Nanodisc comprising angiotensin-converting enzyme 2 and fc fragment, and use thereof

Abstract

The present invention provides: a nanodisc comprising angiotensin-converting enzyme 2 and an Fc fragment; an antiviral use of the nanodisc; and a use of the nanodisc for diseases caused by angiotensin-converting enzyme 2 deficiency. Using the nanodisc of the present invention can improve the pharmacokinetic characteristics of a pharmaceutical composition containing angiotensin-converting enzyme 2 as an active ingredient.

Description

Nanodiscs comprising angiotensin converting enzyme 2 and FC fragments and uses thereof

[0001] The present invention relates to a nanodisc comprising angiotensin converting enzyme 2 and an Fc fragment, and a pharmaceutical composition containing the nanodisc for preventing or treating 'viral infection' or 'disease caused by angiotensin converting enzyme 2 deficiency'.

[0002] Angiotensin-converting enzyme 2 (ACE2) is an enzyme widely found on the surface of human cells, particularly in the lungs, where it cleaves angiotensin II (Ang II) to produce angiotensin (1-7) or angiotensin I (Ang I) to produce angiotensin (1-9). ACE2 is also found in the human heart, liver, digestive tract, kidneys, and vascular endothelium, and is thought to play a protective role in protecting against fibrosis and inflammation in many organs, including the liver, lungs, and heart.

[0003] Angiotensin-converting enzyme 2 (ACE) is part of the renin-angiotensin system. The renin-angiotensin system (RAS) is an endocrine system that traditionally plays a key role in blood pressure regulation. In the renin-angiotensin system, the enzyme renin acts on the circulating AGT protein. Renin cleaves AGT to produce a 10-amino acid fragment known as angiotensin I (Ang I). Angiotensin I is then converted by the angiotensin-converting enzyme (ACE) to the active octapeptide angiotensin II (Ang II), which binds to specific cell-surface angiotensin receptors and exerts its action. Angiotensin II directly has 'adverse effects' on the cardiovascular system, including promoting vascular inflammation and atherosclerosis. Angiotensin-converting enzyme 2 cleaves angiotensin II (Ang II) and converts it to Ang-(1-7), thereby maintaining homeostasis.

[0004] Meanwhile, a nanodisc is a structure formed by wrapping a phospholipid bilayer membrane in a disc shape 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 research on various cell membrane proteins. In addition, they also serve as a vehicle to deliver various physiologically functional substances inside and into the body. As a biologically derived substance, nanodiscs have the advantage of being stable in the body and not causing harmful reactions, making them safe.

[0005] The present invention aims to provide a technology capable of improving the pharmacodynamic characteristics of angiotensin converting enzyme 2.

[0006] The present invention provides a nanodisc comprising a phospholipid bilayer, a membrane-structuring protein, an Fc fragment, and angiotensin converting enzyme 2.

[0007] Specifically, the present invention provides a nanodisc comprising, in a first form, 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 angiotensin-converting enzyme 2 (ACE2) hydrophobicly bonded to the lipid bilayer; wherein an Fc fragment is bound to the membrane scaffold protein, and the Fc fragment protrudes to the outside of the nanodisc.

[0008] 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 angiotensin-converting enzyme 2 (ACE2) hydrophobicly bonded to the lipid bilayer; wherein an Fc fragment is bound to the angiotensin-converting enzyme 2 (ACE2), and the Fc fragment protrudes to the outside of the nanodisc.

[0009] The present invention provides a nanodisc comprising, in a third aspect, 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 angiotensin-converting enzyme 2 (ACE2) 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 angiotensin-converting enzyme 2 (ACE2) is bound to the Fc fragment, such that the Fc fragment protrudes to the outside of the nanodisc.

[0010] The present invention provides a nanodisc in a fourth aspect, 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 angiotensin-converting enzyme 2 (ACE2) bound to one end and an Fc fragment bound to the other end, and wherein the bound angiotensin-converting enzyme 2 (ACE2) and the Fc fragment protrude to the outside of the nanodisc.

[0011] The present invention, in its fifth form, is 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; And a membrane scaffold protein (MSP) surrounding the 'side of the lipid bilayer where the hydrophobic group is exposed to the outside'; wherein the membrane scaffold protein has angiotensin-converting enzyme 2 (ACE2) bound to one end and an Fc fragment bound to the other end, and the 'angiotensin-converting enzyme 2 of the membrane scaffold protein' binds to each other to form a dimer, and the 'Fc fragment of the membrane scaffold protein' binds to each other to form a dimer, thereby forming a membrane scaffold protein in which a plurality of membrane scaffold proteins are interconnected to form an extended length, and the bound angiotensin-converting enzyme 2 (ACE2) and Fc fragments protrude to the outside of the nanodisk.

[0012] The present invention provides a nanodisc in a sixth form, 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 angiotensin-converting enzyme 2 (ACE2) and an Fc fragment bound to one end thereof, and the bound angiotensin-converting enzyme 2 (ACE2) and angiotensin-converting enzyme 2 (Fc fragment) protruding to the outside of the nanodisc.

[0013] In the nanodisk of the present invention, the phospholipid may be at least one selected from phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol, 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).

