Lipid nanoparticles

Abstract

The present invention provides lipid nanoparticles (LNPs) and mRNA vaccines containing lipid nanoparticles. [Solution] The LNP of the present invention is characterized by containing less than 1 mol% of C18-PEG2000 lipid. The present invention provides the use of LNP for immunogenic delivery of nucleic acid molecules, specifically mRNA, thereby making LNP highly suitable for use in vaccines, for example, in the treatment of cancer or infectious diseases.

Description

[Technical Field]

[0001] This invention relates to the field of lipid nanoparticles (LNPs), more specifically, lipid nanoparticles (LNPs) comprising ionic lipids, phospholipids, sterols, PEG lipids, and one or more nucleic acids. The LNPs of this invention are characterized by containing less than about 1 mol% of PEG lipids (e.g., di-C18-PEG2000 lipids). This invention provides the use of LNPs for immunogenic delivery of nucleic acid molecules, specifically mRNA, thereby making LNPs highly suitable for use in vaccines, for example, in the treatment of cancer or infectious diseases. Finally, methods for preparing such LNPs are provided. [Background technology]

[0002] One of the major challenges in the targeted delivery of bioactive substances is often their instability and low cell permeability. This is particularly true for the delivery of nucleic acid molecules, especially (m)RNA molecules. Therefore, suitable packaging is crucial for proper protection and delivery. Consequently, methods and compositions for packaging bioactive substances such as nucleic acids continue to be needed.

[0003] In this regard, lipid-based nanoparticle compositions such as lipoplexes and liposomes are used as packaging vehicles for bioactive substances that enable transport into cells and / or intracellular compartments. These lipid-based nanoparticle compositions typically include mixtures of various lipids such as cationic lipids, ionic lipids, phospholipids, structural lipids (sterols or cholesterol, etc.), and PEG (polyethylene glycol) lipids (as outlined in Non-Patent Document 1).

[0004] Lipid-based nanoparticles, composed of a mixture of four lipids—cationic or ionic lipids, phospholipids, sterols, and PEGylated lipids—are being developed for the non-immunogenic delivery of siRNA and mRNA to the liver after systemic administration. While many such lipid compositions are known in the art, those used for in vivo mRNA delivery typically contain at least 1.5 mol% of PEG lipids and very often contain di-C14-based PEG lipids (DMG-PEG lipids).

[0005] However, we have now discovered, surprisingly, that PEG lipids present in small amounts (i.e., less than approximately 1 mol%) within LNPs result in nanoparticles that are highly suitable for immunogenic delivery of mRNA via systemic injection of LNPs. Furthermore, these effects are even more pronounced for long-chain PEG lipids such as di-C18-PEG lipids. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Reichmuth et al., 2016 [Overview of the Initiative]

[0007] In a first embodiment, the present invention relates to an mRNA vaccine comprising one or more lipid nanoparticles, wherein the lipid nanoparticles are Ionic lipids and Phospholipids and Sterols and PEG lipids and One or more mRNA molecules, Includes, The present invention provides an mRNA vaccine characterized in that the LNP contains less than about 1 mol% of the PEG lipid, preferably about 0.5 mol% to 0.9 mol% of the PEG lipid.

[0008] In a further aspect, the present invention is a lipid nanoparticle (LNP) used for mRNA vaccination, wherein the LNP comprises an ionizable lipid and a phospholipid and a sterol and a PEG lipid and one or more mRNA molecules and and is characterized in that the LNP contains less than about 1 mol% of the PEG lipid, preferably about 0.5 mol% to 0.9 mol% of the PEG lipid, to provide a lipid nanoparticle.

[0009] In yet a further aspect, the present invention is a lipid nanoparticle (LNP) comprising an ionizable lipid and a phospholipid and a sterol and a PEG lipid and one or more nucleic acid molecules and and is characterized in that the PEG lipid is a diC18-PEG2000 lipid and the LNP contains less than about 1 mol% of the PEG lipid, to provide a lipid nanoparticle.

[0010] In a specific embodiment of the present invention, the diC18-PEG2000 lipid is selected from the list comprising (distearoyl-based)-PEG2000 lipids such as DSG-PEG2000 lipid or DSPE-PEG2000 lipid, or (dioleoyl-based)-PEG2000 lipids such as DOG-PEG2000 lipid or DOPE-PEG2000 lipid.

[0011] In a more specific embodiment of the present invention, the LNP contains about 0.5 mol% of the PEG lipid.

[0012] In another specific embodiment of the present invention, the ionizable lipid is ​​1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azandiyl)bis(dodecane-2-ol)(C12-200), Dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or, Compound of formula (I): [ka] (In the formula, RCOO is selected from a list containing myristoyl, α-D-tocopherol succinoyl, linoleyl and oleoyl, and X is [ka] Selected from a list that includes (or a list that includes).

[0013] In a preferred embodiment, the ionic lipid is a lipid of formula (I) (wherein RCOO is α-D-tocopherol succinoyl, and X is [ka] It is.

[0014] In further embodiments of the present invention, the phospholipid is selected from a list including 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and mixtures thereof.

[0015] In further embodiments of the present invention, the sterol is selected from a list including cholesterol, ergosterol, campesterol, oxysterol, antrosterol, desmosterol, nicasterol, sitosterol, and stigmasterol, and is preferably cholesterol.

[0016] Furthermore, in a particular embodiment, the LNP contains 30 mol% to 70 mol% of the ionic lipid, preferably 45 mol% to 65 mol%.

[0017] In yet another embodiment of the present invention, the LNP comprises about 45 mol% or less of the sterol.

[0018] In further embodiments, the LNP contains 5 mol% to 25 mol% of phospholipids, preferably 4 mol% to 15 mol%.

[0019] In a particular embodiment of the present invention, the LNP is Approximately 45 mol% to 65 mol% of the aforementioned ionic lipid, Approximately 4 mol% to 15 mol% of the phospholipid, Approximately 0.5 mol% to 0.9 mol% of the aforementioned PEG lipid, Includes, The balance is maintained by the amount of the aforementioned sterols.

[0020] In a very specific embodiment of the present invention, the LNP is Approximately 64 mol% of the aforementioned ionic lipid, Approximately 8 mol% of the phospholipid, Approximately 0.5 mol% to 0.9 mol% of the aforementioned PEG lipid, Includes, The balance is maintained by the amount of the aforementioned sterols.

[0021] In another very specific embodiment of the present invention, the LNP is Approximately 64 mol% of the aforementioned ionic lipid, Approximately 8 mol% of the phospholipid, Approximately 0.5 mol% of the PEG lipid, Includes, The balance is maintained by the amount of the aforementioned sterols.

[0022] In another very specific embodiment of the present invention, the LNP is Approximately 50 mol% of the aforementioned ionic lipid, Approximately 6 mol% of the phospholipid, Approximately 0.5 mol% to 0.9 mol% of the aforementioned PEG lipid, Includes, The balance is maintained by the amount of the aforementioned sterols.

[0023] In another very specific embodiment of the present invention, the LNP is Approximately 50 mol% of the aforementioned ionic lipid, Approximately 8 mol% of the phospholipid, Approximately 0.5 mol% to 0.9 mol% of the aforementioned PEG lipid, Includes, The balance is maintained by the amount of the aforementioned sterols.

[0024] In another very specific embodiment of the present invention, the LNP is Approximately 60 mol% of the aforementioned ionic lipid, Approximately 12 mol% of the phospholipid, Approximately 0.5 mol% to 0.9 mol% of the aforementioned PEG lipid, Includes, The balance is maintained by the amount of the aforementioned sterols.

[0025] In another embodiment of the present invention, the one or more nucleic acid molecules are selected from a list including mRNA and DNA, and are preferably mRNA.

[0026] In a more specific embodiment, the one or more mRNA molecules are selected from a list that includes mRNA encoding an immunomodulatory polypeptide and / or mRNA encoding an antigen. The mRNA encoding the immunomodulatory polypeptide may be selected from a list that includes, for example, mRNA molecules encoding CD40L, CD70, and caTLR4.

[0027] In a further embodiment, the present invention provides a pharmaceutical composition or vaccine comprising one or more lipid nanoparticles described herein and an acceptable pharmaceutical carrier.

[0028] The present invention also provides lipid nanoparticles, pharmaceutical compositions, or vaccines described herein that are used in human medicine or veterinary medicine, particularly for use in the treatment of cancer or infectious diseases.

