Antibody-bound lipid nanoparticles containing an antibody to which a membrane-structuring protein is bound

Abstract

The present invention relates to antibody-conjugated lipid nanoparticles comprising an antibody bound to a membrane scaffold protein, and more specifically, to antibody-conjugated lipid nanoparticles which are easy to produce and have excellent specific targeting ability by comprising an antibody bound to a lipid and a membrane scaffold protein.

Description

[Technical field]

[0001] The present invention relates to antibody-conjugated lipid nanoparticles comprising an antibody conjugated with a membrane scaffold protein, and more specifically, to antibody-conjugated lipid nanoparticles which are easy to produce and have excellent specific targeting ability by comprising an antibody conjugated with a lipid and a membrane scaffold protein. [Background technology]

[0002] mRNA plays a role in transmitting genetic information from DNA to the organelles that synthesize proteins in the cytoplasm. When such mRNA enters a cell, it synthesizes proteins within the cell and initiates an immune response.

[0003] We are developing an mRNA vaccine using mRNA with the above characteristics. However, there are problems with mRNA, such as the fact that gene expression is suppressed by interference caused by immune responses, and that it is difficult for it to enter the cytoplasm due to its large size.

[0004] To solve these problems, lipid nanoparticle (LNP) technology was developed to help mRNA enter the cytoplasm, and in recent years, it has been used as a delivery vehicle for mRNA vaccines and other applications, and its importance is increasing. However, the technology to selectively deliver lipid nanoparticles (LNPs) to cells has not yet reached a sufficient level.

[0005] Therefore, we are developing lipid nanoparticles with excellent target delivery capabilities by combining the excellent target delivery capabilities of antibodies with lipid nanoparticle technology, but the existing method uses chemical conjugation, which makes the process complicated and reduces the yield.In addition, there are disadvantages in that the chemical properties are unknown and the antibody is attached to the affinity tag, making it difficult to confirm the binding when injected into the body. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides antibody-bound lipid nanoparticles (LNPs) that can be easily produced using an antibody capable of binding to the lipid nanoparticles (LNPs) and have excellent target delivery ability, and a method for producing the same. [Means for solving the problem]

[0007] The present invention provides antibody-bound lipid nanoparticles, characterized in that an "antibody or antibody fragment" bound to a membrane scaffold protein is bound via a hydrophobic bond to a hydrophobic portion of a lipid that constitutes the lipid nanoparticle.

[0008] In the antibody-bound lipid nanoparticle of the present invention, the membrane-structured protein is preferably an amphipathic protein having a helix structure. In this case, the membrane-structured protein may be a fragment of the membrane-structured protein that retains the helix structure and amphipathic properties.

[0009] In the antibody-bound lipid nanoparticles of the present invention, the antibody fragment is preferably scFv or "scFV-Fc in which scFv and Fc are bound to each other."

[0010] In the antibody-bound lipid nanoparticles of the present invention, the "antibody or antibody fragment" to which the membrane scaffold protein is bound is preferably produced by binding a gene encoding the "antibody or antibody fragment" to a gene encoding the membrane scaffold protein, and then expressing the resulting product.

[0011] In the antibody-bound lipid nanoparticle of the present invention, the lipid nanoparticle preferably has a substance to be encapsulated encapsulated therein. In this case, the substance to be encapsulated is preferably a nucleic acid.

[0012] The present invention also provides a method for producing antibody-bound lipid nanoparticles, which comprises mixing an antibody bound to a membrane-structuring protein and a lipid.

[0013] In the method for producing antibody-bound lipid nanoparticles of the present invention, the antibody-bound lipid nanoparticles are preferably mixed with a substance to be encapsulated. Effect of the Invention

