Nanodisc comprising virus receptor and FC fragment, and antiviral use thereof

Abstract

The present invention relates to a nanodisc comprising a virus receptor and a FC fragment. In addition, the present invention relates to a pharmaceutical composition for prevention or treatment of viral infection, the composition containing the nanodisc. In the present invention, the nanodisc comprising a virus receptor and a FC fragment of the present invention has been confirmed to have a long in vivo half-life while also exhibiting excellent antiviral efficacy.

Description

Nanodiscs comprising viral receptors and FC fragments and their antiviral uses

[0001] The present invention relates to a nanodisc comprising a viral receptor and an Fc fragment. The present invention also relates to a pharmaceutical composition for preventing or treating viral infections, characterized by containing the nanodisc.

[0002] Influenza virus is an RNA virus belonging to the family Orthomyxoviridae, and is divided into three serotypes: A, B, and C. Of these, types B and C have only been confirmed to infect humans, while type A has been confirmed to infect humans, horses, pigs, other mammals, and various types of poultry and wild birds. The serotypes of influenza A viruses are classified according to the types of two proteins on the surface of the virus, hemagglutinin (HA) and neuraminidase (NA). 144 types (16 types of HA proteins and 9 types of NA proteins) have been known so far. HA plays a role in the virus's attachment to somatic cells, and NA enables the virus to penetrate into cells.

[0003] Until now, the known treatments for viral infections developed include M2 ​​ion channel inhibitors such as amantadine or rimantadine, and neuraminidase inhibitors such as oseltamivir (trade name Tamiflu) or zanamivir (trade name Relenza). However, these treatments have the problem of limited effectiveness. That is, amantadine or rimantadine derivative compounds rapidly generate resistant mutant viruses, and the H5N1 type influenza virus detected in some regions is resistant to amantadine or rimantadine compounds, and influenza B viruses are known to be insensitive to amantadine derivatives. In addition, it is known that resistant viruses to oseltamivir or zanamivir derivative compounds are increasing, and such resistant viruses are frequently occurring in children.

[0004] Meanwhile, there have been numerous outbreaks of viral diseases, including COVID-19, a coronavirus that has been ongoing since 2020.

[0005] Coronaviruses are RNA viruses belonging to the Coronavirinae subfamily of the Coronaviridae family, which cause respiratory and digestive infections in humans and animals. They are easily transmitted through mucosal infection and droplet transmission, typically causing mild respiratory infections in humans but can also be fatal. They can cause diarrhea in cattle and pigs and respiratory disease in chickens. Coronaviruses are a representative example of viruses that cause fatal infectious diseases in modern civilization. In April 2003, the Severe Acute Respiratory Syndrome (SARS), also known as SARS, broke out in China, killing many people with a mortality rate of 96%. In 2015, the Middle East Respiratory Syndrome (MERS) spread from the Middle East to the rest of the world, resulting in numerous deaths with a mortality rate of approximately 36%. Furthermore, since December 2019, the number of confirmed cases of the novel coronavirus disease (COVID-19) has been increasing worldwide.

[0006] Antibody therapeutics are being developed to treat viral diseases. However, these therapeutics are vulnerable to the emergence of resistant viruses due to mutations. Furthermore, antiviral antibody therapeutics often fail to demonstrate significant therapeutic efficacy. This is because antibodies exert their antiviral activity by inhibiting viral protein function and mediating viral killing by immune cells. Furthermore, antibody therapeutics themselves can potentially provide immunogenicity to the host, increasing the risk of developing resistant viruses.

[0007] Hepatitis B virus (HBV) is a DNA virus that causes significant damage to humanity. The complete form of HBV, called the Dane particle (42 nm in diameter), consists of approximately 32 kb of DNA, a capsid surrounding it, and an outer membrane protein called hepatitis B virus surface antigen (HBSag). Most current HBV treatments are nucleic acid derivatives that work by intercalating into the viral DNA strand during the conversion of pregenomic RNA to DNA by polymerase within the capsid, thereby terminating its synthesis. However, these treatments are susceptible to resistance due to mutations in HBV. Therefore, the development of novel treatments targeting sites other than the HBV polymerase is necessary to overcome resistance to existing antiviral agents.

[0008] A nanodisc is a disc-shaped structure formed by wrapping a phospholipid bilayer membrane with a membrane scaffold protein (MSP), a protein derived from apolipoprotein A1 (Apo-A1), a major component of high-density lipoproteins (HDL) in the body. Nanodiscs are mainly used for structural studies of various cell membrane proteins. They also function as a vehicle to deliver various physiologically functional substances into the body. As a biologically derived substance, nanodiscs are stable in the body and do not cause harmful reactions, making them safe.

[0009]

[0010] The present invention seeks to provide nanodiscs comprising a virus receptor and an Fc fragment in various forms.

[0011] The present invention aims to provide a nanodisc that exhibits excellent pharmacodynamic characteristics and excellent antiviral efficacy.

[0012]

[0013] The present invention provides nanodiscs comprising a phospholipid bilayer, a membrane-structuring protein, an Fc fragment, and a viral receptor in various forms.

[0014] Specifically, the present invention provides a nanodisc comprising, in a first form, a lipid bilayer having a flat, disc-shaped bilayer structure formed from a phospholipid, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; a membrane scaffold protein (MSP) surrounding the 'side of the lipid bilayer where the hydrophobic groups are exposed outward'; and a virus receptor hydrophobicly bonded to the lipid bilayer; wherein an Fc fragment is bound to the virus receptor, and the Fc fragment protrudes to the outside of the nanodisc.

[0015] The present invention provides, in a second aspect, a nanodisc comprising a lipid bilayer having a flat, disc-shaped bilayer structure formed from phospholipids, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; a membrane scaffold protein (MSP) surrounding the 'side of the lipid bilayer where the hydrophobic groups are exposed outward'; and a virus receptor hydrophobicly bonded to the lipid bilayer; wherein an Fc fragment is bound to the membrane scaffold protein, such that the Fc fragment protrudes to the outside of the nanodisc, and an Fc fragment is bound to the virus receptor, such that the Fc fragment protrudes to the outside of the nanodisc.

[0016] The present invention provides a nanodisc comprising a lipid bilayer having a flat disc-shaped bilayer structure formed from a phospholipid, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; and a membrane scaffold protein (MSP) surrounding the 'side of the lipid bilayer where the hydrophobic groups are exposed outward', wherein the membrane scaffold protein has a virus receptor bound to one end and an Fc fragment bound to the other end, and the bound virus receptor and Fc fragment are characterized in that they protrude to the outside of the nanodisc.

[0017] The present invention provides a nanodisk comprising a lipid bilayer having a flat disc-shaped bilayer structure formed from a phospholipid, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; and a membrane scaffold protein (MSP) surrounding the 'side of the lipid bilayer where the hydrophobic groups are exposed to the outside', wherein the membrane scaffold protein has a virus receptor bound to one end and an Fc fragment bound to the other end, and a plurality of membrane scaffold proteins are interconnected to form a dimer by mutually binding the 'virus receptors of the membrane scaffold protein' and the 'Fc fragments of the membrane scaffold protein' to form a dimer, thereby forming a membrane scaffold protein in which the length is extended, and wherein the bound virus receptors and Fc fragments protrude to the outside of the nanodisk.

[0018] The present invention provides a nanodisc comprising a lipid bilayer having a flat disc-shaped bilayer structure formed from a phospholipid, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; and a membrane scaffold protein (MSP) surrounding the 'side of the lipid bilayer where the hydrophobic groups are exposed outward', wherein the membrane scaffold protein has a virus receptor and an Fc fragment bound to one end thereof, and the bound virus receptor and Fc fragment protrude to the outside of the nanodisc.

