Hyperactivated lipid nanoparticles

Abstract

The present disclosure relates to lipid nanoparticles comprising a lysophosphatidylcholine (LPC) compound and at least one additional lipid, and their use in hyperactivating mammalian dendritic cells, such as human dendritic cells.The present disclosure also relates to compositions comprising lipid nanoparticles comprising LPC and at least one additional lipid, the compositions comprising one or more of a pathogen recognition receptor agonist, an antigen, and a mammalian dendritic cell, and methods for producing and using the compositions.

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 / 417,282, filed October 18, 2022, and U.S. Provisional Patent Application No. 63 / 307,569, filed February 7, 2022, each of which is incorporated by reference in its entirety.

[0002] Field The present disclosure relates to lipid nanoparticles comprising a lysophosphatidylcholine (LPC) compound and at least one additional lipid, and their use in hyperactivating mammalian dendritic cells, such as human dendritic cells.The present disclosure also relates to a composition comprising lipid nanoparticles comprising LPC and at least one additional lipid, the composition comprising one or more of a pathogen recognition receptor agonist, an antigen, and a mammalian cell, and a method for producing and using the composition. [Background technology]

[0003] background Lipid nanoparticles (LNPs) have become an important vaccine delivery tool, especially in the context of mRNA vaccines. LNP-based mRNA vaccines and protein subunit vaccines are effective in inducing antigen-specific antibody responses but often exhibit limited antigen-specific T cell responses.

[0004] Dendritic cells (DCs) provide T cells with several signals that are important for the establishment of appropriate T cell responses. The type and magnitude of the signal depends on the activation state of the DCs (Zhivaki and Kagan, Nature Reviews Immunology, 22:322-339, 2022). Naive DCs are resting cells that have the ability to take up antigens. Activated DCs not only have the ability to take up antigens, but also have an enhanced ability to present peptide fragments of antigens on major histocompatibility complex molecules. In addition, activated DCs have increased expression of costimulatory molecules for stimulation of T cells. Hyperactivated DCs not only share activity with their active DC counterparts, but also gain the ability to hypermigrate to lymph nodes and secrete IL-1β. Pyroptotic DCs secrete high levels of IL-1β, similar to hyperactivated DCs. However, pyroptotic DCs are dead cells that rapidly lose their T cell stimulatory capacity. DCs secrete IL-1β without pyroptosis when matured with pathogen-associated molecular pattern (PAMP)-containing molecules, lipopolysaccharide (LPS), and damage-associated molecular pattern (DAMP)-containing molecules, such as PGPC (1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine), characterizing these cells as hyperactivated (Zanoni et al., Science, 352(6290):1232-1236, 2016). Thus, LNP formulations capable of hyperactivating dendritic cells are desirable for inclusion in vaccines. In particular, there is a need in the art for LNPs capable of inducing IL-1β secretion and enhancing the production of long-lived T cell responses. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Zhivaki and Kagan, Nature Reviews Immunology, 22:322-339, 2022 [Non-Patent Document 2] Zanoni et al., Science, 352(6290):1232-1236, 2016 Summary of the Invention [Means for solving the problem]

[0006] A brief summary The present disclosure relates to lipid nanoparticles comprising a lysophosphatidylcholine (LPC) compound and at least one additional lipid, and their use in hyperactivating mammalian dendritic cells.The present disclosure also relates to a composition comprising lipid nanoparticles comprising LPC and at least one additional lipid, further comprising one or more of a pathogen recognition receptor agonist, an antigen, and a mammalian dendritic cell, and a method for producing and using the composition.

[0007] In particular, the present disclosure provides a composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and a TLR7 / 8 agonist, wherein the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl chain, and the LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof. In some embodiments, the acyl chain is a C18-C22 acyl chain, a C21-C24 acyl chain, or a C22 acyl chain. In some embodiments, the composition further comprises an antigen and / or a dendritic cell.

[0008] In some aspects, the present disclosure provides a composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and an antigen, wherein the acyl chain is a C21-C24 acyl chain, and the LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof. In some embodiments, the composition further comprises a dendritic cell and / or a TLR7 / 8 agonist.

[0009] In some aspects, the present disclosure provides a composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and a dendritic cell, wherein the acyl chain is a C21-C24 acyl chain, and the LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof. In some embodiments, the composition further comprises a TLR7 / 8 agonist and / or an antigen.

[0010] In some embodiments of the foregoing aspects, the antigen is present in a biological sample obtained from the individual. In some embodiments, the biological sample comprises biopsy tissue. In some embodiments, the biological sample comprises cells. In other embodiments, the biological sample does not comprise cells. In some embodiments, the biological sample comprises pus from an abscess. In some embodiments, the antigen comprises a protein antigen. In some embodiments, the antigen comprises a tumor antigen. In some embodiments, the tumor antigen comprises a synthetic or recombinant neoantigen. In some embodiments, the tumor antigen comprises a tumor cell lysate. In some embodiments, the antigen comprises a microbial antigen, the microbial antigen comprising one or more of a viral antigen, a bacterial antigen, a protozoal antigen, and a fungal antigen. In some embodiments, the microbial antigen comprises a purified or recombinant surface protein. In some embodiments, the microbial antigen comprises an inactivated whole virus.

[0011] In some embodiments, the composition does not include LPS or MPLA. In some embodiments, the composition does not include oxPAPC or oxPAPC species. In some embodiments, the composition does not include HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC, and / or PGPC. In some embodiments, the composition does not include isolated mRNA. In some embodiments, the composition does not include a surfactant (e.g., a poloxamer). In some embodiments, the composition does not include Poloxamer 407 (KP407), Poloxamer 188 (KP188), and / or Pluronic® P123 (P123).

[0012] In some embodiments, the composition further comprises an adjuvant, the adjuvant comprising an aluminum salt adjuvant, a squalene-in-water emulsion, a saponin, or a combination thereof.

[0013] In some embodiments, the present disclosure provides a pharmaceutical formulation comprising any of the compositions of the preceding aspects and a pharma- ceutical acceptable excipient. In some embodiments, the formulation does not include a surfactant (e.g., a poloxamer). In some embodiments, the formulation does not include Poloxamer 407 (KP407), Poloxamer 188 (KP188), and / or Pluronic® P123 (P123).

[0014] In another aspect, the disclosure provides a method for generating hyperactivated dendritic cells, comprising contacting dendritic cells with an effective amount of a composition comprising an isolated lysophosphatidylcholine (LPC) having a single C13-C22 acyl chain or a C13-C24 acyl chain, at least one additional lipid, and a TLR7 / 8 agonist for generating hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1 beta without undergoing pyroptosis, and wherein the LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof. In some embodiments, the dendritic cells are contacted ex vivo with the composition or pharmaceutical formulation of any one of the preceding embodiments. In other embodiments, the dendritic cells are contacted in vivo with a pharmaceutical formulation comprising the composition of any one of the preceding embodiments. In some aspects, the present disclosure provides a pharmaceutical formulation comprising a plurality of hyperactivated dendritic cells generated according to the preceding embodiments, and a pharma- ceutically acceptable excipient. In some embodiments, the plurality comprises at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 or 10 8 of overactivated DCs.

[0015] In another aspect, the present disclosure provides a composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and a pathogen recognition receptor (PRR) agonist, wherein the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl chain, and the LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof. In some embodiments, the PRR agonist is an agonist of a toll-like receptor (TLR), a NOD-like receptor (NLR), a RIG-I-like receptor (RLR), or a C-type lectin receptor (CLR). In some embodiments, the PRR agonist is an agonist of a cytoplasmic DNA sensor (CDS) or a stimulator of IFN genes (STING). In some embodiments, the PRR agonist comprises a TLR7 / 8 agonist.In some embodiments, the composition further comprises an antigen and / or a dendritic cell.

[0016] In some embodiments of the foregoing aspects, the acyl chain is a C21-C24 acyl chain. In some embodiments, the acyl chain is a C22 acyl chain. In some embodiments, the acyl chain is fully saturated. In some embodiments, the LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

[0017] In some embodiments of the above aspects, the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 Daltons or less. In some embodiments, the TLR7 / 8 agonist comprises an imidazoquinoline compound. In some embodiments, the TLR7 / 8 agonist comprises resiquimod (R848). In some embodiments, the LPC comprises LPC(22:0) and the TLR7 / 8 agonist comprises resiquimod (R848).