[0014] The antibody-bound lipid nanoparticles of the present invention contain an antibody bound to a membrane scaffold protein, and therefore have excellent specific delivery ability to target cells while being easily produced. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram showing a conventional method for producing antibody-bound lipid nanoparticles. [Diagram 2] 2 is a schematic diagram showing a method for producing antibody-bound lipid nanoparticles using the antibody (grabber antibody) of the present invention. In Fig. 2, the upper part shows the case where the antibody (grabber antibody) of the present invention is mixed in the lipid nanoparticle production process, and the lower part shows the process where the antibody (grabber antibody) of the present invention is added to the produced lipid nanoparticles. [Diagram 3] FIG. 3 is a diagram showing the structure of the antibody of the present invention (Grabber antibody). [Figure 4]Fig. 4 shows the results of SDS gel electrophoresis to confirm whether the single chain antibody fragment (Gr-scFv) of the present invention was normally produced after it was produced using an E. coli protein expression system. In Fig. 4, 10B4 and NLDC145 represent the antibodies used, and ApoA1, MSP1E3D1, and ApoeE3 represent the membrane-structured proteins used. [Diagram 5] FIG. 5 shows the results of SDS gel electrophoresis to confirm whether the single-chain antibody fragment (Grabber Single Chain Variable Fragment, Gr-scFv) of the present invention was produced normally after being produced using a mammalian cell protein expression system. [Figure 6] FIG. 6 shows the results of SDS gel electrophoresis to confirm whether the single-chain antibody of the present invention (Grabber Single chain variable fragment-Fc, Gr-scFv-Fc) was produced normally after being produced using a mammalian cell protein expression system. [Figure 7] FIG. 7 shows the results of SDS gel electrophoresis to confirm whether the antibody of the present invention (Grabber Antibody, Gr-Antibody) was produced normally after being produced using a mammalian cell protein expression system. [Figure 8] Figure 8 shows the results of measuring the A280 of membrane structured protein (MSP), lipid nanoparticles (LNP), and a mixture of lipid nanoparticles and membrane structured protein (MSP LNP) using size exclusion chromatography to confirm whether the antibody of the present invention can bind to lipid nanoparticles. [Figure 9] Figure 9 shows the results of confirming the particle size of lipid nanoparticles (LNP) and a mixture of lipid nanoparticles and membrane-structured proteins (MSP LNP) using dynamic light scattering to confirm whether the antibodies of the present invention can bind to lipid nanoparticles. [Figure 10]FIG. 10 shows the results of calculating the mRNA encapsulation efficiency (Encapsulation %) of antibody-bound lipid nanoparticles at different mixing times while mixing the antibody of the present invention during the formation of lipid nanoparticles in order to optimize the mixing time between the lipid nanoparticles and the antibody of the present invention. [Figure 11] FIG. 11 shows the results of calculating the mRNA encapsulation efficiency (Encapsulation %) after antibody-conjugated lipid nanoparticles were prepared by mixing various lipid:protein molar ratios (LP ratios) to optimize the mixing ratio between lipid nanoparticles and the antibody of the present invention. [Figure 12] FIG. 12 shows the results of confirming the particle size of the antibody-bound lipid nanoparticles formed by mixing the lipid nanoparticles and the antibody of the present invention at various lipid:protein molar ratios (LP ratios) to optimize the mixing ratio. [Figure 13] FIG. 13 shows the results of observing the change in encapsulation efficiency after storing the antibody-conjugated lipid nanoparticles of the present invention at 4° C. for 7 days to confirm their storage stability. [Figure 14] FIG. 14 shows the results of examining the size of the antibody-bound lipid nanoparticles of the present invention while storing them at 4° C. for 7 days in order to confirm their storage stability. [Figure 15] FIG. 15 shows the results of confirming the relative binding ability by fluorescence measurement after treating MutuDC1940 cells expressing a target ligand and A549 cells not expressing a target ligand with antibody-conjugated lipid nanoparticles containing a DiD dye in order to confirm whether the antibody-conjugated lipid nanoparticles of the present invention specifically bind to target cells. [Figure 16] FIG. 16 shows the results of examining the relative gene expression levels of MutuDC1940 cells expressing a target ligand and A549 cells not expressing a target ligand after treatment with antibody-conjugated lipid nanoparticles to confirm whether the antibody-conjugated lipid nanoparticles of the present invention specifically deliver mRNA to target cells. [Figure 17]FIG. 17 shows the results of examining the relative gene expression levels of MutuDC1940 cells expressing a target ligand and A549 cells not expressing a target ligand after treatment with antibody-conjugated lipid nanoparticles in the presence of serum, in order to confirm whether the antibody-conjugated lipid nanoparticles of the present invention can specifically deliver mRNA to target cells even under conditions where serum interferes with the delivery. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention provides antibody-bound lipid nanoparticles, characterized in that an "antibody or antibody fragment" bound to a membrane scaffold protein is bound via a hydrophobic bond to a hydrophobic portion of a lipid that constitutes the lipid nanoparticle.

[0017] A known method for producing antibody-conjugated lipid nanoparticles (a form in which an antibody is conjugated to the surface of a lipid nanoparticle) comprises at least three steps, including: 1) a step of producing lipid nanoparticles by mixing a lipid mixture constituting the lipid nanoparticles with a substance to be encapsulated; 2) a step of binding the lipid nanoparticles to a compound capable of binding to an antibody; and 3) a step of binding the antibody to the compound (see Figure 1).

[0018] However, in the present invention, antibody-bound lipid nanoparticles can be produced without a separate antibody binding step by using an antibody bound to a membrane scaffold protein (see FIG. 3). That is, when the substance to be encapsulated, the lipid mixture, and the antibody of the present invention (so-called "grabber antibody") are mixed, it was confirmed that the antibody bound to the membrane scaffold protein is arranged on the surface of the lipid nanoparticles at the same time as the lipid nanoparticles are formed, and antibody-bound lipid nanoparticles are produced (see the upper part of FIG. 2). This means that when an antibody bound to a membrane scaffold protein is used, the existing three-step production process can be simplified into a single-step production process.

[0019] It was also confirmed that antibody-bound lipid nanoparticles could be formed by adding the antibody of the present invention (so-called "grabber antibody") to the lipid nanoparticles thus prepared (see the lower part of Figure 2). This means that it is possible to modify lipid nanoparticles with antibodies and endow the lipid nanoparticles with specificity by simply adding the antibody, without changing the manufacturing process of already developed lipid nanoparticles.

[0020] Meanwhile, in the present invention, the membrane scaffold protein (MSP) is a protein having a helix structure and exhibiting amphipathic characteristics, and serves as a mediator between the lipid and the antibody of the antibody-bound lipid nanoparticle of the present invention. In this case, the membrane scaffold protein binds to the hydrophobic portion of the lipid that constitutes the lipid nanoparticle through hydrophobic bonds.

[0021] A representative example of a membrane-structured protein is apolipoprotein. Apolipoprotein is a protein that is specifically present in plasma fat protein, and is known to stabilize the structure of fat protein, activate enzymes involved in fat protein metabolism, and act as a ligand for fat protein receptors present on the cell surface. Examples of the apolipoprotein include apolipoprotein A1 (ApoA-I), apolipoprotein A2 (ApoA-2), apolipoprotein B (ApoB), apolipoprotein C (ApoC), apolipoprotein E (ApoE), MSP1 (Membrane scaffold protein 1), MSP1D1, MSP1D2, MSP1E1, MSP1E2, MSP1E3, MSP1E3D1, MSP2, MSP2N1, MSP2N2, MSP2N3, etc.