[0029] The following will refer specifically to the drawings, but it is emphasized that the details shown are for illustrative purposes only, as an example, to illustrate various embodiments of the present invention. These drawings are presented to provide what is considered to be the most useful and straightforward explanation of the principles and conceptual aspects of the present invention. In this regard, no attempt has been made to show structural details of the present invention in more detail than is necessary for a basic understanding of the present invention. This explanation, together with the drawings, will make to those skilled in the art how some embodiments of the present invention can be actually implemented. [Brief explanation of the drawing]

[0030] [Figure 1] This figure shows the magnitude of the E7-specific CD8 T cell response measured after initial intravenous immunization with mRNA LNPs formulated with different proportions of DMG-PEG2000 and DSG-PEG2000 in the LNP composition. Two-way ANOVA using Tukey's multiple comparison test. ns, no significant difference; ***p<0.001. [Figure 2] This figure shows the magnitude of the E7-specific CD8 T cell response measured after a second intravenous immunization with mRNA LNPs formulated with a fixed proportion of DMG-PEG2000, DPG-PEG2000, or DSG-PEG2000 LNPs. [Figure 3] This figure shows the magnitude of the E7-specific CD8 T cell response measured after the fourth intravenous immunization with mRNA LNP or synthetic long-chain peptides. One-way ANOVA using Tukey's multiple comparison test. ns, **p<0.01, ****p<0.0001. [Figure 4]This figure shows the DOE-led optimization of LNP composition for maximum T cell response. A. E7-specific T cells in blood after three immunizations (weekly intervals) with E7 mRNA LNPs from the DOE library. B. Graph showing the E7-specific CD8 T cell response as a function of DSG-PEG2000 (%). A very significant negative correlation was observed between PEG-lipid (%) after the third immunization and the magnitude of the E7-specific CD8 T cell response. C. E7-specific T cells in blood after three immunizations (weekly intervals) with predicted optimal (LNP36) and non-optimal DSG-PEG2000 LNPs (LNP37). Mean ± SD is shown. Statistics were evaluated by one-way ANOVA with Sidak's multiple comparison test. ***p<0.001 [Figure 5] This figure shows that the optimized mRNA LNP vaccine induces a qualitative T cell response and a strong antitumor effect. A. Dynamics of E7-specific CD8+ T cells in the blood. B. Increase in serum IFN-γ after repeated immunization. C. Production of IFN-γ and TNF-α by splenic CD8+ E7-specific T cells in the spleen after three immunization cycles. [Figure 5-1] D, Mean TC-1 tumor growth in LNP36-immunized mice. E, Survival of LNP36-immunized mice. F, TC-1 tumor-infiltrating lymphocytes (TILs) after two LNP36 immunizations. G, E7 specificity of TILs. A, F, G: Mean ± SD shown. B: Box plot. D: Mean ± SEM shown. F, G: Statistics evaluated by one-way ANOVA with Tukey's multiple comparison test. E: Statistics evaluated by Mantel-Cox log-rank test. **, p<0.01, *** p<0.001, ns = no significant difference [Figure 6]This figure shows that LNPs are taken up by various (innate) immune cells and activate them. A. Percentage of luciferase activity in the kidney, lung, heart, liver, and spleen relative to total luciferase activity. B. LNP uptake in multiple cell types, measured by the difference in Cy5 MFI in LNP-injected mice compared to TBS buffer-injected mice. C. Percentage of luciferase activity in the kidney, lung, heart, liver, and spleen relative to total luciferase activity. Optimal LNP36 showed increased luciferase activity in the spleen compared to non-optimal LNP37. [Figure 6-1] D. Cell uptake of optimal LNP36 is higher compared to non-optimal LNP37. [Figure 6-2] E. Significant accumulation of E7 mRNA in the spleen. F. Transient increases in serum IFN-α and IP-10 cytokines were observed (compared 6 and 24 hours after LNP administration). G. CD86 expression in cDC1 and cDC2 was slightly upregulated by non-optimal LNP37 and strongly upregulated by optimal LNP36. A, B; C, D, E, F. Mean ± SD are shown. [Modes for carrying out the invention]

[0031] As already described in detail herein, the present invention is an mRNA vaccine comprising one or more lipid nanoparticles, wherein the lipid nanoparticles are Ionic lipids and Phospholipids and Sterols and PEG lipids and One or more mRNA molecules, Includes, The present invention provides an mRNA vaccine characterized in that the LNP contains less than about 1 mol% of the PEG lipid, preferably about 0.5 mol% to 0.9 mol% of the PEG lipid.

[0032] In a particular embodiment, the present invention relates to an mRNA vaccine comprising one or more lipid nanoparticles, wherein the lipid nanoparticles are Approximately 45 mol% to 65 mol% of ionic lipids, Approximately 4 mol% to 15 mol% of phospholipids, Sterols and PEG lipids and One or more mRNA molecules, Includes, The present invention provides an mRNA vaccine characterized in that the LNP contains less than about 1 mol% of the PEG lipid, preferably about 0.5 mol% to 0.9 mol% of the PEG lipid.

[0033] The present invention also relates to lipid nanoparticles (LNPs) used for mRNA vaccination, Ionic lipids and Phospholipids and Sterols and PEG lipids and One or more mRNA molecules, Includes, The present invention provides lipid nanoparticles characterized in that the LNP contains less than about 1 mol% of the PEG lipid, preferably about 0.5 mol% to 0.9 mol% of the PEG lipid.

[0034] In a particular embodiment, the present invention relates to lipid nanoparticles (LNPs) used for mRNA vaccination, wherein the LNPs are Approximately 45 mol% to 65 mol% of ionic lipids, Approximately 4 mol% to 15 mol% of phospholipids, Sterols and PEG lipids and One or more mRNA molecules, Includes, The present invention provides lipid nanoparticles characterized in that the LNP contains less than about 1 mol% of the PEG lipid, preferably about 0.5 mol% to 0.9 mol% of the PEG lipid.

[0035] Therefore, the present invention provides LNPs containing PEG lipids present in relatively small amounts (e.g., less than about 1 mol%, particularly about 0.5 mol% to 0.9 mol%), which have been found to be remarkably suitable for immunogenic delivery of nucleic acids, especially mRNA. In particular, this effect has been found to be more pronounced for LNPs containing long-chain PEG lipids such as C18-PEG lipids, and more specifically, C18-PEG2000 lipids. "Immunogenic delivery of nucleic acid molecules" means the delivery of nucleic acid molecules to cells, thereby resulting in contact with the cells, internalization within the cells, and / or expression of the nucleic acid molecules, which induces an immune response.

[0036] Therefore, in a further embodiment, the present invention relates to lipid nanoparticles (LNPs), Ionic lipids and Phospholipids and Sterols and PEG lipids and One or more nucleic acid molecules, especially mRNA molecules, Includes, The present invention provides lipid nanoparticles characterized in that the PEG lipid is a C18-PEG2000 lipid, and the LNP contains less than approximately 1 mol% of the PEG lipid.

[0037] In certain embodiments, the present invention relates to lipid nanoparticles (LNPs), Approximately 45 mol% to 65 mol% of ionic lipids, Approximately 4 mol% to 15 mol% of phospholipids, Sterols and PEG lipids and One or more nucleic acid molecules, Includes, The present invention provides lipid nanoparticles characterized in that the PEG lipid is a C18-PEG2000 lipid, and the LNP contains less than 1 mol% of the PEG lipid, preferably about 0.5 mol% to 0.9 mol% of the PEG lipid.

[0038] Lipid nanoparticles (LNPs) are generally known as nano-sized particles composed of combinations of different lipids. While many different types of lipids can be included in such LNPs, the LNPs of the present invention are typically composed of combinations of ionic lipids, phospholipids, sterols, and PEG lipids.

[0039] In the context of this application, whereever specific embodiments are provided with respect to the lipid nanoparticles disclosed herein, the limitations provided in such embodiments shall apply equally to lipid nanoparticles intended for use as part of or in mRNA vaccination as claimed mRNA vaccines.

[0040] As used herein, the term “nanoparticles” means any particles having a diameter that makes the particles particularly suitable for systemic administration, especially intravenous administration, of nucleic acids, and typically having a diameter of less than 1,000 nanometers (nm), preferably less than 500 nm, more preferably less than 200 nm, for example, 50 nm to 200 nm, and preferably 80 nm to 160 nm.

