Immunogenic mRNA delivery vehicle

Abstract

The present disclosure relates to a lipid-based delivery vehicle for mRNA vaccines, including a lysophosphatidylcholine (LPC) compound for enhancing vaccine immunogenicity. The present disclosure also relates to a method for the use of mRNA vaccines. Although the rapid production of some effective mRNA COVID-19 vaccines has been possible, lipid-based formulations for the delivery of mRNA vaccines remain to be improved. In particular, more immunogenic and / or less reactogenic formulations may be provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 307,578, filed February 7, 2022, the entirety of which is incorporated herein by reference.

[0002] Field The present disclosure relates to a lipid-based delivery vehicle for mRNA vaccines, comprising a lysophosphatidylcholine (LPC) compound for enhancing vaccine immunogenicity. The present disclosure also relates to methods for the use of mRNA vaccines. [Background technology]

[0003] background mRNA vaccines have several advantages over conventional vaccines. For example, they can be rapidly developed, inexpensively produced, and safely administered (Pardi et al., Nat Reg Drug Discov, 17(4):261-279, 2018). However, naked mRNA is unstable and rapidly degrades after administration. Fortunately, advances in mRNA chemistry and delivery systems have enabled the rapid generation of several effective mRNA COVID-19 vaccines (Hou et al., Nature Review Materials, 6:1078-1094, 2021). Nonetheless, there is room for improvement in lipid-based formulations for the delivery of mRNA vaccines, specifically, more immunogenic and / or less reactogenic formulations would be desirable. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Pardi et al., Nat Reg Drug Discov, 17(4):261-279, 2018 [Non-Patent Document 2] Hou et al., Nature Review Materials, 6:1078-1094, 2021 Summary of the Invention [Means for solving the problem]

[0005] A brief summary The present disclosure relates to a lipid-based delivery vehicle for mRNA vaccines, comprising a lysophosphatidylcholine (LPC) compound for enhancing vaccine immunogenicity. The present disclosure also relates to methods for the use of mRNA vaccines. [Brief description of the drawings]

[0006] [Figure 1] Figures 1A-B show that lipid nanoparticles (LNPs) loaded with both 22:0 Lyso PC and mRNA remain nanoparticle size (<125 nm) and relatively uniform in diameter (PDI<0.3). Figure 1A shows that the mRNA-loaded LNPs are larger than LNPs lacking mRNA, and that the addition of 22:0 LPC to the LNPs increases the size of the mRNA-loaded LNPs. Figure 1B shows that all formulations tested contain LNPs with relatively uniform sizes.

[0007] [Figure 2-1]Figures 2A-2F show that loading LNPs with 22:0 Lyso PC increases the immunogenicity of LNP formulations by enabling hyperactivation of human monocyte-derived dendritic cells (moDCs). The viability of human moDCs cultured with LNPs loaded with 50 μM or 100 μM 22:0 Lyso PC compared to human moDCs treated with R848 alone (an exemplary PAMP) is shown in Figures 2A and 2B, respectively. IL-1β secretion by human moDCs cultured with LNPs loaded with 50 μM or 100 μM 22:0 Lyso PC, with or without the addition of R848, is shown in Figures 2C and 2D, respectively. IL-6 secretion by human moDCs cultured with LNPs loaded with 50 μM or 100 μM 22:0 Lyso PC, with or without the addition of R848, is shown in Figures 2E and 2F, respectively. LNPs were prepared with or without mRNA encoding GFP and with various levels of 22:0 Lyso PC (0%, 30%, 40% molar ratio of 22:0 Lyso PC in LNP). LNPs without 22:0 Lyso PC (LNP0) were administered to obtain similar total lipid levels as LNPs loaded with 22:0 Lyso PC. The mRNA dose was similar across formulation types (approximately 5 μg / mL). [Figure 2-2] Same as above.

[0008] [Diagram 3]Figures 3A-3D show that loading of mRNA-containing LNPs with 22:0 Lyso PC does not prevent translation of the mRNA. Green fluorescent protein (GFP) expression was assessed as the percentage of GFP-positive moDCs after 48 h of culture in the presence of LNPs loaded with 50 μM or 100 μM 22:0 Lyso PC, as shown in Figures 3A and 3B, respectively. GFP expression was also assessed as median fluorescence intensity (MFI) in moDCs after 48 h of culture in the presence of LNPs loaded with 50 μM or 100 μM 22:0 Lyso PC, as shown in Figures 3C and 3D, respectively. LNPs were prepared with or without mRNA encoding GFP, and LNPs were prepared with various levels of 22:0 Lyso PC (0%, 30%, and 40% molar ratio of 22:0 Lyso PC in LNPs). LNPs without 22:0 Lyso PC (LNP0) were administered to obtain similar total lipid levels as LNPs loaded with 22:0 Lyso PC. The mRNA dose was similar across formulation types (approximately 5 μg / mL).

[0009] [Figure 4-1] Figures 4A-4H show that loading of 22:0 Lyso PC into LNPs containing mRNA increases the expression of activation markers and antigen-presenting molecules by moDCs. Specifically, loading of 22:0 Lyso PC into LNPs increases CD86 expression (Figures 4A-4B), CD40 expression (Figures 4C-4D), HLA-DR expression (Figures 4E-4F), and HLA-ABC expression (Figures 4G-4H) in moDCs treated with 50 μM or 100 μM 22:0 Lyso PC. LNPs were prepared with various levels of 22:0 Lyso PC (0%, 30%, and 40% molar ratio of 22:0 Lyso PC in LNPs). LNPs without 22:0 Lyso PC (LNP0) were administered to obtain similar total lipid levels as LNPs loaded with 22:0 Lyso PC. The mRNA dose was similar across formulation types (approximately 5 μg / mL). [Figure 4-2] Same as above.

[0010] [Diagram 5] Figures 5A-5B show that LNPs loaded with 22:0 Lyso PC and OVA mRNA promote activation of naive T cells (OT-I T cells) from transgenic mice expressing T cell receptors that recognize the OVA epitope. Figure 5A shows IL-1β secretion by mouse bone marrow-derived dendritic cells (BMDCs) cultured with LNPs loaded with 50 μM or 100 μM 22:0 Lyso PC and 0, low, or high amounts of OVA mRNA in the presence or absence of R848. Figure 5B shows IFNγ secretion by OVA-specific OT-I T cells cocultured for 72 hours with BMDCs containing OVA mRNA and hyperactivated for 48 hours with LNPs loaded with 50 μM 22:0 Lyso PC, with or without the addition of R848. LNPs were prepared with various levels of 22:0 Lyso PC (0% and 40% molar ratio of 22:0 Lyso PC in LNPs). LNPs without 22:0 Lyso PC (LNP0) were administered to obtain similar total lipid levels as LNPs loaded with 22:0 Lyso PC. OVA mRNA doses tested included 0, a low dose (~0.25 μg / mL), and a high dose (~2.5 μg / mL).

[0011] [Figure 6]Figures 6A-B show that LNPs loaded with 22:0 Lyso PC and OVA mRNA reactivate OVA-specific T cells from mice pre-immunized against OVA. Figure 6A shows IFNγ secretion by T cells co-cultured with BMDCs pre-exposed to OVA and overactivated with LNPs loaded with 50 μM 22:0 Lyso PC for 24 h, with or without R848 for 96 h. Figure 6B shows IFNγ secretion by T cells co-cultured with BMDCs pre-exposed to OVA and overactivated with LNPs loaded with 50 μM 22:0 Lyso PC for 48 h, with or without R848 for 96 h. LNPs were prepared with various levels of 22:0 Lyso PC (0% and 40% molar ratio of 22:0 Lyso PC in LNPs). LNPs without 22:0 Lyso PC (LNP0) were administered to obtain similar total lipid levels as LNPs loaded with 22:0 Lyso PC. OVA mRNA doses tested included 0, a low dose (~0.25 μg / mL), and a high dose (~2.5 μg / mL). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description The present disclosure relates to a lipid-based delivery vehicle for mRNA vaccines, comprising a lysophosphatidylcholine (LPC) compound for enhancing vaccine immunogenicity. The present disclosure also relates to methods for the use of mRNA vaccines. General Techniques and Definitions

[0013] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of one in the art.

[0014] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless specifically stated otherwise. For example, "an" excipient includes one or more excipients.

[0015] The phrase "comprising," as used herein, is open ended and indicates that such embodiments may include additional elements. In contrast, the phrase "consisting of" is closed and indicates that such embodiments do not include additional elements (except for trace impurities). The phrase "consisting essentially of" is partially closed and indicates that such embodiments may include additional elements that do not substantially alter the basic characteristics of such embodiments.

[0016] The term "about" when used herein in reference to a value includes 90% to 110% of that value (e.g., a molecular weight of about 900 daltons refers to a molecular weight of 810 daltons to 990 daltons).

[0017] An "effective amount" or "sufficient amount" of a substance is an amount sufficient to produce beneficial or desired results, including clinical results, and thus the "effective amount" depends on the context in which it is applied. For example, in the context of administering an immunogenic composition, an effective amount contains sufficient antigen to stimulate an immune response to the antigen (e.g., antigen-reactive antibody and / or cellular immune response), and one or both of a lysophosphatidylcholine (LPC) compound and a PRR agonist.

[0018] The terms "individual" and "subject" refer to a mammal. "Mammals" include, but are not limited to, humans, non-human primates (e.g., monkeys), farm animals, sport animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats). In some embodiments, the subject is a human patient, e.g., a human patient suffering from cancer and / or an infectious disease.

[0019] The term "dose," as used herein in reference to an immunogenic composition, refers to a measured portion of an immunogenic composition that is taken by (administered to or received by) a subject at any one time.

[0020] The terms "isolated" and "purified" as used herein refer to a material that is removed from at least one component with which it is naturally associated (e.g., removed from its original environment). By way of example, when used in reference to an LPC, an isolated LPC is at least 90%, 95%, 96%, 97%, 98% or 99% pure as determined by thin layer chromatography or gas chromatography. As a further example, when used in reference to a recombinant protein, an isolated protein refers to a protein that has been removed from the culture medium of a host cell that produced the protein.

[0021] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is present in a form that is effective for the biological activity of the active ingredient and does not contain additional components that would be unacceptably toxic to the individual to whom the formulation or composition may be administered. Such formulations or compositions are intended to be sterile.

[0022] "Excipient" as used herein includes a pharma- ceutically acceptable excipient, carrier, vehicle, or stabilizer that is non-toxic to cells or mammals exposed at the dosages and concentrations used. Often the physiologically acceptable excipient is an aqueous pH buffered solution.

[0023] The term "antigen" refers to a substance that is specifically recognized and bound by an antibody or T cell antigen receptor. Antigens can include peptides, polypeptides, proteins, glycoproteins, polysaccharides, complex carbohydrates, sugars, gangliosides, lipids and phospholipids, portions thereof and combinations thereof. Antigens, when present in the compositions of the present disclosure, can be synthesized or isolated from nature. Antigens suitable for administration in the methods of the present disclosure include any molecule that can induce an antigen-specific B cell or T cell response. Haptens are included within the scope of "antigens." A "hapten" is a low molecular weight compound that is not immunogenic by itself, but is generally made immunogenic when conjugated to a larger immunogenic molecule (carrier).