[0014] In the nanodisk of the present invention, the membrane scaffold protein is an amphipathic protein having a helix structure. In this case, the membrane scaffold protein may be an apolipoprotein or a fragment of an apolipoprotein in which the 'helix structure and amphipathic properties' of the apolipoprotein are maintained.

[0015] In the nanodiscs of the first, second, and third forms of the present invention, the angiotensin converting enzyme 2 may have a hydrophobic transmembrane domain at the C-terminus that is hydrophobicly bound to a hydrophobic region of the lipid bilayer.

[0016] In the nanodisks of the fourth, fifth, and sixth forms of the present invention, the angiotensin converting enzyme 2 is preferably a water-soluble angiotensin converting enzyme 2 from which the transmembrane domain has been removed.

[0017] In the first or third form of the 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.

[0018] In the second and third forms of the nanodisk of the present invention, the Fc fragment bound to angiotensin converting enzyme 2 may be produced by combining a gene encoding angiotensin converting enzyme 2 with a gene encoding an Fc fragment, and then expressing the same.

[0019] In the fourth and fifth forms of the nanodisk of the present invention, the angiotensin converting enzyme 2 and Fc fragments bound to the membrane-structured protein may be produced by binding a gene encoding angiotensin converting enzyme 2 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.

[0020] In the sixth type nanodisk of the present invention, the membrane-structured protein may have angiotensin-converting enzyme 2 (ACE2) 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 angiotensin-converting enzyme 2 to the 5' end of the gene encoding the Fc fragment, and then expressing the same.

[0021] In the sixth type nanodisk of the present invention, the membrane-structured protein may have an Fc fragment and angiotensin-converting enzyme 2 (ACE2) sequentially bound to one end. At this time, the Fc fragment and angiotensin-converting enzyme 2 bound to the membrane-structured protein may be produced by binding a gene encoding angiotensin-converting enzyme 2 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 angiotensin-converting enzyme 2, and then expressing the same.

[0022] The fourth type nanodisk of the present invention may be one in which angiotensin converting enzyme 2 of a membrane-structured protein surrounding one of the bilayers and angiotensin converting enzyme 2 of a membrane-structured protein surrounding another of the bilayers bind to each other to form a dimer, and may be one in which 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 bind to each other to form a dimer.

[0023] In the fifth type nanodisk of the present invention, the membrane-structured protein with an extended length may be a membrane-structured protein with an extended length, in which two 'angiotensin converting enzyme 2 of the membrane-structured protein' are mutually combined to form a dimer, and two 'Fc fragments of the membrane-structured protein' are mutually combined to form a dimer, thereby forming a membrane-structured protein with an extended length by connecting two membrane-structured proteins to each other.

[0024] The fifth type nanodisk of the present invention may be one in which one of the bilayers is surrounded by an elongated membrane-structured protein, and the other layer of the bilayers is surrounded by another elongated membrane-structured protein.

[0025] Meanwhile, the nanodisc of the present invention may have improved substrate conversion ability of angiotensin converting enzyme 2. At this time, the substrate conversion ability may be a substrate conversion ability that converts angiotensin II into angiotensin (1-7) or angiotensin I into angiotensin (1-9).

[0026] The nanodisc of the present invention may increase the half-life of angiotensin converting enzyme 2.

[0027] In addition, the present invention provides a pharmaceutical composition for preventing or treating a viral infection, characterized in that it contains 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, and the Togaviridae family.

[0028] In addition, the present invention provides a pharmaceutical composition for preventing or treating a disease caused by angiotensin converting enzyme 2 deficiency, characterized in that it contains the nanodisc. In this case, the disease caused by angiotensin converting enzyme 2 deficiency may be at least one selected from pneumonia, pulmonary edema, pulmonary fibrosis, cardiac fibrosis, hepatic fibrosis, kidney patterns, kidney failure, bronchopulmonary dysplasia, diabetic nephropathy, and coronavirus disease-19 (COVID-19).

[0029] In the present invention, it was confirmed that the nanodiscs of the present invention comprising angiotensin converting enzyme 2 and an Fc fragment have superior substrate conversion ability compared to angiotensin converting enzyme 2 and a long half-life in the body. Therefore, when using the present invention, the pharmacodynamic characteristics of a pharmaceutical composition comprising angiotensin converting enzyme 2 as an active ingredient can be improved.

[0030] In addition, the nanodisc of the present invention comprising angiotensin converting enzyme 2 and Fc fragments has excellent antiviral efficacy.

[0031] Figures 1 to 4 schematically show the manufacturing process and form of nanodiscs. Figure 1A shows a nanodisc (ND-Fc) containing an Fc fragment, Figure 1B and Figures 2 to 4 schematically show the manufacturing process and form of nanodiscs of the present invention containing an Fc fragment and angiotensin converting enzyme 2 (NDA-Fc, AFc-ND, AFc-ND-Fc, A-Fc-ND, Fc-A-ND, A-ND-Fc).

