VL-li VL CDR2 LGS 8 nker-VH

-hinge-F
c-C)( see VH¨CDR1 DGKEKREA 25 Example VH CDR2 IIPFFDKA 32 8) VH CDR3 ARDGRKDYYGSGSYLHYYGMDV 38 Linker of GGSSRSSSSGGGGSGGGG 64 VL-VH
hinge EPKSSDKTHTSPPCP 65 Fc VVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGK
VH

REAISWVRQAPGQGLEWMGGIIPFFDKANY
AQKFQGRVTITADESTSTAYMELSSLRSEDT
AVYYCARDGRKDYYGSGSYLHYYGMDV
WGQGTTVTVSS
VL

NGYNYLDWYLQRPGQSPQLLIYLGSNRASG
VPDRFSGSGSGTDFTLKIGRVEAEDVGIYYC
MQALQTPLTFGGGTKVEIK

41 [275] [Table 61 Clone/ Part Amino Acid Sequence (N¨>C) SEQ ID
name NO

(N-VL-li nker-VH-hinge-Fc- VH¨CDR1 DGKEKREA 25 C) (see VH CDR2 IIPFFDKA 32 Example VH CDR3 DGRKDYYGSGSYLHYYGMDV 38 8) Linker of VL- GGSSRSSSSGGGGSGGGG 64 VH
hinge EPKSSDKTHTSPPCP 65 Fc VVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLS
LSPGK
VH

