Lipid for enhancing immunogenicity of mrna / LNP vaccine

Abstract

The present invention relates to a lipid for enhancing the immunogenicity of mRNA / LNP vaccines and, more specifically, to a lipid nanoparticle comprising a lipid that includes propionate, butyrate, or both, and a drug delivery carrier utilizing same. The lipid nanoparticle according to the present invention can significantly enhance the immunogenicity of a nucleic acid-based vaccine contained therein.

Description

Lipids for enhancing immunogenicity of mRNA / LNP vaccines

[0001] This application claims the benefit of Republic of Korea Patent Application No. 10-2023-0182290, filed December 14, 2023, which is a continuation-in-part of Republic of Korea Patent Application No. 10-2024-0125876, filed September 13, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a lipid for enhancing the immunogenicity of an mRNA / LNP vaccine, and more specifically, to a lipid nanoparticle comprising a lipid containing propionate, butyrate, or both thereof, and a drug delivery system using the same.

[0003]

[0004] Lipid nanoparticles (LNPs) are particulate carriers composed of lipids that effectively deliver substances with limited intracellular penetration, such as therapeutic nucleic acids, proteins, and peptides. Lipid nanoparticles are one of the most important nucleic acid delivery systems, boasting the advantages of ease of manufacture, biodegradability, and excellent stability. In 2018, the FDA approved the delivery of siRNA via lipid nanoparticles, sparking extensive research into the use of lipid nanoparticles as mRNA vaccine carriers. Since then, mRNA-containing lipid nanoparticle-based vaccines (mRNA / LNP vaccines) have been widely used during the global COVID-19 pandemic.

[0005] However, currently approved mRNA / LNP vaccines are particularly vulnerable to variants of concern (VOC), requiring improvement and optimization of their immunogenicity.

[0006]

[0007] Recent studies have demonstrated enhanced immunogenicity of mRNA vaccines through the integration of Toll-Like Receptor 7 (TLRα) agonists. TLR7 agonist-adjuvanted nanoparticles can enhance the adaptive immune response to antigens through higher IgG titers and variable neutralizing antibodies, ultimately providing protection against VOCs. However, TLR7 / 8 agonists can cause adverse effects in vaccine recipients, such as systemic toxicity. Therefore, the development of novel methods to enhance the immunogenicity of mRNA / LNP vaccines is urgently needed.

[0008]

[0009] Accordingly, the inventor of the present invention conducted extensive research to improve the immunogenicity of an mRNA vaccine based on a lipid nanoparticle carrier, and as a result, formulated a lipid nanoparticle with a lipid containing propionate, butyrate, or both as an auxiliary lipid constituting the lipid nanoparticle, and confirmed that the immunogenicity of the mRNA vaccine based on a lipid nanoparticle carrier was improved, thereby completing the present invention.

[0010]

[0011] Accordingly, it is an object of the present invention to provide lipid nanoparticles comprising a lipid containing butyrate, propionate or both.

[0012]

[0013] Another object of the present invention is to provide a drug delivery composition comprising the lipid nanoparticles and a therapeutic agent.

[0014]

[0015] In order to achieve the above-described object of the present invention, the present invention provides a lipid nanoparticle comprising a lipid containing butyrate, propionate, or both thereof.

[0016]

[0017] In order to achieve another object of the present invention, the present invention provides a drug delivery composition comprising the lipid nanoparticle and a therapeutic agent.

[0018]

[0019] As used herein, the term "lipid particle" or "lipid nanoparticle (LNP)" is intended to refer to a lipid formulation (referred to as "TNA lipid particle," "TNA lipid nanoparticle," or "TNA LNP") that can be used to deliver a therapeutic agent, such as a nucleic acid therapeutic agent (TNA), to a target site of interest (e.g., a cell, tissue, organ, etc.). In one embodiment of any of the aspects or embodiments herein, the lipid particle of the invention is an LNP containing one or more therapeutic nucleic acids, wherein the LNP can typically be comprised of an ionizable lipid, a steroid, a non-cationic lipid, and optionally a PEGylated lipid to prevent aggregation of the particle, and further optionally a tissue-specific targeting ligand to deliver the LNP to the target site of interest. In another preferred embodiment, the therapeutic agent, such as a therapeutic nucleic acid, can be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation.

[0020]

[0021] The term "lipids" as used herein is intended to refer to a group of organic compounds, including but not limited to esters of fatty acids, which are characterized by poor solubility in water but generally soluble in many organic solvents. These are typically classified into at least three classes: (1) "simple lipids", which include fats and oils as well as waxes; (2) "complex lipids", which include phospholipids and glycolipids; and (3) "derived lipids", such as steroids. Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other phosphorus-deficient compounds, such as sphingolipids, glycosphingolipids, diacylglycerols, and β-acyloxyacids, also belong to the group designated as amphipathic lipids. Furthermore, the amphipathic lipids described above can be mixed with other lipids, including triglycerides and sterols.

