TW202304505A - Lipid nanoparticles - Google Patents

Abstract

The present invention relates to the field of lipid nanoparticles (LNP); more specifically comprising an ionizable lipid, a phospholipid, a sterol, a PEG lipid and one or more nucleic acids. The LNP's of the present invention are characterized in comprising less than about 1 mol% of a C14-PEG2000 lipid; as well as particular percentages of the other lipids. The present invention provides use of the LNP's for immunogenic delivery of nucleic acid molecules, specifically mRNA; thereby making them highly suitable for use in vaccines, such as for the treatment of cancer or infectious diseases. Finally, methods are provided for preparing such LNP's.

Description

lipid nanoparticles

The present invention is in the field of lipid nanoparticles (LNPs); more specifically, ionizable lipids, phospholipids, sterols, PEG lipids and one or more nucleic acids. The LNPs of the invention are characterized as comprising less than about 1 mol% C14-PEG2000 lipid; and specified percentages of other lipids. The present invention provides the use of LNPs for the immunological delivery of nucleic acid molecules, in particular mRNA; thus making them very suitable for use in vaccines, for example for the treatment of cancer or infectious diseases. Finally, methods for preparing such LNPs are provided.

One of the main challenges in the field of targeted delivery of bioactive substances is often their instability and low cell penetration potential. This is especially true for the delivery of nucleic acid molecules, especially (m)RNA molecules. Therefore, proper packaging is critical for adequate protection and delivery. Accordingly, there is a continuing need for methods and compositions for packaging biologically active substances such as nucleic acids.

In this regard, lipid-based nanoparticle compositions such as lipoplexes and liposomes have been used as packaging vehicles for biologically active substances to allow transport into cells and/or intracellular compartments. Such lipid-based nanoparticle compositions generally comprise mixtures of different lipids, such as cationic lipids, ionizable lipids, phospholipids, structured lipids (such as sterols or cholesterol), PEG (polyethylene glycol) lipids, etc. (such as Reviewed in Reichmuth et al., 2016).

Lipid-based nanoparticles comprising a mixture of four lipids (cationic or ionizable lipids, phospholipids, sterols, and pegylated lipids) have been developed for non-immune delivery of siRNA to the liver following systemic administration and mRNA. While many such lipid compositions are known in the art, lipid compositions for in vivo mRNA delivery typically contain a PEG lipid concentration of at least 1.5 molar and have a low ratio of ionizable lipid:phospholipids, e.g. About 1:1-about 5:1.

However, we have now surprisingly found that the use of low amounts (ie, less than about 1 mol%) of PEG lipids results in nanoparticles that are highly suitable for immunological delivery of mRNA following systemic injection of LNP. It was found that by combining such low concentrations of PEG lipids with relatively high concentrations of ionizable lipids (i.e., 55-70 mol%) and relatively low concentrations of phospholipids (i.e., less than about 10 mol%), these The effect is more pronounced, so LNPs have a relatively high ionizable lipid:phospholipid ratio (ie 6:1-11:1). Additionally, some embodiments of the invention are characterized by a low percentage of sterols (ie, less than about 30 mol%, eg, about 25 mol%).

In a first aspect, the present invention provides a lipid nanoparticle (LNP), comprising: - ionizable lipids; - Phospholipids; - sterols; - PEG lipids; and - one or more mRNA molecules; Features: The PEG lipid is C14-PEG lipid; The LNP comprises less than about 1 mol% of the PEG lipid; The molar percentage of the ionizable lipid is about and between 50-70 mol %; and The molar percentage of the sterol is about or higher than 25 mol%.

In another aspect, the present invention provides a lipid nanoparticle (LNP), comprising: - ionizable lipids; - Phospholipids; - sterols; - PEG lipids; and - one or more mRNA molecules; Features: The PEG lipid is C14-PEG lipid; The LNP comprises less than about 1 mol% of the PEG lipid; The molar percentage of the ionizable lipid is about and between 50-60 mol %; and The molar percentage of the sterol is about or higher than 30 mol%.

In another embodiment of the present invention, the LNP comprises about 0.5 mol% to about 0.9 mol% of the PEG lipid.

In another specific embodiment, the molar percentage of the phospholipid is less than about 10 mol%; preferably about 5 mol%.

In another embodiment of the present invention, the ratio of ionizable lipid to phospholipid is higher than 5:1; preferably between about 6:1 and 11:1; most preferably about 11:1.

In yet another embodiment of the present invention, the molar percentage of the ionizable lipid is about and between 55-60 mol%.

In a specific embodiment of the present invention, the C14-PEG lipid is dimyristyl lipid, which has two C14 fatty acid tails. For example, the C14-PEG2000 lipid is preferably selected from the following list: 1,2- Dimyrisyl- rac -glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) or 2-dimyristyl-sn-glycero-3-phosphoethanolamine ethylene glycol-2000 (DMPE -PEG2000).

-1-基)乙基)氮烷二基)雙(十二烷基-2-醇)(C12-200)； - 二亞麻油基甲基-4-二甲基胺基丁酸酯(DLin-MC3-DMA)；或 - 式(I)化合物：

其中：RCOO選自包含以下所列：肉豆蔻醯基、α-D-生育酚琥珀醯基、亞油醯基及油醯基；且X選自包含以下所列：

。 In another specific embodiment of the present invention, the ionizable lipid is selected from the group comprising: - 1,1'-((2-(4-(2-((2-(bis(2-hydroxydeca Dialkyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperene

-1-yl)ethyl)azanediyl)bis(dodecyl-2-ol) (C12-200); -Dilinoleylmethyl-4-dimethylaminobutyrate (DLin - MC3-DMA); or - a compound of formula (I):

Wherein: RCOO is selected from the group consisting of myristyl, α-D-tocopheryl succinyl, linoleyl, and oleyl; and X is selected from the group consisting of:

.

。 In a preferred embodiment, the ionizable lipid is a lipid of formula (I), wherein RCOO is α-D-tocopheryl succinyl, and X is

.

In yet another embodiment of the present invention, the phospholipid is selected from the group consisting of: 1,2-dioleyl- sn -glycero-3-phosphoethanolamine (DOPE), 1,2-dioleyl- sn - Glycero-3-phosphocholine (DOPC), 1.2-distearyl- sn -glycero-3-phosphocholine (DSPC) and mixtures thereof; especially mixtures of DOPE, DOPC and the like.

In yet another embodiment of the present invention, the sterol is selected from the group consisting of: cholesterol, ergosterol, campesterol, oxysterol, antrosterol, streptosterol, and nikarsterol (nicasterol), sitosterol and stigmasterol; preferably cholesterol.

In yet another embodiment of the present invention, the LNP comprises about 5-15 mol% of the phospholipid.

In a specific embodiment of the invention, the LNP comprises: - about 50-70 mol% of the ionizable lipid; - about 5-15 mol% of the phospholipid; - about 0.5-0.9 mol% of the PEG lipid; and Balanced with the amount of the sterol.

In a specific embodiment of the invention, the LNP comprises: - about 50-60 mol% of the ionizable lipid; - about 5-15 mol% of the phospholipid; - about 0.5-0.9 mol% of the PEG lipid; and Balanced with the amount of the sterol.

In a very specific embodiment of the invention, the LNP comprises: - about 50 mol% of the ionizable lipid; - About 10 mol% of DOPE; - about 39.5 mol% cholesterol; and - About 0.5 mol% of DMG-PEG2000.

In another very specific embodiment of the invention, the LNP comprises: - about 56.5 mol% of the ionizable lipid; - About 5 mol% of DOPE; - about 38 mol% cholesterol; and - About 0.5 mol% of DMG-PEG2000.

In another very specific embodiment of the invention, the LNP comprises: - about 65 mol% of the ionizable lipid; - About 9.5 mol% DOPE; - about 25 mol% cholesterol; and - About 0.5 mol% of DMG-PEG2000.

In a more specific embodiment, the one or more mRNA molecules are selected from the group comprising: mRNA encoding an immunomodulatory polypeptide and/or mRNA encoding an antigen. The immunomodulatory encoded mRNA can, for example, be selected from the list comprising: mRNA molecules encoding CD40L, CD70 and caTLR4.

In another aspect, the present invention provides a pharmaceutical composition or vaccine comprising one or more lipid nanoparticles as defined herein and an acceptable pharmaceutical carrier.

The invention also provides lipid nanoparticles, pharmaceutical compositions or vaccines as defined herein for use in human or veterinary medicine; in particular for the treatment of cancer or infectious diseases.

With specific reference now to the drawings, it is emphasized that the particulars shown are exemplary and for purposes of illustration only and discussion of various embodiments of the invention. They are presented in order to provide what is considered to be the most useful and easiest to describe the principles and concepts of the present invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The illustrations of the drawings make it easy for those of ordinary skill in the art to see how the several forms of the invention may be embodied in practice. [Simple description of the diagram]

