Antibody-conjugated lipid nanoparticles comprising an antibody bound to a membrane scaffold protein

Abstract

This invention relates to antibody-binding lipid nanoparticles comprising antibodies bound to membrane scaffold proteins, and more specifically, to antibody-binding lipid nanoparticles comprising lipids and antibodies bound to membrane scaffold proteins, which are therefore easy to prepare and have excellent specific targeting capabilities.

Description

Antibody-bound lipid nanoparticles containing antibodies that bind to membrane scaffold proteins Technical Field

[0001] This invention relates to antibody-binding lipid nanoparticles comprising antibodies bound to membrane scaffold proteins, and more specifically, to antibody-binding lipid nanoparticles comprising lipids and antibodies bound to membrane scaffold proteins, which are therefore easy to prepare and have excellent specific targeting capabilities. Background Technology

[0002] Messenger RNA (mRNA) plays a crucial role in transmitting genetic information from deoxyribonucleic acid (DNA) to organelles in the cytoplasm that synthesize proteins. When such messenger RNA enters the cell, it synthesizes proteins and triggers an immune response.

[0003] Messenger RNA vaccines have been developed using messenger RNA with the aforementioned characteristics. However, messenger RNA suffers from problems such as gene expression suppression due to interference caused by immune responses, and its large size makes it difficult for the messenger RNA to enter the cytoplasm.

[0004] To address the aforementioned issues, lipid nanoparticle (LNP) technology has been developed to facilitate the entry of messenger RNA into the cytoplasm. Its value has been enhanced with the recent use of carriers such as messenger RNA vaccines. However, the technology for selectively targeting and delivering lipid nanoparticles to cells still has limitations.

[0005] Therefore, lipid nanoparticles with excellent targeted delivery capabilities have been developed by combining the outstanding targeting and delivery capabilities of antibodies with lipid nanoparticle technology. Existing methods utilize chemical conjugation, which results in complex procedures and low yields. Furthermore, due to the unclear chemical nature and the attachment of antibodies as affinity tags, it is difficult to confirm their binding when injected into living organisms. Summary of the Invention

[0006] Technical issues

[0007] In this invention, it is intended to utilize antibodies that can bind to lipid nanoparticles to provide antibody-binding lipid nanoparticles and their preparation method, which are easy to prepare and have excellent targeted delivery capabilities.

[0008] Methods for solving problems

[0009] The present invention provides an antibody-bound lipid nanoparticle, characterized in that an antibody or antibody fragment bound to a membrane scaffold protein binds to the hydrophobic portion of the lipid constituting the lipid nanoparticle via hydrophobic bonds through the membrane scaffold protein as a medium.

[0010] In the antibody-binding lipid nanoparticles of the present invention, preferably, the membrane scaffold protein is an amphiphilic protein having a helix structure. In this case, the membrane scaffold protein may also be a fragment of a membrane scaffold protein that retains both the helix structure and amphiphilic properties.

[0011] In the antibody-binding lipid nanoparticles of the present invention, preferably, the antibody fragment is scFv or scFV-Fc which is scFv bound to Fc.

[0012] In the antibody-binding lipid nanoparticles of the present invention, preferably, the antibody or antibody fragment bound to the membrane scaffold protein is prepared by expressing the gene encoding the membrane scaffold protein after binding the gene encoding the gene encoding the membrane scaffold protein to the gene encoding the antibody or antibody fragment.

[0013] In the antibody-binding lipid nanoparticles of the present invention, preferably, the lipid nanoparticles contain an encapsulated target substance. In this case, preferably, the encapsulated target substance is nucleic acid.

[0014] Furthermore, the present invention provides a method for preparing antibody-binding lipid nanoparticles, characterized in that an antibody bound to a membrane scaffold protein and lipids are mixed.

[0015] In the preparation method of antibody-binding lipid nanoparticles of the present invention, preferably, the antibody-binding lipid nanoparticles further include an encapsulated target substance for mixing.

[0016] Invention Effects

[0017] The antibody-binding lipid nanoparticles of the present invention contain bound membrane scaffold proteins, exhibit excellent ability to be specifically delivered to target cells, and can be easily prepared. Attached Figure Description

[0018] Figure 1 briefly illustrates existing methods for preparing antibody-binding lipid nanoparticles.

[0019] Figure 2 briefly illustrates the preparation method of antibody-bound lipid nanoparticles using the antibody (Grabber antibody) of the present invention. On the other hand, part (a) of Figure 2 shows the preparation of lipid nanoparticles by mixing them with the antibody (Grabber antibody) of the present invention, and part (b) of Figure 2 shows the process of adding the antibody (Grabber antibody) of the present invention to the lipid nanoparticles.

[0020] Figure 3 briefly illustrates the structure of the antibody (Grabber antibody) of the present invention.

[0021] Figure 4 shows the results of sodium dodecyl sulfate polyacrylamide (SDS) gel electrophoresis to confirm the complete production of the single-chain antibody fragment (Gr-scFv) of the present invention after production using the E. coli protein expression system. Figure 4 shows the antibodies using 10B4 and NLDC 145, and the membrane scaffold proteins using ApoA1, MSP1E3D1, and ApoeE3.

[0022] Figure 5 shows the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis, used to confirm the complete production of the single-chain antibody fragment of the present invention after production using the mammalian cell protein expression system.

[0023] Figure 6 shows the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis, used to confirm the complete production of the single-chain antibody (Graber Single chain variable fragment-Fc, Gr-scFv-Fc) of the present invention after production using the mammalian cell protein expression system.

