Phospholipid analogs for hyperactivation of mammalian dendritic cells

Abstract

The present disclosure relates to phospholipid analogue (PLA) compounds and their use in superactivating mammalian dendritic cells, such as human dendritic cells or canine dendritic cells. The disclosure also relates to compositions comprising PLA and one or more pathogen-recognizing receptor agonists, antigens, and mammalian dendritic cells, as well as methods of producing and using the compositions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 441,702, filed January 27, 2023. The entire contents of these applications are hereby incorporated herein by reference. Technical Field

[0003] The present disclosure relates to phospholipid analog compounds and their use in hyperactivating mammalian dendritic cells (e.g., human dendritic cells or canine dendritic cells). The present disclosure also relates to compositions comprising phospholipid analogs and one or more pathogen recognition receptor agonists, antigens, and human or canine dendritic cells, as well as methods of producing and using the compositions. Background Art

[0004] Typically, dendritic cells (DC) are matured by vaccine adjuvants such as Toll-like receptor agonists and do not result in IL-1 β secretion. In the case of activation of inflammasomes, IL-1 β secretion does occur, but at the expense of DC death caused by the dissolution process of cell death called pyroptosis (Evavold et al., J Mol Biol, 430 (2): 217-237, 2018). However, when DC is matured using molecules containing pathogen-associated molecular patterns (PAMPs), lipopolysaccharides (LPS) and molecules containing damage-associated molecular patterns (DAMPs) (such as PGPC (1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine)), they can produce and secrete IL-1 β in the absence of pyrophosphorylation, and these surviving DCs are characterized as super-activated (Zanoni et al., Science, 352 (6290): 1232-1236, 2016). In fact, in a mouse model, superactivated DCs showed an improved ability to induce immune responses compared to cells activated with LPS alone (Zhivaki et al., Cell Rep, 33(7):108381, 2020). However, little is known about how to effectively stimulate superactivation of human dendritic cells.

[0005] Therefore, there is a need in the art to identify PAMPs and DAMPs suitable for superactivating human DCs. Furthermore, there is a need to identify alternatives to the use of LPS and PGPC for superactivating mammalian DCs. In particular, although LPS (endotoxin) is a potent PAMP, its use in humans is contraindicated due to the potential for septic shock. Furthermore, aqueous-soluble PAMPs and DAMPs would facilitate the preparation of pharmaceutical formulations. Summary of the Invention

[0006] The present disclosure relates to phospholipid analog (PLA) compounds and their use in hyperactivating mammalian dendritic cells (e.g., human dendritic cells or canine dendritic cells). The present disclosure also relates to compositions comprising PLA and one or more pathogen recognition receptor agonists, antigens, and human or canine dendritic cells, as well as methods of producing and using the compositions.

[0007] In some embodiments, the phospholipid analog used in the methods and compositions disclosed herein is a compound of formula (I), wherein R E1 C 17 -C 19 The compound of formula (I) wherein R E2 The compound of formula (I) is a C1-C2 alkyl group, wherein R E1 C 17 -C 19 n-alkyl and wherein R E2 is a C1-C2 alkyl compound of formula (I), wherein the compound is edelfosine (EDEL) compound of formula (I):

[0008]

[0009] The compound of formula (II), wherein R M C 14 -C 18 The compound of formula (II) wherein R M C 15 -C 17 A compound of formula (II) containing an n-alkyl group, wherein the compound is miltefosine (MILT):

[0010]

[0011] The compound of formula (III), wherein R P C 17 -C 19 A compound of formula (III) wherein the compound is n-alkyl or a compound of formula (III) wherein the compound is perifosine (PERIF):

[0012]

[0013] or a protonated form of any of the foregoing, or a pharmaceutically acceptable salt of any of the foregoing.

[0014] In some aspects, the present disclosure provides a composition comprising a compound of formula (I) and a TLR agonist. In some aspects, the present disclosure provides a composition comprising edilfosine and a TLR agonist. In some aspects, the present disclosure provides a composition comprising a compound of formula (II) and a TLR agonist. In some aspects, the present disclosure provides a composition comprising miltefosine and a TLR agonist. In some aspects, the present disclosure provides a composition comprising a compound of formula (III) and a TLR agonist. In some aspects, the present disclosure provides a composition comprising perifosine and a TLR agonist. In any of the above embodiments, the composition may further comprise an antigen and / or dendritic cells. In any of these embodiments, the compound of formula (I), formula (IA) (edilfosine), formula (II), formula (II-A) (miltefosine), formula (III) or formula (III-A) (perifosine) may be a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0015] In some aspects, the present disclosure provides a composition comprising a compound of formula (I) and a TLR7 / 8 agonist. In some aspects, the present disclosure provides a composition comprising edilfosine and a TLR7 / 8 agonist. In some aspects, the present disclosure provides a composition comprising a compound of formula (II) and a TLR7 / 8 agonist. In some aspects, the present disclosure provides a composition comprising miltefosine and a TLR7 / 8 agonist. In some aspects, the present disclosure provides a composition comprising a compound of formula (III) and a TLR7 / 8 agonist. In some aspects, the present disclosure provides a composition comprising perifosine and a TLR7 / 8 agonist. In any of the above embodiments, the composition may further comprise an antigen and / or dendritic cells. In any of these embodiments, the compound of formula (I), formula (IA) (edilfosine), formula (II), formula (II-A) (miltefosine), formula (III) or formula (III-A) (perifosine) may be a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0016] In some aspects, the present disclosure provides a composition comprising a compound of formula (I) and an antigen. In some aspects, the present disclosure provides a composition comprising edilfosine and an antigen. In some aspects, the present disclosure provides a composition comprising a compound of formula (II) and an antigen. In some aspects, the present disclosure provides a composition comprising miltefosine and an antigen. In some aspects, the present disclosure provides a composition comprising a compound of formula (III) and an antigen. In some aspects, the present disclosure provides a composition comprising perifosine and an antigen. In any of the foregoing embodiments, the composition may further comprise a TLR agonist, such as a TLR7 / 8 agonist and / or a dendritic cell. In any of these embodiments, the compound of formula (I), formula (IA) (edilfosine), formula (II), formula (II-A) (miltefosine), formula (III) or formula (III-A) (perifosine) may be a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0017] In some aspects, the present disclosure provides a composition comprising a compound of formula (I) and a dendritic cell. In some aspects, the present disclosure provides a composition comprising edilfosine and a dendritic cell. In some aspects, the present disclosure provides a composition comprising a compound of formula (II) and a dendritic cell. In some aspects, the present disclosure provides a composition comprising miltefosine and a dendritic cell. In some aspects, the present disclosure provides a composition comprising a compound of formula (III) and a dendritic cell. In some aspects, the present disclosure provides a composition comprising perifosine and a dendritic cell. In any of the foregoing embodiments, the composition may further comprise an antigen and / or a TLR agonist, such as a TLR7 / 8 agonist. In any of these embodiments, the compound of formula (I), formula (IA) (edilfosine), formula (II), formula (II-A) (miltefosine), formula (III) or formula (III-A) (perifosine) may be a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0018] In some embodiments of the foregoing aspects or embodiments, the antigen is present in a biological sample obtained from an individual. In some embodiments, the biological sample comprises a biopsy. In some embodiments, the biological sample comprises cells. In other embodiments, wherein 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, and the microbial antigen comprises one or more of a viral antigen, a bacterial antigen, a protozoan 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.

[0019] In some embodiments, the composition does not comprise liposomes. In some embodiments, the composition does not comprise LPS or MPLA. In some embodiments, the composition does not comprise oxPAPC or an oxPAPC. In some embodiments, the composition does not comprise HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC and / or PGPC. In some embodiments, the composition does not comprise lysophosphatidylcholine (LPC). In some embodiments, the composition does not comprise 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

[0020] In some embodiments, the composition further comprises an adjuvant, wherein the adjuvant comprises an aluminum salt adjuvant, a squalene-in-water emulsion, a saponin, or a combination thereof.

[0021] In some embodiments, the present disclosure provides a pharmaceutical formulation comprising the composition of any of the foregoing aspects or embodiments and a pharmaceutically acceptable excipient.

[0022] In other aspects, the present disclosure provides a method for producing hyperactivated dendritic cells, the method comprising contacting dendritic cells with a composition comprising an effective amount of an isolated phospholipid analog (PLA) of Formula (I), Formula (IA), Formula (II), Formula (II-A), Formula (III) or Formula (III-A) or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof, and a TLR agonist, such as a TLR7 / 8 agonist, for producing hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing cell pyroptosis. In some embodiments, the dendritic cells are contacted ex vivo with a composition or pharmaceutical preparation of any of the foregoing embodiments. In other embodiments, the dendritic cells are contacted in vivo with a pharmaceutical preparation comprising a composition of any of the foregoing embodiments. In some aspects, the present disclosure provides a pharmaceutical preparation comprising a plurality of hyperactivated dendritic cells produced by the foregoing embodiments and a pharmaceutically acceptable excipient. In some embodiments, the plurality comprises at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 or 10 8 Hyperactivated DC.

[0023] In other aspects, the present disclosure provides a composition comprising an isolated phospholipid analog (PLA) of Formula (I), Formula (IA), Formula (II), Formula (II-A), Formula (III) or Formula (III-A) or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof, and a pathogen recognition receptor (PRR) agonist. 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 includes a TLR7 / 8 agonist. In some embodiments, the composition further comprises an antigen and / or a dendritic cell.

[0024] In any embodiment of the foregoing aspects or embodiments, the isolated PLA may include a compound of formula (I) or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof. In any embodiment of the foregoing aspects or embodiments, the isolated PLA may include edilfosine or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof. In any embodiment of the foregoing aspects or embodiments, the isolated PLA may include a compound of formula (II) or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof. In any embodiment of the foregoing aspects or embodiments, the PLA may include miltefosine or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof. In any embodiment of the foregoing aspects or embodiments, the isolated PLA may include a compound of formula (III) or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof. In any embodiment of the foregoing aspects or embodiments, the PLA may include perifosine or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0025] In some embodiments of the foregoing aspects or embodiments, the TLR agonist, such as a TLR7 / 8 agonist, is a small molecule with a molecular weight of 900 Daltons or less. In some embodiments, the TLR7 / 8 agonist includes an imidazoquinoline compound. In some embodiments, the TLR7 / 8 agonist includes resiquimod (R848). In some embodiments, the PLA includes a compound of formula (I) or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist includes resiquimod (R848). In some embodiments, the PLA includes edilfosine or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist includes resiquimod (R848). In some embodiments, the PLA includes a compound of formula (II) or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist includes resiquimod (R848). In some embodiments, the PLA comprises miltefosine or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist comprises resiquimod (R848). In some embodiments, the PLA comprises a compound of formula (III) or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist comprises resiquimod (R848). In some embodiments, the PLA comprises perifosine or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist comprises resiquimod (R848).

[0026] The present disclosure further provides a composition for superactivating human dendritic cells, the composition comprising an isolated phospholipid analog (PLA) compound and a pathogen recognition receptor (PRR) agonist, and wherein the composition is effective in achieving higher levels of dendritic cell superactivation than a comparative composition comprising a comparative compound instead of PLA. In some embodiments, superactivation occurs in vitro or ex vivo. In other embodiments, superactivation occurs in vivo. In some embodiments, higher levels of dendritic cell superactivation include inducing human dendritic cells to secrete IL-1β in vitro when contacted with a composition comprising PLA and a PRR agonist at a level of at least 2, 3, or 4 times that when contacted with a comparative composition comprising a comparative compound and a PRR agonist, wherein the PRR agonist is LPS. In some embodiments, the concentration of PLA and the concentration of the comparative compound are the same concentration, optionally in the range of about 10 μM to about 80 μM, and LPS is present in the composition and the comparative composition at a concentration of 1 g / ml. In some embodiments, the higher level of dendritic cell hyperactivation comprises a lipid activity index for IL-1β secretion from human dendritic cells for a composition comprising PLA and a PRR agonist that is at least 4, 5, or 6 times greater in activity units than a comparative composition comprising a comparator compound and a PRR agonist. In some embodiments, the comparator compound is PGPC. In some embodiments, the comparator compound is 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

[0027] In any of the embodiments disclosed herein, the phospholipid analog may be in the form of a pharmaceutically acceptable salt. In any of the embodiments disclosed herein, the phospholipid analog may be in a protonated form or a deprotonated form. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1A Shown is the viability of human monocyte-derived dendritic cells (moDCs) after lipid stimulation (41.25 μM). Figure 1B The amount of IL-1β (IL-1β) secreted by stimulated moDCs is shown.

[0029] Figure 2A Viability of human moDCs after lipid stimulation (20.6 μM) is shown. Figure 2B The amount of IL-6 secreted by stimulated human moDCs is shown. Figure 2C The amount of IL-1β (IL-1β) secreted by stimulated moDCs is shown.

[0030] Figure 3A 、 Figure 3B and Figure 3CShown are the results of an ELISPOT assay of interferon gamma (IFNγ) following stimulation of splenocytes from immunized mice. IFNγ spot-forming cells (SFC) were assessed after overnight incubation of splenocytes with medium alone (unstimulated), an irrelevant peptide control (SARS CoV2 Spike PepTivator cocktail), or with peptides corresponding to the OVA protein (OVAPepTivator and SIINFEKL peptides). Figure 3A The frequency of IFNγ SFCs for each sample and restimulation condition is shown. Figure 3B The frequency of OVA PepTivator-specific IFNγ SFCs per mouse after subtraction of background (unstimulated conditions) is shown. Figure 3C The frequency of SIINFEKL-specific IFNγ SFC per mouse after background (unstimulated condition) subtraction is shown. The graph shows the mean and SD. Each symbol represents one mouse, and n = 5 mice per group. *p < 0.05, **p = 0.01, ***p = 0.001.

[0031] Figure 4A 、 Figure 4B and Figure 4C Shown are IFNγ concentrations measured by ELISA after restimulation of splenocytes from immunized mice. IFNγ levels were quantified in supernatants after 72 hours of incubation of splenocytes with medium alone (unstimulated), an irrelevant peptide control (SARS CoV2 SpikePepTivator), or with peptides corresponding to the OVA protein (OVAPepTivator and SIINFEKL peptides). Figure 4A The IFNγ concentration determined for each sample and restimulation condition is shown. Figure 4B OVA-peptide activator-specific IFNγ secretion of each mouse after subtraction of background (unstimulated conditions) is shown. Figure 4C The SIINFEKL-specific SFC for each mouse after background subtraction (unstimulated condition) is shown. The graph shows the mean and SD. Each symbol represents one mouse, and n = 5 mice per group. *p < 0.05, **p = 0.01.

[0032] Figure 5A and Figure 5B Shown are the results of an IL-5 ELI SPOT assay after stimulation of splenocytes from immunized mice. IL-5 SFC was assessed after overnight incubation of splenocytes with medium alone (unstimulated), an irrelevant peptide control (SARS CoV2 Spike PepTivator), or with a peptide corresponding to the OVA protein (OVA PepTivator). Figure 5A The frequency of IL-5 SFCs for each sample and restimulation condition is shown. Figure 5B The frequency of OVA PepTivator-specific IL-5 SFC per mouse after background (unstimulated condition) subtraction is shown. The graph shows the mean and SD. Each symbol represents one mouse, and n = 5 mice per group. ***p = 0.001, ****p < 0.001.