Figure 1: Shows the results of three intravenous immunizations with E7 mRNA LNPs composed of SS-EC/DOPE/chol/DMG-PEG2000 at the indicated molar ratios. Figure 2: shows the results of three intravenous immunizations with E7 mRNA LNPs composed of SS-EC/DOPE/chol/DMG-PEG2000 at the indicated molar ratios. Figure 3: Shows four intravenous injections of 10 μg ADPGK mRNA encapsulated in low percentage PEG LNP (50/10/39.5/0.5 ionizable lipid/DOPE/cholesterol/PEG-lipid) or 50 μg ADPGK synthetic long peptide (SLP) results of internal administration. Figure 4: DOE-driven optimization of LNP composition for maximizing T cell responses. A, E7-specific T cells in blood after three immunizations (weekly intervals) with E7 mRNA LNPs from the DOE pool. B, E7-specific CD8 T cell responses to the function of %DMG-PEG2000 for 11 LNPs of the DOE pool. C, Mean ± SD of E7-specific T cells in blood after three immunizations (weekly intervals) with predicted optimal LNP34 and non-optimal LNP35 are shown. Statistics were assessed by One-Way ANOVA with Sidak's multiple comparison test. ***p<0.001. Figure 5: Optimized mRNA LNP vaccine induces qualitative T cell responses and potentiates antitumor efficacy. A, Kinetics of E7-specific CD8 ⁺ T cells in blood. B, IFN-γ in serum increases with repeated immunizations. C, IFN-γ and TNF-α production by CD8+E7-specific T cells in the spleen after three immunizations. D, Mean TC-1 tumor growth in LNP34-immunized mice. E, Survival rate of LNP34-immunized mice. F, TC-1 tumor infiltrating lymphocytes (TIL) after two immunizations with LNP34. G, E7-specificity of TILs. A, B, F, G show mean ± SD. C shows mean ± SEM. D, Statistics evaluated by Mantel-Cox log-rank test. F, G, Statistical data were evaluated by One-Way ANOVA and Tukey's multiple comparison test. **, p<0.01, ***p<0.001, ns=not significant. Figure 6: LNPs are taken up and activated by various (innate) immune cells. A. Luciferase activity in kidney, lung, heart, liver and spleen as a percentage of total luciferase activity. B. Uptake of LNPs in various cell types as measured by the difference in Cy5 MFI in LNP-injected mice versus TBS-buffer-injected mice. C. Luciferase activity in kidney, lung, heart, liver, and spleen as a percentage of total luciferase activity. Optimal LNP34 showed higher luciferase activity in spleen compared to non-optimal LNPs (LNP35). D. Cellular uptake of optimal LNP34 is higher compared to non-optimal LNP35. E. A transient increase in serum IFN-α and IP-10 cytokines was observed (6 hours vs. 24 hours after LNP administration). F. CD86 expression on cDC1 and cDC2 is weakly upregulated by non-optimal LNP35 but strongly upregulated by optimal LNP34. AD. Mean ± SD is shown. D, Statistics evaluated by One-Way ANOVA and Sidak's multiple comparison test. **, p<0.01, ***p<0.001, ns=not significant. Figure 7: E7-specific T cells in blood after two immunizations (weekly intervals) with alternative optimal (LNP59) and non-optimal (LNP53) DMG-PEG2000 LNPs, shown as mean ± SD. Statistical data were evaluated by One-Way ANOVA and Sidak's multiple comparison test. ***p<0.001.

As detailed above, the present invention provides LNPs comprising C14-PEG lipids (e.g., C14-PEG2000 lipids) present in relatively low amounts (e.g., less than about 1 mol%), for which we have surprisingly found that they are well suited for use in Immunological delivery of nucleic acids, especially mRNA.

In the context of the present invention, "immunological delivery of a nucleic acid molecule" means the delivery of a nucleic acid molecule into contact with a cell, the internalization and/or intracellular expression of which nucleic acid molecule leads to the induction of an immune response.

Therefore, in the first aspect, the present invention provides a lipid nanoparticle (LNP), which comprises: - ionizable lipids; - Phospholipids; - sterols; - PEG lipids; and - one or more mRNA molecules; Features: The PEG lipid is C14-PEG lipid; The LNP comprises less than about 1 mol% of the PEG lipid; The molar percentage of the ionizable lipid is between about 50-70 mol %; and The molar percentage of the sterol is about 25 mol% or more.

In another specific embodiment, the present invention provides a lipid nanoparticle (LNP), which comprises: - ionizable lipids; - Phospholipids; - sterols; - PEG lipids; and - one or more nucleic acid molecules; in particular mRNA molecules; Features: The PEG lipid is C14-PEG lipid; The LNP comprises less than about 1 mol% of the PEG lipid; The molar percentage of the ionizable lipid is between about 50-60 mol %; and The molar percentage of the sterol is about 30 mol% or more.

In further specific embodiments of the invention, the LNP comprises about 0.5 mol% to about 0.9 mol% of the PEG lipid.

Lipid nanoparticles (LNPs) are generally known as nanoscale particles composed of combinations of different lipids. Although many different types of lipids can be included in such LNPs, the LNPs of the invention are generally composed of a combination of ionizable lipids, phospholipids, sterols, and PEG lipids.

As used herein, the term "nanoparticle" refers to any particle having a diameter that renders the particle suitable, inter alia, for systemic administration, especially intravenous administration, of nucleic acids, typically having a particle size of less than 1000 nanometers (nm). The diameter is preferably less than 500 nm, even more preferably less than 200 nm, for example, between 50 and 200 nm; preferably between 80 and 160 nm.

In the context of the present invention, the term "PEG lipid" or "PEGylated lipid" means any suitable lipid modified with a PEG (polyethylene glycol) group. The PEG lipid of the present invention is characterized in that it is a C14-PEG lipid. These lipids contain a polyethylene glycol moiety, which defines the molecular weight of the lipid, and a fatty acid tail comprising 14 carbon atoms. In certain embodiments, the C14-PEG2000 lipid is dimyrisyl-based, i.e. has 2 C14 tails, for example selected from the group comprising: (dimyrisyl-based)- PEG2000 lipids, such as DMG-PEG2000 lipid (1,2-dimyristoyl- rac -glycerol-3-methoxypolyethylene glycol-2000) or 2-dimyristoyl- sn -glycerol-3- Phosphoethanolamine ethylene glycol-2000 (DMPE-PEG2000).

In the context of the present invention, the term "ionizable" (or cationic) in the context of a compound or lipid means the presence of any uncharged group in the compound or lipid which is capable of generating ions ( usually H ⁺ ions) and are therefore themselves positively charged. Alternatively, any uncharged groups in the compound or lipid can generate electrons and thus be negatively charged.

。 Any type of ionizable lipid may suitably be used in the context of the present invention. In particular, suitable ionizable lipids are ionizable amine-based lipids comprising two identical or different tails linked by SS bonds, each of which tails contain an ionizable amine, for example by means:

.

其中： RCOO選自包含以下所列：肉豆蔻醯基、α-D-生育酚琥珀醯基、亞油醯基及油醯基；且 X選自包含以下所列：

。 In certain embodiments, the ionizable lipid is a compound of formula (I):

wherein: RCOO is selected from the group consisting of myristyl, α-D-tocopheryl succinyl, linoleyl, and oleyl; and X is selected from the group consisting of:

.

。 Such ionizable lipids may specifically be represented by any of the following formulae:

.

The latter of the aforementioned lipids represents Coatsome SS-EC, as used in the Examples section.

， 例如以下所示

。 More specifically, the ionizable lipid is a lipid of formula (I), wherein RCOO is α-D-tocopheryl succinyl, and X is

, for example as shown below

.

-1-基)乙基)氮烷二基)雙(十二烷基-2-醇)(C12-200)；及二亞麻油基甲基-4-二甲基胺基丁酸酯(DLin-MC3-DMA)。

Other suitable ionizable lipids may be selected from 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2- Hydroxydodecyl)amino)ethyl)piperene

-1-yl)ethyl)azanediyl)bis(dodecyl-2-ol) (C12-200); and dilinoleylmethyl-4-dimethylaminobutyrate (DLin -MC3-DMA).

其中： RCOO選自包含以下所列：肉豆蔻醯基、α-D-生育酚琥珀醯基、亞油醯基及油醯基；且X選自包含以下所列：

- 磷脂； - 固醇； - PEG脂質；及 - 一種或多種核酸分子；特別是mRNA分子； 特徵在於： 該PEG脂質為C14-PEG脂質； 該LNP包含小於約1 mol%之該PEG脂質； 該可離子化的脂質之莫耳百分比為約50–70 mol%之間；及 該固醇之莫耳百分比為約25 mol%或25 mol%以上。 Therefore, in a specific embodiment, the present invention provides a lipid nanoparticle comprising: - a compound of formula (I):

wherein: RCOO is selected from the group consisting of myristyl, α-D-tocopheryl succinyl, linoleyl, and oleyl; and X is selected from the group consisting of:

- phospholipids; - sterols; - PEG lipids; and - one or more nucleic acid molecules; in particular mRNA molecules; characterized in that: the PEG lipids are C14-PEG lipids; the LNP comprises less than about 1 mol% of the PEG lipids; the The molar percentage of ionizable lipids is between about 50-70 mol %; and the molar percentage of sterols is about 25 mol % or more.

其中： RCOO選自包含以下所列：肉豆蔻醯基、α-D-生育酚琥珀醯基、亞油醯基及油醯基；且X選自包含以下所列：

- 磷脂； - 固醇； - PEG脂質；及 - 一種或多種核酸分子；特別是mRNA分子； 特徵在於： 該PEG脂質為C14-PEG脂質； 該LNP包含小於約1 mol%之該PEG脂質； 該可離子化的脂質之莫耳百分比為約50–60 mol%之間；及 該固醇之莫耳百分比為約30 mol%或30 mol%以上。 Therefore, in a specific embodiment, the present invention provides a lipid nanoparticle comprising: - a compound of formula (I):

wherein: RCOO is selected from the group consisting of myristyl, α-D-tocopheryl succinyl, linoleyl, and oleyl; and X is selected from the group consisting of:

- phospholipids; - sterols; - PEG lipids; and - one or more nucleic acid molecules; in particular mRNA molecules; characterized in that: the PEG lipids are C14-PEG lipids; the LNP comprises less than about 1 mol% of the PEG lipids; the The molar percentage of ionizable lipid is between about 50-60 mol %; and the molar percentage of the sterol is about 30 mol % or more.

。 In a preferred embodiment, the ionizable lipid is a lipid of formula (I), wherein RCOO is α-D-tocopheryl succinyl, and X is

.

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 phosphate groups . These two components are most often bound together by glycerol molecules, therefore, the phospholipid of the present invention is preferably a glycerol-phospholipid. In addition, phosphate groups are frequently modified with simple organic molecules such as choline (ie, yielding phosphorylcholine) or ethanolamine (ie, yielding phosphoethanolamine).

In the context of the present invention, suitable phospholipids may be selected from the list comprising: 1,2-dioleyl- sn -glycero-3-phosphoethanolamine (DOPE), 1,2-dioleyl- sn- Glyceryl-3-phosphocholine (DOPC), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dilinoleyl-sn-glycero-3- Phosphocholine (DLPC), 1,2-Dimyrisyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-bis Undecyl-sn-glycero-phosphocholine (DUPC), 1-palmityl-2-oleyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-deca Octenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1 - Hexadecyl-sn-glycero-3-phosphocholine (C 16 Lyso PC), 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl base-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanyl-sn-glycerol- 3-Phosphoethanolamine (ME 16.0 PE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinoleyl-sn-glycero-3-phosphoethanolamine, 1, 2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl - sn-glycero-3-phosphoethanolamine, 1,2-dioleyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and mixtures thereof.

In a particular embodiment of the invention, when the phospholipid is chosen as DSPC, the ionizable lipid may advantageously be DLin-MC3-DMA.