[0022]

[0023] In the present invention, the “ionized lipid” refers to a lipid whose charge state changes depending on the pH of the surrounding environment. Preferably, the ionized lipid according to the present invention may be neutral at physiological pH but positive at acidic pH. Therefore, the lipid nanoparticle of the present invention including the ionized lipid maintains a stable structure in blood having a physiological pH, and reaches the target cell without interference from serum proteins, etc., and moves into the endosome within the target cell. Since the late stage endosome is an acidic environment, the ionized lipid of the lipid nanoparticle becomes positively charged and unstable, and eventually the structure of the lipid nanoparticle collapses, so that the component (YAP1 expression or activity inhibitor) loaded in the lipid nanoparticle can be delivered into the cell. In addition, the ionized lipid can enable the component to be encapsulated into the lipid nanoparticle with high efficiency through electrostatic interaction with anionic components (e.g., nucleic acids).

[0024]

[0025] The term "non-cationic lipid" in the present invention refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.

[0026]

[0027] The term "neutral lipid" as used herein is intended to refer to any lipid species that exists in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelins, cephalins, cholesterol, cerebrosides, and diacylglycerols.

[0028]

[0029] The term “anionic lipid” as used herein refers to any lipid that is negatively charged at physiological pH. Such lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups linked to neutral lipids.

[0030]

[0031] The term “encapsulation” as used herein is intended to refer to a lipid particle that completely encapsulates, partially encapsulates, or both, an active agent or therapeutic agent, such as a nucleic acid (e.g., an ASO, mRNA, siRNA, ceDNA, viral vector). In a preferred embodiment, the nucleic acid is completely encapsulated in the lipid nanoparticle (e.g., forms a nucleic acid-containing lipid particle).

[0032]

[0033] The term "nucleic acid" in the present invention is considered to refer to a polymer (i.e., deoxyribonucleotides or ribonucleotides) containing at least two nucleotides in single-stranded or double-stranded form, including DNA, RNA and hybrids thereof. The DNA may be in the form of, for example, an antisense molecule, a plasmid DNA, a DNA-DNA duplex, a pre-condensed DNA, a PCR product, a vector (P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, chromosomal DNA, or derivatives and combinations of these groups. The DNA may be in the form of a minicircle, a plasmid, a vacmid, a minigene, a ministring DNA (a linear covalently closed DNA vector), a closed linear duplex DNA (CELiD or ceDNA), a doggybone TMThe nucleic acid may be in the form of DNA, dumbbell-shaped DNA, minimal immunologically defined gene expression (MIDGE) vectors, viral vectors, or non-viral vectors. The RNA may be in the form of small interfering RNA (siRNA), Dicer-based dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, viral RNA (vRNA), or combinations thereof. The nucleic acid includes nucleic acids that are synthetic, naturally occurring, and non-naturally occurring, and that have similar binding properties to the reference nucleic acid, known nucleotide analogs, or nucleic acids containing modified backbone residues or linkages. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, phosphorodiamidate morpholino oligomers (morpholinos), phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, locked nucleic acids (LNA™), and peptide nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly set forth.

[0034]

[0035] The terms "nucleic acid therapeutic agent," "therapeutic nucleic acid," and "TNA" are used interchangeably herein and refer to any therapeutic modality that utilizes a nucleic acid as the active ingredient of a therapeutic agent for treating a disease or disorder. As used herein, these terms refer to RNA-based therapeutic agents and DNA-based therapeutic agents. Non-limiting examples of RNA-based therapeutic agents include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigenes, viral DNA (e.g., lentiviral or AAV genomes) or nonviral DNA vectors, closed linear duplex DNA (ceDNA / CELiD), plasmids, bacmids, doggybone™ DNA vectors, minimal immunologically defined gene expression (MIDGE) vectors, nonviral ministring DNA vectors (linear, covalently closed DNA vectors), and dumbbell-shaped DNA minimal vectors (“dumbbell DNA”). As used herein, the term “TNA LNP” refers to a lipid particle containing at least one of the TNAs described above.

[0036]

[0037] Hereinafter, the present invention will be described in detail.

[0038]

[0039] The present invention provides lipid nanoparticles comprising a lipid containing butyrate, propionate, or both.