KREAISWVRQAPGQGLEWMGGIIPFFDKA
NYAQKFQGRVTITADESTSTAYMELSSLRSE
DTAVYYCARDGRKDYYGSGSYLHYYGMD
VWGQGTTVTVSS
VL

NGYNYLDWYLQRPGQSPQLLIYLGSNRASG
VPDRFSGSGSGTDFTLKIGRVEAEDVGIYYC
MQAVQTPLTFGGGTKVEIK

42 [276] [Table 71 Clone/ Part Amino Acid Sequence (N¨>C) SEQ ID
name NO

(N-VL-V VL CDR2 LGS 8 H-C) VL

NGYNYLDWYLQRPGQSPQLLIYLGSNRASG
VPDRFSGSGSGTDFTLKIGRVEAEDVGIYYC
MQALQTPLTFGGGTKVEIK
VH

KEAISWVRQAPGQGLEWMGGIIPFFDKAN
YAQKFQGRVTITADESTSTAYMELSSLRSED
TAVYYCARDGRKDYYGSGSYLHYYGMDV
WGQGTTVTVSS

43 [277] [Table 81 Clone/ Part Amino Acid Sequence (N¨>C) SEQ ID
name NO

(N-VL-V

H-C) VL

NGYNYLDWYLQRPGQSPQLLIYEGSNRASG
VPDRFSGSGSGTDFTLKIGRVEAEDVGIYYC
MQALQTPLTFGGGTKVEIK
VH

KEAISWVRQAPGQGLEWMGGIIPFFDKAN
YAQKFQGRVTITADESTSTAYMELSSLRSED
TAVYYCARDGRKDYYGSGSYLHYYGMDV
WGQGTTVTVSS

44 [278] [Table 9]
Clone/ Part Amino Acid Sequence (N¨>C) SEQ ID
name NO

(N-VL-V VL CDR2 SNN 10 H-C) VL

YVYWYQQLPGTAPKLLIYSNNQRPSGVPDR
FSGSKSGTSASLAISGLRSEDEADYYCATWD
DNLSGPVFGGGTKVTVLG
VH

SYYWGWIRQPPGKGLEWIGSIFYIGNTYYN
PSLKSRVTISVDTSKNQFSLRLSSVTAADTA
VYYCARQEGSSIIRFDPWGQGTLVTVSS

45 [279] [Table 101 Clone/ Part Amino Acid Sequence (N¨>C) SEQ ID
name NO

(N-VL-V VL CDR2 WAS 11 H-C) VL

SSNNKNYLAWYQQKPGQPPKLLIYWASTRE
SGVPDRFSGSGSGTDFTLTISSVQTEDVAVY
YCQQYYGSPYTFGQGTKLEIK
VH

SYGISWVRQAPGQGLEWMGRIIPILGIANY
AQKFQGRVTITADKSTSTAYMELSSLRSEDT
AVYYCASLFDSSGYYPYYFDYWGQGTLVT
VSS

46 [280] [Table 111 Clone/ Part Amino Acid Sequence (N¨>C) SEQ ID
name NO

(N-VL-V VL CDR2 WAS 11 H-C) VL

SSNNKNYLAWYQQKPGQPPKLLISWASTRE
SGVPERFSGSGSGTDFTLTISGLQAEDVAVY
YCQQYYSTPPTFGQGTKVDIK
VH

SFTISWVRQAPGQGLEWMGRIIPIFGIANYA
QKFQGRVTITADKSTGTAYMELSSLRSEDTA
VYYCATHFGASGYDPYYFDYWGQGTLVT
VSS

47 [281] [Table 121 Clone/ Part Amino Acid Sequence (N¨>C) SEQ ID
name NO

(N-VL-V VL CDR2 GNS 12 H-C) VL

YDVHWYQQLPGTAPKLLIYGNSNRPSGVPD
RFSGSKSGTSASLAISGLQSEDEGDYYCAA
WDDSLSGPVFGGGTELTVLG
VH

SYYWGWIRQPPGKGLEWIGSIFYIGNTYYN
PSLKSRVTISVDTSKNQFSLKLSSVTAADTA
VYYCARQEGSSIIRFDPWGQGTLVTVSS

48 [282] [Table 131 Clone/ Part Amino Acid Sequence (N¨>C) SEQ ID
name NO

(N-VL-V VL CDR2 SNN 10 H-C) VL

YVYWYQQLPGTAPKLLIYSNNHRPSGVPDR
FSGSKSGTSASLAISGLRSEDEAVYYCAAW
DDSLSGVVFGGGTELTVLG
VH

SYYWGWIRQPPGKGLEWIGSIYYTGNTYYN
PSLKSRLTISVDTSKNQFSLKLSSVTAADTA
VYYCARQVADLGYFDYWGQGTLVTVSS

49 [283] [Table 141 Clone/ Part Amino Acid Sequence (N¨>C) SEQ ID
name NO

(N-VL-V VL CDR2 SNS 13 H-C) VL

NYVYWYQQLPGAAPKLLIYSNSQRPSGVPD
RFSGSKSGTSASLVISGLRSEDEADYYCAAW
DDSLSGPVFGGGTQLTVLG
VH

SYYWGWIRQPPGKGLEWIGSIYYTGNTYYN
PSLKSRLTISVDTSKNQFSLKLSSVTAADTA
VYYCARQVADLGYFDYWGQGTLVTVSS

50 [284] [Table 151 Clone/ Part Amino Acid Sequence (N¨>C) SEQ ID
name NO

(N-VL-V VL CDR2 SNN 10 H-C) VL

YVYWYQQLPGTAPKLLIYSNNQRPSGVPDR
FSGSKSGTSASLAINGLQSEDEADYYCAAW
DDSLNGPVFGGGTKLTVLG
VH

SYYWGWIRQPPGKGLEWIGSIYYTGNTYYN
PSLKSRLTISVDTSKNQFSLKLSSVTAADTA
VYYCARQVADLGYFDYWGQGTLVTVSS

51 [285] [Table 161 Clone/ Part Amino Acid Sequence (N¨>C) SEQ ID
name NO

(N-VL-V VL CDR2 QDS 14 H-C) VL

EKYASWYQQRPGQSPVLVIYQDSRRASGIP
ERFS GSNS GNTATLTIS GT QAMDEADYYCQ
AWDSRRAVFGGGTELTVLG
VH

YSMNWVRQAPGKGLEWVSSISTTGSYIFYA
DSVKGRFTISRDNAKNSLYLQMNTLRPEDT
ALYYCAKGTAFDGGLAFDIWGQGTIVTVS
S

52 [286] [Table 171 Clone/ Part Amino Acid Sequence (N¨>C) SEQ ID
name NO

(N-VL-V VL CDR2 QDT 71 H-C) VL

VFWYQQKPGQSPVLAIYQDTKRPSGIPERFS
GSNSGNTATLTISGTQPMDEADYYCQAWD
NNFYVFGTGTKLTVLG
VH

YAMHWVRQTPGKGLEWVSGISWDSGSIAY
ADS VKGRFTISRDNAKNSLYLQMNSLRAED
TAVYYCAREKQLVPYYYYGMDVWGQGTT
VTVSS
[287]
[288] Example 7. Selection of MERS-CoV neutralizing antibodies [289] We performed a microneutralization assay (Example 5.3) to test the neutralizing activity of the 36 identified scFv clones against MERS-CoV
(MERS-CoV/KOR/KNIH/002 05 2015). The obtained results are shown in Table 18 (CPE inhibition by scFv clones)

53 [290] [Table 181 Clone IC50 (n/mL) C-8 6.45 10 9.61 15 2.78 20 3.22 34 4.43 119 9.61 42 4.67 46 4.67 47 3.03 48 2.40 m336 3.71 [291] as shown in Table 18, among the clones, scFV clones 10, 15, 20, C-8, 34, 42, 46, 47, and 48 potently inhibited MERS-CoV replication, with half-maximal inhibitory con-centration (IC50) values ranging from 2.40 to 9.61 [tg/mL.
[292] Next, the stability of these clones during nebulization was tested.
The inventors nebulized the fusion proteins, each of which comprises the scFv clone and hFc (SEQ
ID NO: 66) (N-VL-(linker)-VH-hinge-Fc-C; hereinafter, "scFv-hFc"), at a con-centration of 100 [tg/mL (each scFv-hFc) in PBS using a vibrating mesh nebulizer and then collected the aerosol. All the collected samples showed clearly visible ag-gregation. After centrifugation to remove the aggregated material, the supernatant (post-nebulized scFv-hFc fusion proteins) and pre-nebulized scFv-hFc fusion proteins were subjected to ELISA using recombinant S glycoprotein-coated microtiter plates (Example 5.1). The amount of bound scFv-hFc fusion protein was determined using HRP-conjugated anti-human IgG antibody and ABTS(2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) by measuring absorbance at 405 nm, the results obtained from post- and pre-nebulized scFv-hFc fusion proteins were compared.
[293] The obtained results are shown in Figures la and lb. Figures la and lb shows re-activity of scFv clones before and after nebulization. As shown in Figures la and lb, all nine scFv-hFc fusion proteins showed significantly reduced reactivity against re-combinant S glycoprotein after nebulization.
[294]