[0033] Figure 6A and Figure 6B Figure 2 shows IL-5 concentrations measured by ELISA after restimulation of splenocytes from immunized mice. IL-5 levels were quantified in supernatants after 72 hours of incubation of splenocytes with medium alone (unstimulated), an irrelevant peptide control (SARS CoV2 Spike PepTivator), or with a peptide corresponding to the OVA protein (OVA PepTivator). Figure 6A The IL-5 concentration determined for each sample and restimulation condition is shown. Figure 6B The OVA-peptide activator-specific IL-5 secretion per mouse is shown after subtracting background (unstimulated conditions). The bar graph shows the mean and SD. Each symbol represents one mouse, and n = 5 mice per group. **p < 0.01, ****p < 0.001.

[0034] Figure 7A and Figure 7B SIINFEKL tetramer staining of CD8+ T cells in blood and splenocytes of immunized mice is shown. Figure 7A The frequencies (left) and counts (right) of SIINFEKL-specific CD8+ T cells in the blood are shown. Figure 7B The frequencies (left) and counts (right) of SIINFEKL-specific CD8+ T cells in the spleen are shown. *p<0.05, **p=0.01, ***p=0.001.

[0035] Figure 8A Shown are the structures of cationic and ionizable lipids suitable for use in lipid nanoparticles (LNPs) of the present disclosure. Figure 8B The structures of other types of lipids suitable for use in LNPs of the present disclosure are shown. See also Hou et al., Nature Review Materials, 6: 1078-1094, 2021, which is incorporated herein by reference. DETAILED DESCRIPTION

[0036] The present disclosure relates to phospholipid analog (PLA) compounds and their use in hyperactivating human dendritic cells. The present disclosure also relates to compositions comprising PLA and one or more pathogen recognition receptor agonists, antigens, and human dendritic cells, as well as methods of producing and using the compositions. In further embodiments, the dendritic cells are non-human dendritic cells, provided that the dendritic cells are not rodent dendritic cells.

[0037] General techniques and definitions

[0038] 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 skills of a person skilled in the art.

[0039] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated. For example, "a" excipient includes one or more excipients.

[0040] As used herein, the phrase "comprising" is open ended, indicating that such embodiments may include additional elements. In contrast, the phrase "consisting of is closed ended, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase "consisting essentially of is partially closed ended, indicating that such embodiments may further include elements that do not materially change the basic characteristics of such embodiments.

[0041] As used herein, the term "about" refers to a value and encompasses 90% to 110% of that value (eg, a molecular weight of about 900 Daltons refers to a molecular weight of 810 Daltons to 990 Daltons).

[0042] An "effective amount" or "sufficient amount" of a substance is an amount sufficient to produce a beneficial or desired result (including a clinical result), and thus, an "effective amount" depends on the context in which it is used. For example, in the case of administering an immunogenic composition, an effective amount contains sufficient antigen, and one or both of a phospholipid analog (PLA) compound and a PRR agonist to stimulate an immune response (e.g., antigen-reactive antibodies and / or a cellular immune response) against the antigen.

[0043] The terms "individual" and "subject" refer to mammals. "Mammals" include, but are not limited to, humans, non-human primates (e.g., monkeys), farm animals, sports 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.

[0044] The term "dose" as used herein with reference to an immunogenic composition refers to the measured portion of the immunogenic composition ingested (administered or received) by a subject at any one time.

[0045] As used herein, the terms "isolated" and "purified" refer to a material that is removed from at least one component with which it was associated during the material's production process (e.g., removed from its original environment). For example, when used in reference to PLA, the isolated PLA has a purity of at least 90%, 95%, 96%, 97%, 98%, or 99%, as determined by thin layer chromatography (TLC), high pressure liquid chromatography (HPLC), or gas chromatography (GC). 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 cells in which the protein was produced. As a further example, when used in reference to a synthetic compound, an isolated compound or purified compound has been removed from the reaction mixture used to synthesize it.

[0046] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to preparations that are in a form that permits the biological activity of the active ingredient to be effective, and contain no additional components that are unacceptably toxic to the subject to which the formulation or composition would be administered. Such preparations or compositions are intended to be sterile.

[0047] As used herein, "excipient" includes pharmaceutically acceptable excipients, carriers, vehicles, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Physiologically acceptable excipients are typically pH buffered solutions.

[0048] 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; parts thereof and combinations thereof. When present in the compositions of the present disclosure, the antigen can be synthetic or isolated from nature. Antigens suitable for administration in the methods of the present disclosure include any molecule that can elicit an antigen-specific B cell or T cell response. Haptens are included within the scope of "antigen". A "hapten" is a low molecular weight compound that is not immunogenic by itself, but becomes immunogenic when conjugated to a generally larger immunogenic molecule (carrier).

[0049] "Polypeptide antigens" can include purified natural peptides, synthetic peptides, recombinant peptides, crude peptide extracts, or partially purified or unpurified peptides in active state (e.g., peptides from attenuated or inactivated viruses, microorganisms, or cells), or fragments of such peptides. The length of a polypeptide antigen is preferably at least eight amino acid residues.

[0050] The term "agonist" is used in the broadest sense to include any molecule that activates signal transduction through a receptor. In some embodiments, the agonist binds to a receptor. For example, a TLR8 agonist binds to a TLR8 receptor and activates the TLR8 signal transduction pathway.

[0051] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbon group. A Cx alkyl group refers to an alkyl group having x carbon atoms. A Cx-Cy alkyl group or a Cx-y alkyl group refers to an alkyl group having from x to y carbon atoms (inclusive). "N-alkyl" refers to a straight chain, i.e., linear, alkyl group.

[0052] "Alkylene" refers to a divalent saturated aliphatic hydrocarbon group.

[0053] "Alkenyl" refers to a monovalent hydrocarbon 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 from x to y carbon atoms, inclusive.

[0054] "Stimulation" of a response or parameter includes initiating and / or enhancing the response or parameter compared to otherwise identical conditions except for the parameter of interest, or compared to another condition (e.g., an increase in TLR signaling when a TLR agonist is present compared to when a TLR agonist is absent). For example, "stimulating" an immune response means an increase in the immune response. Depending on the parameter being measured, 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.

[0055] Conversely, "inhibition" of a response or parameter includes reducing and / or inhibiting the response or parameter when compared to otherwise identical conditions except for the parameter of interest, or alternatively, compared to another condition (e.g., a decrease in abnormal cell proliferation following administration of a composition comprising a PLA 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, "inhibiting" an immune response means a decrease in the response. Depending on the parameter being measured, the decrease can be from 2-fold to 2,000-fold, or from 5-fold to 500-fold or more, or from 2, 5, 10, 50, or 100-fold to 500, 1,000, 2,000, 5,000, or 10,000-fold.

[0056] The relative terms "higher" and "lower" refer to a measurable increase or decrease in a response or parameter, respectively, compared to otherwise identical conditions except for the parameter of interest, or compared to another condition. For example, a "higher level of DC hyperactivation" refers to a level of DC hyperactivation due to a treatment condition (including a PLA compound of the present invention) that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the level of DC hyperactivation due to a control condition (e.g., no PLA, PGPC, oxPAPC, etc.). A "lower level of DC hyperactivation" refers to a level of DC hyperactivation due to a treatment condition (including a PLA compound of the present invention) that is at least 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, or 1 / 10 of the level of DC hyperactivation due to a control condition (e.g., no PLA, PGPC, oxPAPC, etc.). In some embodiments, the control condition comprises a comparative compound of PLA that replaces the treatment condition.

[0057] As used herein, the term "immunization" refers to the process of increasing a mammalian subject's response to an antigen and thereby improving its ability to resist or overcome infection and / or fight disease.

[0058] As used herein, the term "vaccination" refers to the introduction of a vaccine into the body of a mammalian subject.

[0059] "Adjuvant" refers to a substance that, when added to a composition comprising an antigen, enhances or potentiates the immune response of a mammalian recipient to the antigen following exposure to the antigen.

[0060] The term "treat" or "treatment" of a disease refers to the implementation of a regimen that may include administering one or more therapeutic agents to an individual (human or other) in an effort to obtain beneficial or desired results, including clinical results, in the individual. Beneficial or desired clinical results include, but are not limited to, relief or improvement of one or more signs or symptoms of the disease, alleviation of the extent of the disease, stabilization of the disease state (i.e., no worsening), prevention of the spread of the disease, delaying or slowing the progression of the disease, improvement or alleviation of the disease state, and alleviation (whether partial or complete). "Treatment" may also refer to prolonging survival compared to the expected survival of an individual who has not received treatment. In addition, "treatment" and "treatment" can be performed by administering one or more therapeutic agents, or by administering a series of doses of a therapeutic agent. "Treatment" or "treatment" does not require complete relief of signs or symptoms, nor does it require a cure, and specifically includes regimens that only have a relieving effect on the individual. "Relief" of a disease or condition refers to a reduction in the extent of the disease or condition and / or undesirable clinical manifestations and / or a slowing of the progression of the disease or condition compared to the expected untreated result.

[0061] The compounds described herein can be administered in any pharmaceutically acceptable form, for example in the form of a pharmaceutically acceptable salt, or, if the form is pharmaceutically acceptable, in the form of a free base or free acid. The compounds described herein or their pharmaceutically acceptable salts can be administered in a pharmaceutically acceptable carrier or excipient. As used herein, "pharmaceutically acceptable" or "pharmacologically acceptable" means a substance that is not biologically or otherwise unfavorable, for example, the substance can be incorporated into a pharmaceutical composition administered to a patient without causing any significant adverse biological effect or interacting in a harmful manner with any other component of the composition containing it. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicology and manufacturing testing, and / or are included in the inactive ingredient guide compiled by the US Food and Drug Administration. "Pharmaceutically acceptable salts" are those salts that retain at least some of the biological activity of the free (non-salt) compound and can be administered to an individual as a drug or pharmaceutical. Such salts include, for example: (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or with organic acids such as acetic acid, oxalic acid, propionic acid, succinic acid, maleic acid, tartaric acid, etc.; (2) salts formed when the acidic proton present in the parent compound is replaced by a metal ion such as an alkali metal ion, an alkaline earth ion or an aluminum ion; or coordinated with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, etc. Acceptable inorganic bases that can be used to prepare salts include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, etc. Pharmaceutically acceptable salts can be prepared in situ during the manufacturing process, or by reacting the purified compound of the present invention in free acid or base form with a suitable organic or inorganic base or acid, respectively, and isolating the salt thus formed during a subsequent purification process.

[0062] I. Phospholipid analog (PLA) compounds

[0063] The isolated phospholipid analogs useful in the compositions and methods disclosed herein contain at least one phosphate group and an n-alkyl "tail." The isolated phospholipid analogs are described below by Formula (I), Formula (II), and Formula (III). Specific isolated phospholipid analogs useful in the compositions and methods disclosed herein are edelfosine (EDEL) of Formula (IA); miltefosine (MILT) of Formula (II-A); and perifosine (PERIF) of Formula (III-A).

[0064] In some embodiments, the isolated phospholipid analog (PLA) having an alkyl chain is a compound of formula (I):

[0065]

[0066] where RE1 C 16 -C 20 n-alkyl and R E2 is C1-C4 alkyl, or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some embodiments, R E1 It is C 17 -C 19 In some embodiments, R E2 In some embodiments, R E1 It is C 17 -C 19 n-alkyl, and R E2 It is a C1-C2 alkyl group.

[0067] In some embodiments, the isolated phospholipid analog (PLA) having an alkyl chain of formula (I) is edelfosine (EDEL), as shown in formula (IA):

[0068]

[0069] or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0070] In some embodiments, the isolated phospholipid analog (PLA) having an alkyl chain is a compound of formula (II):

[0071]

[0072] where R M C 14 -C 20 n-alkyl or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some embodiments, R M It is C 14 -C 18 In some embodiments, R M It is C 15 -C 17 n-alkyl.

[0073] In some embodiments, the isolated phospholipid analog (PLA) having an alkyl chain of formula (II) is miltefosine (MILT), as shown in formula (II-A):

[0074]

[0075] or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0076] In some embodiments, the isolated phospholipid analog (PLA) having an alkyl chain is a compound of formula (III):

[0077]

[0078] where R P C 16 -C 20 n-alkyl or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some embodiments, R P It is C 17 -C 19 n-alkyl.

[0079] In some embodiments, the isolated phospholipid analog (PLA) having an alkyl chain of formula (III) is perifosine (PERIF), as shown in formula (III-A):

[0080]

[0081] or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0082] Phospholipid analogs can be protonated or deprotonated, that is, protonation means that any or all free phosphates are converted to phosphates. - Protonation refers to the addition of a proton to a group, while deprotonation refers to the removal of a proton from any or all free phosphate OH groups. The phospholipid analog may be in the form of a salt, such as a pharmaceutically acceptable salt.

[0083] II. Pathogen Recognition Receptor Agonists

[0084] The compositions and methods of the present disclosure may also include pathogen recognition receptor (PRR) agonists. In some embodiments, PRR agonists include agonists of toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), or C-type lectin receptors (CLRs). In other embodiments, PRR agonists include cytoplasmic DNA sensors (CDSs) or IFN gene stimulators (STINGs). In some embodiments, PRR agonists include TLR7 / 8 agonists.

[0085] A. TLR agonists and TLR7 / 8 agonists

[0086] The term "TLR agonist" as used herein refers to an agonist of a Toll-like receptor (TLR). The term "TLR7 / 8 agonist" as used herein refers to an agonist of TLR7 and / or TLR8. In one aspect, the TLR7 / 8 agonist is a TLR7 agonist. In another aspect, the TLR7 / 8 agonist is a TLR8 agonist. In another aspect, the TLR7 / 8 agonist is an agonist of TLR7 and TLR8. The TLR7 / 8 agonists of the present disclosure are suitable for superactivating human dendritic cells in the presence of LPC.

[0087] In some aspects, the TLR agonist is a small molecule. In some aspects, the TLR7 / 8 agonist is a small molecule. In some embodiments, the TLR7 / 8 agonist is a small molecule or a salt thereof having a molecular weight of 900 Daltons or less. In other words, small molecule TLR7 / 8 agonists are not macromolecules like recombinant proteins or synthetic oligonucleotides, which are regulated by the U.S. FDA Center for Biologics Evaluation and Research. Small molecule TLR7 / 8 agonists are not regulated by the FDA Center for Drug Evaluation and Research. In some embodiments, the small molecule has a molecular weight of about 90 to about 900 Daltons. In some embodiments, the TLR7 / 8 agonist includes an imidazoquinoline compound. In some preferred embodiments, the TLR7 / 8 agonist includes resiquimod (R848).

[0088] B. Other PRR agonists

[0089] In some aspects, pathogen recognition receptor (PRR) agonists include Toll-like receptor (TLR) agonists, but with the proviso that the TLR agonist does not include a TLR7 / 8 agonist. In some embodiments, the TLR agonist includes an agonist of one or more of TLR2, TLR3, TLR4, TLR5, TLR9, and TLR13. In some embodiments, the PRR agonist is a TLR2 / 6 agonist, such as Pam2CSK4. In other embodiments, the TLR agonist is a TLR4 agonist, such as 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, such as LPS (endotoxin).

