WO2024214905A1 - Antibody-peptide fusion protein - Google Patents

Abstract

The present invention relates to an antibody-peptide fusion protein and use thereof. More specifically, the present invention relates to a composition and a treatment method for treating viral diseases by using an antibody-peptide fusion protein.

Description

Antibody-peptide fusion protein

The present invention relates to antibody-peptide fusion proteins and uses thereof. More specifically, the present invention relates to antibody-peptide fusion proteins; improved virus inhibitory function of the fusion proteins; and compositions and methods for treating viral diseases using the same.

Peptide therapeutics based on amino acids are currently in the spotlight as treatments for cancer, obesity, and diabetes, but peptide drugs that are in the spotlight as antiviral therapeutics are not widely known.

In particular, as we enter the pandemic period due to COVID-19, research on various antiviral peptide treatments is being conducted, but most of the research has focused on immunotherapy and vaccines, and it is known that the level of development of effective antiviral peptide treatments themselves is not yet high.

Among various previously reported studies, there are reports on the development of peptides with a viral envelope lipid disruption mechanism (Lipid Envelope Antiviral Disruption (LEAD)) (Langmuir 2019, 35, 9934-9943 and BBA - Biomembranes 1864 (2022) 183821), but these studies have not yet been reported to have actually led to the development of effective antiviral therapeutics for infectious diseases.

The present inventors have completed the present invention by confirming the effect of significantly improving the actual antiviral function by using a peptide having the above LEAD (Lipid Envelope Antiviral Disruption) function and forming a fusion protein with an antibody.

The present application provides an antibody-peptide fusion protein in which an antiviral peptide and an antibody are linked.

The present application provides a novel antiviral peptide. In this case, the amino acid sequence of the antiviral peptide may be a known amino acid sequence or a newly designed amino acid sequence.

One object of the present application is to provide an antibody-peptide fusion protein.

Another object of the present application is to provide a use of antibody-peptide fusion proteins for the prevention or treatment of viral diseases.

Another object of the present application is to provide a pharmaceutical composition comprising an antibody-peptide fusion protein for the prevention or treatment of a viral disease.

Another object of the present application is to provide a method for treating viral diseases using antibody-peptide fusion proteins.

Figure 1 illustrates the structure of a fusion protein encoded by nucleic acids cloned into a vector and the positions at which it is cleaved.

Figure 2 shows the SDS-PAGE gel analysis result regarding the purification process of 7NAB antibody-mCherry (#14022 + #14023) using Protein A resin. The left side of Figure 2 shows Coomassie staining, and the right side of Figure 2 shows a fluorescence image. The M line represents a molecular weight marker, the FT line represents a solution that passed through the resin, the W line represents a solution rinsed with buffer, and the E1, 2, and 3 lines represent eluted proteins. The R line represents the resin after protein elution.

Figure 3 shows the SDS-PAGE analysis results for the PreScission cleavage reaction of 7NAB antibody-mCherry protein (#14022+#14023). The left side of Figure 3 shows Coomassie staining, and the right side of Figure 2 shows a fluorescence image. Column M indicates a molecular weight marker.

Figure 4 shows the SDS-PAGE gel analysis results regarding the purification process of 7NAB antibody (#14022 + #14023) using Protein A resin after PreScission cleavage. The left side of Figure 4 shows Coomassie staining, and the right side of Figure 4 shows a fluorescence image. The FT column indicates the solution that passed through the resin, E1 to E4 columns indicate the eluted proteins, and M column indicates a molecular weight marker.

Figure 5 shows the SDS-PAGE gel analysis results regarding the purification process of 7NAB antibodies (#14022 + #14023) using cation exchange chromatography. Columns E1 to E5 represent eluted proteins, and column M represents a molecular weight marker.

Figure 6 shows the results of SDS-PAGE gel analysis after using the endotoxin removal kit on the purified 7NAB antibody (#14022 + #14023). Column M represents the molecular weight marker, column B represents before using the endotoxin removal kit, and column A represents after using the endotoxin removal kit.

In FIGS. 2 to 6, H_mcherry refers to a protein in which the heavy chain of 7NAB antibody and mCherry are linked, and the molecular weight of H_mcherry is about 83 kDa. In FIGS. 2 to 6, L_mcherry refers to a protein in which the light chain of 7NAB antibody and mCherry are linked, and the molecular weight of L_mcherry is about 53 kDa. In FIGS. 2 to 6, Heavy chain refers to the heavy chain of 7NAB antibody, and the molecular weight of the Heavy chain is about 55 kDa. In FIGS. 2 to 6, Light chain refers to the light chain of 7NAB antibody, and the molecular weight of the Light chain is about 25 kDa. In FIGS. 2 to 6, mcherry refers to mCherry protein, and the molecular weight of the mCherry protein is about 28 kDa.

Figure 7 shows the SDS-PAGE gel analysis results regarding the purification process of 7NAB antibody_DS-05_mCherry (#14022 + #14065) using Protein A resin. The left side of Figure 7 shows Coomassie staining, and the right side of Figure 7 shows a fluorescence image. The M column represents a molecular weight marker, and the E1 and E2 columns represent eluted proteins.

Figure 8 shows the SDS-PAGE analysis results regarding the PreScission cleavage reaction of 7NAB antibody_DS-05_mCherry (#14022 + #14065). The right side of Figure 8 shows Coomassie staining, and the left side of Figure 8 shows a fluorescence image. Column M represents a molecular weight marker, column B represents before cleavage reaction, and column A represents after cleavage reaction.

Figure 9 shows the process of purifying 7NAB antibody_DS-05 (#14022 + #14065) using Protein A resin after PreScission cleavage, and shows the results of SDS-PAGE gel analysis. The FT column indicates the solution that passed through the resin, the M column indicates the molecular weight marker, and the E1 to E3 columns indicate the eluted proteins.

Figure 10 shows the process of purifying 7NAB antibody_DS-05 (#14022 + #14065) using cation exchange chromatography, and shows the results of SDS-PAGE gel analysis. The FT column indicates the solution that passed through the resin, columns E1 to 5 indicate the eluted proteins, and M indicates a molecular weight marker.

Figure 11 shows the results of SDS-PAGE gel analysis after using the endotoxin removal kit on the purified 7NAB antibody_DS-05 (#14022 + #14065). Column M represents the molecular weight marker, column B represents before using the kit, and column A represents after using the kit.

Figure 12 shows the results of analyzing the concentration of protein linked to 7NAB antibody and DS-05 peptide using SDS-PAGE gel after endotoxin removal. At this time, the concentration of protein linked to 7NAB antibody and DS-05 peptide is 0.16 mg/㎖.

In Figures 7 to 12, H_mcherry refers to a protein in which the heavy chain of 7NAB antibody; DS-05 peptide; and mCherry protein are linked, and the molecular weight of H_mcherry is about 83 kDa. In Figures 7 to 12, L_mcherry refers to a protein in which the light chain of 7NAB antibody and mCherry are linked, and the molecular weight of L_mcherry is about 53 kDa. In Figures 7 to 12, Heavy chain refers to a protein in which the heavy chain of 7NAB antibody and DS-05 peptide are linked, and the molecular weight of the Heavy chain is about 55 kDa. In Figures 7 to 12, Light chain refers to the light chain of 7NAB antibody, and the molecular weight of the Light chain is about 25 kDa. In Figures 7 to 12, mcherry refers to mCherry protein, and the molecular weight of mCherry protein is about 28 kDa.

Figure 13 shows the process of purifying 7TCQ antibody_mCherry (#14252 + #14235) using Protein A resin, and shows the results of SDS-PAGE gel analysis. The left side of Figure 13 shows a fluorescence image, and the right side of Figure 13 shows Coomassie staining. The M column represents a molecular weight marker, the FT column represents a solution that passed through the resin, and the E column represents an eluted protein.

Figure 14 shows the process of purifying 7TCQ antibody (#14252 + #14235) using Protein A resin after PreScission cleavage, and shows the results of SDS-PAGE gel analysis. The left side of Figure 14 shows a fluorescence image, and the right side of Figure 14 shows Coomassie staining. Column B indicates before cleavage reaction, column A indicates after cleavage reaction, M indicates a molecular weight marker, column FT indicates a solution that passed through the resin, columns E1 to E4 indicate eluted proteins, and column R indicates the resin after eluted.

Figure 15 shows the process of purifying 7TCQ antibody (#14252 + #14235) using cation exchange chromatography, and shows the results of SDS-PAGE gel analysis. Column M indicates a molecular weight marker, and column E indicates that the eluted protein is concentrated.

Figure 16 shows the results of SDS-PAGE gel analysis after using the endotoxin removal kit on purified 7TCQ antibody (#14252 + #14235). Column B represents before using the kit, A represents after using the kit, and M represents the molecular weight marker.

In Figures 13 to 16, H_mcherry refers to a protein in which the heavy chain of 7TCQ antibody and mCherry protein are linked, and the molecular weight of H_mcherry is about 83 kDa. In Figures 13 to 16, L_mcherry refers to a protein in which the light chain of 7TCQ antibody and mCherry are linked, and the molecular weight of L_mcherry is about 53 kDa. In Figures 13 to 16, mcherry refers to mCherry protein, and the molecular weight of the mCherry protein is about 28 kDa. In Figures 13 to 16, Heavy chain refers to the heavy chain of 7TCQ antibody, and the molecular weight of the Heavy chain is about 55 kDa. In Figures 13 to 16, Light chain refers to the light chain of 7TCQ antibody, and the molecular weight of the Light chain is about 25 kDa. In Figures 13 to 16, Prescission means PreScission protease.

Figure 17 shows the process of purifying 7TCQ antibody_DS-05_mCherry (#14233 + #14235) using Protein A resin, and shows the results of SDS-PAGE gel analysis. The left side of Figure 17 shows Coomassie staining, and the right side of Figure 17 shows a fluorescence image. The FT column indicates the solution that passed through the resin, W indicates the solution rinsed with buffer, M indicates a molecular weight marker, E1 to 6 indicate eluted proteins, and the R column indicates the resin after the proteins were eluted.

Figure 18 shows the SDS-PAGE analysis results regarding the PreScission cleavage reaction of 7TCQ antibody_DS-05_mCherry (#14233 + #14235). The left side of Figure 18 shows Coomassie staining, and the right side of Figure 18 shows a fluorescence image. Column M indicates a molecular weight marker, column B indicates before cleavage reaction, and column A indicates after cleavage reaction.

Figure 19 shows the process of purifying 7TCQ antibody_DS-05 (#14233 + #14235) using Protein A resin after PreScission cleavage, and shows the results of SDS-PAGE gel analysis. The FT column indicates the solution that passed through the resin, the M column indicates the molecular weight marker, and E1 to E5 indicate the eluted proteins.

In Figures 17 to 19, H_mcherry refers to a protein in which the heavy chain of 7TCQ antibody; DS-05 peptide; and mCherry protein are linked, and the molecular weight of H_mcherry is about 83 kDa. In Figures 17 to 19, L_mcherry refers to a protein in which the light chain of 7TCQ antibody and mCherry are linked, and the molecular weight of L_mcherry is about 53 kDa. In Figures 17 to 19, Heavy chain refers to a protein in which the heavy chain of 7TCQ antibody and DS-05 peptide are linked, and the molecular weight of the Heavy chain is about 55 kDa. In Figures 17 to 19, Light chain refers to the light chain of 7TCQ antibody, and the molecular weight of the Light chain is about 25 kDa. In Figures 17 to 19, Prescission refers to PreScission protease. In Figures 17 to 19, mcherry refers to mCherry protein, and the molecular weight of mCherry protein is about 28 kDa.

Figure 20 shows the process of purifying 7TCQ antibody_YAP-1_mCherry (#14234 + #14235) using Protein A resin, and shows the results of SDS-PAGE gel analysis. The left side of Figure 20 shows a fluorescence image, and the right side of Figure 20 shows Coomassie staining. The M column represents a molecular weight marker, the E column represents the eluted protein, and the R column represents the resin after the protein was eluted.

Figure 21 shows the process of purifying 7TCQ antibody_YAP-1 (#14234 + #14235) using Protein A resin after PreScission cleavage, and shows the results of SDS-PAGE gel analysis. The left side of Figure 21 shows a fluorescence image. The right side of Figure 21 shows Coomassie staining. The M column represents a molecular weight marker, the cut column represents the antibody after the preScission cleavage reaction, the FT column represents the solution that passed through the resin, and the E column represents the eluted protein.

Figure 22 shows the process of purifying 7TCQ antibody_YAP-1 (#14234 + #14235) using cation exchange chromatography, and shows the results of SDS-PAGE gel analysis. The M column represents a molecular weight marker, the E1 column represents the eluted protein, the R1 column represents the resin after the protein was eluted, the E2 column represents additional protein eluted after gel confirmation, and the R2 column represents the resin after the protein was additionally eluted.

In FIGS. 20 to 22, H_mcherry refers to a protein in which the heavy chain of 7TCQ antibody; YAP-1 peptide; and mCherry protein are linked, and the molecular weight of H_mcherry is about 83 kDa. In FIGS. 20 to 22, L_mcherry refers to a protein in which the light chain of 7TCQ antibody and mCherry are linked, and the molecular weight of L_mcherry is about 53 kDa. In FIGS. 20 to 22, Heavy chain refers to a protein in which the heavy chain of 7TCQ antibody and YAP-1 peptide are linked, and the molecular weight of the Heavy chain is about 55 kDa. In FIGS. 20 to 22, Light chain refers to the light chain of 7TCQ antibody, and the molecular weight of the Light chain is about 25 kDa. In Figures 20 to 22, mcherry means mCherry protein, and the molecular weight of mCherry protein is about 28 kDa.

Figure 23 shows the neutralization capacity and IC ₅₀ values calculated based on the measured percentage of infected cells after SARS-CoV2 was incubated with cells together with various concentrations of DS-05 peptide.

Figure 24 shows the neutralization capacity and IC ₅₀ values calculated based on the measured percentage of infected cells after SARS-CoV2 was incubated with cells incubated with various concentrations of YAP-1 peptide.

Figure 25 shows the neutralization capacity and IC ₅₀ values calculated based on the measured percentage of infected cells after SARS-CoV2 was incubated with cells together with various concentrations of 7NAB antibody.

Figure 26 shows the neutralization capacity and IC ₅₀ values calculated based on the measured percentage of infected cells after SARS-CoV2 was incubated with cells incubated with various concentrations of 7TCQ antibody.

Figure 27 shows the neutralization capacity and IC 50 values calculated based on the measured percentage of infected cells after SARS-CoV2 was incubated with cells together with various concentrations of 7NAB_DS-05 (a fusion protein of 7NAB antibody and DS- ₀₅ peptide).

Figure 28 shows the neutralization capacity and IC 50 values calculated based on the measured percentage of infected cells after SARS-CoV2 was incubated with cells together with various concentrations of 7TCQ_DS-05 (a fusion protein of 7TCQ antibody and DS- ₀₅ peptide).

Figure 29 shows the neutralization capacity and IC 50 values calculated based on the measured percentage of infected cells after SARS-CoV2 was incubated with cells together with various concentrations of 7TCQ_YAP-1 (a fusion protein of 7TCQ antibody and YAP- ₁ peptide).

Figure 30 illustrates the positions of the expected amino acid residues when a peptide having an amino acid sequence of DWLRIIWDWVCSVVSDFK (SEQ ID NO: 316) has an alpha helix structure. In SEQ ID NO: 316, the N-terminal is indicated at the first position, aspartic acid (Asp, D), and the C-terminal is indicated at the last position, lysine (Lys K). At this time, lines are connected in order, starting from the N-terminus, D, to the next amino acid residue after D, W, and then to the next amino acid residue, L, and the last position connected by the line is the C-terminus, K. Figure 30 shows that a hydrophilic region and a hydrophobic region are distinguished within a single structure of the peptide, and that each region contains nine amino acid residues. At this time, the types of amino acid residues included in the hydrophilic region are Tryptophan (Trp, W), Valine (Val, V), Leucine (Leu, L), Phenylalanine (Phe, F), and Isoleucine (Ile, I), which are all hydrophilic amino acid residues. The types of amino acid residues included in the hydrophobic region are composed of hydrophobic amino acid residues Lysine (Lys; Lys K), Arginine (Arginine; Arg, R), Serine (Serine; Ser, S), and Aspartic acid (Aspartic acid; Asp, D), and hydrophilic amino acid residues Cysteine (Cysteine; Cys, C) and Isoleucine (Ile, I), and among the nine amino acid residues included in the hydrophobic region, seven amino acid residues are hydrophobic amino acid residues. Among the amino acid residues illustrated in Figure 30, those having a checkered pattern are hydrophilic amino acid residues, and those having other patterns are hydrophobic amino acid residues. Therefore, the characteristics of the amino acid residues distributed in each region can be more easily confirmed.

Some embodiments of the present application provide antibody-peptide fusion proteins.

Some embodiments of the present application provide antibody-peptide fusion proteins comprising:

An antibody or fragment thereof, wherein the antibody is capable of binding to the S2 domain of the spike protein of a coronavirus;

a linker, wherein said linker is linked to a constant region of said antibody or a fragment thereof; and

An antiviral peptide, wherein the antiviral peptide is linked to the antibody or a fragment thereof via the linker,

At this time, the antiviral peptide is an amphipathic peptide,

The number of amino acid residues constituting the above antiviral peptide is 18.