[0022] Apo-A1, given as an example above, refers to a protein composed of a single polypeptide of 243 amino acids with a molecular weight of 28 kDa, having eight repeat unit domains of 11 or 22 amino acids, and having an alpha (α)-helix ratio of the secondary structure constituting HDL of 60 to 75%. The ApoA-I is known to be used mainly as a component of high density lipoprotein (HDL) that plays a role in removing cholesterol from surrounding tissues and transporting it to the liver or other lipoproteins. ApoE is a protein composed of a single polypeptide of 299 amino acids with a molecular weight of 33 kDa, and is a protein involved in transporting cholesterol like ApoA1.

[0023] In addition, in the present invention, a fragment of a membrane-structured protein that retains the helix structure and amphipathic properties can also be used as the membrane-structured protein. That is, a part (fragment) of a membrane-structured protein can be used instead of the entire membrane-structured protein, as long as the "helical structure and amphipathic properties" of the membrane-structured protein are not lost.

[0024] On the other hand, in the present invention, the "antibody or antibody fragment" binds to the lipid nanoparticle and plays a role in imparting specific targeting ability to the target cell to which the "antibody or antibody fragment" can bind. The antibody-bound lipid nanoparticle of the present invention can use various "antibodies or antibody fragments" according to the characteristics of the target cell. In this case, the antibody fragment may be an scFv or an "scFv-Fc in which scFv and Fc are bound" so as to impart specific targeting ability.

[0025] Meanwhile, in the present invention, the "antibody bound to a membrane scaffold protein" may be produced by binding a gene encoding an antibody to a gene encoding a membrane scaffold protein, and then expressing the resulting gene.

[0026] On the other hand, the antibody-bound lipid nanoparticle of the present invention preferably has a substance to be encapsulated therein, and in this case, the substance to be encapsulated is preferably a nucleic acid. One example of the nucleic acid may be mRNA.

[0027] Lipid nanoparticles are mainly used to safely deliver encapsulated substances to targets. Foreign mRNA has problems such as suppression of gene expression due to interference from the human immune response, and is difficult to introduce into cells due to its large size. mRNA vaccines have solved these problems by using lipid nanoparticles, and the antibody-bound lipid nanoparticles developed in the present invention may also be used as a carrier for transporting mRNA vaccines.

[0028] The lipid nanoparticles are preferably made by mixing ionizable lipids, structural helper lipids, cholesterol, and polyethylene glycol. Lipid nanoparticles made in this way are easy to encapsulate drugs such as nucleic acids, easy to pass through cell membranes, and have excellent retention in the body.

[0029] The present invention also provides a method for producing antibody-bound lipid nanoparticles, which comprises mixing an antibody bound to a membrane-structuring protein and a lipid.

[0030] As described above, the present invention can simplify the existing three-step production process into a single-step production process by using an antibody bound to a membrane-structured protein.

[0031] Meanwhile, in the method for producing the antibody-bound lipid nanoparticles of the present invention, the antibody-bound lipid nanoparticles are preferably mixed with a substance to be encapsulated. The substance to be encapsulated can preferably be an mRNA vaccine, and when produced in this manner, the nanoparticles can be used as a carrier for transporting the mRNA vaccine.

[0032] The present invention will now be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following examples and includes modifications in the technical ideas equivalent thereto.

[0033] [Example 1: Production of plasmid for producing antibodies that form lipid nanoparticles]

[0034] In this example, a plasmid capable of producing the "antibody that forms lipid nanoparticles" of the present invention is produced.

[0035] 1-1. Construction of plasmid for producing Grabber single chain variable fragment (Gr-scFv) that forms lipid nanoparticles

[0036] To prepare a plasmid for producing a "single-chain antibody fragment (Gr-scFv, see FIG. 3(a)) that forms lipid nanoparticles" consisting of a heavy chain variable region (VH), a light chain variable region (VL), and a membrane scaffold protein (MSP), pET-22b(+) or pcDNA3.1(+) was used as a vector and cloned using T4 DNA polymerase.

[0037] Specifically, PCR was performed using primers designed to be complementary to pET-22b(+) or pcDNA3.1(+) vector and both ends of the VH-linker-VL-linker-Apolipoprotein insert. Then, to remove the methylated backbone plasmid for PCR, the DpnI enzyme was treated at 37°C for 2 hours. T4 DNA polymerase was added and reacted at 25°C for 150 seconds and on ice for 10 minutes to induce hydrogen bonds between the vector and the insert. 1 μL of the reaction mixture was mixed with 100 μL of Escherichia coli Top10 soluble cells (Competent cells), and then incubated on ice for 30 minutes. For gene transduction, heat shock was applied at 42°C for 45 seconds. After that, 900 μL of SOC (Super Optimal broth with Catabolite repression) liquid medium was further added, and the cells were cultured at 37 ° C for 1 hour and centrifuged (13,000 rpm, 1 minute) to obtain cells. The obtained cells were resuspended in 100 μL of SOC liquid medium, smeared on a carbenicillin LB (Luria-Bertani) solid medium, and cultured at 37 ° C for 16 hours to form colonies. The colonies were inoculated into LB liquid medium with a concentration of 100 μg / mL of carbenicillin and cultured at 37 ° C for 16 hours, and then the plasmid for producing single-chain antibody fragments that form lipid nanoparticles was purified using a plasmid purification kit.

