Nanodiscs comprising fab fragments and fc fragments of antibody and antiviral use thereof

Abstract

The present invention relates to nanodiscs comprising Fab or scFv fragments and Fc fragments of an antibody and a use thereof. The nanodiscs of the present invention comprising Fab or scFv fragments and Fc fragments show higher production yield compared to conventional nanodiscs, exhibit excellent pharmacokinetic properties, and may demonstrate excellent antiviral efficacy. In addition, the nanodiscs of the present invention comprising Fab or scFv fragments and Fc fragments can be used as an antibody-drug conjugate (ADC) platform. In particular, drugs loaded onto the nanodiscs of the present invention comprising Fab or scFv fragments and Fc fragments may exhibit improved pharmacokinetic characteristics such as in vivo half-life, bioavailability, and activity.

Description

Nanodiscs comprising FAB fragments and FC fragments of antibodies and their antiviral uses

[0001] The present invention relates to a nanodisc comprising a 'Fab fragment or scFv fragment' and an Fc fragment of an antibody and a use thereof.

[0002] Antibodies possess exceptional targeting ability, and are being used in the development of antibody-drug conjugates (ADCs) for targeted drug delivery. However, forming an ADC requires an appropriate linker to connect the antibody and drug. This attachment can affect the stability and activity of the antibody, and the drug must also be chemically modified to enable attachment to the linker.

[0003] Another targeted drug delivery method that leverages the unique properties of antibodies involves entrapping hydrophobic drugs in liposomes, then modifying the liposomes with antibodies. This approach capitalizes on the fact that many drugs, including anticancer agents, are hydrophobic and can be dissolved in lipids. However, this approach still faces the challenge of requiring the addition of cholesterol and various chemical modifications to attach the antibodies to the liposomes.

[0004] Furthermore, the ideal linker connecting the antibody-drug complex must maintain stable binding throughout the body's circulation and be capable of dissociation upon reaching the target cell. Cleavable linkers typically exhibit reduced stability in the body. Non-cleavable linkers offer the advantage of high stability during circulation, but also have the disadvantage of potentially reducing the efficacy of the drug itself.

[0005] Meanwhile, antibody therapeutics are being developed to treat viral diseases. However, antibody therapeutics for viral diseases often fail to demonstrate significant therapeutic efficacy. This is because antibodies exert their antiviral activity by inhibiting viral protein function and mediating viral killing by immune cells. Furthermore, because antibody therapeutics for viral diseases only work against specific antigens through antigen-antibody interactions, viruses with high mutation rates are more likely to develop resistance to antibody therapeutics.

[0006] Meanwhile, a nanodisc is a structure formed by wrapping a phospholipid bilayer 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. In a nanodisk, the membrane scaffold protein (MSP) surrounds the side of the '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 disc.

[0007] Nanodiscs are composed of bio-derived materials, demonstrating excellent stability in the body. Furthermore, nanodiscs are known to function as carriers for drug delivery within the body, utilizing the hydrophobic regions of their phospholipid bilayer to stably load various drugs.

[0008] The present invention aims to provide a technology capable of forming an antibody-drug conjugate (ADC) in a simple and stable manner while improving the pharmacodynamic characteristics of a drug.

[0009] In addition, the present invention seeks to provide a pharmaceutical composition for preventing or treating viral infections that exhibits excellent antiviral efficacy.

[0010] The present invention provides a first type nanodisc comprising a lipid bilayer having a flat disc-shaped bilayer structure formed from a phospholipid, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; and a membrane scaffold protein (MSP) surrounding the 'side of the lipid bilayer where the hydrophobic groups are exposed outward', wherein an Fc fragment and a Fab fragment are bound to the membrane scaffold protein, and the Fc fragment and the Fab fragment protrude to the outside of the nanodisc.

[0011] In addition, the present invention provides a second type nanodisc comprising a lipid bilayer having a flat disc-shaped bilayer structure formed from a phospholipid, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; and a membrane scaffold protein (MSP) surrounding the 'side of the lipid bilayer where the hydrophobic groups are exposed outward', wherein an scFv fragment and an Fc fragment are bound to the membrane scaffold protein, and the scFv fragment and the Fc fragment protrude to the outside of the nanodisc.

[0012] Meanwhile, in the first type nanodisc of the present invention, the Fc fragment and Fab fragment may be bound to one end of a membrane-structured protein. In this case, the membrane-structured protein to which the Fc fragment and Fab fragment are bound may be produced by linking a gene encoding the membrane-structured protein with a 'gene encoding the Fc fragment and Fab fragment' and then expressing the same.

[0013] Meanwhile, in the first type nanodisc of the present invention, the Fc fragment and the Fab fragment may be respectively bound to both ends of a membrane-structured protein. In this case, the membrane-structured protein having the Fc fragment and the Fab fragment respectively bound to both ends may be produced by binding a gene encoding an Fc fragment to one end of a gene encoding a membrane-structured protein, binding a gene encoding a Fab fragment to the other end, and then expressing the same.