[0090] In other aspects, PRR agonists include NOD-like receptor (NLR) agonists. In further aspects, PRR agonists include RIG-I-like receptor (RLR) agonists. In additional aspects, PRR agonists include C-type lectin receptor (CLR) agonists. In yet further aspects, PRR agonists include CDS agonists or STING agonists.

[0091] III. Antigens

[0092] The compositions and methods of the present disclosure may also include antigens. 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 to 1800 amino acids, 9 to 1000 amino acids, or 10 to 100 amino acids in length. In some embodiments, the antigen comprises a synthetic protein or a 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.

[0093] In some embodiments, the antigen is a tumor antigen comprising an amino acid sequence of at least one full-length protein 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 a normal cell of a mammalian subject. Neoantigens are considered to be particularly useful in enabling T cells to distinguish between cancer cells and non-cancerous cells (see, for example, Schumacher and Schreiber, Science, 348: 69-74, 2015). In other embodiments, tumor antigens include viral antigens, such as antigens of oncoviruses.

[0094] In some embodiments, the tumor antigen is a fusion protein comprising two or more polypeptides, wherein 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 such embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, wherein each polypeptide comprises a non-contiguous amino acid sequence from the same tumor antigen.

[0095] In some embodiments, the antigen is a microbial antigen. In some embodiments, the microbial antigen includes a viral antigen, a bacterial antigen, a protozoan antigen, a fungal antigen, or a combination thereof. In some embodiments, the microbial antigen includes a surface protein or other antigenic subunit of a microorganism. In other embodiments, the microbial antigen includes an inactivated or attenuated microorganism. For example, the microbial antigen may include an inactivated virus, such as a chemically or genetically inactivated virus. Alternatively, the microbial antigen may include a virus-like particle.

[0096] In some embodiments, the antigen can be present in a biological sample obtained from an individual (e.g., a human patient). For example, the antigen can include a cancer cell. On the other hand, the antigen can include a cell infected by a microorganism, such as a virus-infected cell.

[0097] IV. Dendritic Cells

[0098] The compositions and methods of the present disclosure may also include dendritic cells (DCs), which are antigen-presenting cells that are believed to connect the innate and adaptive immune systems of mammals. In preferred embodiments, the dendritic cells are subpopulation 1 conventional dendritic cells (cDC1, formerly known as myeloid DC1), as opposed to plasmacytoid dendritic cells (pDC).

[0099] In some embodiments, DC is a super-activated DC that expresses high levels of CD40 and IL-12p70. The term "super-activated dendritic cell" as used herein refers to a cell state in which DC is able to secrete IL-1β while maintaining cell viability (e.g., without pyroptosis). In this way, super-activated dendritic cells are able to stimulate powerful T cell immunity, which clearly combines the advantages of activated dendritic cells and pyroptotic dendritic cells (Zhivaki et al., Cell Reports, 33(7), 2020, 108381).

[0100] V. Pharmaceutical Preparations

[0101] Some compositions of the present disclosure are pharmaceutical preparations comprising a pharmaceutically acceptable excipient and a PLA compound. In some embodiments, the pharmaceutical preparation further comprises a PRR agonist, a dendritic cell, an antigen, an adjuvant, or any combination thereof. The pharmaceutical preparation of the present disclosure may be in the form of a solution or a suspension. Alternatively, the pharmaceutical preparation may be a dehydrated solid (e.g., a freeze-dried or spray-dried solid). The pharmaceutical preparation of the present disclosure is preferably sterile and preferably substantially free of endotoxins. The term "pharmaceutical preparation" is used interchangeably herein with the terms "pharmaceutical product" and "drug." In some embodiments, the pharmaceutical preparation comprises various ingredients in specific proportions based on the intended use of the preparation. In some embodiments, the pharmaceutical preparation comprises a PLA compound and a nonionic surfactant. In some embodiments, the nonionic surfactant comprises an ethylene oxide-propylene oxide copolymer, such as poloxamer-407 (CAS registration number 977057-91-2).

[0102] A. Excipients

[0103] Pharmaceutically acceptable excipients of the present disclosure include, for example, solvents, buffers, tension regulators, fillers, and preservatives (see, for example, Pramanick et al., Pharma Times, 45: 65-77, 2013). In some embodiments, the pharmaceutical formulation may include an excipient that acts as one or more of a solvent, a buffer, a tension regulator, and a filler (e.g., sodium chloride in saline can act as both an aqueous vehicle and a tension regulator). Pharmaceutically acceptable excipients of the present disclosure also include detergents, wetting agents, emulsifiers, foaming agents, and dispersants, as well as surfactants.

[0104] Many of the lipids disclosed herein are slightly soluble in water. Surfactants can be used to solubilize lipids in aqueous formulations. There are many types of surfactants, which can be divided into anionic surfactants, nonionic surfactants, cationic surfactants, and zwitterionic surfactants.

[0105] Some examples of nonionic surfactants include poloxamers, which are triblock copolymers of ethylene oxide and propylene oxide with the general formula:

[0106] HO-[CH2CH2-O-] a -[CH2CH(CH3)-O-] b -[CH2-CH2-O-] a -H. Some poloxamers are sold under the trade name (PLURONIC is a registered trademark of BASF SE, Ludwigshafen, Germany). Examples of poloxamers include poloxamer 407 (KP407; a=101, b=56); poloxamer 188 (KP188; a=80, b=27); P84 (P-84; a=19, b=39); and P123 (P-123; a=20, b=70) (the above a and b values ​​may vary slightly).

[0107] Other nonionic surfactants include series (CREMAPHOR is a registered trademark of BASF SE, Ludwigshafen, Germany). Surfactants include EL (KEL), a polyoxyethylene triglyceride mixture formed by reacting castor oil with ethylene oxide in a molar ratio of about 1:35, and RH40 (also known as RH40; KOLLIPHOR is a registered trademark of BASF SE), obtained by reacting 40 mol of ethylene oxide with 1 mol of hydrogenated castor oil.

[0108] In some embodiments, the pharmaceutical formulation comprises an aqueous vehicle as a solvent. Suitable vehicles include, for example, sterile water, saline, phosphate-buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic.

[0109] The pharmaceutical preparation may include a buffer. The buffer controls the pH to inhibit degradation of the active agent during processing, storage, and optional reconstitution. Suitable buffers include, for example, salts of acetate, citrate, phosphate, or sulfate. Other suitable buffers include, for example, amino acids such as arginine, glycine, histidine, and lysine. The buffer may also include hydrochloric acid or sodium hydroxide. In some embodiments, the buffer maintains the pH of the composition within the range of 6 to 9. In some embodiments, the pH is greater than (lower limit) 6, 7, or 8. In some embodiments, the pH is less than (upper limit) 9, 8, or 7. That is, the pH is within the range of about 6 to 9, wherein the lower limit is less than the upper limit.

[0110] The pharmaceutical composition may contain a tonicity adjuster. Suitable tonicity adjusters include, for example, dextrose, glycerol, sodium chloride, glycerol, and mannitol.

[0111] The pharmaceutical preparation may include a filler. When the pharmaceutical composition needs to be lyophilized before administration, a filler is particularly useful. In some embodiments, a filler is a protective agent that helps to stabilize the active agent during freezing or spray drying and / or during storage and prevents its degradation. Suitable fillers are sugars (monosaccharides, disaccharides, and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose, and raffinose.

[0112] The pharmaceutical preparation may contain a preservative. Suitable preservatives include, for example, antioxidants and antimicrobials. However, in a preferred embodiment, the pharmaceutical preparation is prepared under aseptic conditions and is placed in a disposable container and therefore does not need to contain a preservative.

[0113] The pharmaceutical formulations of the present disclosure are suitable for parenteral administration. That is, the pharmaceutical formulations of the present disclosure are not intended for enteral administration (e.g., not by oral, gastric, or rectal administration).

[0114] B. Adjuvant

[0115] Pharmaceutically acceptable adjuvants of the present disclosure include, for example, aluminum salt adjuvants, squalene emulsions in water, saponins, or combinations thereof. In some embodiments, the adjuvant is an aluminum salt adjuvant selected from amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, and combinations thereof. In other embodiments, the adjuvant is a squalene emulsion in water, such as MF59 or AS03. In other embodiments, the adjuvant is a saponin, such as QuilA or QS-21, as in AS01 or AS02.

[0116] VI. Production Method

[0117] In some aspects, the present disclosure relates to methods for preparing hyperactivated dendritic cells, and methods for preparing immunogenic compositions. The immunogenic compositions are suitable for hyperactivation of dendritic cells in vitro, ex vivo, or in vivo.

[0118] In one aspect, the present disclosure provides a method for producing hyperactivated dendritic cells (DCs), the method comprising contacting dendritic cells with an effective amount of an isolated phospholipid analog (PLA) having a normal alkyl chain and a PRR agonist for producing hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing pyroptosis. In some embodiments, DC is isolated, while in other embodiments, DC is present in a biological sample obtained from a mammalian subject (e.g., a human patient). In some embodiments, DC is a monocyte-derived DC, preferably cDC1.

[0119] In another aspect, the present disclosure provides a method for producing an immunogenic composition comprising combining an antigen with an effective amount of an isolated phospholipid analog (PLA) and a PRR agonist to produce an immunogenic composition. In some embodiments, the antigen comprises a protein antigen present in a biological sample obtained from a mammalian subject or purified from the biological sample. In some embodiments, the protein antigen is a synthetic or recombinant protein. In some preferred embodiments, the antigen is a tumor antigen. In some preferred embodiments, the antigen is a microbial antigen.

[0120] In a specific embodiment, the present disclosure provides a method of producing an immunogenic composition, the method comprising:

[0121] a) depleting a cell suspension prepared from a tumor to obtain a suspension enriched in tumor cells;

[0122] b) lysing cells from the tumor cell-enriched suspension to obtain a tumor cell lysate; and

[0123] c) contacting the tumor cell lysate with an isolated phospholipid analog (PLA) and a PRR agonist to obtain an immunogenic composition. In some embodiments, leukocytes are depleted from a cell suspension rich in tumor cells by contacting the tumor cell-rich suspension with specific antibodies against leukocytes. In some embodiments, leukocytes are depleted by contacting the tumor cell-rich suspension with anti-CD45 antibodies. In some embodiments, cells are lysed by a cell lysis method based on physical destruction, such as, but not limited to, mechanical lysis, liquid homogenization, ultrasonic treatment, freeze-thaw, or manual grinding. In some preferred embodiments, cells are lysed by one or more freeze-thaw cycles.

[0124] In any embodiment of the foregoing method, the isolated PLA is a compound of formula (I), formula (II) or formula (III), or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some preferred embodiments, the PLA is edelfosine or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some preferred embodiments, the PLA is miltefosine or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some preferred embodiments, the PLA is perifosine or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some preferred embodiments, the PRR agonist is a TLR7 / 8 agonist. In some preferred embodiments, the TLR7 / 8 agonist is an imidazoquinoline compound, and in particularly preferred embodiments, it is resiquimod (R848).

[0125] VII. Other lipids

[0126] The compositions and methods of the present disclosure comprise at least one additional lipid, wherein the compound of Formula (I), Formula (II), or Formula (III) or a protonated form thereof or a pharmaceutically acceptable salt thereof and the at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid comprises an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structured lipid, or a mixture thereof. In some embodiments, the LNP comprises a compound of Formula (I), wherein R E1 C 17 -C 19 The compound of formula (I) wherein R E2 The compound of formula (I) is a C1-C2 alkyl group, wherein R E1 C 17 -C 19 n-alkyl and wherein R E2 is a compound of formula (I) wherein R is a C1-C2 alkyl group, or edilfosine, or a protonated form thereof or a pharmaceutically acceptable salt thereof; and an ionizable lipid, a second phospholipid, a pegylated lipid, or a structured lipid, or a mixture thereof. In some embodiments, the LNP comprises a compound of formula (II), wherein R M C 14 -C 18 The compound of formula (II) wherein R M C 15 -C 17 In some embodiments, the LNP comprises a compound of formula (III), wherein R P It is C 17 -C19 A compound of formula (III) containing an n-alkyl group, or perifosine, or a protonated form thereof, or a pharmaceutically acceptable salt thereof; and an ionizable lipid, a second phospholipid, a pegylated lipid, or a structured lipid, or a mixture thereof. The structures of other lipids suitable for use in the compositions and methods of the present disclosure are as follows: Figure 8A and Figure 8B As shown (reproduced from Hou et al., Nature Review Materials, 6: 1078-1094, 2021).

[0127] In some embodiments, the at least one additional lipid comprises one or both of an additional phospholipid and a structural lipid, optionally wherein 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 wherein the pegylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoylglycerol [DMG]. In some embodiments, the at least one additional lipid comprises or further comprises an ionizable lipid, optionally wherein the ionizable lipid comprises (6Z,9Z,28Z,31Z)-heptatriacontac-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA) or an analog or derivative thereof. In some embodiments, the at least one additional lipid comprises at least one lipid from the following list (disclosed in Hou et al., Nature Review Materials 6, 1078–1094 (2021) in Figure 2); these lipids include 306Oi10, tetrakis(8-methylnonyl) 3,3′,3″,3″′-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate; 9A1P9, decyl(2-(dioctylammonium)ethyl)phosphate; A2-Iso5-2DC18, ethyl 5,5-di((Z)-heptadeca-8-en-1-yl)-1-(3-( ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; β-sitosterol, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5 BAME-O16B, bis(2-(dodecyldisulfanyl)ethyl) 3,3′-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosanoyl)azanediyl) dipropionate BHEM - cholesterol, 2-(((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethane-1-ammonium bromide;C12-200, 1,1′-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol); cKK-E12, 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione; DC-cholesterol, 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol; DLin-MC3-DMA, (6Z,9Z,28Z,31Z)-heptatriacontac-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOS PA, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanium trifluoroacetate; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; DOTMA, 1,2-di-O-octadecenyl-3-trimethylammonium-propane; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; ePC, ethylphosphatidylcholine; FTT5, hexa(octan-3-yl) 9,9′,9″,9″′,9″″,9″′″-((((phenyl-1,3,5-tricarbonyl)di(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanonanoate; lipid H(SM-102), heptadecan-9 -yl 8-((2-hydroxyethyl)(6-oxo-6-(undecanyloxy)hexyl)amino)octanoate; OF-Deg-Lin, (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azatriyl))tetra(ethane-2,1-diyl)(9Z,9′Z,9″Z,9″′Z,12Z,12′Z,12″Z,12″′Z)-tetra(octadec-9,12-dienoate); PEG2000-DMG, 1,2-dimyristoyl rac-glycero-3-methoxypolyethylene glycol-2000; TT3,N1,N3,N5-tris(3-(didodecanoylamino)propyl)benzene-1,3,5-tricarboxamide, such as; Figure 8A and Figure 8B shown.

[0128] VIII. mRNA encoding antigens

[0129] The compositions and methods of the present disclosure comprise mRNA encoding an antigen, or are suitable for use with a formulation comprising mRNA encoding an antigen. In some embodiments, the antigen is a protein antigen. The terms "polypeptide" and "protein" are used interchangeably herein to refer to an antigen comprising a peptide chain of at least 8 amino acids in length. In some embodiments, the length of the antigen is 8 to 1800 amino acids, 9 to 1000 amino acids, or 10 to 100 amino acids. The polypeptide may be post-translationally modified, for example, by phosphorylation, hydroxylation, sulfonation, palmitoylation, and / or glycosylation.