[0041] In the context of the present invention, the term "PEG lipid" or, alternatively, "PEGylated lipid" means any suitable lipid modified with a PEG (polyethylene glycol) group. Particularly preferred PEG lipids in the context of the present invention are di-C18-PEG lipids. In the context of the present invention, when the term C18-PEG lipid is used, it means a di-C18-PEG lipid, i.e., a lipid having two C18 lipid tails. However, shorter-chain PEG lipids, such as di-C14-PEG lipids (e.g., DMG-PEG, more specifically DMG-PEG2000, or DMPE-PEG, more specifically DMPE-PEG2000) or di-C16-PEG lipids can also be suitably used. Di-C18-PEG lipids contain a polyethylene glycol moiety that defines the molecular weight of the lipid and a fatty acid tail having 18 C atoms. In certain embodiments, the diC18-PEG2000 lipid is selected from a list including (distearoyl-based)-PEG2000 lipids, such as DSG-PEG2000 lipid (2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000) or DSPE-PEG2000 lipid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), or (dioleoil-based)-PEG2000 lipids, such as DOG-PEG2000 lipid (1,2-dioleoil-rac-glycerol) or DOPE-PEG2000 lipid (1,2-dioleoil-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]). [ka]

[0042] In the context of this invention, the term "ionic" (or alternatively, cationic) in relation to compounds or lipids means ion (usually H +This means the presence of any uncharged group in the compound or lipid that can dissociate by obtaining an ion and thus acquiring a positive charge. Alternatively, any uncharged group in the compound or lipid may acquire electrons and thus acquire a negative charge.

[0043] In the context of the present invention, any type of ionic lipid can be suitably used. Specifically, a suitable ionic lipid comprises two identical or different tails linked via an SS bond, each of which tails is [ka] It is an ionic aminolipid containing an ionic amine, as represented by [formula].

[0044] In a particular embodiment, the ionic lipid is of formula (I): [ka] It is a compound of, During the ceremony, RCOO is selected from a list including myristoyl, α-D-tocopherol succinoyl, linoleyl and oleoyl. X is [ka] Selected from a list that includes this item.

[0045] Such ionic lipids are specifically represented by the following formula: [ka] It can be represented by any of the following:

[0046] More specifically, the ionic lipid is the lipid of formula (I), where RCOO is α-D-tocopherol succinoyl and X is [ka] As shown, [ka] That is the case.

[0047] Other suitable ionic lipids may be selected from 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azandiyl)bis(dodecane-2-ol)(C12-200) and dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA). [ka]

[0048] Therefore, in a particular embodiment, the present invention is Ionic lipids of formula (I): [ka] (In the formula, RCOO is selected from a list including myristoyl, α-D-tocopherol succinoyl, linoleyl and oleoyl. X is [ka] (Selected from a list including), in particular, where RCOO is α-D-tocopherol succinoyl and X is [ka] The lipids in equation (I) and Phospholipids and Sterols and DiC18-PEG2000 lipids present in less than approximately 1 mol%, One or more nucleic acid molecules, The present invention provides lipid nanoparticles containing [the specified substance].

[0049] In the context of the present invention, the term "phospholipid" means a lipid molecule consisting of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group. Since these two components are almost always linked by a glycerol molecule, the phospholipids of the present invention are preferably glycerol-phospholipids. Furthermore, the phosphate group is often modified with a simple organic molecule, for example, choline (i.e., phosphocholine) or ethanolamine (i.e., phosphoethanolamine).

[0050] Suitable phospholipids in the context of the present invention include 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine ( DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-difytanol-sn-glycero-3-phosphoethanolamine (ME16.0 You can choose from a list including PE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0051] In a more specific embodiment, the phospholipid is selected from a list including 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and mixtures thereof.

[0052] Therefore, in a particular embodiment, the present invention is Ionic lipids of formula (I): [ka] (In the formula, RCOO is selected from a list including myristoyl, α-D-tocopherol succinoyl, linoleyl and oleoyl. X is [ka] (Selected from a list including), in particular, where RCOO is α-D-tocopherol succinoyl and X is [ka] The lipids in equation (I) and A phospholipid selected from DOPC and DOPE, or a mixture thereof, Sterols and DiC18-PEG2000 lipids present in less than approximately 1 mol%, One or more nucleic acid molecules, The present invention provides lipid nanoparticles containing [the specified substance].

[0053] In the context of the present invention, the term "sterol," also known as steroid alcohol, refers to a subgroup of steroids that are naturally occurring in plants, animals, and fungi, or that can be produced by certain bacteria. In the context of the present invention, any suitable sterol can be used, selected from the list including, for example, cholesterol, ergosterol, campesterol, oxysterol, antrosterol, desmosterol, nicasterol, sitosterol, and stigmasterol, and preferably cholesterol.

[0054] Therefore, in a particular embodiment, the present invention is Ionic lipids of formula (I): [ka] (In the formula, RCOO is selected from a list including myristoyl, α-D-tocopherol succinoyl, linoleyl and oleoyl. X is [ka] (Selected from a list including), in particular, where RCOO is α-D-tocopherol succinoyl and X is [ka] The lipids in equation (I) and A phospholipid selected from DOPC and DOPE, or a mixture thereof, Cholesterol and, DiC18-PEG2000 lipids present in less than approximately 1 mol%, One or more nucleic acid molecules, The present invention provides lipid nanoparticles containing [the specified substance].

[0055] In a very specific embodiment of the present invention, the lipid nanoparticles are Ionic lipids of formula (I): [ka] (In the formula, RCOO is selected from a list including myristoyl, α-D-tocopherol succinoyl, linoleyl and oleoyl. X is [ka] (Selected from a list including), in particular, where RCOO is α-D-tocopherol succinoyl and X is [ka] The lipids in equation (I) and A phospholipid selected from DOPC and DOPE, or a mixture thereof, Cholesterol and, Di-DSG-PEG2000 lipids present in less than approximately 1 mol%, One or more nucleic acid molecules, Includes.

[0056] In another very specific embodiment of the present invention, the lipid nanoparticles are Ionic lipids of formula (I): [ka] (In the formula, RCOO is selected from a list including myristoyl, α-D-tocopherol succinoyl, linoleyl and oleoyl. X is [ka] (Selected from a list including), in particular, where RCOO is α-D-tocopherol succinoyl and X is [ka] The lipids in equation (I) and A phospholipid selected from DOPC and DOPE, or a mixture thereof, Cholesterol and, DSPE-PEG2000 lipids present in less than approximately 1 mol%, One or more nucleic acid molecules, Includes.

[0057] In certain embodiments of the present invention, the LNP comprises an ionic lipid to phospholipid ratio of about 8:1, or alternatively, about 6:1, about 4:1, or about 2:1.

[0058] In a further specific embodiment, the LNP contains about 30 mol% to 70 mol% of the ionic lipid, preferably about 45 mol% to 65 mol%, for example, about 65 mol%, about 45 mol% or more, about 50 mol% or more, about 55 mol% or more, or about 60 mol% or more.

[0059] In a further embodiment, the LNP contains 4 mol% to 25 mol% of phospholipid, preferably 4 mol% to 15 mol%, for example, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol%, and is preferably about 6 mol% to 9 mol%.

[0060] Therefore, in a particular embodiment of the present invention, the following: The LNP contains approximately 45 mol% to 65 mol% of the ionic lipid; The LNP contains approximately 4 mol% to 15 mol% of the phospholipid; The LNP contains approximately 0.5 mol% to 0.9 mol% of the PEG lipid; One or more of the following apply: The balance is maintained by the amount of the aforementioned sterols.

[0061] Therefore, in a very specific embodiment of the present invention, the LNP is Ionic lipids of formula (I) in concentrations of approximately 45 mol% to 65 mol%: [ka] (In the formula, RCOO is selected from a list including myristoyl, α-D-tocopherol succinoyl, linoleyl and oleoyl. X is [ka] (Selected from a list including), in particular, where RCOO is α-D-tocopherol succinoyl and X is [ka] The lipids in equation (I) and A phospholipid selected from approximately 4 mol% to 15 mol% of DOPC and DOPE, or a mixture thereof, Cholesterol to maintain balance, Approximately 0.5 mol% to 0.9 mol% of DSG-PEG2000 lipid or DSPE-PEG2000 lipid, One or more nucleic acid molecules, Includes.

[0062] In the context of the present invention, when mol% is used, it means the mol% of a particular component relative to empty nanoparticles, i.e., nanoparticles that do not contain nucleic acids. This means that the mol% of a component is calculated relative to the total amount of ionic lipids, phospholipids, sterols, and PEG lipids present in the LNP.

[0063] In further specific embodiments, the present invention, The ionic lipids exceeding 60 mol%, Approximately 8 mol% of the phospholipid, Approximately 0.5 mol% to 0.9 mol% of the aforementioned PEG lipid, Includes, The present invention provides lipid nanoparticles in which the balance is maintained by the amount of the aforementioned sterols.

[0064] More specifically, the present invention is Approximately 64 mol% of the aforementioned ionic lipid, Approximately 8 mol% of the phospholipid, Approximately 0.5 mol% to 0.9 mol% of the aforementioned PEG lipid, Includes, The present invention provides lipid nanoparticles in which the balance is maintained by the amount of the aforementioned sterols.