In a more specific embodiment, the phospholipid is selected from the group consisting of: 1,2-dioleyl- sn -glycero-3-phosphoethanolamine (DOPE), 1,2-dioleyl- sn- Glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and mixtures thereof; especially DOPE, DOPC and mixtures thereof.

其中： RCOO選自包含以下所列：肉豆蔻醯基、α-D-生育酚琥珀醯基、亞油醯基及油醯基；且 X選自包含以下所列：

； 特別是，式(I)之脂質，其中RCOO為α-D-生育酚琥珀醯基，且X為

- 磷脂，其選自DOPC及DOPE或其等之混合物； - 固醇； - 以小於約1 mol%存在之C14-PEG2000脂質；及 - 一種或多種核酸分子；特別是mRNA分子； 特徵在於： 該可離子化的脂質之莫耳百分比為約50–70 mol%之間；及 該固醇之莫耳百分比為約25 mol%或25 mol%以上。 Therefore, in a particular embodiment, the invention provides a lipid nanoparticle comprising: - an ionizable lipid of formula (I);

wherein: RCOO is selected from the group consisting of myristyl, α-D-tocopheryl succinyl, linoleyl, and oleyl; and X is selected from the group consisting of:

; especially, the lipid of formula (I), wherein RCOO is α-D-tocopheryl succinyl, and X is

- phospholipids selected from DOPC and DOPE or mixtures thereof; - sterols; - C14-PEG2000 lipids present in less than about 1 mol %; and - one or more nucleic acid molecules; in particular mRNA molecules; characterized in that: the The molar percentage of ionizable lipids is between about 50-70 mol %; and the molar percentage of sterols is about 25 mol % or more.

其中： RCOO選自包含以下所列：肉豆蔻醯基、α-D-生育酚琥珀醯基、亞油醯基及油醯基；且 X選自包含以下所列：

； 特別是，式(I)之脂質，其中RCOO為α-D-生育酚琥珀醯基且X為

- 磷脂，其選自DOPC及DOPE或其等之混合物； - 固醇； - 以小於約1 mol%存在之C14-PEG2000脂質；及 - 一種或多種核酸分子；特別是mRNA分子； 特徵在於： 該可離子化的脂質之莫耳百分比為約50-60 mol%之間；及 該固醇之莫耳百分比為約30 mol%或30 mol%以上。 Therefore, in a specific embodiment, the invention provides a lipid nanoparticle comprising: - an ionizable lipid of formula (I);

wherein: RCOO is selected from the group consisting of myristyl, α-D-tocopheryl succinyl, linoleyl, and oleyl; and X is selected from the group consisting of:

in particular, lipids of formula (I), wherein RCOO is α-D-tocopheryl succinyl and X is

- phospholipids selected from DOPC and DOPE or mixtures thereof; - sterols; - C14-PEG2000 lipids present in less than about 1 mol %; and - one or more nucleic acid molecules; in particular mRNA molecules; characterized in that: the The molar percentage of ionizable lipids is between about 50-60 mol%; and the molar percentage of sterols is about 30 mol% or more.

In the context of the present invention, the term "sterols", also known as sterols, is a subclass of steroids that occur naturally in plants, animals and fungi, or can be produced by some bacteria. In the context of the present invention, any suitable sterol may be used, for example selected from the list comprising: cholesterol, ergosterol, campesterol, oxysterol, antrocardine, streptosterol, nicarsterol , sitosterol and stigmasterol; preferably cholesterol.

其中： RCOO選自包含以下所列：肉豆蔻醯基、α-D-生育酚琥珀醯基、亞油醯基及油醯基；且 X選自包含以下所列：

； 特別是，式(I)之脂質，其中RCOO為α-D-生育酚琥珀醯基，且X為

- 磷脂，其選自DOPC及DOPE，或其等之混合物； - 膽固醇； - 以小於約1 mol%存在之C14-PEG2000脂質；及 - 一種或多種核酸分子；特別是mRNA分子 特徵在於： 該可離子化的脂質之莫耳百分比 為約50–70 mol%之間；及 該固醇之莫耳百分比 為約25 mol%或25 mol%以上。 Therefore, in a particular embodiment, the invention provides a lipid nanoparticle comprising: - an ionizable lipid of formula (I);

wherein: RCOO is selected from the group consisting of myristyl, α-D-tocopheryl succinyl, linoleyl, and oleyl; and X is selected from the group consisting of:

; especially, the lipid of formula (I), wherein RCOO is α-D-tocopheryl succinyl, and X is

- phospholipids selected from DOPC and DOPE, or mixtures thereof; - cholesterol; - C14-PEG2000 lipids present in less than about 1 mol %; and - one or more nucleic acid molecules; in particular mRNA molecules characterized in that: The molar percentage of ionized lipid is between about 50-70 mol %; and the molar percentage of the sterol is about 25 mol % or more.

其中： RCOO選自包含以下所列：肉豆蔻醯基、α-D-生育酚琥珀醯基、亞油醯基及油醯基；及 X選自包含以下所列：

； 特別是，式(I)之脂質，其中RCOO為α-D-生育酚琥珀醯基，且X為

- 磷脂，其選自DOPC及DOPE，或其等之混合物； - 膽固醇； - 以小於約1 mol%存在之C14-PEG2000脂質；及 - 一種或多種核酸分子；特別是mRNA分子 特徵在於： 該可離子化的脂質之莫耳百分比為約50-60 mol%之間；及 該固醇之莫耳百分比為約30 mol%或30 mol%以上。 Therefore, in a specific embodiment, the invention provides a lipid nanoparticle comprising: - an ionizable lipid of formula (I);

wherein: RCOO is selected from the group consisting of myristyl, α-D-tocopheryl succinyl, linoleyl, and oleyl; and X is selected from the group consisting of:

; especially, the lipid of formula (I), wherein RCOO is α-D-tocopheryl succinyl, and X is

- phospholipids selected from DOPC and DOPE, or mixtures thereof; - cholesterol; - C14-PEG2000 lipids present in less than about 1 mol %; and - one or more nucleic acid molecules; in particular mRNA molecules characterized in that: The molar percentage of ionized lipids is between about 50-60 mol%; and the molar percentage of sterols is about 30 mol% or more.

其中： RCOO選自包含以下所列：肉豆蔻醯基、α-D-生育酚琥珀醯基、亞油醯基及油醯基；及 X選自包含以下所列：

； 特別是，式(I)之脂質，其中RCOO為α-D-生育酚琥珀醯基，且X為

- 磷脂，其選自DOPC及DOPE,或其等之混合物； - 膽固醇； - 以小於約1 mol%存在之DMG-PEG2000脂質；及 - 一種或多種核酸分子；特別是mRNA分子； 特徵在於： 該可離子化的脂質之莫耳百分比為約50–70 mol%之間；及 該固醇之莫耳百分比為約25 mol%或25 mol%以上。 In a very specific embodiment of the invention, the lipid nanoparticles comprise: - an ionizable lipid of formula (I);

wherein: RCOO is selected from the group consisting of myristyl, α-D-tocopheryl succinyl, linoleyl, and oleyl; and X is selected from the group consisting of:

; especially, the lipid of formula (I), wherein RCOO is α-D-tocopheryl succinyl, and X is

- phospholipids selected from DOPC and DOPE, or mixtures thereof; - cholesterol; - DMG-PEG2000 lipids present in less than about 1 mol %; and - one or more nucleic acid molecules; in particular mRNA molecules; characterized in that: the The molar percentage of ionizable lipids is between about 50-70 mol %; and the molar percentage of sterols is about 25 mol % or more.

其中： RCOO選自包含以下所列：肉豆蔻醯基、α-D-生育酚琥珀醯基、亞油醯基及油醯基；及 X選自包含以下所列：

； 特別是，式(I)之脂質，其中RCOO為α-D-生育酚琥珀醯基，且X為

- 磷脂，其選自DOPC及DOPE，或其等之混合物； - 膽固醇； - 以小於約1 mol%存在之DMG-PEG2000脂質；及 - 一種或多種核酸分子；特別是mRNA分子； 特徵在於： 該可離子化的脂質之莫耳百分比為約50–60 mol%之間；及 該固醇之莫耳百分比為約30 mol%或30 mol%以上。 In a very specific embodiment of the invention, the lipid nanoparticles comprise: - an ionizable lipid of formula (I);

wherein: RCOO is selected from the group consisting of myristyl, α-D-tocopheryl succinyl, linoleyl, and oleyl; and X is selected from the group consisting of:

; especially, the lipid of formula (I), wherein RCOO is α-D-tocopheryl succinyl, and X is

- phospholipids selected from DOPC and DOPE, or mixtures thereof; - cholesterol; - DMG-PEG2000 lipids present in less than about 1 mol %; and - one or more nucleic acid molecules; in particular mRNA molecules; characterized in that: the The molar percentage of ionizable lipid is between about 50-60 mol %; and the molar percentage of the sterol is about 30 mol % or more.

We have also found that by using low concentrations of PEG lipids with relatively high concentrations of ionizable lipids (ie between 50-70 mol%; eg 50-65 mol% or 55-60 mol%) and relatively low Concentrations of phospholipids (i.e., less than about 10 mol%) are used to further increase the immunogenic effect of the LNPs of the present invention, therefore, for relatively high ionizable lipid:phospholipid ratios (i.e., 5:1-10:1; or LNPs between about 6:1 and about 11:1). Thus, a high concentration of ionizable lipids can be, for example, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%. mol%, about 58 mol%, about 59 mol%, about 60 mol%, about 61 mol%, about 62 mol%, about 63 mol%, about 64 mol%, about 65 mol%; about 66 mol%, about 67 mol%, about 68 mol%, about 69 mol%; about 70 mol%.

Therefore, in another specific embodiment, the molar percentage of the phospholipids is between about 5-15 mol% phospholipids; especially between about 5-10 mol%; more particularly less than about 10 mol%; for example About 9 mol%, about 8 mol%, about 7 mol%, about 6 mol%; about 5 mol%; preferably about 5 mol%.

In certain embodiments of the present invention, the LNP comprises ionizable lipids to phospholipids in a ratio of about 5:1 or greater; preferably about 6:1 or greater; more preferably 8:1 More than 1, preferably about 10:1; or about 6:1 to 11:1; most preferably about 11:1, such as about 10.76:1.