[0024] A "polypeptide antigen" may include purified native peptides, synthetic peptides, recombinant peptides, crude peptide extracts, or partially purified or unpurified active peptides (e.g., peptides that are part of attenuated or inactivated viruses, microorganisms, or cells), or fragments of such peptides. Polypeptide antigens are preferably at least eight amino acid residues in length.

[0025] The term "agonist" is used in the broadest sense and includes any molecule that activates signal transduction through a receptor. In some embodiments, an agonist binds to a receptor. For example, a TLR8 agonist binds to a TLR8 receptor and activates the TLR8-signal transduction pathway.

[0026] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group. Cx alkyl refers to an alkyl group having x carbon atoms. Cx-Cy alkyl or Cx-y alkyl refers to an alkyl group having between x and y carbon atoms, inclusive.

[0027] "Alkylene" refers to a divalent saturated aliphatic hydrocarbyl group.

[0028] "Alkenyl" refers to a monovalent hydrocarbyl group having at least one double bond (>C=C<). Cx alkenyl refers to an alkenyl group having x carbon atoms. Cx-Cy alkenyl or Cx-y alkenyl refers to an alkenyl group having between x and y carbon atoms, inclusive.

[0029] "Stimulation" of a response or parameter includes eliciting and / or enhancing that response or parameter when compared to the same conditions except for the parameter of interest, or alternatively when compared to another condition (e.g., increased TLR signaling in the presence of a TLR agonist compared to the absence of a TLR agonist). For example, "stimulation" of an immune response refers to an increase in the response. The increase can be 2-fold to 2,000-fold, or 5-fold to 500-fold or more, or 2, 5, 10, 50, or 100-fold to 500, 1,000, 2,000, 5,000, or 10,000-fold, depending on the parameter being measured.

[0030] Conversely, "inhibition" of a response or parameter includes reducing and / or suppressing that response or parameter when compared to the same conditions except for the parameter of interest or alternatively when compared to another condition (e.g., a reduction in abnormal cell proliferation after administration of a composition comprising an LPC compound and one or more of a pathogen recognition receptor agonist, an antigen, and human dendritic cells compared to administration of a placebo composition or no treatment). For example, "inhibition" of an immune response refers to a reduction in the response. The reduction can be 2-2,000-fold, or 5-500-fold or less, or 2, 5, 10, 50, or 100-fold to 500, 1,000, 2,000, 5,000, or 10,000-fold, depending on the parameter being measured.

[0031] The relative terms "higher" and "lower" refer to a measurable increase or decrease in a response or parameter, respectively, when compared to the same conditions except for the parameter of interest, or alternatively, compared to another condition. For example, "higher levels of DC hyperactivation" refers to a level of DC hyperactivation resulting from a treatment condition (including the LPC compounds of the present disclosure) that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than the level of DC hyperactivation resulting from a control condition (e.g., without LPC, PGPC, oxPAPC, etc.). Similarly, "lower levels of DC hyperactivation" refers to a level of DC hyperactivation resulting from a treatment condition (including the LPC compounds of the present disclosure) that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times lower than the level of DC hyperactivation resulting from a control condition (e.g., without LPC, PGPC, oxPAPC, etc.).

[0032] As used herein, the term "immunization" refers to the process of increasing a mammalian subject's response to an antigen, thus improving its ability to resist or overcome infection and / or resist disease.

[0033] The term "vaccination," as used herein, refers to the introduction of a vaccine into the body of a mammalian subject.

[0034] "Adjuvant" refers to a substance that, when added to a composition containing an antigen, enhances or potentiates the immune response to the antigen in a mammalian recipient upon exposure.

[0035] The term "treating" a disease or "treatment" of a disease refers to carrying out a protocol that may include administering one or more therapeutic agents to an individual (human or non-human) with the goal of obtaining a beneficial or desired result, including a clinical outcome, in the individual. Beneficial or desired clinical outcomes include, but are not limited to, alleviation or amelioration of one or more signs or symptoms of the disease, reduction in the extent of the disease, stabilization of the disease state (i.e., not worsening), prevention of the spread of the disease, delay or slowing of the progression of the disease, improvement or alleviation of the disease state, and remission (whether partial or complete). "Treatment" can also mean extending survival compared to the expected survival of an individual not receiving treatment. Furthermore, "treating" and "treatment" can occur by administration of a dose of one or more therapeutic agents, or can occur upon administration of a series of doses of one or more therapeutic agents. "Treatment" or "treatment" does not require complete relief of signs or symptoms, does not require a cure, and specifically includes protocols that have only a palliative effect on the individual. "Ameliorating" a disease or disorder means reducing the extent and / or undesirable clinical symptoms of the disease or disorder and / or slowing the time course of progression of the disease or disorder compared to the expected untreated outcome. I. Lysophosphatidylcholine Compounds

[0036] "Lysophosphatidylcholine" (LPC) or "lysophosphatidylcholine molecule" refers to a glycerol molecule bearing one phosphocholine group on a hydroxyl group of glycerol and one acyl group on one of the other two hydroxyl groups of glycerol. The remaining hydroxyl group is unsubstituted.

[0037] In some embodiments, the isolated lysophosphatidylcholine (LPC) having a single acyl chain has the form [ka] [ka] It is of the following.

[0038] In some embodiments, the isolated lysophosphatidylcholine (LPC) having a single acyl chain has the form [ka] It is of the following.

[0039] The alkyl or alkenyl chain, together with the carbonyl carbon, forms an acyl chain that is one carbon atom longer than the alkyl or alkenyl chain. For example, a (C23 alkyl)-C(=O)- group forms a C24 acyl chain. Thus, if the "(alkyl or alkylene)" group is a C12-C23 alkyl group (e.g., a C12-C19 alkyl group or a C20-C23 alkyl group), the (C12-C23 alkyl-C(=O)- group forms a C13-C24 acyl chain (e.g., a C13-C20 acyl chain or a C21-C24 acyl chain). If the "(alkyl or alkylene)" group is a C12-C23 alkenyl group (e.g., a C12-C19 alkenyl group or a C20-C23 alkyl group), the (C12-C23 alkyl-C(=O)- group forms a C13-C24 acyl chain (e.g., a C13-C20 acyl chain or a C21-C24 acyl chain). 3 alkenyl group), the (C12-C23 alkenyl-C(=O)- group forms a C13-C24 acyl chain (e.g., a C13-C20 acyl chain or a C21-C24 acyl chain). The acyl chain can be referred to as saturated or unsaturated acyl to distinguish between alkyl- and alkenyl-containing acyl groups. Standard delta or omega designations can be used to indicate the position of the double bond or bonds in the unsaturated acyl chain.

[0040] The lysophosphatidylcholine (LPC) compounds of the present disclosure have a single acyl chain, where the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl chain. In some embodiments, the acyl chain is a C18-C22 acyl chain or a C21-C24 acyl chain. In some preferred embodiments, the acyl chain is a C22 acyl chain. The names and structures of exemplary LPC compounds for inclusion in the LNPs of the present disclosure, as well as their Chemical Abstracts Service (CAS) registry numbers, are listed as compound numbers 30-43, and optionally numbers 30-42, in Table I of International Application PCT / US2022 / 071664, which is incorporated herein by reference. Several methods for synthesizing lysophospholipids are known (see, e.g., D'Arrigo et al, "Synthesis of lysophospholipids," Molecules, 15(3):1354-77, 2010 and Yang et al., "Lysophosphatidylcholine synthesis by lipase-catalyzed ethanolysis," J Oleo Sci., 64(4):443-7, 2015, and references cited therein). In addition, many lysophospholipids are commercially available. II. Pathogen Recognition Receptor Agonists

[0041] The compositions and methods of the present disclosure may further comprise a pathogen recognition receptor (PRR) agonist. In some embodiments, the PRR agonist comprises a toll-like receptor (TLR), a NOD-like receptor (NLR), a RIG-I-like receptor (RLR), or a C-type lectin receptor (CLR) agonist. In other embodiments, the PRR agonist comprises a cytoplasmic DNA sensor (CDS) or a stimulator of IFN genes (STING). In some embodiments, the PRR agonist comprises a TLR7 / 8 agonist. A. TLR7 / 8 agonists

[0042] The term "TLR7 / 8 agonist" as used herein refers to an agonist of TLR7 and / or TLR8. In one embodiment, the TLR7 / 8 agonist is a TLR7 agonist. In another embodiment, the TLR7 / 8 agonist is a TLR8 agonist. In a further embodiment, the TLR7 / 8 agonist is an agonist of both TLR7 and TLR8. The TLR7 / 8 agonists of the present disclosure are suitable for hyperactivating human dendritic cells in the presence of LPC.

[0043] In some aspects, the TLR7 / 8 agonist is a small molecule. In some embodiments, the TLR7 / 8 agonist is a small molecule or salt thereof having a molecular weight of 900 Daltons or less. That is, the small molecule TLR7 / 8 agonist is not a large molecule such as a recombinant protein or a synthetic oligonucleotide regulable by the Center for Biologics Evaluation and Research of the U.S. FDA. Rather, the small molecule TLR7 / 8 agonist is regulable by the Center for Drug Evaluation and Research of the FDA. In some embodiments, the small molecule has a molecular weight of about 90 to about 900 Daltons. In some embodiments, the TLR7 / 8 agonist comprises an imidazoquinoline compound. In some preferred embodiments, the TLR7 / 8 agonist comprises resiquimod (R848). B. Other PRR agonists

[0044] In some aspects, the pathogen recognition receptor (PRR) agonist comprises a toll-like receptor (TLR) agonist, provided that the TLR agonist does not comprise a TLR7 / 8 agonist. In some embodiments, the TLR agonist comprises one or more agonists of TLR2, TLR3, TLR4, TLR5, TLR9 and TLR13. In some embodiments, the PRR agonist is a TLR2 / 6 agonist, e.g., Pam2CSK4. In other embodiments, the TLR agonist is a TLR4 agonist, e.g., monophosphoryl lipid A (MPLA). However, in preferred embodiments, the TLR agonist is not an agonist of TLR2, TLR4 and / or TLR9. For example, in preferred embodiments, the TLR9 agonist is not a TLR4 ligand, e.g., LPS (endotoxin).

[0045] In other embodiments, the PRR agonist comprises a NOD-like receptor (NLR) agonist. In further embodiments, the PRR agonist comprises a RIG-I-like receptor (RLR) agonist. In further embodiments, the PRR agonist comprises a C-type lectin receptor (CLR) agonist. In yet further embodiments, the PRR agonist comprises a CDS agonist or a STING agonist. III. Antigen-encoding mRNA

[0046] The compositions and methods of the present disclosure are suitable for use with formulations that include an mRNA encoding an antigen or that otherwise include an mRNA encoding an antigen. In some embodiments, the antigen is a protein antigen. The terms "polypeptide" and "protein" are used interchangeably herein to refer to an antigen that includes a peptide chain at least 8 amino acids in length. In some embodiments, the antigen is 8-1800 amino acids, 9-1000 amino acids, or 10-100 amino acids in length. The polypeptide may be post-translationally modified, for example, by phosphorylation, hydroxylation, sulfonation, palmitoylation, and / or glycosylation.