[0065] More specifically, the present invention is Approximately 64 mol% of the aforementioned ionic lipid, Approximately 8 mol% of the phospholipid, Approximately 0.5 mol% of the PEG lipid, Includes, The present invention provides lipid nanoparticles in which the balance is maintained by the amount of the aforementioned sterols.

[0066] In a very specific embodiment of the present invention, the LNP is Approximately 64.4 mol% of the aforementioned ionic lipid, Approximately 8 mol% of the phospholipid, Approximately 27.1 mol% of the sterols, Approximately 0.5 mol% of the PEG lipid, Includes.

[0067] Therefore, in a very specific embodiment of the present invention, the LNP is Approximately 64.4 mol% of ionic lipids of formula (I): [ka] (In the formula, RCOO is α-D-tocopherol succinoyl, and X is [ka] (That is the case) Approximately 8 mol% of phospholipids selected from DOPC and DOPE, or mixtures thereof, Approximately 27.1 mol% of the aforementioned cholesterol, Approximately 0.5 mol% of DSG-PEG2000 lipid or DSPE-PEG2000 lipid, One or more nucleic acid molecules, Includes.

[0068] Table 1 shows other particularly suitable LNP compositions in the context of the present invention.

[0069] Table 1: Suitable LNP compositions [Table 1]

[0070] Other particularly suitable LNPs are, 64.4 / 8 / 27.1 / 0.5 58 / 14.5 / 27 / 0.5 48 / 25.5 / 27 / 0.5 53 / 17.67 / 28.58 / 0.75 It is characterized by the ratio of ionic lipids / phospholipids / sterols / C18-PEG2000 lipids.

[0071] The inventors have found that the LNP of the present invention is particularly suitable for immunogenic delivery of nucleic acids. Therefore, the present invention provides an LNP comprising one or more nucleic acid molecules, such as DNA or RNA, and more specifically mRNA.

[0072] The amount of nucleic acid in the LNP is typically expressed as the N / P ratio, i.e., the ratio of nitrogen atoms in the ionic lipid to phosphate groups in the nucleic acid. In the context of the present invention, the N / P ratio of the LNP is approximately 4:1 to 16:1.

[0073] In the context of the present invention, “nucleic acid” refers to deoxyribonucleic acid (DNA) or preferably ribonucleic acid (RNA), more preferably mRNA. According to the present invention, nucleic acids include genomic DNA, cDNA, mRNA, molecules produced by recombination, and chemically synthesized molecules. According to the present invention, nucleic acids may be single-stranded or double-stranded, and may be in the form of linear or covalently closed circular molecules. Nucleic acids can be used for cell introduction, i.e., cell transfection, in the form of RNA, which can be produced, for example, by in vitro transcription from a DNA template. RNA can be further modified before application by sequence stabilization, capping, and / or polyadenylation.

[0074] In the context of this invention, the term "RNA" refers to a molecule comprising, and preferably entirely or substantially composed of, ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. This term includes double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially purified RNA, essentially pure RNA, synthetic RNA, RNA produced by recombination, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations include, for example, the addition of non-nucleotide substances to or within the RNA, e.g., one or more nucleotides of RNA. Nucleotides in an RNA molecule may also include non-standard nucleotides, e.g., nucleotides not naturally occurring, or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs. Nucleic acids may be contained in a vector. As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phages, viral vectors such as adenovirus vectors or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs) or yeast artificial chromosomes, or analogs of naturally occurring RNA.

[0075] According to the present invention, the term "RNA" includes, and more preferably relates to, "mRNA," where "mRNA" means "messenger RNA" and relates to a "transcript" that can be prepared using DNA as a template and codes for a peptide or protein. mRNA typically includes a 5' untranslated region (5'-UTR), a protein or peptide coding region, and a 3' untranslated region (3'-UTR). mRNA has a limited half-life both intracellularly and in vitro. Preferably, mRNA is prepared by in vitro transcription using a DNA template. In one embodiment of the present invention, RNA is obtained by in vitro transcription or chemical synthesis. Methodologies for in vitro transcription are known to those skilled in the art. For example, there are various commercially available in vitro transcription kits.

[0076] In a particular embodiment of the present invention, the mRNA molecule is an mRNA molecule encoding an immunomodulatory protein.

[0077] In the context of the present invention, the term “mRNA molecule encoding an immunomodulatory protein” means an mRNA molecule encoding a protein that modifies the functionality of antigen-presenting cells, more specifically, dendritic cells. Such molecules may be selected from a list including CD40L, CD70, caTLR4, IL-12p70, EL-selectin, CCR7, and / or 4-1 BBL, ICOSL, OX40L, IL-21, and more specifically one or more of CD40L, CD70, and caTLR4. A preferred combination of immunostimulators used in the methods of the present invention is CD40L and caTLR4 (i.e., “DiMix”). In another preferred embodiment, a combination of CD40L, CD70, and caTLR4 immunostimulatory molecules, also referred to herein as “TriMix”, is used.

[0078] In another specific embodiment, the mRNA molecule is an mRNA molecule encoding an antigen-specific and / or disease-specific protein.

[0079] According to the present invention, the term “antigen” includes any molecule, preferably a peptide or protein, that contains at least one epitope that triggers and / or against which an immune response is directed; therefore, the term “antigen” also means encompassing the minimal epitope from an antigen. “Minimal epitope” as defined herein means the smallest structure capable of triggering an immune response. Preferably, in the context of the present invention, an antigen is a molecule that, optionally after processing, induces an immune response that is preferably specific to an antigen or a cell expressing the antigen. In particular, “antigen” relates to a molecule that, optionally after processing, is presented by an MHC molecule and specifically reacts with T lymphocytes (T cells).

[0080] In certain embodiments, the antigen is a target-specific antigen that may be a tumor antigen, or a bacterial, viral, or fungal antigen. The target-specific antigen may be derived from one of the following: whole mRNA isolated from target cells (which may be more than one), one or more specific target mRNA molecules, protein lysates of target cells (which may be more than one), specific proteins derived from target cells (which may be more than one), or synthetic target-specific peptides or proteins, and synthetic mRNA or DNA encoding a target-specific antigen or a peptide derived therefrom.

[0081] To avoid any misunderstanding, the LNP of the present invention may contain a single mRNA molecule, or it may contain a combination of multiple mRNA molecules, such as one or more mRNA molecules encoding immunomodulatory proteins, and / or one or more mRNA molecules encoding antigen-specific and / or disease-specific proteins.

[0082] In a very specific embodiment, the mRNA molecule encoding an immunomodulatory molecule can be combined with one or more mRNA molecules encoding antigen-specific and / or disease-specific proteins. For example, the LNP of the present invention may include mRNA molecules encoding immunostimulatory molecules CD40L, CD70, and / or caTLR4 (Dimix or Trimix, etc.) combined with one or more mRNA molecules encoding antigen-specific and / or disease-specific proteins. Therefore, in a very specific embodiment, the LNP of the present invention includes mRNA molecules encoding CD40L, CD70, and / or caTLR4 combined with one or more mRNA molecules encoding antigen-specific and / or disease-specific proteins.

[0083] In a further embodiment, the present invention provides a pharmaceutical composition comprising one or more LNPs as defined herein. Such a pharmaceutical composition is particularly suitable as a vaccine. Accordingly, the present invention also provides a vaccine comprising one or more LNPs according to the present invention.

[0084] In the context of the present invention, the term “vaccine” as used herein means any preparation intended to induce adaptive immunity (antibody and / or T-cell response) to a disease. For that purpose, the vaccine intended herein contains at least one mRNA molecule encoding an antigen from which an adaptive immune response is initiated. This antigen may exist in the form of a weakened or dead microorganism, a protein or peptide, or a nucleic acid encoding the antigen. In the context of the present invention, an antigen means a protein or peptide that is recognized as a foreign substance by the host’s immune system and thereby stimulates the production of antibodies against such antigens for the purpose of combating them. A vaccine may be a prophylactic vaccine (e.g., to prevent or mitigate the effects of future infection by any natural or “wild” pathogen) or a therapeutic vaccine (e.g., to actively treat or alleviate the symptoms of an ongoing disease). The administration of a vaccine is called vaccination.

[0085] The vaccine of the present invention can be used to induce an immune response, particularly an immune response against disease-related antigens or cells expressing disease-related antigens, such as an immune response against cancer. Therefore, the vaccine can be used in preventive and / or therapeutic measures for diseases involving disease-related antigens or cells expressing disease-related antigens, such as cancer. The immune response is preferably a T-cell response. In one embodiment, the disease-related antigen is a tumor antigen. The antigen encoded by RNA contained in the nanoparticles described herein is preferably a disease-related antigen or elicits an immune response against disease-related antigens or cells expressing disease-related antigens.