The above antiviral peptides can cause damage to the lipid bilayer of the virus,

At this time, the antibody-peptide fusion protein can bind to the S2 domain of the spike protein of the coronavirus,

At this time, the antibody-peptide fusion protein can cause damage to the lipid bilayer of the virus.

In a particular embodiment, the amino acid sequence of the antiviral peptide can be any one amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 315.

In certain embodiments, the amino acid sequence of the antiviral peptide may be any one of the following amino acid sequences:

SEQ ID NO: 1, SEQ ID NO: 3 to 5, SEQ ID NO: 8 to 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38 to 40, SEQ ID NO: 43, SEQ ID NO: 45 to 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 64 to 73, SEQ ID NO: 75, SEQ ID NO: 78 to 87, SEQ ID NO: 90 to 93, SEQ ID NO: 97 to 102, SEQ ID NO: 104 to 106, SEQ ID NO: 109 to 112, SEQ ID NO: 114 to 116, SEQ ID NO: 118 to 123, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 130 to 132, SEQ ID NO: 136, SEQ ID NO: 138 to 147, SEQ ID NO: 151 to 155, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166 to 168, SEQ ID NO: 170 to 174, SEQ ID NO: 176, SEQ ID NO: 180 to 182, SEQ ID NO: 184 to 186, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 194 to 196, SEQ ID NO: 198, SEQ ID NO: 201, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208 to 211, SEQ ID NO: 215, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 240, SEQ ID NOs: 242 to 246, SEQ ID NOs: 248 to 254, SEQ ID NOs: 256 to 258, SEQ ID NOs: 260 to 270, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NOs: 275 to 288, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NOs: 293 to 296, SEQ ID NO: 298, and SEQ ID NOs: 299 to 315.

In a specific embodiment, the linker is a linker comprising a peptide, wherein the N-terminus of the peptide comprised in the linker may be linked to the C-terminus of the heavy chain of the antibody or fragment thereof.

In a specific embodiment, the linker is a linker comprising a peptide, wherein the C-terminus of the peptide comprised in the linker may be linked to the N-terminus of the heavy chain of the antibody or fragment thereof.

In a specific embodiment, the linker is a linker comprising a peptide, and the number of amino acid residues of the peptide comprised in the linker may be from 5 to 20.

In a specific embodiment, the linker is a linker comprising a peptide, and the amino acid sequence of the peptide comprised in the linker may comprise GGGGSGGGGSGGGGS (SEQ ID NO: 317).

In certain embodiments, the coronavirus may comprise one or more selected from an Alphacoronavirus, a Betacoronavirus, a Gammacoronavirus, and a Deltacoronavirus.

In certain embodiments, the coronavirus is a betacoronavirus,

At this time, the betacoronavirus may include at least one selected from embecovirus, merbecovirus, and sarbecovirus.

In a specific embodiment, the coronavirus is a Sarbecovirus,

At this time, the sarbecovirus may include at least one selected from SARS-CoV and SARS-CoV-2.

In certain embodiments, the coronavirus may be SARS-CoV-2.

In certain embodiments, the antibody or fragment thereof can bind to the S2 domain of the spike protein of a betacoronavirus,

At this time, the antibody-peptide fusion protein may be capable of binding to the S2 domain of the spike protein of betacoronavirus.

In certain embodiments, the antibody or fragment thereof can bind to the S2 domain of the spike protein of SARS-CoV2 and the S2 domain of the spike protein of a variant of SARS-CoV2,

At this time, the antibody-peptide fusion protein may be capable of binding to the S2 domain of the spike protein of SARS-CoV2 and the S2 domain of the mutant spike protein of SARS-CoV2.

In certain embodiments, the antibody fragment can be any one selected from F(ab), F(ab'), F(ab') ₂ , monospecific F(ab') ₂ , bispecific F(ab') ₂ , scFv, ScFv-Fc, and sdAb.

Some embodiments of the present application provide a pharmaceutical composition for treating a viral disease comprising a therapeutically effective amount of the antibody-peptide fusion protein.

At this time, the above viral disease is a disease caused by infection with a virus.

In certain embodiments, the pharmaceutical composition for treating a viral disease may further comprise a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable adjuvant.

In certain embodiments, the viral disease is a respiratory disease resulting from a viral infection, wherein the viral infection may be a betacoronavirus infection.

In certain embodiments, the viral disease is a respiratory disease resulting from a viral infection, wherein the viral infection may be an infection with SARS-CoV2 or a variant thereof.

Some embodiments of the present application provide a method for treating a viral disease, comprising administering to a subject infected with the virus a pharmaceutical composition comprising a therapeutically effective amount of the antibody-peptide fusion protein.

In certain embodiments, the viral disease is a respiratory disease resulting from a viral infection, wherein the viral infection may be a betacoronavirus infection.

In certain embodiments, the viral disease is a respiratory disease resulting from a viral infection, wherein the viral infection may be an infection with SARS-CoV2 or a variant thereof.

Some embodiments of the present application provide uses of the antibody-peptide fusion protein for treating viral diseases.

In certain embodiments, the viral disease is a respiratory disease resulting from a viral infection, wherein the viral infection may be a betacoronavirus infection.

In certain embodiments, the viral disease is a respiratory disease resulting from a viral infection, wherein the viral infection may be an infection with SARS-CoV2 or a variant thereof.

Some embodiments of the present application provide uses of the antibody-peptide fusion protein for the manufacture of a medicament for treating a viral disease.

In certain embodiments, the viral disease is a respiratory disease resulting from a viral infection, wherein the viral infection may be a betacoronavirus infection.

In certain embodiments, the viral disease is a respiratory disease resulting from a viral infection, wherein the viral infection may be an infection with SARS-CoV2 or a variant thereof.

Hereinafter, with reference to the attached drawings, the content of the invention will be described in more detail through specific implementation examples and examples. It should be noted that the attached drawings include some implementation examples of the invention, but not all implementation examples. The content of the invention disclosed by this specification can be implemented in various ways and is not limited to the specific implementation examples described herein. It should be understood that these implementation examples are provided to satisfy the legal requirements applicable to this specification. Those skilled in the art will be able to think of many modifications and other implementation examples of the content of the invention disclosed herein. Therefore, the content of the invention disclosed herein is not limited to the specific implementation examples described herein, and modifications and other implementations thereof are also intended to be included within the scope of the claims.

Definition of terms

The definitions of terms used in this specification are as follows.

approximately

The term "about" as used herein means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

Amino acid sequence notation

Unless otherwise stated, when describing an amino acid sequence in this specification, the amino acid single-letter notation or three-letter notation is used, and is described in the direction from the N-terminal to the C-terminal. For example, in the case of notation as RNVP, it means a peptide in which arginine, asparagine, valine, and proline are sequentially connected in the direction from the N-terminal to the C-terminal. As another example, in the case of notation as Thr-Leu-Lys, it means a peptide in which threonine, leucine, and lysine are sequentially connected in the direction from the N-terminal to the C-terminal. In the case of amino acids that cannot be expressed in the single-letter notation, other letters are used and additional explanations are provided.

The notation for each amino acid is as follows: Alanine (Ala, A); Arginine (Arg, R); Asparagine (Asn, N); Aspartic acid (Asp, D); Cysteine (Cys, C); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Histidine (His, H); Isoleucine (Ile, I); Leucine (Leu, L); Lysine (Lys K); Methionine (Met, M); Phenylalanine (Phe, F); Proline (Pro, P); Serine (Ser, S); Threonine (Thr, T); Tryptophan (Trp, W); Tyrosine (Ty, Y); and Valine (Val, V).

Nucleic acid sequence notation

The symbols A, T, C, G, and U used herein are to be interpreted as having the meanings understood by a person skilled in the art. They may be appropriately interpreted as bases, nucleosides, or nucleotides on DNA or RNA, depending on the context and technology. For example, when referring to a base, they may be interpreted as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U) themselves, respectively, and when referring to a nucleoside, they may be interpreted as adenosine (A), thymidine (T), cytidine (C), guanosine (G), or uridine (U), respectively, and when referring to a nucleotide in a sequence, they should be interpreted to mean a nucleotide including each of the above nucleosides.

In this specification, the symbol N can be appropriately interpreted as a base, a nucleoside, or a nucleotide on DNA or RNA, depending on the context and technology. For example, when it refers to a base, it can be interpreted as any one of adenine (A), thymine (T), cytosine (C), guanine (G), and uracil (U), respectively, and when it refers to a nucleoside, it can be interpreted as any one of adenosine (A), thymidine (T), cytidine (C), guanosine (G), and uridine (U), respectively, and when it refers to a nucleotide in a sequence, it should be interpreted as meaning a nucleotide including each of the above nucleosides.

Antiviral

The term "antiviral" as used herein refers to a property capable of damaging or destroying the outer membrane of a virus, thereby causing a decrease or inactivation of the virus' ability to infect cells. For example, "antiviral peptide" refers to a peptide capable of damaging or destroying the outer membrane of a virus, thereby causing a decrease or inactivation of the virus' ability to infect cells.

Antibody

The structure of the antibody is divided into a heavy chain region and a light chain region, depending on the chain type. The structure of the antibody is divided into a fragment antigen-binding region (Fab region) and a fragment crystallizable region (Fc region), depending on the function of binding to the antigen. The structure of the antibody is divided into a variable region and a constant region, depending on the variability of the amino acid sequence. Other structures of the antibody include a hinge portion and a tail portion.

The above antibody is structurally largely divided into a heavy chain region and a light chain region. The heavy chain region has a polypeptide chain structure and includes four or five immunoglobulin domains. The light chain region has a polypeptide chain structure and includes two immunoglobulin domains. Each immunoglobulin domain includes about 90 to 130 amino acids.

The heavy chain region and the light chain region are functionally largely divided into a fragment antigen-binding region (Fab region) and a fragment crystallizable region (Fc region). The Fab region is a region that includes a portion that binds to an antigen (antigen-binding portion). The Fc region is a portion that can bind to an Fc receptor. The heavy chain region has both a Fab region and an Fc region. The light chain region has a Fab region.

The Fab region of the heavy chain region includes a heavy chain variable region (VH) and a heavy chain constant region 1 (CH1). The Fc region of the heavy chain region includes a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3), or includes CH2, CH3, and a heavy chain constant region 4 (CH4). The Fab region of the light chain region includes a light chain variable region (VL) and a light chain constant region (CL). The light chain region does not have an Fc region. Each of the VH, CH1, CH2, CH3, CH4, VL, and CL is one immunoglobulin domain, and one immunoglobulin domain includes about 100 to 120 amino acids.

The immunoglobulin domains included in the heavy chain region are positioned in the order of VH, CH1, CH2, and CH3 or VH, CH1, CH2, CH3, and CH4 from the N-terminal to the C-terminal. The immunoglobulin domains included in the light chain region are positioned in the order of VL and CL from the N-terminal to the C-terminal.

The heavy chain region and the light chain region are connected by a disulfide bond. Specifically, they are connected by a disulfide bond between CH1 of the heavy chain region and CL of the light chain region. The Fab region and the Fc region are connected by a hinge region. Specifically, the C-terminal portion of CH1 of the heavy chain region and the N-terminal portion of CH2 are connected by a hinge region.

fragment of antibody

As used herein, the term "antibody fragment" refers to a portion of an antibody, wherein the antibody fragment may have any type of form capable of binding to an antigen to which the antibody can bind. In one specific example, the antibody fragment comprises an antigen-binding portion of the antibody.

In one specific example, the type of the antibody fragment may be selected from F(ab), F(ab'), F(ab') ₂ , monospecific F(ab') ₂ , bispecific F(ab') ₂ , scFv, ScFv-Fc, and sdAb.

The above F(ab) means that it has a structure including the VH, CH1, VL, and CL of an antibody, and has only one antigen binding site.

The above F(ab') has a structure including the VH, CH1, VL, and CL of an antibody, and means that, compared to Fab, it additionally has a hinge including one or more cysteine residues at the C-terminal of CH1.

The above F(ab') ₂ means that two F(ab') are connected. At this time, the connection of the two F(ab') is due to a disulfide bond of cysteine residues in the hinge region, and the two connected F(ab') can be the same or different. The above monospecific F(ab') ₂ means that the antigen-binding portions of the two connected F(ab') of F(ab') ₂ can bind to the same antigen. The above bispecific F(ab') ₂ means that the antigen-binding portions of the two connected F(ab') of F(ab') ₂ can bind to different antigens.

The above single-chain variable fragment (scFv) means that it includes only VH and VL, and does not include immunoglobulin domains (CL, CH1, CH2, CH3, and CH4) belonging to the constant region of an antibody. At this time, the VH and VL included in the scFv are generally covalently bonded to each other via a peptide linker. Specifically, the peptide linker connects the C-terminal of VH and the N-terminal of VL, or connects the N-terminal of VH and the C-terminal of VL. When two to three scFvs are connected, there may be types such as tandem di-scFv, diabody, tandem tri-scFv, and tribody. The scFv-Fc means that it includes scFv, Ch2, and CH3. Specifically, the scFv-Fc means that scFv, Ch2, and CH3 are connected in that order.

The above single-domain antibody (sdAB) means an antibody that contains only one of the variable regions of the antibody, VH or VL.

Problems with prior art

The present invention solves the problem of antiviral peptides being difficult to use as therapeutic agents, thereby providing an effective therapeutic agent for viral infectious diseases.

Peptides known as antiviral peptides have an antiviral effect or virus-killing effect that attacks the outer membrane of a virus, but there is a problem in that they do not properly show a therapeutic effect on viral infection when administered to an actual subject. The reason is that the antiviral peptide injected into a subject infected with a virus is decomposed by various biochemical reactions in the body or acts (aggregates, binds, etc.) on the outer membrane of general cells in the subject's body, so the probability of reaching the target viral outer membrane is low. In other words, it is understood that the amount of antiviral peptide that actually reaches the viral outer membrane in the subject's body is small.

The present invention has the advantage of increasing the probability that the antiviral peptide reaches the target viral envelope by conjugating an antibody capable of binding to a virus to the antiviral peptide, thereby enabling it to be used as an effective treatment for viral infectious diseases.

1. Antibody-peptide fusion protein of the present disclosure

1.1 Antibody-peptide fusion protein of the present disclosure

In one aspect of the present disclosure, an antibody-peptide fusion protein is disclosed, wherein an antibody or antibody fragment is linked to an antiviral peptide.

The above antibody-peptide fusion protein comprises an antibody or a fragment thereof and an antiviral peptide, and may further comprise a linker.

In some embodiments, the antibody-peptide fusion protein comprises an antibody and an antiviral peptide.

In some embodiments, the antibody-peptide fusion protein comprises an antibody, a linker and an antiviral peptide.

In some embodiments, the antibody-peptide fusion protein comprises a fragment of an antibody and an antiviral peptide.

In some embodiments, the antibody-peptide fusion protein comprises an antibody fragment, a linker and an antiviral peptide.

The structure of the above antibody-peptide fusion protein may be “antibody-antiviral peptide”, “antibody-linker-antiviral peptide”, “antibody fragment-antiviral peptide” or “antibody fragment-linker-antiviral peptide”.

The above antiviral peptide is a peptide having an antiviral or virus-killing effect. In a specific example, the antiviral effect of the antiviral peptide may include inhibition of viral membrane protein function, inhibition of viral RNA amplification within the nucleus of infected cells, and/or inhibition of infection through damage to the viral envelope. Specifically, the antiviral peptide may cause damage to the outer membrane (or phospholipid bilayer) of the virus. A specific description of the above antiviral peptide is disclosed in Section <<2. Antiviral Peptide>> of the present disclosure.

The above antibody or fragment thereof is mainly used for its function of specifically recognizing a desired target virus. That is, the antibody or fragment thereof refers to an antibody or fragment thereof that can bind to a virus. The type of the antibody fragment may be selected from F(ab), F(ab'), F(ab') ₂ , monospecific F(ab') ₂ , bispecific F(ab') ₂ , scFv, ScFv-Fc, and sdAb. A specific description of the antibody and fragment thereof is disclosed in Section <<3. Antibodies and fragments thereof>> of the present disclosure.

The above linker connects, binds, or conjugates the antibody and the antiviral peptide, and can be designed in various ways considering the structure of the antibody and the antiviral peptide. A specific description of the linker is disclosed in Section <<4. Linker>> of the present disclosure.

1.2 Structure of the fusion protein

In the antibody-peptide fusion protein of the present disclosure, the antiviral peptide may be directly linked to the antibody or indirectly linked via a linker.

“The antibody and the antiviral peptide being linked with” means that the antibody and the antiviral peptide are linked through at least one bond selected from a covalent bond, an ionic bond, a hydrogen bond, a disulfide bond, a peptide bond, and a glycosidic bond to form a single construct. Depending on the type of bond and the bonding substance, the term “fusion” or “conjugation” can be used interchangeably.

The antibody-peptide fusion protein of the present disclosure may have a structure represented by Formula I or Formula II.

[Chemical Formula I]

Ab-P

[Chemical Formula II]

Ab-L-P

In the above chemical formulas I and II, Ab is an antibody.

At this time, the antibody may be a whole antibody or an antibody fragment, and may be referred to as a fused antibody or a fragment of a fused antibody. The antibody or fragment thereof has "target specificity" for a virus, and a detailed description thereof is described in Section <<3. Antibody or fragment thereof>> of the present disclosure.

In the above chemical formulas I and II, P represents an antiviral peptide, which may be referred to as a fused antiviral peptide. The antiviral peptide is capable of causing damage to the phospholipid bilayer of a virus, and a detailed description thereof is described in Section <<2. Antiviral Peptide>> of the present disclosure.