[0032] Figure 5 shows the results of confirming the molecular weight through electrophoresis after producing a membrane-structured protein (MSP-Fc) to which the Fc fragment of the antibody is bound, to confirm whether the membrane-structured protein (MSP-Fc) to which the Fc fragment of the antibody is bound is produced intact.

[0033] Figure 6 shows the results of measuring particle size using dynamic light scattering (DLS) (Figure 6A) and measuring molecular weight using a multi-angle optical scattering (SEC-MALS) detector (Figure 6B) to confirm whether the nanodisc containing the Fc fragment (ND-Fc) was completely manufactured.

[0034] Figure 7 schematically shows the differences in the manufacturing process of nanodiscs (ND) and large nanodiscs (P2N2-ND) containing E. 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 8 shows the results of 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 8), and comparing the purification yield with that of general nanodiscs (ND) (C of Figure 8).

[0036] Figure 9 shows the manufacturing process of the nanodisc (NDA-Fc) of the present invention including the Fc fragment and angiotensin converting enzyme 2 (Figure 9A), the purification process using size exclusion chromatography (SEC) (Figure 9B), the results of SDS-PAGE analysis to confirm whether the nanodisc was manufactured intact (Figure 9C), and the results of dynamic light scattering (DLS) measurement (Figure 9D).

[0037] Figure 10 shows the results of size exclusion chromatography (SEC) performed to confirm the excellent production yield of the nanodisc (NDA-Fc) of the present invention including the Fc fragment and angiotensin converting enzyme 2, compared with the case of the nanodisc (NDA) including angiotensin converting enzyme 2.

[0038] Figure 11 shows the purification process using size exclusion chromatography (SEC) after self-assembly of the nanodisc (AFc-ND, AFc-ND-Fc) of the present invention including the Fc fragment and angiotensin converting enzyme 2 (Figure 11A) and the results of fabrication confirmation using SDS-PAGE to confirm whether the nanodisc was completely fabricated (Figure 11B).

[0039] Figure 12 shows the results of purifying membrane-structured proteins (sACE2-Fc-MSP, sACE2-MSP-Fc) for producing nanodiscs (sA-Fc-ND, sA-ND-Fc) of the present invention including an Fc region and angiotensin converting enzyme 2 (A of Figure 12) 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) (B of Figure 12).

[0040] Figure 13 shows the results of a CPE inhibition experiment conducted to confirm the excellent antiviral efficacy of the nanodiscs (NDA-Fc, AFc-ND, sA-ND-Fc) of the present invention including an Fc fragment and angiotensin converting enzyme 2, compared with the nanodiscs (sAND, NDA) including angiotensin converting enzyme 2.

[0041] Figure 14 shows the results of confirming the concentration distributed in each organ (A of Figure 14) and serum half-life (B of Figure 14) after injecting scFv-Fc antibody (P2B-2FB) or ND-Fc into mice to evaluate the pharmacodynamic properties of the nanodisk (ND-Fc) of the present invention including an Fc fragment.

[0042] Figure 15 shows the results of confirming the concentration distributed in each organ (Figure 15A) and serum half-life (Figure 15B) after intravenous administration (IV) of sACE2-Fc or NDA-Fc to mice to evaluate the pharmacodynamic properties of the nanodisc (NDA-Fc) of the present invention comprising an Fc fragment and angiotensin-converting enzyme 2. In addition, Figure 15 shows the results of confirming the concentration distributed in each organ after intranasal administration (IN) of sACE2-Fc or NDA-Fc to mice (Figure 15C).

[0043] 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.

[0044] The half-life of typical nanodiscs reported in the literature is approximately 0.5 to 2 hours and up to 60 hours, and it is known that almost no nanodiscs are observed in the blood after 96 hours (Denisov, Ilia G et al; Simon-Gracia, Lorena et al; Park, Hyun-Ji et al).

[0045] 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, when the angiotensin converting enzyme 2 was loaded onto the nanodisc and the half-life in the body was confirmed, 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 nanodisc concentration in the serum maintained a high concentration of about 40% of the initial concentration even after 168 hours.

[0046] That is, in the present invention, it was confirmed that the half-life of angiotensin converting enzyme 2 can be increased when angiotensin converting enzyme 2 is loaded onto a nanodisc including an Fc fragment.

[0047] In addition, the inventors of the present invention have confirmed through Korean Patent No. 10-2022-0022501 that the substrate conversion ability of angiotensin converting enzyme 2 can be improved when angiotensin converting enzyme 2 is loaded onto a nanodisk.

[0048] Accordingly, the present invention provides a nanodisc comprising an Fc fragment and angiotensin converting enzyme 2. That is, when the present invention is used, the pharmacodynamic characteristics of a pharmaceutical composition using angiotensin converting enzyme 2 as an active ingredient can be improved.

[0049] 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. In this case, the Fc fragment and angiotensin converting enzyme 2 may be included in the nanodisk in various forms.

[0050] Specifically, angiotensin converting enzyme 2 may be hydrophobicly bound to the lipid bilayer of the nanodisc, and the Fc fragment may be bound to the membrane-structuring protein of the nanodisc (NDA-Fc, see B in Figure 1).

[0051] Angiotensin converting enzyme 2 and Fc fragment-bound proteins can be hydrophobic-bound to the lipid bilayer of nanodiscs (AFc-ND, see C in Fig. 2).