[0047] In some embodiments, the antigen is a tumor antigen comprising at least one full-length protein amino acid sequence or a fragment thereof. In some embodiments, the tumor antigen comprises an amino acid sequence or a fragment thereof from an oncoprotein. In some embodiments, the mammalian antigen is a neoantigen or is encoded by a gene comprising a mutation relative to a gene present in normal cells from a mammalian subject. Neoantigens are believed to be particularly useful in enabling T cells to distinguish between cancer cells and non-cancer cells (see, e.g., Schumacher and Schreiber, Science, 348:69-74, 2015). In other embodiments, the tumor antigen comprises a viral antigen, e.g., an antigen of a virus that causes cancer.

[0048] In some embodiments, the tumor antigen is a fusion protein comprising two or more polypeptides, where each polypeptide comprises an amino acid sequence from a different tumor antigen or a non-contiguous amino acid sequence from the same tumor antigen. In some of these embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, where each polypeptide comprises a non-contiguous amino acid sequence from the same tumor antigen.

[0049] In some embodiments, the antigen is a microbial antigen. In some embodiments, the microbial antigen comprises a viral antigen, a bacterial antigen, a protozoan antigen, a fungal antigen, or a combination thereof. In some embodiments, the microbial antigen comprises a surface protein or other antigenic subunit of a microorganism.

[0050] In some preferred embodiments, the mRNA comprises a 5' untranslated region (5'UTR) at the 5' end of the coding region and a 3' untranslated region (3'UTR) at the 3' end of the coding region. In some preferred embodiments, the mRNA comprises one or both of a 5' cap structure and a polyA tail. IV. Lipid-Based Delivery Vehicles

[0051] The compositions and methods of the present disclosure include lipid-based delivery vehicles for mRNA vaccines. In some embodiments, the vehicle is a lipid nanoparticle (LNP). In other embodiments, the vehicle is a lipid that forms a complex with mRNA (RNA-Lipoplex).

[0052] In some embodiments, the LNPs comprise a first phospholipid (lysophosphatidylcholine with a single C13-C24 acyl chain [LPC:C13-C24] and at least one lipid selected from the group consisting of an ionizable lipid, a cationic lipid, a second phospholipid, a PEGylated lipid, a structural lipid, and mixtures thereof. In some embodiments, at least one lipid comprises an ionizable lipid. In some embodiments, at least one lipid comprises a cationic lipid. In some embodiments, at least one lipid comprises a second phospholipid. In some embodiments, at least one lipid comprises a PEGylated lipid. In some embodiments, at least one lipid comprises a structural lipid. In some embodiments, at least one lipid comprises an ionizable lipid, a second phospholipid, a PEGylated lipid, and a structural lipid.

[0053] In some embodiments, the lipid component of RNA-Lipoplex comprises one or more lipids.In some preferred embodiments, the one or more lipids comprise a first lipid and a second lipid, where the first lipid is different from the second lipid.In some embodiments, the first lipid is a cationic lipid, and the second lipid is a neutral or anionic lipid.

[0054] The structure of a lipid suitable for use in the lipid-based mRNA delivery vehicle of the present disclosure is shown below (reproduced from Figure 2 of Hou et al., Nature Review Materials, 6:1078-1094, 2021). [ka] V. Pharmaceutical Preparations

[0055] Some compositions of the present disclosure are pharmaceutical formulations that contain pharma- ceutical acceptable excipients. The pharmaceutical formulations of the present disclosure can be in the form of a solution or suspension. Alternatively, the pharmaceutical formulations can be anhydrous solids (e.g., lyophilized or spray-dried solids). The pharmaceutical formulations of the present disclosure are preferably sterile and preferably essentially endotoxin-free. The term "pharmaceutical formulation" is used herein interchangeably with the terms "pharmaceutical product" and "medicine". In some embodiments, the pharmaceutical formulations contain specific ratios of various components based on the intended purpose of the formulation.

[0056] Pharmaceutically acceptable excipients of the present disclosure include, for example, solvents, buffers, osmolality adjusters, bulking agents, and preservatives (see, for example, Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, pharmaceutical formulations can include excipients that function as one or more of solvents, buffers, osmolality adjusters, and bulking agents (e.g., sodium chloride in saline can act as both an aqueous vehicle and an osmolality adjuster).

[0057] In some embodiments, the pharmaceutical formulation comprises an aqueous vehicle as a solvent. Suitable vehicles include, for example, sterile water, saline solution, phosphate buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic.

[0058] The pharmaceutical formulation may include a buffering agent. The buffering agent controls the pH to inhibit degradation of the active agent during processing, storage, and, if necessary, reconstitution. Suitable buffering agents include salts, including, for example, acetate, citrate, phosphate, or sulfate. Other suitable buffering agents include, for example, amino acids, such as arginine, glycine, histidine, and lysine. The buffering agent may further include hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the composition within the range of 6-9. In some embodiments, the pH is greater than 6, 7, or 8 (lower limit). In some embodiments, the pH is less than 9, 8, or 7 (upper limit). That is, the pH is in the range of about 6-9, where the lower limit is less than the upper limit.

[0059] The pharmaceutical composition may include a osmolality modifier. Suitable osmolality modifiers include, for example, glucose, glycerol, sodium chloride, glycerin and mannitol.

[0060] Pharmaceutical preparations may contain bulking agents. Bulking agents are particularly useful when pharmaceutical compositions should be freeze-dried before administration. In some embodiments, bulking agents are protective agents that help stabilize and prevent decomposition of active agents during freeze-drying or spray-drying and / or storage. Suitable bulking agents are sugars (monosaccharides, disaccharides and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbital, glucose and raffinose.

[0061] The pharmaceutical formulation may contain a preservative. Suitable preservatives include, for example, antioxidants and antimicrobial agents. However, in a preferred embodiment, the pharmaceutical formulation is prepared under sterile conditions and is contained in a single-use container, and therefore does not need to contain a preservative.

[0062] The pharmaceutical formulations of the present disclosure are suitable for parenteral administration, i.e., they are not intended for enteral administration (e.g., not orally, intragastricly, or rectally). VI.How to use

[0063] In some aspects, the disclosure relates to a method of using any one of the compositions or formulations described herein. The method of use is suitable for multiple uses, including stimulating an immune response. In some embodiments, the method of use includes a method of treating cancer. In some embodiments, the method of use includes a method of inhibiting abnormal cell proliferation. In some embodiments, the method of use includes a method of treating or preventing an infectious disease. The method includes administering an effective amount of the formulation or composition described herein to an individual in need thereof to achieve a particular outcome. The individual is a mammalian subject, such as a human patient. In other embodiments, the individual is a non-human patient. In some embodiments, the individual is a canine patient. That is, in some embodiments, the method of use includes clinical use, while in other embodiments, the method of use includes preclinical and / or veterinary use. For preclinical use, the mammalian subject can be a non-human primate (e.g., monkey or ape) or rodent (e.g., mouse or rat). For veterinary uses, the mammalian subject can be a farm animal (eg, a cow), a sport animal (eg, a horse), or a pet (eg, a companion animal such as a dog or cat). A. Stimulation of the immune response

[0064] Briefly, the present disclosure provides a method of stimulating an immune response in an individual, comprising administering to the individual a composition or formulation described herein in an amount sufficient to stimulate an immune response in the individual. "Stimulating" an immune response (used interchangeably with "eliciting" and immune response) refers to an increase in immune response, which may result from inducing a new immune response (e.g., as a result of a primary vaccination regimen) or enhancing an existing immune response (e.g., as a result of a booster vaccination regimen). In some embodiments, stimulating an immune response includes one or more of the group consisting of stimulating cytokine production, stimulating B lymphocyte proliferation, stimulating interferon pathway-related gene expression, stimulating chemoattractant-related gene expression, and stimulating dendritic cell DC maturation. Methods for measuring the stimulation of an immune response are known in the art.

[0065] For example, the present disclosure provides a method of inducing an antigen-specific immune response in an individual by administering to the individual a composition or formulation described herein in an amount sufficient to induce an antigen-specific immune response in the individual. In a preferred embodiment, the composition or formulation comprises an antigen. In some embodiments, the composition or formulation is administered to a tissue of the individual that contains the antigen. The immune response may comprise one or both of an antigen-specific antibody response and an antigen-specific cytotoxic T lymphocyte (CTL) response. "Inducing" an antigen-specific antibody response means increasing the titer of an antigen-specific antibody above a threshold level, e.g., a baseline titer or a seroprotective antibody level before administration. "Inducing" an antigen-specific CTL response means increasing the frequency at which antigen-specific CTLs are found in peripheral blood above a baseline frequency before administration.

[0066] Analysis of immune responses (both qualitative and quantitative) can be performed by any method known in the art, including, but not limited to, measuring antigen-specific antibody production (including measuring specific antibody subclasses), activation of specific populations of lymphocytes, such as B cells and helper T cells, production of cytokines, such as IFN-alpha, IFN-gamma, IL-6, IL-12, and / or release of histamine. Methods for measuring antigen-specific antibody responses include enzyme-linked immunosorbent assay (ELISA). Activation of specific populations of lymphocytes can be measured by proliferation assays and with fluorescence-activated cell sorting (FACS). Cytokine production can also be measured by ELISA. In some embodiments, the method of stimulating an immune response includes stimulating interleukin-1 beta (IL-1β), interferon-gamma (IFN-γ), and / or tumor necrosis factor-alpha (TNF-α) secretion by monocyte-derived dendritic cells or peripheral blood mononuclear cells. In some preferred embodiments, at least 50%, 55%, 60%, 65%, 70% or 75% of cells contacted with a composition of the present disclosure are still viable 40-56 hours (or about 48 hours) after contact. In some preferred embodiments, at least 75% of cells contacted with a composition of the present disclosure are still viable 40-56 hours (or about 48 hours) after contact.

[0067] In some embodiments, the method is suitable for stimulating an anti-tumor immune response. In other embodiments, the method is suitable for stimulating an anti-microbial immune response. In some embodiments, the anti-microbial response is an anti-bacterial immune response. In some embodiments, the anti-microbial response is an anti-fungal immune response. In some embodiments, the anti-microbial response is an anti-viral immune response. In some embodiments, the anti-microbial response is an anti-protozoan immune response. B. Treatment or Prevention of Disease

[0068] The present disclosure further provides a method of treating or preventing a disease in an individual, comprising administering to the individual a composition or formulation described herein in an amount sufficient to treat or prevent the disease in the individual. In some embodiments, the disease is cancer. In some embodiments, the disease is abnormal cell proliferation. In other embodiments, the disease is an infectious disease. In one aspect, the method can include administering the composition to a subject in need thereof.

[0069] In some embodiments, the method includes treating cancer in an individual or otherwise treating a mammalian subject having cancer. In some embodiments, the cancer is a blood cancer, such as lymphoma, leukemia, or myeloma. In other embodiments, the cancer is a non-blood cancer, such as a sarcoma, carcinoma, or melanoma. In some embodiments, the cancer is malignant.

[0070] In some embodiments, the methods include inhibiting abnormal cell growth in an individual. "Abnormal cell growth" refers to the growth of a benign or malignant tumor. A malignant tumor can be a metastatic tumor.