In the above chemical formula II, L is a linker. At this time, the linker is a substance having a function of connecting the antibody and the antiviral peptide. A specific description of the linker is described in Section <<4. Linker>> of the present disclosure.

In the above chemical formulas I and II,

The position at which the antiviral peptide (P) or linker (L) is linked to the antibody (Ab) is a site or position at which the target specificity of the antibody is not affected.

The antiviral peptide or linker may be linked to any one amino acid residue included in the antibody. In one embodiment, the site on the antibody to which the antiviral peptide or linker is linked may be any one of the light chain region, the heavy chain region, the variable region, the constant region, the Fab region, the Fc region, the VH, the VC, the CH1, the CH2, the CH3, or the CL region of the antibody, and the antiviral peptide or linker may be linked to any one amino acid residue forming these regions. In this case, the amino acid residue included in the antibody may not include an artificial modification, or may include an artificial modification.

For example, any one amino acid residue included in the antibody may be a lysine group or a cysteine group. In this case, it may be linked through an amine group of the lysine group or a thiol group of the cysteine group.

In one specific embodiment, the position at which the antiviral peptide or linker is linked to the antibody may be the N-terminus of the light chain or heavy chain of the antibody.

In one specific embodiment, the position at which the antiviral peptide or linker is linked to the antibody may be the C-terminus of the light chain or heavy chain of the antibody.

In the above chemical formula I, the connection between the antibody and the antiviral peptide is

It may be formed by at least one bond selected from a covalent bond, an ionic bond, a hydrogen bond, a disulfide bond, a peptide bond, and a glycosidic bond. Preferably, the antibody unit and the antiviral peptide unit may be linked via a covalent bond or a peptide bond.

In the above chemical formulas I and II,

The position at which the antibody (Ab) or linker (L) is linked to the above antiviral peptide (P) is a site or position at which the antiviral efficacy of the peptide is not affected.

The antibody or linker may be linked to any one amino acid residue included in the antiviral peptide. In one embodiment, the site or position on the antiviral peptide to which the antibody or linker is linked may be the N-terminal or C-terminal of the antiviral peptide. In this case, the amino acid residue included in the antiviral peptide may not contain an artificial modification, or may contain an artificial modification.

In the above chemical formula I,

The connection between the above antibody and the antiviral peptide can be formed through a covalent bond, for example, an amide covalent bond (peptide bond). The peptide bond is formed through a chemical reaction between the amino group (RNH ₂ ) and the carboxyl group (RCOOH) of two amino acids.

In the above chemical formula II,

The above linker (L) is not limited to a substance that can form a single structure (a fusion protein expressed by chemical formula II) by linking an antibody and an antiviral peptide.

In one specific embodiment, the linker (L) may be a polypeptide consisting of a specific amino acid sequence.

At this time, an amide covalent bond (peptide bond) is formed between the antibody and the linker and between the linker and the antiviral peptide.

At this time, the fusion protein represented by the chemical formula II may be expressed as a single fusion protein using a nucleic acid sequence including [nucleic acid sequence encoding antibody]-[nucleic acid sequence encoding linker]-[nucleic acid sequence encoding antiviral peptide].

In one specific example, the linker (L) may be a compound having a specific chemical structure.

At this time, covalent bonds, ionic bonds, hydrogen bonds, disulfide bonds, etc. can be formed between the antibody and the linker and between the linker and the peptide depending on the structural characteristics of the compound.

At this time, the fusion protein represented by the chemical formula II includes a specific chemical structure between [antibody] and [antiviral peptide], and this specific chemical structure can be formed using various chemical reactions known in the art, for example, click chemistry reaction, etc.

1.3 Effect of fusion proteins

The antibody-peptide fusion protein of the present disclosure has a virus inhibition function. In this case, the virus refers to a target virus targeted by the antibody included in the antibody-peptide fusion protein. The virus inhibition function may refer to a neutralizing ability against the virus, an antiviral effect, a killing effect against the virus, or an effect of inhibiting the infection of a virus into a general cell.

For example, the antibody-peptide fusion protein has neutralizing ability against the virus.

For example, the antibody-peptide fusion protein has the effect of inhibiting infection of the virus.

For example, the antibody-peptide fusion protein causes damage to the outer membrane (or phospholipid bilayer) of the virus.

For example, the antibody-peptide fusion protein can cause destruction of the outer membrane (or phospholipid bilayer) of the virus. Specifically, the antibody-peptide fusion protein can cause destruction of the phospholipid bilayer of the virus by causing damage to the phospholipid bilayer of the virus.

For example, the antibody-peptide fusion protein can cause a decrease or inactivation of the ability of a virus to infect a cell. Specifically, the antibody-peptide fusion protein can cause a decrease or inactivation of the ability of a virus to infect a cell by causing damage (or destruction) of the phospholipid bilayer.

For example, the antibody-peptide fusion protein can cause a reduction or inactivation of the ability of a virus to multiply (or replicate). Specifically, the antibody-peptide fusion protein can cause a reduction or inactivation of the ability of a virus to multiply (or replicate) by causing damage (or destruction) of the phospholipid bilayer.

For example, the antibody-peptide fusion protein may have a superior virus inhibition function compared to the antibody contained in the fusion protein.

For example, the antibody-peptide fusion protein may have a more superior virus inhibition function than the antiviral peptide contained in the fusion protein.

1.4 Advantages of fusion proteins

For treating viral infections, the therapeutic effect is better when using an antibody-peptide fusion protein in which the antiviral peptide is fused or linked to an antibody, compared to when using the antiviral peptide disclosed herein without fusing it to an antibody.

This is because, when an antiviral peptide is injected (or administered) into the body of a living organism infected with a virus for treatment, the antiviral peptide 1) is easily decomposed (degraded) by various biochemical reactions in the body due to its small size, or 2) is easily aggregated (binded, or attached) to the outer membrane of general cells, so that the amount to reach the intended target virus is insufficient. For these reasons, there has been a problem in the relevant technical field that the antiviral peptide cannot be used as a treatment for viral infection.

In order to solve these problems, when an antibody-peptide fusion protein in which the antiviral peptide and an antibody capable of binding to the target virus are linked or fused is injected (or administered) into the body of a living organism infected with the virus, 1) the antiviral peptide is not easily decomposed by various biochemical actions in the body due to the antibody being very large in size compared to the antiviral peptide, 2) furthermore, due to the large size of the antibody, it does not easily aggregate on the outer membrane of general cells, and 3) as the antibody repeatedly binds and dissociates from the target virus, it helps the antiviral peptide to specifically act on the outer membrane of the target virus near the virus, so that the amount is sufficient to reach the intended target virus.

That is, when an antibody-peptide fusion protein is used to treat a target viral infection, there is an advantage in that a therapeutic effect can be observed even when an appropriate effective amount necessary for treatment is administered.

1.5 Fusion proteins of specific embodiments

In certain embodiments, the antibody-peptide fusion protein of the present disclosure may comprise heavy and light chains linked to an antiviral peptide.

In certain embodiments, the antibody-peptide fusion protein of the present disclosure can comprise two heavy chains and two light chains linked to an antiviral peptide, wherein the two heavy chains linked to the peptide can be identical to each other, and the two light chains can be identical to each other.

In certain embodiments, the antibody-peptide fusion protein of the present disclosure comprises a heavy chain and a light chain linked to an antiviral peptide, wherein the amino acid sequence of the light chain may be SEQ ID NO: 318 and the amino acid sequence of the heavy chain linked to the antiviral peptide may be SEQ ID NO: 330.

In certain embodiments, the antibody-peptide fusion protein of the present disclosure comprises a heavy chain and a light chain linked to an antiviral peptide, wherein the amino acid sequence of the light chain may be SEQ ID NO: 320 and the amino acid sequence of the heavy chain linked to the antiviral peptide may be SEQ ID NO: 331.

In certain embodiments, the antibody-peptide fusion protein of the present disclosure comprises a heavy chain and a light chain linked to an antiviral peptide, wherein the amino acid sequence of the light chain may be SEQ ID NO: 320 and the amino acid sequence of the heavy chain linked to the antiviral peptide may be SEQ ID NO: 332.

2. Antiviral peptides

2.1 Overview - Antiviral Peptides of the Present Disclosure

According to one aspect of the present disclosure, an antiviral peptide is disclosed. The antiviral peptide is an amphipathic peptide (or polypeptide) having both hydrophilic (polar, hydrophilicity) amino acids and hydrophobic (nonpolar, hydrophobicity) amino acids.

In some embodiments, the antiviral peptide may be a known antiviral peptide.

In some embodiments, the antiviral peptide may be selected from a known amino acid sequence.

In some embodiments, the antiviral peptide may be designed from a known antiviral peptide.

Details regarding the structure, characteristics, etc. that the antiviral peptide of the present disclosure may have are described below.

2.2 Length of antiviral peptide

The number of amino acid residues constituting the antiviral peptide of the present disclosure is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36. In other words, the antiviral peptide is a 12mer, 13mer, 14mer, 15mer, 16mer, 17mer, 18mer, 19mer, 20mer, 21mer, 22mer, 23mer, 24mer, 25mer, 26mer, 27mer, 28mer, 29mer, 30mer, 31mer, 32mer, 33mer, 34mer, 35mer, or 36mer peptide.

For example, the number of amino acid residues constituting the antiviral peptide may be between the two values selected in the immediately preceding sentence. For example, the number of amino acid residues constituting the antiviral peptide may be 16 to 27.

Preferably, the number of amino acid residues constituting the antiviral peptide may be 18. In other words, the antiviral peptide may be composed of 18 amino acid residues. In another words, the antiviral peptide may be an 18mer peptide.

2.3 Amino acid residues of antiviral peptides

The antiviral peptide of the present disclosure comprises hydrophilic amino acid residues and hydrophobic amino acid residues. Herein, the hydrophilic amino acid residues may include arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), lysine (Lys, K), serine (Ser, S), and/or threonine (Thr, T). The above hydrophobic amino acid residues may include alanine (Ala, A), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), proline (Pro, P), valine (Val, V), phenylalanine (Phe, F), tyrosine (Tyr, Y), and/or tryptophan (Trp, W).

In some embodiments, the number of hydrophilic amino acid residues and hydrophobic amino acid residues included in the antiviral peptide may be the same. In other some embodiments, the number of hydrophilic amino acid residues included in the antiviral peptide may be 1, 2, 3, 4, or 5 more than the number of hydrophobic amino acid residues. In still other some embodiments, the number of hydrophobic amino acid residues included in the antiviral peptide may be 1, 2, 3, 4, or 5 more than the number of hydrophilic amino acid residues. In preferred embodiments, the number of hydrophobic amino acid residues included in the antiviral peptide may be 4 more than the number of hydrophilic amino acid residues.

In some embodiments, when the antiviral peptide is an 18mer peptide, the antiviral peptide may comprise 7 hydrophilic amino acid residues and 11 hydrophobic amino acid residues. In some other embodiments, when the antiviral peptide is an 18mer peptide, the antiviral peptide may comprise 8 hydrophilic amino acid residues and 10 hydrophobic amino acid residues. In some other embodiments, when the antiviral peptide is an 18mer peptide, the antiviral peptide may comprise 9 hydrophilic amino acid residues and 9 hydrophobic amino acid residues. In some other embodiments, when the antiviral peptide is an 18mer peptide, the antiviral peptide may comprise 10 hydrophilic amino acid residues and 8 hydrophobic amino acid residues. In some other embodiments, when the antiviral peptide is an 18mer peptide, the antiviral peptide may comprise 11 hydrophilic amino acid residues and 7 hydrophobic amino acid residues.

2.4 Structure of antiviral peptides - alpha helix structure

The antiviral peptide of the present disclosure can form an alpha helix (α-helix) structure when reacting with a lipid membrane. In this case, the antiviral peptide reacting with a lipid membrane may mean a case where the antiviral peptide comes into contact with a lipid membrane (specifically, a phospholipid bilayer).

When the above antiviral peptide has an alpha helix structure, the positions of amino acid residues can be diagrammed in a direction looking from top to bottom of the alpha helix structure, as in Fig. 30. Fig. 30 is an example of one of the known antiviral peptides to explain the alpha helix structure of the antiviral peptide, and the amino acid sequence of the peptide illustrated in Fig. 30 is N-terminal-DWLRIIWDWVCSVVSDFK (SEQ ID NO: 316)-C-terminal. When the peptide has an alpha helix structure rotating around an imaginary first axis, when the peptide is viewed from one direction parallel to the imaginary first axis, each amino acid can be seen as illustrated in Fig. 30. At this time, if one virtual plane including the virtual first axis (the virtual plane is indicated by a dashed dotted line in FIG. 30) is well selected, the peptide can be divided into a hydrophilic region and a hydrophobic region by the virtual plane. At this time, each of the two regions divided by the virtual plane includes 9 amino acid residues.

In the present specification, the hydrophilic region may mean a region containing a greater number of hydrophilic amino acid residues among the two regions divided by the virtual plane, and the hydrophobic region may mean a region other than the hydrophilic region among the two divided regions.

In some embodiments, the hydrophilic region may comprise only hydrophilic amino acid residues and no hydrophobic amino acid residues. That is, the hydrophilic region may comprise nine hydrophilic amino acid residues.

In some other embodiments, the hydrophilic region may comprise eight hydrophilic amino acid residues and one hydrophobic amino acid residue.

In some other embodiments, the hydrophilic region may comprise seven hydrophilic amino acid residues and two hydrophobic amino acid residues.

In some other embodiments, the hydrophilic region may comprise six hydrophilic amino acid residues and three hydrophobic amino acid residues.

In some embodiments, the hydrophobic region may comprise only hydrophobic amino acid residues and no hydrophilic amino acid residues. That is, the hydrophobic region may comprise nine hydrophobic amino acid residues.

In some other embodiments, the hydrophobic region may comprise eight hydrophobic amino acid residues and one hydrophilic amino acid residue.

In some other embodiments, the hydrophobic region may comprise seven hydrophobic amino acid residues and two hydrophilic amino acid residues.

In some other embodiments, the hydrophobic region may comprise six hydrophobic amino acid residues and three hydrophilic amino acid residues.

In one embodiment, each hydrophilic amino acid or hydrophilic amino acid residue can be any one selected from arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), lysine (Lys, K), serine (Ser, S), and threonine (Thr, T).

In one embodiment, each hydrophobic amino acid or hydrophobic amino acid residue can be any one selected from alanine (Ala, A), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), valine (Val, V), phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W).

2.5 Characteristics of antiviral peptides - Amphipathic

The antiviral peptide of the present disclosure is an amphipathic peptide. Specifically, the antiviral peptide does not have amphipathic characteristics when it has a linear structure, but has amphipathic characteristics when it has an alpha-helical structure. The reason for this is that, due to the characteristics of the amino acid sequence of the antiviral peptide, when it has a linear structure, there is no region in which hydrophilic amino acid residues or hydrophobic amino acid residues are concentratedly distributed within a single structure, but when it has an alpha-helical structure, there is a region in which hydrophilic amino acid residues or hydrophobic amino acid residues are concentratedly distributed within a single structure. That is, the antiviral peptide has amphipathic characteristics when it reacts with a lipid membrane or forms an alpha-helical structure for other reasons.

The above amphipathic characteristic means that when the target peptide has a three-dimensional structure such as an alpha helix structure, one side of the target peptide within the three-dimensional structure exhibits hydrophilic (polar, hydrophilicity) properties, and the other side exhibits hydrophobic (nonpolar, hydrophobicity) properties. In other words, the fact that the target peptide has amphipathicity means that the target peptide has both hydrophobic and hydrophilic properties.

The amphipathic characteristic of the above antiviral peptide can be explained through the distribution of hydrophilic amino acid residues and hydrophobic amino acid residues within the three-dimensional helical structure of the antiviral peptide, which is described in the above section <<2.4 Structure of Antiviral Peptide - Alpha Helix Structure>>. Specifically, since the alpha helix structure is divided into two regions by an imaginary plane, in one region, hydrophilic amino acid residues are distributed in greater quantities than hydrophobic amino acid residues, and in the other region, hydrophobic amino acid residues are distributed in greater quantities than hydrophilic amino acid residues, the antiviral peptide having the alpha helix structure has both hydrophilic and hydrophobic properties within a single structure.

The above amphipathic feature is thought to be one of the reasons for the efficacy of the antiviral peptide in acting on the phospholipid bilayer.

For example, the amphipathic characteristic of the antiviral peptide can be measured by Heliquest software. In some embodiments, when the sequence of the antiviral peptide is input into the Heliquest software, the hydrophobic moment (uH) value can be any one of 0.5 to 0.8. That is, the antiviral peptide can have a hydrophobic moment value (hydrophobic moment value calculated from Heliquest software) of any one of 0.5 to 0.8. In some embodiments, when the sequence of the antiviral peptide is input into the Heliquest software, the hydrophobicity (H) value can be any one of 0.7 to 0.9. That is, the antiviral peptide can have a hydrophobicity value (hydrophobicity value calculated from Heliquest software) of any one of 0.7 to 0.9. In some embodiments, when the sequence of the antiviral peptide is input into the Heliquest software, the net charge can be any value between -2 and 0. That is, the antiviral peptide can have a charge (as calculated from the Heliquest software) between -2 and 0.