[0040]

[0041] The inventors of the present invention formulated a lipid nanoparticle carrier by adding 1 to 40 mol% of a lipid containing butyrate, propionate, or both as an auxiliary lipid in addition to the components of existing known lipid nanoparticles, and confirmed that, as a result, compared to a control lipid nanoparticle that did not contain the lipid nanoparticle, the encapsulation efficiency, mRNA delivery efficiency, and its expression efficiency were significantly increased, and accordingly, the number of immune cells increased and antigen-specific immunogenicity increased.

[0042]

[0043] In the present invention, the lipid containing butyrate, propionate or both thereof means any lipid in which butyrate, propionate or both thereof are chemically bound to a substance known in the art as a lipid or having the properties of a lipid, such as an ionized lipid, a cationic lipid, a neutral lipid, an anionic lipid, a steroid, a PEGylated lipid, etc.

[0044]

[0045] In a preferred embodiment of the present invention, the lipid containing butyrate, propionate or both thereof may be, but is not limited to, cholesteryl propionate, cholesteryl butyrate, tributyrin, 1,2-palmitin-3-butyrin, 1-butyrin-2-olein-3-palmitin or a combination thereof.

[0046]

[0047] In one aspect of the present invention, the lipid containing butyrate, propionate, or both may be included in the lipid nanoparticles in an amount of 1 to 40 mol%. Preferably, it may be included in an amount of 1 to 30 mol%, more preferably, 3 to 30 mol%, even more preferably, 3 to 25 mol%, and most preferably, 5 to 22.5 mol%.

[0048]

[0049] In the present invention, the lipid nanoparticle may further include one or more selected from the group consisting of ionized lipids, non-cationic lipids, steroids, and PEGylated lipids.

[0050]

[0051] In the present invention, the ionizable lipid is not particularly limited in type, but may be selected from the group consisting of, for example, SM-102, CL1, ALC-0315, DOTAP, DDA, DC-Chol, DODAP, DOTMA, 14:1 Ethyl PC, 16:0-18:1 Ethyl PC, 18:1 Ethyl PC, 18:0 Ethyl PC, 16:0 Ethyl PC, 14:0 Ethyl PC, 12:0 Ethyl PC, MVL5, 14:0 DAP, 16:0 DAP, 18:0 DAP, DOBAQ, 18:0 TAP, 16:0 TA, 14:0 TAP, and GL67.

[0052]

[0053] In the lipid nanoparticles provided by the present invention, the ionized lipid may be included in an amount of 30 to 55 mol%, preferably 35 to 50 mol%, and most preferably 40 to 50 mol%.

[0054]

[0055] In the present invention, the steroid may be selected from the group consisting of cholesterol, bile acid derivatives, and cholic acid derivatives, but is not limited thereto.

[0056]

[0057] In the lipid nanoparticles provided by the present invention, the steroid may be included in an amount of 10 to 60 mol%. Preferably, it may be included in an amount of 10 to 50 mol%, more preferably, it may be included in an amount of 15 to 50 mol%, even more preferably, it may be included in an amount of 20 to 50 mol%, and most preferably, it may be included in an amount of 22.5 to 40 mol%.

[0058]

[0059] In the present invention, the non-cationic lipid is distearoyl-sn-glycero-phosphoethanolamine (DSPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), Monomethylphosphatidylethanolamine (e.g., 16-O-monomethyl PE), dimethylphosphatidylethanolamine (e.g., 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), dielaidoylphosphatidylethanolamine (DEPE), 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE); lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof, but are not limited thereto.

[0060]

[0061] In the lipid nanoparticles provided by the present invention, the non-cationic lipid may be included in an amount of 1.5 to 20 mol%, preferably 5 to 20 mol%, and most preferably 5 to 15 mol%.

[0062]

[0063] In the present invention, the PEGylated lipid is PEG-dilauryloxypropyl; PEG-dimyristyloxypropyl; PEG-dipalmityloxypropyl, PEG-distearyloxypropyl; dimyristoyl glycerol polyethylene glycol (DMG-PEG); distearoyl-rac-glycerol-PEG (DSG-PEG); PEG-dilaurylglycerol; PEG-dipalmitoylglycerol; PEG-disterylglycerol; PEG-dilaurylglycamide; PEG-dimyristylglycamide; PEG-dipalmitoylglycamide; PEG-disterylglycamide; 1-[8'-(cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol)(PEG-cholesterol); 3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether (PEG-DMB), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxyl (DSPE-PEG-OH), but is not limited thereto.

[0064]

[0065] In the lipid nanoparticles provided by the present invention, the PEGylated lipid may be included in an amount of 0.1 to 5 mol%, preferably 0.5 to 3 mol%, and most preferably 1 to 3 mol%.