54 [295] The clones C-8 and 48 were selected, as these antibodies exhibited the lowest IC50 values among the antibodies derived from patients P002 and P014, respectively.

Before performing further studies, the mechanism underlying inhibition of viral infection on cells was examined. The antibodies were mixed and incubated with re-combinant S glycoprotein fused with a polyhistidine tag at the C-terminus.
After in-cubation with hDPP4-expres sing Huh-7 cells (hDPP4+), the relative amount of bound recombinant S glycoprotein was measured by flow cytometry analysis (Example 5.4) using FITC-conjugated anti-HIS antibody. Per each sample, 10,000 cells were monitored, and the data were analyzed using FlowJo software.
[296] The obtained results are shown in Figure 2 (C-8 (A), 48 (B), m336 (C), or negative control (D) scFv-hFc). As shown in Figure 2, both C-8 and 48 scFv-hFc nearly completely blocked binding of recombinant S glycoprotein to cells at equimolar con-centration of 100 nM, indicating that the antibodies block the initial interaction of the virus with cells.
[297]
[298] Example 8. Modification of CDR residues to enhance antibody stability [299] To enhance the stability of the C-8 and 48 clones, the inventors sought to introduce mutations in CDRs, except for heavy chain CDR3 (HCDR3), for replacement of hy-drophobic residues with hydrophilic residues. We defined CDRs according to the Inter-national Immunogenetics Information System (IMGT) and targeted Phe, Ile, Leu, Val, Met, Trp, and Tyr which were defined as hydrophobic amino acids. For the C-8 clone, the F29, Y32, 151, 152, F53, and F54 hydrophobic residues in HCDR1 and HCDR2 were selected for randomization (Figure 3A). These six residues were designed to encode the wild-type amino acid, Asp, Glu, or redundant amino acids depending on the degenerate codon in the first scFv phage-display library. The inventors preferred negatively charged amino acids to positively charged amino acids as lowering the iso-electric point of an antibody may reduce the non-specific in vivo clearance.
The randomized scFv phage-display library had a complexity of 2.6 x 109 colony-forming units, which exceeded the theoretical complexity of 1.3 x 105 on the nucleotide level.
After two rounds of biopanning on recombinant MERS-CoV S RBD protein, the inventors randomly rescued phage clones and performed phage ELISA. Eleven scFv clones showed reactivity to recombinant MERS-CoV S RBD protein similar to or higher than that of the original C-8 clone. The C-8-2 clone harbored F29E and replacements, while the other 10 clones had only one residue replaced with either Asp, Glu, or redundant amino acids, depending on the degenerate codon.
[300] Figure 3 shows sequential randomization of CDR residues of the C-8 clone. As shown in Figure 3 (A), in the first randomized library, six hydrophobic amino acid residues (asterisks) in HCDR1 and HCDR2 were targeted. The second library was

55 prepared in the C-8-2 clone by randomizing nine amino acid residues (asterisks) that were not randomized in the first randomized library. As shown in Figure 3 (B), eight amino acid residues (asterisks) in LCDRs of the C-8-2-4B clone selected from the second library were randomized in the third randomized library.
[301]
[302] Biophysical characterization of C-8, C-8-2, and C-8-2-4B clones were conducted.
Following nebulization at a concentration of 100 [tg/mL or 300 [tg/mL for scFv-hFc or IgG1 full length antibody (with kappa light chain) (Example 4), respectively, aerosol was collected and subjected to ELISA (Figure 4A-C) and flow cytometry (Figure 4D).
C-8-2 scFv-hFc (Figure 4A), C-8-2-4B scFv-hFc (Figure 4B), and C-8-2-4B IgG1 (Figure 4C) were serially diluted and incubated with recombinant S
glycoprotein-coated microtiter plates. In Figure 4D, C-8 IgG1 and C-8-2-4B IgG1 were incubated with recombinant S glycoprotein fused with a polyhistidine tag at the C-terminus, and the complex was allowed to react with hDPP4-expressing cells. The amount of bound recombinant S glycoprotein was measured using FITC-conjugated anti-HIS
antibody (absorbance at 405 nm). Data are representative of 10,000 cells for each sample.
[303]
[304] More detailed description on the above biophysical characterization is provided as follows.
[305]
[306] Biophysical characterization of scFv-hFc fusion proteins [307] To test the stability of the C-8-2 clone during nebulization, a scFv-hFc fusion protein was prepared and subjected to ELISA following nebulization as above. As shown in Figure 4A, the reactivity of C-8-2 scFv-hFc to recombinant S glycoprotein was much less affected by nebulization than that of C-8 scFv-hFc; however, the reactivity of the C-8-2 clone was somewhat reduced compared with that of the C-8 clone.
[308] To achieve further stabilization and affinity maturation, a second scFv phage-display library was generated using the same strategy to randomize nine residues in and HCDR2 of the C-8-2 clone to introduce more negatively charged residues (Figure 3A). The proline at H52A was excluded from the randomization, as proline frequently forms a unique structure essential for antibody reactivity. The second randomized scFv phage-display library had a complexity of 1.0 x 109 colony-forming units, which exceeded the theoretical complexity of 4.2 x 106 on the nucleotide level.
After the second round of biopanning on recombinant MERS-CoV S RBD protein, we selected 12 clones that displayed greater reactivity to recombinant MERS-CoV S RBD
protein than the C-8-2 clone in phage ELISA analysis. Clone C-8-2-4B contained replacement at six residues (G26D, T28K, S30K, 531R, G55D, and T56K; Figure 3A) and showed the highest intrinsic solubility score among the 12 tested clones.
Interestingly, only two