2.6 Antiviral Peptide Examples

The amino acid sequence of the above antiviral peptide may be any one amino acid sequence selected from SEQ ID NOs: 1 to 315 of Table 1 below, or a sequence having 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more homology (sequence identity) thereto. At this time, whether the peptides represented by the amino acid sequences of SEQ ID NOs: 1 to 300 have an antiviral effect has been confirmed in another international application of the present inventors (PCT application number: PCT/KR2023/018361). In addition, the antiviral effect of the antiviral peptides represented by the amino acid sequences of SEQ ID NO: 301 to SEQ ID NO: 315 has been confirmed in another international application of the present inventors (PCT application number: PCT/KR2023/013277).

Amino acid sequence Sequence number Amino acid sequence Sequence number ESLKNFWESLWKFFTHFF 1 IEIMDFVIGVLDSIKIF 159 ITKFYKSFQDLWDMVIHI 2 IEQLVEYTKIILDFIISW 160 SFLDIVWSIIDIFKEFCK 3 KIFIGLLELIEDIFHGTF 161 QSFLDIVWSIIDIFKEFC 4 FRYLIEAINSFVKVFELW 162 QDLCLRIFSSLVRILDLF 5 INLLLKIVFELSKLIDDF 163 LHGLIDGLEYWIRGYLDW 6 NFIDRVFTVISDLILKLL 164 LIDGLEYWIRGYLDWVVH 7 SIFELINLQFDDLLVKIL 165 NFIDKTFTVLADIILKIL 8 DIIKDFAKFFTIFFGITF 166 NFIDKTFTIIADILLRII 9 WRGMLIKILSFIEGYIDF 167 VFQWLWDWVKKIFNWLED 10 LKDWLGFWLSLYQRFIEN 168 LWDWVKKIFNWLEDLFLN 11 NDILKVLSEWLTDLFTII 169 WVKKIFNWLEDLFLNFTG 12 DIWVNVLVSFITLLREWR 170 VVIYGIIGFLDDFLKIFK 13 DKIVLFILSILEKIYNLL 171 FFLRWLYSVSDYLSDFWE 14 EDILFSILYLLKKIANLI 172 RWLYSVSDYLSDFWESLL 15 ILADILVKLLDEIKYILK 173 DALWRLWLGFFREHFAAI 16 LLEYIDLVYNILISILKK 174 SEWTGILDRLLKWVGLLV 17 MLESLINKLITLIDFIVD 175 TGILDRLLKWVGLLVDIV 18 DLIHIILDKIIDTLVHFM 176 VELIDDLYRKLYSWFLHI 19 LRLLSNYFESMIDIFVKL 177 IDDLYRKLYSWFLHILTF 20 DVLLRVFVEGWTHLVRLL 178 MGIFQAFADVFKLLFKEI 21 DVMADQFVTFLEKWFKLF 179 AHFGFFWRWFDEIFTLLD 22 DWQALWEELKSIFVFWIN 180 WEALKYWWNLLQYWSKEL 23 ESIWEFIKSLLDAWSGWV 181 FQFIQKLIDDLLHVIEGL 24 FWEIFVVWLKQFIDIENS 182 IQKLIDDLLHVIEGLVLI 25 WFIKKLNTWLDVILDEWK 183 FDFIEKFSHVLFDIVGII 26 EALMLIYKLFWDEFSSWL 184 IEKFSHVLFDIVGIIMKV 27 WDCVQDIWEYIFRTAMKI 185 FDFIKSIIIGIVEGLTEF 28 EEIAVILRIMEKLWHVFM 186 IDILIRFYHLFESIYKYI 29 EDVSEVLAHIGTCLRKIF 187 LIRFYHLFESIYKYIMDL 30 EFWNRWHMSLSFWFRDFV 188 QIGKDISNLLFDIWRVWL 31 EGILSFILISLKDFLRVF 189 ISNLLFDIWRVWLEKLGE 32 IVHLLNELFTKFDRLIGI 190 LLSMFLVMIQWWRDIVRE 33 FTNFHRQLFCWIDKWIDL 191 ILDTSVQFWYEFIDYIMR 34 LWEAIETFIGEIIAIFYK 192 TSVQFWYEFIDYIMRVIE 35 WEKTLSLIGEVIEIWMLV 193 EILGAFFHKFEKFFDLFM 36 EILREFMTLISHIWSQIK 194 FGLVKLWVEKIVSFIEQF 37 QDILSKLYLLLARVFEIW 195 VKLWVEKIVSFIEQFLNF 38 SFIRKGIEAWFTLLMELI 196 LFYLLSRLWFGLLETFRD 39 EMFALIFEYIDRIFSIVR 197 LLSRLWFGLLETFRDILV 40 ENWKDFFLIFWQKYFNVW 198 RFVQGVDMFLETLWKVWM 41 LNKYFFEWFQAIKGFVEW 199 QGVDMFLETLWKVWMELL 42 FGLLQSFADFLKYIVKEI 200 WSEIFRELFSLLGRIWSS 43 KIMFTFVKELLNYLDGFI 201 LALEKYWIEWLEKWQMLL 44 ETLVRLFRFFLGEWYAVL 202 FFHLWLNILAELLRFGDR 45 EVILASWYRIYTKIMDLI 203 ILDFFVGFIDWLVRYLNK 46 IVLEIIHHFVEILDRYFG 204 MDFIRAIILGIIEGITEW 47 KIVIGLLEFVEDIFHGLY 205 DHWHNLFIKLFKYYYDII 48 EYIDNIIEYIINILKLTI 206 YNLFDLLIYIWNKIFDDY 49 IIIITDLLENLKELIIKI 207 DIFQDLLKEVIFVLSKIL 50 FAKDIWRILTAWFAGLAD 208 LEILVQILRYWIKDVSFF 51 LSVAGWLDTIWDAIKRFF 209 LHIISYMGDIFKLIKEFW 52 FAKDIWRILVAWTTTLWD 210 EVWIEGLRSYLLDWWNFL 53 KDIITVIAKYFGFIIEFL 211 LNFIGVLVDSLKEILNEW 54 FEFLSRMVDSLVSFLSEF 212 GGFWDSIKALWRNLIDFL 55 FEIIHGLLDRIMQLLDVL 213 NFIYKELEQIIDLFMVLI 56 MLGLMDELIQMIDALVRI 214 FFDSIRILWTKIINGLVD 57 FFDFFVEDFSRWYIQIIR 215 MLMEQFLDYIGKLLFKIF 58 QLLLRALDAVATCWEKLF 216 EQFLDYIGKLLFKIFMTI 59 ILEWFTNYIRSWEEVFKL 217 KSLNFVLAAWIRILEEIL 60 ILVSQWVEYLKIFENFFK 218 ETFKEFVHVWFKSIFNWI 61 FFEKIMSHIKNLLEWLGV 219 FDFFKIVGEIFGLFENYF 62 LQMLGDFERAWSLILEFI 220 TIVSRVLGKVLDFWVDFI 63 ILVRWLSHFFEYLDRFYT 221 TTLMKFVELWFKDVFNWM 64 FGDLLGEFWLGLKKIFYI 222 QWFDLLKDIFSKFTIFLQ 65 MYAVIGFLDDFLKVFKQI 223 LLKELLDLLQDMVVMLLS 66 VWDEFLVLLSKLMEIVNN 224 AIFTFLHKLVEVLSDYLK 67 MEKILEIIRVILKLYEQW 225 AIMKKIIDFYGQWVDSFI 68 DKIFTDIFHYLEVLFRII 226 ANINDILMKIIKIFDAIL 69 FLDNIDFLFMFIVKFLDK 227 AVTWDILLDIIERLLQQL 70 FLEDVVIYISRLLRLWIS 228 WDILLDIIERLLQQLQNL 71 INDLFSEILMSFLKFLDL 229 LVLYGLIGFLDDFLKVFR 72 LKTFISTVNELFEWVIRL 230 IDFFDKVSHVFFKIIGYI 73 WLSGIYEFFLRYKNIWVE 231 IIELIKGIILGIVEGLTE 74 LKIYYEIMDFIDFVLSKW 232 HEVLDFLDNLMISIFKIL 75 FTYIDDLVEGIVKLIGVI 233 SIWQLVLEQFDDLLVKIL 76 VLVYFWRDYIEIIVKGIT 234 DIIDRFVDWITMTFGGFF 77 FVDIVFGLLEGWWRFADG 235 YEVYRVFSDIILHIFLKLI 78 QLSILFTDWLEWFDKILA 236 VVGLIIKWFDWIYELGKQ 79 FWMGVIGFLDDYMKVVLK 237 DMLGSLYKILENILIIFR 80 GEMASQFVTFLEKWFELF 238 KKLVEYILDIFLKLSEIL 81 GFFFGWIGLLEGIIREIK 239 MTVISLFKNLIEIFEKYL 82 FFSMVRDLLSWMESIIRQ 240 VDLFSDFVTLFRKLMMIL 83 GIHALWRIFDDWADIFVV 241 QDLKEIISYIFDIIKIII 84 GRWIDMLISRIFDIMLAF 242 DLLIDILKYLVRLTEDII 85 HEVMEFIEKLFLYIFKTI 243 DSIIDLFKDFMLTFRQFF 86 HLKDWTDTFVYILEKLI 244 FWKDIINLIKGFILSIID 87 HTLLDFWRLVWEFGVKVI 245 MEFFQEYYDIIRLWSTIL 88 HWSKLLEEIQALIKLLLA 246 DVLVKAWAMWGDYLENIF 89 IDDILTILLRLQKGLLGL 247 KFLKAVFELWEFIKDFLV 90 IEHLEQLFWRIFRNIVLF 248 LKEWLDYYNWLSRLIEFF 91 IQVFADVFKMLIKEIITI 249 TEFIKMFKSFVDLLFEVV 92 VAFGIIGFIDDFIKVILK 250 VLILKTIRELTSFFIDLL 93 IIGWIDHFQNWLEKLHKL 251 EILDFFMTLFGSILTDIR 94 VLYKEVLKTIIDLIENII 252 FTTILSDFQSFLERLLDI 95 IIISLLKQLLKTYGDLID 253 TEILKQFLTHFDTIFEVF 96 IIYQVIDELVNYFKKFLF 254 ELIDELYQKVYRWFMQIF 97 IKIINQWEALLKEFTSLI 255 LEQLMYLIEEFVKYLIRF 98 VTIYEMVLKSLLEIFKIL 256 SELMRWIISEIYAVWKIF 99 ILKDLLHWFKTQFFTWFD 257 IKFLKEFIEFLREYINVI 100 TIWLGFIGFMDDYIKVFK 258 SWKEIVNLLYELLASLIR 101 INALFDVWIDVQRRWVYL 259 YITAILVLWSRMMKEILD 102 IWRRWLYVLWDEVAQLVN 260 IAFGIIGFLDDILKIIHR 103 LNDIVIIMKSIIYEWFDI 261 FWRRHVFIWISFIDSYFE 104 IRVMFDEITRILNHLLGI 262 GKIFKVLYDSFMLLWNEW 105 TTWWNILERIDHKLFMWI 263 GWFGFISDAMELVLKILK 106 LSEWLRIIEFLLQGLKVY 264 KVIFDVIESFSKVIFGVI 107 WLLLFSEWLGNLQKIFDE 265 VILDLFKEIIDEVLKGII 108 RVILGAIERFIGVLIEHF 266 FLDALLKILEGWLYTYAK 109 KILDQWFFRITAYIDELL 267 WSEILREFLNLISLIWSR 110 IYIFFRHWTTIYERLLEI 268 IFLYLVDQIYDIIRMIEK 111 VFWYFWEDLTGIAKGIIK 269 YIYLILKFIQDIYNDIYR 112 KEFTELFILLKSIFSFV 270 FQFIEKFSEIMFELVRMI 113 KELWLNIVNIILTLYEDL 271 FSFWVWLDNIIDLVKKYI 114 KILNQITLFWMDMFKDII 272 YTSFVDVFFKIVDYLNKW 115 KLFSKLIDILDDYYVLLV 273 FTFWTWLEAILDLIKKHI 116 LDKVMKIWTTFVEQIFGV 274 MFVETLWKVWTELLDVLG 117 LDVLSSIGQLIRDILDFI 275 VLVGMIIKWFDWLYEMGK 118 LEKIYDIIGYIYQYLKLL 276 GILINVINDFERQWLRIF 119 LVFFSKVLQYLIEELLEI 277 GLQNFLEWVVDFIRGIIK 120 LISFIDYISEIIANIIDR 278 TRIVDWYDLVYGLLDGWW 121 LITMVLTMIQWWRDVIRE 279 GLLEIIVILWKNIHRSLE 122 LVMIVATMIQWWRDVIRE 280 LIWELIKQILKGISYIHD 123 SLLTLILEQFDDLLVKIL 281 YLWLDLRGFWTQIFEEIV 124 LLKEILNLYLDLGTWLLK 282 LLKIVSALWEKWLSLLEA 125 WEKLMLLIIDLFKFLDQY 283 HWWFEGVKWLGRVLELLY 126 LQELMKVLIDWINDVLVG 284 FSELLRLTRTLLGIIDCI 127 LWLSWEEGLQHFLKYLLD 285 QIIDWYSGLVRRWVIWYE 128 LWNDIMQFVEQFFWRLVE 286 ECAEKILETWGELLSKWH 129 LYEFFQAYKTVWTEIWKI 287 IRTLFDEITRILNHLLWL 130 MLIERLIDAINSWVQLIE 288 TLYAFFLLTLIREIVKGIE 131 QIWNMVIELLDQVVEVFK 289 WKEWWKVFYTIVDYINQI 132 YVVTFVELVWNIIEKLLD 290 WKKWWQVFYTVVDYVDQI 133 REVHEIWASLIEIALSLW 291 VKLGSIYFSFFDDFFRII 134 RVIHQFLELWYRYVVEVL 292 KLMVDFFNILNEIVMKLV 135 TDIIDIIDQIVILLTKFI 293 YKWFAFFKDVTDFISHLF 136 TLTDWKTWISFWKTLIDW 294 LDAIGDLLLDFLLLLNKW 137 WFEAVLGYISAVVELFRE 295 LTLLLLTMVQWWRDIIRE 138 WIESILELIKKHLLSLWN 296 TLLRAVETLFSLWLEFMR 139 WLAWIDWIRGLDGQLIKI 297 LRLWEIIARYVEGIVHLF 140 YEIIKWTSFLLEYLTEII 298 WLDNIIDLVKKYILALWN 141 YGELNIFWLQILEIIRRW 299 WFEALLGYISAVIEYFEK 142 FKLIWGSIFSFLEQIEKF 300 DFWSKLVYFFAEIIRFLS 143 DIFCDWWRWVISVLDSVK 301 AKNIIKLLLELIEVILNV 144 DVVSIWFKWIDCLWDSVR 302 LEEILEVIISLFKKLNLI 145 RLVKSIWDWFCIVVDSWD 303 LLVKILKLIINELSNVME 146 DVWCSWVSWVIKLFRDID 304 ALLIYFRRDWVDLIGGWL 147 DWVDCFWSWLIKVIDRVS 305 IQWFEVLDGLLGRYWKAL 148 SVVCDWWDVIIKWFDRLS 306 ATWQDWFHRWLEILDSSL 149 DWVSKFWRILICVWDSVD 307 LWKSLDSWASLILQWFED 150 DVVDDWWSVLCKWFRIIS 308 DVTRLLELILDVLAYIIS 151 RVFCIVWDWVKSWIDDLS 309 DEFLDFAALLFGWFRKFV 152 SWVDSFWDWLIRVIKCVD 310 DEILGRLLKILFVLFVDL 153 DVVDIVLRWICSWWSDFK 311 FRMFIGDLEKIVNLLLSL 154 RVIDCWVDWWSIVFKSLD 312 ILLKFLESVGGVLLDILR 155 SVFCSLWRWVIDWVDDIK 313 LIVILDDSFLELLRLFIK 156 KLVDSIWSWFICVVRDWD 314 LLFGLLGFLDDYLKVVLR 157 DVFCIWIDWVKSLWRDVS 315 LFYGFIGFLDDFVSIVKK 158

In certain specific embodiments, the amino acid sequence of the antiviral peptide may be any one of the following amino acid sequences:

SEQ ID NO: 1, SEQ ID NO: 3 to 5, SEQ ID NO: 8 to 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38 to 40, SEQ ID NO: 43, SEQ ID NO: 45 to 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 64 to 73, SEQ ID NO: 75, SEQ ID NO: 78 to 87, SEQ ID NO: 90 to 93, SEQ ID NO: 97 to 102, SEQ ID NO: 104 to 106, SEQ ID NO: 109 to 112, SEQ ID NO: 114 to 116, SEQ ID NO: 118 to 123, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 130 to 132, SEQ ID NO: 136, SEQ ID NO: 138 to 147, SEQ ID NO: 151 to 155, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166 to 168, SEQ ID NO: 170 to 174, SEQ ID NO: 176, SEQ ID NO: 180 to 182, SEQ ID NO: 184 to 186, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 194 to 196, SEQ ID NO: 198, SEQ ID NO: 201, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208 to 211, SEQ ID NO: 215, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 240, SEQ ID NOs: 242 to 246, SEQ ID NOs: 248 to 254, SEQ ID NOs: 256 to 258, SEQ ID NOs: 260 to 270, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NOs: 275 to 288, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NOs: 293 to 296, SEQ ID NO: 298 and SEQ ID NO: 299.

In certain specific embodiments, the amino acid sequence of the antiviral peptide may be any one amino acid sequence selected from SEQ ID NO: 301 to SEQ ID NO: 315.

