Binding Molecule Having Neutralizing Activity Against Middle East Respiratory Syndrome-Coronavirus

Abstract

The present invention relates to a binding molecule having neutralizing activity against Middle East Respiratory Syndrome-Coronavirus (MERS-CoV). More particularly, the present invention relates to a binding molecule having strong ability to bind to an S protein of MERS-CoV and neutralizing activity against MERS-CoV and thus being very useful in the prevention, treatment or diagnosis of MERS-CoV infection.

Claims

1. A neutralizing binding molecule, which binds to a spike protein (S protein) on a surface of a Middle East Respiratory Syndrome-Coronavirus (MERS-CoV).

2. The binding molecule of claim 1, wherein the binding molecule comprises at least one of the binding molecules i) to vi) below: i) a binding molecule comprising a) a heavy-chain variable region comprising a CDR1 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR3 region of SEQ ID NO: 3, and b) a light-chain variable region comprising a CDR1 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR3 region of SEQ ID NO: 6; ii) a binding molecule comprising a) a heavy-chain variable region comprising a CDR1 region of SEQ ID NO: 7, a CDR2 region of SEQ ID NO: 8, and a CDR3 region of SEQ ID NO: 9, and b) a light-chain variable region comprising a CDR1 region of SEQ ID NO: 10, a CDR2 region of SEQ ID NO: 11, and a CDR3 region of SEQ ID NO: 12; iii) a binding molecule comprising a) a heavy-chain variable region comprising a CDR1 region of SEQ ID NO: 13, a CDR2 region of SEQ ID NO: 14, and a CDR3 region of SEQ ID NO: 15, and b) a light-chain variable region comprising a CDR1 region of SEQ ID NO: 16, a CDR2 region of SEQ ID NO: 17, and a CDR3 region of SEQ ID NO: 18; iv) a binding molecule comprising a) a heavy-chain variable region comprising a CDR1 region of SEQ ID NO: 19, a CDR2 region of SEQ ID NO: 20, and a CDR3 region of SEQ ID NO: 21, and b) a light-chain variable region comprising a CDR1 region of SEQ ID NO: 22, a CDR2 region of SEQ ID NO: 23, and a CDR3 region of SEQ ID NO: 24; v) a binding molecule comprising a) a heavy-chain variable region comprising a CDR1 region of SEQ ID NO: 25, a CDR2 region of SEQ ID NO: 26, and a CDR3 region of SEQ ID NO: 27, and b) a light-chain variable region comprising a CDR1 region of SEQ ID NO: 28, a CDR2 region of SEQ ID NO: 29, and a CDR3 region of SEQ ID NO: 30; and vi) a binding molecule comprising a) a heavy-chain variable region comprising a CDR1 region of SEQ ID NO: 31, a CDR2 region of SEQ ID NO: 32, and a CDR3 region of SEQ ID NO: 33, and b) a light-chain variable region comprising a CDR1 region of SEQ ID NO: 34, a CDR2 region of SEQ ID NO: 35, and a CDR3 region of SEQ ID NO: 36.

3. The binding molecule of claim 1, wherein the binding molecule comprises at least one of the binding molecules i) to vi) below: i) a binding molecule comprising a) a heavy-chain variable region having a sequence identity of 95% or more to a heavy-chain variable region of a polypeptide sequence of SEQ ID NO: 37, and b) a light-chain variable region having a sequence identity of 95% or more to a light-chain variable region of a polypeptide sequence of SEQ ID NO: 38; ii) a binding molecule comprising a) a heavy-chain variable region having a sequence identity of 95% or more to a heavy-chain variable region of a polypeptide sequence of SEQ ID NO: 39, and b) a light-chain variable region having a sequence identity of 95% or more to a light-chain variable region of a polypeptide sequence of SEQ ID NO: 40; iii) a binding molecule comprising a) a heavy-chain variable region having a sequence identity of 95% or more to a heavy-chain variable region of a polypeptide sequence of SEQ ID NO: 41, and b) a light-chain variable region having a sequence identity of 95% or more to a light-chain variable region of a polypeptide sequence of SEQ ID NO: 42; iv) a binding molecule comprising a) a heavy-chain variable region having a sequence identity of 95% or more to a heavy-chain variable region of a polypeptide sequence of SEQ ID NO: 43, and b) a light-chain variable region having a sequence identity of 95% or more to a light-chain variable region of a polypeptide sequence of SEQ ID NO: 44; v) a binding molecule comprising a) a heavy-chain variable region having a sequence identity of 95% or more to a heavy-chain variable region of a polypeptide sequence of SEQ ID NO: 45, and b) a light-chain variable region having a sequence identity of 95% or more to a light-chain variable region of a polypeptide sequence of SEQ ID NO: 46; and vi) a binding molecule comprising a) a heavy-chain variable region having a sequence identity of 95% or more to a heavy-chain variable region of a polypeptide sequence of SEQ ID NO: 47, and b) a light-chain variable region having a sequence identity of 95% or more to a light-chain variable region of a polypeptide sequence of SEQ ID NO: 48.