In yet another embodiment of the present invention, the molar percentage of the ionizable lipid is between about 50-70 mol%, such as between 50-65 mol%, especially between about 55-60 mol%.

Sterols are generally used as balancing lipids, and in some embodiments are in an amount of about 25 mol % or more, such as about 25 mol %, about 26 mol %, about 27 mol %, about 28 mol %, about 29 mol % mol%. Alternatively, the amount is about 30 mol% or more; for example about 30 mol%; about 31 mol%; about 32 mol%; about 33 mol%; about 34 mol%; about 35 mol%, .... In certain embodiments, the amount of cholesterol is between about 25 mol% and 29 mol%. Therefore, the concentration of sterols is usually weighed against the concentrations of other lipids to make up the complete 100%. Thus, the amount of sterols can be calculated as 100 mol% minus mol% phospholipids minus mol% PEG lipids minus mol% ionizable lipids.

Therefore, in specific embodiments of the present invention, one or more of the following applies: - the LNP comprises between about 50 mol% and 70 mol% of the ionizable lipid; or, between about 50-65 mol%; or 50-60 mol%; for example between about 55-60 mol%; - the LNP comprises between about 5 mol% and 15 mol% of the phospholipid; preferably less than about 10 mol%; optimally about 5 mol%; - the LNP comprises the PEG lipid between about 0.5 mol% to 0.9 mol%; Balanced with the amount of the sterol.

其中： RCOO選自包含以下所列：肉豆蔻醯基、α-D-生育酚琥珀醯基、亞油醯基及油醯基；及 X選自包含以下所列：

； 特別是，式(I)之脂質，其中RCOO為α-D-生育酚琥珀醯基，且X為

- 約5-15 mol%之磷脂，其選自DOPC及DOPE，或其等之混合物； - 膽固醇，用於平衡； - 約0.5-0.9 mol%之DMG-PEG2000脂質；及 - 一種或多種核酸分子，特別是mRNA分子。 Thus, in a very specific embodiment of the invention, the LNP comprises: - about 50-70 mol% of ionizable lipids of formula (I);

wherein: RCOO is selected from the group consisting of myristyl, α-D-tocopheryl succinyl, linoleyl, and oleyl; and X is selected from the group consisting of:

; especially, the lipid of formula (I), wherein RCOO is α-D-tocopheryl succinyl, and X is

- about 5-15 mol% of phospholipids selected from DOPC and DOPE, or mixtures thereof; - cholesterol, for equilibrium; - about 0.5-0.9 mol% of DMG-PEG2000 lipids; and - one or more nucleic acid molecules , especially mRNA molecules.

其中： RCOO選自包含以下所列：肉豆蔻醯基、α-D-生育酚琥珀醯基、亞油醯基及油醯基；及 X選自包含以下所列：

； 特別是，式(I)之脂質，其中RCOO為α-D-生育酚琥珀醯基，且X為

- 約5-15 mol%之磷脂，其選自DOPC及DOPE，或其等之混合物； - 膽固醇，用於平衡； - 約0.5-0.9 mol%之DMG-PEG2000脂質；及 - 一種或多種核酸分子，特別是mRNA分子。 Thus, in a very specific embodiment of the invention, the LNP comprises: - about 50-60 mol% of ionizable lipids of formula (I);

wherein: RCOO is selected from the group consisting of myristyl, α-D-tocopheryl succinyl, linoleyl, and oleyl; and X is selected from the group consisting of:

; especially, the lipid of formula (I), wherein RCOO is α-D-tocopheryl succinyl, and X is

- about 5-15 mol% of phospholipids selected from DOPC and DOPE, or mixtures thereof; - cholesterol, for equilibrium; - about 0.5-0.9 mol% of DMG-PEG2000 lipids; and - one or more nucleic acid molecules , especially mRNA molecules.

When mol % is used in the context of the present invention, it refers to the mol % of a particular component relative to the empty nanoparticles (ie without nucleic acid). This means that the mol% of components is calculated relative to the total amount of ionizable lipids, phospholipids, sterols and PEG lipids present in the LNP.

In a specific embodiment of the invention, the LNP comprises: - about 50-60 mol% of the ionizable lipid; - about 5-15 mol% of the phospholipid; - about 0.5-0.9 mol% of the DMG-PEG2000 lipid; and Balanced with the amount of the sterol.

In a very specific embodiment of the invention, the LNP comprises: - about 56.5 mol% of the ionizable lipid; - About 5.25 mol% of DOPE; - about 37.75 mol% cholesterol; and - About 0.5 mol% of DMG-PEG2000.

In another very specific embodiment of the invention, the LNP comprises: - about 50 mol% of the ionizable lipid; - About 10 mol% of DOPE; - about 39.5 mol% cholesterol; and - About 0.5 mol% of DMG-PEG2000.

In another very specific embodiment of the invention, the LNP comprises: - about 50 mol% of the ionizable lipid; - About 11 mol% of DOPE; - about 38.5 mol% cholesterol; and - About 0.5 mol% of DMG-PEG2000.

In another very specific embodiment of the invention, the LNP comprises: - about 50 mol% of the ionizable lipid; - About 7.76 mol% DOPE; - about 41.66 mol% cholesterol; and - About 0.58 mol% of DMG-PEG2000.

In another very specific embodiment of the invention, the LNP comprises: - about 65 mol% of the ionizable lipid; - About 9.5 mol% DOPE; - about 25 mol% cholesterol; and - About 0.5 mol% of DMG-PEG2000.

其中： RCOO為α-D-生育酚琥珀醯基，且X為

- 約10 mol%之磷脂，其選自DOPC及DOPE，或其等之混合物； - 約39.5 mol%之膽固醇； - 約0.5 mol%之DMG-PEG2000脂質；及 - 一種或多種核酸分子，特別是mRNA分子。 Thus, in a very specific embodiment of the invention, the LNP comprises: - about 50 mol% of ionizable lipids of formula (I);

where: RCOO is α-D-tocopheryl succinyl, and X is

- about 10 mol% of phospholipids selected from DOPC and DOPE, or mixtures thereof; - about 39.5 mol% of cholesterol; - about 0.5 mol% of DMG-PEG2000 lipids; and - one or more nucleic acid molecules, in particular mRNA molecule.

其中： RCOO為α-D-生育酚琥珀醯基，且X為

- 約5.25 mol%之磷脂，其選自DOPC及DOPE，或其等之混合物； - 約37.75 mol%之膽固醇； - 約0.5 mol%之DMG-PEG2000脂質；及 - 一種或多種核酸分子，特別是mRNA分子。 In another very specific embodiment of the invention, the LNP comprises: - about 56.5 mol% of ionizable lipids of formula (I);

where: RCOO is α-D-tocopheryl succinyl, and X is

- about 5.25 mol% of phospholipids selected from DOPC and DOPE, or mixtures thereof; - about 37.75 mol% of cholesterol; - about 0.5 mol% of DMG-PEG2000 lipids; and - one or more nucleic acid molecules, in particular mRNA molecule.

其中： RCOO為α-D-生育酚琥珀醯基，且X為

- 約9.5 mol%之磷脂，其選自DOPC及DOPE，或其等之混合物； - 約25 mol%之膽固醇； - 約0.5 mol%之DMG-PEG2000脂質；及 - 一種或多種核酸分子，特別是mRNA分子。 In another very specific embodiment of the invention, the LNP comprises: - about 65 mol% of ionizable lipids of formula (I);

where: RCOO is α-D-tocopheryl succinyl, and X is

- about 9.5 mol% of phospholipids selected from DOPC and DOPE, or mixtures thereof; - about 25 mol% of cholesterol; - about 0.5 mol% of DMG-PEG2000 lipids; and - one or more nucleic acid molecules, in particular mRNA molecules.

其中： RCOO為α-D-生育酚琥珀醯基，且X為

- 約11 mol%之磷脂，其選自DOPC及DOPE，或其等之混合物； - 約38.5 mol%之膽固醇； - 約0.5 mol%之DMG-PEG2000脂質；及 - 一種或多種核酸分子，特別是mRNA分子。 In another very specific embodiment of the invention, the LNP comprises: - about 50 mol% of ionizable lipids of formula (I);

where: RCOO is α-D-tocopheryl succinyl, and X is

- about 11 mol% of phospholipids selected from DOPC and DOPE, or mixtures thereof; - about 38.5 mol% of cholesterol; - about 0.5 mol% of DMG-PEG2000 lipids; and - one or more nucleic acid molecules, in particular mRNA molecule.

其中： RCOO為α-D-生育酚琥珀醯基，且X為

- 約7.76 mol%之磷脂，其選自 DOPC及DOPE，或其等之混合物； - 約41.66 mol%之膽固醇； - 約0.58 mol%之DMG-PEG2000脂質；及 - 一種或多種核酸分子，特別是mRNA分子。 In another very specific embodiment of the invention, the LNP comprises: - about 50 mol% of ionizable lipids of formula (I);

where: RCOO is α-D-tocopheryl succinyl, and X is

- about 7.76 mol% of phospholipids selected from DOPC and DOPE, or mixtures thereof; - about 41.66 mol% of cholesterol; - about 0.58 mol% of DMG-PEG2000 lipids; and - one or more nucleic acid molecules, in particular mRNA molecule.