2.7 Effect of antiviral peptides

The antiviral peptide of the present disclosure causes damage to the outer membrane (or phospholipid bilayer) of the virus.

The antiviral peptide of the present disclosure can cause destruction of the outer membrane (or phospholipid bilayer) of the virus. Specifically, the antiviral peptide can cause destruction of the phospholipid bilayer of the virus by causing damage to the phospholipid bilayer of the virus.

The antiviral peptide of the present disclosure can cause a reduction or inactivation of the ability of a virus to infect a cell. Specifically, the antiviral peptide can cause a reduction or inactivation of the ability of a virus to infect a cell by causing damage (or destruction) of the phospholipid bilayer.

The antiviral peptide of the present disclosure can cause a reduction or inactivation of the ability of a virus to multiply (or replicate). Specifically, the antiviral peptide can cause a reduction or inactivation of the ability of a virus to multiply (or replicate) by causing damage (or destruction) of the phospholipid bilayer.

3. Antibody or fragment thereof

3.1 Overview

An antibody-peptide fusion protein, which is one embodiment of the present disclosure, comprises an antibody or an antibody fragment. The antibodies or antibody fragments disclosed herein are primarily used for their function of specifically recognizing (or specifically binding to) a target virus or a portion of a target virus of interest.

The antibody or fragment thereof disclosed in this specification can inhibit or interfere with the function of the target virus of interest. The antibody or fragment thereof disclosed in this specification can be a neutralizing antibody or fragment thereof against the target virus of interest. In this case, the antibody or fragment thereof having the neutralizing ability can function together with the antiviral peptide as an antiviral therapeutic agent.

In some embodiments, the antibody or fragment thereof refers to an antibody or fragment thereof capable of specifically binding to a virus. In some other embodiments, the antibody or fragment thereof may be an antibody or fragment thereof having binding affinity to a virus. In some other embodiments, the antibody or fragment thereof refers to an antibody or fragment thereof capable of specifically binding to a specific type of virus.

Below, the structure of antibodies and specific examples of viruses to which the antibodies can bind are described.

3.2 Structure of antibodies

As an example, the antibody used in the present invention may be a whole antibody against a virus. As a fragment of the antibody used in the present invention, a fragment of the whole antibody may be used.

The structure of the above antibody is divided into a heavy chain region and a light chain region depending on the chain type. The structure of the above antibody is divided into a fragment antigen-binding region (Fab region) and a fragment crystallizable region (Fc region) depending on the function of binding to an antigen. The structure of the above antibody is divided into a variable region and a constant region depending on the variability of the amino acid sequence. Other structures of the above antibody include a hinge portion and a tail portion. The heavy chain region and the light chain region are largely divided functionally into a fragment antigen-binding region (Fab region) and a fragment crystallizable region (Fc region). The Fab region is a portion including a portion that binds to an antigen (antigen-binding portion). The Fc region is a portion that can bind to an Fc receptor. The heavy chain region contains both a Fab region and an Fc region. The light chain region contains a Fab region.

The immunoglobulin domains included in the heavy chain region are positioned in the order of VH, CH1, CH2, and CH3 or VH, CH1, CH2, CH3, and CH4 from the N-terminal to the C-terminal. The immunoglobulin domains included in the light chain region are positioned in the order of VL and CL from the N-terminal to the C-terminal.

The heavy chain region and the light chain region are connected by a disulfide bond. Specifically, they are connected by a disulfide bond between CH1 of the heavy chain region and CL of the light chain region. The Fab region and the Fc region are connected by a hinge region. Specifically, the C-terminal portion of CH1 of the heavy chain region and the N-terminal portion of CH2 are connected by a hinge region.

The type of the above antibody fragment may be selected from F(ab), F(ab'), F(ab') ₂ , monospecific F(ab') ₂ , bispecific F(ab') ₂ , scFv, ScFv-Fc, and sdAb.

3.3 Target virus of antibodies

The virus to which the antibody or antibody fragment can bind may be a virus belonging to a family such as Bunyaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Poxviridae, Rhabdoviridae, Retroviridae, Togaviridae, or Herpesviridae. For example, the virus may be Sin Nombre Hantavirus belonging to the Bunyaviridae family; Coronavirus associated with various acute respiratory syndromes belonging to the Coronaviridae family; Ebola virus, Marburg virus in the Filoviridae family; West Nile virus, Yellow Fever virus, Dengue Fever virus, Hepatitis C virus in the Flaviviridae family; Hepatitis B virus in the Hepadnaviridae family; Herpes Simplex 1 virus, Herpes Simplex 2 virus in the Herpesviridae family; Influenza virus in the Orthomyxoviridae family; Smallpox virus, Vaccinia virus, Molluscum contagiosum virus, Monkeypox virus in the Poxviridae family; It may be at least one virus selected from the group consisting of Rabies virus belonging to the Rhabdoviridae family; Human Immunodeficiency virus (HIV) belonging to the Retroviridae family; Chikungunya virus belonging to the Togaviridae family; Pseudorabies virus, HHV virus belonging to the Herpesviridae family; and influenza virus belonging to the Orthomyxoviridae family.

In certain embodiments, it may be a coronavirus or a virus belonging thereto (e.g., SARS-CoV2).

3.4 Examples of target viruses for antibodies - coronaviruses

In a specific embodiment, the target virus targeted by (or capable of binding to) the antibody or fragment thereof may be a coronavirus. In another specific embodiment, the antibody or fragment thereof may be primarily used for its function of specifically recognizing (or specifically binding to) a coronavirus or a part of a coronavirus. At this time, the coronavirus may include at least one selected from Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. Preferably, the target virus targeted by the antibody or fragment thereof may be a betacoronavirus. In another specific embodiment, the antibody or fragment thereof may be used for its function of specifically recognizing (or specifically binding to) a betacoronavirus. At this time, the betacoronavirus may include at least one selected from Embecovirus, Merbecovirus, and Sarbecovirus. The above-mentioned embecovirus may include at least one selected from MHV (mouse hepatitis virus), OC43, and HKU1. The above-mentioned merbecovirus may include at least one selected from MERS-CoV, HKU4, and HKU5. The above-mentioned sarbecovirus may include at least one selected from SARS-CoV and SARS-CoV-2.

In a specific embodiment, the target virus of the antibody or fragment thereof may be SARS-COV-2. In this case, SARS-COV-2 means a variant of SARS-COV-2. Examples of variants of SARS-COV-2 include variants such as Alpha, Delta, Gamma, and Omicron. The antibody or fragment thereof may bind to SARS-COV-2 or an outer membrane protein of SARS-COV-2. In one specific example, the antibody or fragment thereof may bind to the spike protein of SARS-COV-2. In one specific example, the antibody or fragment thereof may inhibit or interfere with the function of SARS-COV-2. In one specific example, the antibody or fragment thereof may be a neutralizing antibody or fragment thereof for SARS-COV-2.

3.5 Example of target protein of antibody - Spike protein

When the target virus of the antibody or fragment thereof is a coronavirus, the antibody or fragment thereof can specifically bind to the spike protein (Spike protein, Spike Glycoprotein, or Peplomer) of the coronavirus.

The spike protein plays an important role in allowing the coronavirus to invade cells (e.g., human cells). In one specific example, the spike protein interacts with the ACE2 receptor (Angiotensin-Converting Enzyme 2 receptor) of the cell, allowing the coronavirus to invade human cells.

The spike protein is one of the four largest structural proteins that make up the coronavirus. The spike protein contains about 1100 to 1500 amino acid residues. The C-terminal portion of the spike protein is inside the coronavirus, and the N-terminal portion is outside the coronavirus. A portion of the spike protein located in the membrane portion of the virus has a transmembrane helix structure. The spike protein is largely composed of an S1 domain (or S1 subunit) and an S2 domain (or S2 subunit). The S1 domain binds to the ACE2 receptor of human cells and is involved in the function of the coronavirus attaching to human cells. The S2 domain is involved in the membrane fusion process in which the coronavirus attaches to the membrane of the human cell after the coronavirus attaches to the human cell.

In one specific example, the antibody or fragment thereof may target the S1 domain of the spike protein. At this time, the antibody or fragment thereof may bind to the S1 domain of the spike protein. At this time, the antibody or fragment thereof is not limited in type as long as it has target specificity for the S1 domain. A known antibody or fragment thereof targeting the S1 domain may be used. The S1 domain is generally known as a region in which viruses are easily mutated, and therefore, the antibody or fragment thereof that may be used in the present invention may be selected according to the type of mutation in the S1 domain.

3.6 Example of antibody target protein - S2 domain

In a preferred specific embodiment, the antibody or fragment thereof may target the S2 domain of the spike protein of a coronavirus. At this time, the antibody or fragment thereof may specifically bind to the S2 domain of the spike protein. At this time, the antibody or fragment thereof is not limited in type as long as it has target specificity for the S2 domain. A known antibody or fragment thereof may be used as the antibody or fragment targeting the S2 domain.

At this time, the amino acid sequence of the S2 domain is relatively less mutated and has a high level of sequence conservation compared to the S1 domain. Therefore, in utilizing the target specificity of the antibody, there is an advantage in that the same antibody or the same fragment thereof targeting the S2 domain can be used for various mutants. That is, even if various types of mutants occur in the coronavirus, the antibody or fragment thereof can have a significant level of binding affinity for the S2 domain of the mutant.

3.7 Examples of antibody types - Antibodies to SARS-CoV-2

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection spreads worldwide, therapeutics to treat SARS-CoV-2 infection have been developed. One of them is an antibody therapeutic with neutralizing ability against SARS-CoV-2. In this regard, antibodies that can bind to the S1 domain of the spike protein of SARS-CoV-2 were initially developed. However, various variants of SARS-CoV-2 have occurred, and as mutations occurred, many mutations also occurred in the S1 domain of the spike protein of SARS-CoV-2. Therefore, the antibodies targeting the S1 domain of the spike protein of SARS-CoV-2 that were initially developed had a reduced ability to bind to the S1 domain of the spike protein of the variants, and were not suitable for use as therapeutics.

However, in many cases, the amino acid sequence of the S2 domain of the spike protein of each variant is well conserved in various variants of SARS-CoV-2. For example, the amino acid sequence of the S2 domain is conserved by more than about 99% even when SARS-CoV-2 is mutated in various ways (alpha, gamma, omicron, etc.).

Accordingly, antibodies targeting the S2 domain of the spike protein, which is highly conserved (and has a low mutation level) even in mutants, have begun to be developed. However, antibodies targeting the S2 domain of the spike protein, such as CC40.8, DH1047, and S2P6, have been developed, but they have been reported to have low neutralizing power and thus low therapeutic efficacy.

Therefore, the present specification discloses an antibody-peptide fusion protein comprising an antibody and an antiviral peptide targeting the S2 domain of the spike protein, wherein the antibody-peptide fusion protein can have a strong antiviral effect with high "target specificity" against a virus comprising the S2 domain. The antibody-peptide fusion protein has the advantage of solving problems caused by various mutations by targeting the conserved S2 domain of the spike protein and having a high therapeutic effect by utilizing a peptide having a strong antiviral effect.

In addition, since the S2 domain is highly conserved in betacoronaviruses, an antibody-peptide fusion protein targeting the S2 domain has the advantage of being able to be used as an effective therapeutic agent even if a pandemic occurs in the future due to a new virus belonging to the betacoronavirus family.

A specific example of the antibody or fragment thereof for the above SARS-CoV-2 is CV3-25 (or 7NAB) or a fragment thereof. A specific description of the CV3-25 (or 7NAB) is described in a known literature (Hurlburt, N.K., Homad, L.J., Sinha, I. et al. Structural definition of a pan-sarbecovirus neutralizing epitope on the spike S2 subunit. Commun Biol 5, 342 (2022)), but the CV3-25 (or 7NAB) is not limited to the interpretation described therein.

In a particular embodiment, the antibody comprises a light chain and a heavy chain, the amino acid sequence of the light chain may be SEQ ID NO: 318, and the amino acid sequence of the heavy chain may be SEQ ID NO: 319.

In a particular embodiment, the antibody comprises a light chain and a heavy chain, the amino acid sequence of the light chain may be SEQ ID NO: 320, and the amino acid sequence of the heavy chain may be SEQ ID NO: 321.

4 linker

The antibody-peptide fusion protein of the present disclosure may include a linker. In this case, the antibody-peptide fusion protein of the present disclosure may have a structure of "antibody-linker-antiviral peptide" or "antibody fragment-linker-antiviral peptide." The linker has a functional role of fusing, connecting, binding, or conjugating the antibody and the antiviral peptide.

The linker may not affect the structure, function, or properties of the antibody or fragment thereof. In one specific example, even if the linker is linked to the antibody or fragment thereof, the antibody or fragment thereof may exhibit a function (or property) of recognizing a target virus, or a function (or property) of specifically binding to a target virus. In another specific example, even if the linker is linked to the antibody or fragment thereof, the physicochemical properties or characteristics of the antibody or fragment thereof may be significantly maintained compared to before the linker is linked.

The linker may not affect the structure, function or properties of the antiviral peptide. In one specific example, even if the linker is linked to the antiviral peptide, the antiviral peptide may exhibit an antiviral or virus-killing effect. In another specific example, even if the linker is linked to the antiviral peptide, the physicochemical properties or properties of the antiviral peptide may be significantly maintained compared to before the linker is linked.

The above linker may be a known linker well known in the art. Or, the linker may be designed in various ways considering the structure of the antibody and the antiviral peptide.

In some embodiments, the linker can be a cleavable linker, a non-cleavable linker, a peptide linker, a compound-based linker, a hydrophilic linker, or a hydrophobic linker.

The above cleavable linker is a linker designed to release an antiviral peptide in the vicinity of a target virus. For example, the cleavable linker may be a linker that can be cleaved by an enzyme in vivo or in a cell or a virus-associated enzyme.

Preferably, the linker may be a peptide linker, wherein the peptide linker comprises a polypeptide. The number of amino acid residues constituting the polypeptide included in the above peptide linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, or a range comprised of two values selected from the above values (e.g., 5 to 20).

In certain embodiments, the linker can comprise a polypeptide having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 317). Alternatively, the linker can be a polypeptide having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 317).

5. Method for producing antibody-peptide fusion protein of the present disclosure

In one aspect of the present disclosure, a method for producing an antibody-peptide fusion protein is disclosed.

The above production method may include a step of transfecting a production cell with a vector and/or a virus comprising a nucleic acid sequence encoding an antibody linked to an antiviral peptide. Alternatively, the production method may include a step of transfecting a production cell with a vector and/or a virus comprising a nucleic acid sequence encoding an antibody heavy chain portion linked to an antiviral peptide. Alternatively, the production method may include a step of transfecting a production cell with a vector and/or a virus comprising a nucleic acid sequence encoding an antibody heavy chain portion linked to an antiviral peptide.

In one specific example, the vector may be pEG10 BacMam. In one specific example, the virus may be baculovirus. In one specific example, the production cell may be expi293F cell.

In one specific example, the above production method may further include a method of purifying an antibody-peptide fusion protein produced by the production cell.

6. Uses of the antibody-peptide fusion proteins of the present disclosure

6.1 Overview of Uses of Antibody-Peptide Fusion Proteins of the Present Disclosure

According to one aspect of the present disclosure, the antibody-peptide fusion protein of the present disclosure can be used to cause destruction or damage of the lipid bilayer of a virus. The destruction or damage of the lipid bilayer can reduce the infectivity of the virus, so that the effect of preventing or treating the virus infection (or related disease) can be expected. That is, the antibody-peptide fusion protein can be used as an effective therapeutic agent for the virus infection (or related disease) by inducing destruction or damage of the lipid bilayer. Here, the virus is a virus having an outer coat (or membrane) of a phospholipid bilayer.

Various embodiments in which antibody-peptide fusion proteins are used in connection with treating viral infections (or related diseases) are described below.

6.2 Target Disease

A target disease for which the antibody-peptide fusion protein of the present disclosure can be used for therapeutic purposes is a viral infection (or related disease).

In some embodiments, the virus that is the target of lipid bilayer disruption (or damage) or target of treatment of the antibody-peptide fusion protein can be a virus belonging to a family such as Bunyaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Poxviridae, Rhabdoviridae, Retroviridae, Togaviridae, or Herpesviridae. For example, the virus is Sin Nombre Hantavirus belonging to the Bunyaviridae family; Coronaviruses, which are responsible for various acute respiratory syndromes, belong to the Coronaviridae family; Ebola virus and Marburg virus, which belong to the Filoviridae family; West Nile virus, Yellow Fever virus, Dengue Fever virus, and Hepatitis C virus, which belong to the Flaviviridae family; Hepatitis B virus, which belongs to the Hepadnaviridae family; Herpes Simplex 1 virus and Herpes Simplex 2 virus, which belong to the Herpesviridae family; Influenza virus, which belongs to the Orthomyxoviridae family; The virus may be at least one virus selected from the group consisting of Smallpox virus, Vaccinia virus, Molluscumcontagiosumvirus, Monkeypox virus belonging to the Poxviridae family; Rabies virus belonging to the Rhabdoviridae family; Human Immunodeficiency virus (HIV) belonging to the Retroviridae family; Chikungunya virus belonging to the Togaviridae family; Pseudorabies virus, HHV virus belonging to the Herpesviridae family; and influenza virus belonging to the Orthomyxoviridae family. In certain embodiments, the virus may be a Betacoronavirus or a virus belonging thereto.

In some embodiments, the target disease may be a respiratory disease due to a viral infection. In certain embodiments, the target disease may be a respiratory disease due to a betacoronavirus infection or coronavirus disease-19 (COVID-19).