[0066]

[0067] In one aspect of the present invention, for a composition suitable for manufacturing lipid nanoparticles, for example, ionized lipid: non-cationic lipid: steroid: PEGylated lipid may be composed in a molar % ratio selected from the group consisting of 60:5:33.5:1.5, 50:10:38.5:1.5, 43.5:10:45:1.5, 40:15:43.5:1.5, 30:20:48.5:1.5, 25:25:48.5:1.5, 20:35:43:1, 20:41:38:1, 60:5:32:3, 50:10:37:3, 40:15:42:3, 30:20:47:3 and 25:25:47:3, wherein the content (mol %) of the steroid is 10 to 70% is replaced with a lipid containing butyrate, propionate or both. Preferably, 10 to 50% of the steroid content (mol%) is replaced with a lipid containing butyrate, propionate or both.

[0068]

[0069] In one aspect of the present invention, the lipid nanoparticle may further include a preventive or therapeutic agent, and the agent may be a gene including RNA, DNA or a mixture thereof composed of a single strand or a double strand, and specifically, one selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), micro ribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribonucleic acid (DNAzyme) and guide ribonucleic acid for gene correction (sgRNA). The above may be, but is not limited to, various anionic peptides, protein drugs, protein-nucleic acid structures, or anionic biopolymer-drug conjugates such as hyaluronic acid-peptide conjugates, hyaluronic acid-protein conjugates, antibodies, etc.

[0070]

[0071] The lipid nanoparticles provided by the present invention can be manufactured by mixing a lipid component dissolved in an organic phase with an oligo dissolved in a buffer solution. Since the polarity of the solvent changes when the organic phase and the aqueous solution are mixed, the lipid component contained in the organic phase spontaneously forms particles in the mixed solution. At this time, when the ionized lipid contained in the organic phase is mixed with an acidic buffer solution containing the oligo, the ionized lipid transitions to a cationic state, so the cationic ionized lipid and the anionic oligo bind through electrostatic attraction, forming nanoparticles in which the oligo is encapsulated within the lipid nanoparticle.

[0072]

[0073] In the method for manufacturing lipid nanoparticles according to the present invention, the mixing ratio (volume ratio) of the organic solution and the buffer solution may be 1:1 to 1 to 100.

[0074]

[0075] The present invention also provides a drug delivery composition comprising the lipid nanoparticles of the present invention described above and a therapeutic agent.

[0076]

[0077] In the present invention, the content of the composition is not particularly limited depending on the purpose or aspect of use, and may be, for example, 0.01 to 99 wt%, preferably 0.5 to 50 wt%, and more preferably 1 to 30 wt%, based on the total weight of the composition. In addition, the drug delivery composition according to the present invention may further include additives such as pharmaceutically acceptable carriers, excipients, or diluents in addition to the active ingredient. The drug delivery composition of the present invention may include 0.1 to 99.9 wt% of the lipid nanoparticles produced by the method of the present invention, and 99.9% to 0.1 wt% of the carrier.

[0078]

[0079] In the present invention, the therapeutic agent may be a peptide drug, a protein drug, a nucleic acid, or a combination thereof, and the therapeutic agent may be encapsulated by being enclosed inside the lipid nanoparticle.

[0080]

[0081] The 'treatment' of the present invention comprehensively refers to improving symptoms caused by cancer or the disease, which may include curing, substantially preventing, or improving the condition of the disease, and includes, but is not limited to, alleviating, curing, or preventing one or most of the symptoms resulting from the disease.

[0082]

[0083] In the present invention, the nucleic acid may be characterized in that the nucleic acid is selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), micro ribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribonucleic acid (DNAzyme), and guide ribonucleic acid for gene correction (sgRNA).

[0084]

[0085] The above drug delivery composition can be administered to mammals including humans by various routes, including parenteral administration, and parenteral administration can be applied intravenously, subcutaneously, intraperitoneally, or locally, and the dosage varies depending on the patient's condition and weight, the degree of disease, the drug form, the route of administration, and the time, but can be appropriately selected by those skilled in the art.

[0086]

[0087] When formulating the above drug delivery composition according to an example, it is manufactured using diluents or excipients such as commonly used fillers, bulking agents, lyophilizing agents, binders, wetting agents, disintegrating agents, and surfactants.

[0088]

[0089] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, etc.

[0090]

[0091] Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.

[0092]

[0093] The drug delivery composition of the present invention can be administered while containing a pharmaceutically effective amount of a preventive or therapeutic agent. The effective dosage level of the preventive or therapeutic agent can be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route and excretion rate, the duration of treatment, factors including concomitant medications, and other factors well known in the medical field. In one embodiment, the composition can be administered as an individual therapeutic agent or in combination with another therapeutic agent, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art. For example, the composition can be administered at 0.01 to 100 mg / kg, 0.1 to 50 mg / kg, or 1 to 10 mg / kg.