[0019] In the nanodisk of the present invention, the phospholipid may include at least one selected from phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol, and preferably DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), and It is recommended to include at least one selected from POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine).

[0020] In the nanodisk of the present invention, the membrane scaffold protein is an amphipathic protein having a helix structure, and may be an apolipoprotein or a fragment of an apolipoprotein in which the 'helix structure and amphipathic properties' of the apolipoprotein are maintained.

[0021] In the second type nanodisk of the present invention, the Fc fragment bound to the membrane-structured protein may be produced by binding a gene encoding the Fc fragment to a gene encoding the membrane-structured protein and then expressing the same.

[0022] In the first or second form of the nanodisk of the present invention, the Fc fragment bound to the viral receptor may be produced by combining a gene encoding the viral receptor with a gene encoding the Fc fragment, and then expressing the same.

[0023] In the third or fourth form nanodisk of the present invention, the viral receptor and Fc fragment bound to the membrane-structured protein may be produced by binding a gene encoding a viral receptor to one end of a gene encoding a membrane-structured protein, binding a gene encoding an Fc fragment to the other end, and then expressing the same.

[0024] In the fifth type nanodisc of the present invention, the membrane-structured protein may have a viral receptor and an Fc fragment sequentially bound to one end. In this case, the angiotensin converting enzyme 2 and Fc fragment bound to the membrane-structured protein may be produced by binding a gene encoding an Fc fragment to the 5' end of a gene encoding a membrane-structured protein, binding a gene encoding a viral receptor to the 5' end of the gene encoding the Fc fragment, and then expressing the resulting product.

[0025] In the fifth type nanodisc of the present invention, the membrane-structured protein may have an Fc fragment and a viral receptor sequentially bound to one end. In this case, the Fc fragment and angiotensin converting enzyme 2 bound to the membrane-structured protein may be produced by binding a gene encoding a viral receptor to the 5' end of a gene encoding a membrane-structured protein, binding a gene encoding an Fc fragment to the 5' end of the gene encoding the viral receptor, and then expressing the resulting product.

[0026] In the third type nanodisk of the present invention, the nanodisk may be formed by forming a dimer by mutually combining a viral receptor of a membrane-structured protein surrounding one of the bilayers and a viral receptor of a membrane-structured protein surrounding another of the bilayers, or by mutually combining an Fc fragment of a membrane-structured protein surrounding one of the bilayers and an Fc fragment of a membrane-structured protein surrounding another of the bilayers to form a dimer, whereby the membrane-structured protein forms a disk shape, thereby forming a nanodisk structure.

[0027] In the fourth type nanodisk of the present invention, the membrane-structured protein with an extended length may be a membrane-structured protein in which two membrane-structured proteins are linked to each other through mutual binding of two 'angiotensin converting enzyme 2 of the membrane-structured protein' to form a dimer and through mutual binding of two 'Fc fragments of the membrane-structured protein' to form a dimer.

[0028] In the fourth type of nanodisk of the present invention, the nanodisk may be such that one of the bilayers is surrounded by a membrane-structured protein having an extended length, and the other layer of the bilayer is surrounded by another membrane-structured protein having an extended length.

[0029] Meanwhile, the present invention provides a pharmaceutical composition for preventing or treating viral infection, characterized by containing the nanodisk. At this time, the virus may be at least one selected from the Coronaviridae family, the Bunyaviridae family, the Filoviridae family, the Flaviviridae family, the Hepadnaviridae family, the Herpesviridae family, the Orthomyxoviridae family, the Poxviridae family, the Rhabdoviridae family, the Retroviridae family, the Togaviridae family, the Picornaviridae family, the Paramyxoviridae family, and the Reoviridae family.

[0030] In the present invention, it was confirmed that the nanodisc of the present invention including a virus receptor and an Fc fragment exhibits excellent antiviral efficacy while having a long half-life in the body.

[0031] FIG. 1 and FIG. 2 schematically show the manufacturing process and form of the nanodisc of the present invention. FIG. 1 A is a nanodisc including an Fc fragment (ND-Fc), FIG. 1 B is a nanodisc including fACE2 (full length of ACE) and an Fc fragment (NDA-Fc), FIG. 1 C is a nanodisc including fACE2-Fc (AFc-ND), FIG. 1 D is a nanodisc including MSP-Fc and fACE2-Fc (AFc-ND-Fc), FIG. 2 E is a nanodisc including ACE2-Fc-MSP (A-Fc-ND), FIG. 2 F is a nanodisc including Fc-ACE2-MSP (Fc-A-ND), and FIG. 2 G is a nanodisc including ACE2-MSP-Fc (LMW A-ND-Fc, MMM A-ND-Fc, HMW A-ND-Fc).

[0032] Figure 3 shows the results of confirming the molecular weight through electrophoresis after producing a membrane-structured protein (MSP-Fc) with an Fc fragment attached to it to confirm whether the membrane-structured protein (MSP-Fc) with an Fc fragment attached to it was produced intact.

[0033] Figure 4 shows the results of measuring particle size using dynamic light scattering (DLS) (Figure 4A) and measuring molecular weight using a multi-angle optical scattering (SEC-MALS) detector (Figure 4B) to confirm whether the nanodisc containing the Fc fragment (ND-Fc) was completely manufactured.

[0034] Figure 5 schematically shows the differences in the manufacturing process of nanodiscs (ND) and large nanodiscs (P2N2-ND) containing Escherichia coli-expressed MSP; nanodiscs (hMSP-ND) and large nanodiscs (hMSP2N2-ND) containing human-derived cell (HEK293)-expressed MSP; nanodiscs (ND-Fc, immunodisc) and large nanodiscs (P2N2-ND-Fc) containing human-derived cell (HEK293)-expressed MSP-Fc.

[0035] Figure 6 shows the results of performing size exclusion chromatography (SEC) using a column (Superose 6, Supedex 200) after producing ND-Fc and P2N2-ND-Fc to confirm the excellent production yield of nanodiscs (ND-Fc) containing Fc fragments (A, B of Figure 6), and comparing the purification yield with that of general nanodiscs (ND) (C of Figure 6).

[0036] Figure 7 shows the manufacturing process of nanodiscs (NDA-Fc) containing Fc fragment and angiotensin converting enzyme 2 (A in Figure 7), the purification process using size exclusion chromatography (SEC) (B in Figure 7), the results of SDS-PAGE analysis to confirm whether the nanodiscs were manufactured intact (C in Figure 7), and the results of dynamic light scattering (DLS) measurement (D in Figure 7).

[0037] Figure 8 shows the results of size exclusion chromatography (SEC) to confirm the excellent production yield of nanodiscs (NDA-Fc) containing Fc fragment and angiotensin converting enzyme 2, compared with the case of nanodiscs (NDA) containing angiotensin converting enzyme 2.

[0038] Figure 9 shows the purification process using size exclusion chromatography (SEC) after self-assembly of nanodiscs (AFc-ND, AFc-ND-Fc) containing Fc fragment and angiotensin converting enzyme 2 (Figure 9A) and the results of fabrication confirmation using SDS-PAGE to confirm whether the nanodiscs were completely fabricated (Figure 9B).

[0039] Figure 10 shows the results of purification of membrane-structured proteins (sACE2-Fc-MSP, sACE2-MSP-Fc) for producing nanodiscs (sA-Fc-ND, sA-ND-Fc) containing Fc fragments and angiotensin converting enzyme 2 (Figure 10A) and the results of purifying nanodiscs using size exclusion chromatography (SEC) after producing nanodiscs by self-assembly using the membrane-structured proteins (sACE2-Fc-MSP, sACE2-MSP-Fc) (Figure 10B).