The compositions of other particularly suitable LNPs in the context of the present invention are shown in Table 1. Table 1 : Composition of suitable LNPs No. Ionizable lipid (mol%) Phospholipids (mol%) Cholesterol (mol%) C14-PEG2000 lipid (mol%) 1 50 10,0 39,5 0,5 2 50 8,3 41,2 0,5 3 50 7,1 42,4 0,5 4 50 6,3 43,3 0,5 5 50 5,6 43,9 0,5 6 50 5,0 44,5 0,5 7 50 10,0 39,3 0,7 8 50 8,3 41,0 0,7 9 50 7,1 42,2 0,7 10 50 6,3 43,1 0,7 11 50 5,6 43,7 0,7 12 50 5,0 44,3 0,7 13 50 10,0 39,1 0,9 14 50 8,3 40,8 0,9 15 50 7,1 42,0 0,9 16 50 6,3 42,9 0,9 17 50 5,6 43,5 0,9 18 50 5,0 44,1 0,9 19 55 11,0 33,5 0,5 20 55 9,2 35,3 0,5 twenty one 55 7,9 36,6 0,5 twenty two 55 6,9 37,6 0,5 twenty three 55 6,1 38,4 0,5 twenty four 55 5,5 39,0 0,5 25 55 11,0 33,3 0,7 26 55 9,2 35,1 0,7 27 55 7,9 36,4 0,7 28 55 6,9 37,4 0,7 29 55 6,1 38,2 0,7 30 55 5,5 38,8 0,7 31 55 11,0 33,1 0,9 32 55 9,2 34,9 0,9 33 55 7,9 36,2 0,9 34 55 6,9 37,2 0,9 35 55 6,1 38,0 0,9 36 55 5,5 38,6 0,9 37 60 12,0 27,5 0,5 38 60 10,0 29,5 0,5 39 60 8,6 30,9 0,5 40 60 7,5 32,0 0,5 41 60 6,7 32,8 0,5 42 60 6,0 33,5 0,5 43 60 12,0 27,3 0,7 44 60 10,0 29,3 0,7 45 60 8,6 30,7 0,7 46 60 7,5 31,8 0,7 47 60 6,7 32,6 0,7 48 60 6,0 33,3 0,7 49 60 12,0 27,1 0,9 50 60 10,0 29,1 0,9 51 60 8,6 30,5 0,9 52 60 7,5 31,6 0,9 53 60 6,7 32,4 0,9 54 60 6,0 33,1 0,9 55 60 12,0 27,5 0,5 56 60 10,0 29,5 0,5 57 60 8,6 30,9 0,5 58 60 7,5 32,0 0,5 59 60 6,7 32,8 0,5 60 60 6,0 33,5 0,5 61 60 12,0 27,3 0,7 62 60 10,0 29,3 0,7 63 60 8,6 30,7 0,7 64 60 7,5 31,8 0,7 65 60 6,7 32,6 0,7 66 60 6,0 33,3 0,7 67 60 12,0 27,1 0,9 68 60 10,0 29,1 0,9 69 60 8,6 30,5 0,9 70 60 7,5 31,6 0,9 71 60 6,7 32,4 0,9 72 60 6,0 33,1 0,9 73 65 10,0 24.5 0,5 74 65 8,3 26.2 0,5 75 65 7,1 27.4 0,5 76 65 6,3 28.2 0,5 77 65 5,6 28.9 0,5 78 65 5,0 29.5 0,5 79 65 10,0 24.3 0.7 80 65 8,3 26.0 0.7 81 65 7,1 27.2 0.7 82 65 6,3 28.0 0.7 83 65 5,6 28.7 0.7 85 65 5,0 29.3 0.7 86 65 10,0 24.1 0.9 87 65 8,3 25.8 0.9 88 65 7,1 27.0 0.9 89 65 6,3 27.8 0.9 90 65 5,6 28.5 0.9 91 65 5,0 29.1 0.9

Other particularly suitable LNPs are characterized by a ratio of ionizable lipid/phospholipid/sterol/C14-PEG2000 lipid of: 50/10/39.5/0.5 56.5/5/38/0.5 50/11/38.5/0.5 50/7.76/41.66/0.58 65/9.5/25/0.5

The inventors of the present case have found that the LNPs of the present invention are particularly suitable for the immunological delivery of nucleic acids. Accordingly, the present invention provides LNPs comprising one or more nucleic acid molecules, such as DNA or RNA, more particularly mRNA.

The amount of nucleic acid in the LNP is usually expressed as a molar ratio, ie, the ratio of cationic lipid (ionizable lipid) to RNA phosphate. In the context of the present invention, the molar ratio of LNP is between about 4:1 and 16:1.

Alternatively, the amount of nucleic acid in the LNP can be expressed as the N/P ratio, ie the ratio of nitrogen atoms in the ionizable lipid to phosphate groups in the nucleic acid. In the context of the present invention, the N/P ratio of LNP is between about 4:1 and 16:1.

In the context of the present invention, "nucleic acid" is deoxyribonucleic acid (DNA) or preferably ribonucleic acid (RNA), more preferably mRNA. According to the present invention, nucleic acids include genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. The nucleic acids according to the invention may be single- or double-stranded and in the form of molecules that are linear or covalently closed to form circles. Nucleic acids can be used, for example, to introduce (ie, transfect) cells in the form of RNA prepared by in vitro transcription from a DNA template. Furthermore, the RNA can be modified by stabilizing the sequence, capping and/or polyadenylation prior to use.

In the context of the present invention, the term "RNA" relates to molecules comprising ribonucleotide residues and preferably consisting entirely or essentially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of β-D-ribofuranosyl. The term includes double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA), substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differ from naturally occurring RNA by Addition, deletion, substitution and/or change of one or more nucleotides. Such alterations may include the addition of non-nucleotide substances, eg, to the ends of the RNA or to the interior, eg, at one or more nucleotides of the RNA. Nucleotides in an RNA molecule may also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. Such altered RNAs can be referred to as analogs. A nucleic acid can be contained in a vector. As used herein, the term "vector" includes any vector known to the skilled person, including plastid vectors, cohesive plastid vectors, bacteriophage vectors (such as lambda phage), viral vectors (such as adenovirus or baculoviral Vectors) or artificial chromosome vectors (such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes, or analogs of naturally occurring RNAs.

According to the present invention, the term "RNA" includes and preferably relates to "mRNA", which means "messenger RNA" and to "transcripts" which can be produced using DNA as a template and encode peptides or proteins. An mRNA generally comprises a 5' untranslated region (5'-UTR), a protein or peptide coding region, and a 3' untranslated region (3'-UTR). mRNA has a finite half-life in cells and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In a specific embodiment of the present invention, the RNA is obtained by in vitro transcription or chemical synthesis. In vitro transcription methods are known to the skilled person. For example, there are many in vitro transcription kits commercially available.

In certain embodiments of the invention, the mRNA molecule is an mRNA molecule encoding an immunomodulatory protein.

In the context of the present invention, the term "mRNA molecule encoding an immunomodulatory protein" refers to an mRNA molecule encoding a protein which modifies the functionality of antigen presenting cells, more particularly dendritic cells. Such molecules may be selected from the list comprising: CD40L, CD70, caTLR4, IL-12p70, L-selectin, CCR7 and/or 4-1BBL, ICOSL, OX40L, IL-21; more particularly CD40L, CD70 and caTLR4 one or more of. A preferred combination of immunostimulatory factors used in the method of the present invention is CD40L and caTLR4 (ie "DiMix"). In another preferred embodiment, a combination of CD40L, CD70 and caTLR4 immunostimulatory molecules, also referred to herein as "TriMix", is used.

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

According to the present invention, the term "antigen" includes any molecule, preferably a peptide or protein, comprising at least one epitope that will elicit an immune response and/or against which an immune response is directed; thus, the term "antigen" also includes the term "antigen" derived from an antigen. minimal epitope. A "minimal epitope" as defined herein means the smallest structure capable of eliciting an immune response. Preferably, an antigen in the context of the present invention is a molecule which, optionally upon treatment, induces an immune response, preferably specific for the antigen or a cell expressing the antigen. In particular, "antigen" refers to a molecule that is selectively presented by MHC molecules after processing and specifically reacts with T lymphocytes (T cells).

In certain embodiments, the antigen is a target-specific antigen, which may be a tumor antigen or a bacterial, viral or fungal antigen. The target-specific antigen can be derived from one of the following: total mRNA isolated from target cells, one or more specific target mRNA molecules, protein lysates of target cells, specific proteins from target cells, or synthetic target-specific antigens. peptides or proteins, and synthetic mRNA or DNA encoding target-specific antigens or their derived peptides.

To avoid any misunderstanding, the LNPs of the present invention may comprise a single mRNA molecule, or they may comprise multiple mRNA molecules, such as one or more mRNA molecules encoding immunomodulatory proteins and/or encoding antigen-specific and/or disease-specific proteins combination of one or more mRNA molecules.

In very specific embodiments, the mRNA molecule encoding an immunomodulatory molecule may be combined with one or more mRNA molecules encoding an antigen-specific protein and/or a disease-specific protein. For example, the LNP of the present invention may comprise mRNA molecules (such as Dimix or Trimix) encoding immunostimulatory molecules CD40L, CD70 and/or caTLR4; combined with one or more mRNA molecules encoding antigen-specific proteins and/or disease-specific proteins. Thus, in specific embodiments, the LNPs of the invention comprise mRNA molecules encoding CD40L, CD70 and/or caTLR4; in combination with one or more mRNA molecules encoding antigen-specific and/or disease-specific proteins.

In another aspect, the invention provides a pharmaceutical composition comprising one or more LNPs as defined herein. Such pharmaceutical compositions are particularly suitable as vaccines. Accordingly, the invention also provides a vaccine comprising one or more LNPs according to the invention.

In the context of the present invention, the term "vaccine" as used herein means any preparation intended to provide adaptive immunity (antibody and/or T cell response) against a disease. To this end, vaccines referred to herein comprise at least one mRNA molecule encoding an antigen, wherein the antigen elicits an adaptive immune response. This antigen may be present in a weakened or killed form of the microorganism, protein or peptide or nucleic acid encoding the antigen. In the context of the present invention, an antigen means a protein or peptide that is recognized as foreign by the host's immune system, thereby stimulating the production of antibodies against it, with the aim of combating such antigens. Vaccines can be prophylactic (eg, to prevent or lessen the effects of future infection with any natural or "wild" pathogen) or therapeutic (eg, to actively treat or alleviate the symptoms of an ongoing disease). The administration of vaccines is called vaccination.

The vaccines of the invention are useful for inducing an immune response, particularly an immune response against a disease-associated antigen or a cell expressing a disease-associated antigen, eg, against cancer. Thus, vaccines are useful in the prophylactic and/or therapeutic treatment of diseases involving disease-associated antigens or cells expressing disease-associated antigens, such as cancer. Preferably, the immune response is a T cell response. In a specific embodiment, the disease-associated antigen is a tumor antigen. The antigen encoded by the RNA contained in the nanoparticles described herein is preferably a disease-associated antigen, or elicits an immune response against the disease-associated antigen or cells expressing the disease-associated antigen.

The LNPs and vaccines of the invention are particularly intended for intravenous administration, ie infusion of a liquid substance directly into a vein. The intravenous route is the fastest method of delivering fluids and drugs throughout the body (ie, systemically). Accordingly, the present invention provides intravenous vaccines, and uses of the disclosed vaccines and LNPs for intravenous administration. Thus, the vaccines and LNPs of the invention can be administered intravenously. The present invention also provides the use of the vaccine and LNP according to the invention; wherein the vaccine is administered intravenously.