[0018] The present disclosure further provides a composition for human dendritic cell hyperactivation, comprising an isolated lysophosphatidylcholine (LPC) compound having a single acyl chain, at least one additional lipid, and a pathogen recognition receptor (PRR) agonist, wherein the acyl chain is a C22 acyl chain, the LPC and at least one additional lipid are part of a lipid nanoparticle (LNP), and the composition is effective for achieving a higher level of dendritic cell hyperactivation than a comparative composition comprising a comparative compound instead of LPC. In some embodiments, the at least one additional lipid is selected from the group consisting of ionizable lipids, cationic lipids, additional phospholipids, PEGylated lipids, structural lipids, and mixtures thereof. In some embodiments, the hyperactivation occurs in vitro or ex vivo. In other embodiments, the hyperactivation occurs in vivo. In some embodiments, the higher level of dendritic cell hyperactivation comprises inducing in vitro IL-1 beta secretion from mammalian (e.g., human) dendritic cells at a level at least 2, 3, or 4 times higher when contacted with a composition comprising LPC and a PRR agonist than when contacted with a comparative composition comprising a comparative compound and a PRR agonist, where the PRR agonist is LPS. In some embodiments, the concentration of LPC and the concentration of the comparative compound are the same, optionally within a range of about 10 μM to about 80 μM, and LPS is present at a concentration of 1 μg / ml in both the composition and the comparative composition. In some embodiments, the higher level of dendritic cell hyperactivation comprises a lipid activity index of IL-1 beta secretion from mammalian (e.g., human) dendritic cells that is at least 4, 5, or 6 times higher in activity units for the composition comprising LPC and a PRR agonist than the activity units of the comparative composition comprising a comparative compound and a PRR agonist. In some embodiments, the comparative compound is PGPC. [Brief description of the drawings]

[0019] [Figure 1]Figures 1A-1B show IL-1β secretion by canine peripheral blood mononuclear cells (PBMCs) 2 days after activation with the indicated stimuli, shown as total concentration (Figure 1A) and fold change per donor compared to R848 alone (Figure 1B), respectively. The results demonstrate that 22:0 LYSO PC, when combined with R848, can stimulate canine PBMCs to secrete IL-1β at levels comparable to or higher than DAMPs, such as PGPC or LPS and Alum. Figure 1C shows the relative viability of canine PBMCs 2 days after activation with the indicated stimuli. The results demonstrate that canine PBMCs are still viable after treatment with 22:0 LYSO PC.

[0020] [Diagram 2] Figures 2A-2B show IL-1β secretion by human PBMCs 2 days after activation with the indicated stimuli, shown as total concentration (Figure 2A) and fold change per donor compared to R848 alone (Figure 2B). The results demonstrate that 22:0 LYSO PC, when combined with R848, can stimulate human PBMCs to secrete IL-1β at levels comparable to or higher than DAMPs such as PGPC or LPS and Alum. Figure 2C shows the relative viability of human PBMCs 2 days after activation with the indicated stimuli. The results demonstrate that human PBMCs are still viable after treatment with 22:0 LYSO PC.

[0021] [Diagram 3] Figures 3A-3B show secretion of IFNγ (Figure 3A) and TNFα (Figure 3B) by human PBMCs 2 days after activation with the indicated stimuli, shown as fold change per donor compared to R848 alone. The results demonstrate that 22:0 LYSO PC, when combined with R848, can stimulate human PBMCs to secrete other immunostimulatory cytokines at levels comparable to or higher than DAMPs such as PGPC or LPS and Alum.

[0022] [Figure 4] Figure 4 shows that inclusion of various concentrations of 22:0 Lyso PC in lipid nanoparticles (LNPs) does not affect the size of the resulting LNPs. In this plot, the vehicle controls for LNP2 and LNP3 were the same.

[0023] [Figure 5-1] Figure 5A shows quantification of 22:0 Lyso PC incorporation into GenVoy LNPs. Figure 5B shows quantification of 22:0 Lyso PC incorporation into LNPs containing ionizable lipids. Figure 5C shows quantification of 22:0 Lyso PC incorporation into LNPs lacking ionizable lipids. Figure 5D shows that increasing input amounts of 22:0 Lyso PC increase the loading of 22:0 Lyso PC into LNPs. [Figure 5-2] Same as above.

[0024] [Figure 6-1] Figure 6A shows the viability of monocyte-derived dendritic cells (moDCs) cultured in the presence or absence of LNPs and in the presence or absence of pathogen-associated molecular pattern-containing molecules (PAMPs). Figure 6B shows IL-1β secretion by moDCs cultured in the presence or absence of LNPs and in the presence or absence of PAMPs. The PAMP utilized in the assays of Figures 6A-6B was resiquimod (R848), a TLR7 / 8 agonist. 22:0 Lyso PC, when present, was included at a concentration of 82.5 μM. [Figure 6-2] Same as above.

[0025] [Figure 7] Figure 7A shows that 22:0 Lyso PC in PBS has a large diameter and a large range of particle sizes, and Figure 7B shows that 22:0 Lyso PC loaded into LNPs results in a much smaller particle size with increased uniformity.

[0026] [Figure 8] Figures 8A-D show that 22:0 Lyso PC LNP induces hyperactivation of mouse bone marrow-derived dendritic cells (BMDCs). Figure 8A shows the viability of BMDCs 48 hours after treatment with different formulations as measured using the Cell Titer Glow assay. Data presented are compared to cells treated with R848. Figure 8B shows IL-6 secretion and Figure 8C shows IL-1β secretion by BMDCs 48 hours after treatment with different formulations as measured by ELISA. Means and SD are shown and are representative of triplicates from one experiment. Figure 8D shows the absolute number of CD11c+MHC-II+DCs in the draining lymph nodes that are CFSE+ as measured by flow cytometry. BMDCs were treated with different formulations for 24 hours before CFSE staining and injection. An unpaired t-test was used. Means and SD are shown and are representative of four mice from one experiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description The present disclosure relates to lipid nanoparticles (LNPs) comprising lysophosphatidylcholine (LPC) compounds and at least one additional lipid, and their use in hyperactivation of mammalian dendritic cells. The present disclosure also relates to compositions comprising LNPs comprising LPC and at least one additional lipid, further comprising one or more of a pathogen recognition receptor agonist, an antigen, and a mammalian dendritic cell, and methods for producing and using the compositions. In some embodiments, the dendritic cells are human dendritic cells. In other embodiments, the dendritic cells are non-human dendritic cells. In some embodiments, the non-human dendritic cells are not rodent dendritic cells. In some embodiments, the at least one additional lipid is selected from the group consisting of ionizable lipids, cationic lipids, additional phospholipids, pegylated lipids, structural lipids, and mixtures thereof.

[0028] In some embodiments of the present disclosure, the LNPs of the composition are enriched in particles having a lipid bilayer (liposomes) compared to particles having a single lipid layer (micelles). Specifically, in some embodiments, the LNPs include liposomes and little or no micelles. General Techniques and Definitions

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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).

[0033] 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.

[0034] 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.

[0035] 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.

[0036] The terms "isolated" and "purified," as used herein, refer to a material that is removed from at least one other associated component (e.g., removed from its native environment) during the production of the material. As an 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 the host cell that produced the protein. In addition, when used in reference to a synthesized compound, an isolated or purified compound has been removed from the reaction mixture in which it was synthesized."

[0037] 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.

[0038] "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.

[0039] 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).

[0040] 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.

[0041] 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.

[0042] "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.

[0043] "Alkylene" refers to a divalent saturated aliphatic hydrocarbyl group.

[0044] "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.

[0045] "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.

[0046] 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.

[0047] The relative terms "higher" and "lower" refer to a measurable increase or decrease in a response or parameter, respectively, when compared to the same condition except for the parameter of interest, or alternatively, compared to another condition. For example, "higher levels of DC hyperactivation" refers to the level of DC hyperactivation resulting from a treatment condition (including the LPC compounds of the present disclosure) being 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., no LPC, PGPC, oxPAPC, etc.). Similarly, "lower levels of DC hyperactivation" refers to the level of DC hyperactivation resulting from a treatment condition (including the LPC compounds of the present disclosure) being 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., no LPC, PGPC, oxPAPC, etc.). In some embodiments, the control condition includes a comparison compound instead of the LPC of the treatment condition.

[0048] 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.

[0049] The term "vaccination," as used herein, refers to the introduction of a vaccine into the body of a mammalian subject.

[0050] "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.

[0051] 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

[0052] "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.

[0053] In some embodiments, the isolated lysophosphatidylcholine (LPC) having a single acyl chain has the form [ka] It is of the following.

[0054] In some embodiments, the isolated lysophosphatidylcholine (LPC) having a single acyl chain has the form [ka] [ka] It is of the following.

[0055] 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.

[0056] 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

[0057] 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

[0058] 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.

[0059] 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

[0060] 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).

[0061] 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

[0062] The compositions and methods of the present disclosure may further include an antigen. In some embodiments, the antigen comprises a protein antigen. The terms "polypeptide" and "protein" are used interchangeably herein to refer to a protein antigen comprising a peptide chain of at least 8 amino acids in length. In some embodiments, the protein antigen is 8-1800 amino acids, 9-1000 amino acids, or 10-100 amino acids in length. In some embodiments, the antigen comprises a synthetic or recombinant protein. In other embodiments, the antigen comprises a protein purified from a biological sample. The polypeptide may be post-translationally modified, for example, by phosphorylation, hydroxylation, sulfonation, palmitoylation, and / or glycosylation.

[0063] 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.

[0064] 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.

[0065] 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 antigen subunit of a microorganism. In other embodiments, the microbial antigen comprises an inactivated or attenuated microorganism. For example, the microbial antigen may comprise an inactivated virus, such as a chemically or genetically inactivated virus. Alternatively, the microbial antigen may comprise a virus-like particle.

[0066] In some embodiments, the antigen may be present in a biological sample obtained from an individual, e.g., a human patient. For example, the antigen may include a cancer cell. In another aspect, the antigen may include a microbially infected cell, e.g., a virally infected cell. IV. Dendritic cells

[0067] The compositions and methods of the present disclosure may further include dendritic cells (DCs), which are antigen-presenting cells that are believed to bridge the innate and adaptive immune systems of mammals. In a preferred embodiment, the DCs are subset-1 conventional DCs (cDC1s, previously known as myeloid DC1s), as opposed to plasmacytoid DCs (pDCs).