[0040] Figure 11 shows the process of manufacturing a nanodisc (NTCP-Fc-ND) containing an Fc fragment and NTCP (A in Figure 11), the process of purifying a protein (NTCP-Fc) to which NTCP and Fc fragments are bound (B in Figure 11), the result of purifying the nanodisc (NTCP-Fc-ND) by size exclusion chromatography (SEC) after manufacturing it (C in Figure 11), and the result of confirming the structure of the nanodisc (NTCP-Fc-ND) by transmission electron microscope (TEM) (D in Figure 11).

[0041] Figure 12 shows the results of a CPE inhibition experiment conducted to confirm the excellent antiviral efficacy of nanodiscs (NDA-Fc, AFc-ND, sA-ND-Fc) containing an Fc fragment and angiotensin converting enzyme 2, compared with nanodiscs (sAND, NDA) containing angiotensin converting enzyme 2.

[0042] Figure 13 shows the results of confirming the concentration distributed in each organ (A in Figure 13) and serum half-life (B in Figure 13) after injecting scFv-Fc antibody (P2B-2FB) or ND-Fc into mice to evaluate the pharmacodynamic properties of nanodiscs (ND-Fc) containing Fc fragments.

[0043] Figure 14 shows the results of confirming the concentration distributed in each organ (Figure 14A) and serum half-life (Figure 14B) after intravenous administration (IV) of sACE2-Fc or NDA-Fc to mice to evaluate the pharmacodynamic properties of nanodiscs (NDAFc) containing Fc fragment and angiotensin-converting enzyme 2. In addition, the results of confirming the concentration distributed in each organ after intranasal administration (IN) of sACE2-Fc or NDA-Fc to mice are shown (Figure 14C).

[0044]

[0045] The present invention provides nanodiscs comprising a phospholipid bilayer, a membrane-structuring protein, an Fc fragment, and a viral receptor in various forms.

[0046] Nanodiscs are structures in which one or more membrane scaffold proteins (MSPs) surround the sides of a lipid bilayer formed from phospholipids with hydrophobic bonds, so that the hydrophilic groups of the phospholipids are oriented outward and the hydrophobic groups are oriented inward, and the lipid bilayer takes the shape of a flat disk. According to previous studies, nanodiscs are widely used as delivery vehicles for hydrophobic drugs and are being utilized in research on the structure and function of proteins.

[0047] The half-life of nanodiscs reported in the literature ranges from 0.5 to 2 hours, with some nanodiscs exhibiting a half-life of up to 60 hours. Furthermore, even for nanodiscs with long half-lives, it has been reported that nanodiscs are barely observed in the blood 96 hours after administration (Park, Hyun-Ji, et al. "High-density lipoprotein-mimicking nanodiscs carrying peptide for enhanced therapeutic angiogenesis in diabetic hindlimb ischemia" Biomaterials 161(2018): 69-80.).

[0048] However, in the present invention, it was confirmed that the nanodisc containing the Fc fragment has a very long half-life, and based on this, the in vivo pharmacodynamic characteristics of the nanodisc containing the virus receptor and the Fc fragment were confirmed, and it was confirmed that almost the same concentration was maintained without a significant difference from the initial concentration even after 48 hours, and it was confirmed that the concentration of the nanodisc in the serum was maintained at about 40% of the initial concentration even after 168 hours, maintaining a high concentration.

[0049] In addition, the inventors of the present invention confirmed through Korean Patent Registration No. 10-2181991 or 10-2438720 that a nanodisc containing a virus receptor acts as a cell membrane mimic and perforates the envelope of the virus, and confirmed that the nanodisc containing a virus receptor can be used as an antiviral agent against a wide range of viruses through this mechanism. In the present invention, by further including an Fc fragment in the nanodisc containing the above virus receptor, it was confirmed that the nanodisc exhibits even better antiviral efficacy and has excellent pharmacodynamic characteristics.

[0050] In addition, the present invention is characterized by providing a nanodisc comprising a phospholipid bilayer, a membrane-structuring protein, an Fc fragment, and a viral receptor in various forms.

[0051] Specifically, the nanodisc of the present invention may be in a form in which a protein bound to a viral receptor and an Fc fragment is hydrophobicly bound to the lipid bilayer of the nanodisc (AFc-ND, see C in FIG. 1).

[0052] A protein with a viral receptor and an Fc fragment bound thereto may be hydrophobicly bound to the lipid bilayer of the nanodisc, and another Fc fragment may be additionally bound to a membrane-structured protein (vRFc-ND-Fc, see D in Figure 1).

[0053] The viral receptor and Fc fragments may be bound to one end of a nanodisc membrane-structured protein (vR-Fc-ND, Fc-vR-ND, see E, F of Fig. 2).

[0054] The viral receptor and Fc fragments may be bound to each end of the nanodisc membrane-structured protein (LMW vRND-Fc, MMW vR-ND-Fc, HMW vR-ND-Fc, see G in Fig. 2).

[0055] Meanwhile, in the present invention, the phospholipid may be, for example, at least one selected from the group consisting of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol.

[0056] 상기 포스파티딜콜린(phosphatidylcholine)은 일 예로 DOPC(1,2-Dioleoyl-sn-glycero-3-phosphocholine), DLPC(1,2-Dilauroyl-sn-glycero-3-phosphocholine), DMPC(1,2-Dimyristoyl-sn-glycero-3-phosphocholine), DPPC(1,2-Dipalmitoyl-sn-glycero-3-phosphocholine), POPC(1-Palmitoyl-2-oleoyl-snglycero-3-phosphocholine), C13PC, DDPC(1,2-Didecanoyl-sn-glycero-3-phosphocholine), DSPC(1,2-Distearoyl-sn-glycero-3-phosphocholine), DEPC(1,2-Dierucoyl-sn-glycero-3-phosphocholine), DLOPC(1,2-Dilinoleoyl-sn-glycero-3-phosphocholine), EPC(Egg phosphatidylcholine), MSPC( 1-Myristoyl-2-stearoylsn-glycero-3-phosphocholine), PMPC(1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine), PSPC(1-Palmitoyl-2- stearoyl-sn-glycero-3-phosphocholine),SMPC(1-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine) 또는 SPPC(1-Stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine)일 수 있다.

[0057] In addition, the phosphatidylglycerol is, for example, DMPG (1,2-Dimyristoylsn-glycero-3[Phospho-rac-(1-glycerol)], DPPG (1,2-Dipalmitoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]), DSPG (1,2-Distearoyl-sn-glycero-3[Phospho-rac-(1-glycerol)), POPG (1-Palmitoyl-2-oleoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]), DEPG (1,2-Dierucoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]), DLPG (1,2-Dilauroyl-sn-glycero-3[Phospho-rac-(1-glycerol)]), It may be DOPG (1,2-Dioleoyl-sn-glycero-3[Phospho-rac-(1-glycerol)]) or DSPG (1,2-Distearoyl-snglycero-3[Phospho-rac-(1-glycerol)]), and the phosphatidylethanolamine may be DMPE (1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine), DPPE(1,2-Dipalmitoylsn-glycero-3-phosphoethanolamine), DSPE(1,2-Distearoyl-sn-glycero-3-phosphoethanolamine), DOPE(1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), DEPE(1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine), DLPE (1,2-Dilauroyl-snglycero-3-phosphoethanolamine) or POPE(1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), the phosphatidylserine, DOPS(1,2-Dioleoyl-sn-glycero-3-phosphoserine), DLPS(1,2-Dilauroyl-sn-glycero-3-phosphoserine), DMPS (1,2-Dimyristoyl-sn-glycero-3-phosphoserine), DPPS (1,2-Dipalmitoyl-sn-glycero-3-phosphoserine), DSPS (1,2-Distearoyl-sn-glycero-3-phosphoserine) or POPS (POPS), the phosphatidylinositol may be phosphatidylinositol-4-phosphate, phosphatidylinositol-4,5-bisphosphate, or phosphatidylinositol-3,4,5-trisphosphate.

