Nanodisc using dimer bonding

Abstract

The present invention relates to a nanodisc manufactured by utilizing the formation of a dimer by monomers bound to both ends of a membrane scaffold protein. In the present invention, when a nanodisc is manufactured by utilizing a membrane scaffold protein having monomer proteins forming a dimer bound to both ends thereof, it was confirmed that the nanodisc was formed intact even though different types of proteins were bound to both ends of the membrane scaffold protein, and that monomer proteins linked to the membrane scaffold protein could protrude outside the nanodisc and exhibit intact activity.

Description

Nanodisks using dimer bonds

[0001] The present invention relates to a nanodisk manufactured by utilizing monomers bound to both ends of a membrane scaffold protein to form a dimer.

[0002] 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 research on various cell membrane proteins. They also serve as a vehicle to deliver various physiologically functional substances into the body. As a bio-derived substance, nanodiscs are stable in the body and do not cause harmful reactions, making them safe.

[0003] Recently, a new application of nanodiscs has been reported for their potential as antiviral agents, such as preventing infection of viruses with a lipid bilayer envelope or inhibiting the proliferation of infected viruses. These viruses infect host cells using membrane-associated proteins in the lipid bilayer envelope. Using nanodiscs can disrupt the infection pathway of the virus or directly create holes in the virus surface to inhibit viral replication. Existing antiviral drugs, such as amantadine, oseltamivir (brand name Tamiflu), and zanamivir (brand name Relenza), have limited efficacy, and their effectiveness is limited due to the development of resistance or emergence of resistant viruses. However, nanodiscs are safe because they do not contain substances that induce specific reactions in the body, and they have the advantage of being universally applicable to various mutant viruses.

[0004] The present invention provides a nanodisc in which two proteins are bound to a membrane-structured protein and loaded, yet the nanodisc maintains a stable shape and the loaded proteins exhibit full activity. Furthermore, the present invention provides a novel method for manufacturing large nanodiscs.

[0005] The present invention 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 is a membrane scaffold protein having a monomer A of a dimer, which is a 'fragment of an antibody or a virus receptor including an antigen binding site', bound to one end and a monomer B of a dimer bound to the other end, and a monomer C forming a dimer with the monomer A is bound to one end and a monomer D forming a dimer with the monomer B is bound to the other end, wherein the membrane scaffold protein is a membrane scaffold protein having a length extended by being connected in multiple units, and the membrane scaffold protein having a length extended by being connected in multiple units, wherein the monomer A and the A nanodisk is provided, characterized in that monomers C are interconnected to form a dimer, monomers B and monomer D are interconnected to form a dimer, and a plurality of such dimers are connected, and the formed dimers protrude to the outside of the nanodisk.

[0006] Meanwhile, in monomer A, monomer B, monomer C, and monomer D of the nanodisk of the present invention, A, B, C, and D are identification letters to more clearly distinguish each monomer.

[0007] In the nanodisk of the present invention, the monomer A and the monomer C may be identical homodimers.

[0008] In the nanodisk of the present invention, the monomer B and the monomer D may be identical homodimers.

[0009] In the nanodisk of the present invention, it is preferable that the antibody fragment including the antigen binding site is an scFv fragment or a Fab fragment.

[0010] In the nanodisk of the present invention, it is preferable that the virus receptor is angiotensin-converting enzyme 2.

[0011] In the nanodisk of the present invention, it is preferable that the monomer B is an Fc fragment.

[0012] In the nanodisc of the present invention, the monomer may be a protein. In this case, the monomer protein bound to the membrane-structured protein may be produced by binding a gene encoding a monomer protein to one end of a gene encoding a membrane-structured protein, binding a gene encoding a monomer protein to the other end, and then expressing the resulting gene.

[0013] The nanodisk of the present invention may be formed by forming a dimer by interconnecting monomer A of a membrane-structured protein surrounding one of the bilayers and monomer C of a membrane-structured protein surrounding another of the bilayers, or by interconnecting monomer B of a membrane-structured protein surrounding one of the bilayers and monomer D of a membrane-structured protein surrounding another of the bilayers.

