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 potent immune response. The present invention provides the use of LNP in the immunogenic delivery of nucleic acid molecules, particularly mRNA; thus they are well suited for use in vaccines, for example in the treatment of cancer or infectious diseases. Finally, methods for preparing such LNPs are provided.

Description

[0001] This application is a divisional application of CN201980015578.5. Field of the Invention

[0002] The present invention relates to the field of lipid nanoparticles (LNPs); more specifically, it comprises 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 potent immune response. The present invention provides the use of LNPs in the immunogenic delivery of nucleic acid molecules, especially mRNA; thus making them very suitable for vaccines, for example for treating cancer or infectious diseases. Finally, a method for preparing such LNPs is provided. Background of the Invention

[0004] One of the main challenges in the field of targeted delivery of bioactive substances is usually 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 crucial for adequate protection and delivery. Accordingly, there is a continuing need for methods and compositions for packaging bioactive substances such as nucleic acids.

[0005] 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 comprise a mixture of different lipids such as cationic lipids, ionizable lipids, phospholipids, structural lipids (such as sterols or cholesterol), PEG (polyethylene glycol) lipids, etc. (as reviewed by Reichmuth et al., 2016). Although many such 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.

[0006] Lipid-based nanoparticles comprising a mixture of four lipids (cationic or ionizable lipids, phospholipids, sterols and polyethylene glycolated lipids) have been developed for delivery of siRNA and mRNA to the liver after systemic administration Non-immunogenic To elicit optimal hepatocyte uptake and expression, these LNPs generally exhibit a small size of 70 - 100 nm (Li et al., Nanoletters 2015; Thess et al., Mol ther 2015; Kauffman et al., Biomaterials 2016).

[0007] In addition to liver targeting, LNPs have also been used to deliver mRNA encoding antigens ImmunogenicDelivered to muscle or dermis (Richner et al., Cell 2017; Liang et al., Mol Ther 2017). In this case, small-sized (80 - 120 nm) LNPs are still commonly used because this small size has been shown to be crucial for immunogenicity and small-sized LNPs effectively reach the draining lymph nodes of the injection, while larger LNPs remain at the injection site (see Reichmuth et al., 2016).

[0008] We have now surprisingly found that, contrary to the commonly held view that small-sized particles are beneficial for LNP-mediated mRNA delivery, nanoparticles with a diameter greater than 140 nm, preferably even greater than 200 nm, are highly suitable for immunogenic delivery of mRNA after systemic injection of LNPs, which seems to be related to enhanced delivery of mRNA to the spleen. Summary of the Invention

[0010] 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 LNP has a minimum average diameter of about 140 nm, more preferably about 200 nm.

[0011] In a specific embodiment, the ionizable lipid is a compound of formula (I):

[0012]

[0013] Wherein:

[0014] RCOO is selected from the list comprising: myristoyl, α-D-tocopherol succinyl, linoleyl, and oleoyl; and

[0015] X is selected from the list comprising:

[0016]

[0017] More specifically, the ionizable lipid is a lipid of formula (I) wherein RCCO is α-D-tocopherol succinyl and X is

[0018]

[0019] 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.

[0020] In another specific embodiment, the phospholipids are 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-docosanoyl-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 succinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0021] In a more specific embodiment, the phospholipids are selected from the list comprising: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and mixtures thereof.

[0022] In yet another embodiment, the PEG lipids are 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.

[0023] In another specific embodiment, one or more of the following are applied:

[0024] - The LNP accounts for about 10 mol% to 60 mol% of the ionizable lipid;

[0025] - The LNP accounts for about 15 mol% to 50 mol% of the sterol;

[0026] - The LNP accounts for about 0.5 mol% to 10 mol% of the PEG lipid; and / or

[0027] - The LNP accounts for about 5 mol% to 40 mol% of the phospholipid.