In particular, it was found that the immunogenicity of the LNPs of the invention increases after multiple immunizations. Thus, in certain embodiments, a LNP as defined herein is used for vaccination purposes, wherein the LNP is administered at least two times, preferably at least three times within a certain interval.

The invention also provides LNPs, pharmaceutical compositions and vaccines according to the invention for use in human or veterinary medicine. Use of the LNPs, pharmaceutical compositions and vaccines of the invention in human or veterinary medicine is also contemplated. Finally, the present invention provides a method for the prevention and treatment of human and veterinary disorders by administering the LNPs, pharmaceutical compositions and vaccines according to the present invention to subjects in need thereof.

The invention further provides the use of the LNP, pharmaceutical composition or vaccine according to the invention for the immunological delivery of the one or more nucleic acid molecules. Thus, the LNPs, pharmaceutical compositions and vaccines of the present invention are very useful in the treatment of many human and veterinary diseases. Accordingly, the present invention provides LNPs, pharmaceutical compositions and vaccines of the present invention for use in the treatment of cancer or infectious diseases.

The lipid nanoparticles of the present invention can be prepared according to the protocol indicated in the Examples section. More generally, LNPs can be prepared using methods including: - preparation of a first alcoholic composition comprising the ionizable lipid, the phospholipid, the sterol, the PEG lipid and a suitable alcoholic solvent; - preparing a second aqueous composition comprising the one or more nucleic acids and an aqueous solvent; - mixing the first and second compositions in a microfluidic mixing device.

In more detail, lipid components are combined in an alcoholic vehicle such as ethanol at suitable concentrations. For this, an aqueous composition containing nucleic acids is added, which is then loaded into a microfluidic mixing device.

The goal of microfluidic mixing is to achieve thorough and rapid mixing of multiple samples (ie, lipid and nucleic acid phases) in a microdevice. This sample mixing is usually achieved by enhancing the diffusion effect between the different species streams. For this, several microfluidic mixing devices can be used, as described eg with reference to Lee et al. (2011). A particularly suitable microfluidic mixing device according to the invention is the NanoAssemblr from Precision Nanosystems.

Other techniques suitable for preparing the LNP of the present invention include dispersing the components in a suitable dispersion medium, such as water solvent and alcohol solvent, and applying one or more of the following methods: ethanol dilution method, simple hydration method, ultrasonic treatment , heating, vortexing, ether injection, French press, bile acid method, Ca ²⁺ fusion method, freeze-thaw method, reversed-phase evaporation method, T-joint mixing, microfluidic hydrodynamic focusing, interleaving human font blending etc. [Example]

for example 1-3 materials and methods

[mouse] Female C57BL/6 mice were purchased from Charles River Laboratories (France) and housed in individually ventilated cages with standard bedding and cage reinforcement. Animals were housed and treated according to the institutional (Vrije Universiteit Brussel) and European Union guidelines for animal experimentation. Mice had ad libitum access to food and water. Experiments were initiated when mice were 6 to 10 weeks old. Mice received an intravenous injection of LNP containing 10 µg mRNA (in a volume of 200 µL) via the tail vein. Control mice were injected with 200 µl TBS (Tris-buffered saline) at the same time intervals. The body weight of the mice was monitored every 2 days.

In the case of vaccination with ADPGK synthetic long peptide (SLP), a vaccine containing 50 µg ADPGK SLP (GIPVHLELASMTNMELMSSIVHQQVFPT, (SEQ ID N° 3) Genscript), 50 µg anti-CD40 Mab (Clone FJK45, BioXCell) and 100 µg pIC HMW (InvivoGen) in 200 µl PBS was injected intraperitoneally into mice at the same time intervals.

[mRNA synthesis and purification] Capped, non-nucleoside-modified E7 and ADPGK mRNAs were prepared by in vitro transcription (IVT) from eTheRNA plasmid pEtherna according to the protocol described in WO2015071295. Sequences encoding HPV16-E7 or ADPGK proteins were cloned in frame between the signal sequence and the transmembrane and cytoplasmic regions of human DC-LAMP. This chimeric gene was cloned into pEtherna plastids, which are enriched at the 5' end for translation enhancers and at the 3' end for RNA stabilizing sequences. After IVT, dsRNA was removed by cellulose purification. Cellulose powder was purchased from Sigma and washed in 1xSTE (NaCl-Tris-EDTA) buffer containing 16% ethanol. IVT mRNA (in 1xSTE buffer containing 16% ethanol) was added to washed cellulose pellets and shaken at room temperature for 20 minutes. This solution was then passed through a vacuum filter (Corning). Lysates contained ssRNA fractions and were used for all experiments. mRNA quality was monitored by capillary gel electrophoresis (Agilent, Belgium).

[Generation of mRNA lipid-based nanoparticles] Lipid-based nanoparticles were produced by microfluidically mixing mRNA solutions and lipid solutions in sodium acetate buffer (100 mM, pH 4) at a volume ratio of 2:1 using a NanoAssemblr Benchtop (Precision Nanosystems). The lipid solution contained a mixture of CoatsomeSS-EC (NOF corporation), DOPE (Avanti), cholesterol (Sigma) and DMG-PEG2000 (C14 lipid) (Sunbright GM-020, NOF corporation). The 4 lipids were mixed in different molar ratios. Using Slide-a-lyzer dialysis cassette (20K MWCO, 3 mL, ThermoFisher), the LNP was dialyzed against TBS (TBS volume is 10000 times of LNP volume).

[Flow Cytometry] About 6 days after immunization, blood was collected from the treated and control mice. According to the manufacturer's instructions (MBL International), erythrocytes were lysed and the remaining leukocytes were labeled with APC-labeled E7 _(RAHYNIVTF) -tetramer (SEQ ID N ° 1) or ADPGK (ASMTNMELM)-tetramer (SEQ ID N ° 2) Dyeing. Wash off excess tetramer. Thereafter, the antibody cocktail for surface molecules (listed in Table 2) was added to the cells and incubated at 4°C for 30 minutes. Data were acquired on an LSR Fortessa cytometer and analyzed with Flow Jo software. result

Example 1. Mice received three intravenous immunization injections of E7 mRNA LNP composed of SS-EC/DOPE/chol/DMG-PEG2000 at specified molar ratios. After each immunization, the percentage of E7-specific CD8 T cells in blood induced by the corresponding mRNA LNP composition was assessed by flow cytometry. As evident from Figure 1, mRNA LNP formulated with 0.5 mol% DMG-PEG2000 induced a higher E7-specific CD8 T cell response compared to mRNA LNP formulated with 1 mol% DMG-PEG2000.

Example 2. Mice received three intravenous immunization injections of E7 mRNA LNP composed of SS-EC/DOPE/chol/DMG-PEG2000 at specified molar ratios. After each immunization, the percentage of E7-specific CD8 T cells in blood induced by the corresponding mRNA LNP composition was assessed by flow cytometry. It is evident from Figure 2 that mRNA LNP formulated with 0.5 mol% DMG-PEG2000 induced a higher E7-specific CD8 T cell response compared to mRNA LNP formulated with 2 mol% DMG-PEG2000, and three immunizations The effect is more obvious after injection.

Example 3. Mice received four intravenous administrations of 10 μg ADPGK mRNA packaged in low percentage PEG LNP (50/10/39.5/0.5 ionizable lipid/DOPE/cholesterol/PEG-lipid), or 50 μg ADPGK Synthetic long peptides (SLP). Six days after the fourth immunization injection, the percentage of ADPGK-specific CD8 ⁺ T cells in the blood was determined. Compared with SLP, mRNA LNP formulated with 0.5 mol% DMG-PEG2000 was superior in inducing antigen-specific immune response (Figure 3).

for example 4-8 materials and methods

Animals All mouse experiments were approved by the Utrecht Animal Welfare Body at UMC Utrecht or the Animal Ethics Committee of Ghent University. Animal care was performed according to established guidelines. All mice had unlimited access to water and standard laboratory animal chow. Female C57Bl/6J mice were obtained from Charles River Laboratories, Inc. (Germany/France).

mRNA Synthesis and Purification Codon- optimized E7, TriMix and luciferase mRNAs were prepared from eTheRNA by in vitro transcription (IVT) from eTheRNA plastids. No nucleotide modifications were used. The E7 mRNA used in DOE was ARCA capped. All subsequent experiments were performed using CleanCapped mRNA. After IVT, dsRNA was removed by cellulose purification. mRNA quality was monitored by capillary gel electrophoresis (Agilent, Belgium). Cleancap® Cy5-labeled Fluc mRNA (5-methoxyuridine modified and silica purified) was purchased from TriLink Biotechnologies.

LNP Production and Characterization For biodistribution and cellular uptake studies, LNPs were loaded with a 1:1 mixture of firefly luciferase (Fluc) encoding mRNA (eTheRNA Immunotherapies NV) and Cleancap® Cy5-labeled Fluc mRNA (TriLink Biotechnologies) . For DoE immunogenicity studies, LNPs were loaded with E7 mRNA. All other studies were performed using a 3:1:1:1 ratio mixture of E7, mouse CD40L, mouse CD70 and constitutively active TLR4 mRNA. mRNA was diluted in 100 mM sodium acetate buffer (pH 4), and lipids were dissolved and diluted in ethanol. The mRNA and lipid solutions were mixed using the NanoAssemblr Benchtop microfluidic mixing system (Precision Nanosystems), and then dialyzed against Tris-buffered saline (TBS, 20 mM Tris, 0.9% NaCl, pH 7.4) overnight. Amicon ultracentrifugal filters (10 kD) were used to concentrate LNP. Size, polydispersity index and zeta potential were measured with a Zetasizer Nano (Malvern). mRNA encapsulation efficiency was determined via RiboGreen assay (ThermoFisher). The compositions of all LNPs are summarized in Table 2.

T Cell Responses Mice were immunized intravenously via the tail vein with selected LNPs containing 10 µg mRNA at weekly intervals. Blood for flow cytometry staining was collected 5 to 7 days after immunization. Following lysis of erythrocytes, cells are incubated with FcR blockers and viability stains. After incubation and washing, APC-tagged E7 _(RAHYNIVTF) -tetramer was added and incubated at room temperature for 30 minutes. Excess tetramers were washed away, and an antibody cocktail for the surface molecules CD3 and CD8 was added to the cells and incubated at 4°C for 30 minutes. Samples were collected on a 3-laser AtuneNxt flow cytometer or a 4-laser BD LSRFortessa flow cytometer.