[0068] In some embodiments, the DC is an activated hyper-DC that expresses high levels of CD40 and IL-12p70. As used herein, the term "activated hyper-dendritic cells" refers to a cell state in which DC can secrete IL-1β while maintaining cell viability (e.g., without undergoing pyroptosis). In this way, hyper-activated dendritic cells can stimulate robust T cell immunity (Figure 1), which clearly combines the benefits of activated and pyroptotic dendritic cells (Zhivaki et al., Cell Reports, 33 (7), 2020, 108381). V. Additional Lipids

[0069] The compositions and methods of the present disclosure include at least one additional lipid, and the LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid includes an ionizable lipid, a cationic lipid, an additional phospholipid, a PEGylated lipid, a structured lipid, or a mixture thereof. In some embodiments, the LNP includes a first phospholipid (lysophosphatidylcholine with a single C13-C24 acyl chain [LPC:C13-C24]), an ionizable lipid, a second phospholipid, a PEGylated lipid, and a structured lipid. The structure of the additional lipid suitable for use in the compositions and methods of the present disclosure is shown below (reproduced from Figure 2 of Hou et al., Nature Review Materials, 6:1078-1094, 2021).

[0070] In some embodiments, the at least one additional lipid comprises one or both of an additional phospholipid and a structural lipid, optionally the additional phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and the structural lipid comprises cholesterol. In some embodiments, the at least one additional lipid comprises or further comprises a pegylated lipid, optionally the pegylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG]. In some embodiments, the at least one additional lipid comprises or further comprises an ionizable lipid, optionally the ionizable lipid comprises (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (also known as 4-(dimethylamino)-butanoic acid), (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester (DLin-MC3-DMA) or an analog or derivative thereof. [ka] VI. Pharmaceutical Preparations

[0071] Some compositions of the present disclosure are pharmaceutical formulations comprising a pharmaceutically acceptable excipient and a lipid nanoparticle (LNP) comprising an LPC compound and at least one additional lipid. In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and a mixture thereof. In some embodiments, the pharmaceutical formulation further comprises a PRR agonist, a dendritic cell, an antigen, an adjuvant, or any combination thereof. Some compositions of the present disclosure are pharmaceutical formulations comprising a pharmaceutically acceptable excipient. The pharmaceutical formulation of the present disclosure may be in the form of a solution or suspension. Alternatively, the pharmaceutical formulation may be an anhydrous solid (e.g., a lyophilized or spray-dried solid). The pharmaceutical formulation of the present disclosure is preferably sterile and preferably essentially endotoxin-free. The term "pharmaceutical formulation" is used interchangeably herein with the terms "pharmaceutical product" and "medicament". In some embodiments, the pharmaceutical formulation comprises a specific ratio of various components based on the intended purpose of the formulation. A. Excipients

[0072] 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).

[0073] 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.

[0074] 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.

[0075] The pharmaceutical composition may include a osmolality modifier. Suitable osmolality modifiers include, for example, glucose, glycerol, sodium chloride, glycerin and mannitol.

[0076] 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.

[0077] 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.

[0078] The pharmaceutical and other compositions of the present disclosure typically do not include surfactants (e.g., poloxamers). In particular, in some embodiments, the pharmaceutical and other compositions do not include Poloxamer 407 (KP407), Poloxamer 188 (KP188), and / or Pluronic® P123 (P123).

[0079] 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). B. Adjuvants

[0080] The pharma- ceutically acceptable adjuvants of the present disclosure include, for example, aluminum salt adjuvants, squalene-in-water emulsions, saponins, or combinations thereof. In some embodiments, the adjuvant is an aluminum salt adjuvant selected from the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, and combinations thereof. In other embodiments, the adjuvant is a squalene-in-water emulsion, such as MF59 or AS03. In other embodiments, the adjuvant is a saponin, such as Quil A or QS-21, such as AS01 or AS02. VII. Methods for Production

[0081] The present disclosure relates in some aspects to methods for preparing hyperactivated dendritic cells and methods for preparing immunogenic compositions suitable for in vitro, ex vivo, or in vivo hyperactivation of dendritic cells.

[0082] In one aspect, the present disclosure provides a method for generating hyperactivated dendritic cells (DCs), the method comprising contacting dendritic cells with an effective amount of a composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and a PRR agonist for generating hyperactivated dendritic cells, the hyperactivated dendritic cells secrete IL-1 beta without undergoing pyroptosis, and the LPC and at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid is selected from the group consisting of ionizable lipids, cationic lipids, additional phospholipids, PEGylated lipids, structural lipids, and mixtures thereof. In some embodiments, the DCs are isolated, while in other embodiments, the DCs are present in a biological sample obtained from a mammalian subject, e.g., a human patient. In some embodiments, the DCs are monocyte-derived DCs, preferably cDC1s.

[0083] In certain embodiments, the present disclosure provides a method for the production of an immunogenic composition, comprising: a) optionally depleting leukocytes from a suspension of cells prepared from a tumor to obtain a suspension enriched in tumor cells; b) lysing cells from the tumor cell enriched suspension to obtain a tumor cell lysate; c) contacting the tumor cell lysate with an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and a PRR agonist to obtain an immunogenic composition, wherein the LPC and at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof. In some embodiments, leukocytes are depleted from the tumor cell enriched cell suspension by contacting the tumor cell enriched suspension with an antibody specific for leukocytes. In some embodiments, leukocytes are depleted by contacting the tumor cell enriched suspension with an anti-CD45 antibody. In some embodiments, the cells are lysed by a cell lysis method based on physical disruption, such as, but not limited to, mechanical lysis, liquid homogenization, sonication, freeze-thawing, or manual grinding. In some preferred embodiments, the cells are lysed by one or more freeze-thaw cycles.

[0084] In some embodiments of the aforementioned method, the acyl chain of the LPC is a C13-C22 acyl chain or a C13-C24 acyl chain. In some embodiments, the acyl chain of the LPC is a C18-C22 acyl chain or a C18-C24 acyl chain. In some preferred embodiments, the acyl chain is fully saturated. In some preferred embodiments, the acyl chain of the LPC is a C22 acyl chain. In some preferred embodiments, the LPC is 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)]. In some embodiments, the PRR agonist is a TLR7 / 8 agonist. In some preferred embodiments, the TLR7 / 8 agonist is an imidazoquinoline compound, in a particularly preferred embodiment, resiquimod (R848). VIII.How to use

[0085] In some aspects, the disclosure relates to a method of using any one of the compositions or formulations described herein. In some embodiments, the composition or formulation comprises an LPC compound and at least one additional lipid, and the LPC and at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof. In some embodiments, the composition or formulation further comprises a PRR agonist, a dendritic cell, an antigen, an adjuvant, or any combination thereof. The method of use is suitable for multiple uses, including stimulating an immune response. In some embodiments, the method of use comprises a method of treating cancer. In some embodiments, the method of use comprises a method of inhibiting abnormal cell proliferation. In some embodiments, the method of use comprises a method of treating or preventing an infectious disease. The method comprises 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 a rodent (e.g., mouse or rat). For veterinary use, the mammalian subject can be a farm animal (e.g., cow), a sport animal (e.g., horse), or a pet (e.g., a companion animal, e.g., dog or cat). A. Stimulation of the immune response

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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

[0090] 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.

[0091] In one aspect, the method can comprise administering to a subject in need thereof a composition comprising an LPC compound and at least one additional lipid, wherein the LPC and at least one additional lipid are part of a lipid nanoparticle (LNP).In another aspect, the method comprises adoptive cell therapy, comprising administering to a subject in need thereof a composition comprising a dendritic cell, for example a hyperactivated dendritic cell, an LPC compound, and an additional lipid, wherein the LPC and at least one additional lipid are part of a lipid nanoparticle (LNP).In some embodiments, the composition further comprises a PRR agonist, an antigen, an adjuvant, or any combination thereof.

[0092] In some embodiments, the method includes treating cancer in an individual or otherwise treating a mammalian subject having cancer. In some embodiments, the method includes a) preparing an immunogenic composition comprising a tumor cell lysate, an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and a toll-like receptor 7 / 8 (TLR7 / 8) agonist, where the tumor cell lysate is prepared or has been prepared from a sample of a tumor obtained from a subject having cancer, the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl chain, and the LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP); and b) administering to the subject an effective amount of the immunogenic composition. In some embodiments, the cancer is a hematological cancer, such as lymphoma, leukemia, or myeloma. In other embodiments, the cancer is a non-hematological cancer, such as a sarcoma, carcinoma, or melanoma. In some embodiments, the cancer is malignant.

[0093] 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.

