ANTIBODY BINDING SPECIFICALLY TO SARS-COV-2 S PROTEIN OR ANTIGEN-BINDING FRAGMENT THEREOF, BISPECIFIC ANTIBODY, AND USES THEREOF

Abstract

The present invention relates to an anti-SARS-CoV-2 S protein-specific antibody or an antigen-binding fragment thereof, and therapeutic and diagnostic uses thereof. The anti-SARS-CoV-2 S protein-specific antibody or antigen-binding fragment thereof according to the present invention can bind specifically to the S protein, which plays an important role in the infiltration of SARS-CoV-2 into host cells, to inhibit the infection of SARS-CoV-2, and thus can be advantageously used as a therapeutic agent for COVID-19 and as a diagnostic agent and diagnostic kit for COVID-19.

Claims

1. An antibody or an antigen binding fragment thereof specifically binding to SARS-CoV-2 S protein, comprising: (i) a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 1, CDR-H2 of SEQ ID NO: 2, and CDR-H3 of SEQ ID NO: 3; and a light chain comprising CDR-L1 of SEQ ID NO: 4, CDR-L2 of SEQ ID NO: 5, and CDR-L3 of SEQ ID NO: 6; (ii) a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 10, CDR-H2 of SEQ ID NO: 11, and CDR-H3 of SEQ ID NO: 12; and a light chain variable region comprising CDR-L1 of SEQ ID NO: 13, CDR-L2 of SEQ ID NO: 14, and CDR-L3 of SEQ ID NO: 15; (iii) a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 19, CDR-H2 of SEQ ID NO: 20, and CDR-H3 of SEQ ID NO: 21; and a light chain variable region comprising CDR-L1 of SEQ ID NO: 22, CDR-L2 of SEQ ID NO: 23, and CDR-L3 of SEQ ID NO: 24; (iv) a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 28, CDR-H2 of SEQ ID NO: 29, and CDR-H3 of SEQ ID NO: 30; and a light chain variable region comprising CDR-L1 of SEQ ID NO: 31, CDR-L2 of SEQ ID NO: 32, and CDR-L3 of SEQ ID NO: 33; or (v) a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 37, CDR-H2 of SEQ ID NO: 38, and CDR-H3 of SEQ ID NO: 39; and a light chain variable region comprising CDR-L1 of SEQ ID NO: 40, CDR-L2 of SEQ ID NO: 41, and CDR-L3 of SEQ ID NO: 42.

2. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or antigen binding fragment thereof comprises: (i) the heavy chain variable region of SEQ ID NO: 7 and the light chain variable region of SEQ ID NO: 8; (ii) the heavy chain variable region of SEQ ID NO: 16 and the light chain variable region of SEQ ID NO: 17; (iii) the heavy chain variable region of SEQ ID NO: 25 and the light chain variable region of SEQ ID NO: 26; (iv) the heavy chain variable region of SEQ ID NO: 34 and the light chain variable region of SEQ ID NO: 35; or (v) the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 44.

3. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or antigen binding fragment thereof comprises: (i) the amino acid sequence of SEQ ID NO: 9; (ii) the amino acid sequence of SEQ ID NO: 18; (iii) the amino acid sequence of SEQ ID NO: 27; (iv) the amino acid sequence of SEQ ID NO: 36; or (v) the amino acid sequence of SEQ ID NO: 45.

4. The antibody or antigen binding fragment thereof according to claim 1, wherein the SARS-CoV-2 S protein is a receptor binding domain (RBD), an 51 domain, or a full-length spike protein.

5. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or the antigen binding fragment thereof is a monoclonal antibody, a polyclonal antibody, scFv, Fab, F(ab), F(ab)2, scFv-Fc, a minibody, a diabody, a triabody, a tetrabody, a bispecific antibody, a multispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, or an antigen binding fragment thereof, each comprising the heavy chain variable region and the light chain variable region.

6. A nucleic acid molecule, comprising a nucleotide sequence coding for the antibody or antigen binding fragment thereof according to claim 1.

7. A recombinant vector carrying the nucleic acid molecule of claim 6.

8. An isolated host cell transformed with the recombinant vector of claim 7.

9. A pharmaceutical composition for prevention or treatment of SARS-CoV-2 infectious disease, the composition comprising the antibody or antigen binding fragment thereof specifically binding to SARS-CoV-2 S protein according to claim 1, and a pharmaceutically acceptable carrier.

10. A composition for detecting SARS-CoV-2 virus, comprising the antibody or the antigen binding fragment thereof specifically binding to SARS-CoV-2 S protein according to claim 1.

11. The composition of claim 10, wherein the composition comprises a pair of the following antibodies or antigen binding fragments thereof specifically binding to SARS-CoV-2 S protein: (i) antibody or antigen binding fragment thereof comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 28, HCDR2 comprising the amino acid sequence of SEQ ID NO: 29, HCDR3 comprising the amino acid sequence of SEQ ID NO: 30, LCDR1 comprising the amino acid sequence of SEQ ID NO: 31, LCDR2 comprising the amino acid sequence of SEQ ID NO: 32, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 33; and (ii) antibody or antigen binding fragment thereof comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 24.

12. A kit for detecting SARS-CoV-2 virus, comprising the antibody or antigen binding fragment thereof according to claim 1.

13. The kit of claim 12, wherein the kit comprises a pair of the following antibodies or antigen binding fragments thereof specifically binding to SARS-CoV-2 S protein: (i) antibody or antigen binding fragment thereof comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 28, HCDR2 comprising the amino acid sequence of SEQ ID NO: 29, HCDR3 comprising the amino acid sequence of SEQ ID NO: 30, LCDR1 comprising the amino acid sequence of SEQ ID NO: 31, LCDR2 comprising the amino acid sequence of SEQ ID NO: 32, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 33; and (ii) antibody or antigen binding fragment thereof comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 24.

14. The kit of claim 13, wherein the kit is a sandwich ELISA kit, and wherein one of the antibodies or antigen-binding fragments thereof of (i) and (ii) is used as a capture antibody and the other as a detection antibody.

15. The kit of claim 14, wherein the kit further comprises a signal-detecting antibody conjugated with a label binding to the detection antibody.

16. A bispecific antibody binding specifically to SARS-CoV-2, wherein the bispecific antibody comprises: (a) an antibody or an antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 28, H-CDR2 having the amino acid sequence of SEQ ID NO: 29, and H-CDR3 having the amino acid sequence of SEQ ID NO: 30; and the light chain variable comprising light chain comprising complementarity determining region 1 (L-CDR1) having the amino acid sequence of SEQ ID NO: 31, L-CDR2 having the amino acid sequence of SEQ ID NO: 32, and L-CDR3 having the amino acid sequence of SEQ ID NO: 33; and (b) an antibody or an antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 having the amino acid sequence of SEQ ID NO: 19, H-CDR2 having the amino acid sequence of SEQ ID NO: 20, and H-CDR3 having the amino acid sequence of SEQ ID NO: 21; and the light chain variable comprising light chain comprising L-CDR1 having the amino acid sequence of SEQ ID NO: 22, L-CDR2 having the amino acid sequence of SEQ ID NO: 23, and L-CDR3 having the amino acid sequence of SEQ ID NO: 24.

17. The bispecific antibody of claim 16, wherein the heavy chain variable region of (a) comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region of (a) comprises the amino acid sequence of SEQ ID NO: 8; and the heavy chain variable region of (b) comprises the amino acid sequence of SEQ ID NO: 17 and the light chain variable region of (b) comprises the amino acid sequence of SEQ ID NO: 18.

18. A pharmaceutical composition comprising the bispecific antibody of claim 16 and a pharmaceutically acceptable carrier for treating SARS-CoV-2 infectious disease.

19. The pharmaceutical composition of claim 18, wherein the SARS-CoV-2 is a variant having, on the amino acid sequence of RBD, a mutation selected from the group consisting of N354D/D364Y, V367F, W436R, R408I, G476S, V483A, V341I, F342L, A435S, and a combination thereof.

20. The pharmaceutical composition of claim 18, wherein the SARS-CoV-2 is selected from the group consisting of a wild type, an alpha variant, a beta variant, a gamma variant, a delta variant, and a kappa variant.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0219] FIG. 1 is a view showing the configuration of S1, S2, and RBD proteins that are constituent proteins of spikes of SARS-CoV-2.

[0220] FIG. 2 depicts SDS-PAGE analysis result of purchased SARS-CoV-2 spike proteins S1, S2, and RBD protein antigens.

[0221] FIG. 3 is a diagram showing results of the scFv binding to RBD and phage ELISA for the selection of human antibodies specific to the RBD antigen of the SARS-CoV-2 virus.

[0222] FIG. 4 illustrates the comparison of the yield after mass production and final purification of the SARS-CoV-2 RBD-specific IgG antibodies of the present disclosure.

[0223] FIG. 5 is an image of the SDS-PAGE result of the five selected SARS-CoV-2 RBD-specific IgG antibodies.

[0224] FIG. 6 shows graphs of the reactivity analysis results for the five selected SARS-CoV-2 RBD-specific IgG antibodies against three types of SARS-CoV-2 antigens (RBD, Spike, and S1) using ELISA.

[0225] FIG. 7 is a graph of cross-reactivity analysis results of the five selected SARS-CoV-2 RBD-specific IgG antibodies against the RBD antigens of both the SARS-CoV-2 and SARS-CoV viruses, using ELISA.

[0226] FIG. 8 shows plots of affinity analysis results of the five selected SARS-CoV-2 RBD-specific IgG selected antibodies against the SARS-CoV-2 S1 antigen.

[0227] FIG. 9 shows plots of affinity analysis results of the five selected SARS-CoV-2 RBD-specific IgG antibodies against the SARS-CoV-2 RBD antigen.

[0228] FIG. 10 is an image of the SDS-PAGE analysis results to verify the molecular weight and purity of the nine purchased RBD variant antigens of SARS-CoV-2.