[0071] In some embodiments, the method includes treating or preventing an infectious disease in an individual. In some embodiments, the infectious disease is caused by a viral infection. In other embodiments, the infectious disease is caused by a bacterial infection. In further embodiments, the infectious disease is caused by a fungal infection. In yet further embodiments, the infectious disease is caused by a protozoan infection. Of particular importance are infectious diseases caused by zoonotic pathogens that infect humans and other animals, such as mammals or birds. In some embodiments, zoonotic pathogens are transmitted to humans via intermediate species (vectors). Enumeration of embodiments 1. A composition comprising an mRNA encapsulated in a lipid nanoparticle (LNP), wherein the mRNA comprises a coding region for an antigen, and the LNP comprises a first phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a second phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof, wherein the first phospholipid comprises a lysophosphatidylcholine (LPC) having a single acyl chain, and the acyl chain is a C13-C24 acyl chain. 2. A composition comprising an mRNA and a TLR7 / 8 agonist encapsulated in a lipid nanoparticle (LNP), wherein the mRNA comprises a coding region for an antigen, and the LNP comprises a first phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a second phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof, wherein the first phospholipid comprises a lysophosphatidylcholine (LPC) having a single acyl chain, and the acyl chain is a C13-C24 acyl chain. 3. A composition comprising an mRNA encapsulated in a lipid nanoparticle (LNP) and a TLR7 / 8 agonist, wherein the mRNA comprises a coding region for an antigen, and the LNP comprises a first phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a second phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof, wherein the first phospholipid comprises a lysophosphatidylcholine (LPC) having a single acyl chain, and the acyl chain is a C13-C24 acyl chain. 4. A composition comprising a TLR7 / 8 agonist encapsulated in a lipid nanoparticle (LNP), wherein the LNP comprises a first phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a second phospholipid, a pegylated lipid, a structured lipid, and mixtures thereof, wherein the first phospholipid comprises a lysophosphatidylcholine (LPC) having a single acyl chain, the acyl chain being a C13-C24 acyl chain. 5. A composition comprising a lipid nanoparticle (LNP) and a TLR7 / 8 agonist, wherein the LNP comprises a first phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a second phospholipid, a pegylated lipid, a structured lipid, and mixtures thereof, wherein the first phospholipid comprises lysophosphatidylcholine (LPC) having a single acyl chain, the acyl chain being a C13-C24 acyl chain. 6. The composition of any one of embodiments 1 to 5, wherein the at least one lipid comprises an ionizable lipid, a second phospholipid, a pegylated lipid, and a structured lipid. 7 The ionizable lipid is i) SM-102 or an analog or derivative thereof, and / or ii) ALC-0315 or an analog or derivative thereof 7. The composition of any one of embodiments 1 to 6, comprising: 8. The composition of any one of embodiments 1 to 7, wherein the pegylated lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglyerol, and combinations thereof. 9. The composition of any one of embodiments 1-7, wherein the pegylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG]. 10. The composition of any one of embodiments 1-9, wherein the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campestrol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and combinations thereof. 11. The composition of any one of embodiments 1 to 9, wherein the structured lipid comprises cholesterol. 12 The second phospholipid is i) a hydrophilic head moiety selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin; and ii) one or more fatty acid tail moieties selected from the group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, arachidic acid, arachidonic acid, phytanoic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. 12. The composition of any one of embodiments 1 to 11, comprising: 13 The second phospholipid is 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-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), Sphingomyelin, and Combinations of these 12. The composition of any one of embodiments 1 to 11, selected from the group consisting of: 14. The composition of any one of embodiments 1-13, wherein the second phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). 15. The composition of any one of embodiments 1 to 14, further comprising at least one excipient. 16. The composition of embodiment 15, wherein the excipient comprises sucrose. 17 i) mRNA complexed with one or more lipids (RNA-Lipoplex), and ii) Lysophosphatidylcholine (LPC) with a single C13-C24 acyl chain A composition comprising: The composition, wherein the mRNA comprises a coding region for an antigen, and the one or more lipids comprises a first lipid and a second lipid. 18 i) mRNA complexed with one or more lipids (RNA-Lipoplex); ii) lysophosphatidylcholine (LPC) with a single C13-C24 acyl chain, and iii) TLR7 / 8 agonists A composition comprising: The composition, wherein the mRNA comprises a coding region for an antigen, and the one or more lipids comprises a first lipid and a second lipid. 19. The composition of embodiment 17 or embodiment 18, wherein the first lipid is a cationic lipid and the second lipid is a neutral or anionic lipid. 20 The cationic lipid is i) 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) or an analog or derivative thereof, and ii) 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) or an analog or derivative thereof 20. The composition of embodiment 19, comprising one or both of the following: 21 The neutral or anionic lipid is i) 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or an analog or derivative thereof, and / or ii) cholesterol or an analog or derivative thereof, and / or iii) 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or an analog or derivative thereof 21. The composition of embodiment 19 or embodiment 20, comprising: 22. The composition of any one of embodiments 1 to 21, wherein the acyl chain of the LPC is a C21 to C24 acyl chain. 23. The composition of any one of embodiments 1 to 21, wherein the acyl chain of the LPC is a C22 acyl chain. 24. The composition of any one of embodiments 1 to 23, wherein the acyl chains of the LPC are fully saturated. 25. The composition of embodiment 24, wherein the LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)]. 26. The composition of any one of embodiments 1 to 25, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 Daltons or less. 27. The composition of any one of embodiments 1-25, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound. 28. The composition of embodiment 27, wherein the TLR7 / 8 agonist comprises resiquimod (R848). 29. The composition of any one of embodiments 1-28, wherein the LPC comprises LPC(22:0) and the TLR7 / 8 agonist comprises resiquimod (R848). 30. The composition of any one of embodiments 1 to 28, wherein the antigen is a tumor antigen. 31. The composition of any one of embodiments 1 to 28, wherein the tumor antigen is a neoantigen. 32. The composition of any one of embodiments 1-28, wherein the antigen comprises a microbial antigen. 33. The composition of embodiment 32, wherein the microbial antigen comprises a viral antigen, a bacterial antigen, a protozoal antigen, or a fungal antigen. 34. The composition of embodiment 32, wherein the microbial antigen comprises a surface antigen. 35. The composition of any one of embodiments 1 to 34, wherein the mRNA comprises a 5' untranslated region (5'UTR) at the 5' end of the coding region and a 3' untranslated region (3'UTR) at the 3' end of the coding region. 36. The composition of any one of embodiments 1 to 35, wherein the mRNA comprises a 5' cap structure. 37. The composition of any one of embodiments 1 to 36, wherein the mRNA comprises a polyA tail. 38. The composition according to any one of the preceding embodiments, which does not contain lipopolysaccharide (LPS) or monophosphoryl lipid A (MPLA). 39. The composition of any one of embodiments 1 to 38, which does not contain oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) or oxPAPC species. 40 2-[[(2R)-2-[(E)-7-carboxy-5-hydroxyhept-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium (HOdiA-PC), [(2R)-2-[(E)-7-carboxy-5-oxohept-6-enoyl]oxy-3-hexadecanoyloxypropyl]2-(trimethylazaniumyl)ethyl phosphate (KOdiA-PC), l-palmitoyl-2-(5 -Hydroxy-8-oxo-octenoyl)-sn-glycero-3-phosphorylcholine (HOOA-PC), 2-[[(2R)-2-[(E)-5,8-dioxooct-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium (KOOA-PC), [(2R)-3-hexadecanoyloxy-2-(5-oxopentanoyloxy)propyl]2-(trimethylazaniumyl)ethyl phosphate (POVPC), [(2R)-2-(4-carboxybutanoyloxy)-3-hexadecanoyloxypropyl]2-(trimethylazaniumyl)ethyl phosphate (PGPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[2-[(Z)-oct-2-enyl]-5-oxocyclopent-3-en-l-ylidene]methyl]oxiran-2-yl]butanoyloxy]propyl]2-(trimethylazaniumyl)ethyl phosphate (PECPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentylidene]methyl]oxiran-2-yl]butanoyloxy]propyl]2-(trimethylazaniumyl)ethyl phosphate (PEIPC), and / or 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PAzePC). 41. The composition according to any one of embodiments 1 to 40, which does not contain an antigen. 42. A pharmaceutical formulation comprising the composition according to any one of embodiments 1 to 41 and a pharma- ceutically acceptable excipient. 43. A method for generating hyperactivated dendritic cells, comprising contacting said dendritic cells with an effective amount of a composition of any of the preceding embodiments to generate hyperactivated dendritic cells, wherein said hyperactivated dendritic cells secrete IL-1 beta within about 48 hours after exposure without undergoing cell death. 44. The method of embodiment 43, wherein the dendritic cells are contacted with the composition in vivo. 45. The method of embodiment 43, wherein the dendritic cells are contacted with the composition ex vivo. 46 At least 10 produced by the method according to embodiment 45 3 , 10 4 , 10 5 or 10 6 and a pharma- ceutically acceptable excipient. 47. A method for stimulating an immune response against an antigen, comprising administering to an individual in need thereof an effective amount of the pharmaceutical preparation of embodiment 42 or embodiment 46 to stimulate said immune response against said antigen. 48. A method for treating cancer, comprising administering an effective amount of the pharmaceutical formulation of embodiment 42 or embodiment 46 to an individual in need thereof to treat said cancer. 49. A method for inhibiting abnormal cell growth, comprising administering to an individual in need thereof an effective amount of the pharmaceutical formulation of embodiment 42 or embodiment 46 to inhibit abnormal cell growth. 50. A method for treating or preventing an infectious disease, comprising administering to an individual in need thereof an effective amount of the pharmaceutical formulation of embodiment 42 to treat or prevent said infectious disease. 51. The method of embodiment 50, wherein the infectious disease is a viral disease. 52. The method of embodiment 51, wherein the infectious disease is a bacterial disease. 53. The method or pharmaceutical preparation according to any one of embodiments 43 to 49, wherein said dendritic cells are mammalian cells. 54. The method or pharmaceutical preparation of embodiment 53, wherein said mammalian cells are human cells. 55. The method of any one of embodiments 47-53, wherein the individual is a mammal. 56. The method of embodiment 55, wherein the mammal is a human. 57. The method of embodiment 55, wherein the mammal is a dog or a cat. 58. The composition, formulation, or method or use according to any one of the preceding embodiments, wherein the composition does not comprise a protein. 59. The composition, formulation, method or use according to any one of embodiments 1 to 158, wherein the LNP has an effective diameter of less than about 500 nanometers, optionally from about 5 to about 500 nanometers, optionally from about 10 to about 400 nanometers, optionally from about 20 to about 300 nanometers, or optionally from about 25 to about 250 nanometers. 60. The composition, formulation, method or use of embodiment 59, wherein the LNP has an effective diameter of less than about 250 nanometers. 61. The composition, formulation, method or use of embodiment 60, wherein said LNPs have an effective diameter of less than about 125 nanometers. 62. The composition, formulation, method or use of embodiment 61, wherein the LNP has an effective diameter of about 20 to about 120 nanometers. EXAMPLES