[0094] 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 isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and a TLR7 / 8 agonist, the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl chain; the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof; The LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP); composition. 2 The composition of embodiment 1, wherein the acyl chain is a C18-C22 acyl chain or a C21-C24 acyl chain. 3. The composition of embodiment 1 or embodiment 2, further comprising an antigen. 4. The composition according to any one of embodiments 1 to 3, further comprising dendritic cells. 5. A composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and an antigen, the acyl chain is a C21 to C24 acyl chain, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof; The LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP); composition. 6. The composition of embodiment 5, further comprising dendritic cells. 7 The composition of embodiment 5 or embodiment 6, further comprising a TLR7 / 8 agonist. 8. A composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and dendritic cells, the acyl chain is a C21 to C24 acyl chain, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof; The LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP); composition. 9 The composition of embodiment 8, further comprising a TLR7 / 8 agonist. 10. The composition of embodiment 8 or embodiment 9, further comprising an antigen. 11. The composition of any one of embodiments 1 to 10, wherein the acyl chain is a C22 acyl chain. 12. The composition of any one of the preceding embodiments, wherein the acyl chains are fully saturated. 13. The composition of any one of embodiments 1 to 12, wherein the LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)]. 14. The composition of any one of embodiments 1 to 13, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 Daltons or less. 15. The composition of embodiment 14, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound. 16. The composition of embodiment 15, wherein the TLR7 / 8 agonist comprises resiquimod (R848). 17. The composition of embodiment 14 or embodiment 15, wherein the TLR7 / 8 agonist does not inhibit NLR family pyrin domain containing 3 (NLRP3). 18. The composition of embodiment 13, wherein the LPC comprises LPC(22:0) and the TLR7 / 8 agonist comprises resiquimod (R848). 19. The composition according to any one of embodiments 1 to 18, wherein the antigen is present in a biological sample obtained from an individual. 20. The composition of embodiment 19, wherein the biological sample comprises biopsy tissue. 21. The composition of embodiment 19, wherein the biological sample comprises cells. 22. The composition of embodiment 19, wherein the biological sample does not contain cells. 23. The composition of embodiment 19, wherein the biological sample comprises pus from an abscess. 24. The composition of any one of embodiments 1 to 23, wherein the antigen comprises a protein antigen. 25. The composition of embodiment 24, wherein the antigen comprises a tumor antigen. 26. The composition of embodiment 25, wherein the tumor antigen comprises a synthetic or recombinant neoantigen. 27. The composition of embodiment 26, wherein the tumor antigen comprises a tumor cell lysate. 28. The composition of embodiment 24, wherein the antigen comprises a microbial antigen, the microbial antigen comprising one or more of a viral antigen, a bacterial antigen, a protozoal antigen, and a fungal antigen. 29. The composition of embodiment 28, wherein the microbial antigen comprises a purified or recombinant surface protein. 30. The composition of embodiment 28, wherein the microbial antigen comprises a whole inactivated virus. 31. A composition according to any one of the preceding embodiments, comprising liposomes. 32. The composition according to any one of the preceding embodiments, which does not contain lipopolysaccharide (LPS) or monophosphoryl lipid A (MPLA). 33. The composition of any one of embodiments 1 to 32, which does not contain oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) or oxPAPC species. 34 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). 35. The composition of any one of the preceding embodiments, further comprising an adjuvant, wherein the adjuvant comprises an aluminum salt adjuvant, a squalene-in-water emulsion, a saponin, or a combination thereof. 36. A pharmaceutical formulation comprising the composition according to any one of embodiments 1 to 35 and a pharma- ceutically acceptable excipient. 37. A method for generating hyperactivated dendritic cells, the method comprising contacting the dendritic cells with an effective amount of a composition comprising isolated lysophosphatidylcholine (LPC) having a single C13-C22 acyl chain or a C13-C24 acyl chain, at least one additional lipid, and a TLR7 / 8 agonist for the generation of hyperactivated dendritic cells; the hyperactivated dendritic cells secrete IL-1 beta without undergoing pyroptosis; the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof; The LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP); method. 38. The method according to embodiment 37, wherein the dendritic cells are contacted with the composition according to any one of embodiments 1 to 35 or the formulation according to embodiment 36 ex vivo. 39. The method of embodiment 37, wherein the dendritic cells are contacted with the formulation of embodiment 36 in vivo. 40 At least 10 produced by the method according to embodiment 38 3 , 10 4 , 10 5 or 10 6 and a pharma- ceutically acceptable excipient. 41. A method for stimulating an immune response to an antigen, comprising administering to an individual in need thereof an effective amount of the formulation of embodiment 36 to stimulate said immune response to said antigen. 42. A method of treating cancer, comprising administering an effective amount of the formulation of embodiment 36 to an individual in need thereof to treat said cancer. 43. A method of inhibiting abnormal cell growth, comprising administering to an individual in need thereof an effective amount of the formulation of embodiment 36 to inhibit abnormal cell growth. 44. A method for treating an infectious disease, comprising administering an effective amount of the formulation of embodiment 36 to an individual in need thereof to treat said infectious disease. 45. Use of the formulation of embodiment 36 for inducing an immune response against said antigen in an individual in need thereof. 46. ​​Use of the formulation according to embodiment 36 for inducing an anti-tumor immune response in an individual in need thereof, who has or has had a tumor. 47. Use of the formulation according to embodiment 36 for inducing an antimicrobial immune response in an individual in need thereof infected with or exposed to a microorganism. 48. The composition, formulation, method or use according to any one of embodiments 19 to 47, wherein the individual is a mammalian subject. 49. The composition, formulation, method or use according to any one of embodiments 19 to 47, wherein the individual is a human subject. 50. A method for preparing an immunogenic composition, said method comprising: a) obtaining an enriched suspension of tumor cells from a tumor; b) lysing cells from the tumor cell enriched suspension to obtain a tumor cell lysate; c) contacting said tumor cell lysate with a composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and a toll-like receptor 7 / 8 (TLR7 / 8) agonist to obtain said immunogenic composition; Including, the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl chain; the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof; The LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP); method. 51. The method of embodiment 50, wherein step a) comprises depleting leukocytes from the tumor cell enriched suspension, optionally wherein the leukocytes are depleted by negative selection using an anti-CD45 antibody. 52. The method according to embodiment 50 or embodiment 51, wherein the cells are lysed in step b) by one or more freeze-thaw cycles. 53. The method of any one of embodiments 50-52, wherein the acyl chain is a fully saturated C18-C22 acyl chain or a fully saturated C18-C24 acyl chain. 54. The method of embodiment 53, wherein the LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)]. 55. The method of any one of embodiments 50-54, wherein said TLR7 / 8 agonist is a small molecule having a molecular weight of 900 Daltons or less. 56. The method of embodiment 55, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound. 57. The method of embodiment 56, wherein the TLR7 / 8 agonist comprises resiquimod (R848). 58. The method of embodiment 55 or embodiment 56, wherein the TLR7 / 8 agonist does not inhibit NLR family pyrin domain containing 3 (NLRP3). 59. The method of embodiment 54, wherein said LPC comprises LPC(22:0) and said TLR7 / 8 agonist comprises resiquimod (R848). 60. The method according to any one of embodiments 50 to 59, further comprising, prior to step a), obtaining a sample from said tumor from a mammalian subject having cancer and preparing a suspension of cells from said sample. 61. An immunogenic composition prepared by the method according to any one of embodiments 50 to 60. 62. A method for inducing an anti-cancer immune response, comprising administering to a mammalian subject having cancer an effective amount of the immunogenic composition of embodiment 61. 63. The method of embodiment 62, wherein the anti-cancer immune response comprises a cellular immune response. 64. The method of embodiment 63, wherein the anti-cancer immune response comprises cancer antigen-induced IL-1 beta secretion and / or activation of CD8+ T lymphocytes. 65. The method of any one of embodiments 62-64, wherein the cancer is a non-hematological cancer. 66. The method of embodiment 65, wherein the non-hematological cancer is carcinoma, sarcoma, or melanoma. 67. The method of any one of embodiments 62-64, wherein the cancer is lymphoma. 68 A method of treating cancer, comprising: a) preparing an immunogenic composition comprising a tumor cell lysate, an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and a toll-like receptor 7 / 8 (TLR7 / 8) agonist; wherein the tumor cell lysate is prepared or has been prepared from a tumor sample obtained from the mammalian subject having cancer; the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl chain; the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof; wherein the LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP); b) administering to the subject an effective amount of the immunogenic composition; A method comprising: 69. The method of any one of embodiments 62-68, wherein said acyl chain is a fully saturated C18-C22 acyl chain or a fully saturated C18-C24 acyl chain. 70. The method of embodiment 68, wherein said LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)]. 71. The method of any one of embodiments 62-70, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 Daltons or less. 72. The method of embodiment 71, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound. 73. The method of embodiment 72, wherein the TLR7 / 8 agonist comprises resiquimod (R848). 74. The method of embodiment 70, wherein the LPC comprises 22:0 LPC and the TLR7 / 8 agonist comprises resiquimod (R848). 75. The method of any one of embodiments 68-74, further comprising administering to the subject an effective amount of an additional therapeutic agent. 76. The method of embodiment 75, wherein the additional therapeutic agent comprises one or more of the group consisting of immune checkpoint inhibitors, anti-cancer agents, and radiation therapy. 77 A composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and a pathogen recognition receptor (PRR) agonist, the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl chain; the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof; The LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP); composition. 78. The composition of embodiment 77, wherein the PRR agonist is an agonist of a toll-like receptor (TLR), a NOD-like receptor (NLR), a RIG-I-like receptor (RLR), or a C-type lectin receptor (CLR). 79. The composition of embodiment 77, wherein the PRR agonist is a cytoplasmic DNA sensor (CDS) or stimulator of IFN genes (STING) agonist. 80. The composition of embodiment 77, wherein the PRR agonist comprises one or more of R848, TL8-506, LPS, Pam2CSK4, and ODN2336. 81. The composition of any one of embodiments 77 to 80, further comprising an antigen. 82. The composition of any one of embodiments 77 to 81, further comprising dendritic cells. 83. A pharmaceutical formulation comprising the composition according to any one of embodiments 77 to 82 and a pharma- ceutically acceptable excipient. 84 A pharmaceutical formulation comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and a pharma- ceutically acceptable excipient, the acyl chain is a C21 to C24 acyl chain, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof; The LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP); Pharmaceutical preparations. 85. A pharmaceutical formulation according to embodiment 83 or embodiment 84, wherein the acyl chain is a fully saturated C22 acyl chain. 86. The pharmaceutical formulation of embodiment 85, wherein the LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)]. 87 A composition for hyperactivation of human dendritic cells comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and a pathogen recognition receptor (PRR) agonist, the acyl chain is a C22 acyl chain, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof; the LPC and the at least one further lipid are part of a lipid nanoparticle (LNP); the composition is effective in achieving a higher level of dendritic cell hyperactivation than a comparative composition that contains PGPC instead of the LPC; composition. 88. The composition of embodiment 87, wherein said higher level of dendritic cell hyperactivation comprises inducing at least 2, 3 or 4 fold higher level of IL-1 beta secretion from human dendritic cells in vitro when contacted with said composition comprising said LPC and said PRR agonist than when contacted with said comparative composition comprising said PGPC and said PRR agonist, wherein said PRR agonist is LPS. 89. The composition of embodiment 88, wherein the concentration of LPC and the concentration of PGPC are the same within the range of about 10 μM to about 80 μM, and the LPS is present at a concentration of 1 μg / ml in both the composition and the comparative composition. 90. The composition of embodiment 88, wherein said higher level of dendritic cell hyperactivation comprises a lipid activity index of IL-1 beta secretion from said human dendritic cells that is at least 4, 5 or 6 times higher in activity units for said composition comprising said LPC and said PRR agonist than in activity units for said comparative composition comprising said PGPC and said PRR agonist. 91. The composition, formulation, method or use according to any one of embodiments 19 to 47, wherein said individual is a canine subject. 92. The composition, formulation, method or use according to any one of embodiments 60 to 90, wherein the mammalian subject is a human patient. 93. The composition, formulation, method or use according to any one of embodiments 60 to 90, wherein said mammalian subject is a non-human patient. 94. The composition, formulation, method or use according to any one of embodiments 60 to 90, wherein the mammalian subject is a canine patient. 95. The composition, formulation, method or use of any one of embodiments 1 to 90 or 92, wherein the dendritic cells are human dendritic cells. 96. The composition, formulation, method or use of any one of embodiments 1 to 91 or 94, wherein the dendritic cells are canine dendritic cells. 97. The composition, formulation, method or use of embodiment 95 or embodiment 96, wherein said dendritic cells are present in a composition comprising peripheral blood mononuclear cells (PBMCs). 98. The composition, formulation, method or use according to any one of embodiments 37 to 49 or embodiment 91, wherein the hyperactivated dendritic cells secrete one or both of IFNγ and TNFα. 99. The composition, formulation, method or use according to any one of the preceding embodiments, wherein the at least one further lipid comprises one or both of a further phospholipid and a structural lipid, optionally wherein the further phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and the structural lipid comprises cholesterol. 100. The composition, formulation, method or use of embodiment 99, wherein the at least one further lipid comprises a pegylated lipid, optionally wherein the pegylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG]. 101. The composition, formulation, method or use of embodiment 99 or embodiment 100, wherein the at least one further lipid comprises an ionizable lipid, optionally wherein the ionizable lipid comprises 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester (DLin-MC3-DMA) or an analogue or derivative thereof. 102 A composition comprising a lipid nanoparticle (LNP), said LNP comprising 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, said first phospholipid comprising a lysophosphatidylcholine (LPC) having a single acyl chain, said acyl chain being a C13-C24 acyl chain. 103 A composition comprising a lipid nanoparticle (LNP), the LNP comprising a first phospholipid, an ionizable lipid, a second phospholipid, a pegylated lipid, and a structural lipid, the first phospholipid comprising lysophosphatidylcholine (LPC) having a single acyl chain, the acyl chain being a C13-C24 acyl chain. 104 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 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminium (ALC-0315) or an analog or derivative thereof, or ii) (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA) or an analog or derivative thereof The composition of any one of embodiments 1 to 103, comprising: 105. The composition of any one of the preceding embodiments, 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. 106. The composition of any one of embodiments 1-104, wherein the pegylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG]. 107. The composition of any one of the preceding embodiments, 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. 108. The composition of any one of the preceding embodiments, wherein the structured lipid comprises cholesterol. 109. The composition of any one of the preceding embodiments, wherein the additional phospholipid or the second phospholipid comprises a hydrophilic head moiety selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. 110. The composition of any one of the preceding claims, wherein the additional phospholipid or the second phospholipid comprises 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. 111 The further phospholipid or the second phospholipid 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 The composition of any one of embodiments 1 to 108, selected from the group consisting of: 112. The composition of any one of embodiments 1-108, wherein the additional phospholipid or the second phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). 113. The composition according to any one of the preceding embodiments, wherein the at least one further lipid comprises i) a cationic lipid and ii) comprises or further comprises a neutral or anionic lipid. 114 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 The composition of embodiment 113, comprising one or both of the following: 115 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 115. The composition of embodiment 113 or embodiment 114, comprising: 116. The composition of any one of embodiments 102-115, wherein the acyl chain of the LPC is a C21-C24 acyl chain. 117. The composition of any one of embodiments 102-115, wherein the acyl chain of the LPC is a C22 acyl chain. 118. The composition of any one of embodiments 102 to 117, further comprising a TLR7 / 8 agonist. 119. The composition of embodiment 118, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound. 120. The composition of embodiment 119, wherein the TLR7 / 8 agonist comprises resiquimod (R848). 121. The composition of embodiment 119, wherein the LPC comprises LPC(22:0) and the TLR7 / 8 agonist comprises resiquimod (R848). 122. The composition of any one of embodiments 102 to 121, further comprising an antigen. 123. The composition of embodiment 122, wherein the antigen is a tumor antigen or a neoantigen. 124. The composition of embodiment 122, wherein the antigen is a microbioal antigen, optionally wherein the microbial antigen is a viral antigen, a bacterial antigen, a protozoal antigen, or a fungal antigen. 125. The composition, formulation, method or use according to any one of the preceding embodiments, wherein said composition does not comprise isolated mRNA. 126. The composition, formulation, method or use according to any one of embodiments 1 to 125, 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. 127. The composition, formulation, method or use of embodiment 126, wherein said LNPs have an effective diameter of less than about 250 nanometers. 128. The composition, formulation, method or use of embodiment 127, wherein the LNP has an effective diameter of less than about 125 nanometers. 129. The composition, formulation, method or use of embodiment 128, wherein the LNP has an effective diameter of about 10 to about 110 nanometers. 130. The composition, formulation, method or use according to any one of the preceding embodiments, wherein the composition does not comprise a surfactant. EXAMPLES