[0229] FIG. 11 shows graphs of the reactivity analysis results of the nine RBD variants of SARS-CoV-2 and the five selected SARS-CoV-2 RBD-specific antibodies.

[0230] FIG. 12 is a schematic view illustrating the direct interaction assay between hACE2 and SARS-CoV-2 RBD protein.

[0231] FIG. 13 is a plot of the changes in direct interaction values between the SARS-CoV-2 RBD protein and hACE2, depending on the concentration of hACE2.

[0232] FIG. 14 is a schematic view illustrating an assay of the antibodies for inhibitory activity against direct interaction between hACE2 and SARS-CoV-2 RBD protein.

[0233] FIG. 15 shows plots of the inhibition activity of the five selected IgG antibodies against direct interaction between hACE2 and SARS-CoV-2 RBD protein, with IC50 measurements given therein.

[0234] FIG. 16 shows views illustrating that the monoclonal antibody RD3 of the present disclosure has binding affinity for various recombinant SARS-CoV-2 RBDs (wild type, Alpha, Beta, Gamma, Delta, or Kappa) as analyzed by Surface Plasmon Resonance (SPR).

[0235] FIG. 17 shows views illustrating that the monoclonal antibody RB6 of the present disclosure has binding affinity for various recombinant SARS-CoV-2 RBDs (wild type, Alpha, Beta, Gamma, Delta, or Kappa) as analyzed by Surface Plasmon Resonance (SPR).

[0236] FIG. 18 shows plots of the neutralizing activity of the monoclonal antibody RD3 of the present disclosure against SARS-CoV-2 pseudovirus infection, with IC50 values given therein.

[0237] FIGS. 19 and 20 are plots of the weight change (FIG. 19) and clinical severity (FIG. 20) when the monoclonal antibody RD3 of the present disclosure was administered to mice infected with the wild-type SARS-CoV-2 virus.

[0238] FIG. 21 is a schematic view illustrating a process of selecting SARS-CoV-2 virus RBD-specific human antibody from an scFv library through a phage display technology.

[0239] FIG. 22 shows results of phage ELISA for scFv binding to RBD to select human antibodies specific for the RBD antigen of SARS-CoV-2 virus.

[0240] FIG. 23 is an image of SDS-PAGE for four selected IgG antibodies specific for SARS-CoV-2 RBD.

[0241] FIG. 24 shows KD values of four selected SARS-CoV-2 RBD-specific IgG antibodies to SARS-CoV-2 RBD antigens as measured by Surface Plasmon Resonance (SPR).

[0242] FIG. 25 shows competition ELISA results between K102.1-HRP and four selected SARS-CoV-2 RBD-specific IgG antibodies to select pairs of antibodies for sandwich ELISA.

[0243] FIG. 26 shows sandwich ELISA results to identify the pairing in the selected antibodies of the present disclosure.

[0244] FIG. 27 shows SPR-based competition binding assay results to determine the presence or absence of different epitopes for the selected antibody pair of the present disclosure.

[0245] FIG. 28 is a schematic representation of sandwich ELISA using a selected antibody pair specific for SARS-CoV-2 RBD antigen.

[0246] FIG. 29 is a plot showing the optimization of concentration for the capture antibody of the selected antibody pair for sandwich ELISA.

[0247] FIG. 30 is a plot showing the optimization of concentration for the detection antibody of the selected antibody pair for sandwich ELISA.

[0248] FIG. 31 is a calibration curve for determining the limit of detection (LOD) of SARS-CoV-2 RBD.

[0249] FIG. 32 is a table showing CV values and recovery of intra- and inter-assay for optimized sandwich ELISA.

[0250] FIG. 33 is a graph showing the ability of optimized sandwich ELISA to detect eight types of SARS-CoV-2 RBD variants according to concentration.

[0251] FIG. 34 is a graph showing results of competition ELISA conducted to identify the recognition of respective independent SARS-CoV-2 RBD epitopes by the separate antibodies.

[0252] FIGS. 35, 36 and 37 are schematic views showing structures of the three types of bispecific antibodies capable of being fabricated with the antibodies K102.1 and K102.2 of the present disclosure and vector structures adapting for expressing same.

[0253] FIG. 38 is a graph showing expression levels of the antibodies (K102.1 and K102.2) and bispecific antibodies (K202.A, K202.B, and K202.C) of the present disclosure.

[0254] FIG. 39 is an image of western blots showing expression of the antibodies (K102.1 and K102.2) and bispecific antibodies (K202.A, K202.B, and K202.C) of the present disclosure with high purity.

[0255] FIGS. 40a, 40b, 40c and 40d show the binding affinity of the antibodies and bispecific antibodies of the present disclosure for wild-type SARS-CoV-2 and alpha, beta, gamma, delta, and kappa variants thereof as measured by SPR analysis.

[0256] FIG. 41 shows results of competition assays conducted to identify the recognition of two different independent epitopes by the bispecific antibody of the present disclosure as measured by SPR.

[0257] FIG. 42 shows binding inhibition efficacy of the bispecific antibody at various antibody concentrations to confirm the neutralizing activity of the bispecific antibody against interaction between hACE2 and SARS-CoV-2 wild-type and variants.

[0258] FIGS. 43a and 43b show binding inhibition efficacy of the bispecific antibody of the present disclosure at various antibody concentrations to confirm the neutralizing activity of the bispecific antibody against interaction between hACE2 and RBD with the mutation N354D/D364Y, V367F, W436R, R408I, G476S, V483A, V341I, F342L, or A435S.

[0259] FIG. 44 shows the establishment of hACE2-overexpresisng 293T stable cell lines (293T/hACE2 cells).

[0260] FIG. 45 shows neutralizing activity of the antibodies against the pseudotyped virus infection of SARS-CoV-2 wild-type and variants.

[0261] FIGS. 46a, 46b, 46c and 46d shows changes in pseudotyped virus infection of various SARS-CoV-2 wild-type and variants in the presence or absence of the bispecific antibody K202.B of the present disclosure.

[0262] FIG. 47 is a plot showing pharmacokinetic properties of the bispecific antibody K202.B of the present disclosure.

[0263] FIG. 48 is a schematic view illustrating an experiment for analyzing in vivo efficacy of the bispecific antibody K202.B of the present disclosure in wild-type SARS-CoV-2.

[0264] FIGS. 49, 50, 51, 52 and 53 show in vivo efficacy of the bispecific antibody K202.B of the present disclosure in response to treatment with SARS-CoV-2 wild-type virus and antibody in terms of daily body weight change (FIG. 49), clinical score (FIG. 50), expression level of viral gene (FIGS. 51 and 52), and pathological examination of lung (FIG. 53).

[0265] FIGS. 54 and 55 show in vitro toxicity of the bispecific antibody K202.B of the present disclosure in terms of cell viability (FIG. 54) and expression levels of cell adhesion molecules (FIG. 55).

[0266] FIG. 56 shows biochemical examination results of in vivo toxicity responsible for hepatic and renal toxicity with sera from mice to which K202.B has been administered.

[0267] FIG. 57 is a schematic view illustrating an experiment for analyzing in vivo efficacy of the bispecific antibody K202.B of the present disclosure in SARS-CoV-2 delta variant.

[0268] FIGS. 58, 59, 60, 61 and 62 show in vivo efficacy of the bispecific antibody K202.B of the present disclosure in response to treatment with SARS-CoV-2 delta variant virus and antibody in terms of daily body weight change (FIG. 58), clinical score (FIG. 59), expression level of viral gene (FIGS. 60 and 61), and pathological examination of lung (FIG. 62).

BEST MODE FOR CARRYING OUT THE INVENTION

[0269] A better understanding of the present disclosure may be obtained through the following examples which are set forth to illustrate, but are not to be construed to limit, the present disclosure.

Examples I

[0270] Throughout the description, the term % used to express the concentration of a specific material, unless otherwise particularly stated, refers to (wt/wt) % for solid/solid, (wt/vol) % for solid/liquid, and (vol/vol) % for liquid/liquid.

Example I-1. Preparation of SARS-CoV-2 Protein Antigen for Antibody Selection

[0271] For use in selecting antibodies binding specifically to SARS-CoV-2, the spike full-length protein of SARS-CoV-2 and its constituent proteins 51 and RBD protein antigens were purchased from Sino Biological. The configuration of 51 and RBD proteins in the spike protein is depicted in FIG. 1. The proteins purchased were measured for purity and molecular weight by SDS-PAGE (FIG. 2).

[0272] As shown in FIG. 2, it was observed that the purchased S1, S2, and RBD proteins of the spike protein were high in purity.

Example I-2: Selection of Human Antibody Specific for RBD Antigen of SARS-CoV-2 Virus by Phage Display Technique

[0273] A certain amount of secured SARS-CoV-2 RBD antigen was conjugated to epoxy-conjugated Dynabead (Invitrogen, USA), and human antibodies specific for RBD were selected using the phage display technique. After five rounds of bio-panning, binding antibody clones were measured for titer and degree of enrichment by round through titration. Subsequently, human antibody clones that had excellent reactivity to the RBD antigen and were specific for the antigen were selected using individual phage ELISA. The DNA was isolated through miniprep and ten types of RBD-specific human antibodies with different CDR sequences were secured after base sequencing. The results of the phage ELISA are presented in FIG. 3.

Example I-3: IgG Conversion, Production, and Purification of Selected Antibodies

[0274] 3-1. Conversion of SARS-CoV-2 RBD Antigen-Specific scFv Antibody into IgG (IgG 4 Type)

[0275] For heavy and light chains of the ten kinds of RBD domain-specific scFv antibodies selected, insert DNAs were obtained. They were subcloned into bicistronic vectors for production full-IgG (IgG4 type) antibodies. For use in producing IgG antibodies in Expi293 cells, the recombinant DNA of each of the ten different scFvs was isolated in a large amount with high purity by using Maxi-prep kit. The purity of DNA was examined by NanoDrop.