[0130] In some embodiments, the antigen is a tumor antigen comprising an amino acid sequence of at least one full-length protein 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 a normal cell of a mammalian subject. Neoantigens are considered to be particularly useful in enabling T cells to distinguish between cancer cells and non-cancerous cells (see, for example, Schumacher and Schreiber, Science, 348: 69-74, 2015). In other embodiments, tumor antigens include viral antigens, such as antigens of oncoviruses.

[0131] In some embodiments, the tumor antigen is a fusion protein comprising two or more polypeptides, wherein 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 such embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, wherein each polypeptide comprises a non-contiguous amino acid sequence from the same tumor antigen.

[0132] In some embodiments, the antigen is a microbial antigen. In some embodiments, the microbial antigen includes a viral antigen, a bacterial antigen, a protozoan antigen, a fungal antigen, or a combination thereof. In some embodiments, the microbial antigen includes a surface protein or other antigenic subunit of a microorganism.

[0133] In some preferred embodiments, the mRNA comprises a 5' untranslated region (5'UTR) located at the 5' end of the coding region and a 3' untranslated region (3'UTR) located at the 3' end of the coding region. In some preferred embodiments, the mRNA comprises one or both of a 5' cap structure and a poly A tail.

[0134] IX. Lipid-Based Delivery Vehicles

[0135] Compositions and methods of the present disclosure include lipid-based delivery vehicles for mRNA encoding antigens. In some embodiments, the vehicle is a lipid nanoparticle (LNP). In other embodiments, the vehicle is a lipid (RNA-liposome complex) that forms a complex with the mRNA.

[0136] In some embodiments, the LNP comprises a compound of formula (I), wherein R E1 C 17 -C 19 The compound of formula (I) wherein R E2 The compound of formula (I) is a C1-C2 alkyl group, wherein R E1 C 17 -C 19 n-alkyl and wherein R E2 is a compound of formula (I) having a C1-C2 alkyl group or a compound of formula (I) having edelfosine; and at least one lipid selected from ionizable lipids, cationic lipids, second phospholipids, pegylated lipids, structured lipids, and mixtures thereof. In some embodiments, the compound of formula (I) is isolated. In some embodiments, the LNP comprises a compound of formula (II), wherein R M C 14 -C 18 The compound of formula (II) wherein R M C 15 -C 17 In some embodiments, the compound of formula (II) is isolated. In some embodiments, the LNP comprises a compound of formula (III), wherein R P It is C 17 -C 19 n-alkyl compound of formula (III), or perifosine, or a protonated form thereof, or a pharmaceutically acceptable salt thereof; and an ionizable lipid, a second phospholipid, a pegylated lipid, or a structured lipid, or a mixture thereof. In some embodiments, the compound of formula (III) is isolated. In some embodiments, at least one lipid comprises an ionizable lipid. In some embodiments, at least one lipid comprises a cationic lipid. In some embodiments, at least one lipid comprises a second phospholipid. In some embodiments, at least one lipid comprises a pegylated lipid. In some embodiments, at least one lipid comprises a structured lipid. In some embodiments, at least one lipid comprises an ionizable lipid, a second phospholipid, a pegylated lipid, and a structured lipid.

[0137] In some embodiments, the lipid component of the RNA-liposome complex comprises one or more lipids. In some preferred embodiments, the one or more lipids include a first lipid and a second lipid, wherein the first lipid is different from the second lipid. In some embodiments, the first lipid is a cationic lipid and the second lipid is a neutral or anionic lipid.

[0138] The structures of lipids suitable for use in lipid-based mRNA delivery vehicles of the present disclosure are shown in Figure 8A and Figure 8B (Reprinted from Hou et al., Nature Review Materials, 6: 1078-1094, 2021).

[0139] X. How to use

[0140] In some aspects, the present disclosure relates to methods of using any of the compositions or formulations described herein, comprising a compound of formula (I), wherein R E1 It is C 17 -C 19 The compound of formula (I) wherein R E2 is a compound of formula (I) wherein R E1 It is C 17 -C 19 n-alkyl and wherein R E2 is a compound of formula (I) having a 1-C2 alkyl group, a compound of formula (I) having edelfosine, a compound of formula (II), wherein R M It is C 14 -C 18 The compound of formula (II) wherein R M It is C 15 -C 17 The compound of formula (II) is a n-alkyl, the compound of formula (II) is miltefosine, the compound of formula (III), wherein R P It is C 17 -C 19n-alkyl compound of formula (III), perifosine or its protonated form or a pharmaceutically acceptable salt of any of the foregoing compounds. 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 applicable to a variety of uses involving stimulating an immune response. In some embodiments, the method of use includes a method for treating cancer. In some embodiments, the method of use includes a method for inhibiting abnormal cell proliferation. In some embodiments, the method of use includes a method for treating infectious diseases (e.g., viral, bacterial, fungal or protozoal diseases). In some embodiments, the method of use includes a method for treating protozoal diseases such as leishmaniasis. The method includes administering an effective amount of the formulation or composition described herein to an individual in need thereof to achieve a specific result. 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 relates to clinical use, while in other embodiments, the method of use relates to preclinical and / or veterinary use. For preclinical use, the mammalian subject can be a non-human primate (e.g., a monkey or ape) or a rodent (e.g., a mouse or rat). For veterinary uses, the mammalian subject can be a farm animal (eg, a cattle), a sport animal (eg, a horse), or a pet (eg, a companion animal such as a dog or cat).

[0141] A. Stimulation of immune response

[0142] In short, the present disclosure provides a method for stimulating an individual immune response, including administering to an individual a composition or formulation described herein in an amount sufficient to stimulate an individual immune response. "Stimulating" an immune response (used interchangeably with "eliciting" an immune response) refers to enhancing an immune response, which can be produced by inducing a de novo immune response (e.g., due to an initial vaccination regimen) or enhancing an existing immune response (e.g., due to a booster vaccination regimen). In some embodiments, stimulating an immune response includes one or more of the following: stimulating cytokine production; stimulating B lymphocyte proliferation; stimulating interferon pathway-related gene expression; stimulating chemokine-related gene expression; and stimulating dendritic cell DC maturation. Methods for measuring immune response stimulation are known in the art.

[0143] For example, the present disclosure provides a method for inducing an antigen-specific immune response in an individual by administering to the individual a composition as described herein or a preparation of an amount sufficient to induce an antigen-specific immune response in the individual. In a preferred embodiment, the composition or preparation comprises an antigen. In some embodiments, the composition or preparation is administered to the tissue of the individual comprising the antigen. The immune response can include one or both of an antigen-specific antibody response and an antigen-specific cytotoxic T lymphocyte (CTL) response. "Inducing" an antigen-specific antibody response refers to increasing the titer of the antigen-specific antibody to more than a threshold level, such as the baseline titer or serum protection level before administration. "Inducing" an antigen-specific CTL response refers to increasing the frequency of the antigen-specific CTL found in the peripheral blood to a baseline frequency higher than before administration.

[0144] The analysis (qualitative and quantitative) of immune response can be carried out by any method known in the art, including but not limited to measuring the generation of antigen-specific antibodies (including measuring specific antibody subclasses), the activation of specific lymphocyte populations (such as B cells and helper T cells), the generation of cytokines (such as IFN-α, IFN-γ, IL-5, IL-6, IL-12) and / or the release of histamine. The method for measuring antigen-specific antibody reaction includes enzyme-linked immunosorbent assay (ELISA). The activation of specific lymphocyte populations can be measured by proliferation assay and fluorescence activated cell sorting (FACS). The production of cytokines can also be measured by ELISA. In some embodiments, the method for stimulating immune response includes stimulating interleukin-1β (IL-1β) secretion, interferon-γ (IFN-γ) secretion and / or tumor necrosis factor-α (TNF-α) secretion by monocyte-derived dendritic cells or peripheral blood mononuclear cells. In some embodiments, the method of stimulating an immune response includes stimulating memory CD4+T cells to secrete one or more of IFN-γ, IL-17a, IL-17f, and IL-22. In some embodiments, the method of stimulating an immune response includes increasing Th1 differentiation of initial CD4+T cells. In some preferred embodiments, at least 50%, 55%, 60%, 65%, 70%, or 75% of cells contacted with the compositions of the present disclosure remain viable 40-56 hours (or about 48 hours) after contact.

[0145] In some embodiments, the methods are suitable for stimulating an anti-tumor immune response. In other embodiments, the methods are 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-protozoal immune response. In some embodiments, the anti-protozoal immune response comprises stimulating IL-5 secretion.

[0146] B. Treat or prevent disease

[0147] The present disclosure also provides methods for 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.

[0148] In one aspect, the method may comprise administering to a subject in need thereof a composition comprising a compound of formula (I), wherein R E1 It is C 17 -C 19 The compound of formula (I) wherein R E2 The compound of formula (I) is a C1-C2 alkyl group, wherein R E1 C 17 -C 19 n-alkyl and wherein R E2 The compound of formula (I) is C1-C2 alkyl, the compound of formula (I) is edelfosine, the compound of formula (II), wherein R M C 14 -C 18 The compound of formula (II) is n-alkyl, wherein R M C 15 -C 17 The compound of formula (II) is a n-alkyl, the compound of formula (II) is miltefosine, the compound of formula (III), wherein R P C 17 -C 19 In some embodiments, the composition further comprises a PRR agonist, an antigen, an adjuvant, or any combination thereof. In another aspect, the method comprises adoptive cell therapy and comprises administering to a subject in need thereof a composition comprising dendritic cells, such as hyperactivated dendritic cells; and a compound of formula (I), wherein R E1 C 17 -C 19 The compound of formula (I) wherein R E2 The compound of formula (I) is a C1-C2 alkyl group, wherein R E1 C 17 -C 19 n-alkyl and wherein R E2 The compound of formula (I) is C1-C2 alkyl, the compound of formula (I) is edelfosine, the compound of formula (II), wherein R M C 14 -C18 The compound of formula (II) wherein R M C 15 -C 17 The compound of formula (II) is a n-alkyl, the compound of formula (II) is miltefosine, the compound of formula (III), wherein R P C 17 -C 19 n-alkyl compounds of formula (III), perifosine or a protonated form thereof, or a pharmaceutically acceptable salt of any of the foregoing compounds. In some embodiments, the composition further comprises a PRR agonist, an antigen, an adjuvant, or any combination thereof.

[0149] In some embodiments, the method comprises treating cancer in an individual or treating a mammalian subject suffering from cancer. In some embodiments, the method comprises: a) preparing an immunogenic composition comprising a tumor cell lysate; an isolated compound selected from the group consisting of a compound of formula (I), wherein R E1 C 17 -C 19 The compound of formula (I) wherein R E2 The compound of formula (I) is a C1-C2 alkyl group, wherein R E1 C 17 -C 19 n-alkyl and wherein R E2 The compound of formula (I) is C1-C2 alkyl, the compound of formula (I) is edelfosine, the compound of formula (II), wherein R M C 14 -C 18 The compound of formula (II) wherein R M C 15 -C 17 The compound of formula (II) is a n-alkyl, the compound of formula (II) is miltefosine, the compound of formula (III), wherein R P C 17 -C 19 n-alkyl compound of formula (III), perifosine or its protonated form or a pharmaceutically acceptable salt of any of the foregoing compounds; and a toll-like receptor (TLR) agonist, such as a toll-like receptor 7 / 8 (TLR7 / 8) agonist, wherein the tumor cell lysate is or has been prepared from a tumor sample obtained from a subject with cancer; and b) administering an effective amount of the immunogenic composition to the subject. In some embodiments, the cancer is a blood cancer, such as a 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.

[0150] In some embodiments, the method involves inhibiting abnormal cell proliferation in an individual. "Abnormal cell proliferation" refers to the proliferation of a benign tumor or a malignant tumor. The malignant tumor can be a metastatic tumor.

[0151] In some embodiments, the method relates to 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 (e.g., leishmaniasis). In some embodiments, the method comprises treating a protozoan disease by inducing the secretion of IL-5. 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 through an intermediate species (vector).

[0152] Listed implementation plans

[0153] The present disclosure is further illustrated by the following embodiments, which may be combined in any manner as far as is practical.

[0154] Embodiment 1. A composition comprising an isolated compound of formula (I):

[0155]

[0156] where R E1 C 16 -C 20 n-alkyl and R E2 is C1-C4 alkyl, or a protonated form thereof or a pharmaceutically acceptable salt thereof; and

[0157] TLR7 / 8 agonists.

[0158] Embodiment 2. The composition of embodiment 1, wherein R E1 C 17 -C 19 n-alkyl.

[0159] Embodiment 3. The composition of embodiment 1 or embodiment 2, wherein R E2 It is a C1-C2 alkyl group.

[0160] Embodiment 4. The composition of embodiment 1, wherein the compound is edilfosine (EDEL):

[0161]

[0162] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0163] Embodiment 5. A composition comprising an isolated compound of formula (II):

[0164]

[0165] where R M C 14 -C 20 n-alkyl or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and

[0166] TLR7 / 8 agonists.

[0167] Embodiment 6. The composition of embodiment 5, wherein R M C 14 -C 18 n-alkyl.

[0168] Embodiment 7. The composition of embodiment 5, wherein R M C 15 -C 17 n-alkyl.

[0169] Embodiment 8. The composition of embodiment 5, wherein the compound is miltefosine (MILT):

[0170]

[0171] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0172] Embodiment 9. A composition comprising an isolated compound of formula (III):

[0173]

[0174] where R P C 16 -C 20 n-alkyl or a protonated form thereof or a pharmaceutically acceptable salt thereof; and

[0175] TLR7 / 8 agonists.

[0176] Embodiment 10. The composition of embodiment 9, wherein R P C 17 -C 19 n-alkyl.

[0177] Embodiment 11. The composition of embodiment 9, wherein the compound is perifosine (PERIF):

[0178]

[0179] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0180] Embodiment 12. The composition of any one of embodiments 1-11, further comprising an antigen.

[0181] Embodiment 13. The composition of any one of embodiments 1-12, further comprising dendritic cells.

[0182] Embodiment 14. A composition comprising an isolated compound of formula (I):

[0183]

[0184] where R E1 C 16 -C 20 n-alkyl and R E2 is C1-C4 alkyl, or a protonated form thereof or a pharmaceutically acceptable salt thereof; and

[0185] antigen.

[0186] Embodiment 15. The composition of embodiment 14, wherein R E1 C 17 -C 19 n-alkyl.

[0187] Embodiment 16. The composition of embodiment 14 or embodiment 15, wherein R E2 It is a C1-C2 alkyl group.

[0188] Embodiment 17. The composition of embodiment 14, wherein the compound is edilfosine (EDEL):

[0189]

[0190]

[0191] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0192] Embodiment 18. A composition comprising an isolated compound of formula (II):

[0193]

[0194] where R M C 14 -C 20 n-alkyl or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and

[0195] antigen.

[0196] Embodiment 19. The composition of embodiment 18, wherein R M C 14 -C 18 n-alkyl.

[0197] Embodiment 20. The composition of embodiment 18, wherein R M C 15 -C 17 n-alkyl.