[0095] Abbreviations: CDS (cytoplasmic DNA sensor), CLR (C-type lectin receptor), DAMP (damage-associated molecular pattern), DC (dendritic cell), dLN (draining lymph node), DLS (dynamic light scattering), DMG-PEG-2000 (polyethylene glycol [PEG] 2000 dimyristoylglycerol [DMG]), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), ELSD (evaporative light scattering detector), FLT3L (Fms-related tyrosine kinase 3 ligand), HOdiA-PC (1-palmitoyl-2-(5-hydroxy-8-oxo-6-octenedioyl)-sn-glycero ... glycero-3-phosphatidylcholine), 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-octene-dioyl)phosphatidylcholine), KOOA-PC (1-palmitoyl-(5-keto-8-oxo-6-octenoyl)-sn-glycero-3-phosphocholine), LNP (Lyso nanoparticles), LPC / Lyso PC (lysophosphatidylcholine), Lyso PC (22:0) (1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine), LPS (lipopolysaccharide), MC3 ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, also known as DLin-MC3-DMA), moDC (monocyte-derived dendritic cells), MPLA (monophosphoryl lipid A), NLR (NOD-like receptor), oxPAPC (oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine), PAMP (pathogen-associated molecular pattern), PBMC (peripheral blood mononuclear cells), PGPC (1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine), 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine), POVPC (1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine), PRR (pathogen recognition receptor), RLR (RIG-I-like receptor), R848 (resiquimod), SC (subcutaneous), STING (stimulator of IFN genes), TNFα (tumor necrosis factor-alpha), and TLR (toll-like receptor).

[0096] Although the present disclosure has been described in some detail by way of illustration or example for clarity and understanding, it is apparent to those skilled in the art that certain changes and modifications can be made. Therefore, the following examples should not be construed as limiting the scope of the present disclosure, which is described by the appended claims. Example 1 Combination of single acyl chain lysophosphatidylcholine (LPC) and TLR7 / 8 agonists hyperactivates mammalian peripheral blood mononuclear cells

[0097] 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

[0098] Isolation of PBMCs from 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.

[0099] 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 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 and 5% CO2 for 2 days. The cell cultures were then used for endpoint analysis.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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

[0104] 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.

[0105] 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 PBMCs 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 (Figures 1A-1B). Consistent with previous studies showing that monocytes, which constitute 5-10% of PBMCs, can release IL-1β in response to activation with R848, canine PBMCs 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.

[0106] IL-1β could be detected in cell culture supernatants the day after canine PBMCs were overactivated, whereas cell viability was assessed 2 days after overactivation, ensuring persistent survival after IL-1β secretion. With 22:0 LYSO+R848, the relative viability of cells was not significantly reduced (Figure 1C). Interestingly, combining PGPC with R848 proved to be somewhat toxic to canine PBMCs, whereas no toxicity was observed for human moDCs or human PBMCs. However, the viability of the specific cell population of interest (in this case, monocytes) cannot be determined from the results obtained with the mixture, making it difficult to interpret the observations made from the testing of mixed cell populations. 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

[0107] 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.