6.3 Pharmaceutical composition comprising an antibody-peptide fusion protein

6.3.1 Overview of Pharmaceutical Compositions

According to one aspect of the present disclosure, a pharmaceutical composition for treating or preventing a viral disease is disclosed. The pharmaceutical composition comprises a therapeutically effective amount of an antibody-peptide fusion protein. In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable adjuvant. In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and an adjuvant. Herein, the pharmaceutically acceptable carrier may be for appropriate formulation of the pharmaceutical composition for treating or preventing a viral disease.

At this time, the antibody-peptide fusion protein refers to the antibody-peptide fusion protein disclosed by the present disclosure.

At this time, the viral disease means a disease caused by infection with a virus disclosed in Section <<6.2 Target Disease>> of the present disclosure.

6.3.2 Examples of Formulations of Pharmaceutical Compositions

As a method for formulating the pharmaceutical composition of the present disclosure, a known formulation method used for antibodies, peptides or antibody-peptide fusion proteins can be used.

In one embodiment, the pharmaceutical composition can be formulated as a troches, lozenges, tablets, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs. In another embodiment, the pharmaceutical composition can be formulated as an injection, a suppository, a powder for respiratory inhalation, an aerosol for spraying, an ointment, a powder for application, an oil or a cream. In yet another embodiment, the pharmaceutical composition can be formulated as an injection. Specifically, a therapeutically effective amount of an antibody-peptide fusion protein can be mixed with a stabilizer or a buffer in water to prepare a solution or suspension, which can be formulated for unit dose in ampoules or vials. In yet another embodiment, the pharmaceutical composition can be formulated as an aerosol by mixing a propellant or the like with an additive to prepare a water-dispersed concentrate or a wet powder. In another embodiment, when the pharmaceutical composition is formulated for transdermal use, an ointment, cream, powder for application, oil, external skin preparation, etc. can be prepared by adding animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. as a carrier to a therapeutically effective amount of the antibody-peptide fusion protein.

6.3.3 Examples of pharmaceutically acceptable carriers

The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc., and may further include other common additives such as antioxidants and buffers as needed. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, the compositions can be preferably formulated according to each ingredient using the methods disclosed in Remington's Pharmaceutical Sciences (19th edition, 1995). In one embodiment, the pharmaceutical composition may include a pharmaceutically acceptable carrier, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; an excipient such as dicalcium phosphate; a disintegrant such as corn starch or sweet potato starch; a lubricant such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax; a sweetener; a flavoring agent; a syrup; a liquid carrier such as a fatty oil; a sterile aqueous solution; propylene glycol; polyethylene glycol; an injectable ester such as ethyl oleate; a suspending agent; an emulsion; a freeze-dried preparation; an external preparation; a stabilizer; a buffering agent; an animal oil; a vegetable oil; a wax; paraffin; a starch; tragacanth; a cellulose derivative; a polyethylene glycol; a silicone; a bentonite; a silica; talc; zinc oxide, or a suitable combination thereof.

6.4 Treatment using antibody-peptide fusion proteins

6.4.1 Overview of Treatment Methods

According to one aspect of the present disclosure, a method for treating a viral disease is disclosed. The method for treating a viral disease comprises administering to a subject a pharmaceutical composition for treating the viral disease using an appropriate administration method, an appropriate administration regimen, and an appropriate administration dosage. In this case, the subject to be administered may mean an individual infected with a virus. In this case, the subject to be administered may be a human or a non-human animal.

The pharmaceutical composition for treating the above viral disease is a pharmaceutical composition disclosed in detail in Section <<6.3 Pharmaceutical Composition Comprising Antibody-Peptide Fusion Protein>> of the present disclosure.

The above viral disease means a disease caused by infection with a virus disclosed in Section <<6.2 Target Disease>> of the present disclosure.

6.4.2 Example of administration method

The treatment method of the present disclosure may be to administer to a subject an appropriately formulated therapeutically effective antibody-peptide fusion protein by an appropriate administration method. In one embodiment, the treatment method may be to administer an appropriately formulated therapeutically effective antibody-peptide fusion protein by one selected from oral administration, parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, and subcutaneous administration.

6.4.3 Dosage examples

The treatment method of the present disclosure may be to administer to a subject an appropriately formulated therapeutically effective antibody-peptide fusion protein by an appropriate administration method. In one embodiment, the administration dosage may be about 0.01 mg to 1000 mg per kg of body weight of the subject, based on the antibody-peptide fusion protein.

6.4.4 Dosage cycle example

The treatment method of the present disclosure may be to administer to a subject an appropriately formulated therapeutically effective antibody-peptide fusion protein by an appropriate administration method. In one embodiment, the pharmaceutical composition comprising the antibody-peptide fusion protein at the appropriate dosage may be administered once a day. In another example, the administration cycle may be to administer the pharmaceutical composition comprising the antibody-peptide fusion protein at the appropriate dosage twice or more a day in divided doses. In another example, the administration cycle may be to administer the pharmaceutical composition comprising the antibody-peptide fusion protein at the appropriate dosage at intervals of 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 1 week, 2 weeks, 1 month, and/or 3 months.

6.5 Therapeutic Uses of Antibody-Peptide Fusion Proteins

According to one aspect of the present disclosure, a use of an antibody-peptide fusion protein for treating a viral disease is disclosed. Here, the antibody-peptide fusion protein means an antibody-peptide fusion protein disclosed by the present disclosure. Here, the viral disease means a disease caused by infection with a virus disclosed in section <<6.2 Target Diseases>> of the present disclosure.

6.6 Use of Antibody-Peptide Fusion Proteins in the Manufacturing of Therapeutic Products

According to one aspect of the present disclosure, a use of an antibody-peptide fusion protein for the manufacture of a medicament for treating a viral disease is disclosed. Here, the antibody-peptide fusion protein means an antibody-peptide fusion protein disclosed by the present disclosure. Here, the viral disease means a disease caused by infection with a virus disclosed in section <<6.2 Target Diseases>> of the present disclosure.

Possible embodiments of the invention

Hereinafter, possible embodiments of the invention provided in this specification are listed. The following embodiments provided in this paragraph are merely examples of the invention. Therefore, the invention provided in this specification should not be interpreted as being limited to the following embodiments. The brief descriptions described with the embodiment numbers are also only for the convenience of distinguishing between each embodiment and should not be interpreted as limitations to the invention disclosed in this specification.

Antibody-peptide fusion protein

Example 1, Antibody-Peptide Fusion Protein

Antibody-peptide fusion proteins comprising:

an antibody or fragment thereof; and

Antiviral peptide.

Antiviral peptides contained in antibody-peptide fusion proteins

Example 2, antiviral peptide 1

An antibody-peptide fusion protein, characterized in that the number of amino acid residues constituting the antiviral peptide in Example 1 is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36.

Example 3, antiviral peptide 2

In any one of embodiments 1 to 2, the antiviral peptide is composed of hydrophilic amino acid residues and hydrophobic amino acid residues,

An antibody-peptide fusion protein, characterized in that the number of hydrophilic amino acid residues included in the antiviral peptide and the number of hydrophobic amino acid residues included in the antiviral peptide are the same, or the number of hydrophilic amino acid residues may be 1, 2, 3, 4, or 5 more than the number of hydrophobic amino acid residues, or the number of hydrophobic amino acid residues may be 1, 2, 3, 4, or 5 more than the number of hydrophilic amino acid residues.

Example 4, antiviral peptide 3

In Example 3, an antibody-peptide fusion protein characterized in that the hydrophilic amino acid residues may include one or more selected from the following:

Arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), lysine (Lys, K), serine (Ser, S), and threonine (Thr, T).

Example 5, antiviral peptide 4

An antibody-peptide fusion protein, characterized in that in any one of embodiments 3 to 4, the hydrophobic amino acid residues may include one or more selected from the following:

Alanine (Ala, A), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), proline (Pro, P), valine (Val, V), phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W).

Example 6, antiviral peptide 5

An antibody-peptide fusion protein according to any one of Examples 1 to 5, wherein the antiviral peptide is capable of forming an alpha helix (α-helix) structure.

Example 7, antiviral peptide 6

An antibody-peptide fusion protein characterized in that, in any one of Examples 1 to 6, the antiviral peptide is capable of forming an alpha helix (α-helix) structure when reacting with a lipid membrane.

Example 8, antiviral peptide 7

An antibody-peptide fusion protein according to any one of Examples 1 to 7, wherein the antiviral peptide is an amphipathic peptide.

Example 9, antiviral peptide 8

An antibody-peptide fusion protein having amphipathic characteristics when the antiviral peptide forms an alpha helical structure in any one of Examples 1 to 8.

Example 10, antiviral peptide 9

An antibody-peptide fusion protein according to any one of Examples 1 to 9, wherein the antiviral peptide is capable of causing damage to the outer membrane (or phospholipid bilayer) of the virus.

Example 11, antiviral peptide 10

An antibody-peptide fusion protein according to any one of Examples 1 to 10, wherein the antiviral peptide is capable of causing destruction of the outer membrane (or phospholipid bilayer) of the virus.

Example 12, antiviral peptide 11

An antibody-peptide fusion protein according to any one of Examples 1 to 11, wherein the antiviral peptide is capable of causing a decrease or inactivation of the ability of a virus to infect a cell.

Example 13, antiviral peptide 12

An antibody-peptide fusion protein according to any one of Examples 1 to 12, wherein the antiviral peptide is capable of causing a reduction or inactivation of the ability of a virus to proliferate (or replicate).

Example 14, antiviral peptide 13

An antibody-peptide fusion protein, characterized in that in any one of Examples 10 to 13, the virus is a virus belonging to a family such as Bunyaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Poxviridae, Rhabdoviridae, Retroviridae, Togaviridae, or Herpesviridae, Betacoronavirus, or SARS-CoV2.

Antibodies contained in antibody-peptide fusion proteins

Example 15, Antibody 1

An antibody-peptide fusion protein, characterized in that in any one of Examples 1 to 14, the antibody or fragment thereof is an anti-viral antibody or an antibody capable of binding to a virus.

Example 16, Antibody 2

In any one of Examples 1 to 15,

An antibody-peptide fusion protein, characterized in that the antibody or fragment thereof can specifically recognize (or specifically bind to) a target virus or a part of the target virus.

Example 17, Antibody 3

In any one of Examples 1 to 16,

An antibody-peptide fusion protein characterized in that the antibody or fragment thereof can bind to a virus belonging to a family such as Bunyaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Poxviridae, Rhabdoviridae, Retroviridae, Togaviridae, or Herpesviridae.

Example 18, Antibody 4

In any one of Examples 1 to 17,

The above antibody or fragment thereof can bind to coronavirus,

At this time, the coronavirus may include at least one selected from Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus.

At this time, the betacoronavirus may include at least one selected from embecovirus, merbecovirus, and sarbecovirus,

An antibody-peptide fusion protein, characterized in that at this time, the sarbecovirus can include at least one selected from SARS-CoV and SARS-CoV-2.

Example 19, Antibody 5

In any one of Examples 1 to 18,

An antibody-peptide fusion protein, characterized in that the antibody or fragment thereof can bind to the Spike protein of a coronavirus.

Example 20, antibody 6

In any one of Examples 1 to 19,

An antibody-peptide fusion protein, characterized in that the antibody or fragment thereof can bind to the S2 domain of the Spike protein.

Linker of antibody-peptide fusion protein

Example 21, Connection

In any one of Examples 1 to 20,

An antibody-peptide fusion protein characterized in that the antibody-peptide fusion protein is a single construct formed by linking, fusion, binding or conjugating the antibody and the antiviral peptide through at least one bond selected from a covalent bond, an ionic bond, a hydrogen bond, a disulfide bond, a peptide bond and a glycosidic bond.

Example 22, Linker 1

In any one of Examples 1 to 21,

The above antibody-peptide fusion protein comprises a linker,

An antibody-peptide fusion protein characterized in that the antibody or a fragment thereof and the antiviral peptide are linked, fused, bound or conjugated through the linker.

Example 23, Linker 2

In Example 22,

An antibody-peptide fusion protein, characterized in that the linker is a cleavable linker, a non-cleavable linker, a peptide linker, a compound-based linker, a hydrophilic linker or a hydrophobic linker.

Example 24, Linker 3

In any one of Examples 22 to 23,

An antibody-peptide fusion protein, characterized in that the linker comprises a polypeptide consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acid residues.

Example 25, Linker 4

In any one of Examples 22 to 24,

An antibody-peptide fusion protein, characterized in that the linker comprises a polypeptide having an amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 317).

Structure of an antibody-peptide fusion protein

Example 26, Structure 1

In any one of Examples 1 to 25,

The above antibody-peptide fusion protein has a structure represented by the following chemical formula I or chemical formula II:

[Chemical Formula I]

Ab-P

[Chemical Formula II]

Ab-L-P

In the above chemical formulas I and II, Ab means the antibody or a fragment thereof,

In the above chemical formulas I and II, P refers to the antiviral peptide,

An antibody-peptide fusion protein, characterized in that in the above chemical formula II, L represents the linker.

Example 27, Structure 2

In any one of Examples 1 to 26,

An antibody-peptide fusion protein, characterized in that the position at which the antiviral peptide or the linker is linked to the antibody is any one of the light chain region, heavy chain region, variable region, constant region, Fab region, Fc region, VH, VC, CH1, CH2, CH3, or CL region of the antibody.

Example 28, Structure 3

In any one of Examples 1 to 27,

An antibody-peptide fusion protein, characterized in that the position at which the antiviral peptide or the linker is linked to the antibody is any one amino acid residue included in the antibody, the N-terminus of the light chain of the antibody, the C-terminus of the light chain of the antibody, the N-terminus of the heavy chain of the antibody, or the C-terminus of the heavy chain of the antibody.

Example 29, Structure 4

In any one of Examples 1 to 28,

An antibody-peptide fusion protein, characterized in that the position at which the antibody or the linker is linked to the antiviral peptide is any one amino acid residue contained in the antiviral peptide, the N-terminus of the antiviral peptide, or the C-terminus of the antiviral peptide.

Effects of antibody-peptide fusion proteins

Example 30, Effect 1

In any one of Examples 1 to 29,

An antibody-peptide fusion protein characterized in that the antibody-peptide fusion protein can cause damage to the outer membrane (or phospholipid bilayer) of a virus.

Example 31, Effect 2

An antibody-peptide fusion protein according to any one of Examples 1 to 30, characterized in that the antibody-peptide fusion protein is capable of causing destruction of the outer membrane (or phospholipid bilayer) of the virus.

Example 32, Effect 3

An antibody-peptide fusion protein according to any one of Examples 1 to 31, characterized in that the antibody-peptide fusion protein can cause a decrease or inactivation of the ability of a virus to infect a cell.

Example 33, Effect 4

An antibody-peptide fusion protein according to any one of embodiments 1 to 32, characterized in that the antibody-peptide fusion protein can cause a reduction or inactivation of the ability of a virus to proliferate (or replicate).

Example 34, Effect 5

An antibody-peptide fusion protein, characterized in that in any one of Examples 1 to 33, the antibody-peptide fusion protein can specifically recognize (or specifically bind to) a target virus or a portion of a target virus.

Uses of antibody-peptide fusion proteins

Example 35, target disease

A viral disease, which means a disease caused by a virus infection.

The above viral diseases are viruses belonging to the families of Bunyaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Poxviridae, Rhabdoviridae, Retroviridae, Togaviridae, or Herpesviridae, or coronaviruses, Alphacoronaviruses, Betacoronaviruses, Gammacoronaviruses, Deltacoronaviruses, embecoviruses, and Merbecoviruses. It may be a disease caused by infection with merbecovirus, sarbecovirus, SARS-CoV or SARS-CoV-2.

Example 36, Pharmaceutical composition 1 comprising an antibody-peptide fusion protein

A pharmaceutical composition for the treatment of a viral disease comprising a therapeutically effective amount of an antibody-peptide fusion protein,

The above antibody-peptide fusion protein is an antibody-peptide fusion protein of any one of Examples 1 to 34,

The above viral disease is the viral disease of Example 35.

Example 37, Pharmaceutical composition 2 comprising an antibody-peptide fusion protein

In Example 36, a pharmaceutical composition for treating a viral disease, characterized in that the pharmaceutical composition for treating a viral disease further comprises a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable adjuvant.

Example 38, therapeutic method using antibody-peptide fusion protein

Methods for treating viral diseases, including:

Administering to a subject infected with a virus a pharmaceutical composition comprising a therapeutically effective amount of an antibody-peptide fusion protein;

At this time, the antibody-peptide fusion protein is an antibody-peptide fusion protein of any one of Examples 1 to 34,

The above viral disease is the viral disease of Example 35.

Example 39, Therapeutic use of antibody-peptide fusion proteins

For use in the treatment of a viral disease of an antibody-peptide fusion protein of any one of Examples 1 to 34,

The above viral disease is the viral disease of Example 35.

Example 40 Use of antibody-peptide fusion proteins in the manufacture of therapeutic agents

For use in the manufacture of a pharmaceutical product for treating a viral disease, the antibody-peptide fusion protein of any one of Examples 1 to 34,

The above viral disease is the viral disease of Example 35.

Hereinafter, the invention provided by this specification will be described in more detail through experimental examples and examples. It will be obvious to those skilled in the art that these experimental examples are only intended to illustrate the content disclosed by this specification, and the scope of the content disclosed by this specification is not to be construed as being limited by these experimental examples.