[0024] Figure 7 shows the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis, used to confirm the complete production of the antibody (Gr antibody, Graber Antibody, Gr-Antibody) of the present invention after production using the mammalian cell protein expression system.

[0025] Figure 8 shows the A-values ​​of membrane scaffold protein (MSP), lipid nanoparticles (LNP), and a mixture of lipid nanoparticles and membrane scaffold protein (MSP LNP) confirmed by size exclusion chromatography to verify whether the antibody of the present invention can bind to lipid nanoparticles. 280 The result.

[0026] Figure 9 shows the results of confirming the particle size of lipid nanoparticles (LNPs) and a mixture of lipid nanoparticles and membrane scaffold proteins (MSP LNPs) by dynamic light scattering to verify whether the antibody of the present invention can bind to lipid nanoparticles.

[0027] Figure 10 shows the results of calculating the messenger ribonucleic acid encapsulation efficiency (Encapsulation%) of the antibody binding to the lipid nanoparticles by mixing the antibody of the present invention during the formation of lipid nanoparticles in order to optimize the mixing timing of the lipid nanoparticles with the antibody of the present invention.

[0028] Figure 11 shows the results of calculating the encapsulation efficiency (Encapsulation%) of messenger ribonucleic acid after preparing antibody-bound lipid nanoparticles by mixing them with different lipid:protein molar ratios (LP ratios) to optimize the mixing ratio of lipid nanoparticles with the antibody of the present invention.

[0029] Figure 12 shows the results of dynamic light scattering to confirm the particle size of the antibody-binding lipid nanoparticles after preparing antibody-binding lipid nanoparticles by mixing them with different lipid:protein molar ratios (LP ratios) in order to optimize the mixing ratio of lipid nanoparticles with the antibody of the present invention.

[0030] Figure 13 shows the results of storing the antibody-binding lipid nanoparticles of the present invention at 4°C for 7 days and observing the changes in encapsulation efficiency to confirm the storage stability of the present invention.

[0031] Figure 14 shows the results of storing the antibody-binding lipid nanoparticles of the present invention at 4°C for 7 days to confirm their storage stability and to confirm the size of the antibody-binding lipid nanoparticles of the present invention.

[0032] Figure 15 shows the results of measuring fluorescence to confirm whether the antibody-binding lipid nanoparticles of the present invention specifically bind to target cells. The antibody-binding lipid nanoparticles containing DiD staining reagent were treated with MutuDC1940 cells expressing the target ligand and A549 cells not expressing the target ligand, respectively, to confirm the relative binding ability.

[0033] Figure 16 shows the results of treating MutuDC1940 cells expressing the target ligand and A549 cells not expressing the target ligand with antibody-binding lipid nanoparticles to confirm whether the antibody-binding lipid nanoparticles of the present invention specifically deliver messenger ribonucleic acid to target cells, and confirming the relative gene expression levels of the cells.

[0034] Figure 17 shows the results of treating MutuDC1940 cells expressing the target ligand and A549 cells not expressing the target ligand with antibody-binding lipid nanoparticles in the presence of serum to confirm whether the antibody-binding lipid nanoparticles of the present invention can specifically deliver messenger ribonucleic acid to target cells even when serum irritation occurs. Detailed Implementation

[0035] The present invention provides an antibody-bound lipid nanoparticle, characterized in that an antibody or antibody fragment bound to a membrane scaffold protein binds to the hydrophobic portion of the lipid constituting the lipid nanoparticle via hydrophobic bonds through the membrane scaffold protein as a medium.

[0036] Existing known methods for preparing antibody-bound lipid nanoparticles (in the form of lipid nanoparticles with antibodies bound to their surface) include: step 1), preparing lipid nanoparticles by mixing a lipid mixture constituting the lipid nanoparticles with an encapsulation target substance; step 2), binding a compound capable of binding to the above-mentioned lipid nanoparticles and antibodies; and step 3), binding antibodies to the above-mentioned compound. Therefore, the process consists of at least three steps (see Figure 1).

[0037] However, by using antibodies bound to membrane scaffold proteins (see Figure 3), antibody-bound lipid nanoparticles can be prepared even without an additional antibody binding step in this invention. That is, when the target material, the lipid mixture, and the antibody of this invention (i.e., the so-called "Gr antibody") are mixed, it can be confirmed that the antibody bound to the membrane scaffold proteins aligns on the surface of the lipid particles simultaneously with the formation of the lipid nanoparticles, thereby preparing antibody-bound lipid nanoparticles (see part (a) of Figure 2). This means that when using antibodies bound to membrane scaffold proteins, the existing three-step preparation process can be simplified to a single-step process.

[0038] Furthermore, it can be confirmed that antibody-bound lipid nanoparticles are formed by adding the antibody of the present invention (i.e., the so-called "Gr antibody") to the lipid nanoparticles (see part (b) of Figure 2). This means that even without changing the developed lipid nanoparticle preparation process, lipid nanoparticles can be endowed with specificity simply by adding an antibody to modify the lipid nanoparticles.

[0039] On the other hand, in this invention, the membrane scaffold protein, as a protein with a helical structure and exhibiting amphiphilic characteristics, acts as a mediator between lipids and antibodies in the antibody-binding lipid nanoparticles of this invention. In this case, the membrane scaffold protein is bound to the hydrophobic portion of the lipid constituting the lipid nanoparticles using hydrophobic bonds.

[0040] Representative examples of membrane scaffold proteins include apolipoproteins. Apolipoproteins, proteins specifically found in plasma lipoproteins, are known to stabilize lipoprotein structure, activate enzymes involved in lipoprotein metabolism, and function as ligands for lipoprotein receptors located on the cell surface. Examples of such apolipoproteins 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, and MSP2N3.