[0072] Abbreviations: BMDC (bone marrow-derived dendritic cells), CDS (cytoplasmic DNA sensor), CLR (C-type lectin receptor), DAMP (damage-associated molecular pattern), DC (dendritic cell), DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), GFP (green fluorescent protein), HOdiA-PC (1-palmitoyl-2-(5-hydroxy-8-oxo-6-octenedioyl)-sn-glycero-3-phosphatidylcholine ... phosphorus), HOOA-PC (1-palmitoyl-2-(5-hydroxy-8-oxooct-6-enoyl)-sn-glycero-3-phosphocholine), IFNγ (interferon-gamma), IL-1b / IL1-beta / IL-1β (interleukin-1 beta), KOdiA-PC (1-(palmitoyl)-2-(5-keto-6-octenedioyl)phosphatidylcholine), KOOA-PC (1-palmitoyl-(5-keto-8-oxo-6-octenoyl)-sn-glycero-3-phosphocholine), LNP (lipid nanoparticles), LPC / Lyso PC (lysophosphatidylcholine), Lyso PC(22:0) (1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine), LPS (lipopolysaccharide), moDC (monocyte-derived dendritic cells), MPLA (monophosphoryl lipid A), NLR (NOD-like receptor), OVA (ovalbumin), oxPAPC (oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine), PAMP (pathogen-associated molecular pattern), PBMC (peripheral blood mononuclear cells), PDI (polydispersity index), PGPC (1-palmitoyl-2-glutaryl-sn-glycero-3-phosphorylcholine), rho-3-phosphocholine), POVPC (1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine), PRRs (pathogen recognition receptors), RLRs (RIG-I-like receptors), R848 (resiquimod), SM102 ((8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid), 1-octylnonyl ester), STING (stimulator of IFN genes), TNFα (tumor necrosis factor-alpha), and TLRs (toll-like receptors).

[0073] Although the present disclosure has been described in some detail by way of illustration or example for clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be made. Therefore, the examples should not be construed as limiting the scope of the present disclosure, which is set forth in the appended claims. Example 1 Combination of single acyl chain lysophosphatidylcholine (LPC) and TLR7 / 8 agonists hyperactivates mammalian peripheral blood mononuclear cells

[0074] This example describes the hyperactivation of canine and human peripheral blood mononuclear cells (PBMCs) using lipid DAMPs in combination with small molecule PAMPs. material and method

[0075] Isolation of PBMCs from human and canine whole blood. PBMCs were isolated from whole blood using density gradient centrifugation with Ficoll-Paque PLUS (Cytivia). Whole blood was diluted 1:1 with PBS, layered on top of Ficoll-Paque PLUS, and centrifuged at 1000×g for 30 minutes at room temperature. PBMCs were collected, washed twice with PBS, and incubated with Ack lysis buffer (Lonza) to remove any remaining red blood cells.

[0076] Cell culture and stimulation. Immediately after isolation, PBMCs were plated in RPMI medium containing 10% FBS, 50 units / mL penicillin, 50 mg / mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 mM beta-mercaptoethanol (R10 medium). Cells were plated at 1 × 10 per well. 5 (dog cells) or 1 × 10 6(human cells) were plated in 96-well flat-bottom tissue culture plates. Lyophilized Vaccigrade R848 (Invivogen) was reconstituted, diluted according to the manufacturer's recommendations, and added to the cells at a final concentration of 1 μg / mL. Immediately after, 22:0 LYSO PC (1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine) was added to the cells at a final concentration of 82.5 μM. Further natural agonists were diluted in R10 medium according to the manufacturer's recommendations and added to the cells as follows: human GM-CSF (Peprotech) was added at a final concentration of 10 ng / mL, 2'3'cGAMP (Invivogen) was added at a final concentration of 15 μg / mL, LPS, serotype O55:B5 (Enzo Life Sciences) was added at a final concentration of 1 μg / mL, and hydroxylated Alum (Invivogen) was added at a final concentration of 30 μg / mL. The cells were incubated at 37° C., 5% CO2 for 2 days. The cell cultures were then used for endpoint analysis.

[0077] Endpoint Analysis. After PBMCs were cultured with PAMPs and DAMPs for 2 days, supernatants and cell samples were collected for analysis. Cells in culture were pelleted by centrifugation at 400×g for 5 minutes. Half of the medium volume in the wells was collected for quantifying cytokines by enzyme-linked immunosorbent assay (ELISA) or Lumit™ bioluminescence assay, while the remaining medium and cells were used to quantify cell viability by assessing metabolic activity.

[0078] Quantification of cytokine secretion. IL-1β secretion from human PBMC was assessed using one of the following kits: ELISA MAX Deluxe Set Human IL-1β Kit (Biolegend), Invitrogen Human IL-1β Kit, or Lumit™ Human IL-1β Immunoassay (Promega). IFNγ secretion from human PBMC was assessed using ELISA MAX Deluxe Set Human IFNγ (Biolegend) and TNFα secretion from human PBMC was assessed using Human TNFα Uncoated ELISA Kit (Invitrogen). ELISAs were performed according to the manufacturer's instructions with the following modifications: i) total sample + buffer volume for incubation was reduced from 100 μL to 50 μL, ii) top standard was prepared at 500 pg / mL and diluted 2-fold to 7.8 pg / mL, and iii) sample incubation was completed overnight at 4° C. on an orbital shaker. Lumit™ assays were performed according to the manufacturer's instructions. IL-1β secretion from canine PBMCs was assessed using the Canine IL-1β / IL-1F2 DuoSet ELISA (R&D) according to the manufacturer's instructions with the following modifications: i) total sample+buffer volume for incubation was reduced from 100 μL to 50 μL, and ii) sample incubation was completed on an orbital shaker overnight at 4° C. For all ELISAs, absorbance was measured at 450 nm using a Spectramax M5e plate reader (Molecular Devices) with 570 nm used for correction. For Lumit™ assays, emission was measured for all wavelengths using a Spectramax M5e plate reader (Molecular Devices) with an integration time of 500 ms. To determine cytokine concentrations in the supernatants, sample concentrations were interpolated using standard curves with 4PL analysis in GraphPad Prism 9 (GraphPad Software). The sample interpolated results were then adjusted for any dilutions made to the supernatant.

[0079] Quantification of cell viability. Cell viability was assessed by quantifying the presence of ATP as an indicator of metabolically active cells using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Metabolic activity was assessed according to the manufacturer's instructions. CellTiter-Glo reagent was mixed with cell pellets and fresh medium, then transferred to a white opaque 96-well plate. Luminescence was measured using a 500 ms integration time for all wavelengths on a Spectramax M5e plate reader (Molecular Devices). Percent viability was calculated relative to the control condition of R848-treated PBMCs.

[0080] Statistical analysis. For each condition, cells from each donor were plated for testing in triplicate. For cytokine quantification, triplicate values ​​were used for interpolation and data were plotted as total concentration (pg / mL) or fold change per donor relative to the R848 alone control condition. For viability quantification, triplicate values ​​from each donor were averaged and the average was used as a single donor measurement. Multiple donors were tested and each data point on the bar graph represents the value for one donor. To test for differences between test conditions, test results were compared to the R848 alone control condition. p values ​​were calculated using mixed-effects one-way ANOVA and corrected for multiple comparisons using Dunnett's test. Results - Treatment with 22:0 LYSO PC and R848 hyperactivates canine PBMCs

[0081] The combination of 22:0 LYSO PC (DAMP) and the TLR7 / 8 agonist R848 (PAMP) has already been found to have potent hyperstimulatory activity in human moDCs. To assess whether this hyperstimulatory activity translates to other clinically relevant species, the ability of 22:0 LYSO PC+R848 to hyperactivate PBMCs isolated from canine whole blood was evaluated. As there were no canine-specific reagents available to induce authentic canine moDCs, PBMCs from multiple donors were used instead of moDCs for each data set. Briefly, PBMCs were isolated from whole blood using density gradient centrifugation and then cultured for 2 days with the hyperactivating stimulus of interest.

[0082] After 2 days of culture, hyperactivation was assessed by quantification of IL-1β in cell culture supernatants and by measuring cell viability. When treated with 22:0 LYSO PC and R848 together, canine PBMC secreted equivalent or higher levels of IL-1β compared to any other stimuli tested, both as concentration per mL and as fold change relative to R848 alone per donor. Consistent with previous studies showing that monocytes, which constitute 5-10% of PBMCs, can release IL-1β in response to activation with R848, canine PBMC secreted elevated levels of IL-1β with R848 alone compared to untreated cells. The pyroptotic LPS+Alum combination induced high levels of IL-1β as expected. Notably, PGPC+R848 induced similar levels of IL-1β compared to R848 alone, whereas neither GM-CSF nor 2′3′cGAMP induced significant IL-1β secretion from canine PBMC compared to untreated cells.

[0083] IL-1β could be detected in cell culture supernatants the day after canine PBMCs were overactivated, while cell viability was assessed 2 days after overactivation, ensuring persistent survival after IL-1β secretion. 22:0 LYSO+R848 did not significantly reduce the relative viability of cells. Interestingly, combining PGPC with R848 proved somewhat toxic to canine PBMCs, but no toxicity was observed for human moDCs or human PBMCs. However, it is important to note that the viability of the specific cell population of interest (in this case, monocytes, which we suspect are responding to IL-1β) cannot be determined from the mixture, making interpretation of observations made from testing of mixed cell populations more challenging given concerns about viability. Taken together, these data demonstrate that 22:0 LYSO+R848 induces high levels of IL-1β secretion from canine PBMCs, which is indicative of overactivation. Results - Treatment with 22:0 LYSO PC and R848 hyperactivates human PBMC

[0084] Hyperactivation experiments were also performed with PBMCs isolated from whole blood obtained from human donors. Briefly, PBMCs were isolated from whole blood of multiple human donors by density gradient centrifugation and cultured for 2 days with the hyperactivation stimuli of interest.

[0085] Human PBMCs, like human moDCs and canine PBMCs, secreted IL-1β at levels higher or comparable to all other stimuli tested. Human PBMCs, like canine PBMCs, secreted IL-1β in response to R848 alone due to monocyte activation, which was elevated by the addition of 22:0 LYSO PC. The pyroptotic combination of LPS+Alum induced high levels of IL-1β as expected. Consistent with the observations in canine PBMCs, PGPC+R848 did not induce substantially higher levels of IL-1β than R848 alone. GM-CSF did not induce IL-1β secretion from human PBMCs significantly above background levels produced by untreated cells.

[0086] Furthermore, the viability of human PBMCs was assessed 2 days after overactivation, ensuring their persistent survival after IL-1β secretion. No significant decrease in the viability of human PBMCs was observed after treatment with either stimuli. However, the observations made from the testing of mixed cell populations are difficult to interpret, since the viability of the specific cell population of interest (in this case, monocytes) cannot be determined from the results obtained with the mixture. Taken together, these data demonstrate that both human and canine PBMCs are overactivated by 22:0 LYSO PC+R848. Interestingly, canine PBMCs are overactivated to a greater extent by 22:0 LYSO PC+R848 than by PGPC+R848.