[0028] 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, such as (plus the list of all potential molecules) and / or mRNA molecules encoding antigens and / or disease-specific mRNA. 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 antigens and / or disease-specific mRNA.

[0029] The present invention also provides a pharmaceutical composition or vaccine comprising one or more LNPs as defined herein.

[0030] Furthermore, the present invention provides LNPs as defined herein or a pharmaceutical composition comprising one or more such LNPs for human or veterinary medicine.

[0031] In another aspect, the present invention provides a method for preparing the LNP according to the present invention, comprising:

[0032] - Preparing a first alcohol composition comprising the ionizable lipid, the phospholipid, the sterol, the PEG lipid, and a suitable alcohol solvent;

[0033] - Preparing a second aqueous composition comprising the one or more nucleic acids and an aqueous solvent;

[0034] - Mixing the first and second compositions in a microfluidic mixing device using the following settings:

[0035] - The total flow rate (FR) is about 0.5 to about 8 ml / min, preferably about 1 to 4 ml / min.

[0036] - The flow rate ratio (FRR) is about 1 / 1 to 5 / 1, preferably about 2 / 1 to about 3 / 1.

[0037] In a specific embodiment, the alcoholic solvent is ethanol and / or the aqueous solvent is water.

[0038] The present invention further provides the use of the LNP, pharmaceutical composition or vaccine according to the present invention in the immunogenic delivery of the one or more nucleic acid molecules.

[0039] Finally, the present invention provides the LNP, pharmaceutical composition or vaccine as described herein for the prevention and / or treatment of cancer or infectious diseases.

[0040] Brief Description of the Drawings

[0041] Now specifically referring to the accompanying drawings, it should be emphasized that the details shown are merely exemplary and are only for the purpose of illustratively discussing different embodiments of the present invention. They are presented 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 has been made to show the structural details of the present invention in more detail than is necessary for a basic understanding of the present invention. The drawings enable those skilled in the art to readily understand how to embody several forms of the present invention in practice.

[0042] Figure 1 : Immunogenicity of 140 nm vs. 230 nm sized mRNA LNPs of the same composition.

[0043] Figure 2 : Immunogenicity of OVA mRNA LNPs of different sizes after intravenous administration.

[0044] Figure 3 : Immunogenicity of OVA mRNA LNPs of different sizes after a single intravenous administration.

[0045] 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).

[0046] Figure 5 : Comparison of the immunogenicity of 140 nm and 230 nm sized mRNA LNPs after one and two intravenous administrations of mRNA LNP.

[0047] Figure 6 : Comparison of the spleen / liver expression ratio of 100 nm and 230 nm sized mRNA LNPs

[0048] Figure 7: Comparison of intravenous LNP mRNA vs. intranodal administration

[0049] Figure 8: The T cell response to LNP mRNA formulations is persistent and augmentable.

[0050] Figure 9 : Compared to LNPs with an average size of only 100 nm, LNPs with an average size of 150 nm or 200 nm are more immunogenic in terms of T cell response. DETAILED DESCRIPTION OF THE INVENTION

[0052] As has been described in detail above, the present invention provides LNPs having a diameter larger than that typically used in the art, and we have surprisingly found that they are very suitable for the immunogenic delivery of nucleic acids, particularly mRNA; and we have found the correlation with enhanced delivery to the spleen. "Immunogenic delivery of a nucleic acid molecule" means delivering a nucleic acid molecule to a cell such that it comes into contact with the cell, and internalization and / or expression of the nucleic acid molecule within the cell results in induction of an immune response.

[0053] Thus, in a first aspect, the present invention provides lipid nanoparticles (LNPs) 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 LNPs is about 140 nm; more preferably about 200 nm.

[0054] 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 an ionizable lipid, a phospholipid, a sterol, and a PEG lipid.

[0055] As used herein, the term "nanoparticle" refers to any particle having a diameter such that the particle is suitable for systemic, particularly intravenous, administration, especially of nucleic acids, and generally has a diameter of less than 1000 nanometers (nm).