[0108] Human PBMCs, like human moDCs and canine PBMCs, secreted IL-1β at levels higher than or comparable to all other stimuli tested (Figures 2A-2B). 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.

[0109] Moreover, the viability of human PBMCs was assessed 2 days after overactivation, ensuring their persistent survival after IL-1β secretion. No significant decrease in human PBMC viability was observed after treatment with either stimuli (Figure 2C). 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.

[0110] Since activated human PBMCs can secrete other cytokines in addition to IL-1β, the secretion of the pro-inflammatory 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 (Figures 3A-3B). Notably, LPS+Alum induced high levels of IL-1β secretion from human PBMCs (Figure 3B), 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 secretion of the pro-inflammatory cytokines IFNγ and TNFα from human PBMCs. Example 2 Preparation of lipid nanoparticles containing lysophosphatidylcholine (LPC)

[0111] This example describes the preparation of lipid nanoparticles (LNPs) loaded with hyperactivated lipids (e.g., 22:0 LYSO PC) using a microfluidic process. material and method

[0112] LNPs were synthesized using a NanoAssemblr® Ignite™ microfluidic device (Precision Nanosystems, Vancouver, BC, Canada). First, LNPs were produced using a kit containing GenVoy-ILM™ ionizable lipid mix (Precision Nanosystems, Vancouver, BC, Canada). Empty LNP vehicles were constructed using a kit without mRNA, and LNPs loaded with excess activator were produced by adding 22:0 Lyso PC to a molar ratio of 10% of the total LNP content. LNPs were also produced using individual components (without a kit) to determine whether the loading of 22:0 Lyso PC into LNPs could be purposefully altered. LNPs were produced using the following components: (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester (CAS Registry Number 1224606-06-7, referred to herein as "DLin-MC3-DMA" or "MC3") (Cayman Chemical); 1,2-distearoyl-sn-glycero-3-phosphocholine (CAS Registry Number 816-94-4, referred to herein as "DSPC") (Avanti); Cholesterol (Sigman), 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 combination with 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine (CAS Registry Number 125146-65-8, referred to herein as "22:0 Lyso PC") (Avanti). Lipids were first dissolved in ethanol and then combined according to the molar percentages shown in Table 2-1. Lipids in ethanol were combined with PBS, pH 7.4 in a volume ratio of 1:3. The NanoAssemblr® Ignite™ microfluidic device was programmed with a flow rate of 12 mL / min, a start waste volume of 0.35 mL, and an end waste volume of 0.05 mL. LNPs were washed with PBS, pH 7.4 to remove residual ethanol, then concentrated by spinning at 2000×g for 30 min using an Amicon 10K MWCO centrifugal filter. Table 2-1. LNP formulations^ [Table 2-1] ^ Percent molar concentrations of components of different LNP formulations. LNP2 and LNP3 formulations share the same vehicle (LNP Vehicle 2).

[0113] Loading of 22:0 Lyso PC into LNPs was assessed using HPLC. LNPs in PBS were frozen at -80°C, then lyophilized and stored at -20°C until they could be quantified. LNPs were reconstituted in ethanol and then mixed with water to dissolve the PBS. A seven-point standard curve of 22:0 Lyso PC was prepared in ethanol with water and PBS added to match the 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 (ELSD). Samples were detected using a Luna 5 μm NH2 100 Å, 150×4.6 mm LC column (Phenomenex, Torrance, CA) 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 a 3 min period before the next sample was run. The flow rate was set at 1 mL / min, and the injection volume was 5 μL for samples and standards. The ELSD used an evaporator temperature of 80 °C, a nebulizer temperature of 30 °C, and a nitrogen gas flow rate of 0.9 standard L / min. Agilent CDS 2.6 software was used for HPLC instrument control, data acquisition, and processing.

[0114] The size of the LNPs was assessed using dynamic light scattering (DLS) on a NanoBrook Omni particle size and zeta potential analyzer (Brookhaven Instruments Corp., Holtsville, NY). Four measurements were taken per sample, each for 120 seconds, with the first measurement taken per sample being excluded from downstream analysis as the time required for sample equilibration. Data points on the size classification graphs represent individual replicate measurements from two preparations of LNPs. result

[0115] The incorporation of 22:0 Lyso PC into LNPs was explored to test the effect of 22:0 Lyso PC on the physical characteristics and biological activity of LNPs. Both the loading level and number of loaded LNPs were considered to be critical variables affecting the payload of 22:0 Lyso PC delivered to cells.

[0116] GenVoy-ILM™ ionizable lipid mix (Precision Nanosystems, Vancouver, BC, Canada) was used to generate LNPs with (based on molar ratio) 10% 22:0 Lyso PC or without 22:0 Lyso PC (blank vehicle LNPs). Additional LNPs were generated by combining or not combining the following components: MC3, DPSC, cholesterol, and DMG-PEG2000 with 22:0 Lyso PC. All LNPs were generated by using NanoAssemblr® Ignite™ microfluidic device (Precision Nanosystems, Vancouver, BC, Canada). LNPs were then purified using spin filtration to remove ethanol and unincorporated material.

[0117] LNP formulations were sized using dynamic light scattering (DLS) to determine their mean effective diameter. Two LNP batches of each formulation were prepared and three sizing measurements were performed per batch. LNPs ranged from 50 to 200 nm in diameter depending on the formulation and the addition of 22:0 Lyso PC (Figure 4). The addition of 22:0 Lyso PC did not appear to significantly affect the effective diameter of LNPs in any of the formulations tested.

[0118] The four LNP formulations tested were started with different 22:0 LPC input molar ratios. To determine whether modulating the input ratio would affect the loading level of 22:0 Lyso PC into the LNPs, the amount of 22:0 Lyso PC present in the LNPs was assessed using HPLC. LNP preparations were lyophilized and then dissolved in a mixture of ethanol and water for quantification. Samples were compared to a standard curve of 22:0 Lyso PC prepared using the same dissolution conditions. 22:0 Lyso PC was successfully detected in preparations where 22:0 Lyso PC was included in the starting input and was not detected in their corresponding empty vehicle controls (Figures 5A-5C). Theoretical values ​​of lipid incorporation were calculated assuming 100% efficiency of incorporation of 22:0 Lyso PC into the LNPs. The actual amount of 22:0 Lyso PC measured by HPLC was within 75% of the theoretical value (Figure 5D). As the input amount of 22:0 Lyso PC increased, the measured loading of 22:0 Lyso PC into the LNPs also increased, indicating that the loading of 22:0 Lyso PC into the LNPs can be tailored depending on the amount of 22:0 Lyso PC required for a particular application. Example 3 Hyperactivation of human dendritic cells using TLR7 / 8 agonists in combination with lipid nanoparticles containing lysophosphatidylcholine (LPC)

[0119] This example describes the hyperactivation of human monocyte-derived dendritic cells (moDCs) using a TLR7 / 8 agonist in combination with LNPs loaded with a hyperactivating lipid (eg, 22:0 LYSO PC). material and method

[0120] 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 fetal bovine serum containing 10% dimethyl sulfoxide. For studies with cultures of monocyte-derived dendritic cells (moDCs), monocytes were thawed and cultured in RPMI 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 (R10 medium). 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 on day 3 the cells were further fed with R10 medium containing GM-CSF and IL-4.

[0121] 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) and with or without hyperactivating lipid (or vehicle control). LNP-induced hyperactivation was measured using two assays: CellTiter-Glo assay (Promega) detects ATP as a measure of cell viability; IL-1β Lumit assay (Promega) measures IL-1β cytokine present in moDC cell culture supernatants. Experimental conditions were tested in triplicate and the average of results from one donor was plotted. Data represent results from six human donor samples tested across two experiments. result

[0122] Activated over-moDCs produce IL-1β, a key cytokine for the generation and reactivation of long-lived memory T cells, while retaining cell viability. The ability of LNPs prepared as described in Example 2 to over-activate human moDCs was also tested. For comparison, 22:0 Lyso PC was simply resuspended in PBS medium, which results in 22:0 Lyso PC becoming a large, flaky, insoluble material. When cell viability was measured using an ATP quantification assay, most experimental conditions had negligible effects on cell viability (Figure 6A). The most obvious exception was the condition in which moDCs were treated with LNPs made with GenVoy-ILM, which resulted in an average viability below 75% (vs. treatment with R848 alone). GenVoy-ILM LNPs induced IL-1β production regardless of whether 22:0 Lyso PC was included in the formulation (Figure 6B). Given that cell viability was decreased, it was presumed that GenVoy-ILM LNPs were causing cell death, and therefore, IL-1β release cannot be the result of moDC overactivation.

[0123] When cells were administered 22:0 Lyso PC directly resuspended in PBS, IL-1β was produced with a concomitant decrease in cell viability (Figure 6A). In addition, the large flaky format of 22:0 Lyso PC in PBS resulted in variable replication when using donor-derived moDCs, as the dose of moDCs was not consistent. In comparison, LNP formulations had a more consistent IL-1β response. 22:0 Lyso PC LNPs induced IL-1β secretion above background measurements from their corresponding empty LNP vehicle controls (Figure 6B). Interestingly, despite the administration of 82.5 μM 22:0 Lyso PC across the different formulations, increased amounts of IL-1β were produced from cells treated with fewer LNPs loaded with higher amounts of 22:0 Lyso PC per LNP. Formulations containing 10% 22:0 Lyso PC (GenVoy and LNP1) did not increase immunogenicity compared to their vehicle controls. In contrast, formulations containing 25% or 40% 22:0 Lyso PC actually induced higher levels of IL-1β secretion compared to their vehicle controls, with LNPs containing 40% 22:0 Lyso PC inducing the highest levels of IL-1β secretion. These data indicate that the payload of 22:0 Lyso PC given per LNP is an important contributor to moDC hyperactivation. Dispersing an equivalent amount of 22:0 Lyso PC over a larger number of LNPs reduces the efficiency of hyperactivation.