[0086] The LNPs and vaccines of the present invention are specifically intended for intravenous administration, i.e., direct injection of liquid substances into a vein. Intravenous routes are the fastest way to deliver fluids and drugs throughout the body, i.e., systemically. Accordingly, the present invention provides intravenous vaccines, as well as the use of the disclosed vaccines and LNPs for intravenous administration. Thus, the vaccines and LNPs of the present invention can be administered intravenously. The present invention also provides the use of the vaccines and LNPs according to the present invention, which are administered intravenously.

[0087] The present invention also provides LNPs, pharmaceutical compositions, and vaccines according to the present invention for use in human medicine or veterinary medicine. The use of LNPs, pharmaceutical compositions, and vaccines according to the present invention for human medicine or veterinary medicine is also intended. Finally, the present invention provides methods for preventing and treating disorders in humans and animals by administering LNPs, pharmaceutical compositions, and vaccines according to the present invention to subjects in need thereof.

[0088] The present invention further provides the use of LNPs, pharmaceutical compositions, or vaccines according to the present invention for immunogenic delivery of one or more nucleic acid molecules. Therefore, the LNPs, pharmaceutical compositions, and vaccines of the present invention are highly useful in the treatment of several human and animal disorders. Accordingly, the present invention provides LNPs, pharmaceutical compositions, and vaccines used in the treatment of cancer or infectious diseases.

[0089] The lipid nanoparticles of the present invention can be prepared according to the protocols specified in the Examples section. More generally, LNPs are, To prepare a first alcohol composition comprising the ionic lipid, the phospholipid, the sterol, the PEG lipid, and a suitable alcohol solvent, To prepare a second aqueous composition comprising one or more nucleic acids and an aqueous solvent, The first composition and the second composition are mixed in a microfluidic mixing device, It can be prepared using a method that includes [a specific component].

[0090] More specifically, the lipid components are combined at suitable concentrations in an alcoholic vehicle such as ethanol. An aqueous composition containing nucleic acids is then added, and subsequently the mixture is introduced into a microfluidic mixing device.

[0091] The objective of microfluidic mixing is to achieve complete and rapid mixing of multiple samples (i.e., lipid phase and nucleic acid phase) in a microscale apparatus. Such sample mixing is typically achieved by enhancing the diffusion effect between different types of flows. Several microfluidic mixing apparatuses can be used for this purpose, such as those outlined in Lee et al., 2011. A particularly preferred microfluidic mixing apparatus according to the present invention is the NanoAssemblr from Precision Nanosystems.

[0092] Other suitable techniques for preparing the LNP of the present invention include dispersing the components in a suitable dispersion medium, such as an aqueous solvent and an alcohol solvent, and the following methods: ethanol dilution, simple hydration, sonication, heating, vortexing, ether injection, French press, cholic acid, Ca 2+This includes applying one or more of the following methods: fusion, freeze-thaw, reverse-phase evaporation, T-junction mixing, microfluidic hydrodynamic focusing, staggered herringbone mixing, etc. [Examples]

[0093] Materials and methods of Examples 1 and 2 mouse Female C57BL / 6 mice were purchased from Charles River Laboratories (France) and housed in individually ventilated cages with standard bedding and cage enrichment. The animals were maintained and treated according to the guidelines of the Institute for Animal Experiments (Vrije Universiteit Brussels) and the European Union. The mice had free access to food and water. The experiment began when the mice were 6 to 10 weeks old. The weight of the mice was monitored every two days.

[0094] In the case of vaccination with ADPGK synthetic long-chain peptide (SLP), mice were intraperitoneally injected with a combination of 50 μg of ADPGK SLP (GIPVHLELASMTNMELMSSIVHQQVFPT (SEQ ID NO: 3), Genscript), 50 μg of anti-CD40 Mab (clone FJK 45, BioXCell), and 100 μg of pIC HMW (InvivoGen) in 200 μl of PBS at the same time intervals.

[0095] mRNA synthesis and purification Capped non-nucleoside-modified E7 and ADPGK mRNAs were prepared by eTheRNA by in vitro transcription (IVT) from the eTheRNA plasmid pEtherna, according to the protocol described in International Publication No. 2015071295. Sequences encoding HPV16-E7 or ADPGK proteins were cloned in-frame between the signal sequence and transmembrane and cytoplasmic regions of human DC-LAMP. This chimeric gene was cloned into a pEtherna plasmid enhanced with a 5' translational enhancer and a 3' RNA stabilization sequence. After IVT, dsRNA was removed by cellulose purification. Cellulose powder was purchased from Sigma and washed in 1×STE (sodium chloride-Tris-EDTA) buffer containing 16% ethanol. IVT mRNA (in 1×STE buffer containing 16% ethanol) was added to the washed cellulose pellet and shaken at room temperature for 20 minutes. The solution was then filtered through a vacuum filter (Corning). The eluate contained the ssRNA fraction and was used in all experiments. mRNA quality was monitored by capillary gel electrophoresis (Agilent, Belgium).

[0096] Generation of mRNA-based lipid nanoparticles Lipid-based nanoparticles were prepared by microfluidic mixing of mRNA solution and lipid solution in sodium acetate buffer (100 mM, pH 4) at a volume ratio of 2:1 and a rate of 9 mL / min using a NanoAssemblr Benchtop (Precision Nanosystems). The lipid solution contained a mixture of Coatsome-EC (NOF Corporation), DOPE (Avanti), cholesterol (Sigma), and one of the following PEG lipids: DMG-PEG2000 (C14 lipid) (Sunbright GM-020, NOF Corporation), DPG-PEG2000 (C16 lipid) (Sunbright GP-020, NOF Corporation), and DSG-PEG2000 (C18 lipid) (Sunbright GS-020, NOF Corporation). The four lipids were mixed in different molar ratios. LNPs were dialyzed against TBS (TBS volume 10,000 times greater than LNP volume) using a slide-a-lyzer dialysis cassette (20K MWCO, 3 mL, ThermoFisher). Size, polydispersity, and zeta potential were measured using a Zetasizer Nano (Malvern). The percentage of mRNA inclusion was measured by a ribogreen assay (ThermoFisher).

[0097] Flow cytometry Approximately six days after immunization, blood was collected from both treated and control mice. Red blood cells were lysed, and the remaining white blood cells were labeled with APC-labeled E7 cells according to the manufacturer's instructions (MBL International). (RAHYNIVTF) Cells were stained with either the -tetramer (SEQ ID NO: 1) or the ADPGK(ASMTNMELM)-tetramer (SEQ ID NO: 2). Excess tetramers were washed away. Subsequently, antibody mixtures against surface molecules (listed in Table 2) were added to the cells and incubated at 4°C for 30 minutes. Data were acquired using an LSR Fortessa or Attune cytometer and analyzed with Flow Jo software.

[0098] Table 2: List of antibodies used for flow cytometry analysis of the number / percentage of E7-specific and Adpgk-specific T cells. [Table 2]

[0099] result Selection of C18-PEG2000 and low %PEG Example 1 - E7 Antigen Mice were administered 10 μg of E7 mRNA, packaged in LNP (50 / 10 / (40-x) / x ionic lipids / DOPE / cholesterol / PEG-lipids), intravenously as a single dose (Figure 1) or double dose (Figure 2). E7-specific CD8 in the blood was then measured. + The percentage of T cells was determined on day 6 after immunization. Figure 1 shows that LNPs with a low percentage of PEG (0.5%) induce a stronger antigen-specific immune response than LNPs with an intermediate percentage of PEG (1.5%) or a high percentage of PEG (4.5%). Both Figures 1 and 2 show that DSG-PEG2000 (C18) is superior to shorter carbon chain PEG lipids such as DMG-PEG2000 (C14) and DPG-PEG2000 (C16) in inducing an immune response.

[0100] Example 2 - ADPGK antigen Mice were intravenously administered four times either 10 μg of ADPGK mRNA or 50 μg of ADPGK synthetic long-chain peptide (SLP) packaged in low-proportion PEG LNP (50 / 10 / 39.5 / 0.5 ionic lipids / DOPE / cholesterol / PEG-lipids). ADPGK-specific CD8 levels in the blood were also evaluated. + The proportion of T cells was determined on day 6 after the fourth immunization cycle. DSG-PEG2000(C18)LNP was superior to DMG-PEG2000(C14)LNP in inducing an antigen-specific immune response (Figure 3). Both LNPs were more immunogenic than SLPs.