4. The binding molecule of claim 1, wherein the binding molecule is a Fab fragment, a Fv fragment, a diabody, a chimeric antibody, a humanized antibody, or a human antibody.

5. The binding molecule of claim 1 further comprising at least one tag bound to the binding molecule to form an immunoconjugate.

6. A nucleic acid molecule encoding a neutralizing binding molecule, which binds to a spike protein (S protein) on a surface of a Middle East Respiratory Syndrome-Coronavirus (MERS-CoV).

7. The nucleic acid molecule of claim 6 further comprising an expression vector receiving the nucleic acid molecule.

8. The nucleic acid molecule of claim 7 further comprising a cell transformed from the expression vector into a host cell to produce a binding molecule that binds to MERS-CoV and thus has neutralizing activity.

9. The nucleic acid molecule of claim 8, wherein the host cell is any one selected from the group consisting of a CHO cell, a F2N cell, a COS cell, a BHK cell, a Bowes melanoma cell, a HeLa cell, a 911 cell, a HT1080 cell, an A549 cell, a HEK 293 cell and a HEK293T cell.

10. A composition for preventing or treating MERS-CoV infection comprising a neutralizing binding molecule, which binds to a spike protein (S protein) on a surface of a Middle East Respiratory Syndrome-Coronavirus (MERS-CoV).

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

12. A method of diagnosing, preventing or treating a disease caused by MERS-CoV infection comprising administering a neutralizing binding molecule, which binds to a spike protein (S protein) on a surface of a Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) in a therapeutically effective amount to a subject having a disease caused by MERS-CoV infection.

13. A kit for diagnosing MERS-CoV comprising a neutralizing binding molecule, which binds to a spike protein (S protein) on a surface of a Middle East Respiratory Syndrome-Coronavirus (MERS-CoV).

Description

BRIEF DESCRIPTION OF DRAWINGS

[0062] FIG. 1 shows the neutralizing activity depending on the antibody concentration against Korean isolate MERS-CoV (MERS-CoV/Korea/KNIH/002_05_2015) by performing a plaque assay with two finally selected antibodies;

[0063] FIG. 2 shows the virus titer through a plaque assay after tissue culture by infecting human lung tissue with Korean isolate MERS-CoV and an antibody in order to evaluate the neutralizing activity of the antibody using a human lung tissue infection model (ex vivo);

[0064] FIG. 3A shows the results of quantitative PCR for evaluation of animal treatment efficacy using an animal model capable of MERS-CoV infection and proliferation (hDPP4 (human dipeptidyl peptidase 4) receptor-overexpressing mouse) and an antibody binding to the virus;

[0065] FIG. 3B shows the results of a plaque assay for evaluation of animal treatment efficacy using an animal model capable of MERS-CoV infection and proliferation (hDPP4 (human dipeptidyl peptidase 4) receptor-overexpressing mouse) and an antibody binding to the virus;

[0066] FIG. 4A shows the results of quantitative PCR for evaluation of preventive efficacy of an antibody against MERS-CoV using an animal model capable of MERS-CoV infection and proliferation (hDPP4 (human dipeptidyl peptidase 4) receptor-overexpressing mouse) (*p<0.05, **p<0.01, ***p<0.001);

[0067] FIG. 4B shows the results of a plaque assay for evaluation of preventive efficacy of an antibody against MERS-CoV using an animal model capable of MERS-CoV infection and proliferation (hDPP4 (human dipeptidyl peptidase 4) receptor-overexpressing mouse) (*p<0.05, **p<0.01, ***p<0.001);