[0198] Embodiment 21. The composition of embodiment 18, wherein the compound is miltefosine (MILT):

[0199]

[0200] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0201] Embodiment 22. A composition comprising an isolated compound of formula (III):

[0202]

[0203] where R P C 16 -C 20 n-alkyl or a protonated form thereof or a pharmaceutically acceptable salt thereof; and

[0204] antigen.

[0205] Embodiment 23. The composition of embodiment 22, wherein R P C 17 -C 19 n-alkyl.

[0206] Embodiment 24. The composition of embodiment 22, wherein the compound is perifosine (PERIF):

[0207]

[0208] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0209] Embodiment 25. The composition of any one of embodiments 14-24, further comprising a TLR7 / 8 agonist.

[0210] Embodiment 26. The composition of any one of embodiments 14-25, further comprising dendritic cells.

[0211] Embodiment 27. A composition comprising an isolated compound of formula (I):

[0212]

[0213] where R E1 C 16 -C 20 n-alkyl and R E2 is C1-C4 alkyl, or a protonated form thereof or a pharmaceutically acceptable salt thereof; and

[0214] Dendritic cells.

[0215] Embodiment 28. The composition of embodiment 27, wherein R E1 C 17 -C 19 n-alkyl.

[0216] Embodiment 29. The composition of embodiment 27 or embodiment 15, wherein R E2 It is a C1-C2 alkyl group.

[0217] Embodiment 30. The composition of embodiment 27, wherein the compound is edilfosine (EDEL):

[0218]

[0219] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0220] Embodiment 31. A composition comprising an isolated compound of formula (II):

[0221]

[0222] where R M C 14 -C 20 n-alkyl or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and

[0223] Dendritic cells.

[0224] Embodiment 32. The composition of embodiment 31, wherein R M C 14 -C 18 n-alkyl.

[0225] Embodiment 33. The composition of embodiment 31, wherein R M C 15 -C 17 n-alkyl.

[0226] Embodiment 34. The composition of embodiment 31, wherein the compound is miltefosine (MILT):

[0227]

[0228] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0229] Embodiment 35. A composition comprising an isolated compound of formula (III):

[0230]

[0231] where R P C 16 -C 20 n-alkyl or a protonated form thereof or a pharmaceutically acceptable salt thereof; and

[0232] Dendritic cells.

[0233] Embodiment 36. The composition of embodiment 35, wherein R P C 17 -C 19 n-alkyl.

[0234] Embodiment 37. The composition of embodiment 35, wherein the compound is perifosine (PERIF):

[0235]

[0236] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0237] Embodiment 38. The composition of any one of Embodiments 27-37, further comprising a TLR7 / 8 agonist.

[0238] Embodiment 39. The composition of any one of embodiments 27-38, further comprising an antigen.

[0239] Embodiment 40. The composition of any one of embodiments 1-39, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 Daltons or less.

[0240] Embodiment 41. The composition of embodiment 40, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

[0241] Embodiment 42. The composition of embodiment 41, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

[0242] Embodiment 43. The composition of embodiment 40 or embodiment 41, wherein the TLR7 / 8 agonist does not inhibit NLR family pyrin-containing domain 3 (NLRP3).

[0243] Embodiment 44. The composition of any one of Embodiments 1-43, wherein the antigen is present in a biological sample obtained from an individual.

[0244] Embodiment 45. The composition of embodiment 44, wherein the biological sample comprises a biopsy tissue.

[0245] Embodiment 46. The composition of embodiment 44, wherein the biological sample comprises cells.

[0246] Embodiment 47. The composition of embodiment 44, wherein the biological sample does not comprise cells.

[0247] Embodiment 48. The composition of embodiment 44, wherein the biological sample comprises pus from an abscess.

[0248] Embodiment 49. The composition of any one of Embodiments 1-48, wherein the antigen comprises a protein antigen.

[0249] Embodiment 50. The composition of any one of Embodiments 1-49, wherein the antigen comprises a tumor antigen.

[0250] Embodiment 51. The composition of embodiment 50, wherein the tumor antigen comprises a synthetic or recombinant neoantigen.

[0251] Embodiment 52. The composition of embodiment 50 or embodiment 51, wherein the tumor antigen comprises a tumor cell lysate.

[0252] Embodiment 53. The composition of any one of embodiments 1-49, wherein the antigen comprises a microbial antigen, and the microbial antigen comprises one or more of a viral antigen, a bacterial antigen, a protozoan antigen, and a fungal antigen.

[0253] Embodiment 54. The composition of embodiment 53, wherein the microbial antigen comprises a purified or recombinant surface protein.

[0254] Embodiment 55. The composition of embodiment 53, wherein the microbial antigen comprises an inactivated whole virus.

[0255] Embodiment 56. The composition of any one of embodiments 1-55, wherein the composition does not comprise liposomes.

[0256] Embodiment 57. The composition of any one of Embodiments 1-56, wherein the composition does not comprise LPS or MPLA.

[0257] Embodiment 58. The composition of any one of embodiments 1-57, wherein the composition does not comprise oxPAPC or oxPAPC species, optionally wherein the composition does not comprise HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC and / or PGPC.

[0258] Embodiment 59. The composition of embodiment 58, wherein the composition does not comprise lysophosphatidylcholine (LPC), optionally wherein the composition does not comprise 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

[0259] Embodiment 60. The composition of any one of embodiments 1-59, further comprising an adjuvant, wherein the adjuvant comprises an aluminum salt adjuvant, a squalene-in-water emulsion, a saponin, or a combination thereof.

[0260] Embodiment 61. A pharmaceutical formulation comprising the composition of any one of embodiments 1-60 and a pharmaceutically acceptable excipient.

[0261] Embodiment 62. A method for producing hyperactivated dendritic cells, the method comprising contacting the dendritic cells with a composition comprising an effective amount of an isolated compound of formula (I) or a protonated form or a pharmaceutically acceptable salt thereof and a TLR7 / 8 agonist for producing hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing pyroptosis.

[0262] Embodiment 63. The method according to embodiment 62, wherein R in formula (I) E1 C 17 -C 19 n-alkyl.

[0263] Embodiment 64. The method of embodiment 62 or embodiment 63, wherein R in formula (I) E2 It is a C1-C2 alkyl group.

[0264] Embodiment 65. The method of embodiment 62, wherein the compound of formula (I) is edelfosine (EDEL):

[0265]

[0266] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0267] Embodiment 66. A method for producing hyperactivated dendritic cells, the method comprising contacting the dendritic cells with a composition comprising an effective amount of an isolated compound of formula (II) or a protonated form or a pharmaceutically acceptable salt thereof and a TLR7 / 8 agonist for producing hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing pyroptosis.

[0268] Embodiment 67. The method according to embodiment 66, wherein R in formula (II) M C 14 -C 18 n-alkyl.

[0269] Embodiment 68. The method according to embodiment 66, wherein R in formula (II) M C 15 -C 17 n-alkyl.

[0270] Embodiment 69. The method according to embodiment 66, wherein the compound of formula (II) is miltefosine (MILT):

[0271]

[0272] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0273] Embodiment 70. A method for producing hyperactivated dendritic cells, the method comprising contacting the dendritic cells with a composition comprising an effective amount of an isolated compound of formula (III) or a protonated form or a pharmaceutically acceptable salt thereof and a TLR7 / 8 agonist for producing hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing pyroptosis.

[0274] Embodiment 71. The method according to embodiment 70, wherein R P C 17 -C 19 n-alkyl.

[0275] Embodiment 72. The method according to embodiment 70, wherein the compound of formula (III) is perifosine (PERIF):

[0276]

[0277] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0278] Embodiment 73. The method of any one of embodiments 62-72, wherein the dendritic cells are contacted ex vivo with the composition of any one of embodiments 1-60 or the formulation of embodiment 61.

[0279] Embodiment 74. The method of any one of embodiments 62-72, wherein the dendritic cells are contacted with the formulation of embodiment 61 in vivo.

[0280] Embodiment 75. A pharmaceutical preparation comprising at least 10 3 , 10 4 , 10 5 or 10 6 A hyperactivated dendritic cell produced by the method described in embodiment 73, and a pharmaceutically acceptable excipient.

[0281] Embodiment 76. A method of stimulating an immune response to an antigen, comprising administering to an individual in need thereof an effective amount of the formulation of embodiment 73 to stimulate an immune response to the antigen.

[0282] Embodiment 77. A method of treating cancer, comprising administering to an individual in need thereof an effective amount of the formulation of embodiment 61 to treat the cancer.

[0283] Embodiment 78. A method of inhibiting abnormal cell proliferation, comprising administering to an individual in need thereof an effective amount of the formulation of embodiment 61 to inhibit abnormal cell proliferation.

[0284] Embodiment 79. A method of treating an infectious disease, comprising administering to an individual in need thereof an effective amount of the formulation of embodiment 61 to treat the infectious disease.

[0285] Embodiment 80. Use of the formulation of embodiment 61 for inducing an immune response against an antigen in an individual in need thereof.

[0286] Embodiment 81. Use of the formulation of embodiment 61 for inducing an anti-tumor immune response in an individual in need thereof, wherein the individual is or was a tumor carrier.

[0287] Embodiment 82. Use of the formulation of embodiment 61 for inducing an anti-microbial immune response in an individual in need thereof, wherein the individual is infected with or has not been exposed to the microorganism.

[0288] Embodiment 83. The composition, formulation, method or use of any one of embodiments 44-82, wherein the individual is a mammalian subject.

[0289] Embodiment 84. The composition, formulation, method or use of any one of embodiments 44-82, wherein the individual is a human subject.

[0290] Embodiment 85. A method of preparing an immunogenic composition, the method comprising:

[0291] a) depleting a cell suspension prepared from a tumor to obtain a suspension enriched in tumor cells;

[0292] b) lysing cells from the tumor cell-enriched suspension to obtain a tumor cell lysate; and

[0293] c) contacting the tumor cell lysate with the isolated compound of formula (I) or a protonated form or a pharmaceutically acceptable salt thereof and a toll-like receptor 7 / 8 (TLR7 / 8) agonist to obtain an immunogenic composition.

[0294] Embodiment 86. The method according to embodiment 85, wherein R in formula (I) E1 C 17 -C 19 n-alkyl.

[0295] Embodiment 87. The method of embodiment 85 or embodiment 86, wherein R in formula (I) E2 It is a C1-C2 alkyl group.

[0296] Embodiment 88. The method of embodiment 85, wherein the compound of formula (I) is edelfosine (EDEL):

[0297]

[0298] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0299] Embodiment 89. A method of preparing an immunogenic composition, the method comprising:

[0300] a) depleting a cell suspension prepared from a tumor to obtain a suspension enriched in tumor cells;

[0301] b) lysing cells from the tumor cell-enriched suspension to obtain a tumor cell lysate; and

[0302] c) contacting the tumor cell lysate with the isolated compound of formula (II) or a protonated form or a pharmaceutically acceptable salt thereof and a toll-like receptor 7 / 8 (TLR7 / 8) agonist to obtain an immunogenic composition.

[0303] Embodiment 90. The method according to embodiment 89, wherein R in formula (II) MC 14 -C 18 n-alkyl.

[0304] Embodiment 91. The method according to embodiment 89, wherein R in formula (II) M C 15 -C 17 n-alkyl.

[0305] Embodiment 92. The method of embodiment 89, wherein the compound of formula (II) is miltefosine (MILT):

[0306]

[0307] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0308] Embodiment 93. A method of preparing an immunogenic composition, the method comprising:

[0309] a) depleting a cell suspension prepared from a tumor for leukocytes to obtain a tumor cell-enriched suspension;

[0310] b) lysing cells from the tumor cell-enriched suspension to obtain a tumor cell lysate; and

[0311] c) contacting the tumor cell lysate with the isolated compound of formula (III) or a protonated form or a pharmaceutically acceptable salt thereof and a toll-like receptor 7 / 8 (TLR7 / 8) agonist to obtain an immunogenic composition.

[0312] Embodiment 94. The method according to embodiment 93, wherein R P C 17 -C 19 n-alkyl.

[0313] Embodiment 95. The method according to embodiment 93, wherein the compound of formula (III) is perifosine (PERIF):

[0314]

[0315] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0316] Embodiment 96. The method of any one of embodiments 85-95, wherein in step a) leukocytes are depleted by negative selection using an anti-CD45 antibody.

[0317] Embodiment 97. The method of any one of embodiments 85-96, wherein in step b) the cells are lysed by one or more freeze-thaw cycles.

[0318] Embodiment 98. The method of any one of embodiments 85-97, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 Daltons or less.

[0319] Embodiment 99. The method of embodiment 98, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

[0320] Embodiment 100. The method of embodiment 99, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

[0321] Embodiment 101. The method of embodiment 98 or embodiment 99, wherein the TLR7 / 8 agonist does not inhibit NLR family pyrin-containing domain 3 (NLRP3).

[0322] Embodiment 102. The method of any one of Embodiments 85-101, further comprising obtaining a sample from a tumor of a mammalian subject suffering from cancer prior to step a) and preparing a cell suspension from the sample.

[0323] Embodiment 103. An immunogenic composition prepared by the method of any one of embodiments 85-102.

[0324] Embodiment 104. A method of eliciting an anti-cancer immune response, the method comprising:

[0325] An effective amount of the immunogenic composition of embodiment 103 is administered to a mammalian subject having cancer.

[0326] Embodiment 105. The method of embodiment 104, wherein the anti-cancer immune response comprises a cellular immune response.

[0327] Embodiment 106. The method of embodiment 105, wherein the anti-cancer immune response comprises cancer antigen-induced IL-1β secretion and / or activation of CD8+ T lymphocytes.

[0328] Embodiment 107. The method of any one of Embodiments 104-106, wherein the cancer is a non-hematological cancer.

[0329] Embodiment 108. The method of embodiment 107, wherein the non-hematological cancer is carcinoma, sarcoma, or melanoma.

[0330] Embodiment 109. The method of any one of Embodiments 104-106, wherein the cancer is lymphoma.

[0331] Embodiment 110. A method of treating cancer, the method comprising:

[0332] a) preparing an immunogenic composition comprising a tumor cell lysate, a compound of formula (I) or a protonated form thereof or a pharmaceutically acceptable salt thereof, and a toll-like receptor 7 / 8 (TLR7 / 8) agonist, wherein the tumor cell lysate is or has been prepared from a tumor sample obtained from a mammalian subject suffering from cancer; and

[0333] b) administering to the subject an effective amount of the immunogenic composition.

[0334] Embodiment 111. The method according to embodiment 110, wherein R in formula (I) E1 C 17 -C 19 n-alkyl.

[0335] Embodiment 112. The method of embodiment 110 or embodiment 111, wherein R in formula (I) E2 It is a C1-C2 alkyl group.

[0336] Embodiment 113. The method of embodiment 110, wherein the compound of formula (I) is edelfosine (EDEL):

[0337]

[0338] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0339] Embodiment 114. A method of treating cancer, the method comprising:

[0340] a) preparing an immunogenic composition comprising a tumor cell lysate, a compound of formula (II) or a protonated form thereof or a pharmaceutically acceptable salt thereof, and a toll-like receptor 7 / 8 (TLR7 / 8) agonist, wherein the tumor cell lysate is or has been prepared from a tumor sample obtained from a mammalian subject suffering from cancer; and

[0341] b) administering to the subject an effective amount of the immunogenic composition.