[0101] Materials and methods of Examples 3-6 animal All mouse experiments were conducted with the approval of the Utrecht Animal Welfare Body of UMC Utrecht or the Animal Ethics Committee of Ghent University. Animal care followed established guidelines. All mice had unlimited access to water and standard laboratory feed. Female C57Bl / 6J mice were obtained from Charles River Laboratories, Inc. (Germany / France). μMT mice were obtained from Jackson Laboratory (USA). Non-GLP studies in non-human primates were conducted at Charles River Laboratories (France) in accordance with local regulations.

[0102] mRNA synthesis and purification Codon-optimized E7, TriMix, and luciferase mRNAs were prepared by eTheRNA via in vitro transcription (IVT) from eTheRNA plasmids. No nucleotide modifications were used. The E7 mRNA used in DoE was ARCA capped. All subsequent experiments were performed using CleanCap-capped mRNAs. After IVT, dsRNA was removed by cellulose purification. mRNA quality was monitored by capillary gel electrophoresis (Agilent, Belgium).

[0103] Manufacturing and characterization of LNPs In in vivo distribution and cell uptake studies, LNPs were loaded with a 1:1 mixture of firefly luciferase (Fluc) mRNA (eTheRNA Immunotherapy NV) and Cleancap® Cy5-labeled Fluc mRNA (TriLink Biotechnologies). In DoE immunogenicity studies, LNPs were loaded with E7 mRNA. All other studies were performed using a 3:1:1:1 mixture of E7, CD40L, CD70, and TLR4 mRNA. mRNA was diluted in 100 mM sodium acetate buffer (pH 4), and lipids were dissolved in ethanol and diluted. The mRNA and lipid solutions were mixed using a NanoAssemblr Benchtop microfluidic mixing system (Precision Nanosystems) and subsequently dialyzed overnight against Tris-buffered saline (TBS, 20 mM Tris, 0.9% NaCl, pH 7.4). An Amicon ultracentrifuge filter (10 kD) was used to concentrate the LNPs. Size, polydispersity index, and zeta potential were measured using Zetasizer Nano (Malvern). mRNA encapsulation efficiency was determined by ribogreen assay (ThermoFisher). The composition of all LNPs is summarized in Table 3 of Example 3.

[0104] In vivo distribution and cellular uptake Mice were intravenously injected with 10 μg of mRNA from a selected LNP formulation via the tail vein. Four hours later, the mice were anesthetized with 250 μL of pentobarbital (6 mg / mL). Blood samples were collected in tubes containing gel coagulation factor (Sarstedt). Subsequently, the thoracic cavity was opened, the portal vein was severed, and the mice were perfused with 7 mL of PBS through the right ventricle. Organs were removed and flash-frozen in liquid nitrogen. For liver and spleen tissue, portions of the organs were stored in ice-cold PBS for flow cytometry analysis.

[0105] Cellular uptake Liver and spleen tissues were placed in petri dishes containing RPMI 1640 medium, each containing 1 mg / mL collagenase A (Roche) or 20 μg / mL liberase® (Roche), and 10 μg / mL DNase I, Grade II (Roche), respectively. The tissues were finely chopped using a surgical scalpel and incubated at 37°C for 30 minutes. Subsequently, the tissue suspensions were passed through a 100 μm nylon cell strainer. The liver suspension was centrifuged at 70 × g for 3 minutes to remove parenchymal cells. The supernatant and spleen suspension were centrifuged at 500 × g for 7 minutes to pellet the cells. Red blood cells were lysed in ACK buffer (Gibco) for 5 minutes, inactivated with PBS, and subsequently passed through a 100 μm cell strainer. The cells were washed with RPMI 1640 containing 1% fetal bovine serum (FBS), mixed with trypan blue, and counted using a Luna-II automated cell counter (Logos Biosystems). 3 x 10 5 (Liver) or 6 x 10 5 Live spleen cells were seeded in a 96-well plate, pelletized at 500×g for 5 minutes, and resuspended in 2% PBSA in PBS (2% PBSA) containing 50% Brilliant Stain Buffer (BD Biosciences) and 2 μg / mL TruStain FcX (BioLegend). Cells were incubated on ice for 10 minutes and mixed twice in a 1:1 ratio with 2% PBSA containing three applicable antibody cocktails. Cells were incubated in a shaker at room temperature for 15 minutes, washed twice with 2% PBSA, and resuspended in 2% PBSA containing 0.25 μg / mL 7-AAD Viability Stain (BioLegend). Samples were acquired using a 4-laser BD LSRFortessa flow cytometer. Analysis was performed using FlowJo software.

[0106] Whole body distribution Approximately 50 mg to 100 mg of each tissue sample was cut, weighed, and placed in a 2 mL microtube containing a layer of approximately 5 mm of 1.4 mm ceramic beads (Qiagen). For each mg of tissue, 3 μL of cold Cell Culture Lysis Reagent (Promega) was added, and the tissue was homogenized at full speed for 60 seconds at 4°C using a Mini-BeadBeater-8 (BioSpec). The homogenate was stored at -80°C, thawed, and centrifuged at 10,000 × g for 10 minutes at 4°C to remove beads and debris. The supernatant was then stored again at -80°C. Ten microliters of each lysate were divided twice into a white 96-well plate. Using a SpectraMax iD3 plate reader equipped with an injector, 50 μL of Luciferase Assay Reagent (Promega) was mixed and dispensed into each well, followed by a 2-second wait and recording of luciferase luminescence for 10 seconds. Luciferase activity was normalized to the background signal obtained from organ lysates of mice injected with TBS.

[0107] T cell response Mice were intravenously immunized via the tail vein with 10 μg of mRNA in selected LNPs at weekly intervals. Blood for flow cytometry staining was collected 5-7 days post-immunization. After erythrocyte lysis, cells were incubated with FcR block and viability dye. After incubation and washing, APC-labeled E7 cells were selected. (RAHYNIVTF) -The tetramer was added and incubated at room temperature for 30 minutes. Excess tetramer was washed away, and an antibody mixture against surface molecules CD3 and CD8 was added to the cells and incubated at 4°C for 30 minutes. Samples were acquired using a 3-laser AtuneNxt flow cytometer or a 4-laser BD LSRFortessa flow cytometer.

[0108] Intracellular cytokine production in the spleen was determined on day 7 after the third immunization. The spleen was disrupted, red blood cells were lysed, and a single cell suspension of splenocytes was prepared by filtering the sample through a 40 μM cell strainer. 200,000 cells / well / sample were seeded twice in a 96-well plate. Before incubating the cells at 37 °C, 4 μg of E7 peptide (Genscript) was added for stimulation. One hour after peptide stimulation, GolgiPlug (BD Cytofix / Cytoperm kit (BD Biosciences)) was added. The cells were incubated for an additional 4 hours. Thereafter, the cells were incubated with FcR block and viability dye. After incubation and washing, APC-labeled E7 (RAHYNIVTF) -tetramer was added and incubated at room temperature for 30 minutes. The excess dextramer was washed away, and an antibody mixture against surface molecules CD3 and CD8 was added to the cells and incubated at 4 °C for 30 minutes. Further steps were according to the manufacturer's instructions of the BD Cytofix / Cytoperm kit (BD Biosciences). After permeabilization, the cells were stained for IFN-γ and TNF-α. Samples were acquired on a 4-laser BD LSRFortessa flow cytometer. Analysis was performed using FlowJo software.

[0109] Immune cell activation 5 μg of mRNA in the selected LNP was intravenously injected into the mice via the tail vein. The spleen was excised 4 hours later for flow cytometry staining. A single cell suspension of splenocytes was prepared and incubated with digestion buffer (DMEM containing DNase-1 and collagenase-III) for 20 minutes with regular shaking. Thereafter, the samples were incubated with Fc block and viability dye. After incubation and washing, the cells were stained with cell lineage markers and activation markers. Samples were acquired on a 3-laser AtuneNxt flow cytometer. Analysis was performed using FlowJo software.

[0110] TC-1 tumor experiment TC-1 cells were obtained from Leiden University Medical Center. 500,000 TC-1 cells in 50 μL of PBS were subcutaneously injected into the right flank of mice. Tumor measurements were performed using calipers. Tumor volume was calculated as (minimum diameter 2 × maximum diameter) / 2. Ant-PD-1 and isotype control antibodies were diluted to a concentration of 200 μg per 200 μL of fresh PBS per mouse and injected intraperitoneally. Mice were administered either anti-PD-1 antibody (monotherapy or in combination with mRNA LNP immunization) or isotype control (in combination with LNP immunization). Antibody injections were started on day 3 after the first mRNA LNP immunization and continued every 3-4 days until 2 weeks after the last LNP injection. For analysis of tumor-infiltrating lymphocytes, tumors were isolated on day 3 after the second mRNA LNP immunization and placed in 24-well plates filled with MACS tissue preservation buffer (Miltenyi Biotec). The tumor was finely chopped and incubated in digestion buffer for 1 hour with regular shaking. The erythrocytes were then lysed, and all samples were filtered through a 70 μM cell strainer. Lymphocytes were concentrated by ficoll-paque density gradient purification before proceeding to staining. Cells were first incubated with FcR block and viability dye. After incubation and washing, APC-labeled E7 (RAHYNIVTF) -The tetramer was added and incubated at room temperature for 30 minutes. Excess tetramer was washed away, and an antibody mixture against surface molecules CD45 and CD8 was added to the cells and incubated at 4°C for 30 minutes. The samples were acquired using a 3-laser AtuneNxt flow cytometer. Analysis was performed using FlowJo software.