[0014] Meanwhile, when the first type nanodisc is manufactured using a membrane-structured protein in which the Fc fragment and Fab fragment are respectively bound to both ends, nanodiscs of a general size and large nanodiscs can be manufactured.

[0015] Specifically, when the Fc fragment of the membrane-structured protein surrounding one of the bilayers of the nanodisc and the Fc fragment of the membrane-structured protein surrounding the other of the bilayers bind to each other to form a dimer, or when the Fab fragment of the membrane-structured protein surrounding one of the bilayers and the Fab fragment of the membrane-structured protein surrounding the other of the bilayers bind to each other to form a dimer, a nanodisc of a general size is formed.

[0016] When multiple membrane-structured proteins are interconnected to form an elongated membrane-structured protein through the mutual binding of 'Fab fragments of membrane-structured proteins' to form a dimer and the mutual binding of 'Fc fragments of membrane-structured proteins' to form a dimer, a nano-disc with an enlarged diameter is formed. That is, one of the bilayers of the nano-disc is surrounded by a membrane-structured protein with an elongated length, and the other layer of the bilayer is surrounded by another membrane-structured protein with an elongated length, thereby forming a large nano-disc.

[0017] Meanwhile, in the second type nanodisc of the present invention, the Fc fragment and scFv fragment may be bound to one end of a membrane-structured protein. In this case, the membrane-structured protein to which the Fc fragment and scFv fragment are bound may be produced by linking a gene encoding the membrane-structured protein with a 'gene encoding the Fc fragment and scFv fragment' and then expressing the same.

[0018] Meanwhile, in the second type nanodisc of the present invention, the Fc fragment and scFv fragment may be respectively bound to both ends of a membrane-structured protein. In this case, the membrane-structured protein having the Fc fragment and scFv fragment respectively bound to both ends may be produced by binding a gene encoding an Fc fragment to one end of a gene encoding a membrane-structured protein, binding a gene encoding an scFv fragment to the other end, and then expressing the same.

[0019] Meanwhile, when a second type nanodisc is manufactured using a membrane-structured protein having the Fc fragment and scFv fragment linked to each of the two ends, a nanodisc of a general size and a large nanodisc can be manufactured.

[0020] Specifically, when the Fc fragment of the membrane-structured protein surrounding one of the bilayers of the nanodisc and the Fc fragment of the membrane-structured protein surrounding the other of the bilayers bind to each other to form a dimer, or when the scFv fragment of the membrane-structured protein surrounding one of the bilayers and the scFv fragment of the membrane-structured protein surrounding the other of the bilayers bind to each other to form a dimer, a nanodisc of a general size is formed.

[0021] When multiple membrane-structured proteins are interconnected to form an elongated membrane-structured protein through the mutual binding of 'scFv fragments of membrane-structured proteins' to form dimers and the mutual binding of 'Fc fragments of membrane-structured proteins' to form dimers, a nano-disc with an enlarged diameter is formed. That is, one of the bilayers of the nano-disc is surrounded by a membrane-structured protein with an elongated length, and the other layer of the bilayer is surrounded by another membrane-structured protein with an elongated length, thereby forming a large nano-disc.

[0022] Meanwhile, in the nanodisk of the present invention, the phospholipid may be, for example, 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).

[0023] Meanwhile, in the nanodisk of the present invention, the membrane scaffold protein may be 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.

[0024] Meanwhile, the nanodisk of the present invention may further include a virus receptor.

[0025] Meanwhile, the nanodisk of the present invention can be loaded with a drug within the phospholipid.

[0026] In addition, the present invention provides a composition for preventing or treating viral infection comprising the nanodisk.

[0027] The nanodisc of the present invention comprising a 'Fab fragment or scFv fragment' and an Fc fragment can be used as an antibody-drug conjugate (ADC) platform.

[0028] The drug loaded on the nanodisc of the present invention including the 'Fab fragment or scFv fragment' and the Fc fragment can have improved pharmacodynamic characteristics such as half-life in the body, bioabsorption rate, and activity.

[0029] In addition, the nanodisc of the present invention comprising a 'Fab fragment or scFv fragment' and an Fc fragment has a higher production yield than a general nanodisc, exhibits excellent pharmacodynamic properties, and can exhibit excellent antiviral efficacy.

[0030] Figure 1 schematically shows the structural differences between a nanodisc (ND) and a nanodisc (ND-Fc) comprising an Fc fragment, and a nanodisc of the present invention comprising a 'Fab fragment or scFv fragment' and an Fc fragment (scFv-Fc-ND, scFv-ND-Fc, Fab-Fc-ND, Fab-ND-Fc).

[0031] Figure 2 shows the results of confirming the molecular weight through electrophoresis after producing a membrane-structured protein (MSP-Fc) with an Fc fragment attached to it to confirm whether the membrane-structured protein (MSP-Fc) with an Fc fragment attached to it was produced intact.