3-2. Mass Production of RBD-Specific IgG (IgG4) Antibody

[0276] The 10 different IgG antibodies were transfected into 1 L or greater of Expi293 cells with the aid of the Expi293 system (Invitrogen) comprising ExpiFectamine for transient expression, followed by incubation for 7 days. In order to purify the antibodies, the cultures were centrifuged and the supernatants (media) were obtained by removing the cell pellets.

3-3. Purification and Production Assay of Antibodies

[0277] The selected antibodies were purified by affinity column chromatography using protein A sepharose beads. In addition, the RBD-specific IgG antibodies were analyzed for yield after mass production and final purification.

[0278] The analysis of the 10 different RBD-specific antibodies for productivity showed that the five antibodies RB4, RB6, RD3, RD10, and RG6 were produced at high yields of 50 mg/L or higher, with the productivity of 140 mg/L or higher given for RD3, RD10, and RG6 clones (FIG. 4).

Example I-4: Physical/Chemical Characterization of Selected Antibodies

4-1. Purity and Molecular Weight of Selected Antibodies

[0279] The five selected SARS-CoV-2 RBD-specific antibodies were loaded at the same content to a polyacrylamide gel. After SDS-PAGE, all the five antibodies were observed to have a purity of 95% or higher and a molecular weight of 50 kDa for heavy chain and 25 kDa for light chain, as analyzed by Coomassie Brilliant Blue staining (FIG. 5).

4-2. Reactivity of Selected Antibodies to SARS-CoV-2 RBD, 51 Domain, and Full-Length Spike Antigens

[0280] In order to examine the reactivity of the five selected SARS-CoV-2 RBD-specific antibodies to SARS-CoV-2 RBD, S1 domain, and full-length spike protein antigens, 0.1 g of each antigen was coated on 96-well high binding plates (Corning, USA) and ELISA was conducted for each antibody. The results are depicted in FIG. 6.

[0281] As can be seen in FIG. 6, all the five selected antibodies were observed to have superb reactivity to SARS-CoV-2 Spike, S1, and RBD antigens.

4-3. Cross-Reactivity of Selected Antibodies to SARS-CoV-2 and SARS-CoV RBD Antigens

[0282] In order to examine the reactivity of the five selected SARS-CoV-2-specific antibodies to SARS-CoV RBD as well as SARS-CoV-2 RBD, 0.1 g of each of purchased SARS-CoV-2 and SARS-CoV RBD was coated on 96-well high binding plates (Corning, USA) and ELISA was conducted for each antibody. The results are depicted in FIG. 7.

[0283] As shown in FIG. 7, all the five selected antibodies were observed to have reactivity to SARS-CoV RBD antigen, too.

4-4. Affinity (K.SUB.D .Value) of Selected Antibodies for RBD and 51 Antigens

[0284] The five selected antibodies were measured for K.sub.D values for SARS-CoV-2 RBD and S1. In this regard, SARS-CoV-2 RBD and S1 purchased were each bound to 96-well high binding plates (Corning, USA), and absorbance (450 nm) was read while increasing the concentrations of the selected antibodies. The results are depicted in FIGS. 8 and 9.

[0285] FIG. 8 shows plots of affinity of the five selected RBD-specific antibodies for SARS-CoV-2 S1 antigen. FIG. 9 shows plots of affinity of the five selected RBD-specific antibodies for SARS-CoV-2 RBD antigen.

[0286] As shown in FIGS. 8 and 9, K.sub.D values for 51 antigen were decreased in the order of RB4, RG6, RD3, RD10, and RB6 and K.sub.D values for RBD antigen were decreased in the order of RB4, RD3, RG6, RD10, and RB6. Of the five selected antigens, the four antigens RB4, RD3, RD10, and RG6 4 exhibited a K.sub.D value of 10.sup.10 M for 51 antigen, and all the five antibodies exhibited a K.sub.D value of 10.sup.10 M for RBD antigen. It was finally confirmed that the selected antibodies had high affinity for each antibody.

4-5. Reactivity of Selected Antibodies to 9 RBD Mutants

[0287] In relation to SARS-CoV-2, nine typical RBD mutant antigens (V431A, F342L, V367F, R408I, A435S, W436R, G476S, V483A, and N354D/D364Y) that were classified worldwide were purchased from Sino Biological. Each of the antigens was measured for purity and molecular weight by SDS-PAGE. As a result, the RBD mutants were measured to have a size of about 30 kDa and a purity of 90% or higher (FIG. 10).

[0288] In addition, in order to examine the reactivity of the five selected antibodies to the nine SARS-CoV-2 RBD mutant antigens, ELISA was performed. As a result, all the five selected RBD-specific antibodies were found to bind to the nine RBD mutant protein antigens, too (FIG. 11).

Example I-5: Inhibitory Activity Against Direction Interaction Between hACE2 and SARS-CoV-2 RBD Protein (Functional Analysis for Deriving Leading Substance)

5-1. Establishment of Basic Technology for Direct Interaction Assay

[0289] The neutralization ability of antibody to inhibit protein-protein interaction between hACE2 receptor and SARS-CoV-2 spike protein was investigated by ELISA using purified proteins. To this end, neutralization ability was analyzed using the Spike RBD (SARS-CoV-2): ACE2 inhibitor screening assay kit (BPS Bioscience, Cat. No. 79931).

[0290] In brief, SARS-CoV-2 RBD protein (Fc-tagged) was coated on 96-well plates included within the assay kit and incubated with the ligand human ACE2 (His-tagged; hACE2-His). Then, anti-His-HRP and HRP substrate were added before measuring chemiluminescence on ELISA reader to analyze binding affinity between RBD domain-ACE2. A schematic diagram for assaying direct interaction between hACE2-His and SARS-CoV-2 RBD is given in FIG. 12. The assay results are depicted in FIG. 13.

[0291] As shown in FIG. 13, chemiluminescence values increased with increasing of hACE2-His levels, indicating that interaction between hACE2 and SARS-CoV-2 RBD increases with increasing of hACE2 concentration. The median value 10 nM on the standard curve was determined as the concentration of hACE2-His to be used for inhibition assay.

5-2. Assay of RBD Neutralizing Antibody for Inhibiting ACE2-Spike RBD Interaction

[0292] Using the established direct interaction assay, the selected antibodies were measured for ability to inhibit interaction between SARS-CoV-2 RBD and hACE2.

[0293] In brief, SARS-CoV-2 RBD protein (Fc-tagged) was coated on 96-well plates included within the assay kit and incubated with the ligand human ACE2-His alone and in combination with the selected antibodies (0.016, 0.08, 0.4, 2, 10, and 50 nM). Then, anti-His-HRP and HRP substrate were added before measuring chemiluminescence on ELISA reader to analyze the ability of the antibodies to inhibit interaction between SARS-CoV-2 RBD and hACE2. A schematic diagram for assaying direct interaction is given in FIG. 12. The assay results are depicted in FIG. 13.

[0294] As shown in FIG. 15, the addition of RB4, RB6, RD3, RD10, and RG6 by concentration effectively inhibited interaction between spike RBD region-hACE2.

[0295] In FIG. 15, the IC.sub.50 value was measured to be 0.8412 nM for RB4, 1.950 nM for RB6, 1.315 nM for RD3, 1.965 nM for RD10, and 84.02 nM RG6. Among them, the four antibodies RB4, RB6, RD3, and RD10 were observed to effectively inhibit interaction between SARS-CoV-2 RBD and hACE2 even when used at very low concentrations.

Example I-6: Surface Plasmon Resonance (SPR) Assay of Neutralizing Antibodies

[0296] The binding kinetics of antibodies (RD3 and RB6) to SARS-CoV-2 RBD were analyzed at 25 C. on an iMSPR-mini instrument (iCLUEBIO, Seongnam, Republic of Korea) using 10 mM HEPES pH 7.4, 700 mM NaCl, 2 mM CaCl.sub.2), 1 mM MnCl.sub.2, and 0.005% (v/v) Tween-20 as a running buffer. The recombinant SARS-CoV-2 RBD (wild-type, Alpha, Beta, Gamma, Delta, or Kappa) was covalently immobilized on the surface of a COOHAu chip (iCLUEBIO) up to 500 response units through standard amine coupling. The monoclonal antibodies (8, 16, 32, 64, and 128 nM) were injected onto the surface of a sensor chip at a flow rate of 50 L/min. Curve fitting and data analysis were performed using the iMSPR analysis software (Tracedrawer; iCLUEBIO). The results are summarized in Tables 1 and 2 and depicted in FIGS. 16 and 17.

TABLE-US-00001 TABLE 1 RD3 RBD types K.sub.a (10.sup.5 M.sup.1) K.sub.d (10.sup.4 M.sup.1S.sup.1) K.sub.D (nM) Wild-type 1.61 2.76 1.72 Alpha (B.1.1.7) 2.06 1.93 0.94 Beta (B.1.351) 0.93 2.95 3.18 Gamma (P.1) 0.70 3.15 4.51 Delta (B.1.617.2) 2.11 2.98 1.41 Kappa (B.1.617.1) 0.95 3.17 3.34

TABLE-US-00002 TABLE 2 RB6 RBD types K.sub.a (10.sup.5 M.sup.1) K.sub.d (10.sup.4 M.sup.1S.sup.1) K.sub.D (nM) Wild-type 1.97 4.34 2.20 Alpha (B.1.1.7) 1.54 5.57 3.62 Beta (B.1.351) 4.92 7.88 1.6 Gamma (P.1) 4.54 9.39 2.07 Delta (B.1.617.2) 3.33 5.11 1.53 Kappa (B.1.617.1) 4.07 6.83 1.68
As understood from the data of Tables 1 and 2, the antibodies RD3 and RB6 of the present disclosure had high binding affinity for various types of SARS-CoV-2 RBD.