[0014] In the nanodisk of the present invention, the membrane-structured protein with an extended length may be a membrane-structured protein with an extended length formed by connecting two membrane-structured proteins.

[0015] It is preferable that the nanodisk of the present invention surrounds one of the bilayers with a membrane-structured protein having an extended length, and surrounds the other of the bilayers with another membrane-structured protein having an extended length.

[0016] In the nanodisk of the present invention, the membrane-structured 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.

[0017] The nanodisk of the present invention may further include a virus receptor that hydrophobically binds to the lipid bilayer.

[0018] In addition, the present invention provides a composition for preventing or treating viral infection containing the nanodisk.

[0019] In the present invention, when a nanodisk is manufactured using a 'membrane-structured protein having monomeric proteins forming dimers bound to both ends', it was confirmed that the nanodisk was formed intact even though different types of proteins were bound to both ends of the membrane-structured protein, and it was confirmed that the monomeric proteins linked to the membrane-structured protein could protrude outside the nanodisk and exhibit full activity.

[0020] In addition, in the present invention, when a nanodisk is produced using a membrane-structured protein having a 'monomer protein forming a dimer' bound to both ends as described above, it was confirmed that not only a nanodisk having a general diameter size but also a large nanodisk with an increased diameter is produced.

[0021] Figure 1 schematically shows the manufacturing process and shape of the large nanodisk of the present invention.

[0022] Figure 2 shows the results of purifying a membrane-bound protein (sACE2-MSP-Fc) in which soluble angiotensin converting enzyme 2 and Fc fragments are bound to each of its two ends.

[0023] Figure 3 shows the results of purification after producing the nanodiscs (Large sACE2-ND-Fc) of the present invention using sACE2-MSP-Fc. The horizontal axis represents the elution volume (mL), and the vertical axis represents A 280 (mAU).

[0024] Figure 4 shows the results of purifying a membrane-structured protein (scFv-MSP-Fc) in which scFv fragments and Fc fragments are linked to each of the two ends.

[0025] Figure 5 shows the results of purification after producing the nanodiscs (Large scFv-ND-Fc) of the present invention using scFv-MSP-Fc. The horizontal axis represents the elution volume (mL), and the vertical axis represents A 280 (mAU).

[0026] Figure 6 shows the results of purifying a membrane-structured protein (Fab-MSP-Fc) in which Fab fragments and Fc fragments are linked to each of the two ends.

[0027] Figure 7 shows the results of purification after fabricating the nanodiscs (Large Fab-ND-Fc) of the present invention using Fab-MSP-Fc. The horizontal axis represents the elution volume (mL), and the vertical axis represents A 280 (mAU).

[0028] Figure 8 shows the results (CPE inhibition (%), CPE inhibition (%)) of confirming the antiviral efficacy of the nanodisc (Large sACE2-ND-Fc) of the present invention to confirm whether the 'dimer-forming monomer protein' (sACE2, Fc fragment) included in the nanodisc (Large sACE2-ND-Fc) of the present invention is fully active.

[0029] Figure 9 shows the results of comparing the antiviral efficacy of scFv-Fc-ND, scFv-ND-Fc, Fab-Fc-ND, and Fab-ND-Fc (% relative infectivity) to confirm whether the 'dimer-forming monomeric proteins' (scFv fragment, Fab fragment, Fc fragment) included in the nanodiscs (scFv-ND-Fc, Fab-ND-Fc) of the present invention are fully active.

[0030] The present invention 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 is a membrane scaffold protein having a monomer A of a dimer, which is a 'fragment of an antibody or a virus receptor including an antigen binding site', bound to one end and a monomer B of a dimer bound to the other end, and a monomer C forming a dimer with the monomer A is bound to one end and a monomer D forming a dimer with the monomer B is bound to the other end, wherein the membrane scaffold protein is a membrane scaffold protein having a length extended by being connected in multiple units, and the membrane scaffold protein having a length extended by being connected in multiple units, wherein the monomer A and the A nanodisk is provided, characterized in that monomers C are interconnected to form a dimer, monomers B and monomer D are interconnected to form a dimer, and a plurality of such dimers are connected, and the formed dimers protrude to the outside of the nanodisk.