[0052] Angiotensin converting enzyme 2 and a protein bound to the Fc fragment may be hydrophobicly bound to the lipid bilayer of the nanodisc, and another Fc fragment may be additionally bound to a membrane-structured protein (AFc-ND-Fc, see D in Figure 2).

[0053] Angiotensin converting enzyme 2 and Fc fragments may be bound to one end of a nanodisc membrane-structured protein (A-Fc-ND, Fc-A-ND, see E, F of Figure 3).

[0054] Angiotensin converting enzyme 2 and Fc fragments may be bound to both ends of the nanodisc membrane-structured protein, respectively (LMW A-ND-Fc, MMW A-ND-Fc, HMW A-ND-Fc, see G in Fig. 4).

[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-sn-glycero-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-stearoyl-sn-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-Dimyristoyl-sn-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-sn-glycero-3[Phospho-rac-(1-glycerol)]), and the phosphatidylethanolamine may be DMPE (1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-Dipalmitoyl-sn-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-sn-glycero-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 plays a role in surrounding the side of the 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 multiple membrane-structuring proteins bound together'. In this case, when a single layer is surrounded by a 'membrane-structuring protein whose length is extended by multiple membrane-structuring proteins bound together', a large nanodisk with an increased 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. Examples of the above apolipoproteins include 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 may be used, which maintains the 'helical structure and amphipathic properties' of the apolipoprotein. In other words, a part (fragment) of the apolipoprotein, rather than the entire apolipoprotein, may be used, as long as the 'helical structure and amphipathic properties' of the apolipoprotein are not lost.

[0063] Meanwhile, in the present invention, angiotensin-converting enzyme 2 (ACE2) is an enzyme widely distributed on the surface of human cells, especially in the lungs, and exhibits substrate conversion ability to cleave angiotensin II (Ang II) to produce angiotensin (1-7) or cleave angiotensin I (Ang I) to produce angiotensin (1-9).

[0064] Meanwhile, in the present invention, the Fc fragment and angiotensin converting enzyme 2 can be included in the nanodisc of the present invention in various forms.

[0065] Angiotensin converting enzyme 2 has a hydrophobic transmembrane domain at the C-terminus, and this region can be included in the nanodisc of the present invention by utilizing the fact that it can hydrophobicly bind to hydrophobic regions of a lipid bilayer or bind to membrane-structuring proteins.

[0066] At this time, in the case where angiotensin converting enzyme 2 is loaded inside the nanodisk lipid bilayer using hydrophobic bonds (NDA-Fc, AFc-ND, AFc-ND-Fc), it is preferable to use full-length angiotensin converting enzyme 2 having a transmembrane domain, which is a hydrophobic region, but in the case where angiotensin converting enzyme 2 is bound to a membrane-structured protein (A-ND-Fc, A-Fc-ND, Fc-A-ND), recombinant angiotensin converting enzyme 2 (sACE2) with the transmembrane domain removed may be used.

[0067] Meanwhile, in the case where angiotensin converting enzyme 2 is bound to a membrane structural protein (A-ND-Fc, A-Fc-ND, Fc-A-ND), a membrane structural protein may be produced by binding a gene encoding angiotensin converting enzyme 2 to a gene encoding a membrane structural protein and then expressing the same.

[0068] Additionally, in the present invention, the Fc fragment may be included in the nanodisc of the present invention in a form bound to 'angiotensin converting enzyme 2 or membrane structural protein'.

[0069] Specifically, the Fc fragment bound to angiotensin converting enzyme 2, such as in the case of AFc-ND and AFc-ND-Fc, may be included in the nanodisk of the present invention by hydrophobic binding of a binding protein (ACE2-Fc) produced by linking a gene encoding angiotensin converting enzyme 2 and a gene encoding an Fc fragment, and then expressing the same to a hydrophobic region of a lipid bilayer using the transmembrane domain, which is a hydrophobic region of angiotensin converting enzyme 2.

[0070] In the case of NDA-Fc and AFc-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.

[0071] Additionally, in the present invention, both the Fc fragment and angiotensin converting enzyme 2 may be included in the nanodisc of the present invention in a form bound to a membrane-structured protein.

[0072] Specifically, a binding protein (ACE2-MSP-Fc, ACE2-Fc-MSP, Fc-ACE2-MSP) manufactured by combining a gene encoding angiotensin converting enzyme 2, 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.

[0073] When manufacturing nanodiscs using the ACE2-Fc-MSP binding protein, nanodiscs in the form of low molecular weight (LMW) sA-ND-Fc, middle molecular weight (MMW) sA-ND-Fc, and high molecular weight (HMW) sA-ND-Fc can be manufactured (see G in Fig. 4).

[0074] Meanwhile, the ACE2-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 ACE2-Fc-MSP binding protein can surround the phospholipid bilayer and form the nanodisc structure of the present invention is because angiotensin-converting enzyme 2 (ACE2) and the Fc fragment are combined in the form of a homodimer, so that the membrane-structured protein (MSP) can form a complete disc shape. That is, multiple ACE2-Fc-MSP binding proteins form a disc structure by combining the angiotensin-converting enzyme 2 (ACE2) and Fc fragments of each other.