[0032] Figure 3 shows the results of measuring particle size by dynamic light scattering (DLS) (Figure 3A) and measuring molecular weight by a multi-angle optical scattering (SEC-MALS) detector (Figure 3B) after fabricating nanodiscs (Fc-ND) containing Fc fragments to confirm whether the nanodiscs (Fc-ND) containing Fc fragments were completely fabricated.

[0033] Figure 4 schematically shows the differences in the production 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) and large nanodiscs (P2N2-Fc-ND) containing human-derived cell (HEK293)-expressed MSP-Fc.

[0034] Figure 5 shows the results of size exclusion chromatography (SEC) using a column (Superose 6, Supedex 200) after manufacturing nanodiscs (Fc-ND) to confirm the excellent production yield of nanodiscs (Fc-ND) containing Fc fragments (A, B of Figure 5), and comparing the purification yield with that of general nanodiscs (ND) (C of Figure 5).

[0035] Figure 6 shows the manufacturing process of nanodiscs (NDA-Fc) containing an Fc fragment loaded with angiotensin converting enzyme 2 (ACE2), a viral receptor (A in Figure 6), the purification process using size exclusion chromatography (SEC) (B in Figure 6), SDS-PAGE analysis to confirm whether NDA-Fc was completely manufactured (C in Figure 6), and the results of dynamic light scattering (DLS) measurement (D in Figure 6).

[0036] Figure 7 shows the results of size exclusion chromatography (SEC) to confirm the excellent production yield of nanodiscs (NDA-Fc) containing an Fc fragment loaded with angiotensin converting enzyme 2 (ACE2), a viral receptor, and compared with the results of nanodiscs (NDA) containing angiotensin converting enzyme 2 (ACE2).

[0037] Figure 8 shows a schematic manufacturing process of a nanodisc (scFv-Fc-ND, Fab-Fc-ND) containing an 'scFv fragment or Fab fragment' and an Fc fragment (A of Figure 8), a purification process of a membrane-structured protein (scFv-Fc-MSP, Fab-Fc-MSP) in which an 'scFv fragment or Fab fragment' and an Fc fragment are linked together at one end (B of Figure 8), and a purification process of scFv-Fc-ND, Fab-Fc-ND (C of Figure 8).

[0038] Figure 9 shows a schematic manufacturing process of nanodiscs (scFv-ND-Fc, Fab-ND-Fc) including 'scFv fragment or Fab fragment' and Fc fragment (A of Figure 9), a purification process of membrane-structured proteins (scFv-MSP-Fc, Fab-MSP-Fc) in which 'scFv fragment or Fab fragment' and Fc fragment are linked to both ends, respectively (B of Figure 9), and a purification process of scFv-ND-Fc, Fab-ND-Fc (C of Figure 9).

[0039] Figure 10 shows the results of confirming the concentration distributed in each organ (A of Figure 10) and serum half-life (B of Figure 10) after injecting P2B-2FB or ND-Fc, which are antibodies in the form of scFv-Fc, into mice to evaluate the pharmacodynamic properties of nanodiscs (ND-Fc) containing Fc fragments.

[0040] Figure 11 shows the results of evaluating the pharmacodynamic properties of nanodiscs (NDA-Fc) containing an Fc fragment loaded with angiotensin converting enzyme 2 (ACE2), after injecting sACE2-Fc or NDA-Fc into mice, and confirming the concentration distributed in each organ (A, C of Figure 11) and serum half-life (B of Figure 11).

[0041] Figure 12 shows an experiment to confirm the neutralizing effect against influenza virus in order to confirm the excellent antiviral efficacy of the nanodisc (scFv-Fc-ND, Fab-Fc-ND, scFv-ND-Fc, Fab-ND-Fc) of the present invention including 'scFv fragment or Fab fragment' and Fc fragment, and shows the results compared with the case of scFv-Fc antibody.

[0042] The present invention provides a nanodisc comprising a lipid bilayer having a flat, disc-shaped bilayer structure formed from a phospholipid, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; and a membrane scaffold protein (MSP) surrounding the 'side of the lipid bilayer where the hydrophobic groups are exposed outward', wherein an Fc fragment and a Fab fragment are bound to the membrane scaffold protein, and the Fc fragment and the Fab fragment protrude to the outside of the nanodisc.

[0043] In addition, the present invention provides a nanodisc comprising a lipid bilayer having a flat disc-shaped bilayer structure formed from a phospholipid, wherein hydrophilic groups are oriented outward and hydrophobic groups are oriented inward; and a membrane scaffold protein (MSP) surrounding the 'side of the lipid bilayer where the hydrophobic groups are exposed outward', wherein an scFv fragment and an Fc fragment are bound to the membrane scaffold protein, and the scFv fragment and the Fc fragment protrude to the outside of the nanodisc.

[0044] Antibody-drug conjugates must maintain stable binding throughout their circulating course, release the loaded drug upon reaching the target cell, and ensure full drug activity. If the linker connecting the antibody-drug conjugate is cleavable, its stability in the body is reduced. While non-cleavable linkers offer the advantage of high stability during circulation, they also carry the disadvantage of potentially reducing the efficacy of the drug itself.