[0031] Nanodiscs are structures in which membrane-structured 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, giving the lipid bilayer a flat, disc-like shape. Previous research has shown that nanodiscs are widely used as delivery vehicles for hydrophobic drugs and are also utilized in studies of protein structure and function.

[0032] The inventors of the present invention, through Korean Patent Publication No. 10-2021-0035753, have discovered that by sequentially linking two genes expressing membrane-associated proteins and then expressing them, a large membrane-associated protein with an extended length can be produced, which can then be utilized to produce large nanodiscs. However, when two genes expressing membrane-associated proteins are sequentially linked and then expressed as described above, the large size of the expressed protein leads to problems such as low yield.

[0033] In addition, the inventor of the present invention confirmed through Republic of Korea Patent No. 10-2610178 that a nanodisc exhibiting excellent antiviral efficacy can be produced by further conjugating a protein that is active against a virus, such as angiotensin converting enzyme 2 (ACE2), to a membrane-structured protein. However, as described above, the nanodisc refers to a form in which the membrane-structured protein (MSP) surrounds the side of the phospholipid bilayer. However, when different proteins are conjugated to both ends of the membrane-structured protein (MSP), the membrane-structured protein cannot stably surround the phospholipid bilayer, and thus a stable nanodisc cannot be produced, and thus there was a problem in that more diverse shapes of nanodiscs could not be produced.

[0034] However, in the present invention, even when other proteins are bound to both ends of the membrane-structuring protein, in the case of a form in which a 'monomer protein forming a dimer' is bound, it was confirmed that multiple membrane-structuring proteins are connected around the phospholipid bilayer to form a complete nanodisk shape.

[0035] In addition, when multiple membrane-structured proteins are linked through dimer formation, it was confirmed that the proteins forming the dimer are oriented so as to protrude out of the nanodisk and exhibit full activity.

[0036] In addition, the inventor of the present invention has confirmed through Republic of Korea Patent Publication No. 10-2021-0035753 that nanodiscs with a large diameter can exhibit better antiviral efficacy than nanodiscs with a general size. In the present invention, when a nanodisc is manufactured using a membrane-structured protein having a 'dimer-forming monomer protein' bound to both ends, it can be confirmed that not only nanodiscs with a general diameter but also nanodiscs with an enlarged diameter are manufactured, and it has been confirmed that a large nanodisc manufactured by connecting multiple membrane-structured proteins through dimer formation has a higher nanodisc manufacturing yield and better stability than a large nanodisc manufactured using a large membrane-structured protein with an extended length.

[0037] Meanwhile, in monomer A, monomer B, monomer C, and monomer D of the nanodisk of the present invention, A, B, C, and D are identification letters to more clearly distinguish each monomer.

[0038] Meanwhile, the nanodisk of the present invention is characterized by being manufactured using a 'membrane-structured protein having monomeric proteins forming dimers bound to both ends', and depending on the form in which each membrane-structured protein within the extended membrane-structured protein is positioned in the nanodisk, a large nanodisk with a large diameter and a general nanodisk with a small diameter can be manufactured.

[0039] Specifically, when the membrane-structuring protein surrounding one layer of the phospholipid bilayer of the nanodisk and the membrane-structuring protein surrounding the other layer of the bilayer are interconnected through dimer bonds, the nanodisk has a general diameter.

[0040] On the other hand, when one layer of the phospholipid bilayer of the nanodisk is surrounded by a 'membrane-structured protein whose length is extended through dimer bonds' and the other layer of the bilayer is surrounded by another 'membrane-structured protein whose length is extended through dimer bonds', a large nanodisk with an increased diameter is formed. In other words, four or more membrane-structured proteins surround the bilayer of the nanodisk.