[0094]

[0095] The lipid nanoparticle according to the present invention can exhibit an effect of significantly increasing the immunogenicity of a nucleic acid-based vaccine contained therein.

[0096]

[0097] FIGS. 1A to 1C show the physicochemical properties of lipid nanoparticles comprising a composition for enhancing immunogenicity according to the present invention, including the Z-average nano-size (FIG. 1A), polydispersity index (PDI) (FIG. 1B), and mRNA encapsulation efficiency (FIG. 1C) of the lipid nanoparticles.

[0098] Figures 2a to 2c show the in vitro mRNA delivery efficiency (Figure 2a) and in vivo mRNA delivery efficiency (Figure 2b) of lipid nanoparticles containing a composition for enhancing immunogenicity according to the present invention (mRNA dose: 0.3 mg / kg, data are presented as mean ± SEM, n=3-5 mice / group).

[0099] FIGS. 3A to 3F show the results of immune profiling of lipid nanoparticles containing the composition for enhancing immunogenicity according to the present invention, including the percentile of total dendritic cells in lymphoid organs ( FIG. 3A ), the percentile of mature dendritic cells in lymphoid organs ( FIG. 3B ), the percentile of monocytes in lymphoid organs ( FIG. 3C ), the percentile of macrophages in lymphoid organs ( FIG. 3D ), the relative number of follicular helper T cells in lymphoid organs ( FIG. 3E ), and the relative number of germinal center B cells in lymphoid organs ( FIG. 3F ) (mRNA dose: 0.3 mg / kg, data are presented as mean ± SEM, n=6-8 / group of mice, and data are collected from 2-3 independent experiments. *P<0.05, **P<0.01, ***P<0.001 were analyzed by One-way ANOVA with Tukey's post-hoc test, and are significant compared to the untreated group.)

[0100] Figures 4a to 4c show the antigen-specific immunogenicity of lipid nanoparticles including the composition for enhancing immunogenicity according to the present invention, and show the experimental plan for immunogenicity study (Figure 4a), anti-OVA IgG endpoint titer (Figure 4b), and OVA antigen peptide-specific CD8+ T cell ratio (Figure 4c) (*P<0.05, **P<0.01 were analyzed by nonparametric analysis, Kruskal Wallis test, and data are expressed as mean ± SEM, n=6-8 mice / group).

[0101] Figures 5a to 5c show the experimental plan for immunogenicity study (Figure 5a), anti-spike IgG endpoint titer (Figure 5b), and spike peptide reactive T cell ratio (Figure 5c) to confirm the spike antigen-specific immunogenicity of lipid nanoparticles including the composition for enhancing immunogenicity according to the present invention (*P<0.05 was analyzed by Mann Whitney test or nonparametric analysis, Kruskal wallis test, and data are expressed as mean ± SEM, n=6-7 mice / group).

[0102] Figures 6a to 6c show the experimental plan for the study (Figure 6a), the tumor growth curve (Figure 6b), and the OVA peptide specific T cell ratio (Figure 6c) to confirm the protective effect of lipid nanoparticles including the composition for enhancing immunogenicity according to the present invention on tumors (**P<0.01, ***P<0.001 were analyzed by 2-way ANOVA and nonparametric analysis, Kruskal Wallis test, and the data are expressed as mean ± SEM, n=5-6 mice / group).

[0103] Figures 7a to 7e are drawings showing the preparation of lipid nanoparticles (TriB-LNP) containing Tributyrin, a lipid to which butyrate (butyric acid) is bound, and the analysis of its characteristics and effects (Figure 7a. Structure of Tributyrin. Figure 7b. Lipid components of lipid nanoparticles containing Tributyrin (Tributyrin used at 5% of the molar ratio of the total lipid, OVA mRNA used / encapsulated). Figure 7c. Nano size analysis. Figure 7d. Uniformity analysis of lipid nanoparticles. Figure 7e. Analysis of mRNA encapsulation rate. (Experimental replicate n=3 / group, data are shown as mean ± sd))

[0104] Figures 8a to 8c show the results of evaluating the effect of lipid nanoparticles (TriB-LNP) containing Tributyrin on enhancing the immunogenicity of mRNA vaccines compared to the control lipid nanoparticles (Con-LNP) (Figure 8a. Experimental summary (PBMCs were analyzed by flow cytometry 8 days after injection of Con-LNP or TriB-LNP loaded with OVA mRNA into the hind leg muscle, mRNA dose: 0.3 mg / kg). Figure 8b. Proportion of CD8+T cells. Figure 8c. Proportion of OVA antigen-specific CD8+T cells. The proportion of OVA antigen-specific CD8+T cells was analyzed by detecting CD8+T cells binding to OVA peptide (SIINFEKL)-tetramer. (mouse n=6 / group, data are shown as mean ± sem, scattered dots, each dot indicates data from a mouse, **P<0.01, ns=non-significant))

[0105]

[0106] Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and the present invention is not limited thereto.