[0075] At this time, when multiple ACE2-MSP-Fc binding proteins are linked to each other, and the angiotensin converting enzyme 2 and Fc regions surrounding one of the lipid bilayers of the nanodisc are mutually linked to the angiotensin converting enzyme 2 and Fc regions surrounding the other lipid bilayer to form a dimer, a nanodisc of a general size is created (see LMW sA-ND-Fc in G of Figure 4).

[0076] When multiple ACE2-MSP-Fc binding proteins are linked to each other and form a membrane-structured protein with an extended length, a large nanodisk is created that forms a large disk and has an increased diameter (see True MMW sA-ND-Fc and HMW sA-ND-Fc in G of FIG. 4).

[0077] When manufacturing nanodiscs using ACE2-Fc-MSP, Fc-ACE2-MSP binding protein, nanodiscs in the form of A-Fc-ND, Fc-A-ND can be manufactured.

[0078] 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.

[0079] Meanwhile, the pharmaceutical composition of the present invention can exhibit antiviral efficacy regardless of the type of virus. Therefore, in the present invention, 'viral infection' may be caused by any one or more viruses selected from the Coronaviridae family, Bunyaviridae family, Filoviridae family, Flaviviridae family, Hepadnaviridae family, Herpesviridae family, Orthomyxoviridae family, Poxviridae family, Rhabdoviridae family, Retroviridae family, and Togaviridae family.

[0080] However, since the nanodisc of the present invention contains angiotensin-converting enzyme 2, which can act as a virus receptor, it is preferably a virus that recognizes angiotensin-converting enzyme 2 as a virus receptor. A specific example of a virus that recognizes angiotensin-converting enzyme 2 as a virus receptor is SARS-CoV-2.

[0081]

[0082] In addition, the present invention provides a pharmaceutical composition for preventing or treating a disease caused by angiotensin converting enzyme 2 deficiency, characterized by containing the nanodisc. The nanodisc of the present invention has excellent substrate conversion ability compared to angiotensin converting enzyme 2 and a long half-life in the body.

[0083] Meanwhile, in the present invention, the disease caused by angiotensin converting enzyme 2 deficiency refers to diseases in which angiotensin converting enzyme 2 can be an active ingredient and exhibit efficacy. More specifically, it may refer to diseases caused by angiotensin converting enzyme 2 deficiency, diseases accompanied by angiotensin converting enzyme 2 deficiency, diseases whose symptoms are prevented or treated when the activity of angiotensin converting enzyme 2 is increased, etc.

[0084] Specific examples of diseases associated with angiotensin-converting enzyme 2 deficiency include pneumonia, pulmonary edema, bronchopulmonary dysplasia, pulmonary fibrosis, cardiac fibrosis, hepatic fibrosis, kidney patterns, kidney failure, diabetic nephropathy, and COVID-19.

[0085] Angiotensin-converting enzyme 2 (ACE2) and lung disease: Angiotensin-converting enzyme 2 deficiency has been reported to exacerbate pulmonary edema following influenza A H5N1 and H7N9 virus infection. Administration of recombinant human ACE2 has been reported to improve lung injury induced by avian influenza H5N1 virus in mice. Bronchopulmonary dysplasia (BPD) is a chronic lung disease of infants that presents as a systemic syndrome and is associated with neurodevelopmental defects, cognitive impairment, growth failure, pulmonary hypertension, and cardiopulmonary dysfunction. In adult animal models of acute lung injury, ACE2 has been reported to suppress pulmonary edema formation, inflammation, and fibrogenesis. Results suggesting that ACE2 may play a similar role have also been reported in a neonatal bronchopulmonary dysplasia model.

[0086] Angiotensin-converting enzyme 2 (ACE2) and fibrosis: In cardiac fibrosis, ACE2 expression has been reported to have a protective effect against cardiac fibrosis induced by angiotensin II or hypertension. In a liver fibrosis model, both ACE2's metabolites, angiotensin (1-7), and ACE2, have been reported to prevent liver damage and progression to cirrhosis. In a mouse model of bile duct ligation, ACE2 supplementation has been reported to prevent liver fibrosis. In fibrotic conditions of adult and neonatal lungs, ACE2 has been reported to be downregulated through a Mas receptor mechanism.

[0087] Angiotensin-converting enzyme 2 (ACE2) and nephropathy: Low levels of ACE2 have been reported in models of renal disease and renal failure. Acquired or genetic ACE2 deficiency has been reported to exacerbate histological damage and increase proteinuria in nephropathy. In diabetic models, attenuated ACE2 expression has been reported in the glomeruli.

[0088] Angiotensin-converting enzyme 2 and COVID-19: The coronavirus (SARS-CoV-2) uses angiotensin-converting enzyme 2 as a viral receptor, causing cell damage, excessive release of immune mediators and viral particles in lung tissue, and coagulation abnormalities. Therefore, angiotensin-converting enzyme 2 (ACE2) is being considered as a potential coronavirus treatment, not only as an inhibitor of coronavirus entry, but also by reducing the inflammatory response and ACE2 deficiency caused by the coronavirus.