[0045] However, in the present invention, a nanodisc (so-called "antibody") to which an antibody is bound was manufactured, and when utilizing this, by loading a drug onto the nanodisc, it was confirmed that an antibody-drug complex that has excellent stability in the body and can stably deliver the drug to the target can be easily manufactured by linking the antibody and the drug as one body.

[0046] In addition, the half-life of nanodiscs reported in the literature is known to be about 0.5 to 2 hours, and some nanodiscs are known to have a half-life of up to 60 hours. Even in the case of nanodiscs with a long half-life, it has been reported that nanodiscs are hardly observed in the blood 96 hours after administration (Park, Hyun-Ji, et al. "High-density lipoprotein-mimicking nanodiscs carrying peptide for enhanced therapeutic angiogenesis in diabetic hindlimb ischemia." Biomaterials 161 (2018): 69-80.), and it was confirmed that the nanodiscs of the present invention have a much longer half-life than existing known nanodiscs.

[0047] In addition, the inventors of the present invention have confirmed through Korean Patent No. 10-2022-0022501 that the activity of the loaded drug can be improved when the drug is loaded onto a nanodisk.

[0048] That is, the nanodisk of the present invention including a 'Fab fragment or scFv fragment' and an Fc fragment can easily and stably implement a drug complex (ADC, antibody-drug conjugate) form, and has the characteristic of improving the pharmacodynamic characteristics of the loaded drug, such as the half-life in the body, bioabsorption rate, and activity, so that the drug can exhibit a more enhanced effect.

[0049] In addition, the present invention provides a composition for preventing or treating viral infection comprising the nanodisk.

[0050] The inventors of the present invention have confirmed through Korean Patent Registration Nos. 10-2181991 and 10-2438720 that nanodiscs can act as cell membrane mimics and perforate the envelope of viruses, and confirmed that nanodiscs can be used as antiviral agents against a wide range of viruses through this mechanism. In the present invention, when a membrane-structured protein in which an antibody Fc fragment and a 'Fab fragment or scFv fragment' are combined was used to produce the nanodiscs of the present invention, it was confirmed that the yield was increased during the production process, the antiviral activity was further improved, and excellent pharmacodynamic properties were exhibited.

[0051] In particular, in order for the existing nanodiscs of the present inventor (Korean Patent Nos. 10-2181991 and 10-2438720) to exhibit excellent antiviral efficacy, they had to be configured to include a viral receptor (e.g., ganglioside or angiotensin-converting enzyme 2). However, the present invention confirmed that a nanodisc including a 'Fab fragment or scFv fragment' of an antibody can exhibit excellent antiviral efficacy even without a separate viral receptor.

[0052] Meanwhile, the nanodisk of the present invention is composed of a phospholipid bilayer, a membrane-structured protein, an Fc fragment, and a 'Fab fragment or scFv fragment', and may be manufactured in various sizes and shapes.

[0053] Specifically, when the nanodisk of the present invention is manufactured using a membrane-structured protein (Fab-Fc-MSP, scFv-Fc-MSP) in which a 'Fab fragment or scFv fragment' and an Fc fragment are linked to one end, nanodisks in the form of scFv-Fc-ND and Fab-Fc-ND are manufactured (see C and E of Figure 1).

[0054] At this time, the membrane-structured protein having the 'Fab fragment or scFv fragment' and the Fc fragment bound to one end may be manufactured by sequentially binding a gene encoding the 'Fab fragment or scFv fragment', a gene encoding the Fc fragment, and a gene encoding the membrane-structured protein, and then expressing the same.

[0055] When the nanodisc of the present invention is manufactured using a membrane-structured protein (Fab-MSP-Fc, scFv-MSP-Fc) in which a 'Fab fragment or scFv fragment' and an Fc fragment are linked to each of both ends, nanodiscs in the form of scFv-ND-Fc, Large scFv-ND-Fc, Fab-ND-Fc, and Large Fab-ND-Fc are manufactured (see D and F of Figure 1).

[0056] Typically, nanodiscs are manufactured in the shape of a disk in which one layer of the phospholipid bilayer is surrounded by a membrane-structuring protein, and the other layer is surrounded by another membrane-structuring protein.

[0057] Meanwhile, in the case of using a membrane-structured protein (Fab-MSP-Fc, scFv-MSP-Fc) in which a 'Fab fragment or scFv fragment' and an Fc fragment are respectively linked to both ends, a homodimer-type bond is formed between the 'Fab fragments or scFv fragments' of the multiple membrane-structured proteins, and a homodimer-type bond is formed between the Fc fragments, so that the multiple membrane-structured proteins are linked to each other.

[0058] Accordingly, when a plurality of membrane-structured proteins (Fab-MSP-Fc) having 'Fab fragments or scFv fragments' and Fc fragments bound to both ends are connected to each other to form a disk shape, and when the Fc fragment of the membrane-structured protein surrounding one of the bilayers of the nanodisc and the Fc fragment of the membrane-structured protein surrounding the other of the bilayers bind to each other to form a dimer, or when the Fab fragment of the membrane-structured protein surrounding one of the bilayers and the Fab fragment of the membrane-structured protein surrounding the other of the bilayers bind to each other to form a dimer, a nanodisc of a general size is formed.