[0107]

[0108] The present invention is described in detail below.

[0109] However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0110]

[0111] Example 1. Analysis of physicochemical properties of lipid nanoparticles containing bio-derived auxiliary lipids.

[0112] mRNA / LNP was composed of SM-102 ionizable lipid (mol ratio 40-50%), cholesterol (mol ratio 48.5-38.5%), DSPC (mol ratio 10%), and DMG-PEG (mol ratio 1.5%) as the lipid components of conventional lipid nanoparticles, and was composed by replacing some of the cholesterol with cholesteryl ester, a bio-derived auxiliary lipid (Table 1). Con-LNP was formulated with the conventional four lipid components (SM-102:cholesterol:DSPC:DMG-PEG=43.5:45:10:1.5 mol%) without bio-derived auxiliary lipids. All lipid nanoparticles were formulated using a microfluidic rapid mixer (IGNITE, Precision Nanosystem). The composition and content of the manufactured lipid nanoparticles are shown in Table 1 below.

[0113]

[0114] [Table 1]

[0115]

[0116] When the manufactured LNPs were analyzed by Dynamic Light Scattering (DLS), all LNPs exhibited uniform sizes below 200 nm and PDI values ​​below 0.2 (see Figs. 1a and 1b). mRNA encapsulation efficiency was measured using RiboGreen analysis, and LNP-C and LNP-F showed lower mRNA encapsulation efficiency compared to Con-LNP, LNP-A, B, D, and E.

[0117]

[0118] Example 2. mRNA delivery and expression of lipid nanoparticles containing biogenic auxiliary lipids in vitro and in vivo.

[0119] To compare mRNA delivery efficiency, three different cell lines (C2C12, Raw264.7, 4T1) were treated with mRNA / LNP containing firefly luciferase expression mRNA (mRNA concentration of 0.1-0.25 ug / mL) and compared with Con-LNP.

[0120] In Fig. 2a, luciferase expression was normalized to Con-LNP, and LNP-C and LNP-F showed lower mRNA delivery efficiency compared to Con-LNP (statistically ns).

[0121] mRNA delivery in vivo was evaluated by intramuscular injection of mRNA / LNP (6 µg / mouse based on mRNA) into C57BL / 6 mice, and LNP-C and F showed mRNA expression that was more than 10-fold lower than Con-LNP (see Fig. 2b).

[0122] These results are consistent with the low mRNA encapsulation efficiency of LNP-C and LNP-F as confirmed in Example 1 above. LNP-C and LNP-F were excluded from subsequent studies.

[0123]

[0124] Example 3. Immunoprofiling of lipid nanoparticles containing biogenic adjuvant lipids in vitro and in vivo.

[0125] To evaluate the immunogenicity of mRNA / LNP, ovalbumin was used as a model antigen. Mice were injected intramuscularly with mRNA / LNP (6 μg / mouse based on mRNA). Inguinal lymph nodes were harvested 1 or 7 days after injection, and the phenotype of immune cells was determined by flow cytometry.

[0126] As a result, compared to Con-LNP, mice injected with adjuvant lipid nanoparticles did not show a significant difference in the proportion of total DC (CD11c++, MHCII++) to immune cells (Fig. 3a). However, the proportion of mature DC (CD80+, CD86+) was increased in the lymph nodes of LNP-B, D, and E-injected mice compared to Con-LNP-injected mice (Fig. 3b), and the proportion of inflammatory monocytes (CD11b+, Ly6c++) but not macrophages (CD11b+, F4 / 80+) was also increased in LNP-B, D, and E-injected mice (Figs. 3c and 3d). As previously reported, follicular helper T cells (FHT cells) and germinal center B cells (GCB cells) play important roles in antigen-specific, adaptive immune responses and vaccination efficacy. As shown in Figures 3e and 3f, the relative numbers of FHT cells and GCB cells were more than doubled in the LNP-D administration group compared to the Con-LNP administration group. This confirmed that lipid nanoparticles containing biogenic adjuvant lipids exhibited a higher immune activation effect than Con-LNP.

[0127]

[0128] Example 4. Antigen-specific adaptive immunogenicity of lipid nanoparticles containing bio-derived adjuvant lipids

[0129] To assess antigen-specific adaptive immune responses, mice were intramuscularly inoculated with OVA mRNA / LNP vaccine (6 μg / mouse based on mRNA). After two vaccinations (prime and boost shots), serum and spleen cells were collected to measure anti-OVA IgG levels and antigen-specific T cell responses (Fig. 4a).