56 residues were replaced with Asp, and four residues were replaced with positively charged amino acids, as allowed by the degenerate codons (Figure 3A). Then, C-8-2-4B scFv-hFc fusion protein was prepared using a eukaryotic expression system.
After nebulization, the reactivity of C-8-2-4B scFv-hFc to recombinant S
glycoprotein was less affected than either C-8 or C-8-2 scFv-hFc (Figure 4A, B). In addition, the re-activity of C-8-2-4B scFv-hFc was enhanced compared to that of C-8-2 scFv-hFc and comparable to that of C-8 scFv-hFc.
[309]
[310] Biophysical characterization of IgG1 antibodies [311] ELISA
[312] Next, C-8 and C-8-2-4B IgGI were prepared using a eukaryotic expression system and compared the reactivity of these immunoglobulins to recombinant S
glycoprotein before and after nebulization. As expected, the reactivity of C-8-2-4B IgGI
was better retained following nebulization than that of C-8 IgGI (Figure 4C). It was tested whether C-8-2-4B IgGI effectively blocked the interaction between recombinant S gly-coprotein and hDPP4-expressing Huh-7 cells after nebulization. In flow cytometry analysis, we found that C-8-2-4B IgGI almost completely blocked the binding of re-combinant S glycoprotein to hDPP4-expressing cells following nebulization, while C-8 IgGI failed to block this interaction after nebulization (Figure 4D).
[313] As C-8-2-4B IgGI showed a somewhat reduced reactivity after nebulization, we sought to confer additional stability by randomizing eight hydrophobic residues in LCDRs using the same randomization scheme. The inventors achieved 2.0 x 109 colony-forming units in the third randomized scFv phage-display library, exceeding the theoretical complexity of 2.1 x 106 (Figure 3B). After two rounds of biopanning on recombinant MERS-CoV S RBD protein, clones in a phage ELISA with reactivity similar to or greater than that of C-8-2-4B were selected. Sanger sequencing revealed that a single clone was repetitively selected. The selected clone, C-8-2-4B-10D, harbored replacements at L27C and L92V with valine (Figure 3B). C-8-2-4B-10D
IgG
1 was prepared using a eukaryotic expression system and analyzed the characteristics using ELISA, size-exclusion high-performance liquid chromatography (SE-HPLC), dynamic light scattering (DLS), and plaque reduction neutralization tests (PRNT50).
[314] Following nebulization at a concentration of 1 mg/mL of each of C-8 IgGI, C-8-2-4B-10D IgGI and m336, aerosol was collected and subjected to ELISA. Re-combinant S glycoprotein-coated microtiter plates were incubated with pre-nebulized and post-nebulized C-8 IgGi (Figure 5A), C-8-2-4B-10D IgGi (Figure 5B), and m336 (Figure 5C). HRP-conjugated anti-human IgG antibody was used as the probe, and ABTS was used as the substrate. All experiments were performed in duplicate, and the data indicate mean SD.