[0038] 1-2. Construction of plasmid for producing single-chain antibody (Grabber single chain variable fragment-Fc, Gr-scFv-Fc) that forms lipid nanoparticles

[0039] In order to prepare a plasmid for producing a "single-chain antibody that forms lipid nanoparticles (Gr-scFv-Fc, see FIG. 3(b))" consisting of a heavy chain variable region (VH), a light chain variable region (VL), a fragment crystallizable region (Fc region), and a membrane scaffold protein (MSP), a plasmid for producing a single-chain antibody that forms lipid nanoparticles was prepared in the same manner as in Example 1-1 above, using the pcDNA3.1(+) vector and a VH-linker-VL-hinge-Fc-linker-Apolipoprotein insert.

[0040] 1-3. Construction of plasmid for producing antibody (Gr-Antibody) that forms lipid nanoparticles

[0041] In order to produce a plasmid for producing an "antibody that forms lipid nanoparticles (Gr-Antibody, see FIG. 3(c))", which is a plasmid consisting of a heavy chain variable region (VH), a heavy chain constant region (CH) and a membrane scaffold protein (MSP), and a plasmid consisting of a light chain variable region (VL) and a light chain constant region (CL), plasmids for producing an antibody that forms lipid nanoparticles were prepared in the same manner as in Example 1-1 above, using the pMAZ vector and a VH-CH-linker-Apolipoprotein or VL-CL insert.

[0042] [Example 2: Production of antibodies that form lipid nanoparticles]

[0043] In this example, the plasmid prepared in Example 1 was used to produce the "antibody that forms lipid nanoparticles" of the present invention.

[0044] 2-1. Production of Grabber Single Chain Variable Fragment (Gr-scFv) that Forms Lipid Nanoparticles

[0045] To produce a single-chain antibody fragment (Grabber Single Chain Variable Fragment, Gr-scFv) forming lipid nanoparticles having the protein sequences of SEQ ID NOs: 1 to 9, a plasmid using the pET-22b(+) vector prepared in Example 1-1 was expressed using an E. coli protein expression system.

[0046] Specifically, the plasmid was introduced into the expression host E. coli BL21(DE3) at 42°C, which was then smeared on a 100μg / mL Cabenicillin LB (Luria-Bertani) solid medium and cultured at 37°C for 16 hours to form colonies, and the transformed E. coli strains were selected. Each transformed strain was inoculated into a 100μg / mL Cabenicillin LB liquid medium and cultured at 37°C for 16 hours, after which it was subcultured. The optical density (OD 600 After culturing until the pH reached 1.5, 0.5 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added and the mixture was cultured at 25°C for 6 hours to express single-chain antibody fragments that form lipid nanoparticles. The culture was then centrifuged (4,000 rpm, 10 minutes) to obtain cells. The obtained cells were pulverized using an ultrasonicator and then separated into water-soluble and insoluble fractions by centrifugation (15,000 rpm, 10 minutes).

[0047] On the other hand, the proteins in each fraction were confirmed by SDS gel electrophoresis, and it was confirmed that the "single-chain antibody fragment forming lipid nanoparticles" of the present invention was produced normally (Figure 4). On the other hand, in Figure 4, 10B4 and NLDC145 represent the antibodies used, and ApoA1, MSP1E3D1, and ApoeE3 represent the membrane-structured proteins used.

[0048] In addition, in order to produce a single-chain antibody fragment that forms lipid nanoparticles, the plasmid using the pcDNA3.1(+) vector prepared in Example 1-1 was expressed using a mammalian cell expression system.

[0049] Specifically, HEK-293F (Human embryonic kidney 293F) was used as the expression host, and the cells were cultured in a water-soluble suspended state at 37°C, 120 rpm, and 8% CO2, and the cell suspension (1.1 × 10 6 A total of 180 mL of culture medium (1.2 mL / mL) was prepared. For transfection, 250 μg of plasmid and 750 μg of PEI (Polyethylenimine) were mixed in 20 mL of medium, and then mixed with the prepared cell solution. The cells were then cultured for 96 hours at 37°C, 120 rpm, and 8% CO2, and centrifuged (8,000 g, 10 minutes) to remove the cells and obtain a supernatant. The supernatant was mixed with a phosphate buffer solution of pH 7.0 in a 1:1 ratio and purified using proL agarose beads. The material not bound to the beads was removed by treatment with a phosphate buffer solution of pH 7.0. The single-chain antibody fragment was purified by removing the bond between the single-chain antibody fragment and the agarose beads by treatment with a solution containing 0.1 M glycine at pH 2.5, and then the pH was restored to neutral pH using 1 M Tris buffer solution of pH 8.0.

[0050] On the other hand, when the purified single-chain antibody fragment was examined by SDS gel electrophoresis, it was confirmed that the "single-chain antibody fragment forming lipid nanoparticles" of the present invention was produced normally (Figure 5).

[0051] 2-2. Production of single-chain antibodies (Grabber Single chain variable fragment-Fc, Gr-scFv-Fc) that form lipid nanoparticles

[0052] To produce a single-chain antibody (Grabber Single Chain Variable Fragment-Fc, Gr-scFv-Fc) that forms lipid nanoparticles having the protein sequences of SEQ ID NOs: 10 to 18, a plasmid using the pcDNA3.1(+) vector prepared in Example 1-2 was expressed using a mammalian cell expression system.