Example I-7: SARS-CoV-2 Pseudovirus Neutralization Assay

[0297] Pseudotyped replication-deficient lentiviral particles carrying the SARS-CoV-2 S protein of the wild-type or B.1 (D614G) variant, and a firefly luciferase reporter gene were prepared using Lenti-X SARS-CoV-2 packaging mix (Takara Bio, Kusatsu, Japan). Briefly, the packaging mix was transiently transfected into Expi293 cells with ExpiFectamine 293 reagent. After culturing for 72 hours, the supernatants containing the pseudotyped viruses were collected and centrifuged briefly (500g for 10 min) to remove cellular debris. Virus titration was measured using Lenti-X GoStix Plus (Takara Bio) according to the manufacturer's instructions.

[0298] The pseudotyped replication-deficient Moloney murine leukemia virus (MLV) particles carrying the SARS-CoV-2 S protein of B.1.1.7 (alpha), B.1.617.2 (delta) or B.1.617 (kappa) variant, and a firefly luciferase reporter gene were obtained from eEnzyme (Gaithersburg, MD, USA).

[0299] To determine the neutralization activity of monoclonal antibodies on pseudotyped virus infection, 110.sup.4 293T/hACE2 cells in 50 L culture medium were seeded in 96-well tissue culture plates overnight. Serial dilutions of the antibodies were pre-incubated at room temperature for 10 minutes with 50 L of pseudotyped virus [110.sup.7 PFU/mL], and the mixture was subsequently incubated with the cells for 24 hours.

[0300] The firefly luciferase reporter gene expression (indicative of viral presence) was measured using ONE-Glo luciferase substrate (Promega, Madison, WI, USA). In brief, the culture medium was removed and incubated with 100 l of ONE-Glo substrate. After 5 minutes, 70 l supernatant was transferred to white flat-bottom 96-well assay plates (Corning; Lowell, MA, USA) and the luminescence signal was measured using the Synergy H1 microplate reader. The recorded relative luminescence units were normalized to those derived from cells infected with each SARS-CoV-2 pseudotyped virus in the absence of antibodies. Dose-response curves for IC.sub.50 values were determined using 4-parameter non-linear regression analysis (Graph Pad Prism 8.0).

[0301] Results are summarized in Table 3 and depicted in FIG. 18.

TABLE-US-00003 TABLE 3 IC.sub.50 (nM) Pseudovirus types RD3 Wild-type 1.94 0.07 D614G (B.1) 1.40 0.01 Alpha 6.16 0.08 Delta 4.48 0.10 Kappa 94.78 0.34

Example I-8: Preparation of True SARS-CoV-2 Virus

[0302] All experiments for true wild-type SARS-CoV-2 viruses were performed in a Biosafety Level 3 laboratory facility. A dilution of 40 l of the patient sample in medium was inoculated into 150,000 VERO E6 cells in a 6-well plate. After 72 hours of infection, the supernatant was collected, centrifuged, and stored at 80 C. After two consecutive passages, RNA samples were prepared from the supernatant, and NGS confirmed that the clinical isolate was wild-type.

Example I-9: In Vivo Infection and Clinical Monitoring

[0303] All procedures for in vivo efficacy studies of monoclonal antibodies were approved by the Institutional Animal Care and Use Committee (IACUC) at KNOTUS (KNOTUS IACUC, Protocol Number: 22-KE-0076) and performed in a biosafety cabinet at the Biosafety Level 3 facilities. Female K18-hACE2 c57BL/6J mice 8-10 weeks old were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and housed in a specific pathogen-free condition and allowed to freely access foods and water. They were randomly assigned into experimental groups.

[0304] The mice (n=7/group) were anesthetized by isoflurane inhalation and intranasally inoculated with 10.sup.4 PFU wild-type SARS-CoV-2 in 30 L of PBS. After viral infection, the monoclonal antibody IgG1 or IgG4 was intravenously injected once (+3 h) at a dose of 30 mg/kg. After infection, the mice were monitored for weight change and the results are depicted in FIG. 19.

[0305] In addition, clinical severity was scored according to the criteria of Table 4, below, and the results are depicted in FIG. 20.

TABLE-US-00004 TABLE 4 Score Description Appearance & Mobility 0 Healthy No observable sign of disease 1 Slightly Slightly ruffled coat ruffled 2 Ruffled Ruffled coat throughout the body and a wet appearance 3 Sick Very ruffled coat and slightly closed, inset eyes 4 Very sick Very ruffled coat; closed, inset eyes; and moribund state

[0306] As shown in FIG. 19, the IgG4-type RD3 monoclonal antibody-administered group experienced less weight loss compared to the PBS-administered group, confirming its effect in alleviating clinical severity. Also, as illustrated in FIG. 20, the clinical severity appeared relatively lower in the RD3 (IgG4)-administered group compared to the PBS- or RD3 (IgG1)-administered groups. From these results, it was evident that the RD3 monoclonal antibody of the present disclosure is more efficacious in its IgG4 form.

Example II

Example II-1. Isolation of Human Antibody Specific for RBD Antigen of SARS-CoV-2 Virus by Using Phage Display Technology

[0307] Selection was made of SARS-CoV-2 RBD antigen-specific human antibodies from the human synthetic single-chain variable fragment (scFv) antibody library. As illustrated in FIG. 21, a selection process using phage display technology was carried out.

[0308] First, SARS-CoV-2 RBD antigen-specific scFv clones were selected through five rounds of bio-panning using magnetic beads Dynabeads M-270 epoxy, Invitrogen) coated with 4 g of the recombinant SARS-CoV-2 RBD antigen.

[0309] Then, 96 clones were randomly selected from output colonies formed on plates and tested for their reactivity to the SARS-CoV-2 RBD antigen by phase ELISA to pick out human antibody clones that are highly reactive to and specific for the corresponding antigen. Results of the phase ELISA are depicted in FIG. 22.

Example II-2. IgG Conversion, Production, and Purification of Selected Antibodies

[0310] 2.1. SARS-CoV-2 RBD Antigen-Specific scFv Antibody Conversion to IgG

[0311] Heavy and light chains of four types of the selected RBD-specific scFv antibodies were amplified by PCR to obtain respective insert DNAs which were then cloned into mammalian expression vector pcDNA3.1 for production of IgG antibodies. Each recombinant DNA of the four types of scFvs was overproduced with high purity in Expi293 cell using a Maxi-prep kit. The purity of DNA was measured using nanodrop 2000 spectrophotometer (Thermo Fisher Scientific).

2.2. Mass Production and Purification of RBD-Specific IgG Antibodies

[0312] Transient expression was performed using the Expi293 system (Invitrogen). In this regard, the four types of IgG antibodies were transfected into Expi293 cells with the ExpiFectamine 293 transfection kit (Gibco) and then incubated for five days. Following centrifugation of the cell culture, the cell pellet was removed. The antibodies were purified from the supernatant (medium).

[0313] The purification of the selected antibodies was conducted by affinity chromatography using protein A sepharose bead (Repligen, Waltha, MA, USA).

Example II-3. Physicochemical Characterization of Selected Antibodies

: Assay of Selected Antibody for Purity and Molecular Weight

[0314] The four SARS-CoV-2 RBD-specific antibodies (K102.1, K102.2, K102.3, and K102.4) were loaded in the same amount onto a polyacrylamide gel and resolved by SDS-PAGE. From Coomassie Brilliant Blue staining, it was observed that each of the four antibodies had a final purity of 90% or higher and molecular weights of approximately 50 kDa for the heavy chain and 25 kDa for the light chain (FIG. 23).

[0315] In addition, the binding affinity (K.sub.D) of the selected antibodies was observed to be 1.64310.sup.9 M for K102.1 and 2.46510.sup.9 M for K102.2 (FIG. 24).

Example II-4. Selection of Monoclonal Antibody Pair Binding to Different Epitopes of SARS-CoV-2 RBD

4.1. Conjugation of Marker to Selected Antibody (K102.1)

[0316] For use as a detection antibody in sandwich ELISA, the antibody K102.1 produced in Example 2 was conjugated with the marker HRP (horseradish peroxidase), using EZ-Link Plus Activated Peroxidase Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

4.2. Selection of Antibody Pair Recognizing Different Epitopes of SARS-CoV-2 RBD

[0317] To select an antibody pair recognizing different epitopes of SARS-CoV-2 RBD from the selected SARS-CoV-2 RBD-specific antibody group, the four selected antibodies were subjected to competition ELISA with the HRP-conjugated antibody (K102.1-HRP).

[0318] The results are depicted in FIG. 25. As shown, K102.1 was observed to have a distinct binding site to the SARS-CoV-2 RBD compared to K102.2 and K102.3. Also, based on the SPR analysis, as shown in FIG. 24, K102.2 exhibited about 5 times higher affinity for the SARS-CoV-2 RBD than K102.3.

[0319] Therefore, K102.1 and K102.2 were selected as a pair of antibodies that can be utilized for the development of Sandwich ELISA.

Example II-5. Analysis of Selected Pair for Sandwich ELISA

5.1. Assay for Suitability of Selected Antibody Pair

[0320] The selected antibody pair (K102.1 and K102.2) determined in Example 4 was evaluated for suitability for use in the sandwich ELISA of the present disclosure by verifying pairing therebetween. The sandwich ELISA was performed using K102.1 or K102.2 as the capture antibody and K102.1-HA or K102.2-HA as the detection antibody.

[0321] As shown in FIG. 26, it was confirmed that K102.1 and K102.2 can be paired as either the capture antibody or the detection antibody for the Sandwich ELISA.

5.2. Verification of Distinct Epitopes of SARS-CoV-2 RBD for Selected Antibody Pair

[0322] Furthermore, SPR (Surface Plasmon Resonance)-based competition binding assay was performed to determine whether the selected antibody pair bind to distinct epitopes on the SARS-CoV-2 RBD. By measuring real-time binding of the selected antibodies to the SARS-CoV-2 RBD an iMSPR mini-instrument (icluebio, South Korea), the competition binding assay was used to evaluate whether K102.1 or K102.2-HA possess unique or overlapping binding sites.