57 [315] The obtained results of ELISA as above are shown in Figure 5. As shown in Figure 5, a noticeable decline in reactivity to recombinant S glycoprotein by C-8 IgGI and m336 IgGI after nebulization was observed; yet, the change in reactivity of C-8-2-4B-10D IgGI after nebulization was negligible.
[316]
[317] SE-HPLC & DLS analysis (Stability) [318] To examine the stability of test antibodies, SE-HPLC and DLS analysis for C-8 IgGI, C-8-2-4B-10D IgGi, and m336 IgGi were conducted referring to Examples 5.5 and 5.6, respectively.
[319] More specifically, to evaluate the size distribution profile of pre-nebulized C-8 IgGI
(Figure 6A), pre-nebulized C-8-2-4B-10D IgGi (Figure 6B), pre-nebulized m336 IgGi (Figure 6C), post-nebulized C-8 IgGI (Figure 6D), post-nebulized C-8-2-4B-10D
IgGI
(Figure 6E), and post-nebulized m336 IgGI (Figure 6F) antibodies, DLS was performed using 633-nm/4-mW laser at a 173 detection angle. PBS was used as the reference solvent, and the results were evaluated with Zetasizer software 7.02. All ex-periments were performed in triplicate, and representative results are shown for each antibody.
[320] The obtained results are shown in Table 19 and Figure 6.
[321] [Table 191 SE-HPLC (% monomer / % DLS (% monomer SD / % aggregates aggregates) SD) Antibody Pre-nebulizat Post-nebulizat Pre-nebulization Post-nebulization ion ion C-8 100.0 / 0 97.9 / 2.1 100.0 0 /0 78.4 3.5 / 21.6 3.5 C-8-2-4B- 100.0 / 0 100.0 / 0 99.2 0.7 / 0.8 98.6 0.4 / 1.4 0.4 10D 0.7 m336 100.0 / 0 99.4 / 0.6 96.6 0.6 / 3.4 77.5 2.3 /
22.5 2.3 0.6 [322] As shown in Table 19, in SE-HPLC analysis, high-molecular weight aggregates were detected in post-nebulization samples of C-8 and m336 IgGi; however, no aggregate was found in post-nebulized samples of C-8-2-4B-10D IgGi as shown om Table 17 above. As shown in Table 18 and Figure 6,in accordance with these SE-HPLC
data, DLS analysis showed that the nebulization process converted 21.6% and 22.5% of and m336 IgGi, respectively, into high-molecular-weight aggregates, while nebu-lization resulted in <1% aggregates for C-8-2-4B-10D IgGi.
[323]

58 [324] Example 9. Neutralizing potency after nebulization [325] The neutralizing activities of pre- and post-nebulized C-8 IgGI and C-IgGI were evaluated in PRNT50 using the live MERS-CoV referring to Example 5.7.
Antibodies were mixed with live MERS-CoV, and then the antibody-virus mixture was allowed to infect Vero cells.
[326] More specifically, culture media containing 100 PFU live MERS-CoV
(MERS-CoV/KOR/KNIH/002 05 2015) was mixed with equal volume of serially diluted C-8 IgGI (Figure 7a), C-8-2-4B-10D IgGI (Figure 7b), and palivizumab (Figure 7c). After incubation for 1 h, the mixture was added to Vero cells (ATCC CCL-81).
After 2 days, the plaques were counted. The inhibition of virus infection was plotted as a function of IgGI antibody concentration, and PRNT50 values were calculated by GraphPad Prism 6. All experiments were performed in quadruplicate, and the data indicate mean SD.
[327] The obtained results (Relative infection (%)) are shown in Figures 7a-7c. As shown in Figures 7a-7c, C-8 IgGI and C-8-2-4B-10D IgGI exhibited effective inhibitory activity against MERS-CoV, with IC50 values of 0.29 and 0.28 [tg/mL, respectively.
After nebulization, C-8-2-4B-10D showed an IC50 value similar to that of pre-nebulized IgGI, but the IC50 value of C-8 IgGI was dramatically increased following nebulization.
[328]
[329] Example 10. Test of binding ability of CDR-modified neutralizing antibody to MERS-CoV mutants [330] In this example, it was tested whether the CDR-modified neutralizing antibodies of Example 8 can retain their ability to bind to MERS-CoV mutants. As several poly-morphisms within S protein (D5 10G and I529T) of MERS-CoV were identified during the MERS outbreak in South Korea, the inventors examined binding activity of C-and C-8-2-4B-10D antibodies against recombinant wild-type or mutant MERS-CoV
RBD (including mutation of D5 10G, I529T, or D5 10G & I529T) protein using ELISA
analysis referring to Example 5.1.
[331] More specifically, recombinant wild-type or mutant MERS-CoV RBD
protein-coated microtiter plates were incubated with varying concentration of C-8 IgGl, C-8-2-4B-10D IgGl, or irrelevant IgGl(negative control). HRP-conjugated anti-human IgG antibody was used as the probe, and ABTS was used as the substrate. All ex-periments were performed in duplicate, and the data indicate mean SD.
[332] The obtained results are shown in Figure 8. As shown in Figure 8, antibody clone C-8 as well as CDR-modified C-8-2-4B-10D successfully bound to recombinant mutant RBD proteins (D5 10G, I529T, or D5 10G & I529T) in a dose-dependent manner without any noticeable decline in reactivity compared to that of recombinant wild-type