Experimental Example 1 Production and Purification of Antibody-Peptide Fusion Proteins

The present inventors attempted to confirm the antiviral effect of a fusion protein of an antibody capable of binding to a virus and an antiviral peptide. At this time, the antibodies used were 7NAB antibody and 7TCQ antibody, and both 7NAB antibody and 7TCQ antibody are antibodies that can specifically bind to the S2 domain of the spike protein of the SARS-CoV2 virus. At this time, the linked antiviral peptides were DS-05 peptide (SEQ ID NO: 305) and YAP-1 peptide (SEQ ID NO: 1).

Experimental Example 1.1 Plasmid Cloning

A nucleic acid encoding the heavy chain of 7NAB antibody linked to a nucleic acid encoding mCherry fluorescent protein (#14023 in Table 3), a nucleic acid encoding the heavy chain of 7TCQ antibody linked to a nucleic acid encoding mCherry fluorescent protein (#14252 in Table 3), a nucleic acid encoding the light chain of 7NAB antibody linked to a nucleic acid encoding mCherry fluorescent protein (#14022 in Table 3), and a nucleic acid encoding the light chain of 7TCQ antibody linked to a nucleic acid encoding mCherry fluorescent protein (#14235 in Table 3) were each cloned into the pEG10 BacMam vector.

In addition, the inventors of the present invention attempted to produce an antibody in which an antiviral peptide is fused to the C-terminus of the heavy chain of the antibody. To this end, a nucleic acid (#14065 in Table 3) in which a nucleic acid encoding a DS-05 peptide and a nucleic acid encoding an mCherry fluorescent protein are linked to a nucleic acid encoding a heavy chain of a 7NAB antibody; a nucleic acid (#14233 in Table 3) in which a nucleic acid encoding a DS-05 peptide and a nucleic acid encoding an mCherry fluorescent protein are linked to a nucleic acid encoding a heavy chain of a 7TCQ antibody; and a nucleic acid (#14234 in Table 3) in which a nucleic acid encoding a YAP-1 peptide and a nucleic acid encoding an mCherry fluorescent protein are linked to a nucleic acid encoding a heavy chain of a 7TCQ antibody were cloned into the pEG10 BacMam vector, respectively.

Table 2 shows the amino acid sequences of 7NAB antibody, 7TCQ antibody, mCherry fluorescent protein, DS-05 peptide, and YAP-1 peptide.

Table 3 shows the amino acid sequences encoded by the nucleic acids cloned into the vector.

Figure 1 shows the structures of the fusion proteins encoded by #14023, #14252, #14022, #14235, #14065, #14233 and #14234, respectively, and the positions cleaved in the fusion proteins by “Experimental Example 1.4 Protein purification process”.

protein Amino acid sequence 7NAB antibody light chain mefglswvflvallrgvqcEIVLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNSFPYTFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 318) 7NAB antibody heavy chain mefglswvflvallrgvqcEVQLVESGAEVKKPGESLKISCKGSGYTFTRYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGHVTISADKSISTAYLQWNSLKASDTAMYYCARLPQYCSNGVCQRWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 319) 7TCQ antibody light chain mefglswvflvallrgvqcQIVLTQSPAIMSASPGEKVTISCSATSSVSYIYWYQQRPGSSPKPWIYRTSNLASGVPVRFSGSGSGTSYSLTISNMEAEDAATYYCQQYQSYPPTFGAGTKLELKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMS STLTLTKDEYERHNSYTCEATHKTSTSSPIVKSFNRNEC (SEQ ID NO: 320) 7TCQ antibody heavy chain mefglswvflvallrgvqcEVQFQQSGTVLARPGASVKMSCKASGYTFTNYWIHWVKQRPGQGLEYIGGTYPGNGDTTYNQKFKGKAKVTAVTPTSTAYMDLSSLTNEDSAVYYCTRTGSYFDYWGQGTTLTVSSASTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVLTTWNSGSLSSGVHTFPAV LQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIE SKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLLSLSLGKHHHHHH (SEQ ID NO: 321) mCherry protein SNSLEVLFQGPTAAAAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKK PVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGGSLVPRGSSAWSHPQFEKSAPSRLEEELRRRLTE (SEQ ID NO: 322) DS-05 Peptide DWVDCFWSWLIKVIDRVS (SEQ ID NO: 305) YAP-1 peptide ESLKNFWESLWKFFTHFF (SEQ ID NO: 1)

fusion protein Amino acid sequence #14022(7NAB antibody light chain_mCherry) mefglswvflvallrgvqcEIVLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNSFPYTFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SNSLEVLFQGPTAAAAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKK PVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGGSLVPRGSSAWSHPQFEKSAPSRLEEELRRRLTE (SEQ ID NO: 323) #14023(7NAB antibody heavy chain_mCherry) mefglswvflvallrgvqcEVQLVESGAEVKKPGESLKISCKGSGYTFTRYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGHVTISADKSISTAYLQWNSLKASDTAMYYCARLPQYCSNGVCQRWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SNSLEVLFQGPTAAAAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKK PVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGGSLVPRGSSAWSHPQFEKSAPSRLEEELRRRLTE (SEQ ID NO: 324) #14065(7NAB antibody heavy chain_DS-05_mCherry) mefglswvflvallrgvqcEVQLVESGAEVKKPGESLKISCKGSGYTFTRYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGHVTISADKSISTAYLQWNSLKASDTAMYYCARLPQYCSNGVCQRWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVESKYGPPCPS CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLLSLSLGKGGGGSGGGGSGGGGSDWVDCFWSWLIKVIDRVS
SNSLEVLFQGPTAAAAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKK PVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGGSLVPRGSSAWSHPQFEKSAPSRLEEELRRRLTE (SEQ ID NO: 325) #14235(7TCQ antibody light chain_mCherry) mefglswvflvallrgvqcQIVLTQSPAIMSASPGEKVTISCSATSSVSYIYWYQQRPGSSPKPWIYRTSNLASGVPVRFSGSGSGTSYSLTISNMEAEDAATYYCQQYQSYPPTFGAGTKLELKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMS STLTLTKDEYERHNSYTCEATHKTSTSSPIVKSFNRNEC
SNSLEVLFQGPTAAAAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKK PVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGGSLVPRGSSAWSHPQFEKSAPSRLEEELRRRLTE (SEQ ID NO: 326) #14252(7TCQ antibody heavy chain_mCherry) mefglswvflvallrgvqcEVQFQQSGTVLARPGASVKMSCKASGYTFTNYWIHWVKQRPGQGLEYIGGTYPGNGDTTYNQKFKGKAKVTAVTPTSTAYMDLSSLTNEDSAVYYCTRTGSYFDYWGQGTTLTVSSASTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVLTTWNSGSLSSGVHTFPAV LQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIE SKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLLSLSLGKHHHHHH
SNSLEVLFQGPTAAAAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKK PVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGGSLVPRGSSAWSHPQFEKSAPSRLEEELRRRLTE (SEQ ID NO: 327) #14233(7TCQ antibody heavy chain_DS-05_mCherry) mefglswvflvallrgvqcEVQFQQSGTVLARPGASVKMSCKASGYTFTNYWIHWVKQRPGQGLEYIGGTYPGNGDTTYNQKFKGKAKVTAVTPTSTAYMDLSSLTNEDSAVYYCTRTGSYFDYWGQGTTLTVSSASTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVLTTWNSGSLSSGVHTFPAV LQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIESKYGPPCPSCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLLSLSLGKHHHHHHGGGGSGGGGSGGGGSDWVDCFWSWLIKVIDRVS
SNSLEVLFQGPTAAAAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKK PVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGGSLVPRGSSAWSHPQFEKSAPSRLEEELRRRLTE (SEQ ID NO: 328) #14234(7TCQ antibody heavy chain_YAP-1_mCherry) mefglswvflvallrgvqcEVQFQQSGTVLARPGASVKMSCKASGYTFTNYWIHWVKQRPGQGLEYIGGTYPGNGDTTYNQKFKGKAKVTAVTPTSTAYMDLSSLTNEDSAVYYCTRTGSYFDYWGQGTTLTVSSASTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVLTTWNSGSLSSGVHTFPAV LQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIESKYGPPCPSCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGKHHHHHHGGGGSGGGGSGGGGSESLKNFWESLWKFFTHFF
SNSLEVLFQGPTAAAAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKK PVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGGSLVPRGSSAWSHPQFEKSAPSRLEEELRRRLTE (SEQ ID NO: 329)

Experimental Example 1.2 Production of recombinant baculovirus

Each nucleic acid (#14023, #14252, #14022, #14235, #14065, #14233, and #14234) encoding the amino acid sequence in Table 3 was transformed into the DH10Bac strain, and the transformed cells were plated on an LB (Luria broth) agar plate supplemented with 50 μg/ml of kanamycin, 7 μg/ml of gentamicin, 10 μg/ml of tetracycline, 0.17 mM of IPTG (isopropyl β-D-1-thiogalactopyranoside), and 20 mg/ml of Bluo-gal, and cultured at 37°C for 24 to 48 hours. After culture, white colonies were selected from the plate, streaked, and then selected once more. Among the selected colonies, those that successfully recombined with the baculovirus genome were selected by taq PCR with the M13 sequence primer. The selected colonies were inoculated onto LB medium supplemented with 50 μg/ml kanamycin, 7 μg/ml gentamicin, and 10 μg/ml tetracycline and cultured overnight at 37°C. The recombinant viral genome was purified according to the QIAGEN plasmid purification protocol. The virus was produced by transfecting the viral genome into Sf9 cells using Cellfectin reagent according to the protocol provided by the manufacturer.

Experimental Example 1.3 Transduction of Expi293F cells

The produced virus was used for protein expression in Expi293F cells. Expi293 medium was used to culture Expi293F cells, which were cultured at 37°C, 125 rpm, and 8% _CO2 . Cell passage was performed every 3–4 days when the number of cells reached 3–5 x ¹⁰⁶ viable cells/mL. Expi293F cells prepared at a concentration of 3 x ¹⁰⁶ viable cells/mL were infected with 10% (V/V) of the total culture medium, and then cultured at 37°C, 8% _CO2 for 18–22 hours. Afterwards, an enhancer (final concentration of sodium butyrate, 10 mM) that promotes transduction was added, and cultured for an additional 6 days to produce proteins. To produce antibodies fused with antiviral peptides, cells were co-infected and cultured with a virus amount of Heavy chain:Light chain = 3:1 (v/v), and to produce antibodies not fused with antiviral peptides, cells were co-infected and cultured with a virus amount of Heavy chain:Light chain = 1:1 (v/v).

Table 4 shows the types and proportions of viruses that infected cells.

Protein Light chain
virus No. Heavy chain
virus No. Virus ratio used (volume)
Light chain : Heavy chain ratio 7NAB 14022 14023 1:1 7NAB_DS-05 14022 14065 1:3 7TCQ 14235 14252 1:1 7TCQ_DS-05 14235 14233 1:3 7TCQ_YAP-1 14235 14234 1:3

Experimental Example 1.4 Protein Purification

The present inventors purified 7NAB antibody, 7NAB antibody_DS-05 fusion protein, 7TCQ antibody, 7TCQ antibody_DS-05 fusion protein and 7TCQ antibody_YAP-1 fusion protein produced from the virus of Experimental Example 1.3. At this time, FIGS. 2 to 6 show the purification process and result of 7NAB antibody. FIGS. 7 to 12 show the purification process and result of 7NAB antibody_DS-05 fusion protein. FIGS. 13 to 16 show the purification process and result of 7TCQ antibody. FIGS. 17 to 19 show the purification process and result of 7TCQ antibody_DS-05 fusion protein. FIGS. 20 to 22 show the purification process and result of 7TCQ antibody_YAP-1 fusion protein.

The specific details of the protein purification process and results shown in Figures 2 to 22 are described below.

After the virus-infected cells were cultured, cell debris was removed by centrifugation, and the supernatant was loaded onto a protein A resin (GoldBio, P-400-5), and antibodies containing the Fc region were attached to the resin. The antibodies attached to the resin were eluted with a 100 mM Glycine (pH 2.5) solution and collected while being neutralized in a tube containing 2 M Tris (pH 8.0) buffer. Through this process, antibodies linked to the mCherry fluorescent protein were purified, and the purification process and results are confirmed through Figs. 2, 7, 13, 17, and 20.

Figure 2 shows the results of purification after expression of proteins (#14022 and #14023) in which mCherry fluorescent protein is linked to the 7NAB antibody.

Figure 7 shows the results of purification after expression of proteins (#14022 and #14065) in which mCherry fluorescent protein is linked to the 7NAB antibody_DS-05 fusion protein.

Figure 13 shows the results of purification after expression of proteins (#14235 and #14252) in which mCherry fluorescent protein is linked to the 7TCQ antibody.

Figure 17 shows the results of purification after expression of proteins (#14235 and #14233) in which mCherry fluorescent protein is linked to the 7TCQ antibody_DS-05 fusion protein.

Figure 20 shows the results of purification after expression of proteins (#14235 and #14234) in which mCherry fluorescent protein is linked to the 7TCQ antibody_YAP-1 fusion protein.

The eluted protein (protein linked to mCherry) was concentrated to a high concentration and then reacted overnight with PreScission protease that recognizes the amino acid sequence LEVLFQ/GP, thereby cleaving the antibody protein and mCherry fluorescent protein. The cleavage position is as shown in Fig. 1. The process and results of cleavage between the antibody protein and mCherry fluorescent protein are as shown in Figs. 3, 8, 14, 18, and 21.

Figure 3 shows the process and result of cleavage of mCherry fluorescent protein (cleavage at #14022 and #14023) linked to 7NAB antibody by PreScission protease.

Figure 8 shows the process and result of cleavage of mCherry fluorescent protein (cleavage at #14022 and #14065) linked to 7NAB antibody_DS-05 fusion protein by PreScission protease.

Figure 14 shows the process and results of cleavage of mCherry fluorescent protein (cleavage at #14235 and #14252) linked to 7TCQ antibody by PreScission protease. (Lane B: before cleavage reaction, A: after cleavage reaction)

Figure 18 shows the process and result of cleavage of mCherry fluorescent protein (cleavage at #14235 and #14233) linked to 7TCQ antibody_DS-05 fusion protein by PreScission protease.

Figure 21 shows the process and result of cleavage of mCherry fluorescent protein (cut at #14235 and #14234) linked to 7TCQ antibody_YAP-1 fusion protein by PreScission protease. (cut: after PreScission cleavage reaction)

After that, the reactant (cleaved product) was flowed through GST resin (GoldBio), and PreScission protease with GST tag was attached to the resin and separated from the antibody protein, and the flowed out solution was flowed through Protein A resin again, so only the antibody protein was attached to the resin, and the mCherry protein flowed out and separated from the antibody protein. The antibody protein attached to the resin was eluted with a 100 mM Glycine pH 2.5 solution and collected while being neutralized in a tube containing 2 M Tris pH 8 buffer (final concentration: 181 mM Tris, 90 mM). The process and results of the antibody protein eluted and purified are as shown in FIGS. 4, 9, 14, 19, and 21.

Figure 4 shows the process and results of purifying the 7NAB antibody protein (the remaining protein after cleaved mCherry protein portion in #14022 and #14023) after PreScission cleavage.

Figure 9 shows the process and results of purifying the 7NAB antibody_DS-05 fusion protein (the remaining protein after cleaved mCherry protein portion from #14022 and #14065) after PreScission cleavage.

Figure 14 shows the process and results of purifying 7TCQ antibody protein (protein remaining after cleaved mCherry protein portion in #14235 and #14252) after PreScission cleavage.

Figure 19 shows the process and results of purifying the 7TCQ antibody_DS-05 fusion protein (the remaining protein after cleaved mCherry protein portion in #14235 and #14233) after PreScission cleavage.

Figure 21 shows the process and results of purifying the 7TCQ antibody_YAP-1 fusion protein (the remaining protein after cleaved mCherry protein portion in #14235 and #14234) after PreScission cleavage.

The eluted antibody protein was purified by cation exchange chromatography. Specifically, after being diluted 10-fold (v/v) with 20 mM pH 5.5 MES buffer, it was flowed over SP resin (Cytiva), and then the protein was eluted by gradually increasing the concentration of NaCl from 100 mM to 500 mM in 100 mM increments in 20 mM pH 5.5 MES buffer. The process and results of purifying the eluted antibody protein by cation exchange chromatography are as shown in FIGS. 5, 10, 15, and 22.

Figure 5 shows the process and results of purifying the eluted 7NAB antibody protein (the remaining protein after cleaved mCherry protein portion in #14022 and #14023) by Cation Exchange chromatography.

Figure 10 shows the process and results of purifying the eluted 7NAB antibody_DS-05 fusion protein (the remaining protein after cleaved mCherry protein portion from #14022 and #14065) by Cation Exchange chromatography.

Figure 15 shows the process and results of purifying the eluted 7TCQ antibody protein (the remaining protein after cleaved mCherry protein portion in #14235 and #14252) by Cation Exchange chromatography.

Figure 22 shows the process and results of purifying the eluted 7TCQ antibody_YAP-1 fusion protein (the remaining protein after cleaved mCherry protein portion in #14235 and #14234) by Cation Exchange chromatography.

After purification by cation exchange chromatography, the buffer of the eluted protein was changed to PBS. The antibody protein was concentrated with an amicon ultra Centrifugal filter kit (Millipore), then diluted up to 5 times (v/v) with PBS and concentrated again. After this process was repeated, Endotoxin Removal Spin Columns (Thermfisher) were used to remove endotoxin that may be present in the antibody protein according to the provided protocol. The results before and after the endotoxin removal kit was used are as shown in Figs. 6, 11, and 16.