[0068] FIG. 5 shows histological changes in mouse lung for evaluation of preventive efficacy of an antibody against MERS-CoV using an animal model capable of MERS-CoV infection and proliferation (hDPP4 (human dipeptidyl peptidase 4) receptor-overexpressing mouse);

[0069] FIG. 6A shows the mouse body weight reduction for evaluation of therapeutic efficacy of an antibody against MERS-CoV using an animal model capable of MERS-CoV infection and proliferation (hDPP4 (human dipeptidyl peptidase 4) receptor-overexpressing mouse) (*p<0.05, **p<0.01, ***p<0.001);

[0070] FIG. 6B shows the mouse survival rate for evaluation of therapeutic efficacy of an antibody against MERS-CoV using an animal model capable of MERS-CoV infection and proliferation (hDPP4 (human dipeptidyl peptidase 4) receptor-overexpressing mouse) (*p<0.05, **p<0.01, ***p<0.001);

[0071] FIG. 6C shows the results of quantitative PCR for evaluation of therapeutic efficacy of an antibody against MERS-CoV using an animal model capable of MERS-CoV infection and proliferation (hDPP4 (human dipeptidyl peptidase 4) receptor-overexpressing mouse) (*p<0.05, **p<0.01, ***p<0.001); and

[0072] FIG. 6D shows the results of a plaque assay for evaluation of therapeutic efficacy of an antibody against MERS-CoV using an animal model capable of MERS-CoV infection and proliferation (hDPP4 (human dipeptidyl peptidase 4) receptor-overexpressing mouse) (*p<0.05, **p<0.01, ***p<0.001).

MODE FOR INVENTION

[0073] A better understanding of the present invention may be obtained via the following examples, which are set forth to illustrate and are not to be construed as limiting the scope of the present invention. The documents cited herein are incorporated by reference into this application.

Example 1: Isolation of PBMCs from Blood of Patients Recovered from MERS-CoV

[0074] Blood donors were those who were confirmed to have been infected with MERS-CoV in 2015 and no longer exhibited viruses as a result of treatment, and the donor selection and blood collection processes were performed under the approval of the Institutional Review Board (IRB). After donor selection, about 30 ml of whole blood was collected, and PBMCs (peripheral blood mononuclear cells) were isolated using a Ficoll-Paque™ PLUS (GE Healthcare) method. The isolated PBMCs were washed two times with a phosphate buffer solution and then stored in a liquid nitrogen tank at a concentration of 1×10.sup.7 cells/ml in a freezing medium (RPMI:FBS:DMSO=5:4:1).

Example 2: Production of Antibody-Displayed Phage Library

[0075] Total RNA was extracted from the PBMCs isolated in Example 1 using a TRIzol reagent (Invitrogen), after which cDNA was synthesized using a SuperScript™ III First-Strand cDNA synthesis system (Invitrogen, USA).

[0076] Production of the antibody library from the synthesized cDNA was performed with reference to the related literature (Barbas C. et. al. Phage Display: A Laboratory Manual. 2001. CSHL Press). Briefly, light-chain and heavy-chain variable regions of the antibody were amplified from the synthesized cDNA through a PCR (polymerase chain reaction) method using high-fidelity Taq polymerase (Roche) and a degenerative primer set (IDT). The isolated light-chain and heavy-chain variable-region fragments were made into a scFv gene through an overlap PCR method so as to be connected as one sequence in random combination, followed by amplification, cleavage with a restriction enzyme, and isolation of scFv using 1% agarose gel electrophoresis and a gel extraction kit (Qiagen). A phage vector was cleaved with the same restriction enzyme, isolated, mixed with the scFv gene, added with T4 DNA ligase (New England Biolabs), and then allowed to react at 16° C. for 12 hr or more. The resulting reaction solution was mixed with ER2738 competent cells, and was then transformed through an electroporation process. The transformed ER2738 was subjected to shaking culture, added with a VCSM13 helper phage (Agilent Technologies) and cultured for 12 hr or more.