[0089] Meanwhile, the pharmaceutical composition of the present invention can be manufactured in a unit dosage form or 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 can be manufactured in various ways such as oral medication, injection, etc., and can be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and can additionally include a dispersing agent or stabilizer.

[0090] 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.

[0091] 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.

[0092]

[0093] In addition, the present invention provides a method for increasing the half-life of angiotensin-converting enzyme 2 (ACE2) in the body, 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 an Fc fragment is bound to the membrane scaffold protein, and the Fc fragment protrudes outward from the side of the nanodisk, thereby loading angiotensin-converting enzyme 2 (ACE2) onto the nanodisk.

[0094] In the present invention, it was confirmed that when angiotensin converting enzyme 2 is hydrophobically bound and loaded into the lipid bilayer (hydrophobic portion) of the nanodisc of the present invention (ND-Fc, simmunodic), the in vivo half-life of angiotensin converting enzyme 2 can be effectively increased. According to the following examples, angiotensin converting enzyme 2 loaded into the nanodisc of the present invention has a very long in vivo half-life of 166.1 hr.

[0095]

[0096] 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.

[0097]

[0098] [Example 1: Fabrication of nanodiscs (ND-Fc, Immunodisc) containing Fc fragments]

[0099] In this example, a nanodisc (ND-Fc, Immunodisc) containing a membrane-structured protein (MSP-Fc) to which an Fc fragment of an antibody is bound was manufactured.

[0100] First, the MSP-Fc protein was expressed and purified in HEK293 cells, and then mixed with phospholipids to produce nanodiscs (ND-Fc) containing the Fc fragment (Fig. 1A).

[0101]

[0102] 1-1. Production and purification of membrane-bound protein (MSP-Fc) containing the Fc fragment of an antibody

[0103] 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 (MSPE3D1-Fc, SEQ ID NO: 2) was prepared.

[0104] HEK293 soluble suspension cells were cultured under conditions of 37°C, 120 rpm, and 8% CO2, and 1.1x10 6A 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.

[0105] 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. 5).

[0106]

[0107] 1-2. Fabrication and verification of nanodiscs (ND-Fc) containing Fc fragments

[0108] 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.

[0109] 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).

[0110] 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 approximately 2.5 nm larger than that of the existing nanodisk (ND, 11.6 nm), confirming that it was manufactured completely (Fig. 6A).

[0111] 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 1.7%), and ND-Fc recorded 317.3 kDa (error range 3.2%), confirming that the nanodisc shape was well formed. (Fig. 6B).

[0112]

[0113] 1-3. Fabrication of large nanodiscs (P2N2-ND-Fc) containing Fc fragments

[0114] 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 disk 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 by the method of Example 1-1, and then a large nanodisc (P2N2-ND-Fc) including an Fc fragment was produced by the method of Example 1-2 (Fig. 7).

[0115]

[0116] 1-4. Confirmation of the production yield of ND-Fc and P2N2-ND-Fc

[0117] 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. 8).

[0118] Looking at A and B of Figure 8, 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 were produced in the form of monomers. The above results indicate that the Fc fragment increased the production yield of nanodiscs.

[0119] Figure 8C is a comparison result of calculating the production yield based on the results of Figures 8A and B, 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.

[0120]

[0121] 1-5. Fabrication of nanodiscs (NDA-Fc) containing angiotensin-converting enzyme 2 (ACE2) as a virus receptor and Fc fragment

[0122] 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.

[0123] 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.

[0124] 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 same amount of bio-beads as the entire mixture was treated twice, once at room temperature for 5 hours and once at 4°C for 16 hours, to produce nanodiscs (NDA-Fc) containing Fc fragments and ACE2 through a self-assembly process (Fig. 9A).

[0125] 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. 9B). 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. 9C). This confirmed that the monomer substance 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 more clearly confirmed that NDA-Fc was successfully formed (Fig. 9D).

[0126] 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. 10).

[0127]

[0128] 1-6. Fabrication of nanodiscs (AFc-ND, AFc-ND-Fc, sA-ND-Fc, sA-Fc-ND, Fc-sA-ND) containing various forms of angiotensin-converting enzyme 2 (ACE2) and Fc fragments

[0129] In this example, nanodiscs containing ACE2 and Fc fragments were fabricated in a more diverse manner.

[0130] In the same manner as in Example 1-1 above, the genes of angiotensin converting enzyme 2 and Fc fragments were combined (SEQ ID NO: 6), and the resulting binding protein (AFc, SEQ ID NO: 7) was prepared by expressing it.

[0131] 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. 2).

[0132] 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. 2).

[0133] 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. 11A). 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-ND-Fc, and that the nanodisk shape was formed intact (Fig. 11B).

[0134] In addition, the genes of soluble angiotensin converting enzyme 2, Fc fragment and membrane structural protein were combined (SEQ ID NO: 8; SEQ ID NO: 10, SEQ ID NO: 12) and expressed to prepare binding proteins (sACE2-Fc-MSP, SEQ ID NO: 9; Fc-sACE2-MSP, SEQ ID NO: 11; sACE2-MSP-Fc, SEQ ID NO: 13).