[0130] Compared with the Con-LNP-administered group, all bio-derived adjuvant lipid nanoparticles exhibited higher anti-OVA IgG titers, which were more evident in the LNP-D and LNP-E groups, which showed a 5- to 7-fold increase in IgG titers (Fig. 4b). OVA antigen peptides were shown to bind to MHC class I and activate antigen-specific CD8+ T cells, and OVA peptide stimulation of spleen cells (peptide concentration 2 ug / mL) increased the proportion of IFNγ+ CD8+ T cells in all vaccinated mice. The LNP-D-injected group, which had the highest proportion of IFNγ+ CD8+ T cells, was ~1.8-fold higher than that of the Con-LNP group (Fig. 4c).

[0131]

[0132] Example 5. Spike antigen-specific, adaptive immunogenicity of lipid nanoparticles containing biogenic adjuvant lipids.

[0133] To generalize the effectiveness of bio-derived adjuvant lipid nanoparticles, we injected spike mRNA / LNP of COVID-19 intramuscularly into mice and measured antigen-specific immunogenicity.

[0134] After two doses (prime and boost shots) on days 0 and 21, serum and splenocytes were collected on day 35 to measure anti-spike IgG levels (see Fig. 5a). In addition, splenocytes were stimulated with a spike peptide pool (2 µg / ml) for 24 h, and IFNγ+ CD8+ and CD4+ T cells were measured by flow cytometry.

[0135] As a result, compared to the Con-LNP-administered group, the LNP-D-administered group showed a four-fold increase in IgG endpoint titer, demonstrating higher anti-OVA IgG levels (see Fig. 5b). Spike peptide stimulation of spleen cells increased the proportion of IFNγ+ CD8+ T cells, and spleen cells in the LNP-D-administered group showed a higher IFNγ response in CD8+ T and CD4+ T cells compared to the Con-LNP-administered group (see Fig. 5c).

[0136]

[0137] Example 6. Protective effect of lipid nanoparticles containing bio-derived auxiliary lipids on tumors

[0138] To generalize the effects of biogenic adjuvant lipid nanoparticles, Con-LNP and LNP-D were prepared using ALC-0315, not SM-102, as the ionizable lipid, and their protective effects against tumors were confirmed.

[0139] OVA-expressing B16F19 cells were subcutaneously implanted into the hind limbs of mice, followed by inoculation with OVA mRNA on days 4 and 9 (see Fig. 6a). Tumor sizes were measured on days 0, 7, 9, 11, and 14, and tumor cells were stained with fluorescent OVA peptide tetramer and antibodies, followed by flow cytometry to determine the infiltration of antigen-specific CD8+ T cells.

[0140] As a result, compared to the control group (LNP-non-administered group), the Con-LNP and LNP-D administration groups showed delayed tumor growth, and on day 14, the LNP-D administration group showed a 65% smaller tumor size compared to the Con-LNP administration group (see Fig. 6b). In addition, the LNP-D administration group showed a greater number of OVA peptide-specific CD8+ T cells infiltrating the tumor compared to the Con-LNP administration group (see Fig. 6c).

[0141]

[0142] Example 7: Preparation and efficacy verification of lipid nanoparticles containing butyrate-containing lipids.

[0143] Lipid nanoparticles containing tributyrin as a lipid with a butyrate group attached were prepared and their effects were evaluated (Fig. 7a).

[0144] Lipid nanoparticles (TriB-LNP) were prepared by including tributyrin at a molar ratio of 5% of the total lipid (Fig. 7b). Lipid nanoparticles were prepared using a microfluidic nanoparticle formulation device, IGNITE (Precision Nanosystem), and OVA mRNA was used / encapsulated. Control lipid nanoparticles (Con-LNP), which contained cholesterol instead of tributyrin in the composition of the above TriB-LNP, were prepared and used as a control (Table 2).

[0145]

[0146] [Table 2]

[0147]

[0148]

[0149] When the size and uniformity of TriB-LNP lipid nanoparticles containing 5 mol% Tributyrin were analyzed by DLS analysis compared to the control Con-LNP lipid nanoparticles made only of general lipid components (ionizable lipid, helper lipid, PEG-lipid, cholesterol), it was confirmed that there was almost no difference between the two lipid nanoparticles (Fig. 7c, Fig. 7d). In addition, it was confirmed that they showed similar values ​​of OVA mRNA encapsulation rates (Fig. 7e).