[0041] As an example mentioned above, Apo-A1 refers to a protein consisting of a single polypeptide of 243 amino acids with a molecular weight of 28 kDa, possessing eight repeating unit domains formed by 11 or 22 amino acids, and comprising 60% to 75% of the α-helices that form the secondary structure of HDL. ApoA-I is known to be a structural element of high-density lipoprotein (HDL), which plays a role in removing cholesterol from peripheral tissues and transporting it to dry or other lipoproteins. Furthermore, ApoE, a protein consisting of a single polypeptide of 299 amino acids with a molecular weight of 33 kDa, is also a protein involved in cholesterol transport, similar to ApoA1.

[0042] Furthermore, in this invention, fragments of membrane scaffold proteins that retain the "helical structure and amphiphilic properties" can also be used as the aforementioned membrane scaffold proteins. That is, within the range that the "helical structure and amphiphilic properties" of the aforementioned membrane scaffold proteins are not lost, a portion (fragment) of the membrane scaffold protein can be used instead of the entirety.

[0043] On the other hand, in this invention, antibodies or antibody fragments, by binding to lipid nanoparticles, impart specific targeting ability to target cells capable of binding to the aforementioned "antibody or antibody fragment". The antibody-binding lipid nanoparticles of this invention can utilize various "antibodies or antibody fragments" by referring to the characteristics of the target cells. In this case, to impart specific targeting ability, preferably, the antibody fragment is scFv or "scFv-Fc formed by scFv and Fc binding".

[0044] On the other hand, in this invention, an "antibody bound to a membrane scaffold protein" can be prepared by expressing a gene that encodes an antibody or antibody fragment after the gene encoding the membrane scaffold protein is combined with the gene encoding the antibody or antibody fragment.

[0045] On the other hand, preferably, the antibody-binding lipid nanoparticles of the present invention are encapsulated with an encapsulated target substance. In this case, preferably, the encapsulated target substance is a nucleic acid. An example of a nucleic acid may be messenger ribonucleic acid (RNA).

[0046] Lipid nanoparticles are primarily used for the safe delivery of encapsulated target substances. Exogenous messenger RNA (RNA) can inhibit gene expression due to interference from the body's immune response, and its large size makes it difficult to enter the cytoplasm. RNA vaccines address these issues by utilizing lipid nanoparticles. The antibody-binding lipid nanoparticles developed in this invention can also be used as carriers for delivering RNA vaccines.

[0047] Preferably, the lipid nanoparticles are prepared by mixing ionizable lipids, structural helper lipids, cholesterol, and polyethylene glycol. Lipid nanoparticles prepared in this way are easily encapsulated with drugs such as nucleic acids, can easily pass through cell membranes, and exhibit excellent in vivo retention.

[0048] Furthermore, the present invention provides a method for preparing antibody-binding lipid nanoparticles, characterized in that an antibody bound to a membrane scaffold protein and lipids are mixed.

[0049] As described above, the present invention can simplify the existing three-step preparation process into a one-step preparation process by utilizing antibodies that bind to membrane scaffold proteins.

[0050] On the other hand, in the method for preparing antibody-binding lipid nanoparticles of the present invention, preferably, the antibody-binding lipid nanoparticles further include an encapsulating target substance for mixing. Preferably, a messenger RNA vaccine can be encapsulated as the encapsulating target substance, and when prepared in the manner described above, a carrier for delivering the messenger RNA vaccine can be used.

[0051] The present invention will now be described in more detail through the following embodiments. However, the scope of the present invention is not limited to the following embodiments, and also includes modifications of equivalent technical concepts.

[0052] [Example 1: Preparation of plasmids for antibody production that form lipid nanoparticles]

[0053] In this embodiment, a plasmid capable of producing the "antibody that forms lipid nanoparticles" of the present invention is prepared.

[0054] 1-1. Preparation of single-chain antibody fragments (Grabersingle chain variable) to form lipid nanoparticles plasmids for fragment (Gr-scFv) production

[0055] To prepare a plasmid for producing a "single-chain antibody fragment (Gr-scFv, see part (a) of Figure 3)" consisting of a variable region of heavy chain (VH), a variable region of light chain (VL), and a membrane sc fold protein (MSP), pET-22b(+) or pcDNA3.1(+) was used as a vector, and T4 DNA polymerase was used for cloning.

[0056] Specifically, polymerase chain reaction (PCR) was performed using primers designed with the pET-22b(+) or pcDNA3.1(+) vector and the VH-linker-VL-linker-apolipoprotein insert complemented at both ends by 15 bp. Then, to remove methylation from the backbone plasmid used for PCR, DpnI enzyme was treated at 37°C for 2 hours. T4 deoxyribonucleic acid polymerase was added and reacted at 25°C for 150 seconds, followed by 10 minutes on ice to induce hydrophobic binding between the vector and the insert. 1 μL of the reaction mixture was mixed with 100 μL of Escherichia coli Top10 competent cells and cultured on ice for 30 minutes. For gene transduction, a heat shock was applied at 42°C for 45 seconds. Then, 900 μL of Super Optimal Broth with Catabolite Repression (SOC) liquid medium was added, and the cells were cultured at 37°C for 1 hour. Cells were obtained by centrifugation (13000 rpm, 1 minute). The obtained cells were resuspended in 100 μL of SOC liquid medium and spread onto Luria-Bertani (LB) solid medium, and cultured at 37°C for 16 hours to form colonies. The colonies were inoculated into 100 μg / mL LB liquid medium and cultured at 37°C for 16 hours. After purification, the plasmid for the production of single-chain antibody fragments to form lipid nanoparticles was purified using a plasmid purification kit.