[0135] 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. 3E).

[0136] 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 (Fig. 3F).

[0137] sACE2-MSP-Fc: The lipid mole ratio was mixed to be 1:120, and through the 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 4).

[0138] 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. 12B). This confirmed that the nanodisk shape was formed intact.

[0139] 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.

[0140]

[0141] [Example 2: Evaluation of the antiviral efficacy of the nanodisc of the present invention comprising the Fc fragment and ACE2]

[0142] In this example, we aimed to confirm the excellent antiviral efficacy of the nanodisc of the present invention comprising the Fc fragment and ACE2 prepared in Example 1. To this end, the antiviral efficacy of NDA-Fc was evaluated by conducting a CPE inhibition assay using an authentic SARS-CoV-2 virus.

[0143] 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 for 1 hour at 37°C. 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.

[0144] 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: 6) are simultaneously expressed, such as in Calu-3 cells (Fig. 13A).

[0145] As shown in FIG. 13A, 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.

[0146] 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, sA-ND-Fc) containing ACE2 and Fc fragments was superior to that of the nanodisc (NDA, sAND) containing ACE2 (B in FIG. 13).

[0147]

[0148] [Example 3: Evaluation of Pharmacodynamic Properties of Nanodiscs of the Present Invention Comprising Fc Fragment and ACE2]

[0149] In this example, the pharmacodynamic properties of the nanodiscs manufactured in Example 1 were examined. To this end, ND-Fc and NDA-Fc were injected into mice, and their in vivo half-lives were examined.

[0150]

[0151] 2-1. Evaluation of the pharmacodynamic properties of ND-Fc

[0152] After 24, 48, 96, and 168 hours of injection of scFv-Fc type antibody P2B-2FB (SEQ ID NO: 13) 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. 14).

[0153] Figure 14A 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.

[0154] Figure 14B 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).

[0155]

[0156] 2-2. Pharmacodynamic properties of NDA-Fc

[0157] After 4.5 mg / kg of sACE2-Fc (SEQ ID NO: 14) 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. 15).

[0158] Figure 15A 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.

[0159] Figure 15B 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). The above results indicate that the in vivo half-life of ACE2 or a formulation utilizing ACE2 can be increased by using the nanodisc form of the present invention containing an Fc fragment and ACE2.

[0160] In particular, the half-life of conventional protein-based nanodiscs reported in the literature ranges from about 0.5 to 2 hours to a maximum of about 60 hours (Denisov, Ilia G et al; Simon-Gracia, Lorena et al; Park, Hyun-Ji et al), and almost no nanodiscs were observed in the blood after 96 hours (Park, Hyun-Ji et al). 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.

[0161] Figure 15C shows the results of confirming the concentration distributed to each organ after sACE2-Fc or NDA-Fc was injected through the nasal cavity. It can be seen that sACE2-Fc was mostly found 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. The above results mean that by using the nanodisk form of the present invention including the Fc fragment and ACE2, ACE2 or a formulation utilizing ACE2 can be distributed throughout the respiratory tract including the upper respiratory tract, and the bioabsorption rate can be increased.

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 with the hydrophobic group exposed to the outside' of the lipid bilayer; and In a nanodisk including angiotensin-converting enzyme 2 (ACE2) hydrophobicly bound to the lipid bilayer, A nanodisk characterized in that an Fc fragment is bound to the membrane-structured protein, and the Fc fragment protrudes to the outside of the nanodisk.

2. A lipid bilayer having a flat, disc-shaped bilayer structure formed from phospholipids, with hydrophilic groups oriented outward and hydrophobic groups oriented inward; Membrane scaffold protein (MSP) surrounding the 'side with the hydrophobic group exposed to the outside' of the lipid bilayer; and In a nanodisk including angiotensin-converting enzyme 2 (ACE2) hydrophobicly bound to the lipid bilayer, A nanodisc characterized in that an Fc fragment is bound to the angiotensin-converting enzyme 2 (ACE2), and the Fc fragment protrudes to the outside of the nanodisc.

3. 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 with the hydrophobic group exposed to the outside' of the lipid bilayer; and In a nanodisk including angiotensin-converting enzyme 2 (ACE2) hydrophobicly bound to the lipid bilayer, The above membrane-structured protein is bound to an Fc fragment, so that the Fc fragment protrudes to the outside of the nanodisk. A nanodisc characterized in that an Fc fragment is bound to the angiotensin-converting enzyme 2 (ACE2), and the Fc fragment protrudes 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 angiotensin-converting enzyme 2 (ACE2) bound to one end and an Fc fragment bound to the other end. A nanodisc characterized in that the above-mentioned combined angiotensin-converting enzyme 2 (ACE2) 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 angiotensin-converting enzyme 2 (ACE2) 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 'angiotensin converting enzyme 2 of membrane-structured proteins' with each other and 'Fc fragments of membrane-structured proteins' with each other to form a dimer, and the length of the membrane-structured protein is extended. A nanodisc characterized in that the above-mentioned combined angiotensin-converting enzyme 2 (ACE2) and Fc fragment protrude to the outside of the nanodisc.