[0059] On the other hand, when multiple membrane-structured proteins are interconnected to form an elongated membrane-structured protein through the mutual binding of 'angiotensin converting enzyme 2 of membrane-structured proteins' to form a dimer and the mutual binding of 'Fc fragments of membrane-structured proteins' to form a dimer, a nano-disc with an enlarged diameter is formed. That is, when one of the bilayers of the nano-disc is surrounded by a membrane-structured protein with an enlarged length and the other layer of the bilayer is surrounded by another membrane-structured protein with an enlarged length, a total of four or more membrane-structured proteins surround the bilayer of the nano-disc to form a large nano-disc with an enlarged diameter.

[0060] Meanwhile, the membrane-structured protein having the 'Fab fragment or scFv fragment' and the Fc fragment linked to each of the two ends may be produced by linking a gene encoding an Fc fragment to one end of a gene encoding a membrane-structured protein, linking a gene encoding a Fab fragment to the other end, and then expressing the same.

[0061] Meanwhile, in the present invention, the Fc fragment refers to the Fc region of an antibody or a fragment thereof, which is composed of the hinge, CH2, and CH3 regions of the heavy chain of an IgG antibody, and may be included in the nanodisc of the present invention to improve pharmacodynamic characteristics. In addition, the Fc fragment may play a role in enabling immune cells, including monocytes, macrophages, neutrophils, eosinophils, dendritic cells, and natural killer cells (NK cells), to recognize the virus when the nanodisc is bound to a virus, thereby enabling the nanodisc to exhibit better antiviral efficacy in vivo. Meanwhile, the N-terminus of the Fc fragment may further include an antibody white sequence.

[0062] 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 contain the antigen-binding site of the antibody, thereby imparting binding ability to a specific antigen. Meanwhile, the C-terminus of the scFv fragment and the Fab fragment may further include an antibody hinge sequence.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] Meanwhile, the nanodisc of the present invention may further include a 'virus receptor'. In the present invention, the virus receptor is a receptor on the cell membrane that a virus uses when adsorbing to a cell to infect the cell, and may be an antibody against a virus surface antigen, a cell membrane-binding protein to which a virus surface antigen can bind, a compound to which a virus surface antigen can bind, etc. The nanodisc of the present invention can exhibit excellent antiviral efficacy even without a separate virus receptor by including a 'Fab fragment or scFv fragment' of an antibody, but can exhibit even stronger antiviral efficacy by further including a virus receptor.

[0072] Meanwhile, in the present invention, the 'virus receptor' may be, for example, angiotensin converting enzyme 2 or a 'compound containing sialic acid at one end'.

[0073] The above-mentioned angiotensin converting enzyme 2 (ACE2) is known to be used as a receptor by several coronaviruses, including SARS-CoV and SARS-CoV-2, to penetrate human cells. In other words, by including angiotensin converting enzyme 2, the ability to adhere to coronaviruses, including SARS-CoV and SARS-CoV-2, can be enhanced, thereby further enhancing antiviral efficacy.

[0074] The above 'compound containing sialic acid at one end' refers to a compound or sialic acid complex containing sialic acid at one end and having the ability to bind to a virus. Examples include sialyllactose and ganglioside. Sialyllactose and ganglioside are known to have the ability to bind to an influenza virus because they contain sialic acid at one end.

[0075] In the present invention, 'viral infection' may be caused by a virus of any one or more selected from among Coronaviridae, Bunyaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Poxviridae, Rhabdoviridae, Retroviridae, Togaviridae, Picornaviridae, Paramyxoviridae, and Reoviridae, but is not limited thereto. However, it is preferable to use the antibody or virus receptor contained in the nanodisk according to the type of virus to which it exhibits binding ability.

[0076] Meanwhile, the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the nanodisk, which is an active ingredient.

[0077] Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0078] 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.

[0079] 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. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg / kg.

[0080] In the present invention, a pharmaceutically effective amount refers to an amount sufficient to prevent or treat the aforementioned disease. "Prevention" in the present invention refers to the prevention or protective treatment of a disease or disease state. "Treatment" in the present invention refers to the reduction, suppression, alleviation, or eradication of a disease state.

[0081] The pharmaceutical composition of the present invention can be manufactured in a unit dosage form or can be manufactured by inserting it into 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 an oral medication or an injection, 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, granules, tablet or capsule, and can additionally include a dispersing agent or stabilizer.

[0082]

[0083] The present invention will be described in more detail below through the following examples and experimental examples. However, the present invention is not limited to the following examples and experimental examples, and includes variations of equivalent concepts.

[0084]

[0085] [Example 1: Preparation of nanodiscs containing Fc fragments]

[0086] In this example, a nanodisc (ND-Fc) containing a membrane-structured protein (MSP-Fc) bound to an Fc fragment was prepared.