[0058] Meanwhile, in the present invention, the membrane scaffold protein (MSP) has a helix structure and has amphipathic characteristics, and through these characteristics, it plays a role in surrounding the side of the nanodisk lipid bilayer.

[0059] A plurality of membrane-structuring proteins can surround the sides of the nanodisk lipid bilayer. Each layer of the lipid bilayer may be individually surrounded by a membrane-structuring protein, or a single layer of the lipid bilayer may be surrounded by a 'membrane-structuring protein whose length is extended by combining multiple membrane-structuring proteins.' In this case, when a single layer is surrounded by a 'membrane-structuring protein whose length is extended by combining multiple membrane-structuring proteins,' a large nanodisk with a large diameter is formed.

[0060] An example of a membrane-structuring protein is apolipoprotein. Apolipoprotein is a protein specifically present in plasma lipoproteins. It is known to stabilize the structure of lipoproteins, activate enzymes involved in lipoprotein metabolism, and function as a ligand for lipoprotein receptors present on the cell surface. The above apolipoproteins include, for example, apolipoprotein A1 (ApoA-I), apolipoprotein A2 (ApoA-2), apolipoprotein B (ApoB), apolipoprotein C (ApoC), and apolipoprotein E (ApoE), Membrane scaffold protein 1 (MSP1), MSP1D1, MSP1D2, MSP1E1, MSP1E2, MSP1E3, MSP1E3D1, MSP2, MSP2N1, MSP2N2, MSP2N3, etc.

[0061] ApoA-I, mentioned as an example above, is known to be a component of high-density lipoprotein (HDL), which primarily plays a direct role in removing cholesterol from surrounding tissues and transporting it to the liver or other lipoproteins. Apo-A1 is composed of a single polypeptide of 243 amino acids with a molecular weight of 28 kDa. It is a protein with eight repeating unit domains of 11 or 22 amino acids, and the proportion of alpha-helices in the secondary structure that forms HDL is 60 to 75%. In addition, ApoE, like ApoA1, is known to be involved in the transport of cholesterol, and is a protein composed of a single polypeptide of 299 amino acids with a molecular weight of 33 kDa.

[0062] In addition, in the present invention, as the membrane-structured protein, a fragment of an apolipoprotein that maintains the helix structure and amphipathic properties of the apolipoprotein may be used.

[0063] That is, a part (fragment) of the apolipoprotein, rather than the entire apolipoprotein, may be used within the range where the 'helical structure and amphipathic properties' of the apolipoprotein are not lost.

[0064] Meanwhile, in the present invention, the virus receptor is a receptor on the cell membrane that a virus uses when adsorbing to a cell to infect the cell, and may be an antibody to a virus surface antigen, a cell membrane-binding protein capable of binding to a virus surface antigen, a compound capable of binding to a virus surface antigen, etc. The nanodisc of the present invention exhibits the ability to bind to a virus by including a virus receptor.

[0065] In the present invention, the viral receptor can be used in various ways depending on the type of target virus for which antiviral efficacy is desired. For example, but not limited to, SARS-CoV is known to bind to cell membranes using angiotensin-converting enzyme 2 as a viral receptor. Influenza viruses are known to use compounds containing sialic acid as viral receptors. Hepatitis B virus (HBV) is known to use Na+-taurocholate cotransporting polypeptide (NTCP) as a viral receptor.

[0066] In the present invention, the Fc fragment refers to the Fc region of an antibody or a fragment thereof, which is composed of the hinge, CH2, and CH3 regions of the heavy chain of an IgG antibody, and is included in the nanodisc of the present invention to improve the pharmacodynamic characteristics of the nanodisc. In addition, when the nanodisc is combined with a virus, it may play a role in enabling immune cells, including monocytes, macrophages, neutrophils, eosinophils, dendritic cells, and natural killer cells (NK cells), to recognize the virus, thereby enabling the nanodisc to exhibit better antiviral efficacy in vivo.

[0067] That is, the virus receptor of the nanodisk of the present invention performs the role of an antigen-binding portion of an antibody, and the Fc fragment performs the role of a constant portion of an antibody, so that the nanodisk of the present invention may perform a role similar to an antibody.

[0068] In addition, drugs can be loaded onto the phospholipid bilayer of the nanodisc of the present invention, and by utilizing this, the nanodisc of the present invention can be used as a drug delivery system for viruses.

[0069] Meanwhile, the nanodisk of the present invention is composed of a phospholipid bilayer, a membrane-structuring protein, an Fc fragment, and angiotensin converting enzyme 2, wherein the Fc fragment and angiotensin converting enzyme 2 may be included in the nanodisk of the present invention in various ways.

[0070] Specifically, the viral receptor is a type of receptor on the cell membrane and contains a hydrophobic region for binding to the phospholipid bilayer of the cell membrane. Through this hydrophobic region, it can hydrophobicly bind to and be included in the nanodisk phospholipid bilayer, and can be included in the form of a peptide bond to a membrane-structuring protein.

[0071] At this time, in the case where the virus receptor is loaded into the interior of the nanodisk lipid bilayer using hydrophobic bonding (NDvR-Fc, vRFc-ND, vRFc-ND-Fc), it is preferable to use a virus receptor having a hydrophobic region, but in the case of a form in which a peptide is bonded to a membrane-structured protein, a recombinant virus receptor from which the hydrophobic region has been removed may be used.

[0072] Meanwhile, in cases where the viral receptor is bound to a membrane-structured protein (vR-ND-Fc, vR-Fc-ND, Fc-vR-ND), a membrane-structured protein may be produced by binding a gene encoding the viral receptor to a gene encoding the membrane-structured protein and then expressing the same.

[0073] The Fc fragment may be included in the nanodisc of the present invention in the form of a peptide bond to a viral receptor or a membrane-bound protein.

[0074] Specifically, in the case of vRFc-ND and vRFc-ND-Fc, the Fc fragment bound to the virus receptor may be included in the nanodisk of the present invention by hydrophobic binding of a binding protein (Virus receptor-Fc) produced by combining a gene encoding a virus receptor with a gene encoding an Fc fragment, and then expressing the same to a hydrophobic region of a lipid bilayer using a hydrophobic region of the virus receptor.

[0075] In the case of NDvR-Fc and vRFc-ND-Fc, the Fc fragment bound to the membrane-structured protein may be included in the nanodisk of the present invention by using a binding protein (MSP-Fc) manufactured by linking a gene encoding the Fc fragment to a gene encoding the membrane-structured protein and then expressing it.

[0076] Additionally, in the present invention, both the Fc fragment and the viral receptor may be included in the nanodisc of the present invention in a form bound to a membrane-structured protein.

[0077] Specifically, a binding protein (vR-MSP-Fc, vR-Fc-MSP, Fc-vR-MSP) manufactured by combining a gene encoding a viral receptor, a gene encoding a membrane structural protein, and a gene encoding an Fc fragment, and then expressing the combined protein may be included in the nanodisk of the present invention.

[0078] When manufacturing nanodiscs using vR-Fc-MSP, Fc-vR-MSP binding proteins, nanodiscs in the form of vR-Fc-ND, Fc-vR-ND can be manufactured.

[0079] When producing nanodiscs using vR-Fc-MSP binding protein, nanodiscs in the form of low molecular weight (LMW) vR-ND-Fc, middle molecular weight (MMW) vR-ND-Fc, and high molecular weight (HMW) vR-ND-Fc can be produced (see G in Fig. 2).

