Lipid Nanoparticles

Abstract

The present invention relates to the field of lipid nanoparticles (LNPs); more specifically, they comprise ionizable lipids, phospholipids, sterols, PEG lipids and one or more nucleic acids. The LNPs of the present invention are characterized by having a minimum average diameter of about 140 nm, thereby inducing a more powerful immune response. The present invention provides the use of LNPs in the immunogenic delivery of nucleic acid molecules, particularly mRNA; thereby making them very suitable for vaccines, for example, for the treatment of cancer or infectious diseases. Finally, a method for preparing such LNPs is provided.

Description

Field of the Invention

[0001] The present invention relates to the field of lipid nanoparticles (LNPs); more specifically, they comprise ionizable lipids, phospholipids, sterols, PEG lipids and one or more nucleic acids. The LNPs of the present invention are characterized by having a minimum average diameter of about 140 nm, thereby inducing a more powerful immune response. The present invention provides the use of LNPs in the immunogenic delivery of nucleic acid molecules, particularly mRNA; thereby making them very suitable for vaccines, for example, for the treatment of cancer or infectious diseases. Finally, a method for preparing such LNPs is provided. Background of the Invention

[0003] One of the main challenges in the field of targeted delivery of bioactive substances is generally their instability and low cell penetration potential. This is especially true for the delivery of nucleic acid molecules, especially (m) RNA molecules. Therefore, appropriate packaging is essential for adequate protection and delivery. Therefore, there is a continuous need for methods and compositions for packaging bioactive substances such as nucleic acids.

[0004] In this regard, lipid-based nanoparticle compositions such as lipid complexes and liposomes have been used as packaging carriers for bioactive substances to allow transport into cells and / or intracellular compartments. These lipid-based nanoparticle compositions generally include mixtures of different lipids such as cationic lipids, ionizable lipids, phospholipids, structural lipids (such as sterols or cholesterol), PEG (polyethylene glycol) lipids, etc. (such as Reichmuth et al., 2016 review). Although many of these lipid compositions are known in the art, they generally have a small diameter, i.e., less than 200 nm, most often even less than 140 nm.

[0005] Lipid-based nanoparticles containing a mixture of four lipids (cationic or ionizable lipids, phospholipids, sterols, and PEGylated lipids) have been developed for delivery to the liver after systemic administration. Non-immunogenic Delivery of siRNA and mRNA. To elicit optimal hepatocyte uptake and expression, these LNPs typically display a small size of 70-100 nm (Li et al., Nanoletters 2015; Thess et al., Mol ther 2015; Kauffman et al., Biomaterials 2016).

[0006] In addition to liver targeting, LNPs have been used to deliver mRNA encoding antigens to ImmunogenicityDelivery to muscle or dermis (Richner et al. Cell 2017; Liang et al., Mol Ther 2017). In this case, small-sized LNPs (80-120 nm) are still commonly used, because this small size has been shown to be crucial for immunogenicity, and small-sized LNPs effectively reach the injection-draining lymph nodes, while larger LNPs remain at the injection site (see Reichmuth et al., 2016).

[0007] We have now surprisingly found that, contrary to the common belief that small size of particles is beneficial for LNP-mediated mRNA delivery, nanoparticles with diameters greater than 140 nm, preferably even greater than 200 nm, are very suitable for immunogenic delivery of mRNA after systemic injection of LNPs, which appears to be associated with enhanced delivery of mRNA to the spleen. SUMMARY OF THE INVENTION

[0009] In a first aspect, the present invention provides lipid nanoparticles (LNPs) comprising ionizable lipids, phospholipids, sterols, PEG lipids and one or more nucleic acid molecules, characterized in that the LNPs have a minimum average diameter of about 140 nm, more preferably about 200 nm.

[0010] In a specific embodiment, the ionizable lipid is a compound of formula (I):

[0011]

[0012] in:

[0013] RCOO is selected from the list comprising: myristoyl, alpha-D-tocopheryl succinyl, linoleoyl, and oleoyl; and

[0014] X is selected from the list consisting of:

[0015]

[0016] More specifically, the ionizable lipid is a lipid of formula (I) wherein RCCO is α-D-tocopheryl succinyl, and X is

[0017]

[0018] In another embodiment of the present invention, the sterol is selected from the list comprising cholesterol, ergosterol, campesterol, oxysterol, antrosterol, streptosterol, nicasterol, sitosterol and stigmasterol; preferably cholesterol.

[0019] In another specific embodiment, the phospholipid is selected from the list comprising: 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), 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-heneicosanoyl-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-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C 16Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-docosahexanoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dialinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexanoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and mixtures thereof.