Intracellular cytokine production in the spleen was measured 7 days after the third immunization. Single cell suspensions of splenocytes were prepared by crushing the spleen, lysing red blood cells, and filtering the sample through a 40 µM cell strainer. 200.000 cells/well/sample were plated in duplicate in 96-well plates. Cells were stimulated by adding 4 µg of E7 peptide (Genscript) before incubating at 37°C. One hour after peptide stimulation, GolgiPlug (BD Cytofix/Cytoperm Kit (BD Biosciences)) was added. Cells were incubated for another 4 hours. Afterwards, cells were incubated with FcR blockers and viability stains. After incubation and washing, APC-tagged E7 _(RAHYNIVTF) -tetramer was added and incubated at room temperature for 30 minutes. Excess dextramers were washed away and antibody cocktails for the surface molecules CD3 and CD8 were 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, cells were stained for IFN-γ and TNF-α. Samples were collected on a 4-laser BD LSRFortessa flow cytometer. Analysis was performed using FlowJo software.

Inflammatory Cytokines Blood samples were collected in tubes with gel coagulation factors (Sarstedt) 6 hours after each immunization injection (days 0, 7, 14 and 50). The clotted blood samples were centrifuged at 10.000 g for 5 minutes to obtain serum. Serum samples were stored at -80°C until analysis. ProcartaPlex multiplex assay (ThermoFisher) was used to determine the concentrations of IFN-α, IFN-γ, IP-10. Serum samples were diluted 3-fold in assay buffer and incubated with fluorescently labeled beads for 120 minutes. Proceed to further steps according to the protocol. Samples were collected on a MagPix instrument (Luminex). Data were analyzed using ProcartaPlex Analyst software.

TC-1 Tumor Assay TC-1 cells were obtained from Leiden University Medical Center. 50 μL of PBS containing 500,000 TC-1 cells was subcutaneously injected into the right abdomen of the mouse. Tumor measurements were taken using calipers. Tumor volume was calculated as (minimum ^diameter2 x maximum diameter)/2. Ant-PD-1 and isotype control antibodies were freshly diluted in PBS to a concentration of 200 µg in 200 µL per mouse and injected intraperitoneally. Mice received anti-PD-1 antibody (monotherapy or in combination with mRNA LNP immunization) or isotype control (combination with LNP immunization). Antibody was injected every 3 to 4 days starting 3 days after the first mRNA LNP immunization injection and ending 2 weeks after the last LNP injection. For analysis of tumor-infiltrating lymphocytes, tumors were isolated 3 days after the second mRNA LNP immunization injection and placed in 24-well flat dishes filled with MACS tissue storage buffer (Miltenyi Biotec). Tumors were minced and incubated in digestion buffer for 1 hour with regular shaking. Afterwards, erythrocytes were lysed and all samples were filtered through a 70 µM cell strainer. Lymphocytes were enriched by ficoll-paque density gradient purification before staining. First, cells are incubated with an FcR blocker and a viability stain. After incubation and washing, APC-tagged E7 _(RAHYNIVTF) -tetramer was added and incubated at room temperature for 30 minutes. Excess tetramers were washed away and the antibody cocktail for the surface molecules CD45 and CD8 was added to the cells and incubated at 4°C for 30 min. Samples were collected on a 3-laser AtuneNxt flow cytometer or a 4-laser BD LSRFortessa flow cytometer. Analysis was performed using FlowJo software.

Biodistribution and cellular uptake In selected LNP formulations, mice were injected intravenously with 10 µg of mRNA via the tail vein. After 4 h, mice were anesthetized with 250 µL pentobarbital (6 mg/mL). Blood samples were collected in tubes with gel coagulation factors (Sarstedt). Subsequently, the chest cavity was opened, the portal vein was cut off, and the mouse was perfused with 7 mL of PBS through the right ventricle. Organs were removed and snap frozen in liquid nitrogen. For liver and spleen tissues, a section of the organ was preserved in ice-cold PBS for flow cytometry analysis.

Cellular uptake Liver and spleen tissues were plated in Petri dishes with RPMI 1640 medium containing 1 mg/mL Collagenase A (Roche) or 20 µg/mL Liberase TM (Roche), respectively, and 10 µg/mL DNAse I , Class II (Roche). Mince the tissue using a scalpel blade and incubate at 37°C for 30 min. Subsequently, the tissue suspension was passed through a 100 µm nylon cell strainer. Centrifuge the liver suspension at 70 x g for 3 min to remove parenchymal cells. Centrifuge the supernatant and spleen suspension at 500 x g for 7 min to pellet the cells. Red blood cells were lysed in ACK buffer (Gibco) for 5 min, deactivated with PBS, and passed through a 100 µm cell strainer. 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). Seed 3 x 10 ⁵ (liver) or 6 x 10 ⁵ (spleen) living cells in a 96-well plate, pellet at 500 xg for 5 minutes, and resuspend in 50% Brilliant Stain Buffer (BD Biosciences) and 2 µg/ mL of TruStain FcX (BioLegend) in 2% BSA in PBS (2% PBSA). Cells were incubated on ice for 10 minutes and mixed 1:1 in 2% PBSA containing the appropriate antibody mix (three in total) in duplicate. Cells were incubated on a shaker at room temperature for 15 minutes, washed twice with 2% PBSA, and then resuspended in 2% PBSA containing 0.25 µg/mL 7-AAD Viability Stain (BioLegend). Samples were collected on a 4-laser BD LSRFortessa flow cytometer. Analyzed using FlowJo software.

Systemic Distribution Approximately 50-100 mg of each tissue was dissected, weighed and placed in 2 mL microtubes with an approximately 5 mm layer of 1.4 mm ceramic beads (Qiagen). For each milligram of tissue, 3 µL of cold Cell Culture Lysis Reagent (Promega) was added and homogenized using a Mini-BeadBeater-8 (BioSpec) at full speed for 60 seconds at 4°C. Store the homogenate at -80 °C, thaw, centrifuge at 10.000 x g for 10 min at 4 °C to remove beads and debris, and store the supernatant again at -80 °C. Aliquot 10 microliters of each lysate in a white 96-well plate in duplicate. Using a SpectraMax iD3 plate reader equipped with an injector, 50 µL of Luciferase Assay Reagent (Promega) was dispensed into each well while mixing, followed by a 2 second delay and a 10 second recording of luciferase emission. Luciferase activity was normalized to background signal obtained from TBS-injected mouse organ lysates.

Immune Cell Activation Mice received intravenous injection of selected LNPs containing 5 µg mRNA via the tail vein. Spleens were harvested 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, samples were incubated with Fc blockers and viability stains. After incubation and washing, cells were stained for cell lineage and activation markers. Samples were collected on a 3-laser AtuneNxt flow cytometer. Analysis was performed using FlowJo software.

Example 4 - DOE Driven Optimization of LNP Composition for Maximal T Cell Responses A LNP library was generated by combining the commercially available ionizable lipid Coatsome SS-EC with cholesterol, DOPE and PEGylated lipids . DOPE is already part of several approved liposomal products and an investigational mRNA vaccine. For the current experiment, different LNP compositions comprising DMG-PEG2000 were explored.

The first LNP library was designed to address whether the lipid molar ratio affects T cell responses elicited by intravenous mRNA-LNP-vaccination and thus represents a variable that can be optimized to improve vaccine efficacy. The molar percentages of SS-EC, DOPE, and PEG-lipids were considered as independent variables, while cholesterol was considered as filler lipid to balance the molar percentages to 100%. By using the DOE methodology, an experimental design involving 11 LNPs (see composition in Table 3 for details) was established. Table 2: Composition of DMG-PEG2000 LNPs in DOE experiments LNP number Lipid ratio CoatsomeSS-EC/DOPE/Cholesterol/DMG-PEG2000 LNP1 50/15/33.75/1.25 LNP2 50/5/43.75/1.25 LNP3 40.5/12.24/46.48/0.78 LNP4 59.5/12.24/46.48/0.78 LNP5 40.5/12.24/45.54/1.72 LNP6 59.5/12.24/26.54/1.72 LNP7 36.6/7.76/54.39/1.25 LNP8 63.4/7.76/27.59/1.25 LNP9 50/7.76/41.66/0.58 LNP10 50/7.76/40.32/1.92 LNP11 50/10/38.75/1.25

The 11 lipid ratios were evenly distributed in the experimental area (data not shown). For the immunogenicity screen, the percentage of E7-specific CD8 T cells in the blood after three i.v. immunization injections was considered as the response variable to be maximized. To this end, all LNPs package mRNA encoding human papillomavirus 16 (HPV16) oncoprotein E7 as an antigen. The results confirm our hypothesis that the robust magnitude of CD8 T cell responses is dependent on LNP composition. Several mRNA-LNP vaccines elicited more than 50% E7-specific CD8 T-cell responses, whereas other mRNA-LNP-vaccines elicited hardly any responses (Fig. 4a). The molar percentage of DMG-PEG2000 was identified as a key parameter related to the magnitude of the E7-specific CD8 T cell response. A low molar percentage of PEG-lipid was required to achieve a maximal T cell response (Fig. 4c).

Bayesian regression modeling was applied to the data to create a reaction surface model that could predict the immunogenicity of certain LNP components (data not shown). To validate the predictive value of the model, 2 new LNP compositions were evaluated (Table 3). Table 3: Composition of DMG-PEG2000 LNPs in DOE experiments LNP number Lipid ratio CoatsomeSS-EC/DOPE/Cholesterol/DMG-PEG2000 LNP34 56.5/5.25/37.75/0.5 LNP35 (comparative example) 42/12/44.5/1.5

Mice immunized with LNP34 (DMG-PEG2000) had a greater than 90% probability of eliciting >30% E7-specific CD8 T cells (optimal LNP), whereas LNP35 (DMG-PEG2000) was predicted to produce poorer T cell responses (non- - optimal LNP). The experimental data largely matched the predictions, thus successfully validating the model. All mice immunized with the predicted optimal LNP did generate an E7-specific CD8 T cell response above 30%, whereas none of the mice immunized with LNP35 elicited a T cell response above this threshold (Fig. 4b).