[0342] Embodiment 115. The method according to embodiment 114, wherein R in formula (II) M C 14 -C 18 n-alkyl.

[0343] Embodiment 116. The method according to embodiment 114, wherein R in formula (II) M C 15 -C 17 n-alkyl.

[0344] Embodiment 117. The method of embodiment 114, wherein the compound of formula (II) is miltefosine (MILT):

[0345]

[0346] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0347] Embodiment 118. A method of treating cancer, the method comprising:

[0348] a) preparing an immunogenic composition comprising a tumor cell lysate, a compound of formula (III) or a protonated form thereof or a pharmaceutically acceptable salt thereof, and a toll-like receptor 7 / 8 (TLR7 / 8) agonist, wherein the tumor cell lysate is or has been prepared from a tumor sample obtained from a mammalian subject suffering from cancer; and

[0349] b) administering to the subject an effective amount of the immunogenic composition.

[0350] Embodiment 119. The method of embodiment 118, wherein R P C 17 -C 19 n-alkyl.

[0351] Embodiment 120. The method according to embodiment 118, wherein the compound of formula (III) is perifosine (PERIF):

[0352]

[0353] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0354] Embodiment 121. The method of any one of embodiments 104-120, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 Daltons or less.

[0355] Embodiment 122. The method of embodiment 121, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

[0356] Embodiment 123. The method of embodiment 122, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

[0357] Embodiment 124. The method of any one of claims 110-123, further comprising administering to the subject an effective amount of an additional therapeutic agent.

[0358] Embodiment 125. The method of embodiment 124, wherein the additional therapeutic agent comprises one or more of the group consisting of an immune checkpoint inhibitor, an anti-tumor agent, and radiation therapy.

[0359] Embodiment 126. A composition comprising a compound of formula (I) or a protonated form or a pharmaceutically acceptable salt thereof, and a pathogen recognition receptor (PRR) agonist.

[0360] Embodiment 127. The composition of embodiment 126, wherein R in formula (I) E1 C 17 -C 19 n-alkyl.

[0361] Embodiment 128. The composition of embodiment 126 or embodiment 127, wherein R in formula (I) E2 It is a C1-C2 alkyl group.

[0362] Embodiment 129. The composition of embodiment 126, wherein the compound is edilfosine (EDEL):

[0363]

[0364] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0365] Embodiment 130. A composition comprising a compound of formula (II) or a protonated form or a pharmaceutically acceptable salt thereof, and a pathogen recognition receptor (PRR) agonist.

[0366] Embodiment 131. The composition of embodiment 130, wherein R in formula (II) M C 14 -C 18 n-alkyl.

[0367] Embodiment 132. The composition of embodiment 130, wherein R in formula (II) M C 15 -C 17 n-alkyl.

[0368] Embodiment 133. The composition of embodiment 130, wherein the compound is miltefosine (MILT):

[0369]

[0370] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0371] Embodiment 134. A composition comprising a compound of formula (III) or a protonated form or a pharmaceutically acceptable salt thereof, and a pathogen recognition receptor (PRR) agonist.

[0372] Embodiment 135. The composition of embodiment 134, wherein R in formula (III) P C 17 -C 19 n-alkyl.

[0373] Embodiment 136. The composition of embodiment 134, wherein the compound is perifosine (PERIF):

[0374]

[0375] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0376] Embodiment 137. The composition of any one of embodiments 126-136, 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).

[0377] Embodiment 138. The composition of any one of embodiments 126-136, wherein the PRR agonist is an agonist of cytoplasmic DNA sensor (CDS) or stimulator of IFN genes (STING).

[0378] Embodiment 139. The composition of any one of Embodiments 126-136, wherein the PRR agonist comprises one or more of R848, TL8-506, LPS, Pam2CSK4, and ODN 2336.

[0379] Embodiment 140. The composition of any one of Embodiments 126-139, further comprising an antigen.

[0380] Embodiment 141. The composition of any one of Embodiments 126-140, further comprising dendritic cells.

[0381] Embodiment 142. A pharmaceutical formulation comprising the composition of any one of embodiments 126-141 and a pharmaceutically acceptable excipient.

[0382] Embodiment 143. A pharmaceutical formulation comprising an isolated compound of formula (I) or a protonated form or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0383] Embodiment 144. The composition of embodiment 143, wherein R in formula (I) E1 C 17 -C 19 n-alkyl.

[0384] Embodiment 145. The composition of embodiment 143 or embodiment 144, wherein R in formula (I) E2 It is a C1-C2 alkyl group.

[0385] Embodiment 146. The composition of embodiment 143, wherein the compound is edilfosine (EDEL):

[0386]

[0387] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0388] Embodiment 147. A pharmaceutical formulation comprising an isolated compound of formula (II) or a protonated form or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0389] Embodiment 148. The formulation of embodiment 147, wherein R in formula (II) M C 14 -C 18 n-alkyl.

[0390] Embodiment 149. The formulation of embodiment 147, wherein R in formula (II) M C 15 -C 17 n-alkyl.

[0391] Embodiment 150. The formulation of embodiment 147, wherein the compound is miltefosine (MILT):

[0392]

[0393] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0394] Embodiment 151. A pharmaceutical formulation comprising an isolated compound of formula (III) or a protonated form or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0395] Embodiment 152. The composition of embodiment 151, wherein R in formula (III) P C 17 -C 19 n-alkyl.

[0396] Embodiment 153. The composition of embodiment 151, wherein the compound is perifosine (PERIF):

[0397]

[0398] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0399] Embodiment 154. A composition for hyperactivating human dendritic cells, comprising a compound of formula (I) or a protonated form or a pharmaceutically acceptable salt thereof, and a pathogen recognition receptor (PRR) agonist, wherein the composition is effective in achieving a higher level of dendritic cell hyperactivation than a comparative composition comprising PGPC instead of the compound of formula (I).

[0400] Embodiment 155. The composition of embodiment 154, wherein R in formula (I) E1 C 17 -C 19 n-alkyl.

[0401] Embodiment 156. The composition of embodiment 154 or embodiment 155, wherein R in formula (I) E2 It is a C1-C2 alkyl group.

[0402] Embodiment 157. The composition of embodiment 154, wherein the compound is edilfosine (EDEL):

[0403]

[0404] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0405] Embodiment 158. A composition for hyperactivating human dendritic cells, comprising a compound of formula (II) or a protonated form or a pharmaceutically acceptable salt thereof, and a pathogen recognition receptor (PRR) agonist, wherein the composition is effective in achieving a higher level of dendritic cell hyperactivation than a comparative composition comprising PGPC instead of the compound of formula (II).

[0406] Embodiment 159. The composition of embodiment 158, wherein R in formula (II) M C 14 -C 18 n-alkyl.

[0407] Embodiment 160. The composition of embodiment 158, wherein R in formula (II) M C 15 -C 17 n-alkyl.

[0408] Embodiment 161. The composition of embodiment 158, wherein the compound is miltefosine (MILT):

[0409]

[0410] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0411] Embodiment 162. A composition for hyperactivating human dendritic cells, comprising a compound of formula (III) or a protonated form or a pharmaceutically acceptable salt thereof, and a pathogen recognition receptor (PRR) agonist, wherein the composition is effective in achieving a higher level of dendritic cell hyperactivation than a comparative composition comprising PGPC instead of the compound of formula (III).

[0412] Embodiment 163. The composition of embodiment 162, wherein R in formula (III) P C 17 -C 19 n-alkyl.

[0413] Embodiment 164. The composition of embodiment 162, wherein the compound is perifosine (PERIF):

[0414]

[0415] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0416] Embodiment 165. A composition as described in any of Embodiments 154-164, wherein the higher level of dendritic cell hyperactivation comprises inducing IL-1β secretion from human dendritic cells in vitro at a level that is at least 2, 3, or 4 times higher when contacted with a composition comprising the compound and a PRR agonist than when contacted with a comparative composition comprising PGPC and a PRR agonist, wherein the PRR agonist is LPS.

[0417] Embodiment 166. The composition of embodiment 165, wherein the concentration of the compound and the concentration of the PGPC are the same concentration in the range of about 10 μM to about 80 μM, and the LPS is present in the composition and the comparative composition at a concentration of 1 g / ml.

[0418] Embodiment 167. A composition as described in Embodiment 165, wherein the higher level of dendritic cell hyperactivation comprises a lipid activity index for IL-1β secretion from human dendritic cells for the composition comprising the compound and the PRR agonist that is at least 4, 5, or 6 times the lipid activity index of a comparative composition comprising the PGPC and the PRR agonist.

[0419] Embodiment 168. The composition, formulation, method or use of any one of Embodiments 44-82, wherein the individual is a canine subject.

[0420] Embodiment 169. The composition, formulation, method or use of any one of embodiments 44-82 or 102-167, wherein the mammalian subject is a human patient.

[0421] Embodiment 170. The composition, formulation, method or use of any one of Embodiments 44-82 or 102-167, wherein the mammalian subject is a non-human patient.

[0422] Embodiment 171. The composition, formulation, method or use of any one of Embodiments 44-82 or 102-167, wherein the mammalian subject is a canine patient.

[0423] Embodiment 172. The composition, formulation, method or use of any one of Embodiments 1-167 or 169, wherein the dendritic cells are human dendritic cells.

[0424] Embodiment 173. The composition, formulation, method or use of any one of Embodiments 1-168 or 171, wherein the dendritic cells are canine dendritic cells.

[0425] Embodiment 174. The composition, method or use of Embodiment 172 or Embodiment 173, wherein the dendritic cells are present in a composition comprising peripheral blood mononuclear cells (PBMCs).

[0426] Embodiment 175. The composition, method, or use of any one of Embodiments 62-84, 168, or 169 or Embodiment 91, wherein the hyperactivated dendritic cells secrete one or both of IFNγ and TNFα.

[0427] Embodiment 176. The composition, formulation, method or use of any one of Embodiments 1-175, comprising a surfactant.

[0428] Embodiment 177. The composition, formulation, method or use of Embodiment 176, wherein the surfactant comprises a nonionic surfactant.

[0429] Embodiment 178. The composition, formulation, method or use of embodiment 177, wherein the nonionic surfactant comprises an ethylene oxide-propylene oxide copolymer.

[0430] Embodiment 179. The composition, formulation, method or use of Embodiment 177, wherein the nonionic surfactant comprises one or more of Poloxamer 407, Poloxamer 188 and P123.

[0431] Embodiment 180. The composition, formulation, method or use of embodiment 177, wherein the nonionic surfactant comprises poloxamer 407.

[0432] Embodiment 181. A composition, formulation, method, or use according to any one of embodiments 177-180, wherein i) PLA is dissolved in alcohol to form a PLA alcohol solution; ii) the PLA alcohol solution is mixed with a nonionic surfactant to form a mixture; and iii) the alcohol is evaporated from the mixture to form particles comprising PLA and the nonionic surfactant.

[0433] Embodiment 182. The composition, formulation, method or use of any one of embodiments 177-181, wherein the nonionic surfactant is present in an amount of about 2.5% to 25% (w / w), optionally about 5% to 20% (w / w), optionally about 15% (w / w).

[0434] Embodiment 183. The composition, formulation, method, or use of any one of Embodiments 177-182, wherein the PLA and the nonionic surfactant are present in particles having a diameter of about 1000 to 2000 nanometers, optionally about 1500 nanometers in diameter.

[0435] Embodiment 184. A composition comprising a compound of formula (I) or a protonated form or a pharmaceutically acceptable salt thereof; and at least one additional lipid, wherein the at least one additional lipid is selected from the group consisting of ionizable lipids, cationic lipids, additional phospholipids, PEGylated lipids, structured lipids, and mixtures thereof.

[0436] Embodiment 185. The composition of embodiment 184, wherein R in formula (I) E1 C 17 -C 19 n-alkyl.

[0437] Embodiment 186. The composition of embodiment 184 or embodiment 185, wherein R in formula (I) E2 It is a C1-C2 alkyl group.

[0438] Embodiment 187. The composition of embodiment 183, wherein the compound is edilfosine (EDEL):

[0439]

[0440] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0441] Embodiment 188. A composition comprising a compound of formula (II) or a protonated form or a pharmaceutically acceptable salt thereof; and at least one additional lipid, wherein the at least one additional lipid is selected from the group consisting of ionizable lipids, cationic lipids, additional phospholipids, PEGylated lipids, structured lipids, and mixtures thereof.

[0442] Embodiment 189. The composition of embodiment 188, wherein R in formula (II) M C 14 -C 18 n-alkyl.

[0443] Embodiment 190. The composition of embodiment 188, wherein R in formula (II) M C 15 -C 17 n-alkyl.

[0444] Embodiment 191. The composition of embodiment 188, wherein the compound is miltefosine (MILT):

[0445]

[0446] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0447] Embodiment 192. A composition comprising a compound of formula (III) or a protonated form or a pharmaceutically acceptable salt thereof; and at least one additional lipid, wherein the at least one additional lipid is selected from the group consisting of ionizable lipids, cationic lipids, additional phospholipids, PEGylated lipids, structured lipids, and mixtures thereof.

[0448] Embodiment 193. The composition of embodiment 192, wherein R in formula (III) P C 17 -C 19 n-alkyl.

[0449] Embodiment 194. The composition of embodiment 192, wherein the compound is perifosine (PERIF):

[0450]

[0451] or a protonated form thereof or a pharmaceutically acceptable salt thereof.

[0452] Embodiment 195. The composition of any one of embodiments 183-193, wherein the compound and the at least one additional lipid are part of a lipid nanoparticle (LNP).

[0453] Embodiment 196. The composition of any one of Embodiments 184-195, further comprising an antigen.

[0454] Embodiment 197. The composition of any one of Embodiments 184-196, further comprising dendritic cells.

[0455] Embodiment 198. The composition of any one of Embodiments 184-197, further comprising a TLR agonist.

[0456] Embodiment 199. The composition of any one of Embodiments 184-197, further comprising a TLR7 / 8 agonist.

[0457] Example

[0458] Abbreviations: CDS (cytoplasmic DNA sensor); CLR (C-type lectin receptor); DAMP (damage-associated molecular pattern); DC (dendritic cell); dLN (draining lymph node); EDEL (edilfosine); HOdiA-PC (1-palmitoyl-2-(5-hydroxy-8-oxo-6-octenedioyl)-sn-glycero-3-phosphatidylcholine); HOOA-PC (1-palmitoyl-2-(5-hydroxy-8-oxooct-6-enoyl) -sn-glycero-3-phosphocholine); IFNγ (interferon-γ); IL-1b / IL1-beta / IL-1β (interleukin-1β); KOdiA-PC (1-(palmitoyl)-2-(5-keto-6-octenedioyl)phosphatidylcholine); KOOA-PC (1-palmitoyl-(5-keto-8-oxo-6-octenoyl)-sn-glycero-3-phosphocholine); KP407 (Poloxamer 407); LPC / Lyso PC (lysophosphatidylcholine); Lyso PC(22:0) (1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine); LPS (lipopolysaccharide); moDC (monocyte-derived dendritic cell); MILT (miltefosine); MPLA (monophosphoryl lipid A); NLR (NOD-like receptor); OVA (ovalbumin); oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine); PAMP (pathogen-associated molecular pattern); PBMCs (peripheral blood mononuclear cells); PG PC (1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine); POVPC (1-palmitoyl-2-(5'-oxo-pentanoyl)-sn-glycero-3-phosphocholine); PRR (pathogen recognition receptor); RLR (RIG-I-like receptor); R848 (resiquimod); SFC (spot-forming cell); STING (stimulator of IFN genes); TNFα (tumor necrosis factor-α); TLR (toll-like receptor); and WTL (whole tumor lysate).