[0124] Taken together, the data demonstrate that 22:0 Lyso PC can be incorporated into LNPs. This new formulation method is believed to be clinically meaningful as it allows for fine tuning of the amount of 22:0 Lyso PC incorporated into the particles, thereby having a significant impact on the efficiency of overactivation. 22:0 Lyso PC in PBS cannot provide the same level of tunability when overactivated. In addition, another concern with preparing 22:0 Lyso PC in PBS is that it results in the formation of very large visible particles that are not uniformly distributed in the solution. Large uniformly distributed particles would present a challenge with regard to accurate dosing. Also, since visible microparticles are significantly larger than cells, very large particles are likely to limit the biodistribution of 22:0 Lyso PC in vivo. Large particles, when delivered in vivo, may potentially be sequestered by the immune system, thereby limiting the ability of 22:0 Lyso PC to reach dendritic cells for overactivation. Example 4 Hyperactivation of mouse dendritic cells using TLR7 / 8 agonists in combination with lipid nanoparticles containing lysophosphatidylcholine (LPC)

[0125] This example describes the hyperactivation of mouse bone marrow-derived dendritic cells (BMDCs) using a TLR7 / 8 agonist in combination with lipid nanoparticles (LNPs) loaded with a hyperactivating lipid (e.g., 22:0 Lyso PC). material and method

[0126] Synthesis of LNPs. LNPs were synthesized from the following components: 1,2-distearoyl-sn-glycero-3-phosphocholine (CAS Registry Number 816-94-4, referred to herein as "DSPC") (Avanti); Cholesterol (Sigman), 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 in combination 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). LNPs were prepared without 22:0 Lyso PC or loaded with 20% or 40% molar ratio of 22:0 Lyso PC to determine whether the loading of 22:0 Lyso PC could be purposefully varied. Lipid nanoparticles (LNPs) were synthesized using a NanoAssemblr Ignite instrument (Precision Nanosystems). First, lipids were dissolved in ethanol and then added according to the molar percentages shown in Table 4-1 to reach a total lipid concentration of 12.5 mM. Lipids in ethanol were combined with sodium citrate buffer, pH 4, in a volume ratio of 1:3 at a flow rate of 12 mL / min. The LNPs were washed with 10 volumes of phosphate-buffered saline (PBS), pH 7.4, to remove residual ethanol and then concentrated using an Amicon 10K MWCO centrifugal filter.

[0127] 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.

[0128] The size of the LNPs was assessed using dynamic light scattering (DLS) on a NanoBrook Omni (Brookhaven) device. LNPs were diluted 1:10 in PBS before DLS. Three measurements of 90 seconds were recorded for each sample. The size of 22:0 Lyso PC in PBS was assessed using a Mastersizer 3000 (Malvern) equipped with a Hydro SV small volume dispersion unit set at a spin speed of 1500 rpm. Five readings of 5 seconds were recorded for each sample.

[0129] Generation of mouse bone marrow-derived FLT3L-DCs. Femurs and tibias were removed from mice, cut with scissors, and poured into sterile tubes. Bone marrow suspension was 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 per well in P12 plates. 6 The cultures were plated with bone marrow cells. Recombinant mouse FLT3L (Miltenyi) was added to the cultures at 200ng / mL. Differentiated cells were used for subsequent assays on day 8. Differentiation efficiency was monitored by flow cytometry using a BD Symphony A3 and showed CD11c + MHC-II + Cells routinely exceeded 80% viable cells. Per experiment, 5–15 mice were used to generate bone marrow-derived dendritic cells (BMDCs).

[0130] Hyperactivation of mouse bone marrow-derived FLT3L-DCs. BMDCs were harvested on day 8 after differentiation, washed with PBS, and then transplanted into complete IMDM medium (I10) containing FLT3L at 2 × 10 5 Cells were replated at a concentration of 1000 cells / mL. Cells were cultured in the presence or absence of 1 μg / mL R848 and then treated with or without 82 μM 22:0 Lyso PC or 22:0 Lyso PC LNP in PBS. Supernatants were collected 48 hours after stimulation for cytokine measurement. Viability was measured using the CellTiter-Glo assay (Promega), which measures ATP content from cells. Fifty microliters of CellTiter-Glo reagent was added to 50 μL of cells. Luminescence was quantified using a SpectraMax M5e plate reader with an integration time of 500 ms. Viability data was set against the control condition where cells were treated with R848. IL-1β and IL-6 cytokine secretion was measured using a sandwich ELISA (Invitrogen).

[0131] 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 the cell pellet and remaining supernatant and 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 DCs treated with R848.

[0132] Quantification of IL-1β and IL-6 secretion. IL-1β and IL-6 secretion were assessed using ELISA mouse IL-1β and IL-6 kits (Invitrogen). ELISA was performed according to the manufacturer's instructions. Absorbance was measured at 450 nm using a Spectramax M5e plate reader (Molecular Devices) with 570 nm used for correction. To determine IL-1β and IL-6 concentrations in the supernatants, the IL-1β or IL-6 concentrations of the samples were interpolated using standard curves by 4PL analysis in GraphPad Prism 9 (GraphPad Software). The interpolated results of the samples were then adjusted for any dilutions made to the supernatants.

[0133] Hyperactivation of FLT3L-DCs for migration assay. Mouse bone marrow-derived dendritic cells (BMDCs) were harvested on day 8 after differentiation, washed with PBS, and diluted with 10 × 10 6 DCs were then replated at a concentration of 1×10 cells / mL. For hyperactivation, 500 μl of R848 was added at a final concentration of 1 μg / mL and 500 μL of lipid (22:0 Lyso PC or 22:0 Lyso PC LNP prepared in PBS) was added at a final concentration of 82 μM. Cells were incubated for 24 hours at 37°C on a tube rotator. 24 hours after stimulation, cells were washed with PBS and stained with CFSE (1:1000) for 30 minutes at 37°C in the dark. DCs were then counted and 1×10 6Cells were injected subcutaneously (SC) at 100 μL per mouse. 24 hours after injection, the skin-draining lymph nodes (dLN) were dissected from the injection side. Single cell suspensions were prepared and cells were stained with fixable live / dead cell dye (ThermoFisher) in PBS for 20 min at 4°C. Cells were then washed again and stained for 20 min at 4°C with MACS buffer (PBS with 1% FCS and 2 mM EDTA) containing the following fluorescently conjugated antibodies: anti-CD11c, and anti-IA / IE (MHC-II). CD11c in live cells + MHC-II + To determine absolute numbers of , countBright counting beads (ThermoFisher) were used according to the manufacturer's protocol. Data were acquired on a BD FACS Symphony (Becton-Dickenson). Data were analyzed using FlowJo software (Tree Star). Four mice were used per experimental group. result

[0134] 22:0 Lyso PC can be loaded into LNPs. 22:0 Lyso PC was efficiently incorporated into LNPs containing DSPC, cholesterol and DMG-PEG2000. Using the LNP synthesis process described above, 87.4% of the 22:0 Lyso PC added during synthesis was collected from the LNPs and detected by HPLC as shown in Table 4-1. Table 4-1. Molar concentration percentages of LNP components [Table 4-1]

[0135] 22:0 Lyso PC in LNP preparations is more uniform. One concern with preparing 22:0 Lyso PC in PBS is that 22:0 Lyso PC is insoluble, thus resulting in larger particles that are not uniformly distributed in solution. The particles are as large as 130 μm in diameter (FIG. 7A), with a large polydispersity index, indicating a wide particle size range. This particle size is almost 10 times larger than the size of a cell, making it too large to be taken up by cells (e.g., phagocytes). As a result, these large particles cannot reach dendritic cells (or other cells of interest) in vivo. In contrast, when 22:0 Lyso PC is incorporated into LNPs, the LNPs are approximately 50 nm in size, with the largest particles (unfiltered) being less than 1 μm in diameter (FIG. 7B). In addition, the polydispersity index (PDI) of these particles is significantly smaller, indicating a more uniform suspension. LNPs of this size can be readily taken up by cells. Therefore, 22:0 Lyso PC is expected to be more bioavailable in vivo. Uniformity of 22:0 Lyso PC distribution in LNP suspensions is expected to lead to more repeatable and precise dosing levels.

[0136] 22:0 Lyso PC LNP induces IL-1β secretion from mouse DC in vitro. FLT3L-DC were stimulated with medium alone, empty LNP, or 82 μM 22:0 Lyso PC in PBS or 22:0 Lyso PC loaded in LNP. Alternatively, FLT3L-DC were treated with 1 μg / ml R848 in combination with empty LNP, 22:0 Lyso PC LNP, or 22:0 Lyso PC in PBS. 48 hours after stimulation, cell supernatants were collected for ELISA and cell viability was measured by Cell Titer Glow assay, thereby measuring the level of ATP release from cells. When cells were treated with R848 in combination with a hyperactivating lipid formulation (22:0 Lyso PC in PBS or LNP) or in combination with empty LNP, FLT3L DC were viable as revealed by the percent cell viability compared to R848 alone (Figure 8A). In addition, stimulation with R848 in combination with empty LNPs or 22:0 Lyso PC induced high levels of the proinflammatory cytokine IL-6, regardless of whether 22:0 Lyso PC was formulated in PBS or LNPs (Figure 8B). Furthermore, R848 and 22:0 Lyso PC in PBS did not induce IL-1β secretion from viable cells, whereas treatment of DCs with R848 in combination with 22:0 Lyso PC in LNPs did indeed induce IL-1β secretion from viable cells (Figure 8C), indicating that 22:0 Lyso PC in LNPs induces DC hyperactivation and has an advantage over 22:0 Lyso in PBS.