Figure 6 shows the SDS-PAGE gel analysis results before and after the endotoxin removal kit was used on the eluted 7NAB antibody protein (the remaining protein after cleaved mCherry protein portion in #14022 and #14023).

Figure 11 shows the SDS-PAGE gel analysis results before and after the endotoxin removal kit was used on the eluted 7NAB antibody_DS-05 fusion protein (the remaining protein after cleaved mCherry protein portion in #14022 and #14065).

Figure 16 shows the SDS-PAGE gel analysis results before and after the endotoxin removal kit was used on the eluted 7TCQ antibody protein (the remaining protein after cleaved mCherry protein portion in #14235 and #14252).

After the endotoxin was removed, the antibody or antibody-peptide fusion protein whose protein concentration was analyzed was used to evaluate the antiviral efficacy. The results of the protein concentration analysis are shown in Fig. 12.

Figure 12 shows the results of analyzing the concentration of the 7NAB antibody protein from which endotoxin was removed (the remaining protein after cleaving off the mCherry protein portion in #14022 and #14023).

Experimental Example 2 Neutralization Test against SARS-CoV-2

^1.5x104 Vero cells were seeded per well in a 96-well plate and cultured for one day.

DS-05 peptide and YAP-1 peptide were each serially diluted 2-fold with DPBS to prepare solutions with various concentrations (highest concentration: 40 μM, lowest concentration: 0.08 uM).

The 7NAB antibody was serially diluted two-fold with DPBS to produce solutions with various concentrations (highest concentration: 800 μg/mL, lowest concentration: 1.56 μg/mL).

7TCQ antibody was serially diluted two-fold with DPBS to produce solutions with various concentrations (highest concentration: 400 μg/mL, lowest concentration: 0.78 μg/mL).

7NAB antibody_DS-05 peptide fusion protein (hereinafter referred to as '7NAB_DS-05'), 7TCQ antibody_DS-05 peptide fusion protein (hereinafter referred to as '7TCQ_DS-05'), and 7TCQ antibody_YAP-1 peptide fusion protein (hereinafter referred to as '7TCQ_YAP-1') were each serially diluted 2-fold with DPBS to prepare solutions with various concentrations (highest concentration: 80 μg/mL, lowest concentration: 0.16 μg/mL).

At this time, 7NAB_DS-05 is a fusion protein in which the DS-05 peptide is linked to the C-terminus of the heavy chain of the 7NAB antibody, and the heavy chain of the 7NAB antibody and the DS-05 peptide are linked via the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 317). Therefore, 7NAB_DS-05 includes a light chain and a heavy chain in which the DS-05 peptide is linked to the C-terminus, and the amino acid sequence of the light chain is SEQ ID NO: 318, and the amino acid sequence of the heavy chain in which the DS-05 peptide is linked to the C-terminus is mefglswvflvallrgvqcEVQLVESGAEVKKPGESLKISCKGSGYTFTRYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGHVTISADKSISTAYLQWNSLKASDTAMYYCARLPQYCSNGVCQRWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVESKYGPPCPS CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSDWVDCFWSWLIKVIDRVS (SEQ ID NO: 330).

At this time, 7TCQ_DS-05 is a fusion protein in which the DS-05 peptide is linked to the C-terminus of the heavy chain of the 7TCQ antibody, and the heavy chain of the 7TCQ antibody and the DS-05 peptide are linked via the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 317). Therefore, 7TCQ_DS-05 includes a light chain and a heavy chain in which the DS-05 peptide is linked to the C-terminus, and the amino acid sequence of the light chain is SEQ ID NO: 320, and the amino acid sequence of the heavy chain in which the DS-05 peptide is linked to the C-terminus is mefglswvflvallrgvqcEVQFQQSGTVLARPGASVKMSCKASGYTFTNYWIHWVKQRPGQGLEYIGGTYPGNGDTTYNQKFKGKAKVTAVTPTSTAYMDLSSLTNEDSAVYYCTRTGSYFDYWGQGTTLTVSSASTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVLTTWNSGSLSSGVHTFPAV LQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIESKYGPPCPSCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGKHHHHHHGGGGSGGGGSGGGGSDWVDCFWSWLIKVIDRVS (SEQ ID NO: 331).

At this time, 7TCQ_YAP-1 is a fusion protein in which a YAP-1 peptide is linked to the C-terminus of the heavy chain of the 7TCQ antibody, and the heavy chain of the 7TCQ antibody and the YAP-1 peptide are linked through the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 317). Therefore, 7TCQ_YAP-1 includes a light chain and a heavy chain in which a YAP-1 peptide is linked to the C-terminus, and the amino acid sequence of the light chain is SEQ ID NO: 320, and the amino acid sequence of the heavy chain in which a YAP-1 peptide is linked to the C-terminus is mefglswvflvallrgvqcEVQFQQSGTVLARPGASVKMSCKASGYTFTNYWIHWVKQRPGQGLEYIGGTYPGNGDTTYNQKFKGKAKVTAVTPTSTAYMDLSSLTNEDSAVYYCTRTGSYFDYWGQGTTLTVSSASTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVLTTWNSGSLSSGVHTFPAV LQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIESKYGPPCPSCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGKHHHHHHGGGGSGGGGSGGGGSESLKNFWESLWKFFTHFF (SEQ ID NO: 332).

The prepared solution and 200 TCID50 of SARS-CoV-2 virus were mixed in a volume ratio of 1:1 (v/v), and incubated at 37°C for 30 minutes to prepare a mixture. The prepared mixture was treated to 4 wells inoculated with cells at each concentration (40 μL) and incubated at 37°C and 5% _CO2 for 4 days. After incubation, the solution was fixed with 4% paraformaldehyde and stained with crystal violet solution to observe CPE (cytopathic effect), and the observed CPE was used to calculate the neutralizing activity %. The neutralizing activity % was calculated by normalizing with the positive control (mock) and the negative control (0.5% DMSO), and the 50% inhibitory concentration (IC50) was calculated.

The neutralizing ability according to the concentration of each substance is graphically depicted in Figures 23 to 29, through which the IC ₅₀ value of each substance was derived.

Fig. 23 is a graph regarding the DS-05 peptide. Fig. 24 is a graph regarding the YAP-1 peptide. Fig. 25 is a graph regarding the 7NAB antibody. Fig. 26 is a graph regarding the 7TCQ antibody. Fig. 27 is a graph regarding 7NAB_DS-05. Fig. 28 is a graph regarding 7TCQ_DS-05. Fig. 29 is a graph regarding 7TCQ_YAP-1.

Table 5 shows the neutralizing capacity and IC ₅₀ values of each substance (antibody, peptide, and antibody-peptide fusion protein) at each concentration.

Sample Results DS-05 Concentration (μM) 20 10 5 2.5 1.3 0.6 0.3 0.2 0.1 0.04 PBS % Neutralization 100 50 50 0 0 0 0 0 0 0 0 IC ₅₀ (μM) 6.961 YAP-1 Concentration (μM) 20 10 5 2.5 1.3 0.6 0.3 0.2 0.1 0.04 PBS % Neutralization 0 0 0 0 0 0 0 0 0 0 0 IC ₅₀ (μM) >20 7NAB Concentration (μg/ml) 400 200 100 50 25.0 12.5 6.3 3.1 1.6 0.8 PBS % Neutralization 75 50 25 0 0 0 0 0 0 0 0 IC ₅₀ (μg/ml) 159.7 7TCQ Concentration (μg/ml) 200 100 50 25 12.5 6.3 3.1 1.6 0.80 0.40 PBS % Neutralization 100 100 50 50 25 0 0 0 0 0 0 IC ₅₀ (μg/ml) 29.77 7NAB_DS-05 Concentration (μg/ml) 40 20 10 5 2.5 1.3 0.6 0.3 0.2 0.1 PBS % Neutralization 75 75 25 0 0 0 0 0 0 0 0 IC ₅₀ (μg/ml) 10.2 7TCQ_DS-05 Concentration (μg/ml) 40 20 10 5 2.5 1.3 0.6 0.3 0.2 0.08 PBS % Neutralization 100 100 25 0 0 0 0 0 0 0 0 IC ₅₀ (μg/ml) 10.32 7TCQ_YAP-1 Concentration (μg/ml) 40 20 10 5 2.5 1.3 0.6 0.3 0.2 0.08 PBS % Neutralization 100 100 50 0 0 0 0 0 0 0 0 IC ₅₀ (μg/ml) 10.0

Through Table 5, it can be seen that the IC ₅₀ value of the DS-05 peptide is 6.961 uM, the IC ₅₀ value of the 7NAB antibody is 159.7 ug/mL, and the IC ₅₀ value of 7NAB_DS-05, a fusion protein in which the DS-05 peptide is linked to the C-terminus of the 7NAB antibody, is 10.5 ug/mL. At this time, since the molecular weight of the fusion protein is about 150 kDa, if the concentration is calculated based on this, the IC ₅₀ value of the fusion protein is about 0.07 uM. In other words, it can be seen that the IC ₅₀ value of the fusion protein is very low compared to the IC ₅₀ values of the antibody and peptide.

Through Table 5, it can be seen that the IC ₅₀ value of the DS-05 peptide is 6.961 uM or higher, the IC ₅₀ value of the 7TCQ antibody is 29.77 ug/mL, and the IC ₅₀ value of 7TCQ_DS-05, a fusion protein in which the DS-05 peptide is linked to the C-terminus of the 7TCQ antibody, is 10.32 ug/mL. At this time, since the molecular weight of the fusion protein is about 150 kDa, if the concentration is calculated based on this, the IC ₅₀ value of the fusion protein is about 0.069 uM. In other words, it can be seen that the IC ₅₀ value of the fusion protein is very low compared to the IC ₅₀ values of the antibody and peptide.

Through Table 5, it can be seen that the IC ₅₀ value of the YAP-1 peptide is 20uM or higher, the IC ₅₀ value of the 7TCQ antibody is 29.77 ug/mL, and the IC ₅₀ value of 7TCQ_YAP-1, a fusion protein in which the YAP-1 peptide is linked to the C-terminus of the 7TCQ antibody, is 10.0 ug/mL. At this time, since the molecular weight of the fusion protein is about 150 kDa, if the concentration is calculated based on this, the IC ₅₀ value of the fusion protein is about 0.067 uM. In other words, it can be seen that the IC ₅₀ value of the fusion protein is very low compared to the IC ₅₀ values of the antibody and peptide.

Through the above results, it was confirmed that the antiviral effect was higher when the antibody and antiviral peptide were combined and used compared to when the antibody or antiviral peptide was used alone.

Claims (27)

Antibody-peptide fusion proteins comprising: An antibody or fragment thereof, wherein the antibody is capable of binding to the S2 domain of the spike protein of a coronavirus; a linker, wherein said linker is linked to a constant region of said antibody or a fragment thereof; and An antiviral peptide, wherein the antiviral peptide is linked to the antibody or a fragment thereof via the linker, At this time, the antiviral peptide is an amphipathic peptide, The number of amino acid residues constituting the above antiviral peptide is 18. The above antiviral peptides can cause damage to the lipid bilayer of the virus, At this time, the antibody-peptide fusion protein can bind to the S2 domain of the spike protein of the coronavirus, At this time, the antibody-peptide fusion protein can cause damage to the lipid bilayer of the virus. In the first paragraph, An antibody-peptide fusion protein, characterized in that the amino acid sequence of the antiviral peptide is any one amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 315. In the first paragraph, An antibody-peptide fusion protein, characterized in that the amino acid sequence of the above antiviral peptide is any one of the following amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 3 to 5, SEQ ID NO: 8 to 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38 to 40, SEQ ID NO: 43, SEQ ID NO: 45 to 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 64 to 73, SEQ ID NO: 75, SEQ ID NO: 78 to 87, SEQ ID NO: 90 to 93, SEQ ID NO: 97 to 102, SEQ ID NO: 104 to 106, SEQ ID NO: 109 to 112, SEQ ID NO: 114 to 116, SEQ ID NO: 118 to 123, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 130 to 132, SEQ ID NO: 136, SEQ ID NO: 138 to 147, SEQ ID NO: 151 to 155, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166 to 168, SEQ ID NO: 170 to 174, SEQ ID NO: 176, SEQ ID NO: 180 to 182, SEQ ID NO: 184 to 186, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 194 to 196, SEQ ID NO: 198, SEQ ID NO: 201, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208 to 211, SEQ ID NO: 215, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 240, SEQ ID NOs: 242 to 246, SEQ ID NOs: 248 to 254, SEQ ID NOs: 256 to 258, SEQ ID NOs: 260 to 270, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NOs: 275 to 288, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NOs: 293 to 296, SEQ ID NO: 298, and SEQ ID NOs: 299 to 315. In the first paragraph, An antibody-peptide fusion protein, characterized in that the linker is a linker including a peptide, and the N-terminus of the peptide included in the linker is linked to the C-terminus of the heavy chain of the antibody or a fragment thereof. In the first paragraph, An antibody-peptide fusion protein, characterized in that the linker is a linker including a peptide, and the C-terminus of the peptide included in the linker is linked to the N-terminus of the antiviral peptide. In the first paragraph, An antibody-peptide fusion protein, characterized in that the linker is a linker including a peptide, and the number of amino acid residues of the peptide included in the linker is 5 to 20. In the first paragraph, An antibody-peptide fusion protein, characterized in that the linker is a linker including a peptide, and the amino acid sequence of the peptide included in the linker includes GGGGSGGGGSGGGGS (SEQ ID NO: 317). In the first paragraph, An antibody-peptide fusion protein, characterized in that the coronavirus comprises at least one selected from Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. In Article 8, The above coronavirus is a betacoronavirus. An antibody-peptide fusion protein, characterized in that at this time, the betacoronavirus comprises at least one selected from embecovirus, merbecovirus, and sarbecovirus. In Article 9, The above coronavirus is Sarbecovirus, An antibody-peptide fusion protein, characterized in that the sarbecovirus comprises at least one selected from SARS-CoV and SARS-CoV-2. In the first paragraph, An antibody-peptide fusion protein, characterized in that the coronavirus is SARS-CoV-2. In the first paragraph, The above antibody or fragment thereof can bind to the S2 domain of the spike protein of betacoronavirus, At this time, the antibody-peptide fusion protein is characterized in that it can bind to the S2 domain of the spike protein of betacoronavirus. In the first paragraph, The above antibody or fragment thereof can bind to the S2 domain of the spike protein of SARS-CoV2 and the S2 domain of the spike protein of a variant of SARS-CoV2, At this time, the antibody-peptide fusion protein is characterized in that it can bind to the S2 domain of the spike protein of SARS-CoV2 and the S2 domain of the mutant spike protein of SARS-CoV2. In the first paragraph, An antibody-peptide fusion protein, characterized in that the antibody fragment is any one selected from F(ab), F(ab'), F(ab') ₂ , monospecific F(ab') ₂ , bispecific F(ab') ₂ , scFv, ScFv-Fc, and sdAb. A pharmaceutical composition for the treatment of a viral disease comprising a therapeutically effective amount of an antibody-peptide fusion protein, The above antibody-peptide fusion protein is an antibody-peptide fusion protein of any one of claims 1 to 14, The above viral diseases are diseases caused by viral infection. In Article 15, A pharmaceutical composition for treating a viral disease, characterized in that the pharmaceutical composition for treating a viral disease further comprises a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable adjuvant. In Article 15, A pharmaceutical composition for treating a viral disease, characterized in that the viral disease is a respiratory disease caused by a viral infection, and the viral infection is a betacoronavirus infection. In Article 15, A pharmaceutical composition for treating a viral disease, wherein the viral disease is a respiratory disease caused by a viral infection, and the viral infection is characterized by infection with SARS-CoV2 or a variant thereof. Methods for treating viral diseases, including: Administering a pharmaceutical composition comprising a therapeutically effective amount of an antiviral peptide to a subject infected with a virus; At this time, the antiviral peptide is an antiviral peptide according to any one of claims 1 to 14. In Article 19, A method for treating a viral disease, wherein the viral disease is a respiratory disease caused by a viral infection, and the viral infection is characterized by being a betacoronavirus infection. In Article 19, A method for treating a viral disease, wherein the viral disease is a respiratory disease caused by a viral infection, and the viral infection is characterized by infection with SARS-CoV2 or a variant thereof. Use of an antiviral peptide according to any one of claims 1 to 14 for treating a viral disease. In Article 22, The above viral disease is a respiratory disease caused by a viral infection, and the viral infection is characterized by being a betacoronavirus infection, for use in the treatment of a viral disease. In Article 22, The above viral disease is a respiratory disease caused by a viral infection, and the viral infection is characterized by infection with SARS-CoV2 or a variant thereof, for use in the treatment of a viral disease. Use of an antiviral peptide according to any one of claims 1 to 14 for the manufacture of a medicament for treating a viral disease. In Article 25, The above viral disease is a respiratory disease caused by a viral infection, and the above viral infection is characterized by being a betacoronavirus infection, the use of an antiviral peptide for the manufacture of a pharmaceutical product for treating a viral disease. In Article 25, The above viral disease is a respiratory disease caused by a viral infection, and the viral infection is characterized by infection with SARS-CoV2 or a variant thereof, use of an antiviral peptide for manufacturing a pharmaceutical product for treating a viral disease.

Images