[0020] In a more specific embodiment, the phospholipid is selected from the list comprising 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and mixtures thereof.

[0021] In yet another embodiment, the PEG lipid is selected from the list comprising PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

[0022] In another specific embodiment, one or more of the following are used:

[0023] - the LNPs comprise about 10 mol% to 60 mol% of the ionizable lipids;

[0024] - said LNPs comprise from about 15 mol% to 50 mol% of said sterol;

[0025] - the LNPs comprise about 0.5 mol% to 10 mol% of the PEG lipids; and / or

[0026] - said LNPs comprise about 5 mol% to 40 mol% of said phospholipids.

[0027] In another specific embodiment, the one or more nucleic acid molecules are selected from (m)RNA and DNA molecules; in particular one or more mRNA molecules. In a specific embodiment, the one or more mRNA molecules are selected from the list comprising: mRNA molecules encoding immunomodulatory polypeptides, for example (plus the list of all potential molecules) and / or mRNA molecules encoding antigenic and / or disease-specific mRNAs. In a very specific embodiment, the one or more mRNA molecules are selected from the list comprising: mRNA molecules encoding CD40L, CD70 and caTLR4; and / or mRNA molecules encoding antigenic and / or disease-specific mRNAs.

[0028] The invention also provides a pharmaceutical composition or vaccine comprising one or more LNPs as defined herein.

[0029] Furthermore, the present invention provides an LNP as defined herein or a pharmaceutical composition comprising one or more such LNPs for use in human or veterinary medicine.

[0030] In another aspect, the present invention provides a method for preparing the LNP according to the present invention, comprising:

[0031] - preparing a first alcohol composition comprising the ionizable lipid, the phospholipid, the sterol, the PEG lipid and a suitable alcohol solvent;

[0032] - preparing a second aqueous composition comprising the one or more nucleic acids and an aqueous solvent;

[0033] - Mixing the first and second compositions in a microfluidic mixing device using the following setup:

[0034] - The total flow rate (FR) is about 0.5 to about 8 ml / min, preferably about 1 to 4 ml / min.

[0035] - The flow rate ratio (FRR) is about 1 / 1 to 5 / 1, preferably about 2 / 1 to about 3 / 1.

[0036] In a specific embodiment, the alcoholic solvent is ethanol and / or the aqueous solvent is water.

[0037] The present invention further provides the use of a LNP, a pharmaceutical composition or a vaccine according to the present invention for the immunogenic delivery of said one or more nucleic acid molecules.

[0038] Finally, the present invention provides LNPs, pharmaceutical compositions or vaccines as described herein for use in preventing and / or treating cancer or infectious diseases.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Now with specific reference to the accompanying drawings, it is emphasized that the details shown are exemplary only and are only for the purpose of illustrative discussion of different embodiments of the present invention. They are presented in order to provide what is considered to be the most useful and easiest content for describing the principles and concepts of the present invention. In this regard, no attempt is made to show the structural details of the present invention in more details than necessary for a basic understanding of the present invention. The accompanying drawings make it easy for those skilled in the art to know how to embody several forms of the present invention in practice.

[0041] Figure 1 : Immunogenicity of 140 nm versus 230 nm sized mRNA LNPs of the same composition.

[0042] Figure 2 : Immunogenicity of OVA mRNA LNPs of different sizes after intravenous administration.

[0043] Figure 3 : Immunogenicity of OVA mRNA LNPs of different sizes after a single intravenous administration.

[0044] Figure 4 : Percentage of OVA-specific T cells after intravenous administration of large-sized LNPs containing OVA mRNA (10 μg) and TriMix mRNA (15 μg).

[0045] Figure 5 : Comparison of the immunogenicity of 140 nm and 230 nm sized mRNA LNPs after one and two intravenous administrations of mRNA LNPs.

[0046] Figure 6 : Comparison of spleen / liver expression ratios of 100 nm and 230 nm sized mRNA LNPs

[0047] Figure 7 : Comparison of intravenous versus intranodal administration of LNP mRNA

[0048] Figure 8: T cell responses to LNP mRNA formulations are durable and boostable. DETAILED DESCRIPTION OF THE INVENTION

[0050] As already described in detail above, the present invention provides LNPs having a diameter larger than that commonly used in the art, for which we have surprisingly found that they are very suitable for immunogenic delivery of nucleic acids, especially mRNA; for which we have found relevance for enhanced delivery to the spleen. "Immunogenic delivery of nucleic acid molecules" refers to the delivery of nucleic acid molecules to cells whereby they come into contact with the cells, the internalization of the nucleic acid molecule and / or the expression within the cell leading to the induction of an immune response.