[0041] Therefore, since the nanodiscs of the present invention are manufactured not only as nanodiscs of a normal diameter as described above, but also as large nanodiscs with a large diameter, they may be manufactured through a process of manufacturing nanodiscs by mixing a membrane-structured protein with an extended length and a phospholipid, and then separately purifying the nanodiscs of a normal diameter and the large nanodiscs with a large diameter through size exclusion chromatography (SEC) or the like.

[0042] Meanwhile, the nanodisc of the present invention may be manufactured using a membrane-structured protein comprising monomers that form homodimers at both ends. In order to manufacture the nanodisc of the present invention, it is necessary to manufacture the membrane-structured protein in two forms: a membrane-structured protein comprising dimer monomers at both ends, and a membrane-structured protein comprising a monomer capable of forming a dimer by pairing with the monomer. However, when a monomer that forms a homodimer is used, there is an advantage in that only a membrane-structured protein manufactured in one form can be used.

[0043] In the present invention, at least one of the monomers bound to the membrane-structured protein is preferably a fragment of an antibody including a viral receptor or antigen-binding portion capable of forming a dimer.

[0044] In the present invention, the viral receptor is a receptor on the cell membrane that a virus uses when adsorbing to a cell to infect the cell, and refers to an antibody against a viral surface antigen, a cell membrane-binding protein to which a viral surface antigen can bind, a compound to which a viral surface antigen can bind, etc., and it plays a role in attaching the nanodisc to the virus. A preferred example of a viral receptor is angiotensin-converting enzyme 2, which allows the nanodisc to develop binding ability to the SARS-coronavirus, thereby enabling it to exhibit excellent antiviral efficacy.

[0045] Examples of antibody fragments containing an antigen-binding site include scFv fragments (Single-chain Variable Fragment) and Fc fragments (Fragment crystallizable). In the present invention, the Fab fragment refers to an antibody fragment composed of the VL, VH, and CL CH1 domains of the antibody, and the scFv fragment refers to a protein in which the VL and VH domains of the antibody are linked to each other. The Fab fragment and the scFv fragment can have binding ability to a specific antigen by including the antigen-binding site of the antibody. Meanwhile, the nanodisc can load a hydrophobic drug onto the phospholipid bilayer. When the nanodisc includes a Fab fragment or scFv fragment as described above, an antibody-drug conjugate (ADC) form can be easily and stably implemented.

[0046] In addition, another preferred example of a monomer bound to a membrane-structured protein in the present invention is an Fc fragment. 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. When the Fc fragment is included, the nanodisc has a very long half-life. In addition, when the nanodisc is bound to a virus, it can play a role in enabling immune cells, including monocytes, macrophages, neutrophils, eosinophils, dendritic cells, and natural killer (NK) cells, to recognize the virus, thereby enabling the nanodisc to exhibit even better antiviral efficacy in vivo.

[0047] Meanwhile, in the present invention, the monomer protein bound to the membrane structural protein may be produced by binding a gene encoding a monomer protein to one end of a gene encoding a membrane structural protein, binding a gene encoding a monomer protein to the other end, and then expressing the same.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] Meanwhile, the membrane scaffold protein (MSP) of the present invention has a helix structure and amphipathic characteristics, and plays a role in surrounding the side of the lipid bilayer. An example of a membrane scaffold protein is apolipoprotein. Apolipoprotein is a protein that exists specifically in plasma lipoproteins, and 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.

[0052] 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.

[0053] 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.

[0054]

[0055] 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.

[0056]

[0057] [Example 1: Preparation of nanodiscs using sACE2-MSP-Fc]

[0058] 1-1. Expression and purification of sACE2-MSP-Fc

[0059] To produce a membrane-bound protein (sACE2-MSP-Fc) in which soluble angiotensin converting enzyme 2 and Fc fragments are linked to each of the two terminals, a plasmid containing a sequence (sACE2-MSP-Fc, SEQ ID NO: 1) was prepared by linking the soluble angiotensin converting enzyme 2 (sACE2) sequence to the 5'-terminal of the sequence encoding the membrane-bound protein (MSP1E3D1) and linking the sequence encoding the Fc fragment to the 3'-terminal.