[0323] In brief, 128 nM K102.1 in a HEPES buffered Steinberg's solution containing 0.005% (v/v) Tween-20. was injected onto the SARS-CoV-2 RBD surface (ca. 1,000 RU) at a flow rate of 50 l/min for 240 seconds. Subsequently, 128 nM K102.2-HA was introduced under the same conditions onto the surface where K102.1 and SARS-CoV-2 RBD were bound. The resulting curves were obtained as sensorgrams using the iMSPR analysis software.

[0324] As shown in FIG. 27, it was confirmed that K102.1 has a distinct epitope from K102.2 for the SARS-CoV-2 RBD.

Example II-6. Development of Sandwich ELISA Method Using Selected Antibody Pair

[0325] A Sandwich ELISA method for detecting SARS-CoV-2 was developed using the SARS-CoV-2 specific monoclonal antibodies K102.1 (capture antibody) and K102.2 (detection antibody). The sandwich ELISA method of the present disclosure is schematically illustrated in FIG. 28.

6.1. Optimization of Conditions for Sandwich ELISA

6.1.1. Optimal Concentration of Selected Antibody Pair

[0326] To determine the optimal conditions therefor, sandwich ELISA was carried out with increasing concentrations of either the capture antibody or the detection antibody.

[0327] As a result, it was confirmed that 5 g/ml of the capture antibody (K102.1) and 1 g/ml of the detection antibody (K102.2-HA) are optimal concentrations for sandwich ELISA method (FIGS. 29 and 30).

6.1.2. Calibration Curve for Limit of Detection (LOD)

[0328] A calibration curve was derived to determine the limit of detection of SARS-CoV-2 RBD in the sandwich ELISA of the present disclosure. The reproducibility of the calibration curve was validated through six independent analyses, and the linear dynamic range of the derived calibration curve was determined to be between 0 ng/ml and 12 ng/ml (equivalent to 0 pM to 480 pM) for the SARS-CoV-2 RBD (FIG. 31).

[0329] The limit of detection (LOD) for the sandwich ELISA of the present disclosure was derived by calculating the standard deviation (SD) and slope (S) of the calibration curve according to Equation 1. The determined LOD was found to be 0.55 ng/ml (equivalent to 22 pM).

Limit of Detection (LOD)=3(standard deviation (SD)/slope (S) of calibration curve)[Equation 1]

[0330] In addition, the sensitivity was derived by calculating standard deviation (SD) and mean of the blank according to the following Equation 2. The sensitivity was determined to be 0.48 ng/ml (19.2 pM).

Sensitivity)=3standard deviation (SD)+mean of blank[Equation 2]

[0331] Table 5 shows a comparison of the performance of the Sandwich ELISA of the present disclosure with two types of Sandwich ELISA kits previously developed and sold on the market.

TABLE-US-00005 TABLE 5 Trade Company Name Cat. No. Sensitivity Source Eaglebio GENLISATM KBVH015- 12 ng/ml https://eaglebio. SARS- 12 com/wp-content/ CoV-2 uploads/2021/06/ (2019-nCoV) KBVH015-12- Spike RBD SARS-CoV-2- Antigen Spike-RBD- Quantitative Antigen- ELISA Quantitative- ELISA-Package- Insert.pdf Biozol SARS-CoV- ACM- 0.58 ng/ml https://www. 2 Spike S1 55030 biozol.de/en/ Protein product/acm- ELISA Kit 55030

[0332] From the data of Table 5, it is understood that the kit of the present disclosure with a sensitivity of 48 ng/ml (19.2 pM) is superior in terms of sensitivity to the previously approved and commercially available kits with respective sensitivities of 12 ng/ml (Eaglebio) and 0.58 ng/ml (Biozol).

6.2. Validation of Sandwich ELISA

: Measurement of Coefficient of Variation (CV) and Recovery

[0333] To validate the Sandwich ELISA method developed using the selected antibody pair of the present disclosure, the coefficient of variation (CV) and recovery were measured by performing both intra- and inter-assays. Intra-assay precision was determined by measuring samples six times in triplicate within the same assay run. Inter-assay precision was determined by measuring a sample in triplicate in six separate assay runs.

[0334] The mean and standard deviation (SD) was calculated. The coefficient of variation (CV) was calculated according to the following equation 3:

CV(%)=(SD/mean)100.[Equation 3]

[0335] Recovery was calculated according to the following equation 4:

Recovery (%)=Average measured concentration/expected concentration]100[Equation 4]

[0336] As shown in FIG. 32, the intra- and inter-assay CVs for 5 ng/mL SARS-CoV-2 RBD were 8.46% and 9.52%, respectively. The intra- and inter-assay recoveries for 5 ng/mL SARS-CoV-2 RBD were 105.57% and 98.56%, respectively.

[0337] Therefore, it was confirmed that the sandwich ELISA method of the present disclosure is a sensitive, accurate, and reliable technique for detecting SARS-CoV-2 RBD.

Example II-7. Analysis for Performance of Sandwich ELISA

: Ability of Sandwich ELISA to Detect Eight RBD Mutants

[0338] The optimized sandwich ELISA method of the present disclosure was evaluated for ability to detect SARS-CoV-2 RBD mutants.

[0339] Regarding SARS-CoV-2, representative RBD mutants categorized by countries worldwide were secured, comprising eight types: A435S (Finland), N354D (China), G476S and V483A (USA), F342L, V341I, and N501Y (UK), and L452R/T478K (India). The Sandwich ELISA was performed in the presence of increasing concentrations of these antigens to 32 pM, 96 pM, and 200 pM.

[0340] Briefly, 96-well high-binding microplates (Corning) were coated with the capture antibody K102.1 and then blocked using 3% (w/v) bovine serum albumin (BSA) in PBS for 2 hours at 37 C. Next, 100 L of increasing concentrations of the RBDs of wild-type SARS-CoV-2 mutants (A435S, N354D, G476S, V483A, F342L, V341I, N501Y, and L452R/T478K) were added to each well, and the microplates were incubated for 3 hours at 37 C. The plates were washed thrice with 0.05% (v/v) PBST and incubated with the detection antibody HA-tagged K102.2 (K102.2-HA). Subsequently, the plates were washed thrice with 0.05% (v/v) PBST and incubated with HRP-conjugated anti-HA antibody to detect K102.2-HA. To this end, the plates were washed thrice and a TMB substrate solution (Thermo Fisher Scientific) was added to each well, followed by reaction with HRP for 15 minutes. The reaction was terminated by adding 1 M H2504. (100 100 L/well). The absorbance of each sample was read at 450 nm on a microplate reader.

[0341] Consequently, as shown in FIG. 33, the optimized sandwich ELISA method of the present disclosure can detect all the eight RBD mutant protein antigens in the picomolar range.

Example III

Example III-1: Design, Generation, and Characterization of bsAbs

[0342] Four SARS-CoV-2 RBD-specific human scFvs with a complementarity determining region (CDR) sequence were isolated using phage-display technology from the human synthetic scFv library. To prevent Fab arm exchange that results in an unwanted heterogeneous mixture of antibodies by half molecule exchanged with endogenous IgG4, IgG4-based mAbs with S228P mutations [IgG4 (S228P)] were constructed. Among the antibodies, a noncompeting pair of mAbs, K102.1 and K102.2, which recognize independent epitopes of the SARS-CoV-2 RBD, was identified using a competition ELISA (FIG. 34).

[0343] Based on these parental mAbs, three forms of IgG4 (5228P)-(scFv).sub.2 bispecific antibody (bsAb), such as structures of K202.A (FIG. 35), K202.6 (FIG. 36), and K202.0 (FIG. 37), were designed. As a result of expressing the three forms of the bispecific antibodies, the K202.0 bispecific antibody was expressed at a significantly poor rate (FIG. 38).

[0344] Of the designed bispecific antibody forms, the two IgG4(S228P)-(scFv).sub.2 bsAb forms K202.A and K202.B were produced with a purity of 90% or higher (FIG. 39). Then, surface plasmon resonance (SPR) analysis was conducted to compare binding affinity for purified RBDs of wild-type SARS-CoV-2 and SARS-CoV-2 variants comprising alpha, beta, gamma, delta, and kappa between the bsAbs and mAb. The results are depicted in FIG. 40a-d.

[0345] As can be seen in FIG. 40a-d and Table 6, K202.B exhibited as strong binding affinity for RBDs of all the SARS-CoV-2 variants tested in the subnanomolar concentration range as comparable with that of the parental mAb for wild-type SARS-CoV-2.

[0346] To further confirm whether K202.B could recognize two independent epitopes of SARS-CoV-2 RBD, competition assays were performed using SPR. The results are depicted in FIG. 41.

[0347] As shown in FIG. 41, K202.B could bind to the RBD even after saturation with K102.1 or K102.2 (FIGS. 41A and 41B). In contrast, neither K102.1 nor K102.2 bound to the RBD after saturation with K202.B (FIGS. 41C and 41D), suggesting that the bsAb K202.B specifically recognized two independent binding sites.