[0087] Since activated human PBMCs can secrete other cytokines in addition to IL-1β, the secretion of the proinflammatory cytokines IFNγ and TNFα in cell culture supernatants was measured 2 days after hyperactivation. The combination of 22:0 LYSO PC+R848 induced the highest fold change per donor compared to R848 alone in both IFNγ and TNFα secretion compared to all other stimuli tested. Notably, LPS+Alum induced high levels of IL-1β secretion from human PBMCs, but this combination of stimuli did not induce a fold increase in IFNγ or TNFα secretion. Furthermore, neither GM-CSF nor 2'3'cGAMP induced a substantial fold change in IFNγ secretion over R848 alone. These data indicate that the combination of 22:0 LYSO PC+R848 is superior in inducing the secretion of the proinflammatory cytokines IFNγ and TNFα from human PBMCs. Example 2 Incorporation of LPC with a single acyl chain into lipid nanoparticles in the presence of mRNA for hyperactivation of mammalian dendritic cells

[0088] This example describes the preparation and testing of lipid nanoparticles (LNPs) containing a single acyl chain lysophosphatidylcholine (LPC) compound (e.g., 22:0 Lyso PC) and antigen-encoding mRNA. LPC / mRNA-loaded LNPs, in combination with small molecule PAMPs (e.g., R848), are suitable for hyperactivation of mammalian dendritic cells. material and method

[0089] LNP Synthesis. LNPs were synthesized using the following components: (8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid), 1-octylnonyl ester (CAS Registry Number 2089251-47-6, referred to herein as "SM102") (Cayman Chemical); 1,2-distearoyl-sn-glycero-3-phosphocholine (CAS Registry Number 816-94-4, referred to herein as "DSPC") (Avanti); Cholesterol (Sigma), and 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (CAS Registry Number 160743-62-4, referred to herein as "DMG-PEG2000") (Avanti) was prepared with or without 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine (CAS Registry Number 125146-65-8, referred to herein as "22:0 Lyso PC") (Avanti). Table 2-1 details the molar percentage of each component for each LNP formulation tested. LNPs were prepared without 22:0 Lyso PC or loaded with 20%, 30% or 40% molar ratio of 22:0 Lyso PC in the LNP to determine whether the loading of 22:0 Lyso PC could be purposefully altered. LNPs were made without mRNA, with mRNA encoding green fluorescent protein (GFP), or with various loading levels of mRNA encoding ovalbumin (OVA) to determine whether the inclusion of 22:0 Lyso PC could affect mRNA loading into the LNP.

[0090] LNPs were synthesized using a NanoAssemblr Ignite instrument (Precision Nanosystems). First, lipids were dissolved in ethanol and then combined according to the molar composition shown in Table 2-1. Lipids in ethanol were combined with sodium citrate buffer (pH 4) containing mRNA in a volume ratio of 1:3 at a flow rate of 12 mL / min. Enhanced GFP mRNA (Trilink) was added to a concentration of 0.053 mg / mL in sodium citrate buffer for loading into the LNPs. OVA mRNA (Trilink) was added to a concentration of 0.035 mg / mL or 0.004 mg / mL in sodium citrate buffer for loading into the LNPs. LNPs were washed with 10 volumes of PBS, pH 7.4, to remove residual ethanol, and then concentrated using an Amicon 10K MWCO centrifugal filter. Table 2-1. Molar Percentage of Lipids in LNP Formulations [Table 2-1]

[0091] Characterization of LNPs. Loading of 22:0 Lyso PC into LNPs was assessed using HPLC. LNPs in PBS were frozen at -20°C until quantification. LNPs were dissolved by adding one part ethanol to LNPs in PBS. A seven-point standard curve of 22:0 Lyso PC was prepared in 1:1 ethanol:PBS with additions made to match sample preparation. Standards and samples were filtered through a 0.45 μm filter before being run on the HPLC. HPLC quantification was performed using an Agilent 1260 Infinity II HPLC equipped with a 1260 Infinity II evaporative light scattering detector. Samples were detected using a Luna 5 μm NH2 100 Å, 150×4.6 mm LC column (Phenomenex) with a column temperature of 30°C. Two eluents were used: A, 100% water, and B, 100% acetonitrile. The column was loaded with an initial mobile phase consisting of 5% / 95% A / B with a gradient reaching 24% / 76% A / B after 2.5 min. A narrower gradient was used from 2.5 to 6 min, during which time frame A / B slowly reached 25% / 75%. The gradient was then returned to starting conditions over 3 min before the next sample was run. The flow rate was set at 1 mL / min, and the injection volume was 2.5 μL for samples and standards. The evaporative light scattering detector (ELSD) used an evaporator temperature of 50 °C, a nebulizer temperature of 30 °C, and a gas flow rate of 0.9 standard L / min. Agilent CDS 2.6 software was used for HPLC instrument control, data acquisition, and processing.

[0092] Loading of mRNA into LNPs was quantified using RiboGreen assay (ThermoFisher) according to the manufacturer's protocol. Samples were diluted to fall within the range of the standard curve. LNPs were dissolved using Triton® X-100 to assess encapsulation of mRNA into LNPs. Both total and encapsulated mRNA were quantified. Size of LNPs was assessed using dynamic light scattering (DLS) on a NanoBrook Omni (Brookhaven). LNPs were diluted 1:10 in PBS before running on DLS. Three measurements of 90 seconds were recorded for each sample.

[0093] Generation of human monocyte-derived dendritic cells (moDCs). Human monocytes were isolated from Leukopaks purchased from Miltenyi Inc. (San Jose, CA) using the StraightFrom® Leukopak® CD14 MicroBead Kit according to the manufacturer's instructions. Monocytes were then aliquoted and frozen in FBS containing 10% dimethyl sulfoxide. Prior to testing, monocytes were thawed and cultured in RPMI medium (R10 medium) containing 10% FBS, 50 units / mL penicillin, 50 mg / mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 mM beta-mercaptoethanol, 10 mM HEPES, and Gibco MEM non-essential amino acids. To differentiate monocytes into moDCs, recombinant human GM-CSF (50 ng / mL) and IL-4 (25 ng / mL) were added to the R10 medium. Cells were cultured with GM-CSF and IL-4 for 6 days and further fed on day 3 with R10 medium containing GM-CSF and IL-4.

[0094] Hyperactivation of human monocyte-derived dendritic cells (moDCs). moDCs were harvested and counted on day 6 after differentiation. Cells were cultured at 1 × 10 5 Cells were plated at 1000 cells / well. Cells were treated with or without 1 μg / mL R848 (final) in the presence or absence of eGFP mRNA, with or without LNPs loaded with hyperactivating lipids (or vehicle control). LNP-induced hyperactivation was measured after 48 hours in culture with LNPs. Cell viability was assessed using the LDH CyQuant kit (Invitrogen) according to the manufacturer's instructions. IL-1β and IL-6 Lumit assays (Promega) were used to measure IL-1β and IL-6 present in moDC cell culture supernatants. Experimental conditions were tested in triplicate, and the average of results from two human donors was plotted. Data represent results from two experiments.

[0095] In addition to hyperactivation, GFP expression in moDCs was quantified using flow cytometry on a BD FACS Symphony A3 device. After 48 hours in culture with LNPs, moDCs were harvested and stained with a live / dead dye to identify live cells, followed by staining with antibodies specific for CD11c, CD40, CD86, HLA-DR, and HLA-ABC. Live cells were selected for analysis and then stained with antibodies specific for CD11c. + Cells were evaluated for GFP expression to determine whether LNP mRNA can be translated into GFP protein when LNP is loaded with 22:0 Lyso PC. Antigen presentation was evaluated using antibodies specific for HLA-DR and HLA-ABC to determine whether overactivation can interfere with antigen presentation. Activation was evaluated by staining for CD40 and CD86 to determine whether overactivation can increase the expression of activation markers.

[0096] Generation of mouse bone marrow-derived FLT3L-DCs (BMDCs). Femurs and tibias were removed from mice, cut with scissors, and poured into sterile tubes. Bone marrow suspensions were treated with ACK lysis buffer for 1 min and then passed through a 40 μm cell strainer. Cells were counted and resuspended in medium (I10) consisting of complete IMDM containing 10% FBS, penicillin and streptomycin, and L-glutamine and sodium pyruvate supplements. Cells were then plated at 8×10 in P12 plates. 6 Cells were plated at 1000 x g / well. Recombinant mouse FLT3L (Miltenyi) was added to the cultures at 200 ng / mL. Differentiated cells were used for subsequent assays on day 8. Differentiation efficiency was monitored by flow cytometry using a BD Symphony A3 device and CD11c + MHC-II + Cells were routinely greater than 80% viable. Per experiment, 5–15 mice were used to generate BMDCs.

[0097] Hyperactivation of mouse bone marrow-derived FLT3L-DCs and T cell coculture. BMDCs were harvested on days 8 and 9 after differentiation, washed with PBS, and resuspended in complete IMDM medium (I10) at 2 × 10 5 The BMDCs were then replated at a concentration of 100 x 10 cells / well. Cells were cultured in the presence or absence of LNPs loaded with 50 μM 22:0 Lyso PC LNPs, with or without OVA mRNA, either high or low dose of mRNA (Table 2-2). Supernatants and BMDCs were collected 24 and 48 hours after stimulation. IL-1β cytokine secretion by BMDCs was measured using a sandwich ELISA (Invitrogen) according to the manufacturer's instructions. Approximately 2 x 10 4 of hyperactivated BMDCs were seeded into wells of round-bottom 96-well plates for coculture with T cells.

[0098] CD8+ T cells, H2-K b CD8 T cells were collected from the spleens and lymph nodes of transgenic OT-I mice expressing a T cell receptor (TCR) specific for the ovalbumin peptide (residues 257-264) presented by BMDCs. All CD8 T cells from OT-I mice are naive cells specific for the ovalbumin peptide. T cell co-cultures were performed by adding 6 × 10 T cells to each well containing BMDCs. 4 The hyperactivated BMDCs were co-cultured with T cells for 72 h, and then the co-culture supernatants were collected.

[0099] In addition, CD8+ T cells were collected from the spleens and lymph nodes of mice pre-immunized with ovalbumin (OVA) in incomplete Freund's adjuvant (IFA) using a mouse CD8 T cell isolation kit (Miltenyi) according to the manufacturer's instructions. Immunization induced physiologically relevant levels of CD8+ T cell populations containing OVA-specific T cells pre-exposed to antigen. T cell co-cultures were performed by adding 1.6 × 10 5The overactivated BMDCs and CD8+ T cells were co-cultured for 96 hours, and the co-culture supernatant was collected.

[0100] IFNγ secretion by CD8+ T cells in response to OVA presented by BMDCs was assessed using the Mouse Lumit IFNγ Kit (Promega) according to the manufacturer's instructions. Luminescence was measured for 500 ms across all wavelengths using a Spectramax M5e plate reader (Molecular Devices). To determine IFNγ concentrations in the supernatants, sample concentrations were interpolated using a standard curve via 4PL analysis in GraphPad Prism 9 (GraphPad Software). Sample interpolation results were then adjusted for any dilutions made to the supernatants. result

[0101] Lipid nanoparticles (LNPs) have become an important vaccine delivery tool, especially in the context of mRNA vaccines, and have played a major role globally in the fight against COVID-19. LNPs are particularly useful for delivering mRNA cargo to cells. However, while LNP-based mRNA vaccines are effective in inducing antibody responses to the antigens they encode, mRNA vaccines often elicit limited antigen-specific T cell responses, thereby negatively impacting their efficacy and longevity. To address this issue, the present disclosure describes the addition of a lysophosphatidylcholine (LPC) compound with a single acyl chain, e.g., 22:0 Lyso PC, to an LNP formulation containing an antigen-encoding mRNA to enhance its immunogenicity.