[0459] Although the present disclosure has been described in detail by way of illustration and example for the purpose of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be implemented. Therefore, the following examples should not be construed as limiting the scope of the present disclosure, which is defined by the appended claims.

[0460] Example 1: Superactivated activity of edelfosine and miltefosine

[0461] Materials and Methods

[0462] Human monocytes were isolated from Leukopaks purchased from Miltenyi using the StraightFrom Leukopak CD14 microbead kit (Miltenyi). Isolation was performed according to the manufacturer's instructions. The monocytes were then aliquoted and frozen in fetal bovine serum containing 10% dimethyl sulfoxide. For monocyte-derived dendritic cell (moDC) culture studies, monocytes were thawed and cultured in RPMI medium (R10 medium) containing 10% FBS, 50 units / mL penicillin, 50 mg / mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 mM β-mercaptoethanol, 10 mM HEPES, and Gibco MEM non-essential amino acids. In order to differentiate monocytes into moDCs, recombinant human GM-CSF (50 ng / mL) and IL-4 (25 ng / mL) were added to the R10 medium. The cells were cultured with GM-CSF and IL-4 for 6 days and additionally fed on day 3 with R10 medium containing GM-CSF and IL-4.

[0463] After six days of differentiation, moDCs were collected and counted. Cells were plated at 1x10 5 Cells were seeded into 96-well flat-bottom plates at 1 μg / mL per well. Cells were treated with or without 1 μg / mL R848 (final) and with or without super-activated lipids (or vehicle control). In some cases, MCC950 was also treated at a final concentration of 10 μM. R10 medium contained 50 ng / mL GM-CSF and 25 ng / mL IL-4. The final total volume of cells and stimuli was 200 μL / well.

[0464] Two methods are used to prepare super-activated lipids: directly resuspended in R10 culture medium or dissolved in Kolliphor P407 (KP407) micelles. For direct resuspension in R10 culture medium, the lyophilized lipid stock solution is resuspended in R10 by vortexing. Subsequently, more R10 is used to adjust the concentration so that the final concentration added to the well is 41.25 μM. In order to prepare lipids in KP407 micelles, 1-2 mg of 22:0 Lyso PC, edilfosine and miltefosine are first dissolved in 1 mL of ethanol. Then, a magnetic stirring bar is used in a beaker to mix lipids with 4.5 mL of cold 2.78% KP407 at 200 rpm for 1.5 hours to evaporate ethanol. The lost KP407 solution volume is compensated with water, and 10X PBS is added to obtain a 5 mL stock solution containing 2.5% KP407 and 1X PBS. The stock solution was then further diluted with PBS so that the final concentration of lipid added to the culture was 41.25 μM.

[0465] After overnight incubation, cells and culture supernatants were used for downstream readouts. 150 μL of cell supernatant was collected. Viability was measured using the CellTiter-Glo assay (Promega), which measures the ATP content from cells. 50 μL 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 milliseconds. Viability data are set relative to a control condition in which cells were treated with R848 alone. To measure IL-1β secretion, cell culture supernatants were assayed using the Human IL-1β Lumit Kit (Promega). Culture supernatant samples were incubated with enzyme-linked antibodies in 384-well plates for 1 hour, and then luminescent substrate was added. Luminescence of the samples was measured with an integration time of 500 milliseconds. 4-parameter logistic regression analysis was used to determine the IL-1β concentration of the samples by interpolation from the standard curve. Three different human donor samples were studied, and each biological condition was tested in triplicate. The graph shows data from a representative human sample.

[0466] In subsequent studies, the superactivation activity of miltefosine, perifosine, and edilfosine was tested at a lower concentration of 20.6 μM. The lipids were also formulated differently using a slightly higher percentage of KP407. In this case, the lipids were suspended in a 4.44% KP407 solution for 1 hour, and then a 10X PBS concentrate was added. The final concentration of the lipid stock in 4% KP407, 1X PBS was 0.65 mg / mL. The lipid stock was then further diluted in culture medium to reach a target concentration of 20.6 μM in cell culture. Otherwise, human moDCs were incubated overnight with combinations of lipids, R848, and MCC950 to study cellular responses similar to those in the initial studies described above.

[0467] result

[0468] Dendritic cell hyperactivation is a cell state in which these highly specialized antigen-presenting cells are able to secrete IL-1β while maintaining cell viability. This is a special cell state because it was previously believed that IL-1β was only secreted by cells undergoing pyroptotic cell death. By avoiding cell death, hyperactivated DCs can play key functions such as antigen presentation, co-stimulation, and inflammatory cytokine production. In addition, IL-1β has been identified as a key cytokine for initiating T cell responses and forming T cell memory (Ben-Sasson et al., J. Exp Med, 210(3): 491-502, 2013; Ben-Sasson et al., Cold Spring Harbor Symp Quant Biol, 78: 117-124, 2013; and Sarkar et al., J Immunol, 201(12): 3641-3650, 2018). Therefore, hyperactivated DCs play a key role in initiating powerful adaptive immune responses. We sought to determine whether edelfosine and miltefosine could hyperactivate dendritic cells.

[0469] Human monocytes were differentiated into monocyte-derived dendritic cells (moDCs) by culturing in medium containing GM-CSF and IL-4. The moDCs were then collected and plated. The cells were treated with or without R848 and with or without the NLRP3 inhibitor MCC950. To test superactivation, edilfosine and miltefosine were added to the cells using two different preparation methods. First, edilfosine and miltefosine were suspended directly in R10 cell culture medium. Compared to the extremely poorly soluble 22:0Lyso PC, edilfosine and miltefosine were soluble in the culture medium. Despite the difference in solubility, these lipids were comparable in terms of IL-1β secretory activity. Second, edilfosine and miltefosine were prepared using the solvent evaporation method previously used for 22:0lyso PC. The lipids were first dissolved in ethanol and then combined with KP407 in a cold water solution. The solution was warmed to room temperature and the ethanol evaporated, allowing the lipids to be incorporated into the KP407 nanoparticles. Independent of the first preparation, the three lipids formulated in this preparation were compared. After one day of stimulation, supernatants were collected to measure cytokine output. Cell viability was measured using CellTiter-Glo. Under almost all conditions, moDCs remained viable during stimulation ( Figure 1A An exception was the use of edilfosine dissolved directly in the cell culture medium to treat cells. In contrast, edilfosine formulated in KP407 improved cell viability.

[0470] Considering the overall viability of the cell culture conditions, we next quantified the amount of IL-1β secreted by the cells ( Figure 1B). In both formulations, 22:0lyso PC superactivation stimulated IL-1β secretion, while the lipid vehicle control did not, indicating that the assay performed as expected. When resuspended directly in R10 medium, edilfosine induced IL-1β secretion. Given that this treatment condition had poor cell viability, analysis of the second set of data is important, in which edilfosine was formulated in KP407 and had improved viability. In the second edilfosine formulation in KP407, cell viability was improved by approximately 10%, and IL-1βb output was also comparable. These data indicate that edilfosine superactivates moDCs. Miltefosine did not cause cytotoxicity, and stimulation in both formulations also resulted in IL-1β secretion. Treatment of cells with MCC950 inhibited the formation of the NLRP3 inflammasome, thereby preventing the cleavage of pro-IL-1β to its mature form and inhibiting the formation of the gasdermin D pore, through which IL-1β is secreted. Administration of MCC950 reduced the amount of IL-1β detected, confirming that IL-1β secretion is mediated by NLRP3. Finally, the absence of R848 during lipid stimulation resulted in no IL-1β secretion, as expected. Thus, without additional signals from TLR agonists, edelfosine and miltefosine (such as 22:0LPC) were unable to induce IL-1β secretion. The requirement for R848 was confirmed by measuring the NF-κB-dependent cytokines IL-6 and TNFα. These two cytokines were only produced when R848 was added to the cell culture (not shown).

[0471] Together, these data suggest that both edelfosine and miltefosine are hyperactivating lipids and induce moDCs to secrete IL-1β at levels comparable to 22:0 lyso PC when combined with TLR agonists such as R848. Edelfosine and miltefosine also exhibit enhanced solubility in aqueous solution compared to 22:0 Lyso PC.

[0472] At a lipid concentration of 20.6 μM, the activity of moDCs remained above 75% ( Figure 2A ), indicating that this concentration does not result in poor cell viability. Under conditions where cells were incubated with R848, the TLR agonist successfully induced IL-6 production ( Figure 2B When R848 was not included, IL-6 was barely detectable ( Figure 2B As expected, the NLRP3 inhibitor MCC950 did not affect IL-6 production in response to R848 stimulation. Given that R848 is potent and that moDCs respond by producing IL-6, IL-1β was measured to assess DC hyperactivation. When combined with R848, miltefosine, perifosine, and edilfosine induced significantly higher levels of IL-1β secretion than the vehicle control ( Figure 2C, p < 0.0001 by two-way ANOVA). Furthermore, MCC950 treatment of moDCs showed that IL-1β secretion was dependent on NLRP3, as is known to be the case for hyperactivated lipids ( Figure 2C ).

[0473] When evaluating the data overall, we identified optimal lipid concentrations of miltefosine, edilfosine, and perifosine at which human moDCs maintained cell viability while producing inflammatory cytokines such as IL-6 and IL-1β. IL-1β release was dependent on activation of the NLRP3 inflammasome. These data suggest that miltefosine, edilfosine, and perifosine are hyperactivating lipids.

[0474] Example 2: Preparation of lipid nanoparticles containing phospholipid analogs

[0475] This example describes the preparation of lipid nanoparticles (LNPs) loaded with a superactivated phospholipid analog (PLA) compound of Formula (I), Formula (II), or Formula (III) in a microfluidic process.

[0476] Materials and methods

[0477] LNP is used Ignite TM Microfluidics instruments (Precision Nanosystems, Vancouver, BC, Canada) were synthesized using a GenVoy-ILM TM The kit (Precision Nanosystems, Vancouver, BC, Canada) of ionizable lipid mixture is used to produce LNP. The kit without mRNA is used to construct empty LNP vehicle, and the LNP of load super activator is generated by adding the appropriate PLA compound in a mol ratio of 10% of the total LNP content. Also use a separate component (not using a kit) to prepare LNP, to determine whether the PLA compound load in the LNP can be intentionally changed. First, lipid is dissolved in ethanol, then merged according to the molar percentage shown in Table III. The lipid in ethanol is merged with PBS (pH 7.4) with a volume ratio of 1:3. Ignite TM The microfluidics instrument was programmed for a flow rate of 12 mL / min, a starting waste of 0.35 mL, and a final waste of 0.05 mL. LNPs were washed in PBS, pH 7.4, to remove residual ethanol and then concentrated using an Amicon 10K MWCO centrifugal filter at 2000 x g for 30 minutes.

[0478] Table III. LNP Formulations

[0479]

[0480] ^Molar concentration percentages of components of different LNP formulations. LNP 2 and LNP 3 formulations shared the same vehicle (LNP carrier 2).

[0481] The loading of PLA compounds into LNPs was evaluated using HPLC. LNPs in PBS were frozen at -80°C, then lyophilized and stored at -20°C until quantification. LNPs were reconstituted in ethanol and then mixed with water to dissolve the PBS. A seven-point standard curve of PLA compounds was prepared in ethanol and water, and PBS was added to match the sample preparation. Standards and samples were filtered through a 0.45 μM filter before HPLC runs. HPLC quantification was performed using an Agilent 1260 Infinity II HPLC equipped with a 1260 Infinity II evaporative light scattering detector (ELSD). Luna 5 μM NH2 was used with a column temperature of 30°C. Samples were run on a 150 × 4.6 mm LC column (Phenomenex, Torrance, CA). Two eluents were used: A, 100% water; B, 100% acetonitrile. An initial mobile phase consisting of 5% / 95% A / B was used to load the column, with a gradient reaching 24% / 76% A / B after 2.5 minutes. A shallower gradient was applied from 2.5 to 6 minutes, during which A / B slowly reached 25% / 75%. A 3-minute post time was used to return the gradient to the starting conditions before the next sample run. The flow rate was set at 1 mL / min, and the injection volume for both samples and standards was 5 μL. An evaporator temperature of 80°C, a nebulizer temperature of 30°C, and a nitrogen flow rate of 0.9 standard liters / minute were used for the ELSD. Agilent CDS 2.6 software was used for HPLC instrument control, data acquisition, and processing.

[0482] 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 for each sample, each lasting 120 seconds, and the first measurement of each sample was not included in downstream analysis because this was the time required for sample equilibration.

[0483] Example 3: Effects of Miltefosine and Perifosine in vivo

[0484] As described in Example 1, when human monocyte-derived dendritic cells (moDCs) were cultured in vitro in combination with miltefosine or perifosine and R848, the moDCs exhibited characteristics indicative of hyperactivation. To test whether miltefosine and perifosine had the ability to hyperactivate dendritic cells and induce T cell immunity in vivo, a mouse study was conducted. Specifically, the induction of antigen-specific T cell responses was assessed in mice immunized with ovalbumin (OVA) in combination with R848 and two different doses of perifosine or R848 and miltefosine.

[0485] Materials and methods

[0486] Immunization and Study Design. Ten groups of five mice (C57BL / 6) were immunized subcutaneously on days 0, 7, and 14 with ovalbumin (OVA) alone or in combination with the stimulants listed in Table 3-1 below. On day 21, spleens were harvested and processed into single-cell suspensions for detection of antigen-specific T cells by ELISPOT, ELISA, and flow cytometry with SIINFEKL (OVA peptide) tetramer staining. Whole blood was also collected on day 21 for SIINFEKL tetramer staining.

[0487] Table 3-1. Study groups

[0488] Group PAMP DAMP antigen 1 - - - 2 - - OVA 50 μg 3 R848 50μg - OVA 50 μg 4 R848 50μg 100 μg DGP in 4% KP407 OVA 50 μg 5 R848 50μg 100 μg HMC in 4% KP407 OVA 50 μg 6 R848 50μg 100 μg Perifosine in 4% KP407 OVA 50 μg 7 R848 50μg 10 μg perifosine in 4% KP407 OVA 50 μg 8 R848 50μg 10 μg miltefosine in 4% KP407 OVA 50 μg 9 - 100 μg Perifosine in 4% KP407 OVA 50 μg 10 - 10 μg miltefosine in 4% KP407 OVA 50 μg

[0489] DGP is 1-docosyl-sn-glycerol-3-phosphate:

[0490]

[0491] HMC is (S)-1-hydroxy-3-(octadecyloxy)propan-2-ylmethylcarbamate:

[0492]

[0493] Enzyme-linked immunospot (ELISPOT) assay. IFNγ and IL-5 ELISPOT plates (R&D Systems) were blocked for 45 minutes with 200 μL of R10 medium (RPMI-1640 medium supplemented with 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 54 μM β-mercaptoethanol). At the end of the blocking period, the medium was discarded, and 100 μL of R10 medium alone or R10 medium containing 1 μg / mL SARS-CoV-2 spike protein PepTivator (irrelevant peptide control), 1 μg / mL ovalbumin PepTivator, or 2 μg / mL SIINFEKL peptide was added to the corresponding wells. Splenocytes were seeded at 250,000 (IFNγ) or 500,000 (IL-5) cells / well in 100 μL R10 medium and the plates were incubated at 37°C for 20 hours. After incubation, the plates were developed according to the manufacturer's instructions and analyzed using a SmartCount TM Function was read on an S6 Universal M2 ELISPOT plate reader.