[0080] Meanwhile, the vR-Fc-MSP binding protein, unlike the membrane-structured protein form used in the production of existing nanodiscs, contains different proteins at both ends of the membrane-structured protein (MSP). Nevertheless, the reason why the vR-Fc-MSP binding protein can surround the phospholipid bilayer and form the nanodisc structure of the present invention is because the membrane-structured protein forms a dimer bond between the viral receptors and the membrane-structured protein forms a dimer bond between the Fc segments, so that the membrane-structured protein (MSP) can form a complete disc shape. Therefore, when using the vR-Fc-MSP binding protein, it is preferable to use a viral receptor capable of forming a dimer, such as angiotensin-converting enzyme 2 (ACE2).

[0081] At this time, when multiple vR-MSP-Fc binding proteins are linked to each other, and the viral receptor and Fc fragments surrounding one layer of the lipid bilayer of the nanodisc are mutually linked to the viral receptor and Fc fragments surrounding the other layer of the lipid bilayer to form a dimer, a nanodisc of a general size is created (see LMW vR-ND-Fc in G of Fig. 2).

[0082] When multiple vR-MSP-Fc binding proteins are linked to each other to form an elongated membrane-structured protein, and the elongated membrane-structured protein surrounds one layer and the other layer of the nanodisc bilayer, a large nanodisc with an increased diameter is created (see MMWvR-ND-Fc and HMW vR-ND-Fc in G of FIG. 2).

[0083] Furthermore, the present invention provides a pharmaceutical composition containing the nanodiscs for preventing or treating viral infections. The present invention confirms that the nanodiscs of the present invention function as "cell membrane mimics" and exhibit excellent antiviral efficacy.

[0084] Meanwhile, the pharmaceutical composition of the present invention can exhibit antiviral efficacy regardless of the type of virus. It is preferable to use it according to the type of viral receptor contained in the nanodisc. For example, if the viral receptor includes angiotensin-converting enzyme 2, it is suitable for use in inhibiting the SARS coronavirus. If it includes a compound containing sialic acid, such as ganglioside, it is suitable for use in inhibiting the influenza virus.

[0085] In the present invention, 'viral infection' may be caused by a virus of any one or more selected from among Coronaviridae, Bunyaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Poxviridae, Rhabdoviridae, Retroviridae, Togaviridae, Picornaviridae, Paramyxoviridae, and Reoviridae, but is not limited thereto.

[0086] Meanwhile, the pharmaceutical composition of the present invention may be manufactured in a unit dosage form or may be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. At this time, the dosage form may be manufactured in various forms such as oral medication and injection, and may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0087] The pharmaceutical composition of the present invention can be administered orally or parenterally, and can be administered by, for example, intrathecal administration, intravenous administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, intrasternal administration, intratumoral administration, intranasal administration, intracranial administration, intrapulmonary administration, and rectal administration, but is not limited thereto.

[0088] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. A skilled physician can easily determine and prescribe a dosage (pharmaceutically effective amount) effective for the desired treatment or prevention. For example, the daily dosage of the pharmaceutical composition of the present invention may be 0.0001-100 mg / kg.

[0089]

[0090] The present invention will be described in more detail below through the following examples. However, the present invention is not limited to the following examples and includes variations of equivalent concepts.

[0091]

[0092] [Example 1: Fabrication of nanodiscs containing virus receptors and Fc fragments]

[0093] In this example, a nanodisc containing an Fc fragment and a nanodisc containing a virus receptor and an Fc fragment were manufactured.

[0094] Meanwhile, in the examples below, angiotensin-converting enzyme 2 (ACE2) was used as an example of a viral receptor. Angiotensin-converting enzyme 2 is known to have the ability to bind to coronaviruses.

[0095]

[0096] 1-1. Production and purification of membrane-bound protein (MSP-Fc) containing the Fc fragment of an antibody

[0097] For the production of MSP-Fc binding protein, a plasmid consisting of 'a sequence encoding MSP1E3D1 and a sequence encoding the Fc fragment of the antibody (MSP1E3D1-Fc, SEQ ID NO: 2)' was prepared. Here, SEQ ID NO: 1 is the sequence encoding MSP1E3D1.

[0098] HEK293 soluble suspension cells were cultured under conditions of 37°C, 120 rpm, and 8% CO2, and 1.1x10 6 A culture medium of 180 mL cells / mL was prepared. Afterwards, 250 μg of the prepared plasmid and 750 μg of PEI were mixed in 20 mL of culture medium and transfected into the prepared suspension cells. After culturing the cells for 96 hours in an incubator at 37°C, 120 rpm, and 8% CO2, the cells were removed by centrifugation at 8000 × g for 10 minutes and only the supernatant was obtained. The entire supernatant was poured onto Protein G resin, and then the protein was purified from the resin by pouring Elution buffer (0.1 M Glycine, pH 2.8). Then, the pH of the protein was adjusted to pH 7.4 by treating with Neutralization buffer (1 M Tris, pH 9.0) to stabilize it.

[0099] Afterwards, the obtained protein was electrophoresed on an SDS-PAGE gel, and it was confirmed that the measured molecular weight was the same as expected (58 kDa) (Fig. 3).

[0100]

[0101] 1-2. Fabrication and verification of nanodiscs (ND-Fc) containing Fc fragments

[0102] Nanodiscs containing Fc fragments could be prepared by mixing the above-obtained MSP-Fc protein with phospholipids (Fig. 1A).

[0103] Specifically, as a phospholipid, POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) was dissolved in chloroform to prepare a POPC solution at a concentration of 25 mg / mL. 304 μL of the POPC solution was transferred to a glass tube so that the total lipid concentration was 10 mM and the volume was 1 mL. Afterwards, the solvent was removed using nitrogen gas and vacuum, and 1 mL of ND buffer (40 mM Tris-Cl, 300 mM NaCl, 0.5 mM EDTA, 50 mM NaC, pH 7.4) was mixed to hydrate, and then sonicated at 55°C for 30 minutes to obtain a POPC lipid film.

[0104] MSP-Fc and PCPC lipid films were mixed so that the molar ratio of MSP-Fc:lipid was 1:120. After treating the entire mixture with the same amount of bio-beads at room temperature for 5 hours, the bio-beads were removed, and ND-Fc (Immunodisc) was purified through size exclusion chromatography (SEC).

[0105] To confirm whether the above-mentioned ND-Fc was manufactured completely, the particle size was measured using dynamic light scattering (DLS), and the size of ND-Fc was 14.2 nm, which was about 2.5 nm larger than the existing nanodisk (ND, 11.6 nm), confirming that it was manufactured completely (Fig. 4A).

[0106] In addition, to distinguish between liposomes and nanodiscs of the same size, the molecular weights of general nanodiscs (ND) and nanodiscs containing Fc fragments (ND-Fc) were measured using a multi-angle light scattering (SEC-MALS) detector. The theoretical molecular weight of ND is approximately 246 kDa, and the theoretical molecular weight of ND-Fc is 296.4 kDa. As a result of absolute molecular weight measurement using SEC-MALS, ND recorded 259.6 kDa (error range 17%), and ND-Fc recorded 317.3 kDa (error range 32%), confirming that the nanodisc shape was well formed (Fig. 4B).

[0107]

[0108] 1-3. Fabrication of large nanodiscs (P2N2-ND-Fc) containing Fc fragments

[0109] Increasing the size of the nanodisc has the advantage of allowing more drugs to be loaded inside or of stronger antiviral activity. In this example, a sequence encoding a membrane-structured protein (MSP2N2) that can double the size of the disc by repeating MSP twice and a sequence encoding an Fc fragment of an antibody (MSP2N2-Fc, SEQ ID NO: 4) were used to produce a large membrane-structured protein (MSP2N2-Fc) to which an Fc fragment was bound using the method of Example 1-1, and then a large nanodisc (P2N2-NDFc) including an Fc fragment was produced using the method of Example 1-2 (Fig. 5). At this time, SEQ ID NO: 3 is a sequence encoding MSP2N2.