[0060] CHO soluble suspension cells were cultured under conditions of 37°C, 120 rpm, and 8% CO2, and 6 x 10 6A 200 mL cell culture medium was prepared at a concentration of 10 cells / mL. 160 μg of the plasmid containing the sACE2-MSP-Fc sequence prepared above was transfected into the gastric suspension cells using the Expifectamine CHO Transfect (A29130) kit from Themofisher. After culturing for 24 h at 37°C, 120 rpm, and 8% CO2, the enhancer and feed were added and cultured for 11 days at 32°C, 120 rpm, and 5% CO2. After completion of the culture, the culture medium was centrifuged at 8000 g for 30 minutes to remove the cells and obtain the supernatant. After pouring the entire supernatant onto Protein A resin, sACE2-MSP-Fc was purified from the resin by pouring Elution buffer (0.1 M Glycine, pH 2.8), and then the pH was adjusted to pH 7.4 by treating with Neutralization buffer (1 M Tris, pH 9.0) to stabilize it.

[0061] Afterwards, the obtained proteins were divided into a reduced group (reduced group, 'R' in Figure 2) and a non-reduced group (non-reduced group, 'NR' in Figure 2) and electrophoresed on an SDS-PAGE gel. At this time, for the reducing group, 2 μL of 6X Sample reducing buffer (300 mM Tris-HCl, pH 6.8, 0.6 M dithiothreitol, 12% SDS, 60% Glycerol, 0.05% Bromophenol blue) was added to 10 μL of a solution containing protein at a concentration of approximately 0.6 mg / mL and incubated in boiling water for 10 minutes. For the non-reducing group, 2 μL of 6X Sample non-reducing buffer (300 mM Tris-HCl, pH 6.8, 12% SDS, 60% Glycerol, 0.05% Bromophenol blue) was added to 10 μL of a solution containing protein at a concentration of approximately 0.6 mg / mL. In the reducing group, it was confirmed that sACE2-MSP-Fc was detected at the expected molecular weight (146 kDa) of the monomer size. In addition, looking at the results of the non-reducing group, it was possible to confirm the dimer form in which multiple sACE2-MSP-Fc were combined with each other through dimer formation (Fig. 2).

[0062]

[0063] 1-2. Manufacturing of nanodiscs using sACE2-MSP-Fc

[0064] 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 with concentrations of 25 mg / mL and 10 mg / mL, respectively. After dissolving in a buffer solution (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 solution was left in a vacuum for at least 4 hours to remove the solvent, and a lipid film was obtained. 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.

[0065] sACE2-MSP-Fc: The lipid mole ratio was mixed to be 1:120, and 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 nanodisks through a self-assembly process.

[0066] Afterwards, the nanodiscs produced above were separated through Size Exclusion Chromatography, and it was confirmed that nanodiscs of LMW A-ND-Fc, MMW A-ND-Fc, and HMW A-ND-Fc were produced (Fig. 3).

[0067]

[0068] [Example 2: Preparation of nanodiscs using scFv-MSP-Fc]

[0069] 2-1. Expression and purification of scFv-MSP-Fc

[0070] To produce a membrane-bound protein (scFv-MSP-Fc) in which scFv fragments and Fc fragments are linked to each of the two ends, a plasmid containing a sequence (SEQ ID NO: 2, scFv-MSP1E3D1-Fc) was prepared by linking the MEDI8852 scFv sequence to the 5' end of the sequence encoding the membrane-bound protein (MSP1E3D1) and linking the sequence encoding the Fc fragment to the 3' end.

[0071] HEK293 soluble suspension cells were cultured under conditions of 37℃, 120 rpm, and 8% CO2, and 1.1*10 6 cells / mL, 180 mL. Afterwards, 250 μg of the plasmid containing the scFv-MSP1E3D1-Fc sequence and 750 μg of PEI were mixed in 20 mL of culture medium, and then transfected into the prepared HEK293F cells. After culturing the cells for 120 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 extracted from the resin by pouring the elution buffer (0.1 M Glycine, pH 2.8). Afterwards, the pH of the protein was stabilized to pH 7.4 using neutralization buffer (1 M Tris, pH 9.0).