[0051] Therefore, in a first aspect, the present invention provides a lipid nanoparticle (LNP) comprising an ionizable lipid, a phospholipid, a sterol, a PEG lipid and one or more nucleic acid molecules, characterized in that the minimum diameter of the LNP is about 140 nm; more preferably about 200 nm.

[0052] Lipid nanoparticles (LNPs) are generally known as nanoscale particles comprising a combination of different lipids. Although many different types of lipids can be included in such LNPs, the LNPs of the present invention generally comprise a combination of ionizable lipids, phospholipids, sterols, and PEG lipids.

[0053] As used herein, the term "nanoparticle" refers to any particle having a diameter that makes the particle suitable for systemic, especially intravenous administration, especially of nucleic acids, typically having a diameter of less than 1000 nanometers (nm).

[0054] In some embodiments, the average diameter of nanoparticles is less than 600nm. In some embodiments, the average diameter of nanoparticles is less than 400nm, but in any case, the average diameter of nanoparticles of the present invention is greater than about 140nm, more preferably greater than about 200nm. In a specific embodiment, LNP of the present invention has a minimum average diameter of about 140nm, about 150nm, about 160nm, about 170nm, about 180nm, about 190nm or about 200nm. For the sake of clarity, in the case of a mixture using a plurality of LNPs, the minimum average diameter refers to the minimum average diameter of the plurality of LNPs. In the case, for example, the mixture can contain some LNPs with an average diameter less than 140nm, as long as the average diameter of the remaining LNPs is greater than 140nm to cause the average minimum diameter of all LNPs together to be 140nm. Whenever used in this application, even if not specifically noted, the term "diameter" means "average diameter".

[0055] In the context of the present invention, the term "ionizable" (or alternatively cationic) in the context of a compound or lipid refers to the presence of any uncharged group in the compound or lipid that is capable of being ionized by generating an ion (usually H + ions) and thus become positively charged themselves. Alternatively, any uncharged group in the compound or lipid may generate electrons and thus become negatively charged.

[0056] In the context of the present invention, any type of ionizable lipid may be suitably used. In particular, a suitable ionizable lipid is an ionizable amino lipid comprising two identical or different tails connected by an SS bond, each of which comprises an ionizable amine, such as shown below:

[0057]

[0058] In a specific embodiment, the ionizable lipid is a compound of formula (I):

[0059]

[0060] in:

[0061] RCOO is selected from the list comprising: myristoyl, alpha-D-tocopheryl succinyl, linoleoyl, and oleoyl; and

[0062] X is selected from the list consisting of:

[0063]

[0064] Such ionizable lipids are specifically represented by any one of the following formulae:

[0065]

[0066] More specifically, the ionizable lipid is a lipid of formula (I) wherein RCCO is α-D-tocopheryl succinyl, and X is

[0067]

[0068] For example, as shown in the following formula:

[0069]

[0070] In the context of the present invention, the term "sterol" (also referred to as sterol) is a subclass of sterols, which occur naturally in plants, animals and fungi, or may 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, antrosterol, streptosterol, nicasterol, sitosterol and stigmasterol; preferably cholesterol.

[0071] In the context of the present invention, the term "phospholipid" refers to a lipid molecule consisting of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group. These two components are most often bound together by a glycerol molecule, and therefore, among the phospholipids of the present invention, glycerol-phospholipids are preferred. In addition, the phosphate group is often modified by simple organic molecules such as choline (i.e., producing phosphorylcholine) or ethanolamine (i.e., producing phosphoethanolamine).

[0072] In the context of the present invention, suitable phospholipids may be selected from the list comprising: 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), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DL ...DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 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-heneicosanoyl-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-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C 16Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-docosahexanoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dialinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexanoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and mixtures thereof.

[0073] In a more specific embodiment, the phospholipid is selected from the list comprising 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and mixtures thereof.

[0074] In the context of the present invention, the term "PEG lipid" or alternatively "PEGylated lipid" refers to any suitable lipid modified with a PEG (polyethylene glycol) group. In a specific embodiment, the PEG lipid is selected from a list comprising: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol and mixtures thereof. More specific examples of such PEG lipids include C14-PEG2000 (1,2-dimyristoyl-rac-glycerol, methoxypolyethylene glycol-2000 (DMG-PEG2000)) and C18-PEG5000 (1,2-distearoyl-rac-glycerol, methoxypolyethylene glycol-5000 (DSG-PEG5000)).