[0110]

[0111] 1-4. Confirmation of the production yield of ND-Fc and P2N2-ND-Fc

[0112] In this example, the production yields of ND-Fc and P2N2-ND-Fc were examined. The production yield of ND-Fc was examined. To this end, the results obtained when ND-Fc was purified by size exclusion chromatography (SEC) in Examples 1-2 and 1-3 were compared and analyzed. Meanwhile, Superose 6 and Supedex 200 were used as the size exclusion chromatography (SEC) columns, and a case was set to compare the case of using a membrane structured protein (hMSP, hMSP2N2) without an Fc fragment of an antibody produced in animal cells (HEK293 soluble suspension cells) (Fig. 6).

[0113] Looking at A and B of Figure 6, when general MSP was used, it can be confirmed that most of them were eluted at an elution volume of about 9 mL and were produced in the form of aggregates. On the other hand, it can be confirmed that most of ND-Fc was eluted at an elution volume of about 14 mL and was produced in the form of monomers. The above results indicate that the Fc fragment increased the production yield of nanodiscs.

[0114] Figure 6C is a comparison result of calculating the production yield based on the results of Figures 6A and 6B, and it can be confirmed that the production yield increased by about 2.5 times when MSP-Fc was used compared to general MSP. In addition, the production yield of P2N2-ND was about 5%, and the production yield of P2N2-ND-Fc was about 15%, confirming that the production yield increased by about 3 times.

[0115]

[0116] 1-5. Fabrication of nanodiscs (NDA-Fc) containing the viral receptor angiotensin-converting enzyme 2 (ACE2) and Fc fragments

[0117] In this example, we attempted to produce NDA-Fc by loading ACE2 (Angiotensin-converting enzyme 2, sequence number 5), which is used as an infection receptor of coronavirus, onto ND-Fc.

[0118] As lipids, POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) were dissolved in chloroform to prepare lipid solutions at concentrations of 25 mg / mL and 10 mg / mL, respectively. After dissolving in nanodisk (ND) buffer (40 mM Tris-Cl, 300 mM NaCl, 0.5 mM EDTA, 50 mM NaC, pH 7.4), 243 μL of the POPC solution and 65 μL of the DOPS solution were transferred to a glass tube so that the total lipid concentration was 10 mM, the volume was 1 mL, and the molar ratio of POPC:DOPS was 8:2. After that, nitrogen gas was added and the solvent was removed by leaving it in a vacuum for at least 4 hours to obtain a lipid film. The lipid film obtained above was mixed with 1 mL of the ND buffer solution to hydrate the lipid film, and ultrasonic treatment was performed at 55°C for 30 minutes to obtain a lipid suspension in which lipids were evenly distributed.

[0119] ACE2 (molecular weight 94.2 kDa):MSP-Fc (molecular weight 57 kDa):lipid were mixed at a molar ratio of 0.5:1:120. Then, the entire mixture was treated twice, once at room temperature for 5 hours and once at 4°C for 16 hours, with the same amount of biobeads as the entire mixture, to produce nanodiscs (NDA-Fc) containing Fc fragments and ACE2 through a self-assembly process (Fig. 7A).

[0120] The manufactured NDA-Fc was analyzed by size exclusion chromatography (SEC), and NDA-Fc was found in an elution volume of 13 to 14 mL (Fig. 7B). The fractions obtained in an elution volume of 13 to 14 mL were subjected to SDS-PAGE analysis, and bands of ACE2 and ND-Fc were observed (Fig. 7C). This confirmed that the monomer material corresponding to 13 to 14 mL was NDA-Fc containing ACE2. In addition, the DLS analysis results confirmed that the diameter of NDA-Fc (19.5 nm) was 5.3 nm larger than that of ND-Fc without ACE2 (14.2 nm), which clearly confirmed that NDA-Fc was successfully formed (Fig. 7D).

[0121] Meanwhile, as a result of comparing the production yield of nanodiscs manufactured using a general membrane-structured protein (MSP) with that of size-exclusion chromatography (SEC), it was confirmed that in the nanodisc form including the Fc fragment, most were formed in the monomer form (elution volume 13-14 mL) containing ACE2, but in the general nanodisc form, it was confirmed that some nanodiscs containing ACE2 (NDA, elution volume 14 mL) and some nanodiscs not containing ACE2 (ND, elution volume 16 mL) were formed. The above results mean that while many of the existing nanodiscs did not contain ACE2, in the case of the nanodisc form including the Fc fragment, most of the ACE2 was loaded and uniformly formed. Through this, it was confirmed that when the nanodisc includes the Fc fragment, the ACE2 loading ability is improved, so that nanodiscs can be manufactured with a higher yield (Fig. 8).

[0122]

[0123] 1-6. Fabrication of nanodiscs (AFc-ND, AFc-ND-Fc, sA-ND-Fc, sA-Fc-ND, FcsA-ND) containing the viral receptor angiotensin-converting enzyme 2 (ACE2) and Fc fragments in various forms.

[0124] In this example, nanodiscs containing angiotensin converting enzyme 2 (ACE2) and Fc fragments in more diverse forms were fabricated.

[0125] In the same manner as in Example 1-1 above, the genes for angiotensin converting enzyme 2 and Fc fragments were combined (AFc, SEQ ID NO: 6), and a binding protein (AFc, SEQ ID NO: 7) produced by expressing the same was prepared.

[0126] AFc:MSP:lipid were mixed in a molar ratio of 0.5:1:120, and AFc-ND was manufactured through a self-assembly process as in Example 1-5 (C in Fig. 1).

[0127] AFc:MSP-Fc:lipid were mixed in a molar ratio of 0.5:1:120, and AFc-ND-Fc was manufactured through a self-assembly process as in Example 1-5 (D in Fig. 1).

[0128] After the self-assembly process of AFc-ND and AFc-ND-Fc, it was confirmed that a peak was detected at the elution volume corresponding to the expected molecular weight in the SEC results (Fig. 9A). In addition, when the corresponding peak fraction was analyzed by SDS-PAGE, it was confirmed that membrane-structured proteins (MSP or MSP-Fc) and AFc were found in the fractions of AFc-ND and AFC-NDFc, and that the nanodisk shape was formed intact (Fig. 9B).

[0129] In addition, the genes of soluble angiotensin converting enzyme 2, Fc fragment, and membrane structural protein were combined to produce sACE2-Fc-MSP nucleic acid sequence (SEQ ID NO: 8), Fc-sACE2-MSP nucleic acid sequence (SEQ ID NO: 10), and sACE2-MSP-Fc nucleic acid sequence (SEQ ID NO: 12), respectively, and expressed to prepare the recombinant proteins sACE2-Fc-MSP (SEQ ID NO: 9), Fc-sACE2-MSP (SEQ ID NO: 11), and sACE2-MSP-Fc (SEQ ID NO: 13), respectively.

[0130] sACE2-Fc-MSP: The lipids were mixed at a molar ratio of 1:120, and A-Fc-ND was manufactured through a self-assembly process as in Example 1-5 (Fig. 2E).

[0131] Fc-sACE2-MSP: The lipids were mixed at a molar ratio of 1:120, and A-Fc-ND was manufactured through a self-assembly process as in Example 1-5 (F in Fig. 2).

[0132] sACE2-MSP-Fc: The lipids were mixed at a molar ratio of 1:120, and through a self-assembly process as in Example 1-5, LMW A-ND-Fc, MMW A-ND-Fc, and HMW A-ND-Fc were manufactured (G in Figure 2).