TABLE-US-00006 TABLE 6 Equilibrium dissociation constant of parental mAbs and bsAbs to RBDs of SARS-CoV-2 wild-type and variants K102.1 K102.2 RBD type K.sub.a (M.sup.1) K.sub.d (M.sup.1S.sup.1) K.sub.D (nM) K.sub.a (M.sup.1) K.sub.d (M.sup.1S.sup.1) K.sub.D (nM) Wild-type 1.61 10.sup.5 2.76 10.sup.4 1.72 1.97 10.sup.5 4.34 10.sup.4 2.20 B.1.1.7 2.06 10.sup.5 1.93 10.sup.4 0.94 1.54 10.sup.5 5.57 10.sup.4 3.62 B.1.351 0.93 10.sup.5 2.95 10.sup.4 3.18 4.92 10.sup.5 7.88 10.sup.4 1.60 P.1 0.70 10.sup.5 3.15 10.sup.4 4.51 4.54 10.sup.5 9.39 10.sup.4 2.07 B.1.617.2 2.11 10.sup.5 2.98 10.sup.4 1.41 3.33 10.sup.5 5.11 10.sup.4 1.53 B.1.617.1 0.95 10.sup.5 3.17 10.sup.4 3.34 4.07 10.sup.5 6.83 10.sup.4 1.68 K202.A K202.B RBD type K.sub.a (M.sup.1) K.sub.d (M.sup.1S.sup.1) K.sub.D (nM) K.sub.a (M.sup.1) K.sub.d (M.sup.1S.sup.1) K.sub.D (nM) Wild-type 1.65 10.sup.5 1.29 10.sup.4 0.78 1.47 10.sup.5 9.98 10.sup.5 0.68 B.1.1.7 1.89 10.sup.5 1.08 10.sup.4 0.57 8.31 10.sup.5 7.93 10.sup.4 0.95 B.1.351 0.78 10.sup.5 2.49 10.sup.4 3.17 2.22 10.sup.5 2.00 10.sup.4 0.90 P.1 2.03 10.sup.5 3.81 10.sup.4 1.88 0.87 10.sup.5 1.86 10.sup.4 2.14 B.1.617.2 2.47 10.sup.5 2.26 10.sup.4 0.92 1.50 10.sup.5 1.17 10.sup.4 0.78 B.1.617.1 2.48 10.sup.5 2.82 10.sup.4 1.14 2.78 10.sup.5 1.60 10.sup.4 0.58 K.sub.a, Association constant K.sub.d, Dissociation constant K.sub.D, Equilibrium dissociation constant

Example III-2: Neutralizing Activity of bsAb in hACE2-RBD Interaction and SARS-CoV-2 Pseudotyped and Live Virus Infection In Vitro

[0348] 2-1. Neutralizing Activity of bsAbs in hACE2-RBD Interaction

[0349] To assess the neutralizing activity of the bsAbs in hACE2-RBD interactions, ELISA-based neutralization assays were performed with microtiter plates to which the recombinant RBD proteins of wild-type SARS-CoV-2 and SARS-CoV-2 variants comprising alpha, beta, gamma, delta, and kappa variants. The microtiter plates were incubated with recombinant hACE2 in the presence or absence of parental mAb, an mAb cocktail containing parental mAb, or bsAbs. The results are depicted in FIG. 42 and summarized in FIG. 7.

TABLE-US-00007 TABLE 7 IC.sub.50 values of inventive antibodies in direct interaction between hACE2 and RBDs of the wild-type and variant SARS-CoV-2 IC.sub.50 (nM) K102.1 RBD type K102.1 K102.2 K102.2 K202.A K202.B Wild-type 1.33 0.04 4.94 0.06 1.55 0.07 1.57 0.20 1.85 0.09 B.1.1.7 23.10 ND 16.79 2.04 0.07 1.21 0.10 0.06 0.16 B.1.351 ND 0.86 0.06 1.94 0.06 0.75 0.03 0.45 0.15 P.1 ND 3.23 0.16 5.52 0.13 1.19 0.12 1.73 0.10 B.1.617.2 1.83 0.09 1.14 0.11 1.08 0.08 0.73 0.09 0.18 0.12 B.1.617.1 24.32 0.40 0.10 0.71 0.06 0.34 0.08 0.19 0.11 0.09 ND, Not determined

[0350] As is understood from data of FIG. 42 and Table 7, in the case of hACE2 binding to the wild-type RBD, all the antibodies exhibited similar inhibitory effects, but K202.B bispecific antibody more potently inhibits the hACE2 binding than the mAb or mAb cocktail. In addition, as demonstrated in the subnanomolar range of IC.sub.50 values, K202.B was observed to have a little advantageous inhibitory effect on hACE2 binding to the RBDs of the beta, delta, and kappa variants, compared to K202.A.

[0351] Furthermore, K202.B also exhibited a strong inhibitory effect on hACE2 binding to RBDs with N354D/D364Y, V367F, W436R, R408I, G476S, V483A, V341I, F342L, or A435S mutations (FIG. 43a, b).

2-2. Neutralizing Activity of bsAbs Against SARS-CoV-2 Pseudotyped Virus Infection

[0352] To assess the neutralizing ability of the bsAbs of the present disclosure against SARS-CoV-2 pseudotyped virus infection, SARS-CoV-2 pseudotyped virus neutralization assays were conducted using hACE2-overexpressing 293T stable cell line (293T/hACE2 cells) (FIG. 44) in the presence or absence of parental mAbs, mAb cocktail, and bsAbs. The results are depicted in FIG. 45 and summarized in Table 8.

TABLE-US-00008 TABLE 8 IC.sub.50 values of antibodies against pseudotyped virus infection of SARS-CoV-2 wild-type and variants. IC.sub.50 (nM) Pseudovirus K102.1 type K102.1 K102.2 K102.2 K202.A K202.B Wild-type 1.94 0.07 ND 1.96 0.08 0.27 0.07 0.16 0.04 B.1 2.22 0.09 ND 2.05 0.14 0.39 0.14 0.12 0.11 B.1.1.7 6.16 0.08 ND 3.12 0.07 0.18 0.03 0.15 0.03 B.1.351 ND ND ND 0.46 0.04 0.10 0.04 P.1 ND ND ND 1.74 0.10 1.04 0.09 B.1.617.2 4.48 0.10 ND 3.04 0.08 1.83 0.13 0.13 0.09 B.1.617.1 ND ND 15.63 0.24 0.04 0.05 0.04 0.08 ND, Not determined

[0353] As shown in FIG. 45, it was observed that the parental antibody K102.1 of the present disclosure significantly inhibited the pseudotyped virus infection of wild-type, B.1(D614G), alpha, and delta variants in the nanomolar range, but did not inhibit the infection of the other variants. On the other hand, K102.2 had no effects on any of the tested pseudoviruses.

[0354] In contrast, K202.B exhibited stronger inhibitory effects on the infection of almost all the tested pseudotyped viruses than parental mAbs or the mAb cocktail with the IC.sub.50 values of mostly subnanomolar or nanomolar concentrations. The inhibitory effect of the bispecific antibody K202.A was similar to or slightly lower than that of the bispecific antibody K202.B (FIG. 45 and Table 8).

2-3. Neutralizing Activity of bsAbs in Live Virus Infection

[0355] Next, the effect of the bispecific antibody K202.B of the present disclosure on the antibody-dependent enhancement (ADE) was evaluated using permissive cells (293T/hACE2 cells) and Fc gamma receptor-bearing cells (293T, K562, and THP-1 cells). Changes in the pseudotyped virus infection rate of various SARS-CoV-2 wild-type and variants were monitored in the presence of K202.B. The results are depicted in FIG. 46a-d.

[0356] As can be seen in FIG. 46a-d, no significant changes were observed in any pseudotyped virus infection in the presence of K202.B, indicating that the bispecific antibody K202.B of the present disclosure may not induce ADE in vivo.

Example III-3: Assay for In Vivo Efficacy and Toxicity of Bispecific Antibody K202.B in Wild-Type SARS-CoV-2-Infected Animal Models

3-1. In Vivo Seropharmacokinetic Assay

[0357] To investigate the pharmacokinetics of the bispecific antibody K202.B, K202.B was intravenously injected at a dose of 5 mg/kg into ICR mice from which blood was then sampled at various times. Serum levels of K202.B were measured by ELISA. The results are depicted in FIG. 47.

[0358] As shown in FIG. 47, it was found that K202.B exhibited an in vivo half-life of approximately 78 hours in mice.

3-2. In Vivo Efficacy Assay

[0359] Next, an analysis was made of in vivo efficacy of the bispecific antibody K202.B on wild-type SARS-CoV-2.

[0360] Briefly, wild-type SARS-CoV-2 viruses were intranasally administered to the K18-hACE2 transgenic (TG) mice. After 3 hours, the mice were intravenously injected with two doses (5 and 30 mg/kg) of K202.B, or a single dose (30 mg/kg) of K102.1. At 6 days post-infection, the mice were sacrificed and analyzed as illustrated in FIG. 48. The results are depicted in FIGS. 49 and 50.

[0361] As shown in FIG. 49, no significant body weight losses were detected in the wild-type SARS-CoV-2-infected mice injected with the bispecific antibody K202.B over the observation period of time while the mice treated with PBS and K102.1 underwent notable weight loss at 6 days post-injection (dpi).

[0362] In addition, as shown in FIG. 50, each K202.6-treated group exhibited a clinical severity score of 1 or less (mostly score 0), whereas PBS- or K102.1-treated groups displayed scores greater than 3, characterized by a very ruffled coat, slightly closed eyes, and/or a moribund state.

[0363] In addition, lung samples from all mice sacrificed at 6 dpi were subjected to RT-qPCR to determine the relative expression of viral E and RNA-dependent RNA polymerase (RdRp) genes. The results are depicted in FIGS. 51 and 52.

[0364] As shown in FIGS. 51 and 52, the expression of both viral genes was significantly reduced in a dose-dependent manner in each bispecific antibody K202.6-treated group when compared with that in the PBS-treated group. However, the K102.1-treated group underwent only a slight reduction.

[0365] Furthermore, histopathological examination made of lungs from the infected mice at 6th dpi and the results are depicted in FIG. 53 and summarized in Table 9.

TABLE-US-00009 TABLE 9 Pathological score analyses of lungs from the wild-type SARS-CoV-2-infected mice Number of specimen (n = 7) Pathological K102.1 (30 K202.B (5 K202.B (30 score PBS mg/kg) mg/kg) mg/kg) 0 0 0 2 (28.57%) 3 (42.86%) 0.5 0 0 0 0 1 2 (28.57%) 3 (42.86%) 1 (14.29%) 4 (57.14%) 1.5 3 (42.86%) 4 (57.14%) 0 0 2 2 (28.57%) 0 4 (57.14%) 0 Mean* 1.43 1.07 1.29 0.57 *Mean = (pathological score numbers of specimen)/total numbers of specimen Pathological score = (0, 0%; 1, 10%; 2, 10%-50%; 3, 50%; +0.5, pulmonary edema or alveolar hemorrhage)

[0366] As can be seen in Table 9, PBS- and K102.1-treated mice scored 1 or more due to significant pulmonary lesions. In contrast, a high proportion of the K202.6-treated group showed a score of 0 at both 5 and 30 mg/kg doses.