[0053] Specifically, HEK-293F (Human embryonic kidney 293F) was used as the expression host, and the cells were cultured in a water-soluble suspended state at 37°C, 120 rpm, and 8% CO2, and the cell suspension (1.1 × 10 6 A total of 180 mL of medium (cells / mL) was prepared. For transfection, 250 μg of plasmid and 750 μg of PEI (Polyethylenimine) were mixed in 20 mL of medium, and then mixed with the prepared cell solution. The cells were then cultured for 96 hours under conditions of 37°C, 120 rpm, and 8% CO2, and then centrifuged (8,000 g, 10 minutes) to remove the cells and obtain a supernatant. The supernatant was mixed with a phosphate buffer solution of pH 7.0 in a 1:1 ratio and purified using proL agarose beads. The material not bound to the beads was removed by treatment with a phosphate buffer solution of pH 7.0. The single-chain antibody was purified by removing the bond between the single-chain antibody and the agarose beads by treatment with a solution containing 0.1 M glycine at pH 2.8, and then the pH was restored to neutral pH using 1 M Tris buffer solution of pH 8.0.

[0054] On the other hand, when the produced single-chain antibody was confirmed by SDS gel electrophoresis, it was confirmed that the "single-chain antibody forming lipid nanoparticles" of the present invention was produced normally (Figure 6).

[0055] 2-3. Production of antibodies (Gr-Antibodies) that form lipid nanoparticles

[0056] In order to produce an antibody (Gr-Antibody) that forms lipid nanoparticles having the IgL sequences of SEQ ID NOs: 19 to 21 and the IgH sequences of SEQ ID NOs: 22 to 30, a plasmid using the pcDNA3.1(+) vector prepared in Example 1-3 was expressed using a mammalian cell expression system.

[0057] Specifically, HEK-293F (Human embryonic kidney 293F) was used as the expression host, and the cells were cultured in a water-soluble suspended state at 37°C, 120 rpm, and 8% CO2, and the cell suspension (1.1 × 10 6 A total of 180 mL of medium (cells / mL) was prepared. For transfection, 250 μg of plasmid and 750 μg of PEI (Polyethylenimine) were mixed in 20 mL of medium, and then mixed with the prepared cell solution. After that, the cells were cultured for 144 hours under conditions of 37°C, 120 rpm, and 8% CO2, and then centrifuged (8,000 g, 10 minutes) to remove the cells and obtain a supernatant. The supernatant was mixed with a phosphate buffer solution of pH 7.0 in a 1:1 ratio and purified using proL agarose beads. The material not bound to the beads was removed by treating with a phosphate buffer solution of pH 7.0. The antibody was purified by removing the binding between the antibody and the agarose beads by treating with a solution containing 0.1 M glycine of pH 2.8, and then the pH was restored to neutral pH using 1 M Tris buffer solution of pH 8.0.

[0058] On the other hand, when the produced antibodies were confirmed by SDS gel electrophoresis, it was confirmed that the "antibodies that form lipid nanoparticles" of the present invention were produced normally (Figure 7).

[0059] On the other hand, in FIG. 7, 83.6 kDa represents a protein consisting of a heavy chain variable region (VH), a heavy chain constant region (CH), and an apolipoprotein, and 23.4 kDa represents a protein consisting of a light chain variable region (VL) and a light chain constant region (CL).

[0060] [Example 3: Confirmation of adhesion of membrane-structured proteins to lipid nanoparticles]

[0061] In this example, the adhesion of a membrane-structured protein to lipid nanoparticles was confirmed, and whether the "antibody that forms lipid nanoparticles" of the present invention can be attached to the antibody-bound lipid nanoparticles by mixing it with the lipid nanoparticles was confirmed.

[0062] 3-1. Production of lipid nanoparticles

[0063] A lipid solution was prepared by dissolving SM102 (CAS No. 2089251-47-6), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, and DMG-PEG-2000 (1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) in ethanol. On the other hand, an RNA solution was prepared by mixing 75 μL of messenger RNA (mRNA) with an aqueous phase mRNA buffer (10.25 mM citric acid, 2.25 mM trisodium citrate, pH 3.0). The prepared lipid solution and RNA solution were mixed using a microfluidic device such that the molar ratio of SM102:DSPC:cholesterol:DMG-PEG-2000 was 50:10:38.5:1.5, and the molar ratio (N / P ratio) of the amino group of the ionized lipid SM102 to the phosphate group of the mRNA was 6. Then, PBS was added to the mixture to dilute it to a 4-fold volume ratio, producing a lipid nanoparticle solution.

[0064] 3-2. Confirmation of adhesion of membrane-structured proteins to lipid nanoparticles

[0065] The lipid nanoparticle solution prepared in Example 3-1 was mixed with apolipoprotein AI-derived MSP1E3D1 as a membrane-structured protein at a lipid:protein molar ratio (LP ratio) of 240:1, and incubated at 4°C for 16 hours to attach the membrane-structured protein to the lipid nanoparticles.

[0066] Size exclusion chromatography was then used to separate the A and B of lipid nanoparticles (LNPs), membrane structuring proteins (MSPs), and lipid nanoparticles with membrane structuring proteins attached (MSP-LNPs). 280 We confirmed this (Figure 8).

[0067] Referring to FIG. 8, when lipid nanoparticles (LNPs) were mixed with membrane-structured proteins (MSPs), A 280It can be seen that the pick moves, which means that the membrane structuring protein (MSP) is successfully attached to the lipid nanoparticle (LNP).

[0068] In addition, the particle size of lipid nanoparticles (LNPs) and lipid nanoparticles with membrane-structured proteins attached (MSP-LNPs) was measured using dynamic light scattering (Figure 9).

[0069] Referring to Figure 9, it can be seen that the lipid nanoparticles with membrane-structured proteins attached (MSP-LNP) have a relatively large particle size, which means that the membrane-structured proteins (MSP) are successfully attached to the lipid nanoparticles (LNP).