[0056] In some embodiments, the average diameter of the nanoparticles is less than 600 nm. In some embodiments, the average diameter of the nanoparticles is less than 400 nm, but in any case, the average diameter of the nanoparticles of the present invention is greater than about 140 nm, more preferably greater than about 200 nm. In a specific embodiment, the LNP of the present invention has a minimum average diameter of about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, or about 200 nm. For clarity, in the case of using a mixture of multiple LNPs, the minimum average diameter refers to the minimum average diameter of the multiple LNPs. In such a case, for example, the mixture may contain some LNPs having an average diameter less than 140 nm, as long as the average diameter of the remaining LNPs is greater than 140 nm such that the average minimum diameter of all the LNPs together is 140 nm. Whenever used in this application, even if not specifically indicated, the term "diameter" means "average diameter".

[0057] In the context of the present invention, the term "ionizable" (or alternatively cationic) in the context of a compound or lipid means that there is any uncharged group present in the compound or lipid that is capable of dissociating by generating ions (usually H + ions) and thus becoming positively charged itself. Alternatively, any uncharged group in the compound or lipid can generate electrons and thus become negatively charged.

[0058] In the context of the present invention, any type of ionizable lipid can be appropriately used. Specifically, suitable ionizable lipids are ionizable amino lipids that comprise two identical or different tails connected by an S-S bond, each of the tails comprising an ionizable amine, such as those shown by the following:

[0059]

[0060] In a specific embodiment, the ionizable lipid is a compound of formula (I):

[0061]

[0062] Wherein:

[0063] RCOO is selected from the list comprising: myristoyl, α-D-tocopherol succinyl, linoleyl, and oleoyl; and

[0064] X is selected from the list comprising:

[0065]

[0066] Such ionizable lipids are specifically represented by any of the following formulas:

[0067]

[0068] More specifically, the ionizable lipid is a lipid of formula (I), wherein RCCO is α-D-tocopheryl succinyl, and X is

[0069]

[0070] such as represented by the following formula:

[0071]

[0072] In the context of the present invention, the term "sterol" (also known as steroid) 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 can be used, such as selected from the list comprising: cholesterol, ergosterol, campesterol, oxysterol, antrosterol, streptosterol, nicasterol, sitosterol, and stigmasterol; preferably cholesterol.

[0073] In the context of the present invention, the term "phospholipid" refers to a lipid molecule composed of two hydrophobic fatty acid "tails" and a hydrophilic "head" composed of a phosphate group. These two components are most often joined together by a glycerol molecule, and thus, in the phospholipids of the present invention, glycero-phospholipids are preferred. In addition, the phosphate group is often modified by simple organic molecules such as choline (i.e., producing phosphocholine) or ethanolamine (i.e., producing phosphoethanolamine).

[0074] 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 (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-docosanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenoyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl succinyl-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-docosahexaenoyl-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-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and mixtures thereof.

[0075] In a more specific embodiment, the phospholipids are selected from the list comprising: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and mixtures thereof.

[0076] 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 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. 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)).

[0077] In a specific embodiment of the present invention, one or more of the following are applied:

[0078] - The LNP accounts for about 35 mol% to 65 mol% of the ionizable lipid;

[0079] - The LNP accounts for about 15 mol% to 60 mol% of the sterol;

[0080] - The LNP accounts for about 0.5 mol% to 2 mol% of the PEG lipid; and / or

[0081] - The LNP accounts for about 5 mol% to 15 mol% of the phospholipid.

[0082] In another specific embodiment of the present invention, one or more of the following are applied:

[0083] - The LNP accounts for about 10 mol% to 60 mol% of the ionizable lipid;

[0084] - The LNP accounts for about 15 mol% to 50 mol% of the sterol;

[0085] - The LNP accounts for about 0.5 mol% to 10 mol% of the PEG lipid; and / or

[0086] - The LNP accounts for about 5 mol% to 40 mol% of the phospholipid.