[0102] Based on the exemplary lipid structures, we considered incorporating 22:0 Lyso PC into LNPs, and could affect the physical characteristics and biological activity of 22:0 Lyso PC-loaded LNPs by modulating the various LNP components. Both the loading level and number of loaded LNPs were considered to be critical variables that affect the payload of 22:0 Lyso PC delivered to cells.

[0103] 22:0 Lyso PC can be loaded into LNP. LNP was prepared by combining the following SM102, DPSC, cholesterol, and DMG-PEG2000 with or without 22:0 Lyso PC. The input molar ratios for these LNP formulations are listed in Table 2-1. 22:0 Lyso PC is most structurally similar to DSPC, so when the amount of 22:0 Lyso PC added to LNP is increased, DSPC in the formulation is replaced with 22:0 Lyso PC. To determine whether the loading level of 22:0 Lyso PC can be intentionally changed and to understand how loading level can affect the bioactivity of LNP, several different LNP compositions containing various levels of 22:0 Lyso PC were prepared. LNPs were prepared without 22:0 Lyso PC (LNP0) or loaded with 20% (LNP20), 30% (LNP30) or 40% (LNP40) molar ratios of 22:0 Lyso PC. LNPs were also made without mRNA, with mRNA encoding GFP (GFP mRNA), or with mRNA encoding OVA (OVA mRNA) at various loading levels to determine whether inclusion of 22:0 Lyso PC could affect the loading of mRNA into LNPs.

[0104] Table 2-2 details the loading ratio of 22:0 Lyso PC to mRNA for the formulations tested. Various loading levels of mRNA / 22:0 Lyso PC were tested to identify conditions under which both mRNA and 22:0 Lyso PC could be biologically active. In all cases, both mRNA and 22:0 Lyso PC were loaded into the LNPs as assessed by Ribogreen and HPLC, respectively. Except for 22:0 Lyso PC LNP20, the mRNA loading for all LNP formulations was >75% and the 22:0 Lyso PC loading was >80%. Table 2-2. Relative Loading Levels of LNP Formulations^ [Table 2-2] ^LNPs containing no mRNA (LNP 0, LNP 30, LNP 40) were prepared and loaded with 22:0 Lyso PC with similar efficiency to the mRNA-containing LNPs.

[0105] Importantly, both 22:0 Lyso PC and mRNA were loaded into LNPs without dramatically increasing the size or polydispersity index (PDI) of the LNPs (Figure 1A-B). In all cases, LNPs prepared with mRNA were larger, with a slightly larger effective diameter when 22:0 Lyso PC was added to the LNPs (Figure 1A). All LNPs were still nanoparticle sized, with all diameters <125 nm. The addition of 22:0 Lyso PC did not affect the PDI of the LNPs (Figure 1B), and all LNP formulations produced relatively uniform particle populations (PDI<0.3). In most cases, all size readings for LNPs by dynamic light scattering (DLS) were relatively close, except for the LNP20 formulation, which does not contain mRNA. Therefore, in some embodiments, 22:0 Lyso PC is included in the LNP formulation at a molar percent concentration of greater than 20%, preferably greater than 25%, and more preferably at least 30%.

[0106] 22:0 Lyso PC LNPs allow hyperactivation of human moDCs. Human monocytes isolated from Leukopaks were differentiated into monocyte-derived DCs (moDCs) in culture with GM-CSF and IL-4 for 6 days. moDCs were harvested on day 6 after differentiation and plated at 1 × 10 in 96-well flat-bottom plates. 5 moDCs were plated at 1000 cells / well. moDCs were treated with or without 1 μg / mL R848, with or without hyperactivating lipid-loaded LNPs (or LNP vehicle control) in the presence or absence of eGFP mRNA. LNPs were dosed based on the loading content of LNPs to a total of 50 μM or 100 μM 22:0 Lyso PC. LNP-induced hyperactivation was measured after 48 hours in culture with LNPs by assessing cytokine secretion from live moDCs.

[0107] Cell viability was assessed by LDH present in the supernatant and normalized to wells treated with R848 alone (no LNPs). At the 50 μM treatment condition, all wells had similar viability to the R848 alone treatment condition, regardless of the presence of LNPs or 22:0 Lyso PC in the LNPs (Figure 2A). At the 100 μM dose, most treatments containing R848 were associated with >75% cell viability (Figure 2B). Viability in the LNP40 condition dropped below 75% when cultured in the absence of R848, whereas cell viability was above 75% when R848 was added to the wells. To measure the ability of LNP-delivered 22:0 Lyso PC to hyperactivate moDCs, IL-1β cytokine secretion was measured in cell culture supernatants. Importantly, only live LNP-treated cells containing R848 and 22:0 Lyso PC were able to produce IL-1β (Figures 2C-2D). IL-1β secretion was dose-dependent, with cells treated with 100 μM 22:0 Lyso PC producing more IL-1β than cells treated with 50 μM 22:0 Lyso PC. Importantly, IL-1β secretion was not affected by the presence of mRNA in the LNPs, indicating that LNPs loaded with 22:0 Lyso PC and mRNA had increased immunogenicity compared to LNPs lacking 22:0 Lyso PC. In addition, increasing the molar ratio of 22:0 Lyso PC in the LNPs increased IL-1β secretion, indicating that the immunogenicity of LNPs can be altered by varying the amount of 22:0 Lyso PC in the LNPs, even when delivering the same total dose of 22:0 Lyso PC.

[0108] In addition to IL-1β, IL-6 secretion was measured in cell culture supernatants after 48 h. Interestingly, the addition of 22:0 Lyso PC to the LNPs increased the amount of IL-6 secreted in response to R848 (Figures 2E-2F). moDCs treated with LNPs without 22:0 Lyso PC produced IL-6 at levels similar to cells treated with R848 alone, indicating that LNPs themselves are not necessarily strongly immunogenic. However, when LNPs were also loaded with 22:0 Lyso PC, the amount of secreted IL-6 was significantly increased. In addition, this response was dose-dependent, with cells treated with 100 μM 22:0 Lyso PC being able to produce more IL-6 than cells treated with 50 μM 22:0 Lyso PC.

[0109] 22:0 Lyso PC LNPs allow translation of mRNA and increase surface expression of activation markers in human moDCs. To determine whether overactivation of moDCs could affect mRNA expression, the levels of GFP were measured. Only cells treated with LNPs containing GFP mRNA produced GFP expression (Figures 3A-3B). Treatment with LNPs containing 22:0 Lyso PC (LNP mRNA 30 and LNP mRNA 40) indeed reduced the percentage of cells positive for GFP, but most of the cells treated with mRNA LNPs were able to translate GFP mRNA into protein. Importantly, although the median fluorescence intensity (MFI) of GFP was lower for LNPs containing 22:0 Lyso PC (Figures 3C-3D), the percentage of cells expressing mRNA remained high, indicating that cells continue to express GFP, albeit at a lower rate. Considering that these moDCs produce GFP as soon as they are overactivated, it is unlikely that lower levels of GFP expression would translate to significantly lower levels of antigen (GFP) presentation at the cell surface. Interestingly, this was found to be dose-dependent, as increasing amounts of 22:0 Lyso PC resulted in lower levels of GFP expression while simultaneously increasing the levels of IL-6 and IL-1β secretion. Importantly, moDCs treated with LNPs loaded with GFP mRNA are able to express GFP, regardless of the loading of the LNPs with 22:0 Lyso PC.

[0110] In parallel with the aforementioned study, moDCs were harvested after 48 h in culture with LNPs and expressed live CD11c +Cells were assessed for surface expression of CD40, CD86, HLA-DR, and HLA-ABC. Levels of CD86 and CD40 on moDCs were measured to determine whether overactivation could affect expression of activation markers. In moDCs prepared from samples of two human donors, treatment with LNPs loaded with 22:0 Lyso PC increased the median fluorescence intensity (MFI) of both CD86 (Figures 4A-4B) and CD40 (Figures 4C-4D), indicating increased expression of costimulatory and activation markers after overactivation. In addition, treatment with 100 μM 22:0 Lyso PC further increased surface expression of CD86 and CD40, so the increase in CD86 and CD40 was dose-dependent. Levels of HLA-DR and HLA-ABC on moDCs were measured to determine whether overactivation could interfere with expression of major histocompatibility complex (MHC) and therefore antigen presentation. Similar to the expression of activation markers, treatment of moDCs with 22:0 Lyso PC-loaded LNPs increased the MFI of both HLA-DR (MHC-II) (Figures 4E-4F) and HLA-ABC (MHC-I) (Figures 4G-4H), indicating increased expression of antigen-presenting molecules after hyperactivation. This increase was also observed to be dose-dependent, as treatment with 100 μM 22:0 Lyso PC further increased the expression of HLA-DR and HLA-ABC.

[0111] Co-culture of mouse DC:T cells allows activation of antigen-specific T cells with LNPs loaded with 22:0 LPC. To expand on the observations made in the human moDC system, studies were also performed in the mouse system. LNPs were loaded with 22:0 Lyso PC and two different concentrations of OVA mRNA (Table 2-2). Mouse bone marrow-derived dendritic cells (BMDCs) were then hyperactivated with 50 μM or 100 μM 22:0 Lyso PC delivered via LNPs containing R848 and low or high doses of OVA mRNA (Table 2-2). Approximately 48 hours after stimulation, supernatants were collected to measure IL-1β secretion. Importantly, only BMDCs treated with LNPs containing R848 and 22:0 Lyso PC were able to produce high levels of IL-1β (Figure 5A). Cells treated with LNPs that did not contain 22:0 Lyso PC (LNP0 formulation) actually produced more IL-1β than cells treated with R848 alone. Addition of 22:0 Lyso PC to LNPs resulted in significantly more IL-1β production. As seen with human moDCs, IL-1β secretion was dose-dependent, with cells treated with 100 μM 22:0 Lyso PC producing more IL-1β than cells treated with 50 μM. In addition, IL-1β secretion was not affected by the presence or dose of mRNA in the LNPs. This indicates that LNPs loaded with 22:0 Lyso PC and mRNA are more immunogenic than LNPs lacking 22:0 Lyso PC.

[0112] In addition, BMDCs overactivated for 48 h with LNPs containing 22:0 Lyso PC and different doses of OVA mRNA were cocultured with OT-I CD8+ T cells collected from the spleen and lymph nodes of OT-I mice. All CD8+ T cells from OT-I mice were naive (antigen naive) and specific for OVA peptide. Overactivated DCs and CD8+ T cells were cocultured for 72 h, after which the coculture supernatants were collected. IFNγ secretion by CD8+ T cells in response to OVA presented by BMDCs was evaluated. As expected, IFNγ secretion by CD8+ T cells was driven by the presence of OVA mRNA in the LNPs (Figure 5B). Importantly, IFNγ secretion by activated CD8+ T cells was not affected by the presence of 22:0 Lyso PC in the LNPs. Interestingly, the 10-fold dose difference in mRNA between low-dose and high-dose OVA mRNA did not affect IFNγ secretion. Taken together, these results indicate that loading of LNPs with 22:0 Lyso PC does not reduce antigen expression to a level that would negatively affect antigen presentation on the DC cell surface, thus allowing activation of antigen-specific naive CD8+ T cells.