[0070] These results indicate that the antibody forming the lipid nanoparticle of the present invention containing a membrane-structuring protein has excellent adhesion to the lipid nanoparticle.

[0071] [Example 4: Preparation of antibody-bound lipid nanoparticles of the present invention using antibodies that form lipid nanoparticles]

[0072] In this example, lipid nanoparticles to which an antibody is attached (antibody-bound lipid nanoparticles) were prepared using the single-chain antibody that forms the lipid nanoparticles of the present invention prepared in Example 2-2 above.

[0073] In addition, lipid nanoparticles were produced by varying the mixing time and mixing ratio of the single chain antibody forming the lipid nanoparticle of the present invention, and the optimized mixing time and ratio were selected.

[0074] 4-1. Selection of the timing of mixing lipid nanoparticles and single-chain antibodies forming the lipid nanoparticles of the present invention

[0075] A lipid solution was prepared by dissolving SM102 (CAS No. 2089251-47-6), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, and DMG-PEG-2000 (1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) in ethanol. On the other hand, an RNA solution was prepared by mixing 75 μL of messenger RNA (mRNA) with an aqueous phase mRNA buffer (10.25 mM citric acid, 2.25 mM trisodium citrate, pH 3.0). The prepared lipid solution and RNA solution were mixed using a microfluidic device so that the molar ratio of SM102:DSPC:cholesterol:DMG-PEG-2000 was 50:10:38.5:1.5, and the molar ratio (N / P ratio) of the amino group of the ionized lipid SM102 to the mRNA phosphate group was 6. The mixture was then diluted to a 4-fold volume ratio using PBS to form lipid nanoparticles, but the dilution was performed three times, and a single-chain antibody that forms lipid nanoparticles of the present invention was added so that the lipid:protein molar ratio (LP ratio) was 240:1, and the reaction was carried out at 37°C for 1 hour to form antibody-bound lipid nanoparticles.

[0076] In this case, the experimental group in which the single-chain antibody forming lipid nanoparticles of the present invention was added to the first dilution was named "early," the experimental group in which it was added to the second dilution was named "middle," the experimental group in which it was added to the third dilution was named "late," and the experimental group in which it was added evenly divided into a total of three dilutions was named "total."

[0077] Thereafter, a RiboGreen assay was performed to confirm whether the antibody-bound lipid nanoparticles of the present invention were successfully formed.

[0078] Specifically, 20 μL of lipid nanoparticles and antibody-bound lipid nanoparticles were each diluted in 180 μL of dPBS. 200 μL of dPBS or "1% Triton X-100 in dPBS" was further mixed and reacted for 10 minutes to prepare a lipid nanoparticle dilution solution and an antibody-bound lipid nanoparticle dilution solution. 100 μL of RNA solution in the standard concentration range (dPBS standard and 0.5% Triton X-100 in dPBS standard), the lipid nanoparticle dilution solution and the antibody-bound lipid nanoparticle dilution solution prepared above were placed in a 96-well plate, and 100 μL of RiboGreen solution was further added, followed by reaction at 25° C. for 5 minutes. The mRNA concentration was then measured by measuring excitation / emission = 485 / 530 nm using a fluorospectrophotometer (FIG. 10).

[0079] On the other hand, in Figure 10, the value of the experimental group diluted with dPBS was taken as the unencapsulated free mRNA concentration, and the value of the experimental group diluted with "1% Triton X-100 in dPBS" was taken as the total mRNA concentration. The encapsulation efficiency was calculated as (1-(Free mRNA / Total mRNA)) x 100%.

[0080] This confirmed that antibody-bound lipid nanoparticles were normally formed even if the single chain antibody forming the lipid nanoparticles of the present invention was mixed during the formation of the lipid nanoparticles. It was also confirmed that the later the single chain antibody forming the lipid nanoparticles of the present invention was added, the higher the efficiency of encapsulating mRNA. Therefore, in the following experiments, the antibody-bound lipid nanoparticles of the present invention were prepared by adding the single chain antibody forming the lipid nanoparticles of the present invention after the lipid nanoparticles were normally formed.

[0081] 4-2. Selection of the mixing ratio of lipid nanoparticles and single-chain antibodies forming the lipid nanoparticles of the present invention

[0082] A lipid solution was prepared by dissolving SM102 (CAS No. 2089251-47-6), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, and DMG-PEG-2000 (1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) in ethanol. On the other hand, an RNA solution was prepared by mixing 75 μL of messenger RNA (mRNA) with an aqueous phase mRNA buffer (10.25 mM citric acid, 2.25 mM trisodium citrate, pH 3.0). The prepared lipid solution and RNA solution were mixed using a microfluidic device so that the molar ratio of SM102:DSPC:cholesterol:DMG-PEG-2000 was 50:10:38.5:1.5, and the molar ratio (N / P ratio) of the amino group of the ionized lipid SM102 to the mRNA phosphate group was 6. The mixture was diluted 4 times by volume using PBS to form lipid nanoparticles. Single-chain antibodies that form lipid nanoparticles of the present invention at various lipid:protein molar ratios (LP ratios) were added to the lipid nanoparticles, and the reaction was carried out at 4°C for 16 hours to form antibody-bound lipid nanoparticles.

[0083] Thereafter, the encapsulation efficiency was calculated by performing the RiboGreen assay in the same manner as in Example 4-1 (FIG. 11), and the size of the antibody-bound lipid nanoparticles of the present invention formed was confirmed by performing the dynamic light scattering method in the same manner as in Example 3-2 (FIG. 12).