[0057] 1-2. Preparation of single-chain antibodies (Grabersinglechain variable fragments) forming lipid nanoparticles plasmids for production of ment-Fc and Gr-scFv-Fc

[0058] To prepare a plasmid for producing a "single-chain antibody forming lipid nanoparticles (Gr-scFv-Fc, see part (b) of Figure 3)" composed of a heavy chain variable region, a light chain variable region, a fragment crystallizable region (Fc region), and a membrane scaffold protein, a plasmid for producing a single-chain antibody forming lipid nanoparticles was prepared using the pcDNA3.1(+) vector and a VH-linker-VL-hinge-Fc-linker-apolipoprotein insert in the same manner as in Examples 1-1 above.

[0059] 1-3. Preparation of plasmids for producing lipid nanoparticles (Gr antibodies)

[0060] To prepare plasmids for producing "antibodies that form lipid nanoparticles (Gr antibodies, see part (c) of FIG3)" consisting of a heavy chain variable region, a heavy chain constant region (CH) and a membrane scaffold protein, and plasmids consisting of a light chain variable region and a light chain constant region (CL), plasmids for producing antibodies that form lipid nanoparticles were prepared using the pMAZ vector, VH-CH-linker-apolipoprotein or VL-CL insert in the same manner as in Examples 1-1 above.

[0061] [Example 2: Production of antibodies that form lipid nanoparticles]

[0062] In this embodiment, the plasmid prepared in Example 1 above is used to produce the "antibody that forms lipid nanoparticles" of the present invention.

[0063] 2-1. Production of single-chain antibody fragments (Graber single-chain variable) to form lipid nanoparticles fragment, Gr-scFv)

[0064] To produce a single-chain antibody fragment (Gr-scFv) forming lipid nanoparticles having protein sequences 1 to 9, the plasmid prepared in Example 1-1 using the pET-22b(+) vector was expressed using an E. coli expression system.

[0065] Specifically, plasmids were introduced into *E. coli* BL21(DE3), the expression host, at 42°C. The plasmids were then spread onto Luria-Bertany solid medium containing 100 μg / mL carbenicillin and cultured at 37°C for 16 hours to form colonies for screening of transformed *E. coli* strains. The transformed strains were then inoculated onto Luria-Bertany liquid medium containing 100 μg / mL carbenicillin and cultured at 37°C for 16 hours before subculturing. The optical density (OD) of the subculture medium was measured at a wavelength of 600 nm after subculturing. 600 After the concentration of the antibody was reduced to 1.5, 0.5 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) was added, and the cells were cultured at 25°C for 6 hours to express the single-chain antibody fragment that forms lipid nanoparticles. Cells were then obtained by centrifugation (4000 rpm, 10 min) of the culture medium. After the cells were disrupted using an ultrasonic sonicator, the water-soluble and water-insoluble fractions were separated by centrifugation (15000 rpm, 10 min).

[0066] On the other hand, the results of protein analysis in each fraction were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, confirming the complete production of the "single-chain antibody fragment forming lipid nanoparticles" of the present invention (Figure 4). In Figure 4, 10B4 and NLDC 145 represent the antibodies used, and ApoA1, MSP1E3D1, and ApoeE3 represent the membrane scaffold proteins used.

[0067] Furthermore, in order to produce single-chain antibody fragments that form lipid nanoparticles, a mammalian cell expression system was used to express plasmids using the pcDNA3.1(+) vector prepared in Example 1-1 above.

[0068] Specifically, HEK-293F (human embryonic kidney 293F) was used as the expression host and cultured in water-soluble mammalian cells at 37°C, 120 rpm, and 8% CO2 to prepare 180 mL of cell culture (1.1 × 10⁻⁶ cells / mL). 6For transfection, 250 μg of plasmid and 750 μg of polyethyleneimine (PEI) were mixed in 20 mL of culture medium and then mixed with the prepared cell slurry. After culturing at 37°C, 120 rpm, and 8% CO2 for 96 hours, the cells were removed by centrifugation (8000 g, 10 min) to obtain the supernatant. The supernatant was mixed with phosphate buffer (pH 7.0) at a 1:1 ratio and purified using proL agarose beads. The pH 7.0 phosphate buffer was treated to remove unbound material. The single-chain antibody fragment was purified by treating a solution containing 0.1 M glycine at pH 2.5 to detach the binding between the single-chain antibody fragment and the agarose beads. The pH was then restored to neutral using 1 M Tris buffer at pH 8.0.

[0069] On the other hand, the results of the purified single-chain antibody fragment confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed that the "single-chain antibody fragment forming lipid nanoparticles" of the present invention was produced intact (Figure 5).

[0070] 2-2. Production of single-chain antibodies (Graber-single-chain variable fragments) forming lipid nanoparticles ment-Fc, Gr-scFv-Fc)

[0071] To produce single-chain antibodies (Graber single-chain variable fragment-Fc, Gr-scFv-Fc) that form lipid nanoparticles having protein sequences 10 to 18, a mammalian cell protein expression system was used to express plasmids using the pcDNA3.1(+) vector prepared in Examples 1-2 above.