[0075] In a specific embodiment of the present invention, one or more of the following are applied:

[0076] - the LNPs comprise about 35 mol% to 65 mol% of the ionizable lipids;

[0077] - said LNPs comprise from about 15 mol% to 60 mol% of said sterol;

[0078] - the LNPs comprise about 0.5 mol% to 2 mol% of the PEG lipids; and / or

[0079] - said LNPs comprise about 5 mol% to 15 mol% of said phospholipids.

[0080] In another specific embodiment of the present invention, one or more of the following are applied:

[0081] - the LNPs comprise about 10 mol% to 60 mol% of the ionizable lipids;

[0082] - said LNPs comprise from about 15 mol% to 50 mol% of said sterol;

[0083] - the LNPs comprise about 0.5 mol% to 10 mol% of the PEG lipids; and / or

[0084] - said LNPs comprise about 5 mol% to 40 mol% of said phospholipids.

[0085] In a more specific embodiment, one or more of the following apply:

[0086] - the LNPs comprise about 40 mol% to 60 mol% of the ionizable lipids;

[0087] - said LNPs comprise from about 20 mol% to 40 mol% of said sterol;

[0088] - the LNPs comprise between about 0.5 mol% and 5 mol% of the PEG lipids; and / or

[0089] - said LNPs comprise about 5 mol% to 15 mol% of said phospholipids.

[0090] Thus, in a specific embodiment, the LNPs comprise about 10 mol% to 60 mol% of the ionizable lipids; preferably about 40 mol% to 60 mol%.

[0091] In yet another specific embodiment, said LNPs comprise about 15 mol% to 50 mol% of said sterol; preferably about 20 mol% to 40 mol%.

[0092] In another embodiment, the LNP comprises about 0.5 mol% to 10 mol% of the PEG lipid; preferably about 0.5 mol% to 5 mol%.

[0093] In another specific embodiment, said LNP comprises about 5 mol% to 40 mol% of said phospholipid; preferably about 5 mol% to 15 mol%.

[0094] Therefore, in a more specific embodiment, the LNP of the present invention comprises about 10 mol% to 60 mol% of said ionizable lipid; and / or about 15 mol% to 50 mol% of sterol; and / or about 0.5 mol% to 10 mol% of said PEG lipid; and / or about 5 mol% to 40 mol% of said phospholipid.

[0095] In another specific embodiment, the LNP of the present invention comprises about 40 mol% to 60 mol% of said ionizable lipid; and about 20 mol% to 40 mol% of sterol; and about 0.5 mol% to 5 mol% of said PEG lipid; and about 5 mol% to 15 mol% of said phospholipid.

[0096] The compositions of LNPs that are particularly suitable in the context of the present invention are listed in Table 1:

[0097] Table 1: Composition of particularly suitable LNPs

[0098]

[0099] The inventors have found that the LNPs of the present invention are particularly suitable for immunogenic delivery of nucleic acids; this is due to increased spleen targeting compared to small LNPs known in the prior art that are primarily targeted to the liver.

[0100] Thus, the present invention provides LNPs comprising one or more nucleic acid molecules, such as DNA or RNA, more particularly mRNA.

[0101] 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 acid includes genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. Nucleic acid according to the present invention can be single-stranded or double-stranded and linear or covalently closed to form a ring of molecular forms. Nucleic acid can be used for example to introduce (i.e. transfect) cells in the form of RNA, which can be prepared by in vitro transcription from a DNA template. In addition, RNA can be modified by stabilizing sequences, capping and polyadenylation before application.

[0102] In the context of the present invention, the term "RNA" refers to a molecule comprising ribonucleotide residues and preferably consisting entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of a β-D-ribofuranosyl group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA, such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA, which differs from naturally occurring RNA in the addition, deletion, substitution and / or change of one or more nucleotides. Such changes may include adding non-nucleotide substances to, for example, the end or interior of the RNA, for example, at one or more nucleotides of the RNA. The nucleotides in the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs. The nucleic acid may be contained in a vector. As used herein, the term "vector" includes any vector known to the skilled person, including a plasmid vector, a cosmid vector, a phage vector such as lambda phage, a viral vector such as an adenovirus or baculovirus vector, or an artificial chromosome vector such as a bacterial artificial chromosome (BAC), a yeast artificial chromosome, or an analog of naturally occurring RNA.

[0103] According to the present invention, the term "RNA" includes and preferably relates to "mRNA", which means "messenger RNA" and relates to a "transcript" that can be produced using DNA as a template and encodes a peptide or protein. 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 limited half-life in cells and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the invention, RNA is obtained by in vitro transcription or chemical synthesis. In vitro transcription methods are known to technicians. For example, there are many commercially available in vitro transcription kits.

[0104] In a specific embodiment, the mRNA molecule is an mRNA molecule encoding an immunomodulatory protein.