[0084] This confirmed that the encapsulation efficiency was high at approximately 95% when the LP ratio was between 5,000:1 and 100,000:1, and that the size of the lipid nanoparticles increased due to the attachment of the single-chain antibodies that form the lipid nanoparticles of the present invention to the lipid nanoparticles.

[0085] [Example 5: Confirmation of storage stability of antibody-bound lipid nanoparticles of the present invention]

[0086] In this example, the storage stability of the antibody-bound lipid nanoparticles of the present invention was confirmed.

[0087] 5-1. Change in mRNA encapsulation efficiency of antibody-conjugated lipid nanoparticles with storage time

[0088] The antibody-bound lipid nanoparticles of the present invention, which contain firefly luciferase mRNA and a portion of the NLDC145 antibody that binds to the ligand of MutuDC1940 and a membrane-structured protein (MSP1E3D1, ApoA1, or ApoE3), were prepared by the method of Example 4-2, and changes in mRNA encapsulation efficiency were confirmed while storing at 4° C. On the day of preparation of the antibody-bound lipid nanoparticles and 7 days later, the encapsulation efficiency was calculated by performing a RiboGreen assay using the same method as in Example 4-1 (FIG. 13).

[0089] 13, it can be seen that the mRNA encapsulation efficiency of the antibody-bound lipid nanoparticles of the present invention does not decrease even after 7 days from preparation. This result means that mRNA does not leak from the lipid nanoparticles even when the antibody forming the lipid nanoparticles of the present invention is attached to the lipid nanoparticles.

[0090] 5-2. Size change of antibody-conjugated lipid nanoparticles due to storage time

[0091] The antibody-bound lipid nanoparticles of the present invention prepared in Example 5-1 were stored at 4° C., and the size change of the antibody-bound lipid nanoparticles was observed for 7 days by performing dynamic light scattering in the same manner as in Example 3-2 ( FIG. 14 ). Meanwhile, in FIG. 14, the upper and lower panels show the same experimental results, but the upper panel shows the results including the experimental group with an LP ratio of 500:1, and the lower panel shows the results excluding the experimental group with an LP ratio of 500:1.

[0092] 14, it was confirmed that the antibody-bound lipid nanoparticles of the present invention maintain a constant size over time when the LP ratio is greater than 5,000:1, but the size increases dramatically when the LP ratio is between 500:1 and 1,000:1. This confirmed that stable antibody-bound lipid nanoparticles are formed only when the LP ratio exceeds 5,000:1.

[0093] [Example 6: Confirmation of the binding ability of the antibody-bound lipid nanoparticles of the present invention to target cells]

[0094] In this example, it was confirmed whether the antibody-bound lipid nanoparticles of the present invention specifically and effectively bind to target cells.

[0095] The antibody-bound lipid nanoparticles of the present invention were prepared by the method of Example 5-1, but DiD (DiIC18(5); 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt) dye was added at a molar ratio of 1% of the lipid to prepare DID-antibody-bound lipid nanoparticles.

[0096] A549 cells were grown in RPMI-1640 medium, and MutuDC1940 cells were grown in IMDM medium supplemented with 10 mM HEPES and 50 μM β-mercaptoethanol. A549 cells were used as an experimental group that did not express the target ligand, and MutuDC1940 cells were used as an experimental group that constantly expressed the target ligand.

[0097] Plate 1.5 x 10 A549 cells per well in a 96 well Ferulac plate. 5 MutuDC1940 cells were seeded at a concentration of 3.5 × 10 cells / mL in 100 μL of culture. MutuDC1940 cells were coated with collagen I at a concentration of 50 μg / mL to ensure good adhesion to the plate, and then seeded at 3.5 × 10 cells / mL in 100 μL of culture. 5100 μL was seeded at a concentration of 100 μg / mL of cells. After culturing for one day so that the cells would adhere well to the plate, the antibody-bound lipid nanoparticles of the present invention were treated so that the final lipid concentration in the well was 10 μg / mL. After culturing for 30 minutes under conditions of 37°C and 5% CO2, the supernatant was removed. After washing twice with PBS (phosphate buffered saline), 100 μL of cell lysis buffer was added and treated at 37°C for 5 minutes to lyse the cells. Then, fluorescence was measured at Ex / Em=644 / 663 nm using a plate reader to confirm the relative binding ability of the antibody-bound lipid nanoparticles of the present invention (FIG. 15).

[0098] 15, in the experimental group in which A549 cells not expressing a target ligand were treated, no difference was observed between the antibody-conjugated lipid nanoparticles of the present invention and general lipid nanoparticles, but in the experimental group in which MutuDC1940 cells expressing a target ligand were treated, it was confirmed that the antibody-conjugated lipid nanoparticles of the present invention exhibited a higher fluorescence value. Such results indicate that the antibody-conjugated lipid nanoparticles of the present invention specifically and better bind to target cells expressing a target ligand.

[0099] [Example 7: Confirmation of mRNA transfer ability of antibody-bound lipid nanoparticles of the present invention]

[0100] In this example, it was confirmed whether the antibody-conjugated lipid nanoparticles of the present invention efficiently and specifically delivered captured mRNA to target cells.

[0101] 7-1. Confirmation of mRNA transfer in the absence of serum

[0102] A luciferase assay was performed to confirm whether the antibody-bound lipid nanoparticles of the present invention prepared in Example 5-1 successfully delivered mRNA to target cells.

[0103] A549 cells were grown in RPMI-1640 medium, and MutuDC1940 cells were grown in IMDM medium supplemented with 10 mM HEPES and 50 μM β-mercaptoethanol. A549 cells were used as an experimental group in which the target ligand was not expressed in the absence of serum, and MutuDC1940 cells were used as an experimental group in which the target ligand was constantly expressed in the absence of serum.