Example 5 - mRNA Vaccines Induce Qualitative T Cell Responses The success of cancer immunotherapy is influenced by several factors, including T cell phenotype, function, and tumor infiltration. We first assessed the quality and enhanceability of T cell responses elicited by LNP34. To this end, mice received three initial immunization injections on days 0, 7, and 14, followed by a final immunization injection on day 50. Supplementation of E7 mRNA with TriMix, a mixture of 3 immunostimulatory mRNAs (Bonehill et al., 2008), increases the strength of T cell responses.

After 3 immunization injections with E7-TriMix, more than 70% of E7-specific T cells were present in the blood (Fig. 5a). Five weeks after the third immunization, the percentage of E7-specific CD8 T cells remained highly elevated. A rapid expansion of E7-specific effector T cells was observed after the final booster injection, thus confirming that the vaccine was boostable (Fig. 5a). Higher concentrations of IFN-γ were measured in serum with each immunization injection (Fig. 5b), reflecting increased numbers of E7-specific T cells.

To assess T cell function, we performed intracellular cytokine staining after three immunization injections. Multifunctional CD8 T cells simultaneously producing more than one cytokine were associated with better control of infectious diseases and tumors, and for optimal LNP, contributed about 28% of CD8 E7-specific T cells (Fig. 5c).

Example 6 - mRNA Vaccine Induces Tumor Regression Therapeutic antitumor efficacy was assessed in the congenital mouse tumor model TC-1 generated by retroviral transduction with HPV16 E6/E7 antigens. Treatment with 5 μg of E7-TriMix delivered by LNP34 was started when the average tumor diameter reached 55 mm ³ . Additionally, mice were treated with anti-PD-1 (or an isotype control antibody). PD-1 is expressed on activated T cells and interacts with PD-L1 to inhibit T cell function and induce tolerance. PD-1 checkpoint blockade maintains T cell reactivity and is approved for the first-line treatment of patients with metastatic or unresectable recurrent HNSCC. LNP34 vaccination resulted in complete regression of TC-1 tumors (Fig. 5d) and significantly prolonged survival (Fig. 5e), but tumors recurred after cessation of treatment. Anti-PD1 monotherapy did not provide any therapeutic benefit in TC-1-bearing mice.

Finally, we assessed the ability of vaccine-primed T cells to reach the tumor bed. Two immunizations with the respective mRNA-LNP-vaccine resulted in a strong infiltration of CD8 ⁺ tumor-infiltrating T cells into the tumor (Fig. 5f), of which more than 70% were specific for E7 (Fig. 5g). Adding anti-PD-1 to vaccine therapy did not significantly alter the percentage of E7-specific CD8 T cells entering the tumor.

Example 7 - Optimal LNP Increases Uptake and Activation of Immune Cells in the Spleen Tagged firefly luciferase mRNA was encapsulated in DMG-PEG2000 LNPs that were previously screened for immunogenicity. Four hours after LNP injection, luciferase activity was measured in isolated livers, spleens, lungs, hearts and kidneys. As expected, LNP composition had a strong effect on the strength and organ specificity of mRNA expression. The liver was the main target organ, followed by the spleen, but the ratio of liver to spleen varied widely between LNPs (Fig. 6a). The magnitude of the E7-specific CD8 T cell response after the third immunization was positively correlated with spleen presentation. The lack of correlation between total performance and T cell responses further underscores the importance of delivery to the spleen (data not shown). A significant correlation (strongly entangled with lipid composition) was also confirmed between LNP size.

We next assessed whether immunogenicity was associated with early mRNA uptake and activation of specific immune cell types in the spleen. LNP mainly accumulated in macrophages and monocytes (Fig. 6b). There was a strong overall correlation between T cell responses and uptake of LNP by splenic macrophages, monocytes, plasmacytoid DC (pDC), and B cells (data not shown).

To further test the importance of mRNA uptake and expression in the spleen, we compared the biodistribution and cellular uptake profiles of optimal, highly immune LNP34 and non-optimal, less immune LNP35. Relative to LNP35, LNP34 significantly increased relative mRNA expression in spleen (Fig. 6c), as well as uptake by macrophages, B cells and DC in spleen (Fig. 6d).

The optimal mRNA LNP composition, LNP34, elicited higher concentrations of inflammatory cytokines in the blood, indicating increased innate activation, compared to the next-best counterpart (Fig. 6e).

Example 8 - Other DMG-PEG2000 LNPs of the Invention In this example, more LNPs of interest were tested (see Table 4). Mice were given 2 intravenous immunizations, 1 week apart. E7-specific T cells in the blood were analyzed 5 days after the second immunization injection. The data shown in Figure 7 demonstrates that LNP59 performs significantly better than LNP53; and is therefore also well suited for use in the context of the present invention. LNP59 was again characterized by a low percentage of PEG lipids, ie 0.5 mol%, but also a significantly lower concentration of cholesterol, ie less than 30 mol%; in particular around 25 mol%. Table 4: Composition of further DMG-PEG2000 LNPs LNP number Lipid ratio CoatsomeSS-EC/DOPE/Cholesterol/DMG-PEG2000 LNP53 (comparative example) 50/10/38.5/1.5 LNP59 65/9.5/25/0.5

Conclusion LNP composition can be transformed into strong immunogenicity by adjusting the lipid ratio. Optimal LNP compositions showed increased expression in the spleen and enhanced uptake across multiple APC populations. Optimal LNPs induce high concentrations of type I IFN, which was found to be critical for the induction of T cell responses. Surprisingly, most of the injected doses of mRNA were associated with B cells. B cells display an activated phenotype and are critical for the induction of antigen-specific CD8 T cells, pointing to a previously undocumented role for B cells.

The DoE method successfully predicted LNP compositions with high or low immunity. Optimal LNP composition promotes A) mRNA uptake and expression by splenic APCs, mainly B cells, B) innate activation, evidenced by increased release of inflammatory cytokines and expression of activation markers on APCs, C ) high-scale and qualitative T-cell responses capable of regressing established TC-1 tumors. The induction of type I interferons was found to be critical for the efficacy of i.v. administered mRNA vaccines. Furthermore, B cells are critical for the induction of T cell responses, possibly due in part to the production of anti-PEG antibodies. Importantly, the presence of antibodies against LNP did not interfere with priming T cell responses. This is very relevant considering that many people will acquire PEG antibodies after vaccination with PEGylated LNP.

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Claims (16)

A lipid nanoparticle (LNP) comprising: - ionizable lipids; - Phospholipids; - sterols; - PEG lipids; and - one or more mRNA molecules; It is characterized by: The PEG lipid is C14-PEG lipid; The LNP comprises less than about 1 mol% of the PEG lipid; The mole percentage of the ionizable lipid is between about 50-70 mol%; and The molar percentage of the sterol is about 25 mol% or more. The lipid nanoparticle (LNP) of claim 1, which comprises: - ionizable lipids; - Phospholipids; - sterols; - PEG lipids; and - one or more mRNA molecules; It is characterized by: The PEG lipid is C14-PEG lipid; The LNP comprises less than about 1 mol% of the PEG lipid; The molar percentage of the ionizable lipid is between about 50-60 mol%; and The molar percentage of the sterol is about 30 mol% or more. The lipid nanoparticle according to claim 1 or 2, wherein the LNP comprises about 0.5 mol% to about 0.9 mol% of the PEG lipid; preferably about 0.5 mol%. The lipid nanoparticle according to any one of claims 1 to 3, wherein the molar percentage of the phospholipid is less than about 10 mol%; preferably about 5 mol%. The lipid nanoparticle according to any one of claims 1 to 4, wherein the ratio of the ionizable lipid to the phospholipid is higher than 5:1; preferably between about 6:1 and 11:1; most The best is about 11:1. The lipid nanoparticle according to any one of claims 1 to 5, wherein the mole percentage of the ionizable lipid is about 55-60 mol%. The lipid nanoparticle according to any one of claims 1 to 6, wherein the C14-PEG lipid is C14-PEG2000 lipid, preferably selected from the following list: DMG-PEG2000 and DMPE-PEG2000, most preferably DMG -PEG2000. The lipid nanoparticle according to any one of claims 1 to 7, wherein the ionizable lipid is selected from the group consisting of: - 1,1'-((2-(4-(2-((2- (bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperene

-1-yl)ethyl)azanediyl)bis(dodecyl-2-ol) (C12-200); -Dilinoleylmethyl-4-dimethylaminobutyrate (DLin - MC3-DMA); or - a compound of formula (I):

wherein: RCOO is selected from the group consisting of myristyl, α-D-tocopheryl succinyl, linoleyl, and oleyl; and X is selected from the group consisting of:

; Preferably, the ionizable lipid is a lipid of formula (I), wherein RCOO is α-D-tocopheryl succinyl, and X is

. The lipid nanoparticle according to any one of claims 1 to 8, wherein the phospholipid is selected from the following list: mixtures of DOPE, DOPC, DSPC and the like; especially mixtures of DOPE, DOPC and the like. The lipid nanoparticle according to any one of claims 1 to 9, wherein the sterol is selected from the following list: cholesterol, ergosterol, campesterol, oxysterol, antrosterol, chain desmosterol, nicasterol, sitosterol and stigmasterol; preferably cholesterol. The lipid nanoparticle according to any one of claims 1 to 10, wherein the LNP comprises: - about 56.5 mol% of the ionizable lipid; - About 5 mol% of DOPE; - about 38 mol% cholesterol; and - About 0.5 mol% of DMG-PEG2000. The lipid nanoparticle according to any one of claims 1 to 10, wherein the LNP comprises: - about 65 mol% of the ionizable lipid; - About 9.5 mol% DOPE; - about 25 mol% cholesterol; and - About 0.5 mol% of DMG-PEG2000. The lipid nanoparticle according to any one of claims 1 to 12, wherein the one or more mRNA molecules are selected from mRNA molecules encoding immunomodulatory polypeptides, such as mRNA molecules encoding CD40L, CD70 and caTLR4; and/or encoding antigens group of mRNAs. A pharmaceutical composition or vaccine, comprising one or more lipid nanoparticles according to any one of claims 1 to 13 and an acceptable pharmaceutical carrier. The lipid nanoparticle according to any one of claims 1 to 13 or the pharmaceutical composition or vaccine according to claim 14, which is used for human or veterinary medicine. The lipid nanoparticle according to any one of claims 1 to 13 or the pharmaceutical composition or vaccine according to claim 14, which is used for treating cancer or infectious diseases.

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