Example 3: Selection Using Phage Enzyme Immunoassay

[0077] The phage library culture solution prepared in Example 2 was centrifuged to thus remove host cells, added with 4% PEG and 0.5 M NaCl, and centrifuged, so the phage was precipitated and the supernatant was removed. The precipitated phage was diluted with 1% BSA/TBS to afford a phage library, after which panning was independently performed through binding to and dissociation from various MERS-CoV spike proteins (S proteins), thereby isolating an scFv-phage having the ability to bind to MERS-CoV S protein. For example, the phage library was added to an ELISA plate to which an RBD region (residues 367 to 588 on 51 glycoprotein), which is a portion of the MERS-CoV S protein, was attached, followed by reaction at room temperature for 2 hr. The reaction solution was removed, after which the ELISA plate was washed with PBS containing 0.05% Tween 20 and then added with 60 μl of 0.1 M glycine-HCl (pH 2.2), so the scFv-phage was detached from the antigen, and neutralized using 2M Tris (pH 9.1). The scFv-phage thus neutralized was infected with ER2738, cultured with a helper phage and used for subsequent panning. A portion of the infected ER2738 was spread on an LB plate before the addition of the helper phage, and a colony was obtained the next day.

[0078] A total of 1,200 colonies, formed each time panning was performed, were added to a culture medium in a 96-well deep well plate (Axygen), subjected to shaking culture, and added with a helper phage when OD.sub.600 reached 0.7 or more, followed by shaking culture at 37° C. for 12 hr or more. The culture solution was centrifuged, so the host cells were removed and the supernatant containing the scFv-phage was prepared.

[0079] The scFv-phage supernatant thus prepared was diluted at 1:1 with 6% BSA/PBS, placed in each well of a 96-well microtiter plate to which MERS-CoV S proteins were adsorbed and then blocked, and allowed to stand at 37° C. for 2 hr. Each well was washed three times with PBS containing 0.05% Tween 20, added with an anti-M13 antibody labeled with HRP (horseradish peroxidase), and allowed to stand at 37° C. for 1 hr. Each well was washed three times with PBS containing 0.05% Tween 20 and then added with ABTS (2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt), after which absorbance was measured at 405 nm, whereby 444 scFv-phages having the ability to bind to MERS-CoV S proteins were selected.

Example 4: Evaluation of Binding Capacity of Antibody Fragment (scFv-Fc)

[0080] For the 444 scFv-phages selected in Example 3, DNA was obtained through shaking culture of colonies, after which sequences for antibody variable regions were analyzed. Among these, 118 scFv-phages, selected by excluding clones with overlapping amino acid sequences, were cloned into a vector containing an Fc region and converted in the form of an antibody fragment (scFv-Fc) in order to evaluate the expression capacity and neutralizing activity in the candidate antibody animal cell lines. After transfection and expression in F2N cells (Korean Patent No. 10-1005967, Patentee: Celltrion Inc.) using a transfection reagent, the culture solution thereof was used, and the ability of the antibody fragment (scFv-Fc) to bind to three S proteins of MERS-CoV was measured through ELISA. Briefly, MERS-CoV S proteins were attached to the ELISA plate and the expressed antibody fragments were added thereto. After washing the unbound antibody with PBS containing 0.05% Tween 20, antibody fragments bound to the antigen were selected using an anti-human Fc antibody linked with HRP (horseradish peroxidase). Therefore, it was confirmed that 111 antibody fragments specifically bound to S proteins of MERS-CoV.

Example 5: Evaluation of Neutralizing Activity of Antibody Fragment Against MERS-CoV (First In-Vitro Neutralizing Activity Evaluation)

[0081] 1) Measurement of Neutralizing Activity Against Saudi Isolate Virus

[0082] The 111 antibody fragment culture solutions expressed through the method of Example 4 were sequentially diluted 2-fold to thus prepare samples of 12 concentrations. For Saudi isolate virus (MERS/HCoV/KSA/EMC/2012), a virus stock was dissolved, diluted to a concentration of 25 of TCID.sub.50/well, mixed with each of the antibody fragment culture solutions in the 12 concentrations prepared above, allowed to stand at 37° C. for 2 hr, transferred to a 96-well plate containing cultured Vero cells, and allowed to stand at 37° C. for 1 hr to thus induce infection. The mixed solution of the virus and the antibody fragment culture solution was removed, a culture medium was placed in each well, and culture was performed in a 5% CO.sub.2 incubator at 37° C. for 3 days, after which a cytopathic effect in each well was observed using a microscope, and virus-neutralizing activity (the antibody concentration of the well at which 50% neutralizing activity was observed) was calculated (Reed & Muench method). The amount of the virus used per well (25 of TCID.sub.50/well) was determined through back calculation. The lower the antibody concentration showing the 50% virus-neutralizing effect, the better the neutralizing activity.