[0133] After the self-assembly process of sA-Fc-ND and sA-ND-Fc, it was confirmed that a peak was detected at the elution volume corresponding to the expected molecular weight in the SEC results (Fig. 10B). This confirmed that the nanodisk shape was formed intact.

[0134] Additionally, in the case of nanodiscs manufactured using sACE2-MSP-Fc, it was confirmed that nanodiscs were formed in two forms: nanodiscs with a normal diameter and large nanodiscs with a large diameter.

[0135]

[0136] 1-7. Fabrication of nanodiscs containing NTCP and Fc fragments as virus receptors

[0137] In this example, nanodiscs containing NTCP and Fc fragments as virus receptors were fabricated.

[0138] Specifically, in the same manner as in Example 1-1 above, the genes of NTCP and Fc fragments were combined (NTCP-Fc, SEQ ID NO: 14), and the resulting binding protein (NTCP-Fc, SEQ ID NO: 15) was prepared by expressing it (B in Fig. 11).

[0139] NTCP-Fc:MSP:lipid were mixed in a molar ratio of 0.5:1:120, and NTCP-Fc-ND was manufactured through a self-assembly process as in Example 1-5.

[0140] After the self-assembly process of NTCP-Fc-ND, it was confirmed that a peak was detected at the elution volume corresponding to the expected molecular weight in the SEC results (C in Figure 11). Afterwards, a peak at an elution volume of 14 to 15 mL was obtained, and the structure was analyzed using a transmission electron microscope (TEM), confirming that it was successfully formed in the form of a nanodisk (D in Figure 11).

[0141]

[0142] [Example 2: Evaluation of the antiviral efficacy of the nanodisc of the present invention comprising a virus receptor and an Fc fragment]

[0143] In this example, we aimed to confirm the superior antiviral efficacy of the nanodiscs of the present invention, which include a viral receptor and an Fc fragment. To this end, a CPE inhibition assay using an authentic SARS-CoV-2 virus was conducted to confirm the antiviral efficacy of NDA-Fc, AFc-ND, and sA-ND-Fc prepared in Example 1.

[0144] Specifically, Calu-3, Vero E6 cells were cultured at 2 X 10 5 100 μL was dispensed into each well of a 96-well cell culture plate at a concentration of 100 cells / mL and cultured in a 5% CO2 incubator at 37°C for 24 hours. The medium was removed from the cells, and 50 μL of SARS-CoV-2 virus at 100 TCID50 (Tissue cell infectious dose) and 50 uL of antiviral agents diluted at various concentrations were mixed. 100 μL of the mixture was treated to each well and infected at 37°C for 1 hour. The supernatant was removed, and 100 μL of the medium containing the antiviral agents at each concentration was dispensed to each well and cultured in a 5% CO2 incubator at 37°C for 48 to 72 hours.

[0145] After removing the cell supernatant, 100 μL of 4% formaldehyde was dispensed per well and fixed at 25°C for 1 hour. Then, 100 μL of 0.5% crystal violet solution was dispensed per well and stained at 25°C for 1 hour. The crystal violet solution was removed, and 100 μL of methanol was treated per well to dissolve the dried crystal violet. The absorbance at a wavelength of 570 nm was measured using a spectrophotometer to determine the cytopathic effect. Meanwhile, SARS-CoV-2 infects cells through two major routes. The first is the endocytic pathway infection, which is the infection route when only ACE2 is expressed on the surface, such as Vero E6 cells. The second is direct fusion infection, which is an infection route in cases where ACE2 and TMPRSS2 (SEQ ID NO: 16) are simultaneously expressed, such as in Calu-3 cells (Fig. 12A).

[0146] As shown in FIG. 12A, it can be confirmed that the antiviral efficacy of the nanodisc (NDA-Fc) of the present invention containing ACE2 and Fc fragments is superior to that of the nanodisc (NDA) containing ACE2.

[0147] In addition, as a result of comparing the antiviral efficacy of each NDA, AFc-ND, sAND, and sA-ND-Fc using Vero E6 cells in the same manner as described above, it was confirmed that the antiviral efficacy of the nanodisc of the present invention (AFc-ND, sAND-Fc) containing ACE2 and Fc fragments was superior to that of the nanodisc (NDA, sAND) containing ACE2 (B in FIG. 12).

[0148] The above results indicate that the nanodisc of the present invention comprising a virus receptor and an Fc fragment exhibits excellent antiviral efficacy.

[0149]

[0150] [Example 3: Evaluation of Pharmacodynamic Properties of Nanodiscs of the Present Invention Comprising Viral Receptor and Fc Fragment]

[0151] In this example, the pharmacodynamic properties of the nanodiscs of the present invention, including a viral receptor and an Fc fragment, were examined. To this end, ND-Fc and NDA-Fc prepared in Example 1 were injected into mice, and their in vivo half-lives were determined.

[0152]

[0153] 3-1. Evaluation of the pharmacodynamic properties of ND-Fc

[0154] After 24, 48, 96, and 168 hours of injection of scFv-Fc type antibody P2B-2F6 (SEQ ID NO: 17) or ND-Fc through the tail vein of mice, blood and each organ (Heart, Larynx, Trachea, Lung, Liver, Spleen, Kidney) were collected from each experimental group of mice, ground, and the concentrations of the injected antibodies and ND-Fc in the body were confirmed through ELISA (Fig. 13).

[0155] Figure 13A shows the concentration of antibody or ND-Fc distributed in each organ. In the case of antibodies, it was confirmed that they were widely distributed in various organs. On the other hand, ND-Fc of the present invention showed a distribution concentrated in the liver. This result is presumed to be due to the protein-lipid structure of ND-Fc having a structure similar to HDL recognized by hepatocytes.

[0156] Figure 13B shows the concentration of antibody or ND-Fc in mouse serum, and it can be confirmed that both antibody (scFv-Fc) and ND-Fc exhibit similar levels of half-life. The above results indicate that ND-Fc has excellent pharmacodynamic properties similar to those of antibody (scFv-Fc).

[0157]

[0158] 3-2. Pharmacodynamic properties of NDA-Fc

[0159] After 4.5 mg / kg of sACE2-Fc (SEQ ID NO: 18) or NDA-Fc containing ACE2 was injected into the mouse tail vein (Intravenous administration, IV) or nasal cavity (Intranasal administration, IN), blood and each organ (Heart, Larynx, Trachea, Lung, Liver, Spleen, Kidney) were collected from each experimental group of mice after 1, 12, 24, 48, and 168 hours for intravenous injection, and after 1, 6, 12, 24, and 96 hours for intranasal injection. These were then ground and the concentrations of sACE2-Fc and NDA-Fc injected into the body were confirmed through ELISA (Fig. 14).

[0160] Figure 14A shows the results of confirming the concentration distributed to each organ after intravenous injection of sACE2-Fc or NDA-Fc. It can be confirmed that sACE2-Fc and NDA-Fc were present in the majority of serum and moved primarily to the upper respiratory tract, including the larynx and trachea.

[0161] Figure 14B shows the results of analyzing the in vivo half-life in mouse serum after intravenous injection of sACE2-Fc or NDA-Fc. It can be confirmed that the in vivo half-life of NDA-Fc containing ACE2 is 166.1 hr, which is a higher value than the half-life of sACE2-Fc (23.5 hr).

[0162] Meanwhile, the half-life of conventional protein-based nanodiscs reported in the literature is known to be about 0.5 to 2 hours and up to 60 hours, and even in the case of nanodiscs with a long half-life, almost no nanodiscs were observed in the blood after 96 hours (Park, Hyun-Ji, et al "High-density lipoprotein-mimicking nanodiscs carrying peptide for enhanced therapeutic angiogenesis in diabetic hindlimb ischemia" Biomaterials 161 (2018): 69-80). However, it was confirmed that the NDA-Fc of the present invention maintained almost the same concentration without a significant difference from the initial concentration even after 48 hours, and it was confirmed that the concentration of NDA-Fc in the serum maintained a high concentration of about 40% of the initial concentration even after 168 hours. This means that the half-life of the nanodisc of the present invention is very long.