[0087]

[0088] 1-1. Production and purification of membrane-bound Fc fragment-bound protein (MSP-Fc)

[0089] For the production of MSP-Fc binding protein, a plasmid consisting of a sequence encoding MSP1E3D1 and a sequence encoding the Fc fragment of the antibody (MSP1E3D1-Fc, SEQ ID NO: 2) was prepared.

[0090] 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.

[0091] 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. 2).

[0092]

[0093] 1-2. Fabrication and verification of nanodiscs (ND-Fc) containing Fc fragments

[0094] 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.

[0095] 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).

[0096] 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. 3A).

[0097] 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. 3B).

[0098]

[0099] 1-3. Fabrication of large nanodiscs (P2N2-ND-Fc) containing Fc fragments

[0100] 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. 4).

[0101]

[0102] 1-4. Confirmation of the production yield of ND-Fc and P2N2-ND-Fc

[0103] 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. 5).

[0104] Looking at A and B of Figure 5, 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.

[0105] Figure 5C is a comparison result of calculating the production yield based on the results of Figures 5A and 5B, 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.

[0106]

[0107] 1-5. Fabrication of nanodiscs (NDA-Fc) containing physiologically functional substances, angiotensin-converting enzyme 2 (ACE2) and Fc fragments.

[0108] In this example, we attempted to fabricate a nanodisc (NDA-Fc) loaded with ACE2 (angiotensin-converting enzyme 2, SEQ ID NO: 5), which 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).

[0109] 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.

[0110] ACE2 (molecular weight 94.2 kDa):MSP-Fc (molecular weight 57 kDa):lipid were mixed at a molar ratio of 0.5:1:120. Then, the entire mixture was treated twice, once at room temperature for 5 hours and once at 4°C for 16 hours, with the same amount of bio-beads as the entire mixture, to produce nanodiscs (NDA-Fc) containing Fc fragments and ACE2 through a self-assembly process (Fig. 6A).

[0111] 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. 6B). 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. 6C). This confirmed that the monomer material corresponding to 13 to 14 mL was NDA-Fc containing ACE2. In addition, the DLS analysis results confirmed that the diameter of NDA-Fc (19.5 nm) was 5.3 nm larger than that of ND-Fc without ACE2 (14.2 nm), which more clearly confirmed that NDA-Fc was successfully formed (Fig. 6D).

[0112] Meanwhile, as a result of comparing the production yield with that of nanodiscs produced using a general membrane-structured protein (MSP) using 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, some nanodiscs containing ACE2 (NDA, elution volume 14 mL) and some nanodiscs not containing ACE2 (ND, elution volume 16 mL) were formed separately (Fig. 7).

[0113] The above results mean that while existing nanodiscs often did not contain ACE2, in the case of nanodiscs containing Fc fragments, they were formed uniformly with ACE2 mostly loaded.

[0114] Through this, it was confirmed that when the nanodisk includes an Fc fragment, the loading ability of a physiologically functional substance with a hydrophobic region, such as ACE2, is improved, and thus the physiologically functional substance can be loaded at a higher yield.

[0115]

[0116] [Example 2: Manufacturing of nanodiscs of the present invention comprising 'Fab fragment or scFv fragment' and Fc fragment]

[0117] In this example, a membrane-structured protein (scFv-Fc-MSP, scFv-MSP-Fc, Fab-Fc-MSP, Fab-MSP-Fc) in which an 'scFv (Single-chain variable fragment) or Fab (Fragment antigen-binding)' and an Fc fragment are combined was used to manufacture a nanodisc (scFv-Fc-ND, scFv-ND-Fc, Fab-Fc-ND, Fab-ND-Fc) containing an 'scFv fragment or Fab fragment' and an Fc fragment.

[0118]

[0119] 2-1. Production and purification of membrane-structured proteins (scFv-Fc-MSP, scFv-MSP-Fc, Fab-Fc-MSP, Fab-MSP-Fc) combined with ‘Fab fragment or scFv fragment’ and Fc fragment

[0120] To produce a membrane-bound protein (scFv-Fc-MSP) in which scFv fragments and Fc fragments are linked together at one end, a plasmid containing a sequence (SEQ ID NO: 6, scFv-Fc-MSP1E3D1) was prepared by sequentially linking a sequence encoding a membrane-bound protein (MSP1E3D1) and a sequence encoding an scFv-Fc antibody (MEDI8852).

[0121] 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: 7, scFv-MSP1E3D1-Fc) was prepared by linking the 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.

[0122] To produce a membrane-bound protein (Fab-Fc-MSP) in which Fab fragments and Fc fragments are linked together at one end, a plasmid containing a sequence (SEQ ID NO: 8, VH-CH1-Fc-MSP2N2) was prepared by sequentially linking a sequence encoding a membrane-bound protein (MSP2N2) and a sequence encoding MEDI8852 heavy chain. In addition, a plasmid containing a MEDI8852 light chain sequence (SEQ ID NO: 9, MEDI8852 LC) was prepared.