[0072] Specifically, HEK-293F was used as the expression host and cultured in water-soluble mammalian cells at 37°C, 120 rpm, and 8% CO2 to prepare 180 mL of cell culture (1.1 × 10⁻⁶ cells / mL). 6(cells / mL). For transfection, 250 μg of plasmid and 750 μg of polyethyleneimine were mixed in 20 mL of culture medium and then mixed with the prepared cell slurry. After culturing at 37°C, 120 rpm, and 8% CO2 for 96 hours, the cells were removed by centrifugation (8000g, 10 min) to obtain the supernatant. The supernatant was mixed with phosphate buffer (pH 7.0) at a 1:1 ratio and purified using proL agarose beads. The pH 7.0 phosphate buffer was treated to remove unbound material. The single-chain antibody fragment was purified by treating a solution containing 0.1 M glycine at pH 2.8 to detach the binding between the single-chain antibody fragment and the agarose beads. The pH was then restored to neutral using 1 M Tris buffer (pH 8.0).

[0073] On the other hand, the results of the purified single-chain antibody were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, confirming the complete production of the "single-chain antibody forming lipid nanoparticles" of the present invention (Figure 6).

[0074] 2-3. Production of antibodies that form lipid nanoparticles (Gr antibodies)

[0075] In order to produce antibodies (Gr antibodies) that form lipid nanoparticles having IgL sequences of sequences 19 to 21 and IgH sequences of sequences 22 to 30, a mammalian cell protein expression system was used to express plasmids using the pcDNA3.1(+) vector prepared in Examples 1-3 above.

[0076] Specifically, HEK-293F was used as the expression host and cultured in water-soluble mammalian cells at 37°C, 120 rpm, and 8% CO2 to prepare 180 mL of cell culture (1.1 × 10⁻⁶ cells / mL). 6 (cells / mL). For transfection, 250 μg of plasmid and 750 μg of polyethyleneimine were mixed in 20 mL of culture medium and then mixed with the prepared cell slurry. After culturing at 37°C, 120 rpm, and 8% CO2 for 144 hours, the cells were removed by centrifugation (8000g, 10 min) to obtain the supernatant. The supernatant was mixed with phosphate buffer (pH 7.0) at a 1:1 ratio and purified using proL agarose beads. The pH 7.0 phosphate buffer was treated to remove unbound material. The single-chain antibody fragment was purified by treating a solution containing 0.1 M glycine at pH 2.8 to detach the binding between the single-chain antibody fragment and the agarose beads. The pH was then restored to neutral using 1 M Tris buffer (pH 8.0).

[0077] On the other hand, the results of the purified single-chain antibody were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, confirming the complete production of the "antibody forming lipid nanoparticles" of the present invention (Figure 7).

[0078] On the other hand, in Figure 7, 83.6 kDa represents a protein composed of heavy chain variable region, heavy chain constant region, and apolipoprotein, while 23.4 kDa represents a protein composed of light chain variable region and light chain constant region.

[0079] [Example 3: Confirmation of lipid nanoparticle adhesion of membrane scaffold proteins]

[0080] In this embodiment, in order to confirm the adhesion of the membrane scaffold protein to the lipid nanoparticles, the method of mixing the "antibody that forms lipid nanoparticles" of the present invention with the lipid nanoparticles was used to confirm whether the lipid nanoparticles could be attached as an antibody-bound lipid nanoparticle.

[0081] 3-1. Preparation of lipid nanoparticles

[0082] Lipid solutions were prepared by dissolving SM102 (CAS No. 2089251-47-6), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyris toyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000) in ethanol. Conversely, 75 μL of messenger RNA (mRNA) was mixed in an aqueous phase of messenger RNA buffer (10.25 mM citric acid, 2.25 mM trisodium citrate, pH 3.0) to prepare an RNA solution. The prepared lipid solution and ribonucleic acid solution were mixed using a microfluidic device at a molar ratio of SM102:DSPC:cholesterol:DMG-PEG-2000 of 50:10:38.5:1.5, with a molar ratio (N / P ratio) of 6 for the amino group of SM102 (the ionized lipid) to the phosphate group of messenger ribonucleic acid. The mixture was then diluted to a 4-fold volume ratio with phosphate-buffered saline (PBS) to prepare a lipid nanoparticle solution.

[0083] 3-2. Confirm the adhesion of membrane scaffold proteins to lipid nanoparticles.

[0084] MSP1E3D1, a membrane scaffold protein derived from apolipoprotein AI, was mixed into the lipid nanoparticle solution prepared in step 3-1 at a lipid-protein molar ratio (LP ratio) of 240:1 and cultured at 4°C for 16 hours to allow the membrane scaffold protein to attach to the lipid nanoparticles.

[0085] Then, size exclusion chromatography was used to confirm the A-type of lipid nanoparticles (LNP), membrane scaffold protein (MSP), and lipid nanoparticles with membrane scaffold protein (MSP-LNP). 280 (Figure 8)

[0086] As shown in Figure 8, it can be confirmed that when membrane scaffold proteins (MSPs) are mixed in lipid nanoparticles (LNPs), A 280 The shift in the wave peak indicates that the membrane scaffold protein (MSP) is intactly attached to the lipid nanoparticle (LNP).

[0087] Furthermore, the particle size of lipid nanoparticles (LNPs) with attached lipid nanoparticles (LNPs) and membrane scaffold proteins (MSP-LNPs) was measured by performing dynamic light scattering (Figure 9).

[0088] As shown in Figure 9, it can be confirmed that the lipid nanoparticles (MSP-LNP) with attached membrane scaffold proteins are larger in size, which indicates that the membrane scaffold proteins (MSP) are completely attached to the lipid nanoparticles (LNP).

[0089] The above results demonstrate that the antibody of the present invention, which contains membrane scaffold proteins, exhibits excellent adhesion to lipid nanoparticles.