[0105] In the context of the present invention, the term "mRNA molecule encoding an immunomodulatory protein" refers to an mRNA molecule encoding a protein that modifies the functionality of antigen presenting cells, more particularly dendritic cells. Such a molecule can be selected from a list comprising: CD40L, CD70, caTLR4, IL-12p70, EL-selectin, CCR7 and / or 4-1BBL, ICOSL, OX40L, IL-21; in particular one or more of CD40L, CD70 and caTLR4. 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 is used, which is also referred to herein as "TriMix".

[0106] In another specific embodiment, the mRNA molecule is an mRNA molecule encoding an antigen and / or a disease-specific protein.

[0107] According to the present invention, the term "antigen" includes any molecule, preferably a peptide or protein, which contains at least one epitope that will trigger an immune response and / or to which an immune response is directed. Preferably, in the context of the present invention, an antigen is a molecule that induces an immune response, optionally after processing, which immune response is preferably specific to the antigen or a cell expressing the antigen. In particular, "antigen" relates to a molecule that is presented by an MHC molecule and specifically reacts with a T lymphocyte (T cell), optionally after processing.

[0108] In a specific embodiment, the antigen is a target-specific antigen, which may be a tumor antigen, or a bacterial, viral or fungal antigen. The target-specific antigen may be derived from any of the following: total mRNA isolated from (one or more) target cells, one or more specific target mRNA molecules, protein lysates of (one or more) target cells, specific proteins from (one or more) target cells, or synthetic target-specific peptides or proteins and synthetic mRNA or DNA encoding target-specific antigens or peptides derived therefrom.

[0109] 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 a combination of one or more mRNA molecules encoding immunomodulatory proteins and / or one or more mRNA molecules encoding antigens and / or disease-specific proteins.

[0110] In very specific embodiments, the mRNA molecules encoding immunomodulatory molecules can be combined with one or more mRNA molecules encoding antigen-specific proteins and / or disease-specific proteins. For example, the LNPs of the present invention can include mRNA molecules encoding immunostimulatory molecules CD40L, CD70 and / or caTLR4 (e.g., Dimix or Trimix); combined with one or more mRNA molecules encoding antigen-specific proteins and / or disease-specific proteins. Therefore, in very specific embodiments, the LNPs of the present invention include mRNA molecules encoding CD40L, CD70 and / or caTLR4; combined with one or more mRNA molecules encoding antigen-specific proteins and / or disease-specific proteins.

[0111] In another aspect, 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. Therefore, the present invention also provides a vaccine comprising one or more LNPs according to the present invention.

[0112] In the context of the present invention, the term "vaccine" as used herein refers to any preparation intended to provide adaptive immunity (antibodies and / or T cell responses) against a disease. To this end, the vaccine referred to herein comprises at least one mRNA molecule encoding an antigen, wherein the antigen triggers an adaptive immune response. The antigen may be present in a weakened or killed form of a microorganism, protein or peptide or an antigen encoding nucleic acid. In the context of the present invention, an antigen refers to a protein or peptide that is recognized as heterologous by the host's immune system, thereby stimulating the production of antibodies against it, with the aim of combating such antigens. Vaccines can be preventive (e.g., preventing or alleviating the effects of future infections with any natural or "wild" pathogens) or therapeutic (e.g., actively treating or alleviating the symptoms of an ongoing disease). The administration of a vaccine is called vaccination.

[0113] The vaccine of the present invention can be used to induce an immune response, particularly an immune response to a disease-associated antigen or a cell expressing a disease-associated antigen, such as an immune response to cancer. Therefore, the vaccine can be used for the preventive and / or therapeutic treatment of a disease such as cancer involving a disease-associated antigen or a cell expressing a disease-associated antigen. Preferably, the immune response is a T cell response. In one embodiment, the disease-associated antigen is a tumor antigen. The antigen encoded by the RNA contained in the nanoparticle as described herein is preferably a disease-associated antigen or induces an immune response to a disease-associated antigen or a cell expressing a disease-associated antigen.

[0114] The LNPs and vaccines of the present invention are particularly contemplated for intravenous administration, i.e., the infusion of liquid substances directly into a vein. The intravenous route is the fastest method for delivering liquids and drugs throughout the body (i.e., systemically). Therefore, the present invention provides intravenous vaccines, and the use of the disclosed vaccines and LNPs for intravenous administration. Therefore, the vaccines and LNPs of the present invention can be administered intravenously. The present invention also provides the use of vaccines and LNPs according to the present invention; wherein the vaccine is administered intravenously.