[0163] Figure 14C shows the results of confirming the concentration distributed to each organ after nasal injection of sACE2-Fc or NDA-Fc. sACE2-Fc was found mostly in the lungs, whereas NDA-Fc was evenly distributed in the larynx, trachea, and lungs. In addition, it can be confirmed that the area under the curve (AUC) of NDA-Fc is relatively very large.

[0164] The above results indicate that the nanodiscs of the present invention, including a viral receptor and an Fc fragment, can exhibit excellent efficacy in vivo with a low half-life.

Claims

1. A lipid bilayer, a flat disc-shaped bilayer structure formed from phospholipids, in which the hydrophilic groups are oriented outward and the hydrophobic groups are oriented inward; Membrane scaffold protein (MSP) surrounding the 'side where the hydrophobic group is exposed to the outside' of the lipid bilayer; and In a nanodisk comprising a virus receptor hydrophobicly bonded to the lipid bilayer; A nanodisc characterized in that an Fc fragment is bound to the virus receptor and the Fc fragment protrudes to the outside of the nanodisc.

2. A lipid bilayer, a flat disc-shaped bilayer structure formed from phospholipids, in which the hydrophilic groups are oriented outward and the hydrophobic groups are oriented inward; Membrane scaffold protein (MSP) surrounding the 'side where the hydrophobic group is exposed to the outside' of the lipid bilayer; and In a nanodisk comprising a virus receptor hydrophobicly bonded to the lipid bilayer; An Fc fragment is bound to the above membrane-structured protein, and the Fc fragment protrudes to the outside of the nanodisk. A nanodisc characterized in that an Fc fragment is bound to the virus receptor and the Fc fragment protrudes to the outside of the nanodisc.

3. A lipid bilayer having a flat disc-shaped bilayer structure formed from phospholipids, with the hydrophilic groups oriented outward and the hydrophobic groups oriented inward; and In a nanodisk comprising a membrane scaffold protein (MSP) surrounding the 'side where the hydrophobic group is exposed to the outside' of the lipid bilayer, The above membrane-structured protein has a virus receptor bound to one end and an Fc fragment bound to the other end. A nanodisc characterized in that the above-mentioned combined virus receptor and Fc fragment protrude to the outside of the nanodisc.

4. A lipid bilayer having a flat disc-shaped bilayer structure formed from phospholipids, with hydrophilic groups oriented outward and hydrophobic groups oriented inward; and In a nanodisk comprising a membrane scaffold protein (MSP) surrounding the 'side where the hydrophobic group is exposed to the outside' of the lipid bilayer, The above membrane-structured protein has a virus receptor bound to one end and an Fc fragment bound to the other end. It is a membrane-structured protein in which multiple membrane-structured proteins are interconnected to form a dimer by combining 'virus receptors of membrane-structured proteins' with each other and forming a dimer by combining 'Fc fragments of membrane-structured proteins' with each other, thereby forming a dimer. A nanodisc characterized in that the above-mentioned combined virus receptor and Fc fragment protrude to the outside of the nanodisc.

5. A lipid bilayer having a flat disc-shaped bilayer structure formed from phospholipids, with hydrophilic groups oriented outward and hydrophobic groups oriented inward; and In a nanodisk comprising a membrane scaffold protein (MSP) surrounding the 'side where the hydrophobic group is exposed to the outside' of the lipid bilayer, The above membrane-structured protein has a virus receptor and an Fc fragment bound to one end, A nanodisc characterized in that the above-mentioned combined virus receptor and Fc fragment protrude to the outside of the nanodisc.

6. In any one of the clauses 1 to 5, The above phospholipids are, A nanodisk characterized by comprising at least one selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethalolamine, phosphatidylglycerol, and phosphatidylinositol.

7. In any one of the clauses 1 to 5, The above phospholipids are, A nanodisk characterized by comprising at least one selected from the group consisting of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), and POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine).

8. In any one of the clauses 1 to 5, The above membrane scaffold protein is, A nanodisk characterized by being an amphipathic protein having a helix structure.

9. In paragraph 8, The above membrane scaffold protein is, A nanodisc characterized by being an apolipoprotein or an apolipoprotein fragment that maintains the 'helix structure and amphipathic properties' of the apolipoprotein.

10. In paragraph 2, The Fc fragment bound to the above membrane structural protein is, A nanodisc characterized by being manufactured by linking a gene encoding an Fc fragment to a gene encoding a membrane-structured protein and then expressing the same.

11. In paragraph 1 or 2, The Fc fragment bound to the above viral receptor is, A nanodisc characterized by being manufactured by combining a gene encoding a viral receptor and a gene encoding an Fc fragment and then expressing the same.

12. In paragraph 3 or 4, The viral receptor and Fc fragments bound to the above membrane structural proteins are, A nanodisc characterized by being manufactured by linking a gene encoding a viral receptor to one end of a gene encoding a membrane-structured protein and linking a gene encoding an Fc fragment to the other end, and then expressing the same.

13. In paragraph 5, The above membrane-structuring protein is, A nanodisc characterized by having a virus receptor and an Fc fragment sequentially bound to one end.

14. In paragraph 13, The viral receptor and Fc fragments bound to the above membrane structural proteins are, A nanodisc characterized by being manufactured by linking a gene encoding an Fc fragment to the 5' end of a gene encoding a membrane structural protein, linking a gene encoding a viral receptor to the 5' end of the gene encoding the Fc fragment, and then expressing the same.

15. In paragraph 5, The above membrane-structuring protein is, A nanodisc characterized by having an Fc fragment and a virus receptor sequentially bound to one end.

16. In paragraph 15, The Fc fragment and viral receptor bound to the above membrane structural protein, A nanodisc characterized by being manufactured by linking a gene encoding a viral receptor to the 5' end of a gene encoding a membrane structural protein, linking a gene encoding an Fc fragment to the 5' end of the gene encoding the viral receptor, and then expressing the same.

17. In paragraph 3, The above nanodisks are, A nanodisk characterized in that a dimer is formed by mutual binding of a viral receptor of a membrane-structured protein surrounding one layer of the above bilayers and a viral receptor of a membrane-structured protein surrounding the other layer of the above bilayers.

18. In paragraph 3, The above nanodisks are, The Fc fragment of a membrane-structured protein surrounding one of the above bilayers, and A nanodisc characterized in that the Fc fragments of membrane-structured proteins surrounding another layer of the above bilayer are mutually combined to form a dimer.

19. In paragraph 4, The above-mentioned membrane-structured protein having an extended length is, A nanodisc characterized by being a membrane-structured protein whose length is extended by interconnecting two membrane-structured proteins through the mutual binding of two 'viral receptors of membrane-structured proteins' to form a dimer and through the mutual binding of two 'Fc fragments of membrane-structured proteins' to form a dimer.

20. In paragraph 4, The above nanodisks are, One of the above bilayers is surrounded by a membrane-structured protein whose length is extended, A nanodisc characterized in that another layer of said bilayer is surrounded by another elongated membrane-structured protein.

21. A pharmaceutical composition for preventing or treating viral infection, characterized by containing a nanodisk selected from any one of claims 1 to 5.

22. In paragraph 21, The above virus, Coronaviridae, Bunyaviridae, and Phyllostaviruses A pharmaceutical composition for preventing or treating a viral infection, characterized in that it is at least one selected from the following families: Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Poxviridae, Rhabdoviridae, Retroviridae, Togaviridae, Picornaviridae, Paramyxoviridae, and Reoviridae.

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