[0090] [Example 4: Preparation of antibody-bound lipid nanoparticles of the present invention using antibodies that form lipid nanoparticles]

[0091] In this embodiment, the single-chain antibody for forming lipid nanoparticles of the present invention prepared in Examples 2-2 above is used to prepare antibody-attached lipid nanoparticles (antibody-bound lipid nanoparticles).

[0092] Furthermore, lipid nanoparticles were prepared by mixing them with the single-chain antibodies that form lipid nanoparticles according to the present invention at different mixing times and in different mixing ratios, so as to select the optimal mixing time and ratio.

[0093] 4-1. Choosing the timing of mixing lipid nanoparticles with the single-chain antibody of the present invention for forming lipid nanoparticles

[0094] Lipid solutions were prepared by dissolving SM102 (CAS No. 2089251-47-6), DSPC, cholesterol, and DMG-PEG-2000 in ethanol. Meanwhile, a ribonucleic acid (RNA) solution was prepared by mixing 75 μL of messenger RNA (mRNA) in an aqueous messenger RNA buffer (10.25 mM citric acid, 2.25 mM trisodium citrate, pH 3.0). The prepared lipid and RNA solutions were mixed using a microfluidic device at a molar ratio of SM102:DSPC:cholesterol:DMG-PEG-2000 of 50:10:38.5:1.5, with a molar ratio (N / P ratio) of 6 for the amino groups of SM102 (the ionized lipid) to the phosphate groups of the messenger RNA. Then, phosphate-buffered saline (PBS) was added to the mixture to dilute it to a 4-fold volume ratio to form lipid nanoparticles. The dilution was performed in 3 times. The single-chain antibody for forming lipid nanoparticles of the present invention was added at a lipid:protein molar ratio (LP ratio) of 240:1 and reacted at 37°C for 1 hour to form antibody-bound lipid nanoparticles.

[0095] In this case, the experimental group in which the single-chain antibody of the present invention forming lipid nanoparticles was added during the first dilution is named early, the experimental group in which it was added during the second dilution is named middle, the experimental group in which it was added during the third dilution is named late, and the experimental group in which it was added evenly distributed in the total of 3 dilutions is named total.

[0096] Then, in order to confirm whether the antibody-binding lipid nanoparticles of the present invention were successfully formed, a RiboGreen assay was performed.

[0097] Specifically, 20 μL of lipid nanoparticles and antibody-bound lipid nanoparticles were diluted in 180 μL of dPBS, respectively. Then, 200 μL of dPBS or dPBS containing 1% Triton X-100 was mixed and reacted for 10 minutes to prepare the lipid nanoparticle dilution solution and the antibody-bound lipid nanoparticle dilution solution. 100 μL of standard concentration range ribonucleic acid solutions (dPBS standard and dPBS containing 0.5% Triton X-100 standard) were added to each of the prepared lipid nanoparticle dilution solutions and antibody-bound lipid nanoparticle dilution solutions to a 96-well plate. After adding 100 μL of RiboGreen solution, the plates were reacted at 25°C for 5 minutes. Then, the messenger ribonucleic acid concentration was measured using a Fluoro spectrophotometer with an excitation / emission ratio of 485 / 530 nm (Figure 10).

[0098] On the other hand, in Figure 10, the values ​​for the experimental group diluted with dPBS are expressed as the concentration of unencapsulated free mRNA, and the values ​​for the experimental group diluted with dPBS containing 1% Triton X-100 are expressed as the concentration of total mRNA. Encapsulation efficiency is calculated as (1 - (Free mRNA / Total mRNA)) × 100%.

[0099] This confirms that even when the single-chain antibody of the present invention for forming lipid nanoparticles is mixed during the formation of lipid nanoparticles, antibody-binding lipid nanoparticles can still be completely formed. Furthermore, it confirms that the later the single-chain antibody for forming lipid nanoparticles is added, the higher the encapsulation efficiency of the messenger ribonucleic acid. Therefore, in the following experiments, the antibody-binding lipid nanoparticles of the present invention were prepared by adding the single-chain antibody for forming lipid nanoparticles of the present invention after the complete formation of lipid nanoparticles.

[0100] 4-2. Selecting the mixing ratio of lipid nanoparticles and the single-chain antibody used to form lipid nanoparticles in this invention.

[0101] Lipid solutions were prepared by dissolving SM102 (CAS No. 2089251-47-6), DSPC, cholesterol, and DMG-PEG-2000 in ethanol. Meanwhile, a ribonucleic acid (RNA) solution was prepared by mixing 75 μL of messenger RNA (mRNA) in an aqueous messenger RNA buffer (10.25 mM citric acid, 2.25 mM trisodium citrate, pH 3.0). The prepared lipid and RNA solutions were mixed using a microfluidic device at a molar ratio of SM102:DSPC:cholesterol:DMG-PEG-2000 of 50:10:38.5:1.5, with a molar ratio (N / P ratio) of 6 for the amino group of SM102 (the ionized lipid) to the phosphate group of the messenger RNA. The mixture was then diluted 4 times by volume with phosphate-buffered saline (PBS) to form lipid nanoparticles. The single-chain antibody of the present invention, which forms lipid nanoparticles, was added to lipid nanoparticles at different lipid:protein molar ratios (LP ratios) and reacted at 4°C for 16 hours to form antibody-bound lipid nanoparticles.

[0102] Then, the encapsulation efficiency was calculated by performing the RiboGreen assay in the same manner as in Example 4-1 above (Figure 11), and the size of the antibody-binding lipid nanoparticles of the present invention was confirmed by performing the dynamic light scattering method in the same manner as in Example 3-2 above (Figure 12).