[0087] In a more specific embodiment, one or more of the following are applied:

[0088] - The LNP accounts for about 40 mol% to 60 mol% of the ionizable lipid;

[0089] - The LNP accounts for about 20 mol% to 40 mol% of the sterol;

[0090] - The LNP accounts for about 0.5 mol% to 5 mol% of the PEG lipid; and / or

[0091] - The LNP accounts for about 5 mol% to 15 mol% of the phospholipid.

[0092] Thus, in one specific embodiment, the LNP accounts for about 10 mol% to 60 mol% of the ionizable lipid; preferably about 40 mol% to 60 mol%.

[0093] In yet another specific embodiment, the LNP accounts for about 15 mol% to 50 mol% of the sterol; preferably about 20 mol% to 40 mol%.

[0094] In another embodiment, the LNP accounts for about 0.5 mol% to 10 mol% of the PEG lipid; preferably about 0.5 mol% to 5 mol%.

[0095] In another specific embodiment, the LNP accounts for about 5 mol% to 40 mol% of the phospholipid; preferably about 5 mol% to 15 mol%.

[0096] Thus, in a more specific embodiment, the LNP of the present invention comprises about 10 mol% to 60 mol% of the ionizable lipid; and / or about 15 mol% to 50 mol% of the sterol; and / or about 0.5 mol% to 10 mol% of the PEG lipid; and / or about 5 mol% to 40 mol% of the phospholipid.

[0097] In another specific embodiment, the LNP of the present invention comprises about 40 mol% to 60 mol% of the ionizable lipid; about 20 mol% to 40 mol% of the sterol; about 0.5 mol% to 5 mol% of the PEG lipid; and about 5 mol% to 15 mol% of the phospholipid.

[0098] The composition of LNP that is particularly suitable in the context of the present invention is listed in Table 1:

[0099] Table 1: Composition of Particularly Suitable LNP

[0100]

[0101] The inventors of the present invention have found that the LNP of the present invention is particularly suitable for the immunogenic delivery of nucleic acids; this is due to the increased spleen targeting compared to small LNPs that mainly target the liver known in the prior art.

[0102] Accordingly, the present invention provides LNPs comprising one or more nucleic acid molecules such as DNA or RNA, more particularly mRNA.

[0103] 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 present invention can be in the form of single-stranded or double-stranded and linear or covalently closed to form a circular molecule. Nucleic acids 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, the RNA can be modified by stabilizing sequences, capping, and polyadenylation prior to application.

[0104] In the context of the present invention, the term "RNA" refers to a molecule comprising ribonucleotide residues and preferably consisting entirely or substantially entirely 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, such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA, which differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide substances to, for example, the ends or interior of the RNA, such as at one or more nucleotides of the RNA. Nucleotides in the RNA molecule can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs. Nucleic acids can be contained in a vector. As used herein, the term "vector" includes any vector known to the person skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes, or analogs of naturally occurring RNA.

[0105] According to the present invention, the term "RNA" includes and preferably refers to "mRNA", which means "messenger RNA" and refers 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 present invention, RNA is obtained by in vitro transcription or chemical synthesis. In vitro transcription methods are known to the person skilled in the art. For example, there are many commercially available in vitro transcription kits.

[0106] In a specific embodiment, the mRNA molecule is an mRNA molecule encoding an immunomodulatory protein.

[0107] In the context of the present invention, the term "mRNA molecule encoding an immunomodulatory protein" refers to an mRNA molecule encoding a functional protein that modifies antigen-presenting cells, more particularly dendritic cells. Such molecules can be selected from the list comprising: CD40L, CD70, caTLR4, IL-12p70, EL-selectin, CCR7 and / or 4-1BBL, ICOS-L, 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 (i.e., "DiMix"). In another preferred embodiment, a combination of CD40L, CD70 and caTLR4 immunostimulatory molecules is used, which is also referred to herein as "TriMix".