59 RBD protein.
[333] Furthermore, it was tested whether the antibody can inhibit the interaction between RBD mutants and DPP4.
[334] Flow cytometry analysis of the inhibition of recombinant mutant MERS-CoV RBD
protein binding to hDPP4-expressing cells were performed referring to Example 5.4.
Antibodies C-8 IgGI, C-8-2-4B-10D IgGI, m336 IgGI, or negative control IgGI
(each 1 or 4 [LM) were mixed and incubated with recombinant wild type and mutant (D5 10G, I529T, or D510G/I529T) MERS-CoV RBD protein (each 1 [LM) fused with a poly-histidine tag at the C-terminus. After incubation with Huh-7 (hDPP4+) cells, the relative amount of bound recombinant mutant MERS-CoV RBD protein was measured using FITC-conjugated anti-HIS antibody. Per each sample, 10,000 cells were monitored, and the data were analyzed using FlowJo software.
[335] The obtained results are shown in Figures 9a and 9b. As shown in Figures 9a and 9b, both C-8 IgGI and C-8-2-4B-10D IgGI nearly completely blocked binding of re-combinant mutant MERS-CoV RBD protein (D5 10G and I529T) to cells at equimolar concentration of 1000 nM (Figure 2), indicating that the antibodies block the initial in-teraction of the virus with cells regardless of mutations (D5 10G or I529T) in the RBD.
In case of double mutant carrying both D5 10G and I529T, this mutant did not bind to hDPP4-expressing Huh-7 cells which prevent us from conducting further studies.
[336]
[337] Example 11. Conformational epitope mapping [338] Conformational epitope mapping was performed by hydrogen/deuterium exchange mass spectrometry. To elucidate the site where C-8 binds, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was performed and the kinetics of hydrogen/
deuterium exchange at protein backbone amides was evaluated. More specifically, the proteins were deglycosylated by PNGase-F and diluted to 40 pmole in a buffer composed of 10mM potassium phosphate (pH 7.0). Hydrogen/deuterium exchange was performed by mixing 2.5[cL of proteins with 37.5 [LL of D 20 buffer (10 mM
potassium phosphate pD 7.0) and incubating for 0, 0.33 min (20 sec), 10 min,

60 min and 240 min on ice. The incubation of samples was ended by adding 40 [LL of ice-cold quench buffer (1 M TCEP, 2 M Urea, pH 2.66). The quenched samples were quickly thawed and digested by treating pepsin enzyme for 5 min on ice. Rapidly, pepsin treated samples were injected into nanoACQUITY with HDX Technology (Waters, Milford, MA, USA) and online-digested by passing through an Enzymate pepsin column (2.1 mm x 30 mm, 300A, 5 [cm, Waters). Peptide fragments were subsequently trapped and desalted in VanGuard BEH C18 trap column (2.1 mm x 5 mm, 1.7 [cm, Waters) at a flow rate of 100 [cUmin 5% ACN for 3 min and then separated by analytical column (1.0 x 100 mm, 1.7 [cm, Waters, Milford, MA, USA) at a flow rate of 40 [tt/min with gradient for 13 min (started with 5% B for 1 min and increased to 40% B for 7 min). The mobile phase A was 0.1% formic acid in H 2 0, and B was ACN containing 0.1 % formic acid. To minimize the back-exchange of deuterium to hydrogen, the sample, solvents, trap and analytical columns were all maintained at pH
of 2.66 and 0 C during analysis. Mass spectrometric analyses was performed with a SYNAPT G2-Si (Waters, Milford, MA, USA) equipped with Ion Mobility Separation (IMS, Waters, Milford, MA, USA) and standard ESI source. The mass spectra were acquired in the range of m/z 50-2000 in the positive ion mode for 10.5 min.
[339] The obtained HDX profiles of free- and C-8 IgGl-bound MERS-CoV RBD
were shown in Figures 10a-10c, wherein Figure 10b shows a graph corresponding to (A) of Figure 10a and Figure 10c shows graphs corresponding to (B) of Figure 10a. The deuterium uptake graphs (Figures 10b and 10c) for peptides show the relative deuterium incorporation as a function of exposure time. The peptides that had reduction in deuterium incorporation in RBD and C-8 IgG1 complex are highlighted in (A) (5er498-Ala520) and (B) (Tyr540-Leu554) and on the X-ray crystal structure of MERS-CoV RBD (PDB: 4KQZ) (Figure 10a).
[340] Then, mapping of the C-8 epitope and DPP4 binding site on MERS-CoV
RBD
sequence was performed. By monitoring the exchange of backbone amide hydrogen with deuterium within MERS-CoV RBD, the inventors observed reduction in deuterium incorporation in the RBD-C-8 IgGI complex in residues between 5er498-Ala520 (A in Figure 10a) and Tyr540-Leu554 ((B) in Figure 10a), indicating that these residues are involved in C-8 binding. The RBD residues interacting with C-8 and DPP4 are marked in Figure 11 with orange and blue dots, respectively. In contrast, other regions shared almost identical deuterium levels in free and bound RBD, suggesting that these regions do not engage in protein-protein interactions.
Then, we compared the C-8 epitope with the DPP4 binding site that was reported as shown in Figure 11. Remarkably, the C-8 epitope overlapped extensively with the DPP4 binding site indicating that the C-8 block the initial interaction of the virus with natural receptor DPP4 (Figure 11).
[341]
[342] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were indi-vidually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
[343] The use of the terms "a" and "an" and "the" and "at least one" and "one or more" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the