[0150]

[0151] To determine whether TriB-LNP exhibits enhanced immunogenicity compared to Con-LNP, Con-LNP or TriB-LNP loaded with OVA mRNA were injected into the hind limb muscles of mice (C57BL / 6, wild type) (mRNA dose: 0.3 mg / kg), and after 8 days, peripheral blood mononuclear cells (PBMC) were collected and the proportion of antigen-specific CD8+T cells was compared and analyzed (Fig. 8a).

[0152] Analysis of PBMCs from each group delivered with OVA mRNA using Con-LNP or TriB-LNP showed that the proportion of CD8+ T cells in the blood did not differ significantly between the groups (Fig. 8b), but the proportion of OVA antigen-specific CD8+ T cells in the TriB-LNP group increased 1.72-fold compared to the Con-LNP group (T test results, **P<0.01) (Fig. 8c).

[0153]

[0154] The lipid nanoparticles according to the present invention can exhibit an effect of significantly increasing the immunogenicity of a nucleic acid-based vaccine contained therein, and thus have very high potential for industrial use.

Claims

1. A lipid nanoparticle comprising a lipid containing butyrate, propionate or both.

2. A lipid nanoparticle according to claim 1, characterized in that the lipid is an ionized lipid, a non-cationic lipid, a steroid or a PEGylated lipid, such as butyrate, propionate or a combination of both.

3. A lipid nanoparticle according to claim 1, characterized in that the lipid containing butyrate, propionate or both is selected from the group consisting of cholesteryl butyrate, cholesteryl propionate, tributyrin, 1,2-palmitin-3-butyrin and 1-butyrin-2-olein-3-palmitin.

4. A lipid nanoparticle according to claim 1, characterized in that the lipid nanoparticle further comprises at least one selected from the group consisting of ionized lipids, non-cationic lipids, steroids, and PEGylated lipids.

5. A lipid nanoparticle in claim 4, characterized in that the ionized lipid is selected from the group consisting of SM-102, CL1, ALC-0315, DOTAP, DDA, DC-Chol, DODAP, DOTMA, 14:1 Ethyl PC, 16:0-18:1 Ethyl PC, 18:1 Ethyl PC, 18:0 Ethyl PC, 16:0 Ethyl PC, 14:0 Ethyl PC, 12:0 Ethyl PC, MVL5, 14:0 DAP, 16:0 DAP, 18:0 DAP, DOBAQ, 18:0 TAP, 16:0 TA, 14:0 TAP, and GL67.

6. A lipid nanoparticle according to claim 4, characterized in that the ionized lipid is contained in an amount of 30 to 55 mol%.

7. In the fourth paragraph, the non-cationic lipid is distearoyl-sn-glycero-phosphoethanolamine (DSPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), Monomethylphosphatidylethanolamine (e.g., 16-O-monomethyl PE), dimethylphosphatidylethanolamine (e.g., 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), dielaidoylphosphatidylethanolamine (DEPE), 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE); A lipid nanoparticle characterized by being selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, and dilinoleoylphosphatidylcholine.

8. A lipid nanoparticle according to claim 4, characterized in that the non-cationic lipid is contained in an amount of 1.5 to 20 mol%.

9. A lipid nanoparticle according to claim 4, characterized in that the steroid is selected from the group consisting of cholesterol, bile acid, and cholic acid.

10. A lipid nanoparticle characterized in that the steroid is contained in an amount of 10 to 60 mol% in the fourth paragraph.

11. In the fourth paragraph, the PEGylated lipid is PEG-dilauryloxypropyl; PEG-dimyristyloxypropyl; PEG-dipalmityloxypropyl, PEG-distearyloxypropyl; dimyristoyl glycerol polyethylene glycol (DMG-PEG); distearoyl-rac-glycerol-PEG (DSG-PEG); PEG-dilaurylglycerol; PEG-dipalmitoylglycerol; PEG-disterylglycerol; PEG-dilaurylglycamide; PEG-dimyristylglycamide; PEG-dipalmitoylglycamide; PEG-disterylglycamide; 1-[8'-(cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol)(PEG-cholesterol); A lipid nanoparticle characterized by being selected from the group consisting of 3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether (PEG-DMB), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxyl (DSPE-PEG-OH).

12. A lipid nanoparticle characterized in that the lipid containing butyrate, propionate or both of them is contained in an amount of 1 to 40 mol% in the first paragraph.

13. A drug delivery composition comprising a lipid nanoparticle according to any one of claims 1 to 9; and a therapeutic agent.

14. A drug delivery composition according to claim 10, characterized in that the therapeutic agent is a peptide drug, a protein drug, a nucleic acid or a combination thereof.

15. A drug delivery composition according to claim 11, wherein the nucleic acid is selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), micro ribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribonucleic acid (DNAzyme), and guide ribonucleic acid for gene correction (sgRNA).

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