[0123] 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: 10, VH-CH1-MSP1E3D1-Fc) was prepared by linking the 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: 9, MEDI8852 LC) was prepared.

[0124] Afterwards, the plasmid containing the scFv sequence was transfected into HEK293F cells. Specifically, HEK293 soluble suspension cells were cultured under conditions of 37°C, 120 rpm, and 8% CO2, and 1.1*10 6 cells / mL, 180 mL. Afterwards, 250 μg of the plasmid containing the scFv-Fc-MSP1E3D1 or 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.

[0125] For plasmids containing the Fab sequence or MEDI8852 LC sequence, they were transfected into CHO cells. Specifically, CHO soluble suspension cells were cultured under conditions of 37°C, 120 rpm, and 8% CO2, and 1.1*10 6cells / mL, 180 mL. Afterwards, 125 μg of the plasmid containing the 'VH-CH1-Fc-MSP2N2 or 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.

[0126] 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).

[0127] As a result of analyzing the proteins obtained in this way by electrophoresis on an SDS-PAGE gel (B in Fig. 8, B in Fig. 9), bands were found at 82.5 kDa for scFv-Fc-MSP, 84.6 kDa for scFv-MSP-Fc, 121.3 kDa for Fab-Fc-MSP, and 105.4 kDa for Fab-MSP-Fc, confirming that they were completely purified.

[0128]

[0129] 2-2. Production of nanodiscs (scFv-Fc-ND, scFv-ND-Fc, Fab-Fc-ND, Fab-ND-Fc) of the present invention comprising 'Fab fragment or scFv fragment' and Fc fragment

[0130] 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 (scFv-Fc-MSP, scFv-MSP-Fc, Fab-Fc-MSP, or Fab-MSP-Fc) prepared above was treated at a molar ratio of 1:120: lipid. 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 respectively through size exclusion chromatography (SEC) to obtain nanodiscs (scFv-Fc-ND, scFv-ND-Fc, Fab-Fc-ND, Fab-ND-Fc).

[0131] As a result, it was confirmed that the nanodisks were found at an elution volume of approximately 11 to 15 mL, which is a location that matches the expected molecular weight, and that the nanodisks were produced intact (C in Fig. 8, C in Fig. 9).

[0132] Meanwhile, in the case of scFv-ND-Fc or Fab-ND-Fc type nanodiscs, it was confirmed that they were produced in two forms: a normal-sized nanodisc (scFv-ND-Fc or Fab-ND-Fc) was found at an elution volume of approximately 11 to 15 mL, and a large-sized nanodisc (Large scFv-ND-Fc or Large Fab-ND-Fc) was found at an elution volume of 8 to 11 mL (C in Figure 9).

[0133]

[0134] [Experimental Example 1: Evaluation of Pharmacodynamic Properties of Nanodiscs Containing Fc Fragment of Antibody]

[0135] In this experimental example, the pharmacodynamic properties of nanodiscs containing the Fc fragment prepared 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.

[0136]

[0137] 1-1. Evaluation of the pharmacodynamic properties of ND-Fc

[0138] After 24, 48, 96, and 168 hours of injection of scFv-Fc-type antibody P2B-2F6 (SEQ ID NO: 11) or ND-Fc through the tail vein of mice, blood and organs (Heart, Larynx, Trachea, Lung, Liver, Spleen, Kidney) were collected from each experimental group of mice. The collected organs were ground and the concentrations of the injected antibodies and ND-Fc in the body were confirmed through ELISA (Fig. 10).

[0139] Figure 10A 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.

[0140] Figure 10B 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).

[0141]

[0142] 2-2. Pharmacodynamic properties of NDA-Fc

[0143] After 4.5 mg / kg of sACE2-Fc (SEQ ID NO: 12) 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. 11).

[0144] Figure 11A 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.

[0145] Figure 11B 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.

[0146] In particular, the half-life of conventional protein-based nanodiscs reported in the prior literature is known to be about 0.5 to 2 hours and up to 60 hours, and even in the case of nanodiscs with a long half-life, almost no nanodiscs were observed in the blood after 96 hours (Park, Hyun-Ji, et al. "High-density lipoprotein-mimicking nanodiscs carrying peptide for enhanced therapeutic angiogenesis in diabetic hindlimb ischemia." Biomaterials 161 (2018): 69-80.), but 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.

[0147] Figure 11C 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 indicate that the pharmacodynamic characteristics of the nanodisc of the present invention including the Fc fragment are very excellent.

[0148]

[0149] [Experimental Example 2: Evaluation of the antiviral efficacy of the nanodisc of the present invention comprising a 'Fab fragment or scFv fragment' and an Fc fragment]

[0150] In this experimental example, we aimed to evaluate the excellent antiviral efficacy of nanodiscs (scFv-Fc-ND, scFv-ND-Fc, Fab-Fc-ND, Fab-ND-Fc) containing the 'Fab fragment or scFv fragment' and Fc fragment prepared in Example 2. To this end, a neutralizing efficacy test against the A / Puerto Rico / 8 / 1934 H1N1 influenza virus of the nanodiscs was conducted and compared with the MEDI8852 scFv-Fc antibody.