[0108] In another specific embodiment, the mRNA molecule is an mRNA molecule encoding an antigen and / or a disease-specific protein.

[0109] According to the present invention, the term "antigen" includes any molecule, preferably a peptide or a protein, which contains at least one epitope that will elicit an immune response and / or to which the immune response is directed. Preferably, in the context of the present invention, an antigen is a molecule that optionally induces an immune response after processing, which immune response is preferably specific for the antigen or the cell expressing the antigen. In particular, an "antigen" refers to such a molecule that is optionally presented by MHC molecules after processing and reacts specifically with T lymphocytes (T cells).

[0110] In a specific embodiment, the antigen is a target-specific antigen, which can be a tumor antigen, or a bacterial, viral or fungal antigen. The target-specific antigen can 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 the target-specific antigen or its derived peptides.

[0111] To avoid any misunderstanding, the LNPs of the present invention can contain a single mRNA molecule, or they can contain multiple mRNA molecules, such as a combination of one or more mRNA molecules encoding an immunomodulatory protein and / or one or more mRNA molecules encoding an antigen and / or a disease-specific protein.

[0112] 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 comprise mRNA molecules encoding the immunostimulatory molecules CD40L, CD70, and / or caTLR4 (e.g., Dimix or Trimix); in combination with one or more mRNA molecules encoding antigen-specific proteins and / or disease-specific proteins. Thus, in very specific embodiments, the LNPs of the present invention comprise mRNA molecules encoding CD40L, CD70, and / or caTLR4; in combination with one or more mRNA molecules encoding antigen-specific proteins and / or disease-specific proteins.

[0113] In another aspect, the present invention provides a pharmaceutical composition comprising one or more LNPs as defined herein. Such pharmaceutical compositions are particularly suitable as vaccines. Thus, the present invention also provides a vaccine comprising one or more LNPs according to the present invention.

[0114] In the context of the present invention, the term "vaccine" as used herein refers to any preparation intended to provide an adaptive immune response (antibody and / or T cell response) against a disease. To this end, the vaccines referred to herein comprise at least one mRNA molecule encoding an antigen, wherein the antigen elicits an adaptive immune response. The antigen can be in the form of a microorganism, protein or peptide, or a weakened or killed form of an antigen of a coding nucleic acid. In the context of the present invention, an antigen refers to a protein or peptide recognized as foreign by the immune system of a host, thereby stimulating the production of antibodies against it, with the aim of combating such antigens. Vaccines can be prophylactic (e.g., preventing or reducing the effects of future infections by any natural or "wild" pathogen) or therapeutic (e.g., actively treating or alleviating the symptoms of an ongoing disease). The administration of a vaccine is called vaccination.

[0115] The vaccines of the present invention can be used to induce an immune response, particularly an immune response against disease-related antigens or cells expressing disease-related antigens, such as an immune response against cancer. Thus, the vaccines can be used for the prophylactic and / or therapeutic treatment of diseases involving disease-related antigens or cells expressing disease-related antigens, such as cancer. Preferably, the immune response is a T cell response. In one embodiment, the disease-related antigen is a tumor antigen. The antigen encoded by the RNA contained in the nanoparticles described herein is preferably a disease-related antigen or elicits an immune response against a disease-related antigen or cells expressing a disease-related antigen.

[0116] The LNPs and vaccines of the present invention are particularly contemplated for intravenous administration, i.e., directly infusing a liquid substance into a vein. The intravenous route is the fastest way to deliver liquids and drugs throughout the body (i.e., systemically). Accordingly, the present invention provides intravenous vaccines, as well as the use of the disclosed vaccines and LNPs for intravenous administration. Thus, the vaccines and LNPs of the present invention can be administered intravenously. The present invention also provides the use of the vaccines and LNPs according to the present invention; wherein the vaccine is administered intravenously.