[0115] The present invention also provides LNPs, pharmaceutical compositions and vaccines according to the present invention for use in human or veterinary medicine. It is also contemplated that the LNPs, pharmaceutical compositions and vaccines of the present invention are used in human or veterinary medicine. Finally, the present invention provides a method for preventing and treating human and veterinary diseases by administering LNPs, pharmaceutical compositions and vaccines according to the present invention to subjects in need thereof.

[0116] The present invention further provides the use of LNP, pharmaceutical composition or vaccine according to the present invention for immunogenic delivery of one or more nucleic acid molecules. This effect is particularly achieved using the larger LNP of the present invention because they preferentially target the spleen. It was found that the small-sized LNP of the prior art preferentially targets the liver and therefore has little or no immunogenic effect. Therefore, the LNP, pharmaceutical composition and vaccine of the present invention are very useful in treating several human and veterinary diseases. Therefore, the present invention provides LNP, pharmaceutical composition and vaccine of the present invention for treating cancer or infectious diseases.

[0117] In another aspect, the present invention provides a method for preparing an LNP according to the present invention, comprising:

[0118] - preparing a first alcohol composition comprising the ionizable lipid, the phospholipid, the sterol, the PEG lipid and a suitable alcohol solvent;

[0119] - preparing a second aqueous composition comprising the one or more nucleic acids and an aqueous solvent;

[0120] - Mixing the first and second compositions in a microfluidic mixing device using the following setup:

[0121] - The total flow rate (FR) is about 0.5 to about 8 ml / min, preferably about 1 to 4 ml / min.

[0122] - The flow rate ratio (FRR) is about 1 / 1 to 5 / 1, preferably about 2 / 1 to about 3 / 1.

[0123] In more detail, the lipid components are combined in an alcohol vehicle such as ethanol at appropriate concentrations. Additionally, an aqueous composition comprising nucleic acids is added and then loaded into a microfluidic mixing device.

[0124] The purpose of microfluidic mixing is to achieve thorough and rapid mixing of multiple samples (i.e., lipid phase and nucleic acid phase) in a microdevice. This sample mixing is usually achieved by enhancing the diffusion effect between different material flows. In addition, several microfluidic mixing devices can be used, such as those reviewed in Lee et al., 2011. A particularly suitable microfluidic mixing device according to the present invention is the NanoAssemblr from Precision Nanosystems.

[0125] The mixing parameters of this microfluidic mixing device have a great influence on the characteristics of the obtained LNPs, especially on their size. Therefore, in order to obtain large LNPs (i.e., with an average diameter of at least 140 nm, preferably at least 200 nm), the FR and FRR parameters are highly correlated.

[0126] The total FR (Flow Rate) is a measure of the speed at which the microfluid is mixed, and is typically set to 8-12 ml / min in the prior art to allow adequate mixing, thereby obtaining small LNPs for delivery to the liver. In contrast, in the present invention, the FRW is set much lower, thereby obtaining larger LNPs suitable for use in the present invention.

[0127] FRR (Flow Rate Ratio) is the ratio between lipids in the alcohol phase and nucleic acids in the aqueous phase, and is generally set to about 2 / 1 to about 3 / 1 in the present invention. Example

[0128] Example 1.

[0129] In this first embodiment, the effect of particle size on the immunogenicity of mRNA LNP was evaluated. For this reason, mRNA LNPs of 140nm and 230nm sizes of the same composition were prepared. LNPs contained 50mol% of α-D-tocopherol hemisuccinyl (ionizable lipid), 10mol% of DOPE, 38.5mol% of cholesterol and 1.5mol% of DSG-PEG5000 (PEGylated lipid) with a specified lipid ratio (mol%). LNPs were produced using different flow rates (FR; ml / min) and water / ethanol volume ratios (FRR) on Nano-Assemblr. LNPs of 140nm size were produced with FR 4ml / min, while LNPs of 230nm size were produced with FR 1ml / min.

[0130] Figure 1 The percentage of OVA-specific CD8+ T cells after a single intravenous administration of OVA / TriMix mRNA LNPs (OVA: 10 μg; TriMix mRNA: 15 μg) in mice is shown. Figure 1It can be seen very clearly that the immunogenicity of the larger LNPs is much higher compared to the immunogenicity of the smaller LNPs.

[0131] Example 2.

[0132] In this example, the immunogenicity of OVA mRNA LNPs of different sizes and compositions following intravenous administration in mice was determined. Figure 2 As defined above, all LNPs contain α-D-tocopherol hemisuccinyl (ionizable lipid), DOPE, cholesterol, and DSG-PEG5000 at the specified lipid ratios (mol %). Figure 2 As defined above, LNPs were produced on the Nano-Assemblr using different flow rates (FR; ml / min) and water / ethanol volume ratios (FRR). Six days after immunization, the percentage of OVA-specific CD8 T cells was quantified by flow cytometry. Again, from Figure 2 It can be very clearly seen that the larger LNPs are more immunogenic compared to the smaller LNPs.