[0111] Inflammatory cytokines Blood samples were collected in tubes containing gel clotting factor (Sarstedt). The coagulated blood samples were centrifuged at 10,000 g for 5 minutes to obtain serum. Serum samples were stored at -80°C until analysis. The concentrations of inflammatory cytokines such as IFN-γ, TNF-α, and IP-10 were determined using a ProcartaPlex multiplex assay (ThermoFisher). Serum samples were diluted 3-fold with assay buffer and incubated with fluorescently labeled beads for 120 minutes. Further steps were performed according to the protocol. Samples were acquired using a MagPix instrument (Luminex). Data were analyzed using ProcartaPlex Analyst software.

[0112] Example 3 - DOE-driven optimization of LNP composition for maximum T cell response The LNP library was prepared by combining the commercially available ionic lipid Coatsome SS-EC with cholesterol, DOPE, and PEGylated lipids. DOPE is already part of several approved liposomal products and mRNA vaccines under investigation. In this experiment, different LNP compositions, including DSG-PEG2000 lipids, were investigated. It has been reported that the different behaviors of PEG-lipids strongly influence the pharmacokinetics and pharmacodynamics of siRNA LNPs upon intravenous administration.

[0113] The initial LNP library was designed to investigate whether the molar ratios of lipids and the chemical properties of PEG-lipids actually influence the T cell response induced by intravenous mRNA-LNP vaccination, and therefore represent variables that can be optimized to improve vaccine efficacy. The molar percentages of SS-EC, DOPE, and PEG-lipids were considered independent variables, while cholesterol was considered a filler lipid to maintain a balance of molar percentages up to 100%. Using the DOE method, experimental designs containing 11 LNPs were created (see composition in Table 3).

[0114] Table 3: Composition of DSG-PEG2000 LNP in DoE experiments [Table 3]

[0115] The lipid ratios of the 11 LNPs were uniformly distributed across the experimental area (data not shown). In immunogenicity screening, the percentage of E7-specific CD8 T cells in the blood after three intravenous immunizations was considered the maximum response variable. For this purpose, all LNPs were packaged with mRNA encoding the human papillomavirus 16 (HPV16) oncoplastic protein E7 as the antigen. The results support the hypothesis that the magnitude of the CD8 T cell response is strongly dependent on the LNP composition. Some mRNA-LNP vaccines induced an E7-specific CD8 T cell response of over 50%, while others induced little to no response (Figure 4a). The chemical properties of the PEG-lipids and their molar percentage were identified as important parameters related to the magnitude of the E7-specific CD8 T cell response. Low molar percentages of PEG-lipids were required to achieve the maximum T cell response (Figure 4b), and for DSG-PEG2000-based LNPs, the proportion of ionic lipids also had a significant impact on immunogenicity.

[0116] Bayesian regression modeling was applied to the data to create a response surface model (data not shown) that can predict the immunogenicity of a particular LNP composition. The quality of the response surface model for each of the chemical properties of PEG-lipids was determined by the coefficient of determination R 2 This was reflected in the model's ability to explain the variability in T cell responses based on input variables (SS-EC, DOPE, and PEG-lipid percentages). For DSG-PEG2000 LNP, the mean R was 0.74. 2 To obtain the values ​​and validate the model's predictions, two new LNP compositions (Table 4) were evaluated.

[0117] Table 4: Composition of DSG-PEG2000 LNP in DoE experiments [Table 4]

[0118] Mice immunized with LNP36 (DSG-PEG2000) had a probability of over 90% that they would induce more than 30% of E7-specific CD8 T cells (optimal LNP), while LNP37 (DSG-PEG2000) was predicted to produce an insufficient T cell response (non-optimal LNP) (Figure 4c). The experimental data closely matched the predictions, thus successfully validating the model. All mice immunized with the predicted optimal LNP did indeed show more than 30% of E7-specific CD8 T cell responses, but mice immunized with LNP37 did not induce a T cell response exceeding this threshold (Figure 4c).

[0119] Example 4 - Optimal mRNA LNP vaccine that induces a high-magnitude T cell response The success of cancer immunotherapy is influenced by numerous factors, including T cell phenotype, functionality, and tumor infiltration. First, we evaluated the quality and boostability of the T cell response induced by optimal LNPs. For this purpose, mice were prime-immunized three times on days 0, 7, and 14, followed by final immunization on day 50. E7 mRNA was supplemented with TriMix (Bonehill et al., 2008), a mixture of three immunostimulatory mRNAs that enhance the intensity of the T cell response.

[0120] After three immunizations with E7-TriMix, over 70% of E7-specific T cells were present in the blood (Figure 5a). Five weeks after the third immunization, the percentage of E7-specific CD8 T cells remained very high. A rapid proliferation of E7-specific effector T cells was observed after the final booster immunization, demonstrating the vaccine's boostability (Figure 5a). Higher concentrations of IFN-y in the serum were measured after each immunization (Figure 5b), reflecting the increase in the number of E7-specific T cells.

[0121] To evaluate T cell functionality, intracellular cytokine staining was performed after three immunizations with LNP36. Multifunctional CD8 T cells, which simultaneously produce two or more cytokines, are associated with better control of infectious diseases and tumors and account for approximately 30% of E7-specific CD8 T cells (Figure 5c).

[0122] Example 5 - Optimal mRNA LNP vaccine that induces tumor reduction The therapeutic antitumor effect was evaluated in the syngeneic mouse tumor model TC-1, created by retroviral transduction using HPV16 E6 / E7 antigens. Treatment with 5 μg of E7-TriMix delivered via LNP36 was initiated when the tumor reached an average diameter of 55 mm3. Furthermore, mice were treated with anti-PD-1 (or isotype control antibody). PD-1 is expressed on activated T cells and inhibits T cell function and induces tolerance through interaction with PD-L1. PD-1 checkpoint inhibition maintains T cell responsiveness and is approved as a first-line treatment for patients with metastatic or unresectable recurrent HNSCC. LNP36 vaccination resulted in significant tumor regression (Figure 5d) and a substantial extension of survival (Figure 5e), but tumor recurrence occurred after discontinuation of treatment. Anti-PD1 monotherapy had no therapeutic effect on TC-1-carrying mice. LNP36 immunization in combination with anti-PD-1 improved tumor growth control.

[0123] Finally, we evaluated the ability of vaccine-induced T cells to reach the tumor bed. Two doses of each mRNA-LNP vaccine were used to increase CD8 + This resulted in strong tumor infiltration of tumor-invading T cells (Figure 5f), with over 70% being E7 specific (Figure 5g). Addition of anti-PD-1 to vaccine therapy did not significantly alter the proportion of E7-specific CD8 T cells invading tumors.

[0124] Example 6 - Optimal LNP that increases uptake in the spleen and activates immune cells To investigate whether there is a correlation between the magnitude of the induced T cell response and the in vivo distribution of mRNA uptake and expression at the organ and cell type levels, Cy5-labeled firefly luciferase mRNA was encapsulated in DSG-PEG2000 LNPs that had been previously screened for immunogenicity. Luciferase activity was measured in liver, spleen, lung, heart, and kidney excised 4 hours after LNP injection. As predicted, LNP composition strongly influenced the intensity and organ specificity of mRNA expression. The liver was the primary target organ, followed by the spleen, but the liver-to-spleen ratio differed significantly among LNPs (Figure 6a). The magnitude of the E7-specific CD8 T cell response after the third immunization was positively correlated with spleen expression (data not shown).

[0125] Next, we evaluated whether immunogenicity was associated with early mRNA uptake and activation of specific immune cell types in the spleen. LNPs accumulated primarily in macrophages and monocytes (Figure 6b). A strong overall correlation existed between T cell response and LNP uptake by splenic macrophages, monocytes, plasmacytoid dendritic cells (pDCs), and B cells (data not shown).

[0126] To further investigate the importance of mRNA uptake and expression in the spleen, the in vivo distribution and cellular uptake profiles of an optimally immunogenic LNP (LNP36) were compared with those of a suboptimal, less immunogenic LNP (LNP37). Compared to the suboptimal LNP, LNP36 dramatically increased mRNA expression in the spleen (Figure 6c) and uptake by splenic monocytes, macrophages, and dendritic cells (Figure 6d).