[0151] Specifically, antiviral agents were mixed with A / Puerto Rico / 8 / 1934 H1N1 influenza virus at various concentrations (MOI 0.01) and allowed to react 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. 12).

[0152] Specifically, antiviral agents were mixed with A / Puerto Rico / 8 / 1934 H1N1 influenza virus at various concentrations (MOI 0.01) and allowed to react 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. 12).

[0153] Looking at A of Figure 12, the group treated with MEDI8852 scFv-Fc had an IC50 of 56.41 nM, but the group treated with the nanodisk (scFv-Fc-ND) of the present invention including MEDI8852 scFv-Fc had an IC50 of 2.34 nM, confirming that it exhibited even better antiviral efficacy.

[0154] In addition, looking at B of Figure 12, it can be confirmed that nanodiscs (scFv-Fc-ND, scFv-ND-Fc, Fab-Fc-ND, Fab-ND-Fc) containing 'Fab fragment or scFv fragment' and Fc fragment all exhibit excellent antiviral efficacy at a similar level.

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, A nanodisc characterized in that a Fab fragment and an Fc fragment are bound to the membrane-structured protein, and the Fab fragment and the Fc fragment protrude to the outside of the nanodisc.

2. 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, A nanodisc characterized in that an scFv fragment and an Fc fragment are bound to the membrane-structured protein, and the scFv fragment and the Fc fragment protrude to the outside of the nanodisc.

3. In paragraph 1, The above Fc fragment and Fab fragment, A nanodisc characterized by being bound to one end of a membrane-structuring protein.

4. In paragraph 1, The above Fc fragment and Fab fragment, A nanodisc characterized by being bound to each end of a membrane-structuring protein.

5. In paragraph 4, The above nanodisks are, A nanodisk characterized in that a dimer is formed by mutual binding of an Fc fragment of a membrane-structured protein surrounding one layer of the above bilayers and an Fc fragment of a membrane-structured protein surrounding the other layer of the above bilayers.

6. In paragraph 4, The above nanodisks are, A nanodisc characterized in that a Fab fragment of a membrane-structured protein surrounding one layer of the bilayer and a Fab fragment of a membrane-structured protein surrounding the other layer of the bilayer are mutually associated to form a dimer.

7. In paragraph 4, The membrane-structured protein in which the above Fc fragment and Fab fragment are combined is, A nanodisk characterized by being a membrane-structured protein in which multiple membrane-structured proteins are interconnected to form a dimer by mutually combining 'Fc fragments of membrane-structured proteins' and 'Fab fragments of membrane-structured proteins' to form a dimer by mutually combining, thereby forming a membrane-structured protein in which the length is extended.

8. In paragraph 7, The above nanodisks are, One of the above bilayers is surrounded by a membrane-structured protein whose length is extended, A nanodisc characterized in that another layer of said bilayer is surrounded by another elongated membrane-structured protein.

9. In paragraph 2, The above Fc fragment and scFv fragment, A nanodisc characterized by being bound to one end of a membrane-structuring protein.

10. In paragraph 2, The above Fc fragment and scFv fragment, A nanodisc characterized by being bound to each end of a membrane-structuring protein.

11. In paragraph 10, The above nanodisks are, A nanodisk characterized in that a dimer is formed by mutual binding of an Fc fragment of a membrane-structured protein surrounding one layer of the above bilayers and an Fc fragment of a membrane-structured protein surrounding the other layer of the above bilayers.

12. In paragraph 10, The above nanodisks are, A nanodisc characterized in that a dimer is formed by mutual binding of an scFv fragment of a membrane-structured protein surrounding one layer of the bilayer and an scFv fragment of a membrane-structured protein surrounding the other layer of the bilayer.

13. In paragraph 10, The membrane-structured protein in which the above Fc fragment and scFv fragment are combined is, A nanodisk characterized by being a membrane-structured protein whose length is extended by interconnecting multiple membrane-structured proteins through mutually combining 'Fc fragments of membrane-structured proteins' to form dimers and 'scFv fragments of membrane-structured proteins' to form dimers.

14. In paragraph 13, The above nanodisks are, One of the above bilayers is surrounded by a membrane-structured protein whose length is extended, A nanodisc characterized in that another layer of said bilayer is surrounded by another elongated membrane-structured protein.

15. In paragraph 1 or 2, The above membrane-structuring protein is, A nanodisk characterized by being an amphipathic protein having a helix structure.

16. In paragraph 15, 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.

17. In paragraph 1 or 2, The above nanodisks are, A nanodisc further characterized by comprising a virus receptor.

18. In paragraph 1 or 2, The above nanodisks are, A nanodisc characterized by having a drug loaded within a phospholipid.

19. A composition for preventing or treating viral infection, characterized by comprising a nanodisk of claim 1 or 2.

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