[0117] The present invention also provides the LNPs, pharmaceutical compositions and vaccines according to the present invention for human or veterinary medicine. The use of the LNPs, pharmaceutical compositions and vaccines of the present invention for human or veterinary medicine is also contemplated. Finally, the present invention provides a method for preventing and treating human and veterinary diseases by administering the LNPs, pharmaceutical compositions and vaccines according to the present invention to a subject in need thereof.

[0118] The present invention further provides the use of the LNPs, pharmaceutical compositions or vaccines according to the present invention for immunogenic delivery of said one or more nucleic acid molecules. This effect is particularly achieved using the larger LNPs of the present invention because they preferentially target the spleen. It has been found that the small-sized LNPs of the prior art preferentially target the liver and thus have little or no immunogenic effect. Accordingly, the LNPs, pharmaceutical compositions and vaccines of the present invention are very useful in treating several human and veterinary diseases. Thus, the present invention provides the LNPs, pharmaceutical compositions and vaccines of the present invention for treating cancer or infectious diseases.

[0119] In another aspect, the present invention provides a method for preparing the LNPs according to the present invention, comprising:

[0120] - preparing a first alcoholic composition comprising the ionizable lipid, the phospholipid, the sterol, the PEG lipid and a suitable alcoholic solvent;

[0121] - preparing a second aqueous composition comprising the one or more nucleic acids and an aqueous solvent;

[0122] - mixing the first and second compositions in a microfluidic mixing device using the following settings:

[0123] - a total flow rate (FR) of about 0.5 to about 8 ml / min, preferably about 1 to 4 ml / min.

[0124] - a flow rate ratio (FRR) of about 1 / 1 to 5 / 1, preferably about 2 / 1 to about 3 / 1.

[0125] More specifically, the lipid components are combined at a suitable concentration in an alcoholic vehicle such as ethanol. Additionally, an aqueous composition containing the nucleic acid is added and then loaded into a microfluidic mixing device.

[0126] 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 typically achieved by enhancing the diffusion effect between different material flows. Additionally, 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.

[0127] The mixing parameters of such microfluidic mixing devices have a great impact on the characteristics of the obtained LNPs, especially on their size. Therefore, for obtaining 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 relevant.

[0128] The total FR (Flow Rate) is a measure of the speed of microfluidic mixing and is typically set to 8 - 12 ml / min in the prior art to allow sufficient 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 the present invention.

[0129] The FRR (flow rate ratio) is the ratio of lipid in the alcohol phase to nucleic acid in the aqueous phase and is typically set to about 2 / 1 to about 3 / 1 in the present invention. Examples

[0130] Example 1.

[0131] In this first example, the effect of particle size on the immunogenicity of mRNA LNPs was evaluated. For this purpose, mRNA LNPs with sizes of 140 nm and 230 nm of the same composition were prepared. The LNPs contained 50 mol% of α-D-tocopherol hemisuccinate (ionizable lipid), 10 mol% of DOPE, 38.5 mol% of cholesterol, and 1.5 mol% of DSG-PEG5000 (PEGylated lipid) at a specified lipid ratio (mol%). The LNPs were produced on a Nano-Assemblr using different flow rates (FR; ml / min) and water / ethanol volume ratios (FRR). LNPs with a size of 140 nm were produced at FR 4 ml / min, while LNPs with a size of 230 nm were produced at FR 1 ml / min.

[0132] 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. From Figure 1It can be clearly seen that the immunogenicity of larger LNPs is much higher compared to that of smaller LNPs.

[0133] Example 2.

[0134] In this example, the immunogenicity of OVA mRNA LNPs of different sizes and compositions in mice after intravenous administration was determined. As Figure 2 defined, all LNPs contained α-D-tocopherol hemisuccinate (ionizable lipid), DOPE, cholesterol, and DSG-PEG5000 at the specified lipid ratios (mol%). Also as Figure 2 defined, the LNPs were produced on a 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, it can be clearly seen from Figure 2 that the immunogenicity of larger LNPs is higher compared to that of smaller LNPs.