[0083] As shown in Table 3 below, it was confirmed that 36 antibody fragments had neutralizing activity superior to positive control antibodies 1 and 2 (binding to MERS-CoV S protein and having known neutralizing effect).

TABLE-US-00003 TABLE 3 50% Vitus neutralization Classification concentration (μg/mL) Antibody 1 0.022 Antibody 2 <=0.01 Antibody 3 0.014 Antibody 4 0.010 Antibody 5 0.003 Antibody 6 0.021 Antibody 7 0.014 Antibody 8 0.056 Antibody 9 0.022 Antibody 10 0.061 Antibody 11 0.034 Antibody 12 0.051 Antibody 13 0.050 Antibody 14 0.011 Antibody 15 0.086 Antibody 16 0.016 Antibody 17 0.047 Antibody 18 0.011 Antibody 19 0.021 Antibody 20 0.023 Antibody 21 0.024 Antibody 22 0.053 Antibody 23 0.019 Antibody 24 0.067 Antibody 25 0.031 Antibody 26 0.011 Antibody 27 0.020 Antibody 28 0.035 Antibody 29 0.048 Antibody 30 0.007 Antibody 31 0.021 Antibody 32 0.060 Antibody 33 0.010 Antibody 34 0.061 Antibody 35 0.033 Antibody 36 0.031 Positive control antibody 1 0.09 Positive control antibody 2 1.69

[0084] 2) Measurement of Neutralizing Activity Against Korean Isolate Virus

[0085] The 36 antibody fragments having neutralizing activity against Saudi virus confirmed in 1) above were subjected to a PRNT (plaque reduction neutralization test) with Korean isolate virus (MERS-CoV/Korea/KNIH/002_05_2015).

[0086] For PRNT, the antibody sample was diluted, mixed with 100 PFU virus in equal amounts, allowed to react at 37° C. for 1 hr, used to infect a cell line, and subjected to a plaque assay. After culture in a 5% CO.sub.2 incubator at 37° C. for 3 days and then staining using crystal violet, the number of formed plaques was comparatively analyzed, and the neutralizing activity of the antibody sample was evaluated.

[0087] Based on the results of comparative analysis, it was confirmed that the selected antibody fragments reduced plaque formation compared to two positive control antibodies, indicating that the 36 antibody fragments of the present invention had neutralizing activity superior to the two positive control antibodies (the results are not shown).

Example 6. Evaluation of Antibody Expression Rate and Antibody Binding Specificity after Conversion into Fully Human Antibody

[0088] The selected antibody fragments were converted into fully human antibodies using the genetic information thereof, an antibody culture solution was prepared through the method of Example 4, and the antigen-binding sites, antibody expression rates, etc. in the fully human antibodies were confirmed. Based on comprehensive results including the above results of evaluation of virus-neutralizing activity, 18 fully human antibodies were selected out of 36.

Example 7. Evaluation of Neutralizing Activity of Fully Human Antibody Against MERS-CoV (Second In-Vitro Neutralizing Activity Evaluation)

[0089] 1) Evaluation of Neutralizing Activity Against Saudi Isolate Virus

[0090] The 18 antibodies selected in Example 6 were converted into fully human antibodies, after which the virus-neutralizing activity thereof against Saudi isolate virus was evaluated in the same manner as in 1) of Example 5. As shown in Table 4 below, it was confirmed that 16 of the 18 antibodies had virus-neutralizing activity superior to positive control antibody 1 (binding to MERS-CoV S protein and having known neutralizing effect).

TABLE-US-00004 TABLE 4 50% Virus neutralization 50% Virus neutralization concentration concentration (μg/ml) (Virus back (μg/ml) (Virus back titration 36.1 titration 114.8 Classification TCID.sub.50/well) TCID.sub.50/well) Antibody 1 0.08 0.16 Antibody 2 0.07 0.07 Antibody 3 0.07 0.38 Antibody 4 0.08 0.12 Antibody 5 0.02 0.04 Antibody 6 0.42 ND Antibody 7 0.06 0.13 Antibody 9 0.08 0.18 Antibody 14 0.15 0.25 Antibody 18 0.19 0.20 Antibody 21 0.11 0.16 Antibody 26 0.08 0.11 Antibody 27 0.24 0.31 Antibody 29 0.39 0.78 Antibody 33 2.43 ND Antibody 34 0.38 ND Antibody 35 0.11 0.20 Antibody 36 ND ND Positive control 1.75 2.94 antibody 1

[0091] 2) Evaluation of Neutralizing Activity of Fully Human Antibody Against Korean Isolate Virus

[0092] The 18 antibodies selected in Example 6 were converted into fully human antibodies, after which the virus-neutralizing activity thereof against Korean isolate virus was evaluated in the same manner as in 2) of Example 5.