[0072] The protein thus obtained was analyzed by electrophoresis on an SDS-PAGE gel, and it was confirmed that scFv-MSP-Fc was completely purified, with a band found at 84.6 kDa (Fig. 4).

[0073]

[0074] 2-2. Manufacturing of nanodiscs using scFv-MSP-Fc

[0075] As phospholipids, POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) was dissolved in chloroform to prepare a solution with a concentration of 25 mg / mL, and DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) was dissolved in chloroform to prepare a solution with a concentration of 25 mg / mL. Then, 258.43 μL of POPC solution and 48.6 μL of DOPS solution were transferred to glass tubes so that the total lipid concentration was 10 mM and the volume was 1 mL. Afterwards, the solvent was sufficiently removed using nitrogen gas and vacuum to obtain a lipid film composed of POPC and DOPS. The obtained film was hydrated with 1 mL of ND buffer (40 mM Tris-Cl, 300 mM NaCl, 0.5 mM EDTA, 50 mM NaC, pH 7.4) containing sodium cholate (NaC), and sonicated at 55°C for 30 minutes. Thereafter, the membrane-structured protein protein (scFv-MSP-Fc) prepared above was treated so that the molar ratio of protein:lipid was 1:120. Thereafter, bio-beads in the same amount as the entire mixture were treated at room temperature for 5 hours, and then the bio-beads were removed and purified individually through size exclusion chromatography (SEC) to produce nanodisks.

[0076] Afterwards, the fabricated nanodisks were separated through Size Exclusion Chromatography, and it was confirmed that scFv-ND-Fc and Large scFv-ND-Fc nanodisks were fabricated (Fig. 5).

[0077]

[0078] [Example 3: Fabrication of Nanodiscs Using Fab-MSP-Fc]

[0079] 3-1. Expression and purification of Fab-MSP-Fc

[0080] To produce a membrane-bound protein (Fab-MSP-Fc) in which Fab fragments and Fc fragments are linked to each of the two ends, a plasmid containing a sequence (SEQ ID NO: 3, VH-CH1-MSP1E3D1-Fc) was prepared by linking the MEDI8852 VH and CH1 sequences to the 5' end of the sequence encoding the membrane-bound protein (MSP1E3D1) and linking the Fc sequence to the 3' end. In addition, a plasmid containing the MEDI8852 light chain sequence (SEQ ID NO: 4, MEDI8852 LC) was prepared.

[0081] CHO soluble suspension cells were cultured under conditions of 37℃, 120 rpm, and 8% CO2, and 1.1*10 6 cells / mL, 180 mL. Afterwards, 125 μg of the plasmid containing the 'VH-CH1-MSP1E3D1-Fc' sequence and 125 μg of the plasmid containing the MEDI8852 LC sequence and 750 μg of PEI were mixed in 20 mL of culture medium, and then co-transfected into the prepared CHO cells.

[0082] After culturing the cells for 120 hours in an incubator at 37°C, 120 rpm, and 8% CO2, the cells were centrifuged at 8000 g for 10 minutes to remove the cells and collect only the supernatant. The entire supernatant was poured onto Protein G resin, and then Elution buffer (0.1 M Glycine, pH 2.8) was poured to extract the protein from the resin. Afterwards, the pH of the protein was stabilized to pH 7.4 using Neutralization buffer (1 M Tris, pH 9.0).

[0083] The protein thus obtained was analyzed by electrophoresis on an SDS-PAGE gel, and it was confirmed that Fab-MSP-Fc was completely purified, with a band found at 105.4 kDa (Fig. 6).