6. 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 angiotensin-converting enzyme 2 (ACE2) and an Fc fragment bound to one end, A nanodisc characterized in that the above-mentioned combined angiotensin-converting enzyme 2 (ACE2) and Fc fragment protrude to the outside of the nanodisc.

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

8. In any one of the clauses 1 to 6, 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).

9. In any one of the clauses 1 to 6, The above membrane scaffold protein is, A nanodisk characterized by being an amphipathic protein having a helix structure.

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

11. In any one of the clauses 1 to 3, The above angiotensin converting enzyme 2 is, A nanodisc characterized in that a hydrophobic transmembrane domain at the C-terminus is hydrophobicly bound to a hydrophobic region of the lipid bilayer.

12. In any one of the clauses 4 to 6, The above angiotensin converting enzyme 2 is, A nanodisc characterized by being a soluble angiotensin converting enzyme 2 with the transmembrane domain removed.

13. In paragraph 1 or paragraph 3, The Fc fragment bound to the above membrane-structured 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.

14. In paragraph 2 or 3, The Fc fragment bound to the above angiotensin converting enzyme 2 is A nanodisc characterized by being manufactured by combining a gene encoding angiotensin converting enzyme 2 and a gene encoding an Fc fragment and then expressing the same.

15. In paragraph 4 or 5, Angiotensin converting enzyme 2 and Fc fragments bound to the above membrane structural protein, A nanodisc characterized by being manufactured by linking a gene encoding angiotensin converting enzyme 2 to one end of a gene encoding a membrane structural protein and linking a gene encoding an Fc fragment to the other end, and then expressing the same.

16. In paragraph 6, The above membrane-structuring protein is, A nanodisc characterized in that angiotensin-converting enzyme 2 (ACE2) and an Fc fragment are sequentially bound to one end.

17. In paragraph 6, The above membrane-structuring protein is, A nanodisc characterized in that an Fc fragment and angiotensin-converting enzyme 2 (ACE2) are sequentially bound to one end.

18. In paragraph 16, Angiotensin converting enzyme 2 and Fc fragments bound to the above membrane structural protein, 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 angiotensin converting enzyme 2 to the 5' end of the gene encoding the Fc fragment, and then expressing the same.

19. In paragraph 17, The Fc fragment and angiotensin converting enzyme 2 bound to the above membrane structural protein are, A nanodisc characterized by being manufactured by linking a gene encoding angiotensin converting enzyme 2 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 angiotensin converting enzyme 2, and then expressing the same.

20. In paragraph 4, The above nanodisk is, Angiotensin converting enzyme 2 of the membrane structural protein surrounding one of the above bilayers, and A nanodisk characterized in that angiotensin converting enzyme 2 of a membrane-structured protein surrounding another layer of the above bilayer is mutually combined to form a dimer.

21. In the above paragraph 4 The above nanodisk is, The Fc fragment of a membrane-structured protein surrounding one of the above bilayers, 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.

22. In paragraph 5, The above-mentioned membrane-structured protein having an extended length is, A nanodisk characterized by being a membrane-structured protein in which two membrane-structured proteins are interconnected to form a dimer by combining two 'angiotensin converting enzyme 2 of membrane-structured protein' and two 'Fc fragments of membrane-structured protein' to form a dimer by combining two 'Fc fragments of membrane-structured protein', thereby forming a membrane-structured protein in which two membrane-structured proteins are interconnected to form a dimer.

23. In paragraph 5, The above nanodisk is, One of the above bilayers is surrounded by an elongated membrane-structured protein, A nanodisc characterized in that another layer of the above bilayers is surrounded by another elongated membrane-structured protein.

24. In any one of the clauses 1 to 6, The above nanodisk is, A nanodisc characterized by improving the substrate conversion ability of angiotensin converting enzyme 2.

25. In paragraph 24, The above substrate conversion ability is, Converts angiotensin II to angiotensin (1-7), A nanodisc characterized by converting angiotensin I to angiotensin (1-9).

26. In any one of the clauses 1 to 6, The above nanodisk is, A nanodisc characterized by increasing the half-life of angiotensin converting enzyme 2.

27. A pharmaceutical composition for preventing or treating viral infection, characterized in that it contains a nanodisk selected from any one of claims 1 to 6.

28. In paragraph 27, The above virus, A pharmaceutical composition for preventing or treating a viral infection, characterized in that it is 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, and the Togaviridae family.

29. A pharmaceutical composition for preventing or treating a disease caused by angiotensin converting enzyme 2 deficiency, characterized in that it contains a nanodisk selected from any one of claims 1 to 6.

30. In paragraph 29, The disease caused by the above angiotensin converting enzyme 2 deficiency is A pharmaceutical composition for preventing or treating a disease caused by angiotensin converting enzyme 2 deficiency, characterized by at least one selected from the group consisting of pneumonia, pulmonary edema, pulmonary fibrosis, cardiac fibrosis, hepatic fibrosis, kidney patterns, kidney failure, bronchopulmonary dysplasia, diabetic nephropathy, and coronavirus disease-19 (COVID-19).

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