[0133] Example 3.

[0134] In this example, the immunogenicity of OVA mRNA LNPs of different sizes and compositions in mice was determined after a single intravenous administration. The graph depicts the increase in IFN-γ secreting CD8+ T cells / 10 cells after restimulation of splenocytes with the OVA MHC I epitope SIINFEKL (5 μg / ml). 6 The number of spleen cells. Figure 3 As defined above, all LNPs contained α-D-tocopherol hemisuccinyl (ionizable lipid), DOPE, cholesterol, and DSG-PEG5000 at the specified lipid ratios (mol %). Figure 3 As defined, LNPs were produced on the Nano-Assemblr using different flow rates (FR; ml / min) and water / ethanol volume ratios (FRR). Six days after immunization, the percentage of OVA-specific CD8+ T cells was quantified by flow cytometry.

[0135] Example 4.

[0136] Next, we evaluated the effect of varying the PEGylated lipids, and for this purpose, two different compositions were prepared with either DSG-PEG5000 or DMG-PEG2000. The percentage of OVA-specific T cells was determined after intravenous injection of large-sized LNPs containing OVA mRNA (10 μg) and TriMix mRNA (15 μg) and is shown in Figure 4Mice were immunized on days 0 and 20. Blood samples were collected on days 15 and 39 to assess the percentage of OVA-specific T cells. Figure 4 It can be clearly seen that the type of PEGylated lipid has no effect on the immunogenicity of the LNPs.

[0137] Example 5.

[0138] In this example, we evaluated the immunogenicity of small and large LNPs after repeated immunizations using 140 nm and 230 nm sized mRNA LNPs ( Figure 5 ). The LNPs contained 50 mol % α-D-tocopherol hemisuccinyl (ionizable lipid), 10 mol % DOPE (phospholipid), 38 mol % cholesterol, and 1.5 mol % DSG-PEG5000 at the specified lipid ratios (mol %), as Figure 5 The LNPs contained 10 μg of OVA mRNA and 15 μg of TriMix mRNA (CD40L, CD70, and caTLR4). The percentage of OVA-specific CD8 T cell responses was measured five days after immunization.

[0139] Example 6.

[0140] In this example, we compared mRNA expression in the spleen and liver of mice injected intravenously with Fluc mRNA LNPs of different sizes ( Figure 6 ). LNPs contain α-D-tocopherol hemisuccinyl (ionizable lipid), DOPC, cholesterol, and DSG-PEG5000 in lipid ratios of 50 / 10 / 38.5 / 1.5, respectively. Different sizes of LNPs were generated by microfluidic mixing using different flow rates (FR1 vs. FR4). The figure depicts the changes in pH / s / cm 2 The ratio of spleen to liver expression of FluC mRNA measured by in vivo bioluminescence at 100 μg / sr.

[0141] Example 7.

[0142] In this example, we demonstrated that formulated intravenous mRNA LNPs elicited superior immunogenicity and anti-tumor efficacy compared to the intranodal approach.

[0143] The characteristics of the API used are as follows:

[0144] -E7 mRNA, column purified, 10 μg / administration

[0145] -Immunization schedule: 3 times a week

[0146] The results of this experiment are shown in Figure 7In the study, it was clearly shown that the intravenous approach using mRNA LNPs was superior to the intranodal approach in both immunogenicity and anti-tumor efficacy. In addition, there appeared to be a correlation between the anti-tumor efficacy and the magnitude of the T cell response.

[0147] Example 8.

[0148] In this example, we demonstrate that T cell responses to mRNA LNP formulations are durable and boostable.

[0149] For this purpose, three different preparations were used in the immunization protocol, and the results were as follows Figure 8 Based on these results, the following observations / conclusions can be made:

[0150] - Prolonged contraction period with high percentage of E7-specific T cells at 40 days after immunization 3

[0151] -Memory conversion

[0152] -Excellent recall response: IV vaccines can be boosted.

[0153] Example 9.

[0154] In this example, further experiments were performed which demonstrated the effect of the size of the LNPs.

[0155] For this purpose, the following experimental setup was used:

[0156] - Ratio of ionizable lipid / cholesterol / DOPE / DMG-PEG2000 – 50 / 38.5 / 10 / 1.5

[0157] - Three intravenous immunizations; Days 0, 7, and 14

[0158] -mRNA: mRNA encoding OVA, 10 μg / immunization

[0159] - Evaluation of OVA-specific CD8 T cell percentages on days 5, 12, 19, and 50 after the first immunization

[0160] The results are Figure 8 , and clearly showed that LNPs with an average size of 150 nm or 200 nm were significantly more immunogenic in terms of T cell responses compared to LNPs with an average size of only 100 nm.