[0127] Compared to LNP37 formulated with 1.5% DSGPEG2000, LNP36 with the optimal mRNA LNP composition induced higher levels of inflammatory cytokines in the blood, increased CD86 expression in the spleen DC subset (Figure 6g), and showed increased innate activation (Figure 6f).

[0128] Recently, a pilot study in non-human primates (NHPs) was conducted to evaluate the translational value of optimal LNPs (LNP36), and in NHPs, the spleen showed the highest accumulation of E7 mRNA per g of tissue, followed by the liver and bone marrow (Figure 6e).

[0129] conclusion LNP composition is a critical determinant of the T cell response induced by systemic administration of mRNA vaccines. LNPs containing DSG-PEG2000 as the LNP-stabilized PEG-lipid induced an increased T cell response compared to LNPs containing DPG-PEG2000 and DMG-PEG2000. Furthermore, reducing the molar percentage of DSG-PEG2000 to 0.5%–0.9% significantly increased the T cell response. mRNA vaccines delivered with such optimized LNP compositions induce a high-magnitude / high-quality T cell response that can be boosted by repeated administration and confer an antitumor effect in a mouse syngeneic tumor model. Mechanistically, the optimal LNP composition is characterized by increased mRNA expression in the spleen, involving increased mRNA uptake by various antigen-presenting cell types. The optimal LNP formulation induces increased activation of splenic dendritic cells and enhances the release of IFN-α and IP-10 into the blood.

[0130] Drawing translation Figure 1 % E7 specific CD8 + T Cells E7-specific CD8 + T cells (%) % PEG-lipid (%) Figure 2 % E7 specific CD8 + T Cells E7-specific CD8 + T cells (%) Figure 3 % ADPGK specificCD8 + cells ADPGK-specific CD8 + cell(%) Figure 4 A % E7-specific CD8 + T cells E7-specific CD8 + T cells (%) LNP number LNP number B % E7 specific CD8 + T cells E7-specific CD8 + T cells (%) % PEG-lipid PEG-lipid (%) p value p value C % E7-specific CD8 + T cells E7-specific CD8 + T cells (%) Figure 5 A % E7 specific CD8 + T Cells E7-specific CD8 + T cells (%) Time (days) Time (days) B Time (days) Time (days) C Cytokine production Cytokine production % of E7 specific splenicCD8 + T cells E7-specific splenic CD8[[ID=六十二]] + T cells (%) D Tumor volume (mm 3 ) Tumor volume (mm 3 ) No treatment No treatment anti-PD-1 anti-PD-1 LNP36 + isotype LNP36 + isotype LNP36 + anti-PD-1 Days post TC-1 inoculation E Percent survival No treatment anti-PD-1 LNP36 + isotype LNP36 + anti-PD-1 Start treatment Days post TC-1 inoculation F % CD45 + CD3 + (TILs) of live cells + CD3 + (TIL)(%) Untreated anti-PD-1 LNP36 + isotype LNP36 + anti-PD-1 G % E7 specific cells of CD8 + TILs CD8 + TIL's E7 specific cells(%) LNP36 + isotype LNP36 + anti-PD-! Figure 6 A Relative luciferase activity (% of total luciferase activity in all analyzed organs) Spleen Kidneys Liver lungs Heart B Cellular LNP association Resident monocytes Inflammatory monocytes Granulocytes T cells T cells B cells B cells Macrophages C Relative luciferase activity (% of total luciferase activity in all analyzed organs) Spleen Kidneys Liver lungs Heart LNP36 (optimal) LNP36 (optimal) LNP37 (non-optimal) LNP37 (non-optimal) D Cellular association Macrophages LNP36 (optimal) LNP36 (optimal) LNP37 (not optimal) LNP37 (not optimal) B cells B cells E E7 (pg mRNA / g tissue) E7(pg mRNA / g tissue) Liver Spleen Lung Heart Brain Kidney Bone Marrow Adrenal gland Lymph node F 6 hours 24 hours 24 hours

Claims

1. An mRNA vaccine comprising one or more lipid nanoparticles, wherein the lipid nanoparticles are Approximately 45 mol% to 65 mol% of ionic lipids, Approximately 4 mol% to 15 mol% of phospholipids, Sterols and PEG lipids and One or more mRNA molecules, Includes, The mRNA vaccine is characterized in that the LNP contains less than about 1 mol% of the PEG lipid, preferably about 0.5 mol% to 0.9 mol% of the PEG lipid.

2. Lipid nanoparticles (LNPs) used for mRNA vaccination, wherein the LNPs are Approximately 45 mol% to 65 mol% of ionic lipids, Approximately 4 mol% to 15 mol% of phospholipids, Sterols and PEG lipids and One or more mRNA molecules, Includes, Lipid nanoparticles characterized in that the LNP contains less than about 1 mol% of the PEG lipid, preferably about 0.5 mol% to 0.9 mol% of the PEG lipid.

3. Lipid nanoparticles (LNPs), Approximately 45 mol% to 65 mol% of ionic lipids, Approximately 4 mol% to 15 mol% of phospholipids, Sterols and PEG lipids and One or more nucleic acid molecules, Includes, Lipid nanoparticles characterized in that the PEG lipid is a C18-PEG2000 lipid, and the LNP contains less than about 1 mol% of the PEG lipid, preferably about 0.5 mol% to 0.9 mol% of the PEG lipid.

4. The lipid nanoparticle according to claim 3, wherein the C18-PEG2000 lipid is selected from a list including (distearoyl-based)-PEG2000 lipids, such as DSG-PEG2000 lipids or DSPE-PEG2000 lipids, or (dioleoyl-based)-PEG2000 lipids, such as DOG-PEG2000 lipids or DOPE-PEG2000 lipids.

5. The ionic lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azandiyl)bis(dodecane-2-ol) (C12-200), Dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or, Compound of formula (I): 【Chemistry 1】 (In the formula, RCOO is selected from a list containing myristoyl, α-D-tocopherol succinoyl, linoleyl and oleoyl, and X is 【Chemistry 2】 Selected from a list that includes) Preferably, the ionic lipid is a lipid of formula (I) (wherein RCOO is α-D-tocopherol succinoyl, and X is 【Transformation 3】 Lipid nanoparticles according to claim 3 or 4, wherein the lipid nanoparticles are as follows:

6. The lipid nanoparticle according to any one of claims 3 to 5, wherein the phospholipid is selected from a list including DOPE, DOPC and mixtures thereof.

7. The lipid nanoparticle according to any one of claims 3 to 6, wherein the sterol is selected from a list including cholesterol, ergosterol, campesterol, oxysterol, antrosterol, desmosterol, nicasterol, sitosterol and stigmasterol, and is preferably cholesterol.

8. Lipid nanoparticles, The ionic lipids exceeding 60 mol%, Approximately 8 mol% of the phospholipid, Approximately 0.5 mol% to 0.9 mol% of the PEG lipid, Includes, Lipid nanoparticles whose balance is maintained by the amount of the aforementioned sterols.

9. Lipid nanoparticles, Approximately 64 mol% of the aforementioned ionic lipid, Approximately 8 mol% of the phospholipid, Approximately 0.5 mol% to 0.9 mol% of the PEG lipid, Includes, Lipid nanoparticles whose balance is maintained by the amount of the aforementioned sterols.

10. Lipid nanoparticles, Approximately 64 mol% of the aforementioned ionic lipid, Approximately 8 mol% of the phospholipid, Approximately 0.5 mol% of the PEG lipid, Includes, Lipid nanoparticles whose balance is maintained by the amount of the aforementioned sterols.

11. The lipid nanoparticle according to any one of claims 3 to 10, wherein the one or more nucleic acid molecules are selected from a list including mRNA and DNA, and are preferably mRNA.

12. The lipid nanoparticle according to any one of claims 3 to 11, wherein one or more mRNA molecules are selected from the group consisting of mRNA encoding an immunomodulatory polypeptide and / or mRNA encoding an antigen.

13. The lipid nanoparticle according to claim 12, wherein the mRNA encoding the immunomodulatory polypeptide is selected from a list including mRNA molecules encoding CD40L, CD70, and caTLR4.

14. A pharmaceutical composition or vaccine comprising one or more lipid nanoparticles according to any one of claims 3 to 13 and an acceptable pharmaceutical carrier.

15. Lipid nanoparticles according to any one of claims 3 to 13, or a pharmaceutical composition or vaccine according to claim 14, used in human medicine or veterinary medicine, for example, for the treatment of cancer or infectious diseases.

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