[0093] Based on the results of PRNT, as shown in Table 5 below regarding 18 antibodies upon first evaluation (7 antibodies) and upon second evaluation (11 antibodies), it was confirmed that all of the 18 antibodies exhibited low numerical values and thus superior virus-neutralizing activity compared to positive control antibodies 1 and 2 (binding to MERS-CoV S protein and having known neutralizing effect).

TABLE-US-00005 TABLE 5 Classification IC.sub.50 (μg/ml) First experiment Positive control antibody 1 0.03875 Positive control antibody 2 0.1215 Antibody 1 0.0004 Antibody 2 0.0009 Antibody 7 0.00065 Antibody 9 0.0011 Antibody 14 0.0015 Antibody 18 0.0076 Antibody 21 0.00095 Second experiment Positive control antibody 1 0.1038 Positive control antibody 2 0.5195 Antibody 3 0.0032 Antibody 4 0.00165 Antibody 5 0.00025 Antibody 6 0.0008 Antibody 26 0.0039 Antibody 27 0.0018 Antibody 29 0.00375 Antibody 33 0.0232 Antibody 34 0.0006 Antibody 35 0.00275 Antibody 36 0.03325

Example 8. Selection of Clone for Cell Line Development

[0094] In order to select an antibody for cell line development, property evaluation was further performed in addition to the evaluation of neutralizing activity. The property evaluation was performed for antibody target site, antibody expression rate, and heat resistance. In order to evaluate heat resistance, briefly, an antibody and Sypro Orange (Thermo Fisher Scientific) were diluted to appropriate concentrations and mixed, after which the prepared sample was placed in a PCR 96-well plate, and the fluorescence value was measured by setting a melt curve from 25° C. to 99° C. using a 7500 Real-Time PCR System (Thermo Fisher Scientific). Based on the comprehensive results of the above neutralizing activity evaluation and the property evaluation shown in Table 6 below, the 6 antibodies shown in Table 5 were selected and cloned into a vector suitable for cell line development.

TABLE-US-00006 TABLE 6 Antibody Antibody Heat Classification target site expression rate (μg/ml) resistance (° C.) Antibody 1 MERS-RBD 3.142 53 Antibody 2 MERS-RBD 6.16 59 Antibody 3 MERS-RBD 4.296 57 Antibody 4 MERS-RBD 3.687 55 Antibody 5 MERS-RBD 3.558 57 Antibody 6 MERS-Other 4.014 57 Antibody 7 MERS-RBD 2.556 N/A Antibody 9 MERS-RBD 2.088 N/A Antibody 14 MERS-RBD 5.611 N/A Antibody 18 MERS-RBD 3.6 55 Antibody 21 MERS-RBD 2.494 55 Antibody 26 MERS-RBD 3.753 53 Antibody 27 MERS-RBD 6.894 57 Antibody 29 MERS-RBD 3.897 N/A Antibody 33 MERS-S2 6.894 N/A Antibody 34 MERS-Other 3.897 53 Antibody 35 MERS-RBD 4.014 N/A Antibody 36 MERS-S2 3.558 51

Example 9. Evaluation of Neutralizing Activity Against Saudi and Jordan Virus (Third In-Vitro Neutralizing Activity Evaluation)

[0095] For the 6 antibodies selected in Example 8, the neutralizing activity thereof against Saudi isolate virus and Jordan isolate virus (MERS-HCoV/Jordan/01) was evaluated in the same manner as in 1) of Example 5. As shown in Table 7 below, the virus-neutralizing activity thereof was superior to four positive control antibodies (binding to MERS-CoV S protein and having known neutralizing effect).