[0113] We also co-cultured BMDCs overactivated for 48 h with LNPs containing 22:0 Lyso PC and different doses of OVA mRNA with CD8+ T cells collected from the spleens and lymph nodes of mice pre-immunized with ovalbumin in IFA. The OVA-IFA immunization scheme produces a population of antigen-experienced, OVA-specific CD8+ T cells at physiologically relevant levels. Overactivated BMDCs and CD8+ T cells were co-cultured for 96 h, after which co-culture supernatants were collected. IFNγ secretion by reactivated CD8+ T cells in response to OVA presented by BMDCs was evaluated. Importantly, IFNγ secretion by reactivated CD8+ T cells was largely driven by the dose of OVA mRNA delivered and was unaffected by the presence of 22:0 Lyso PC in LNPs (Figures 6A-6B). Again, this data indicates that loading LNPs with 22:0 Lyso PC does not interfere with antigen expression and presentation from antigen-encoding mRNA.

[0114] Taken together, these results indicate that the use of more immunogenic LNPs loaded with LPCs with a single acyl chain (e.g., 22:0 Lyso PC) in addition to antigen-encoding mRNA has advantages over LNPs that do not contain hyperactivated lipids. The effects of hyperactivated LNPs are expected to become more evident in vivo. In particular, the increased migration to lymph nodes and IL-1β secretion by DCs loaded with hyperactivated LNPs is expected to lead to the production of new antigen-specific memory T cells and the reactivation of antigen-specific T cells more potently.

Claims

1. A composition comprising mRNA encapsulated in a lipid nanoparticle (LNP) and a TLR7 / 8 agonist, wherein the mRNA comprises a coding region for an antigen, and the LNP comprises a first phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a second phospholipid, a pegylated lipid, a structured lipid, and a mixture thereof, wherein the first phospholipid comprises lysophosphatidylcholine (LPC) having a single acyl chain, and the acyl chain is a C13 to C24 acyl chain.

2. The composition described in claim 1, wherein the TLR7 / 8 agonist is encapsulated in the lipid nanoparticle (LNP).

3. 10. The composition of claim 1, wherein the at least one lipid comprises an ionizable lipid, a second phospholipid, a pegylated lipid, and a structural lipid.

4. The ionizable lipid is i) 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102) or an analog or derivative thereof, and / or ii) 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminium (ALC-0315) or an analog or derivative thereof The composition of claim 3 comprising:

5. 4. The composition of claim 3, wherein the PEGylated lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglyerol, and combinations thereof.

6. 4. The composition of claim 3, wherein the pegylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoylglycerol [DMG].

7. 4. The composition of claim 3, wherein the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and combinations thereof.

8. The composition of claim 7 , wherein the structural lipid comprises cholesterol.

9. The second phospholipid is i) a hydrophilic head moiety selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin; and ii) lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, arachidic acid, arachidonic acid, phytanoic acid, eicosapentaenoic acid, behenic acid, dodecanoic acid, hydroxybenzo ... one or more fatty acid tail moieties selected from the group consisting of cosapentaenoic acid, and docosahexaenoic acid The composition of claim 3 comprising:

10. The second phospholipid is 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-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and The composition of claim 3 selected from the group consisting of:

11. i) mRNA complexed with one or more lipids (RNA-Lipoplex); ii) lysophosphatidylcholine (LPC) with a single C13-C24 acyl chain, and iii) TLR7 / 8 agonists A composition comprising: The composition, wherein the mRNA comprises a coding region for an antigen, and the one or more lipids comprise a first lipid and a second lipid.

12. 12. The composition of claim 11, wherein the first lipid is a cationic lipid and the second lipid is a neutral or anionic lipid.

13. 2. The composition of claim 1, wherein the acyl chain of the LPC is a C21-C24 acyl chain and / or the acyl chain of the LPC is fully saturated.

14. The composition of claim 13, wherein the LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

15. 15. The composition of claim 14, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 daltons or less.

16. 16. The composition of claim 15, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

17. 17. The composition of claim 16, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

18. The composition of claim 1 , wherein the antigen is a tumor antigen or a microbial antigen.

19. A pharmaceutical formulation comprising the composition of any one of claims 1 to 18 and a pharmaceutically acceptable excipient.

20. 20. A method for generating hyperactivated dendritic cells, comprising contacting dendritic cells ex vivo with an effective amount of the pharmaceutical preparation of claim 19.

21. The pharmaceutical formulation of claim 19 for use in a method for generating hyperactivated dendritic cells, the method comprising contacting dendritic cells in vivo with an effective amount of the pharmaceutical formulation to generate hyperactivated dendritic cells.

22. At least 10 produced by the method of claim 20 ３ , 10 ４ , 10 ５ or 10 ６ and a pharmaceutically acceptable excipient.

23. 20. The pharmaceutical formulation of claim 19 for stimulating an immune response to an antigen, wherein an effective amount of said pharmaceutical formulation is administered to an individual in need thereof to stimulate an immune response to said antigen, thereby stimulating said immune response to said antigen.

24. 20. The pharmaceutical preparation of claim 19 for treating cancer, wherein an effective amount of the pharmaceutical preparation is administered to an individual in need of cancer treatment to treat the cancer.

25. 20. The pharmaceutical preparation of claim 19 for inhibiting abnormal cell growth, wherein an effective amount of the pharmaceutical preparation is administered to an individual in need thereof to inhibit abnormal cell growth, thereby inhibiting abnormal cell growth.

26. 20. The pharmaceutical preparation of claim 19 for treating or preventing an infectious disease, wherein an effective amount of the pharmaceutical preparation is administered to an individual in need thereof to treat or prevent the infectious disease, thereby treating or preventing the infectious disease.

27. The composition of any one of claims 1 to 18, wherein the composition is protein-free.

28. A method for generating hyperactivated dendritic cells, comprising: i) contacting dendritic cells with a TLR7 / 8 agonist ex vivo; and ii) contacting dendritic cells ex vivo with a composition comprising mRNA encapsulated in lipid nanoparticles (LNPs); wherein the mRNA comprises a coding region for an antigen; the LNPs comprise a first phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a second phospholipid, a pegylated lipid, a structured lipid, and mixtures thereof; the first phospholipid comprises lysophosphatidylcholine (LPC) having a single acyl C13-C24 acyl chain; method.

29. A combination for use in a method for generating hyperactivated dendritic cells, the method comprising: (a) a TLR7 / 8 agonist; and (b) a composition comprising mRNA encapsulated in a lipid nanoparticle (LNP), the composition comprising: i) contacting dendritic cells in vivo with (a) said TLR7 / 8 agonist; and ii) contacting dendritic cells in vivo with the composition comprising (b) mRNA encapsulated in lipid nanoparticles (LNPs). wherein the mRNA comprises a coding region for an antigen; the LNPs comprise a first phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a second phospholipid, a pegylated lipid, a structured lipid, and mixtures thereof; the first phospholipid comprises lysophosphatidylcholine (LPC) having a single acyl C13-C24 acyl chain; Combination.

30. A combination for use in a method of stimulating an immune response to an antigen in an individual in need thereof, the method comprising: (a) a TLR7 / 8 agonist; and (b) a composition comprising mRNA encapsulated in a lipid nanoparticle (LNP), the method comprising: i) (a) administering said TLR7 / 8 agonist to said individual; and ii) (b) administering to said individual said composition comprising mRNA encapsulated in lipid nanoparticles (LNPs). wherein the mRNA comprises a coding region for the antigen; the LNPs comprise a first phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a second phospholipid, a pegylated lipid, a structured lipid, and mixtures thereof; the first phospholipid comprises lysophosphatidylcholine (LPC) having a single acyl C13-C24 acyl chain; Combination.

31. A composition for the manufacture of a medicament for stimulating an immune response in an individual, comprising mRNA encapsulated in a lipid nanoparticle (LNP), wherein the mRNA comprises a coding region for an antigen, and the LNP comprises a first phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a second phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof, wherein the first phospholipid comprises lysophosphatidylcholine (LPC) having a single acyl chain, and the acyl chain is a C13 to C24 acyl chain.

32. A composition for the manufacture of a medicament for stimulating an immune response in an individual, comprising: i) mRNA complexed with one or more lipids (RNA-Lipoplex); and ii) lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain, wherein the mRNA comprises a coding region for an antigen, and the one or more lipids comprise a first lipid and a second lipid.

33. A composition for use in a method for generating hyperactivated dendritic cells, comprising a TLR7 / 8 agonist, said method comprising: i) contacting dendritic cells in vivo with said TLR7 / 8 agonist; and ii) contacting dendritic cells in vivo with a composition comprising mRNA encapsulated in lipid nanoparticles (LNPs); wherein the mRNA comprises a coding region for an antigen; the LNPs comprise a first phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a second phospholipid, a pegylated lipid, a structured lipid, and mixtures thereof; the first phospholipid comprises lysophosphatidylcholine (LPC) having a single acyl C13-C24 acyl chain; composition.

34. A composition for use in a method for generating hyperactivated dendritic cells, comprising mRNA encapsulated in a lipid nanoparticle (LNP), said method comprising: i) contacting dendritic cells in vivo with a TLR7 / 8 agonist; and ii) contacting dendritic cells in vivo with the composition comprising mRNA encapsulated in lipid nanoparticles (LNPs); wherein the mRNA comprises a coding region for an antigen; the LNPs comprise a first phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a second phospholipid, a pegylated lipid, a structured lipid, and mixtures thereof; the first phospholipid comprises lysophosphatidylcholine (LPC) having a single acyl C13-C24 acyl chain; composition.

35. A composition for use in a method of stimulating an immune response to an antigen in an individual in need thereof, the composition comprising a TLR7 / 8 agonist, the method comprising: i) administering said TLR7 / 8 agonist to said individual; and ii) administering to said individual a composition comprising mRNA encapsulated in lipid nanoparticles (LNPs). wherein the mRNA comprises a coding region for the antigen; the LNPs comprise a first phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a second phospholipid, a pegylated lipid, a structured lipid, and mixtures thereof; the first phospholipid comprises lysophosphatidylcholine (LPC) having a single acyl C13-C24 acyl chain; composition.

36. A composition for use in a method of stimulating an immune response to an antigen in an individual in need thereof, the composition comprising mRNA encapsulated in a lipid nanoparticle (LNP), the method comprising: i) administering a TLR7 / 8 agonist to said individual; and ii) administering to said individual said composition comprising mRNA encapsulated in lipid nanoparticles (LNPs). wherein the mRNA comprises a coding region for the antigen; the LNPs comprise a first phospholipid and at least one lipid selected from the group consisting of an ionizable lipid, a second phospholipid, a pegylated lipid, a structured lipid, and mixtures thereof; the first phospholipid comprises lysophosphatidylcholine (LPC) having a single acyl C13-C24 acyl chain; composition.

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