61 term "at least one" (or "one or more") followed by a list of one or more items (for example, "at least one of A and B") is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "comprising, but not limited to,") unless otherwise noted.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the speci-fication as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
[344] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
[345] The present disclosure is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods which are functionally equivalent are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure.
Thus, it is intended that the present disclosure cover the modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.
[346] All publications and patent applications mentioned in this specification are herein in-corporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

Claims [Claim 11 An anti-MERS-CoV antibody or an antigen-binding fragment thereof comprising:
a VL-CDR1 comprising an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7;
a VL-CDR2 comprising an amino acid sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14 or 71;
a VL-CDR3 comprising an amino acid sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, or 72;
a VH-CDR1 comprising an amino acid sequence of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, or 73;
a VH-CDR2 comprising an amino acid sequence of SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, or 74; and a VH-CDR3 comprising an amino acid sequence of SEQ ID NO: 38, 39, 40, 41, 42, 43, or 75.
[Claim 21 The anti-MERS-CoV antibody or an antigen-binding fragment thereof according to claim 1, comprising:
(1) a VL-CDR1 of SEQ ID NO: 1, a VL-CDR2 of SEQ ID NO: 8, a VL-CDR3 of SEQ ID NO: 15, a VH-CDR1 of SEQ ID NO: 24, a VH-CDR2 of SEQ ID NO: 31, and a VH-CDR3 of SEQ ID NO: 38;
(2) a VL-CDR1 of SEQ ID NO: 1, a VL-CDR2 of SEQ ID NO: 8, a VL-CDR3 of SEQ ID NO: 15, a VH-CDR1 of SEQ ID NO: 25, a VH-CDR2 of SEQ ID NO: 32, and a VH-CDR3 of SEQ ID NO: 38;
(3) a VL-CDR1 of SEQ ID NO: 2, a VL-CDR2 of SEQ ID NO: 8, a VL-CDR3 of SEQ ID NO: 16, a VH-CDR1 of SEQ ID NO: 25, a VH-CDR2 of SEQ ID NO: 32, and a VH-CDR3 of SEQ ID NO: 38;
(4) a VL-CDR1 of SEQ ID NO: 1, a VL-CDR2 of SEQ ID NO: 8, a VL-CDR3 of SEQ ID NO: 15, a VH-CDR1 of SEQ ID NO: 26, a VH-CDR2 of SEQ ID NO: 32, and a VH-CDR3 of SEQ ID NO: 38;
(5) a VL-CDR1 of SEQ ID NO: 1, a VL-CDR2 of SEQ ID NO: 9, a VL-CDR3 of SEQ ID NO: 15, a VH-CDR1 of SEQ ID NO: 26, a VH-CDR2 of SEQ ID NO: 32, and a VH-CDR3 of SEQ ID NO: 38;
(6) a VL-CDR1 of SEQ ID NO: 3, a VL-CDR2 of SEQ ID NO: 10, a VL-CDR3 of SEQ ID NO: 17, a VH-CDR1 of SEQ ID NO: 27, a VH-CDR2 of SEQ ID NO: 33, and a VH-CDR3 of SEQ ID NO: 39;
(7) a VL-CDR1 of SEQ ID NO: 4, a VL-CDR2 of SEQ ID NO: 11, a VL-CDR3 of SEQ ID NO: 18, a VH-CDR1 of SEQ ID NO: 28, a VH-CDR2 of SEQ ID NO: 34, and a VH-CDR3 of SEQ ID NO: 40;
(8) a VL-CDR1 of SEQ ID NO: 4, a VL-CDR2 of SEQ ID NO: 11, a VL-CDR3 of SEQ ID NO: 19, a VH-CDR1 of SEQ ID NO: 29, a VH-CDR2 of SEQ ID NO: 35, and a VH-CDR3 of SEQ ID NO: 41;
(9) a VL-CDR1 of SEQ ID NO: 5, a VL-CDR2 of SEQ ID NO: 12, a VL-CDR3 of SEQ ID NO: 20, a VH-CDR1 of SEQ ID NO: 27, a VH-CDR2 of SEQ ID NO: 33, and a VH-CDR3 of SEQ ID NO: 39;
(10) a VL-CDR1 of SEQ ID NO: 3, a VL-CDR2 of SEQ ID NO: 10, a VL-CDR3 of SEQ ID NO: 21, a VH-CDR1 of SEQ ID NO: 27, a VH-CDR2 of SEQ ID NO: 36, and a VH-CDR3 of SEQ ID NO: 42;
(11) a VL-CDR1 of SEQ ID NO: 6, a VL-CDR2 of SEQ ID NO: 13, a VL-CDR3 of SEQ ID NO: 20, a VH-CDR1 of SEQ ID NO: 27, a VH-CDR2 of SEQ ID NO: 36, and a VH-CDR3 of SEQ ID NO: 42;
(12) a VL-CDR1 of SEQ ID NO: 3, a VL-CDR2 of SEQ ID NO: 10, a VL-CDR3 of SEQ ID NO: 22, a VH-CDR1 of SEQ ID NO: 27, a VH-CDR2 of SEQ ID NO: 36, and a VH-CDR3 of SEQ ID NO: 42;
(13) a VL-CDR1 of SEQ ID NO: 7, a VL-CDR2 of SEQ ID NO: 14, a VL-CDR3 of SEQ ID NO: 23, a VH-CDR1 of SEQ ID NO: 30, a VH-CDR2 of SEQ ID NO: 37, and a VH-CDR3 of SEQ ID NO: 43; or (14) a VL-CDR1 of SEQ ID NO: 7, a VL-CDR2 of SEQ ID NO: 71, a VL-CDR3 of SEQ ID NO: 72, a VH-CDR1 of SEQ ID NO: 73, a VH-CDR2 of SEQ ID NO: 74, and a VH-CDR3 of SEQ ID NO: 75.
[Claim 31 The anti-MERS-CoV antibody or an antigen-binding fragment thereof according to claim 1, comprising:
a light chain variable region comprising or consisting essentially of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 76; and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, 63, or 77.