[0103] This confirms that when the lipid:protein molar ratio (LP ratio) is 5000:1 to 100000:1, the encapsulation efficiency reaches as high as about 95%, and it can be confirmed that the lipid nanoparticles are enlarged due to the attachment of the single-chain antibody of the present invention to form lipid nanoparticles.

[0104] [Example 5: Confirmation of the storage stability of the antibody-binding lipid nanoparticles of the present invention]

[0105] In this embodiment, the storage stability of the antibody-binding lipid nanoparticles of the present invention was confirmed.

[0106] 5-1. Changes in the efficiency of antibody-bound lipid nanoparticle-based messenger ribonucleic acid encapsulation with storage time

[0107] Following the preparation of the antibody-bound lipid nanoparticles of the present invention, comprising a portion of NLDC145 antibody containing firefly luciferase mRNA and bound to a ligand of MutuDC1940, and the preparation of the antibody-bound lipid nanoparticles containing membrane scaffold proteins (MSP1E3D1, ApoA1, or ApoE3), using the method of Examples 4-2, the nanoparticles were stored at 4°C to confirm changes in the encapsulation efficiency of the messenger ribonucleic acid. On the day of preparation of the antibody-bound lipid nanoparticles and 7 days later, the encapsulation efficiency was calculated using the RiboGreen assay performed in the same manner as in Example 4-1 (Figure 13).

[0108] As shown in Figure 13, it can be confirmed that the messenger ribonucleic acid encapsulation efficiency of the antibody-binding lipid nanoparticles of the present invention did not decrease even after 7 days. These results indicate that even when the antibody of the present invention, which forms the lipid nanoparticles, is attached to the lipid nanoparticles, the messenger ribonucleic acid does not leak out from the lipid nanoparticles.

[0109] 5-2. Changes in the size of antibody-bound lipid nanoparticles over storage time

[0110] The antibody-binding lipid nanoparticles of the present invention prepared in Example 5-1 were stored at 4°C, and dynamic light scattering was performed in the same manner as in Example 3-2 to confirm the size change of the antibody-binding lipid nanoparticles over 7 days (Figure 14). On the other hand, parts (a) and (b) of Figure 14 show the results of the same experiment, with part (a) including the experimental group with a lipid:protein molar ratio (LP ratio) of 500:1, and part (b) excluding the experimental group with a lipid:protein molar ratio (LP ratio) of 500:1.

[0111] As shown in Figure 14, it can be confirmed that the antibody-binding lipid nanoparticles of the present invention maintain a specified size even after time when the lipid:protein molar ratio (LP ratio) is greater than 5000:1, but the size increases dramatically when the ratio is between 500:1 and 1000:1. This confirms that a lipid:protein molar ratio (LP ratio) greater than 5000:1 is required to form stable antibody-binding lipid nanoparticles.

[0112] [Example 6: Confirmation of the binding ability of the antibody-binding lipid nanoparticles of the present invention to target cells]

[0113] In this embodiment, it was confirmed whether the antibody-binding lipid nanoparticles of the present invention specifically bind to target cells.

[0114] The antibody-binding lipid nanoparticles of the present invention were prepared by adding DiD (DiIC18(5); 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt) staining reagent at a molar ratio of 1% of lipids to prepare DID-antibody-binding lipid nanoparticles.

[0115] A549 cells were cultured in RPMI-1640 medium, and MutuDC1940 cells were cultured in IMDM medium supplemented with 10 mM HEPES and 50 μM β-mercaptoethanol. A549 cells were used as the experimental group that did not express the target ligand, while MutuDC1940 cells were used as the experimental group that consistently expressed the target ligand.

[0116] With 1.5×10 per hole 5 100 μL of A549 cells were seeded into 96-well black culture plates at a concentration of cells / mL. To ensure good attachment of MutuDC1940 cells to the culture plate, type I collagen was coated at a concentration of 50 μg / mL, and then seeded at 3.5 × 10⁶ cells / well. 5 100 μL of the antibody-binding lipid nanoparticles were seeded at a concentration of cells / mL. After one day of culture to allow for good cell attachment, the antibody-binding lipid nanoparticles of the present invention were treated to achieve a final lipid concentration of 10 μg / mL per well. After culturing at 37°C and 5% CO2 for 30 minutes, the supernatant was removed. After washing twice with phosphate-buffered saline (PBS), the cells were placed in 100 μL of cell lysis buffer and treated at 37°C for 5 minutes to induce cell lysis. The relative binding capacity of the antibody-binding lipid nanoparticles of the present invention was then confirmed by measuring fluorescence at Ex / Em = 644 / 663 nm using a plate reader (Figure 15).

[0117] As shown in Figure 15, in the experimental group treated with A549 cells that do not express the target ligand, the antibody-binding lipid nanoparticles of the present invention showed no difference from ordinary lipid nanoparticles. However, in the experimental group treated with MutuDC1940 cells that express the target ligand, the antibody-binding lipid nanoparticles of the present invention showed a higher fluorescence value. These results indicate that the antibody-binding lipid nanoparticles of the present invention specifically and better bind to target cells expressing the target ligand.

[0118] [Example 7: Confirmation of the messenger RNA delivery capability of the antibody-binding lipid nanoparticles of the present invention]

[0119] In this embodiment, the antibody-binding lipid nanoparticles of the present invention specifically and effectively deliver the captured messenger ribonucleic acid to the target cells.

[0120] 7-1. Confirm messenger RNA delivery in the absence of serum.

[0121] To confirm whether the antibody-binding lipid nanoparticles of the present invention prepared in Example 5-1 above successfully delivered messenger ribonucleic acid to target cells, a luciferase assay was performed.