TABLE-US-00007 TABLE 7 Neutralizing activity Neutralizing activity against Saudi virus against Jordan virus (MERS-CoV_KSA/ (MERS-HCoV/Jordan/ Classification EMC/2012) IC.sub.50 (μg/ml) 01) IC.sub.50 (μg/ml) Antibody 1 0.44 0.11 Antibody 2 0.09 0.06 Antibody 3 0.13 0.11 Antibody 4 0.06 0.05 Antibody 5 0.06 0.09 Antibody 6 1.11 1.54 Positive control 0.76 0.43 antibody 1 Positive control N/D N/D antibody 2 Positive control 0.26 0.13 antibody 3 Positive control 0.22 0.26 antibody 4

Example 10. Evaluation of Neutralizing Activity Against Korean Isolate Virus (Third In-Vitro Neutralizing Activity Evaluation)

[0096] For the two antibodies selected in Example 9, a plaque assay was performed in the same manner as in 2) of Example 5, and the neutralizing activity of the antibodies at different concentrations against Korean isolate MERS-CoV (MERS-CoV/Korea/KNIH/002_05_2015) was evaluated. Therefore, it was confirmed that the two antibodies had superior virus-neutralizing activity compared to positive control antibody 4 (binding to MERS-CoV S protein and having known neutralizing effect) (FIG. 1).

Example 11. Evaluation of Virus-Neutralizing Activity (Ex Vivo)

[0097] The neutralizing activity of an antibody for cell line development was evaluated using a human lung tissue infection model (ex vivo) (Table 8). Briefly, Korean isolate MERS-CoV (MERS-CoV/Korea/KNIH/002_05_2015) and an antibody sample for evaluation were prepared based on the IC.sub.50 values and allowed to react for 1 hr, after which human lung tissue was infected with the reaction product of the virus and the antibody and tissue culture was carried out. Then, culture was performed for 3 days, during which the culture supernatant was collected at intervals of 24 hr, and the virus titer was measured through a plaque assay. When 100 ng of each of the two antibodies having the efficacy confirmed in Example 10 was allowed to react with MERS-CoV and human lung tissue was then infected therewith, as shown in Table 8 below, it was confirmed that the growth of MERS-CoV was inhibited in the supernatant at 24, 48, and 72 hr compared to negative and positive control antibodies not treated with the antibody (FIG. 2).

TABLE-US-00008 TABLE 8 Lung Virus content (log10PFU/ml/ng) weight 24 hr after 48 hr after 72 hr after Classification (mg) infection infection infection Antibody 3 (100 ng) 4.1 0 0 0 Antibody 5 (100 ng) 5.4 0 0 0 Positive control antibody 3.9 2.1 3.1 3.2 4 (100 ng) Negative control antibody 4.2 3.4 3.9 3.5 (100 ng)

Example 12. Evaluation of Antibody Properties and Binding Characteristics

[0098] Based on the results of measurement of antibody properties such as virus-neutralizing activity, etc., two final antibodies for cell line development were selected, and the properties thereof were evaluated. As evaluation items, aggregate formation evaluation, sequence analysis, disulfide bond maintenance analysis, signal peptide cleavage evaluation, and heavy-chain and light-chain normal binding were measured. As shown in Table 9 below, there were no special issues in the antibody properties.

TABLE-US-00009 TABLE 9 Evaluation Binding DSF SEC-HPLC CE-SDS method ELISA evaluation Main Peptide mapping LC/MS Intact Evaluation Binding Onset peak HMW LMW Cleavage Bond Cleavage IgG Impurity unit site (° C.) (%) (%) (%) (%) formation (%) (%) (%) Antibody 3 MERS- 57 97.5 2.5 0 100 Normal H(99.6), 80.31 19.69 RBD L(100) Antibody 5 MERS- 57 99.61 0.39 0 100 Normal H(99.98), 81.86 18.14 RBD L(99.96)

[0099] Also, antigen-binding capacity to MERS-CoV surface protein (RBD) was evaluated (SPR, Surface Plasmon Resonance). As shown in Table 10 below, the neutralizing activity of the antibody and antibody properties were comprehensively judged, and the binding characteristics of the two finally selected antibodies were evaluated in comparison with positive control antibody 4. It was confirmed that antibody 5 had superior binding characteristics compared to positive control antibody 4.