[0104] Plate 2 x 10 A549 cells per well in a 96 well Ferulac plate. 5 MutuDC1940 cells were seeded at a concentration of 4 × 10 cells / mL in 500 μL of culture. MutuDC1940 cells were coated with collagen I at a concentration of 50 μg / mL to ensure good adhesion to the plate, and then seeded at 4 × 10 cells / mL in 500 μL of culture. 5 500μL was seeded at a concentration of 100 cells / mL. After culturing for 1 day so that the cells would adhere well to the plate, the antibody-bound lipid nanoparticles of the present invention were treated so that 125ng of total mRNA was treated per well. After culturing for 12 hours under 37℃ and 5% CO2 conditions, the supernatant was removed. After adding 200μL of cell lysis buffer and treating for 5 minutes at 37℃ to lyse the cells, 100μL of the supernatant was transferred to a white 96-well plate. Then, 50μL of luciferase substrate was added, and luminescence was measured using a plate reader to confirm the relative expression level of firefly luciferase gene based on the experimental group treated with general lipid nanoparticles (Figure 16).

[0105] 16, in the experimental group treated with A549 cells not expressing the target ligand, it was confirmed that the antibody-bound lipid nanoparticles of the present invention had lower gene expression levels in most cases compared to general lipid nanoparticles. It was also confirmed that the lower the amount of the antibody of the present invention contained, the higher the expression level in cells not expressing the target ligand.

[0106] On the other hand, in the experimental groups in which MutuDC1940 cells expressing the target ligand were treated, it was confirmed that gene expression levels increased by approximately 5- to 20-fold at LP ratios of 5,000:1 to 10,000:1 for MSP1E3D1, 500:1 to 10,000:1 for ApoA1, and 500:1 to 1,000:1 for ApoE3.

[0107] These results indicate that while general lipid nanoparticles randomly deliver the encapsulated mRNA, the antibody-conjugated lipid nanoparticles of the present invention contain a membrane-structured protein, which inhibits random delivery of mRNA to cells, and because they contain an antibody, they improve delivery of mRNA to cells to which the antibody can bind, thereby providing more specific delivery ability than general antibody-conjugated lipid nanoparticles.

[0108] 7-2. Confirmation of mRNA transfer in the presence of serum

[0109] It was confirmed whether mRNA could be specifically delivered as in Example 7-1 without being interfered with even in the presence of serum.

[0110] To this end, luciferase assays were performed in the same manner as in Example 5-1 above to confirm gene expression levels, but experiments were performed using A549 cells grown in RPMI-1640 medium supplemented with 10% FBS and MutuDC1940 cells grown in IMDM medium supplemented with 10% FBS, 10 mM HEPES, and 50 μM β-mercaptoethanol (Figure 17).

[0111] Referring to Figure 17, in the experimental group treated with A549 cells in which the target ligand was not expressed, it was confirmed that in most cases the antibody-conjugated lipid nanoparticles of the present invention showed lower gene expression levels compared to general lipid nanoparticles.

[0112] In contrast, when treated with MutuDC1940 cells expressing the target ligand, the group in which the lipid nanoparticles contained the antibody scFv (NLDC145) showed an approximately 5- to 20-fold increase in expression level at ratios of 5,000:1 to 100,000:1 for MSP1E3D1, 1,000:1 to 100,000:1 for ApoA1, and 500:1 to 100,000:1 for ApoE3.

[0113] These results indicate that even in the presence of serum, such as body fluids, conventional lipid nanoparticles randomly deliver encapsulated mRNA to any cell, whereas the antibody-conjugated lipid nanoparticles of the present invention contain a membrane-structured protein, thereby inhibiting random delivery of mRNA to cells, and contain an antibody, thereby improving delivery of mRNA to cells to which the antibody can bind, and thus having a more specific delivery ability than conventional antibody-conjugated lipid nanoparticles.

Claims

1. An antibody-bound lipid nanoparticle, characterized in that an antibody or antibody fragment bound to a membrane scaffold protein is bound via a hydrophobic bond to a hydrophobic portion of a lipid that constitutes the lipid nanoparticle.

2. The membrane-structured protein is The antibody-bound lipid nanoparticle according to claim 1, which is an amphiphilic protein having a helix structure.

3. The membrane-structured protein is The antibody-bound lipid nanoparticle according to claim 2, which is a fragment of a membrane-structured protein that retains its helical structure and amphipathic properties.

4. The antibody fragment comprises: The antibody-bound lipid nanoparticle according to claim 1, which is an scFv or an scFV-Fc in which an scFv and an Fc are bound to each other.

5. The antibody or antibody fragment to which the membrane scaffold protein is bound is The antibody-bound lipid nanoparticle according to claim 1, which is produced by binding a gene encoding an antibody or an antibody fragment to a gene encoding a membrane-structured protein, and then expressing the resulting product.

6. The lipid nanoparticles are 2. The antibody-bound lipid nanoparticle according to claim 1, wherein a substance to be encapsulated is encapsulated inside.

7. The substance to be encapsulated is The antibody-bound lipid nanoparticle according to claim 6, which is a nucleic acid.

8. A method for producing antibody-bound lipid nanoparticles, comprising mixing an antibody bound to a membrane-structured protein and a lipid.

9. The antibody-bound lipid nanoparticles are The method for producing antibody-bound lipid nanoparticles according to claim 8, further comprising mixing a substance to be encapsulated.

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