[0084]

[0085] 3-2. Fabrication of nanodisks using Fab-MSP-Fc

[0086] As phospholipids, POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) was dissolved in chloroform to prepare a solution with a concentration of 25 mg / mL, and DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) was dissolved in chloroform to prepare a solution with a concentration of 25 mg / mL. Then, 258.43 μL of POPC solution and 48.6 μL of DOPS solution were transferred to glass tubes so that the total lipid concentration was 10 mM and the volume was 1 mL. Afterwards, the solvent was sufficiently removed using nitrogen gas and vacuum to obtain a lipid film composed of POPC and DOPS. The obtained film was hydrated with 1 mL of ND buffer (40 mM Tris-Cl, 300 mM NaCl, 0.5 mM EDTA, 50 mM NaC, pH 7.4) containing sodium cholate (NaC), and sonicated at 55°C for 30 minutes. Thereafter, the molar ratio of the membrane-structured protein protein (Fab-MSP-Fc): lipid was treated to be 1:120. Thereafter, the same amount of bio-beads as the entire mixture was treated at room temperature for 5 hours, and the bio-beads were removed and purified through size-exclusion chromatography (SEC) to produce nanodisks.

[0087] Afterwards, the fabricated nanodiscs were separated through Size Exclusion Chromatography, and it was confirmed that Fab-MSP-Fc and Large Fab-MSP-Fc nanodiscs were fabricated (Fig. 7).

[0088]

[0089] [Experimental Example 1: Confirmation of the Excellent Antiviral Efficacy of the Nanodisk of the Present Invention]

[0090] In this experimental example, we aimed to confirm whether the dimers (ACE2, Fc fragment, scFv fragment, Fab fragment) included in the nanodisc of the present invention can impart excellent efficacy to the nanodisc and exhibit complete activity. To this end, the antiviral efficacy of the nanodisc of the present invention (sACE2-ND-Fc, scFv-ND-Fc, Fab-ND-Fc) was confirmed.

[0091]

[0092] 1-1. Confirmation of the antiviral efficacy of sACE2-ND-Fc

[0093] The antiviral efficacy of the nanodiscs (sACE2-ND-Fc) of the present invention manufactured through Example 1 was examined. To this end, a CPE inhibition assay using authentic SARS-CoV-2 virus was performed, and the antiviral efficacy of sAND and sACE2-ND-Fc was compared.

[0094] Specifically, 2 X 10 Vero E6 cells expressing ACE2 on their surface 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 nanodiscs 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 nanodiscs at each concentration was dispensed to each well and cultured in a 5% CO2 incubator at 37°C for 48 to 72 hours.

[0095] 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. Then, the absorbance at a wavelength of 570 nm was measured using a spectrophotometer to determine the cytotoxicity reduction effect (Fig. 8).

[0096] As shown in Fig. 8, it can be confirmed that the nanodisc of the present invention (sACE2-ND-Fc) in which sACE2 and Fc fragments are respectively bound to both ends of a membrane-structured protein exhibits a superior antiviral efficacy than the nanodisc (sAND) in which only sACE2 is bound to a membrane-structured protein.

[0097] The above results imply that sACE2 and the Fc fragment bound to the membrane-structured protein were linked through dimerization and could protrude outside the nanodisc, thereby exhibiting full activity.

[0098]

[0099] 1-2. Confirmation of the antiviral efficacy of scFv-ND-Fc and Fab-ND-Fc

[0100] The antiviral efficacy of the nanodiscs (scFv-ND-Fc, Fab-ND-Fc) of the present invention manufactured through the above Example 2 was examined. To this end, the neutralizing efficacy against the A / Puerto Rico / 8 / 1934 H1N1 influenza virus was examined, and the antiviral efficacy of scFv-Fc-ND, scFv-ND-Fc, Fab-Fc-ND, and Fab-ND-Fc was compared.

[0101] Specifically, antiviral agents were mixed with A / Puerto Rico / 8 / 1934 H1N1 influenza virus at various concentrations (MOI 0.01) and incubated at room temperature for 1 hour. Then, MDCK cells (2 x 10 ) were seeded in 96-well black plates. 4 The mixture was treated on the well (cells / well). After culturing for 24 hours in a 5% CO2 incubator at 37°C, MUNANA was treated to a final concentration of 100 μM and incubated at 37°C for 1 hour. Afterwards, fluorescence was measured with a spectrophotometer at an excitation wavelength of 355 nm and an emission wavelength of 460 nm, and the neutralizing efficacy was compared (Fig. 9).