[Claim 41 The anti-MERS-CoV antibody or an antigen-binding fragment thereof according to claim 1, comprising:
(1) a light chain variable region comprising or consisting essentially of SEQ ID NO: 44 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 55;
(2) a light chain variable region comprising or consisting essentially of SEQ ID NO: 44 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 56;
(3) a light chain variable region comprising or consisting essentially of SEQ ID NO: 45 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 56;
(4) a light chain variable region comprising or consisting essentially of SEQ ID NO: 44 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 57;
(5) a light chain variable region comprising or consisting essentially of SEQ ID NO: 46 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 55;
(6) a light chain variable region comprising or consisting essentially of SEQ ID NO: 47 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 58;
(7) a light chain variable region comprising or consisting essentially of SEQ ID NO: 48 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 59;
(8) a light chain variable region comprising or consisting essentially of SEQ ID NO: 49 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 60;
(9) a light chain variable region comprising or consisting essentially of SEQ ID NO: 50 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 61;
(10) a light chain variable region comprising or consisting essentially of SEQ ID NO: 51 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 62;
(11) a light chain variable region comprising or consisting essentially of SEQ ID NO: 52 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 62;
(12) a light chain variable region comprising or consisting essentially of SEQ ID NO: 53 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 62;
(13) a light chain variable region comprising or consisting essentially of SEQ ID NO: 54 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 63; or (14) a light chain variable region comprising or consisting essentially of SEQ ID NO: 76 and a heavy chain variable region comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 77.
[Claim 5] The anti-MERS-CoV antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or an antigen-binding fragment thereof is in an IgG form, a scFv form, or a scFv-Fc form.
[Claim 61 The anti-MERS-CoV antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or an antigen-binding fragment thereof binds to an epitope positioned at least partially overlapping or adjacent to a receptor binding site of S protein of MERS-CoV.
[Claim 71 The anti-MERS-CoV antibody or an antigen-binding fragment thereof according to claim 6, wherein the S protein of MERS-CoV is wild type or mutant having D510G, I529T, or both thereof.
[Claim 81 The anti-MERS-CoV antibody or an antigen-binding fragment thereof according to claim 6, wherein the epitope is:
(a) an epitope comprising at least one selected from amino acid residues of SEQ ID NO: 67 or SEQ ID NO: 68;
(b) an epitope comprising at least one selected from amino acid residues of SEQ ID NO: 69; or (c) both of (a) and (b).
[Claim 91 An anti-MERS-CoV antibody or an antigen-binding fragment thereof, which binds to:
(a) an epitope comprising at least one selected from amino acid residues of SEQ ID NO: 67 or SEQ ID NO: 68;
(b) an epitope comprising at least one selected from amino acid residues of SEQ ID NO: 69; or (c) both of (a) and (b).
[Claim 101 A pharmaceutical composition for treating or preventing MERS-CoV
infection or a disease associated with MERS-CoV infection comprising the anti-MERS-CoV antibody or an antigen-binding fragment thereof according to any one of claims 1-4 or 9.
[Claim 11] The pharmaceutical composition according to claim 10, wherein the antibody or an antigen-binding fragment thereof is in an IgG form, a scFv form, or a scFv-Fc form.
[Claim 121 A polynucleotide encoding the anti-MERS-CoV antibody or an antigen-binding fragment thereof according to any one of claims 1-4.
[Claim 131 A recombinant vector comprising the polynucleotide according to claim 12.
[Claim 141 A recombinant cell comprising the polynucleotide according to claim 12.
[Claim 151 A method of treating or preventing MERS-CoV infection or a disease associated with MERS-CoV infection, comprising administering the anti-MERS-CoV antibody or an antigen-binding fragment thereof according to any one of claims 1-4 to a subject in need thereof.