[0135] Example 3.

[0136] In this example, the immunogenicity of OVA mRNA LNPs of different sizes and compositions in mice after a single intravenous administration was determined. The graph depicts the number of CD8+ T cells secreting IFN-γ per 10 6 spleen cells after restimulation of spleen cells with the OVA MHC I epitope SIINFEKL (5 μg / ml). As Figure 3 defined, all LNPs contained α-D-tocopherol hemisuccinate (ionizable lipid), DOPE, cholesterol, and DSG-PEG5000 at the specified lipid ratios (mol%). Also as Figure 3 defined, the LNPs were produced on a 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.

[0137] Example 4.

[0138] Next, we evaluated the effect of changing the PEGylated lipid. For this purpose, two different compositions were prepared with 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 4In the middle. Mice were immunized on day 0 and day 20. Blood samples on day 15 and day 39 were evaluated for the percentage of OVA-specific T cells. From Figure 4 It can be clearly seen that the type of polyethylene glycolated lipid has no effect on the immunogenicity of LNP.

[0139] Example 5.

[0140] In this example, we evaluated the immunogenicity of small and large LNPs after repeated immunization using mRNA LNPs with sizes of 140 nm and 230 nm ( Figure 5 ). The LNP contains 50 mol% of α-D-tocopherol hemisuccinate (ionizable lipid), 10 mol% of DOPE (phospholipid), 38 mol% of cholesterol, and 1.5 mol% of DSG-PEG5000 at the specified lipid ratio (mol%), as Figure 5 defined. The LNP contains 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.

[0141] Example 6.

[0142] In this example, we compared the mRNA expression in the spleen and liver after intravenous injection of mice with Fluc mRNA LNPs of different sizes ( Figure 6 ). The LNP contains α-D-tocopherol hemisuccinate (ionizable lipid), DOPC, cholesterol, and DSG-PEG5000 at a lipid ratio of 50 / 10 / 38.5 / 1.5 respectively. LNPs of different sizes were generated by microfluidic mixing at different flow rates (FR1 vs FR4). The figure depicts the ratio of spleen to liver expression of Fluc mRNA measured by in vivo bioluminescence in ph / s / cm 2 / sr.

[0143] Example 7.

[0144] In this example, we demonstrated that formulated intravenous mRNA LNPs elicit better immunogenicity and anti-tumor efficacy compared to intranodal methods.

[0145] The characteristics of the API used are as follows:

[0146] - E7 mRNA, column purified, 10 μg / dose

[0147] - Immunization protocol: 3 times a week

[0148] The results of this experiment are shown in Figure 7, which clearly shows that, in terms of both immunogenicity and anti-tumor efficacy, the intravenous method using mRNA LNP is superior to the intranodal method. In addition, there appears to be a correlation between anti-tumor efficacy and the magnitude of the T cell response.

[0149] Example 8.

[0150] In this example, we demonstrated that the T cell response to the mRNA LNP formulation is persistent and can be enhanced.

[0151] For this purpose, three different formulations were used in the immunization protocol, and the results are as Figure 8 shown. Based on these results, the following observations / conclusions can be drawn:

[0152] - An extended contraction phase with a high percentage of E7-specific T cells 40 days after immunization 3

[0153] - Memory conversion

[0154] - Excellent recall response: The intravenous formulation vaccine can be boosted.

[0155] Example 9.

[0156] In this example, further experiments were conducted, which demonstrated the effect of the size of the LNP.

[0157] For this purpose, the following experimental setup was used:

[0158] - Ratio of ionizable lipid / cholesterol / DOPE / DMG-PEG2000 – 50 / 38.5 / 10 / 1.5

[0159] - Three intravenous immunizations; days 0, 7, 14

[0160] - mRNA: mRNA encoding OVA, 10 μg / immunization

[0161] - Percentage of OVA-specific CD8 T cells was evaluated on days 5, 12, 19, and 50 after the first immunization

[0162] The results are detailed in Figure 9 and clearly show that, compared to LNPs with an average size of only 100 nm, LNPs with an average size of 150 nm or 200 nm are significantly more immunogenic in terms of T cell response.