[0367] As shown in FIG. 53, further histopathological analyses revealed normal features in the K202.B-treated lungs, whereas PBS- and K102.1-treated mice exhibited severe pulmonary edema or alveolar hemorrhage.

3-3. In Vitro Cytotoxicity Assay

[0368] To assay in vitro cytotoxicity of the bispecific antibody of the present disclosure, endothelial cells were measured for cell viability after treatment with 20 g/ml K202.B or 36 g/ml 5-fluorouracil. The results are given in FIG. 54.

[0369] As shown in FIG. 54, there was no significant effect of K202.B on endothelial cell viability in the K202.B-treated group compared with the control group, suggesting no severe endothelial toxicity.

[0370] Moreover, to confirm the effect of the bispecific antibody on endothelial activation, the cells were treated with inflammatory cytokine (hTNF-) or bispecific antibody K202.B and measured for expression levels of cell adhesion molecules (ICAM-1 and VCAM-1). The results are depicted in FIG. 55.

[0371] As shown in FIG. 55, the expression of cell adhesion molecules (ICAM-1 and VCAM-1) was activated in the hTNF--treated group whereas the cell group treated with the bispecific antibody K202.B of the present disclosure remained unchanged in the expression of cell adhesion molecules (ICAM-1 and VCAM-1), indicating that the bispecific antibody K202.B of the present disclosure has no effect on endothelial activation and thus does not cause endothelial toxicity.

3-4. In Vivo Cytotoxicity Assay

[0372] To evaluate in vivo cytotoxicity of the bispecific antibody K202.B, K202.B was intravenously injected to mice and hepatic and renal toxicity were assayed using the sera.

[0373] The results are depicted in FIG. 56.

[0374] As shown in FIG. 56, no changes were observed in either body weight or biochemical indices indicative of hepatic and renal toxicity, implying that the bispecific antibody K202.B of the present disclosure does not provoke in vivo toxicity.

Example III-4: In Vivo Assay for Efficacy and Toxicity of Bispecific Antibody K202.B in SARS-CoV-2 Delta Variant-Infected Animal Models

4-1. In Vivo Efficacy Assay

[0375] In vivo efficacy of bispecific K202.B against SARS-CoV-2 delta variant was analyzed. To evaluate the in vivo efficacy of K202.B against SARS-CoV-2 delta variant, K18-hACE2 TG mice was intranasally challenged with SARS-CoV-2 delta virus. After 3 hours, the mice received intravenous injections of a dose of 5 or 30 mg/kg of K202.B (FIG. 57).

[0376] As shown in FIG. 58, the PBS-treated group decreased in body weight at 6th dpi with statistical significance whereas no drastic weight loss was observed in the bispecific antibody K202.6-treated group.

[0377] In addition, as shown in FIG. 59, the bispecific antibody K202.B-treated group exhibited a clinical severity score of 1 or less (mostly score 0).

[0378] Lung samples from all mice sacrificed at 6 dpi were subjected to RT-qPCR to determine the relative expression of viral E and RNA-dependent RNA polymerase (RdRp) genes. As can be seen in FIGS. 60 and 61, the K202.B-treated group was observed to effectively reduce the expression of both viral E and RdRp genes, compared to the PBS-treated group, as measured by RT-qPCR. Particularly, almost no viral gene was detected in the 30 mg/kg K202.B-treated group at 6 dpi.

[0379] Furthermore, histopathological examination made of lungs from the infected mice at 6 dpi and the results are depicted in FIG. 62 and summarized in Table 10. As can be seen in Table 10, the pathological score in the K202.B-treated group was increased in a dose-dependent manner from 0 point whereas the PBS-treated mice scored 1 to 2 (mostly 2) due to significant pulmonary lesions.

[0380] As shown in FIG. 62, histopathological analyses revealed normal features in the K202.B-treated lungs whereas PBS-treated mice exhibited pulmonary edema or alveolar hemorrhage.

TABLE-US-00010 TABLE 10 Pathological score analyses of lungs from the SARS-CoV delta variant-infected mice Numbers of specimen (n = 7) Pathological K202.B (5 K202.B (30 score PBS mg/kg) mg/kg) 0 0 2 (28.57%) 5 (71.43%) 0.5 0 0 0 1 2 (28.57%) 5 (71.43%) 2 (28.58%) 1.5 1 (14.29%) 0 0 2 4 (57.14%) 0 0 Mean* 1.57 0.71 0.29 *Mean = (pathological score numbers of specimen)/total numbers of specimen Pathological score = (0, 0%; 1, 10%; 2, 10%-50%; 3, 50%; +0.5, pulmonary edema or alveolar hemorrhage)

Example III: Materials and Methods

III-1. Cell Culture

[0381] The cell lines 293T, K562, and THP-1 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). A purchase was made of CHOZN-GS cells (derived from CHO-K1 and adapted to serum-free and suspension conditions) from Merck (Merck, Whitehouse Station, NJ, USA), Expi293 cells from Thermo Fisher Scientific (Waltham, MA, USA), and human umbilical vein endothelial cells (HUVEC) from Lonza (Basel, Switzerland). 293T cells were cultured in DMEM (Thermo Fisher Scientific) whereas K562 and THP-1 cells were cultured in RPMI media (Thermo Fisher Scientific) media supplemented with 10% (v/v) FBS (Thermo Fisher Scientific) and 100 U/mL penicillin-streptomycin (Thermo Fisher Scientific) at 37 C. in 5% CO.sub.2. HUVECs were maintained in EGM-2 (Lonza). Expi293 cells were cultured in Expi293 Expression Media in shaking incubators at 37 C., 125 rpm, and 8% CO.sub.2. CHOZN-GS cells were in EX-CELL Advanced CHO Fed-batch medium (Sigma-Aldrich, Burlington, MA, USA) in a microscale bioreactor Ambr 15 (Sartorius, Gottingen, Germany) at 37 C.

III-2. Surface Plasmon Resonance (SPR)

[0382] The binding kinetics of antibodies to SARS-CoV-2 RBDs were analyzed at room temperature on an iMSPR-mini instrument (iCLUEBIO, Seongnam, Republic of Korea) using 10 mM HEPES pH 7.4, 700 mM NaCl, 2 mM CaCl.sub.2), 1 mM MnCl.sub.2, and 0.005% (v/v) Tween-20 as a running buffer. The recombinant SARS-CoV-2 RBDs (wild-type and variants comprising B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta), and B.1.617 (kappa)) were covalently immobilized on the surface of a COOHAu chip (iCLUEBIO) up to 500 response units through standard amine coupling. Antibodies (8, 16, 32, 64, and 128 nM, respectively) were injected onto the surface of a sensor chip at a flow rate of 50 L/min. Kinetics evaluation data was obtained using a 1:1 binding model.

[0383] To evaluate the ability of K202.B to bind to different regions of the RBD, competition experiments were performed. After the immobilization of 5 nM WT-RBD-His on the surface of a COOHAu chip, a high concentration (512 nM) of K102.1 or K102.2 antibody was added to saturate the corresponding binding sites on the RBD. Then, 128 nM K202.B was added. Conversely, following the addition of 512 nM K202.B to the surface of recombinant WT-RBD-His-immobilized sensor chip, 256 nM K102.1 or K102.2 was subsequently added. Curve fitting and data analysis were performed using the iMSPR analysis software (Tracedrawer; iCLUEBIO).

III-3. SARS-CoV-2 RBD-Human ACE2 Interaction Neutralization Assay

[0384] The ability of antibodies to inhibit the interaction of the SARS-CoV-2 RBD with hACE2 was investigated using ELISA. 50 ng of purified Fc-tagged hACE2 (hACE2-Fc) (R&D Systems, Minneapolis, MN, USA) was coated in each well of a 96-well plate and incubated for 2 hours at room temperature. After washing with immunobuffer (BPS Bioscience, San Diego, CA, USA), the plates were blocked with blocking buffer (BPS bioscience) for 1 hour at room temperature. Simultaneously, 25 nM of purified SARS-CoV-2 WT- or variant-RBD-His (alpha, beta, gamma, delta, and kappa) (Sino Biological) was pre-incubated in the presence or absence of mAb or bsAbs (0.006, 0.024, 0.097, 0.39, 1.56, 6.25, 25, and 100 nM) for 1 hour at room temperature.

III-4. Establishment of hACE2-Overexpressing 293T Stable Cell Line (293T/hACE2 Cell Line)

[0385] To generate stable 293T/hACE2 cell lines, a pUNO1-hACE2 plasmid (InvivoGen, San Diego, CA, USA) was transfected into 293T cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 48 hours of transfection, the cells were cultured in a medium containing 20 g/mL blasticidin (InvivoGen) to select positive cell populations. The expression of hACE2 was determined using immunoblot and immunocytochemical analysis. For immunoblot analysis, 293T and 293T/hACE2 cell lines were lysed with SDS sample buffer, and subjected to immunoblot analysis using a polyclonal anti-hACE2 antibody (R&D Systems). The distribution of hACE2 in the cell membrane was studied through immunocytochemical analysis. Briefly, 293T and 293T/hACE2 cells were plated on Nunc Lab-Tek II chamber slides (Thermo Fisher Scientific) coated with poly-L-lysine (0.1 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA). After 24 hours, the cells were fixed with 4% formaldehyde for 10 min and washed twice with PBS.

[0386] The cells were blocked using PBS containing 1% (w/v) BSA and incubated with polyclonal anti-hACE2 antibody overnight at 4 C. After washing thrice with PBS, the cells were subsequently incubated with Alexa Fluor 488-labeled anti-goat secondary antibody (Invitrogen) at room temperature for 1 hour, then mounted with mounting solution (Dako North America, Carpinteria, CA, USA). The stained cells were imaged using confocal microscopy (LSM510; Carl Zeiss, Oberkochen, Germany).