TABLE-US-00010 TABLE 10 ka1 kd1 ka2 kd2 Rmax Chi.sup.2 KD % % Sample (1/Ms) (1/s) (1/s) (1/s) (RU) (RU.sup.2) (M) Average SD CV Rel Antibody3 1.137E+06 1.379E−03 8.156E−03 4.380E−03 77.63 1.310 1.21E−09 1.03E−09 2.55E−10 24.7 78 1.277E+06 1.089E−03 7.265E−03 4.515E−03 71.71 0.751 8.53E−10 Antibody5 2.115E+06 2.830E−04 2.837E−02 2.329E−03 87.69 0.539 1.34E−10 1.36E−10 2.90E−12 2.1 589 2.060E+06 2.841E−04 2.420E−02 2.142E−03 86.82 0.463 1.38E−10 Positive 1.674E+05 1.343E−04 9.841E−03 8.071E−03 75.88 0.041 8.02E−10 8.01E−10 1.77E−12 0.2 100 control 1.723E+05 1.378E−04 1.241E−02 1.082E−02 74.87 0.031 8.00E−10 antibody 4

Example 13. Evaluation of Virus-Neutralizing Activity (In Vivo)

[0100] The neutralizing activity of the two antibodies for cell line development against MERS-CoV was evaluated using an animal model. Specifically, the antibody was administered to hDPP4 (human dipeptidyl peptidase 4) receptor-overexpressing mice (TG mice), required for infection of the human body with the virus, and infection with Korean isolate MERS-CoV (MERS-CoV/Korea/KNIH/002_05_2015) was conducted, and thus the preventive and therapeutic efficacies of the two finally selected antibodies were evaluated.

[0101] Specifically, the infection conditions of hDPP4 TG mice with MERS-CoV and the effect of positive control antibody 4 were established using antibodies 3 and 5 and the negative control antibody (FIG. 3). For this, animals were infected with the virus, and on the next day, each antibody was intraperitoneally injected thereto in the corresponding dose. A certain number of days after infection, the lung tissue of the mice was extracted, and the virus was quantified through quantitative PCR (FIG. 3A) and a plaque assay (FIG. 3B). It was confirmed in advance that the animal infection experiment was properly progressed and also that antibody 3 and antibody 5 were capable of having superior or similar therapeutic efficacy compared to positive control antibody 4.

[0102] In order to evaluate the preventive efficacy, each of antibody 5, positive control antibody 4 and the negative control antibody was intraperitoneally injected to hDPP4 (human dipeptidyl peptidase 4) receptor-overexpressing mice (hDPP4 TG mice), required for infection of the human body with MERS-CoV, and on the next day, infection with Korean isolate MERS-CoV (MERS-CoV/Korea/KNIH/002_05_2015) was conducted. A certain number of days after infection, the lung tissue of the mice was extracted, and the virus was quantified (FIG. 4). Based on the MERS-CoV quantification results through quantitative PCR (FIG. 4A) and a plaque assay (FIG. 4B), it was confirmed that antibody 5 had superior preventive efficacy compared to positive control antibody 4.

[0103] Moreover, the mouse lung was extracted on the 7.sup.th day of infection, after which changes in the tissue were directly observed through H&E staining (FIG. 5). Therefore, in the group treated with the negative control antibody, the airway diaphragm was thickened, and many inflammatory cells were introduced, indicating severe histopathological changes (“a” and “d” of FIG. 5). Although the group treated with the positive control antibody was not as severely affected as the group treated with the negative control antibody, the introduction of inflammatory cells and thickened airway diaphragm were confirmed (“b” and “e” of FIG. 5). In contrast, in the group treated with antibody 5, it was confirmed that there were fewer histopathological changes compared to the other treated groups (“c” and “f” of FIG. 5).

[0104] Furthermore, for the evaluation of therapeutic efficacy, animals were infected with the above virus, and on the next day, each of antibody 5, positive control antibody 4 and the negative control antibody was intraperitoneally injected thereto. A certain number of days after infection, the mouse body weight reduction and the mouse survival rate were observed, and also, the mouse lung tissue was extracted and thus the virus was quantified (FIG. 6). Based on the results of evaluation of mouse body weight reduction (FIG. 6A) and mouse survival rate (FIG. 6B), weight loss and mortality were observed only at the lowest dose (2 μg) of the negative control antibody and antibody 5. Also, based on the results of MERS-CoV quantification through quantitative PCR (FIG. 6C) and a plaque assay (FIG. 6D), it was confirmed that antibody 5 had superior or similar therapeutic efficacy compared to positive control antibody 4.