[0163] References

[0164] Reichmuth et al., 2016 - mRNA vaccine delivery using lipid nanoparticles - Therapeutic Delivery Vol.7, N°5.

[0165] Li et al., 2015 - An Orthogonal Array Optimization of Lipid - like Nanoparticles for mRNA Delivery in Vivo - Nano Letters 15(12) pg.8099 - 8107.

[0166] 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.

[0167] Kauffman et al., 2016 - Materials for non - viral intracellular delivery of messenger RNA therapeutics - Journal of Controlled Release, Vol.240, pg.227 - 234.

[0168] Richner et al., 2017 - Modified mRNA vaccines protect against Zika virus infection - Cell, Vol.168, Issue 6 pg.1114 - 1125

[0169] 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.

[0170] Lee et al., 2011 - Microfluidic Mixing: A Review - Int. J. Mol. Sci. 12(5): pg. 3263 - 3287.

Claims

1. A lipid nanoparticle (LNP) comprising: - Ionizable lipid; - From about 5 mol% to about 15 mol% of a phospholipid selected from the list consisting of DOPE, DOPC, and mixtures thereof; - Sterol; - PEG lipid; and - One or more nucleic acid molecules; Characterized in that, The LNP has a minimum average diameter of about 140 nm.

2. The LNP according to claim 1, wherein the ionizable lipid is a compound of formula (I): Wherein: RCOO is selected from the list consisting of: myristoyl, α-D-tocopherol succinyl, linoleoyl, and oleoyl; and X is selected from the list consisting of: Preferably, the ionizable lipid is a lipid of formula (I) wherein RCCO is α-D-tocopherol succinyl, and X is 3. The LNP according to any one of claims 1-2, wherein the sterol is selected from the list consisting of: cholesterol, ergosterol, campesterol, oxysterol, antrosterol, streptosterol, nicasterol, sitosterol, and stigmasterol; preferably cholesterol.

4. The LNP according to any one of claims 1-3, wherein the PEG lipid is selected from the list consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

5. The LNP according to any one of claims 1-4, wherein the LNP accounts for about 10 mol% to 60 mol% of the ionizable lipid and / or the LNP accounts for about 15 mol% to 50 mol% of the sterol.

6. The LNP according to any one of claims 1-5, wherein the one or more nucleic acid molecules are selected from the list consisting of: mRNA and DNA, preferably mRNA.

7. The LNP according to claim 6, wherein the one or more mRNA molecules are selected from the list consisting of: immunomodulatory polypeptide-encoding mRNA and / or antigen-encoding mRNA.

8. The LNP according to any one of claims 6-7, wherein the immunomodulatory-encoding mRNA is selected from the list consisting of: mRNA molecules encoding (including a broader list) CD40L, CD70, and caTLR4.

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

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

11. A method for preparing an LNP containing nucleic acid having a minimum average diameter of about 140 nm, the method comprising the steps of: - Preparing a first alcoholic composition comprising an ionizable lipid, a phospholipid, a sterol, a PEG lipid, and a suitable alcoholic solvent; - Preparing a second aqueous composition comprising one or more nucleic acids and an aqueous solvent supplemented with a suitable buffer; - Mix the first and second compositions in a microfluidic mixing device using the following settings: - The total flow rate (FR) is from about 0.5 to about 8 ml / min, preferably from about 1 to 4 ml / min. - The flow rate ratio (FRR) is from about 1 / 1 to 5 / 1, preferably from about 2 / 1 to about 3 / 1.

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

13. Use of the LNP according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 for inducing an immune response against an antigen encoded by a nucleic acid in a mammal.

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

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