[0102] In addition, as shown in FIG. 9, it can be confirmed that both the nanodisc (scFv-Fc-ND, Fab-Fc-ND) manufactured using a membrane-structured protein having a 'Fab fragment or scFv fragment' and an Fc fragment bound to one end and the nanodisc of the present invention (scFv-ND-Fc, Fab-ND-Fc) manufactured using a membrane-structured protein having a 'Fab fragment or scFv fragment' and an Fc fragment bound to both ends exhibit excellent virus neutralization efficacy at a similar level.

[0103] The above results imply that the 'Fab fragment or scFv fragment' and the Fc fragment bound to the membrane-structured protein were linked through dimer bonding and could protrude outside the nanodisc, thereby exhibiting complete activity.

Claims

1. A lipid bilayer having a flat disc-shaped bilayer structure formed from phospholipids, with hydrophilic groups oriented toward the outside and hydrophobic groups oriented toward the inside; 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 is a membrane-structured protein having a monomer A of a dimer, which is a 'fragment of an antibody or a virus receptor including an antigen-binding site', bound to one end and a monomer B of a dimer bound to the other end, and a membrane-structured protein having a monomer C, which forms a dimer with the monomer A, bound to one end and a monomer D, which forms a dimer with the monomer B, bound to the other end, which are connected in multiple units to form an extended length. The above-mentioned membrane-structured protein having an extended length is a plurality of connected proteins in which the monomer A and the monomer C are interconnected to form a dimer, and the monomer B and the monomer D are interconnected to form a dimer. A nanodisk characterized in that the formed dimer protrudes to the outside of the nanodisk.

2. In paragraph 1, The above monomer A and the above monomer C, A nanodisc characterized by being a homodimer.

3. In paragraph 1, The above monomer B and the above monomer D, A nanodisc characterized by being a homodimer.

4. In paragraph 1, A fragment of an antibody containing the above antigen binding site, A nanodisc characterized by being a scFv fragment or a Fab fragment.

5. In paragraph 1, The above viral receptors are, A nanodisc characterized by being angiotensin-converting enzyme 2.

6. In paragraph 1, The above monomer B is, A nanodisc characterized by an Fc fragment.

7. In paragraph 1, The above monomers are, Nanodisks characterized by being proteins.

8. In paragraph 7, A monomeric protein bound to the above membrane-structuring protein is, A nanodisc characterized by being manufactured by linking a gene encoding a monomeric protein to one end of a gene encoding a membrane-structured protein and linking a gene encoding a monomeric protein to the other end, and then expressing the same.

9. In paragraph 1, The above nanodisks are, A monomer A of a membrane-structuring protein surrounding one of the above bilayers, and A nanodisk characterized in that monomer C of a membrane-structured protein surrounding another layer of the above bilayer is interconnected to form a dimer.

10. In paragraph 1, The above nanodisks are, A monomer B of a membrane-structuring protein surrounding one of the above bilayers, and A nanodisk characterized in that monomers D of a membrane-structured protein surrounding another layer of the above bilayer are interconnected to form a dimer.

11. In paragraph 1, The above-mentioned membrane-structured protein having an extended length is, A nanodisk characterized by a membrane-structured protein having an extended length formed by linking two membrane-structured proteins.

12. In paragraph 1, 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 the above bilayers is surrounded by another elongated membrane-structured protein.

13. In paragraph 1, The above membrane-structuring protein is, A nanodisk characterized by being an amphipathic protein having a helix structure.

14. In paragraph 13, 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.

15. In paragraph 1, The above nanodisks are, A nanodisk further characterized by comprising a virus receptor hydrophobicly bonded to the lipid bilayer.

16. A composition for preventing or treating viral infection, characterized by containing the nanodisk of clause 1.

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