[0122] A549 cells were cultured in RPMI-1640 medium, and MutuDC1940 cells were cultured in IMDM medium supplemented with 10 mM HEPES and 50 μM β-mercaptoethanol. A549 cells were used as the experimental group that did not express the target ligand, while MutuDC1940 cells were used as the experimental group that consistently expressed the target ligand.

[0123] With 2×10 per hole 5 500 μL of A549 cells were seeded into 96-well black culture plates at a concentration of cells / mL. To ensure good attachment of MutuDC1940 cells to the culture plate, type I collagen was coated at a concentration of 50 μg / mL, and then seeded at a concentration of 4 × 10⁶ cells / well. 5500 μL of the solution was seeded at a concentration of cells / mL. After one day of culture to allow for good cell attachment, the antibody-bound lipid nanoparticles of this invention were treated to ensure a total messenger RNA concentration of 125 ng per well. After culturing at 37°C and 5% CO2 for 12 hours, the supernatant was removed. 200 μL of cell lysis solution was added and the cells were lysed at 37°C for 5 minutes. 100 μL of the supernatant was then transferred to a white 96-well plate. 50 μL of luciferase substrate was then added, and luminescence was measured using a microplate reader to confirm the relative expression level of the firefly luciferase gene compared to the experimental group treated with ordinary lipid nanoparticles (Figure 16).

[0124] As shown in Figure 16, it can be confirmed that in the experimental group treated with A549 cells that do not express the target ligand, in most cases, the gene expression levels of the antibody-binding lipid nanoparticles of the present invention were lower compared with ordinary lipid nanoparticles. Furthermore, it can be confirmed that the less antibody contained in the present invention, the higher the expression level in cells that do not express the target ligand.

[0125] Conversely, it can be confirmed that in the experimental group treated with MutuDC1940 cells expressing the target ligand, gene expression increased by approximately 5 to 20-fold when the lipid-to-protein molar ratio (LP ratio) of MSP1E3D1 was 500:1 to 10000:1, that of ApoA1 was 500:1 to 10000:1, and that of ApoE3 was 500:1 to 1000:1.

[0126] The above results indicate that ordinary lipid nanoparticles randomly deliver the messenger ribonucleic acid (RNA) they contain. However, the antibody-binding lipid nanoparticles of the present invention inhibit random delivery of RNA to cells by including membrane scaffold proteins and enhance delivery of RNA to cells that can bind to antibodies by including antibodies, thus exhibiting superior specific delivery capabilities compared to ordinary antibody-binding lipid nanoparticles.

[0127] 7-2. Confirm messenger RNA delivery in the presence of serum.

[0128] To confirm whether messenger ribonucleic acid can be delivered specifically as in Example 7-1 above without hindrance in the presence of serum.

[0129] Therefore, the gene expression level was confirmed by luciferase assay using the same method as described in 5-1 above. A549 cells cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS) and MutuDC1940 cells cultured in IMDM medium with 10% fetal bovine serum, 10 mM HEPES, and 50 μM β-mercaptoethanol were used for the experiment (Figure 17).

[0130] As shown in Figure 17, it can be confirmed that in the experimental group treated with A549 cells that do not express the target ligand, in most cases, the antibody-binding lipid nanoparticles of the present invention have lower gene expression levels compared with ordinary lipid nanoparticles.

[0131] Conversely, it can be confirmed that in the experimental group treated with MutuDC1940 cells expressing the target ligand, the expression level increased by approximately 5 to 20 times when the ratio of MSP1E3D1 was 5000:1 to 10000:1, the ratio of ApoA1 was 500:1 to 10000:1, and the ratio of ApoE3 was 500:1 to 1000:1.

[0132] The above results indicate that, similar to body fluids, even in the presence of serum, ordinary lipid nanoparticles randomly deliver the messenger RNA they contain to any cell. However, the antibody-binding lipid nanoparticles of the present invention inhibit the random delivery of messenger RNA to cells by including membrane scaffold proteins and enhance the delivery of messenger RNA to cells capable of binding to antibodies by including antibodies, thus exhibiting a more specific delivery capability than ordinary antibody-binding lipid nanoparticles.

Claims

1. A method for preparing antibody-bound lipid nanoparticles, characterized in that, include: The process involves mixing and encapsulating a target substance, a lipid mixture constituting lipid nanoparticles, and an antibody or antibody fragment bound to a membrane scaffold protein. The target substance is a nucleic acid. After binding a gene encoding a membrane scaffold protein to a gene encoding an antibody or antibody fragment, the antibody or antibody fragment bound to the membrane scaffold protein is prepared by expression. In the antibody-bound lipid nanoparticles, the antibody or antibody fragment, which is an amphiphilic protein with a helical structure, binds to the lipid nanoparticles via the membrane scaffold protein. The membrane scaffold protein binds in a diffused shape on the surface of the lipid nanoparticles. The shape of the membrane scaffold protein diffused on the surface of the lipid nanoparticles is such that one side of the helix of the membrane scaffold protein faces the hydrophobic inner lipid layer of the lipid nanoparticle, and the other side of the helix faces the hydrophilic outer lipid layer of the lipid nanoparticle.

2. The method for preparing antibody-bound lipid nanoparticles according to claim 1, characterized in that, The aforementioned membrane scaffold proteins are fragments of membrane scaffold proteins that maintain a helical structure and amphiphilic properties.

3. The method for preparing antibody-bound lipid nanoparticles according to claim 1, characterized in that, The antibody fragments mentioned above are scFv or scFV-Fc formed by the binding of scFv and Fc.

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