III-5. SARS-CoV-2 Pseudotyped Virus Neutralization Assay

[0387] Pseudotyped replication-deficient lentiviral particles carrying the SARS-CoV-2 spike (S) protein of the wild-type or D614G variant, and a firefly luciferase reporter gene were prepared using Lenti-X SARS-CoV-2 packaging mix according to the manufacturer's instruction (Takara Bio, Kusatsu, Japan). Briefly, the packaging mix was transiently transfected into Expi293 cells with ExpiFectamine 293 reagent. After culturing for 72 hours, the supernatants containing the pseudotyped viruses were collected and centrifuged briefly (500g for 10 min) to remove cellular debris. Virus titration was measured using Lenti-X GoStix Plus (Takara Bio) according to the manufacturer's instructions. The pseudotyped replication-deficient Moloney murine leukemia virus particles carrying the SARS-CoV-2 S protein of alpha, beta, gamma, delta, or kappa variants and a firefly luciferase reporter gene were obtained from eEnzyme (Gaithersburg, MD, USA).

[0388] To determine the neutralization activity of mAbs or bsAbs against pseudotyped virus infection, 110 4 293T/hACE2 cells in 50 L culture medium were seeded in 96-well tissue culture plates overnight. Serial dilutions of the antibodies were pre-incubated at room temperature for 10 min with 50 L of each pseudotyped virus (110.sup.7 PFU/mL), and the mixture was subsequently incubated with the cells for 24 hours. The firefly luciferase reporter gene expression (which is indicative of viral presence) was measured using ONE-Glo luciferase substrate (Promega, Madison, WI, USA). Next, the culture medium was removed and incubated with 100 L of ONE-Glo substrate. After 5 min, 70 L supernatant was transferred to white flat-bottom 96-well assay plates (Corning) and the luminescence signal was measured using the Synergy H1 microplate reader. The recorded relative luminescence units were normalized to those derived from cells infected with each SARS-CoV-2 pseudotyped virus in the absence of antibodies. Dose-response curves for IC.sub.50 values were determined by nonlinear regression (GraphPad Prism 8.0 software).

III-6. In Vivo Mouse Study

[0389] For in vivo efficacy studies, 8-week-old female B6.Cg-Tg(K18-ACE2).sub.2Prlmn/J (hACE2) mice (The Jackson Laboratory, CA, USA), were housed in a certified A/BSL3 facility (Korea Zoonosis Research Institute, lksan, Republic of Korea). All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at KNOTUS (No. 22-KE-0076), and all experimental protocols requiring biosafety were approved by the Institutional Biosafety Committee of Jeonbuk National University (approval number: JBNU 2020-11-003-003) and performed in a biosafety cabinet at the BL3 and ABL3 facilities of Korea Zoonosis Research Institute at Jeonbuk National University.

[0390] The hACE2-transgenic (hACE2-TG) mice (n=7) was intranasally inoculated with 30 L of wild-type or delta variant virus (110 4 PFU) under anesthesia. Three hours after infection, PBS, mAbs, or bsAbs were injected intravenously.

[0391] The mice were monitored daily for weight change and clinical severity based on the criteria as shown in the table 11.

TABLE-US-00011 TABLE 11 Score Description Appearance & Mobility 0 Healthy No observable sign of disease 1 Slightly Slightly ruffled coat ruffled 2 Ruffled Ruffled coat throughout the body and a wet appearance 3 Sick Very ruffled coat and slightly closed, inset eyes 4 Very sick Very ruffled coat; closed, inset eyes; and moribund state

[0392] The SARS-CoV-2 burden in lung tissues was determined via RT-qPCR.

[0393] Lung tissues were harvested from hACE2-TG mice 6 days after SARS-CoV-2 wild-type or delta variant infection, and total RNAs were extracted from the collected tissues using Wizol Reagent (Wizbiosolutions, Seongnam, Republic of Korea). The samples were subjected to RT-qPCR using a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA).

[0394] Following the reverse transcription of total RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster, CA, USA), the reaction mixture (20 L total) contained 2 L of template cDNA, 10 L of 2 Premix Ex Taq, 200 nM primer, and a probe (E gene: forward primer 5-ACAGGTACGTTAATAGTTAATAGCGT-3 (SEQ ID NO: 67), reverse primer 5-ATATTGCAGCAGTACGCACACA-3 (SEQ ID NO: 68), probe 5-FAM-ACACTAGCCATCCTTACTGCGC TTCG-BHQ1-3 (SEQ ID NO: 69); RdRp gene: forward primer 5-ATGAGCTTAGTCCTGTTG-3 (SEQ ID NO: 70), reverse primer 5-CTCCCTTTGTTGTGTTGT-3 (SEQ ID NO: 71), probe 5-HEX-AGATTGTCTTGTGCTGCCGGTA-BHQ1-3 (SEQ ID NO: 72)). These reactions were denatured at 95 C. for 30 seconds, and then subjected to 45 cycles of 95 C. for 5 seconds and 60 C. for 20 seconds. After completion of the reaction cycles, the temperature was increased from 65 to 95 C. at a rate of 0.2 C./15 seconds and fluorescence was measured every 5 seconds to construct a melting curve. A control sample lacking template DNA was run with each assay. All measurements were performed in duplicate to ensure reproducibility. The authenticity of the amplified product was determined using melting curve analysis. All data were analyzed using Bio-Rad CFX Manager analysis software version 2.1 (Bio-Rad Laboratories). The viral burden was expressed by the copy number of viral RNA per nanogram of total RNA after calculating the absolute copy number of viral RNA in comparison with the standard cDNA template.

Histology

[0395] Excised mouse lung tissues were fixed with 4% (v/v) paraformaldehyde (PFA) in PBS and processed for paraffin embedding. The paraffin blocks were sliced into 3 m-thick sections using a microtome (HistoCore MULTICUT R; Leica, Germany) and mounted on silane-coated glass slides (5116-20F; Muto, Tokyo, Japan). Hematoxylin and eosin, periodic acidSchiff, and modified Masson's trichrome stains were used to identify histopathological changes in all the organs. The histopathology of the lung tissue was observed using light microscopy (Axio Scope A1; Carl Zeiss). Pathological scores were determined based on the percentage of inflammation area for each section in each group using the following scoring system: 0, no pathological change; 1, affected area (10%); 2, affected area (10-50%); 3, affected area (50%); an additional 0.5 point was added when pulmonary edema and/or alveolar hemorrhage was observed.

III-7. In Vitro Antibody-Dependent Enhancement Assay

[0396] Fifty microliters of each SARS-CoV-2 pseudotyped virus (110 7 PFU/mL) was preincubated with different concentrations of K202.B (0.044, 0.138, 0.42, 1.24, 3.7, 11.1, 33.3, and 100 nM) in culture medium. After 30 minutes of incubation at room temperature, the mixture was added to 293T, 293T/hACE2, K562, or THP-1 cells (110 4 cells in a 96-well plate). The cells were cultured for 24 hours, and the luciferase activity of infected cells was measured as described in pseudotyped virus neutralization assay.

III-8. Endothelial Cell Viability Assay

[0397] A total of 510.sup.3 HUVECs were plated in 96-well plates and incubated in the presence or absence of 20 g/mL K202.B or 36 g/mL 5-fluorouracil for 24 hours at 37 C. Cell viability was determined using the Cell Counting Kit-8 (Sigma) according to the manufacturer's instructions. The final absorbance was measured at 450 nm using a spectrophotometer (BioTek).

III-9. Flow Cytometry

[0398] The effect of K202.B on endothelial cell activation was determined by treating cells with 20 ng/ml of human tumor necrosis factor- (hTNF-; Millipore), 20 g/ml of K202.B, or PBS for 24 hours and fixing with 4% (v/v) PFA. The cells were fixed with 4% (v/v) PFA in PBS and incubated with 10 g/well of intercellular cell adhesion molecule-1 (ICAM-1; Abcam, Cambridge, MA, USA) or vascular cell adhesion molecule-1 (VCAM-1; Abcam) antibody for 1 hour at 25 C. Then, Alexa Fluor 647-conjugated anti-mouse IgG or anti-rabbit IgG (1:1000; Invitrogen) was incubated for 1 hour at 25 C. All samples were analyzed using flow cytometry with the aid of FlowJo software (TreeStar, Ashland, OR, USA).

III-10. In Vivo Toxicity and Serum Pharmacokinetic Analysis

[0399] In vivo toxicity and serum pharmacokinetic studies using animals were approved by the IACUC (Approval No. NCC-21-693) of the National Cancer Center, Republic of Korea. Eight-week-old female Institute of Cancer Research (ICR) mice (Orient Bio Inc., Seongnam, Republic of Korea) were intravenously injected with 5 or 30 mg/kg of K202.B (n=3 per group). At 4, 8, 24, 72, 120, 168, 264, 384, and 504 hours post-inoculation, blood samples (50 L) were collected from each mouse and centrifuged at 5000g for 20 min at 4 C. The serum was stored at 80 C. for evaluation of biochemical parameters. Serum levels of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), creatinine (CRE), and blood urea nitrogen (BUN) were measured using a Fuji Dri-Chem 3500 Biochemistry Analyzer (Fujifilm, Tokyo, Japan).

[0400] Serum levels of K202.B were determined using a human IgG ELISA kit (Abcam) according to the manufacturer's instructions. Optical density was measured using a Synergy H1 microplate reader, and values were compared to those from a concurrently analyzed standard curve.

III-11. Statistical Analysis

[0401] Data were analyzed with GraphPad Prism 8.0 software using two-tailed Student's t-test for comparisons between two groups, and one-way analysis of variance (ANOVA) with Bonferroni's correction for multiple comparisons. All data represent the meanstandard deviation (S.D.). A P-value less than 0.05 was considered statistically significant (*P<0.05, **P<0.01, ***P<0.001).

[0402] This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted concurrently herewith as the sequence listing XML file entitled 000352uscoa_SequenceListing.XML, file size 72.2 kilobytes, created on 25 Oct. 2023. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. 1.52(e)(5).