[0161] References

[0162] Reichmuth et al., 2016 – mRNA vaccine delivery using lipid nanoparticles - Therapeutic Delivery Vol.7, N°5.

[0163] Li et al., 2015 – An Orthogonal Array Optimization of Lipid-like Nanoparticles for mRNA Delivery in Vivo - Nano Letters 15(12) pg.8099 - 8107.

[0164] Thess et al., 2015 – Sequence-engineered mRNA without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals – Molecules Therapy Vol.23, Issue 9 pg.1456 - 1464.

[0165] Kauffman et al., 2016 – Materials for non-viral intracellular delivery of messenger RNA therapeutics – Journal of Controlled Release, Vol.240, pg.227 - 234.

[0166] Richner et al., 2017 – Modified mRNA vaccines protect against Zika virus infection – Cell, Vol.168, Issue 6 pg.1114 - 1125

[0167] Liang et al., 2017 – Efficient Targeting and Activation of Antigen-Presenting Cells In Vivo after Modified mRNA Vaccine Administration in Rhesus Macaques – Molecular Therapy, vol.25 Issue 12 pg.2635 - 2647.

[0168] Lee et al., 2011 – Microfluidic Mixing: A Review – Int. J. Mol. Sci. 12(5): pg. 3263-3287.

Claims

1. A lipid nanoparticle (LNP), comprising: -50 mol % to 65 mol % ionizable lipids; - 5 mol% to 15 mol% of a phospholipid selected from the list comprising DOPE, DOPC and mixtures thereof; - 15 mol % to 40 mol % of sterols; - 0.5 mol% to 2 mol% PEG lipid; and - one or more mRNA molecules; It is characterized in that The LNPs have a minimum average diameter of 160 nm to 230 nm, and wherein the LNPs are formulated for intravenous administration, and The ionizable lipid is a compound of formula (I): in: RCOO is selected from the list comprising: myristoyl, alpha-D-tocopheryl succinyl, linoleoyl, and oleoyl; and X is selected from the list consisting of:

2. The LNP of claim 1, wherein the ionizable lipid is a lipid of formula (I), wherein RCCO is α-D-tocopheryl succinyl, and X is 3. The LNP according to any one of claims 1-2, wherein the sterol is selected from the list comprising cholesterol, ergosterol, campesterol, oxysterol, antrosterol, streptosterol, nicasterol, sitosterol and stigmasterol.

4. The LNP according to any one of claims 1-2, wherein the sterol is cholesterol.

5. The LNP according to any one of claims 1-2, wherein the PEG lipid is selected from the list comprising: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

6. The LNP according to any one of claims 1-2, wherein the LNP comprises 50 mol% to 60 mol% of the ionizable lipid.

7. The LNP according to claim 1, wherein the mRNA molecule is selected from the list comprising: immunomodulatory polypeptide encoding mRNA and / or antigen encoding mRNA.

8. The LNP according to claim 7, wherein the immunomodulatory polypeptide encoding mRNA is selected from the list comprising: mRNA molecules encoding CD40L, CD70 and caTLR4.

9. A pharmaceutical composition or vaccine comprising one or more LNPs according to any one of claims 1 to 8 and an acceptable pharmaceutical carrier.

10. The LNP according to any one of claims 1 to 8 or the pharmaceutical composition or vaccine according to claim 9 for use in human or veterinary medicine.

11. A method for preparing a LNP as defined in any one of claims 1 to 8 comprising a nucleic acid, the method comprising the following steps: - preparing a first alcohol composition comprising an ionizable lipid, a phospholipid, a sterol, a PEG lipid, and an alcohol solvent; - preparing a second aqueous composition comprising one or more nucleic acids and an aqueous solvent supplemented with a buffer; - Mixing the first alcoholic composition and the second aqueous composition in a microfluidic mixing device using the following setup: - Total flow rate (FR) is 1 to 4 ml / min. -Flow rate ratio (FRR) from 1 / 1 to 5 / 1.

12. The method according to claim 11, wherein the flow rate ratio (FRR) is 2 / 1 to 3 / 1.

13. The method according to claim 11 or 12, wherein the alcohol solvent is ethanol and / or the aqueous solvent is water.

14. The LNP according to any one of claims 1 to 8 or the pharmaceutical composition or vaccine according to claim 9 for use in treating cancer or infectious diseases.

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