[0137] 22:0 Lyso PC LNP induces DC hypermigration in vivo. Another hallmark of hyperactivation is the ability of hyperactivating lipids to induce DC hypermigration from the skin to the draining lymph nodes (dLNs). To evaluate whether 22:0 Lyso PC in LNP can induce DC migration, FLT3L-DC were incubated overnight on a tube rotator with empty LNP, 22:0 Lyso PC in LNP, or R848 combined with empty LNP, 22:0 Lyso PC in LNP, or 22:0 Lyso PC in PBS. The next day, cells were washed and stained with CFSE. 1 × 10 per mouse 6 Cells were injected subcutaneously in the right dorsum. 24 hours after injection, dLNs were harvested and single cell suspensions were prepared. dLNs from uninjected mice were used as negative controls. Cells were stained with live / dead dyes to identify live cells, CD11c and MHC-II. CFSE + , CD11c + MHC-II + The percentage of DC was measured by flow cytometry. As expected, DC treated with R848 in combination with empty LNP or 22:0 Lyso PC LNP alone did not induce any migration of DC from skin to dLN (Figure 8D). Similarly, DC treated with R848 in combination with empty LNP or R848 in combination with 22:0 Lyso PC in PBS did not induce migration of DC to dLN. Interestingly, DC treated with R848 in combination with 22:0 Lyso PC in LNP enhanced migration of DC to dLN (Figure 8D).

[0138] These data demonstrate that LNPs containing 22:0 Lyso PC are superior hyperactivating lipid formulations compared to 22:0 Lyso PC in aqueous buffers, such as PBS. DCs treated with 22:0 Lyso PC delivered in LNPs showed increased IL-1β secretion and increased migration to draining lymph nodes compared to LNPs without 22:0 Lyso PC and 22:0 Lyso PC formulated in PBS. Thus, it is believed that 22:0 Lyso PC delivered in LNPs, when delivered with antigen in vivo, results in the production of new T cells (particularly memory T cells) more potently than antigen delivered with 22:0 Lyso PC in PBS (or LNPs without 22:0 Lyso PC).

Claims

1. 1. A composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and a TLR7 / 8 agonist, the acyl chain is a C13 to C24 acyl chain; the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof; The LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP); composition.

2. The composition of claim 1, wherein the acyl chain is a C21 to C24 acyl chain.

3. The composition of claim 1 , further comprising an antigen and / or a dendritic cell.

4. The composition of claim 1 , further comprising dendritic cells.

5. 1. A composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and an antigen, the composition does not contain RNA, the acyl chain is a C21 to C24 acyl chain; the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof; The LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP); composition.

6. The composition of claim 5 , further comprising dendritic cells and / or a TLR7 / 8 agonist.

7. 1. A composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and dendritic cells, the acyl chain is a C21 to C24 acyl chain; the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof; The LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP); composition.

8. The composition of claim 7, further comprising a TLR7 / 8 agonist and / or an antigen.

9. 2. The composition of claim 1, wherein the acyl chain is a C22 acyl chain and / or the acyl chain of the LPC is fully saturated.

10. 10. The composition of claim 9, wherein the LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

11. The composition of claim 10, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 daltons or less.

12. The composition of claim 11 , wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

13. 13. The composition of claim 12, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

14. The composition of claim 1 , wherein the antigen comprises a protein antigen.

15. The composition of claim 14 , wherein the antigen comprises a tumor antigen.

16. 15. The composition of claim 14, wherein the antigen comprises a microbial antigen, the microbial antigen comprising one or more of a viral antigen, a bacterial antigen, a protozoal antigen, and a fungal antigen.

17. A pharmaceutical formulation comprising the composition of any one of claims 1 to 16 and a pharmaceutically acceptable excipient.

18. 20. A method for generating hyperactivated dendritic cells, said method comprising contacting dendritic cells ex vivo with an effective amount of the pharmaceutical preparation of claim 17.

19. 20. The pharmaceutical formulation of claim 17 for stimulating an immune response to an antigen, wherein an effective amount of the formulation is administered to an individual in need thereof to stimulate an immune response to the antigen, thereby stimulating the immune response to the antigen.

20. 20. The pharmaceutical formulation of claim 17 for treating cancer, wherein an effective amount of the formulation is administered to an individual in need of cancer treatment to treat the cancer.

21. 20. The pharmaceutical formulation of claim 17 for inhibiting abnormal cell growth, wherein an effective amount of the formulation is administered to an individual in need thereof to inhibit abnormal cell growth, thereby inhibiting abnormal cell growth.

22. 20. The pharmaceutical formulation of claim 17 for treating an infectious disease, wherein an effective amount of the formulation is administered to an individual in need of treatment for an infectious disease to treat the infectious disease.

23. 1. A method for preparing an immunogenic composition, said method comprising: a) lysing cells from a tumor cell enriched suspension previously obtained from the tumor to obtain a tumor cell lysate; b) contacting the tumor cell lysate with a composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain, at least one additional lipid, and a toll-like receptor 7 / 8 (TLR7 / 8) agonist to obtain the immunogenic composition; Including, the acyl chain is a C13 to C24 acyl chain; the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof; The LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP); method.

24. 24. An immunogenic composition prepared by the method of claim 23 for eliciting an anti-cancer immune response, wherein an effective amount of the immunogenic composition is administered to a mammalian subject having cancer.

25. A composition described in any one of claims 1 to 16, wherein the at least one additional lipid comprises the ionizable lipid, the additional phospholipid, the PEGylated lipid, and the structural lipid.

26. 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 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminium (ALC-0315) or an analog or derivative thereof; or ii) (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA) or an analog or derivative thereof 26. The composition of claim 25, comprising:

27. 26. The composition of claim 25, 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 dialkylglycerol, and combinations thereof.

28. 26. The composition of claim 25, 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.

29. the additional phospholipid being and / or comprising a hydrophilic head moiety selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin; comprising 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, phytanic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid; 26. The composition of claim 25.

30. A method for generating hyperactivated dendritic cells, said method comprising: i) contacting dendritic cells with a TLR7 / 8 agonist ex vivo; and ii) contacting dendritic cells ex vivo with an effective amount of a composition comprising lipid nanoparticles (LNPs). wherein the LNPs comprise an isolated lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain, and at least one additional lipid selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a PEGylated lipid, a structural lipid, and mixtures thereof. method.

31. 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 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 an effective amount of (b) the composition comprising lipid nanoparticles (LNPs). wherein the LNPs comprise an isolated lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain, and at least one additional lipid selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a PEGylated lipid, a structural lipid, and mixtures thereof. Combination.

32. 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 a lipid nanoparticle (LNP), the method comprising: i) administering to said individual (a) said TLR7 / 8 agonist; and ii) administering to said individual (b) said composition comprising lipid nanoparticles (LNPs). wherein the LNPs comprise an isolated lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain, and at least one additional lipid selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a PEGylated lipid, a structural lipid, and mixtures thereof. Combination.

33. A composition for the manufacture of a medicament for stimulating an immune response in an individual, said composition comprising a lipid nanoparticle (LNP), said LNP comprising 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, said first phospholipid comprising lysophosphatidylcholine (LPC) having a single acyl chain, said acyl chain being a C13 to C24 acyl chain.

34. The composition described in claim 33, wherein the LNP comprises the first phospholipid, the ionizable lipid, the second phospholipid, the PEGylated lipid and the structural lipid.

35. 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 an effective amount of a composition comprising lipid nanoparticles (LNPs); wherein the LNPs comprise an isolated lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain, and at least one additional lipid selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a PEGylated lipid, a structural lipid, and mixtures thereof. composition.

36. A composition for use in a method for generating hyperactivated dendritic cells, comprising: a lipid nanoparticle (LNP), the method comprising: i) contacting dendritic cells in vivo with a TLR7 / 8 agonist; and ii) contacting dendritic cells in vivo with an effective amount of said composition comprising lipid nanoparticles (LNPs); wherein the LNPs comprise an isolated lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain, and at least one additional lipid selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a PEGylated lipid, a structural lipid, and mixtures thereof. composition.

37. 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 to said individual said TLR7 / 8 agonist; and ii) administering to said individual a composition comprising lipid nanoparticles (LNPs). wherein the LNPs comprise an isolated lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain, and at least one additional lipid selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a PEGylated lipid, a structural lipid, and mixtures thereof. composition.

38. 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 lipid nanoparticle (LNP), the method comprising: i) administering to said individual a TLR7 / 8 agonist; and ii) administering to said individual said composition comprising lipid nanoparticles (LNPs). wherein the LNPs comprise an isolated lysophosphatidylcholine (LPC) having a single C13-C24 acyl chain, and at least one additional lipid selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a PEGylated lipid, a structural lipid, and mixtures thereof. composition.

39. A composition described in any one of claims 35 to 38, wherein the additional lipids include the ionizable lipid, the additional phospholipid, the PEGylated lipid and the structural lipid.

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