[0494] Enzyme-linked immunosorbent assay (ELISA). Splenocytes were seeded at 500,000 cells / well in 100 μL of R10 medium in a 96-well round-bottom tissue culture-treated plate. 100 μL of R10 medium alone or R10 medium containing 1 μg / mL SARS-CoV-2 Spike protein PepTivator (irrelevant peptide control), 1 μg / mL ovalbumin PepTivator, or 2 μg / mL SIINFEKL peptide was added to the corresponding wells, and the plates were incubated at 37°C for 72 hours. After incubation, cell culture supernatants were collected and cytokine secretion was measured using IL-5 or IFNγ ELISA kits from Invitrogen according to the manufacturer's instructions.

[0495] Flow cytometry. The single cell suspension was washed and stained with Live Dead Fixable dye (ThermoFisher) in PBS for 20 minutes at 4°C. The cells were then washed and stained with Fc blocker in FACS buffer (PBS + 0.5% bovine serum albumin) for 10 minutes at 4°C. Subsequently, the cells were washed and stained with PE-H2-KbOva (SIINFEKL peptide) iTag tetramer in FACS buffer at 37°C on a shaker for 1 hour. The cells were then washed and stained in FACS buffer containing the following fluorescent conjugated antibodies: anti-mouse CD3, anti-mouse CD8a, anti-mouse CD4. The cells were incubated at room temperature for 30 minutes and then washed with FACS buffer. Before running the cells, BD liquid counting beads were added to the wells according to the manufacturer's instructions.

[0496] Statistical Analysis. Data were analyzed and graphed using Microsoft Excel and GraphPad Prism software. Data are reported as bar graphs, representing the mean plus standard deviation. Each data point in the bar graph represents a single mouse. Statistical significance was determined using one-way analysis of variance and Dunnett's multiple comparison test.

[0497] result

[0498] When administered with antigen and R848, neither miltefosine nor perifosine induced significant antigen-specific IFNγ secretion. When the injection material was tested on human moDCs in vitro, R848 in combination with miltefosine or perifosine induced strong IL-1β secretion while maintaining cell viability, indicating that these lipids lead to hyperactivation of human cells in vitro (see Figures 1 and 2). To evaluate how these lipids affect the generation of antigen-specific T cells in vivo, mice were subcutaneously immunized with the model antigen ovalbumin (OVA) alone or in combination with R848 and miltefosine or R848 and perifosine. To compare the effects of each agent alone, mice were also immunized with OVA+R848, OVA+miltefosine, and OVA+perifosine. As a positive control, mice were also immunized with OVA+R848+DGP, which has previously been shown to elicit strong antigen-specific T cell responses in mice. As an additional comparison, mice were also immunized with OVA+R848+HMC, an immunization that had not been previously tested in mice but has been shown to superactivate human moDCs in vitro.

[0499] All mice were immunized on day 0 and boosted on days 7 and 14. Spleens were harvested from mice on day 21, and total splenocytes were plated into medium alone (unstimulated), SARS-CoV-2 spike protein PepTivator (irrelevant peptide control), ovalbumin PepTivator, or SIINFEKL peptide for IFNγ ELISPOT assay. Background levels of IFNγ+ spot-forming cells (SFC) were low, averaging 29 SFC / 1x10 when unstimulated or stimulated with an irrelevant peptide control (SpikePepTivator). 6 splenocytes, while when stimulated with OVA PepTivator, the average number of SFC / 1x10 6 spleen cells ( Figure 3A To correct for any background IFNγ secretion, the number of SFCs produced under unstimulated conditions was subtracted from the number of SFCs produced when cells were stimulated with OVA PepTivator or SIINFEKL peptide to obtain OVA-specific IFNγ+ SFCs.

[0500] Splenocytes from mice immunized with OVA alone did not form any IFNγ+ SFCs above background when restimulated with OVA PepTivator, and this number was not significantly increased when mice were immunized with OVA+R848+perifosine (either dose) or OVA+R848+miltefosine ( Figure 3B Similarly, mice immunized with OVA+R848, OVA+perifosine, or OVA+miltefosine did not induce significantly more IFNγ+SFCs than OVA alone. In contrast, OVA+DGP induced significantly higher IFNγ+SFCs than OVA alone (p=0.002), while OVA+R848+HMC tended to produce higher IFNγ+SFCs than OVA alone (p=0.057) ( Figure 3B )

[0501] Splenocytes from mice immunized with OVA alone did not form any IFNγ+ SFCs above background when restimulated with SIINFEKL peptide, and this was not significantly increased when mice were immunized with OVA+R848+perifosine (either dose) or OVA+R848+miltefosine ( Figure 3CSimilarly, mice immunized with OVA+R848, OVA+perifosine, or OVA+miltefosine did not induce significantly more IFNγ+SFCs than OVA alone. In contrast, OVA+R848+DGP and OVA+R848+HMC induced significantly higher numbers of IFNγ+SFCs than OVA alone (p=0.0007, p=0.013, respectively). Figure 3C In response to SIINFEKL peptide restimulation, OVA+R848+DGP was the only group that induced a significantly higher number of IFNγ+SFCs compared to OVA+R848 (p=0.018). Figure 3C ).

[0502] As an additional readout, and to assess the ability of antigen-specific T cells to secrete IFNγ long-term, splenocytes were also inoculated with the stimulators listed above and cultured for 72 hours. IFNγ in the cell culture supernatant was quantified by ELISA. Similar to the ELISPOT assay results, background levels of IFNγ secretion were low, averaging 52 pg / mL when cells were unstimulated or stimulated with an irrelevant peptide (Spike PepTivator) and 1180 pg / mL when stimulated with OVA PepTivator ( Figure 4A To correct for any background IFNγ secretion, the IFNγ concentration under unstimulated conditions was subtracted from the IFNγ concentration when the cells were stimulated with OVA peptide or SIINFEKL peptide to obtain OVA-specific IFNγ secretion. Splenocytes from mice immunized with OVA alone did not induce any IFNγ secretion above background when restimulated with OVA PepTivator or SIINFEKL peptide. Similarly, there was no significant increase in IFNγ secretion after the addition of R848+perifosine (at either dose) or R848+miltifilone ( Figure 4B , Figure 4C ). It is noteworthy that although OVA+R848+HMC did not induce high levels of IFNγ secretion, OVA+R848+DGP induced significantly higher levels of IFNγ secretion compared to OVA alone (p=0.04, p=0.005) ( Figure 4B , Figure 4C ).

[0503] Taken together, these data indicate that perifosine (10 μg / mouse or 100 μg / mouse) and miltefosine (10 μg / mouse) did not induce antigen-specific IFNγ+-secreting T cells when combined with R848 and OVA antigens. In contrast, OVA+R848+DGP induced significantly more IFNγ+-secreting T cells and higher levels of IFNγ secretion compared to OVA alone, while OVA+R848+HMC was less effective in inducing IFNγ+-secreting T cells.

[0504] When administered together with antigen, miltefosine and perifosine induced significant antigen-specific IL-5 secretion. + Helper T cells can be divided into different subsets based on cytokine secretion and function. T helper type 1 (Th1) cells promote pro-inflammatory type 1 immunity characterized by high levels of IFNγ and are primarily responsible for the clearance of intracellular pathogens. T helper type 2 (Th2) cells secrete IL-4, IL-5, and IL-13 and drive the production of antibodies and the elimination of extracellular pathogens. In parallel with the above IFNγ ELISPOT assay, total splenocytes were also inoculated in the IL-5 ELISPOT assay. Splenocytes were cultured for 20 hours with the same stimuli as in the IFNγ ELISPOT assay, with the exception of the SIINFEKL peptide, as it is a CD8+ T cell epitope. Background levels of IL-5+ SFC were low, averaging 3 SFC / 1x10 when unstimulated or stimulated with an irrelevant peptide control (Spike PepTivator). 6 splenocytes, while when stimulated with OVA PepTivator, the average was 102 SFC / 1x10 6 spleen cells ( Figure 5A To correct for any background IL5+ SFC, the number of SFC generated under unstimulated conditions was subtracted from the number of SFC generated when cells were stimulated with OVA PepTivator or SIINFEKL peptide to obtain OVA-specific IFNγ+ SFC.

[0505] When restimulated with OVA PepTivator, splenocytes from mice immunized with OVA alone did not form significantly more IL5+ SFCs compared with mice immunized with PBS, OVA+R848+perifosine (either dose), or OVA+R848+miltefosine ( Figure 5B Similarly, mice immunized with OVA+R848 did not produce higher numbers of IL-5+SFCs compared to OVA alone. However, both OVA+perifosine and OVA+miltefosine induced significantly higher numbers of IL-5+SFCs compared to OVA alone (p<0.0001, p=0.0002) ( Figure 5BAs expected, neither OVA+R848+DGP nor OVA+R848+HMC induced significant changes in IL-5+SFC compared to OVA alone ( Figure 5B ).

[0506] As an additional readout, and to assess the ability of antigen-specific T cells to prolong IL-5 secretion, splenocytes were also inoculated with the same stimulants and cultured for 72 hours. IL-5 was quantified in the cell culture supernatant by ELISA. Similar to the ELISPOT assay results, background levels of IL-5 secretion were low, averaging 0.13 pg / mL when splenocytes were unstimulated or stimulated with an irrelevant peptide (SpikePepTivator), and 66.8 pg / mL when stimulated with OVA peptide ( Figure 6A Mice immunized with OVA alone induced significantly more IL-5 secretion than mice immunized with PBS (p=0.002), and the addition of perifosine further increased IL-5 secretion (p<0.001) ( Figure 6A All other groups of mice that received R848 alone or in combination with lipid DAMPs induced significantly less IL-5 secretion compared to mice immunized with OVA alone (all p=0.02) ( Figure 6B Due to technical failure, OVA+miltefosine was not evaluated in this assay.

[0507] Taken together, these data suggest that perifosine and miltefosine induce significant antigen-specific IL-5 secretion, but this response is attenuated by the addition of R848. Furthermore, no IL-5 secretion was detected in groups immunized with OVA+R848+DGP or OVA+R848+HMC.

[0508] When administered with antigen and R848, neither miltefosine nor perifosine induced significant numbers of SIINFEKL tetramer-stained T cells. In addition to assessing antigen-specific cytokine secretion by ELISPOT assay and ELISA, antigen-specific T cells were quantified in the blood and spleen of immunized mice by flow cytometry with SIINFEKL tetramer staining. When evaluating antigen-specific T cells in the blood, neither OVA+R848+perifosine (at either dose) nor OVA+R848+miltefosine induced higher percentages or counts of SIINFEKL-specific CD8+ T cells compared to OVA immunization alone ( Figure 7A In contrast, OVA+R848+DGP induced significantly higher frequencies (p=0.007) and counts (p=0.04) of SIINFEKL-specific CD8+ T cells in the blood compared with OVA immunization alone ( Figure 7AFurthermore, mice immunized with OVA+R848+HMC tended to have higher frequencies (p=0.07) and significantly higher SIINFEKL-specific CD8+ T cell counts (p=0.03).

[0509] Similarly, when antigen-specific T cells were assessed in the spleen, neither OVA+R848+perifosine (at either dose) nor OVA+R848+miltefosine induced higher frequencies or counts of SIINFEKL-specific CD8+ T cells compared with OVA immunization alone. Figure 7B As observed in the blood, OVA+R848+DGP induced higher frequencies (p=0.02) and counts (p=0.0001) of SIINFEKL-specific CD8+ T cells in the spleen compared to OVA immunization alone ( Figure 7B ). In addition, mice immunized with OVA+R848+HMC had significantly higher counts of SIINFEKL-specific CD8+ T cells in the spleen (p=0.03).

[0510] These data are consistent with the IFNγ results (see Figure 3A and Figure 4A ), and showed that when administered in combination with R848 and OVA antigens, neither perifosine nor miltefosine induced significant SIINFEKL-specific CD8+ T cell frequencies or counts. In contrast, immunization with OVA+R848+DGP or OVA+R848+HMC resulted in significantly more SIINFEKL-specific CD8+ T cells in the blood and spleen of immunized mice.

[0511] in conclusion

[0512] It has been previously determined that perifosine and miltefosine, as well as edilfosine, are superactivators of human monocyte-derived dendritic cells (moDCs) when combined with R848 stimulation (Example 1). Specifically, all three lipids induced IL-1β secretion from live moDCs when combined with R848, similar to the known superactivating lipid (22:0LPC). However, in mice immunized with perifosine or miltefosine in combination with antigen and R848, antigen-specific T cell responses were not elevated. This could be addressed by particulate formulations of perifosine and miltefosine, such as micron-sized particle formulations, which could increase dendritic cell uptake in vivo and potentially induce elevated antigen-specific T cell responses.

Claims

1. A composition comprising an isolated compound of formula (I): where R E1 C 16 -C 20 n-alkyl and R E2 is C1-C4 alkyl, or a protonated form thereof or a pharmaceutically acceptable salt thereof; and TLR7 / 8 agonists.

2. The composition of claim 1, wherein R E1 C 17 -C 19 n-alkyl.

3. The composition of claim 1 or claim 2, wherein R E2 It is a C1-C2 alkyl group.

4. The composition of claim 1, wherein the compound is edelfosine (EDEL): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

5. A composition comprising an isolated compound of formula (II): where R M C 14 -C 20 n-alkyl or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and TLR7 / 8 agonists.

6. The composition of claim 5, wherein R M C 14 -C 18 n-alkyl.

7. The composition of claim 5, wherein R M C 15 -C 17 n-alkyl.

8. The composition of claim 5, wherein the compound is miltefosine (MILT): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

9. A composition comprising an isolated compound of formula (III): where R P C 16 -C 20 n-alkyl or a protonated form thereof or a pharmaceutically acceptable salt thereof; and TLR7 / 8 agonists.

10. The composition of claim 9, wherein R P C 17 -C 19 n-alkyl.

11. The composition of claim 9, wherein the compound is Perifosine (PERIF): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

12. The composition of any one of claims 1-11, further comprising an antigen.

13. The composition of any one of claims 1-12, further comprising dendritic cells.

14. A composition comprising an isolated compound of formula (I): where R E1 C 16 -C 20 n-alkyl and R E2 is C1-C4 alkyl, or a protonated form thereof or a pharmaceutically acceptable salt thereof; and antigen.

15. The composition of claim 14, wherein R E1 C 17 -C 19 n-alkyl.

16. The composition of claim 14 or claim 15, wherein R E2 It is a C1-C2 alkyl group.

17. The composition of claim 14, wherein the compound is edelfosine (EDEL): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

18. A composition comprising an isolated compound of formula (II): where R M C 14 -C 20 n-alkyl or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and antigen.

19. The composition of claim 18, wherein R M C 14 -C 18 n-alkyl.

20. The composition of claim 18, wherein R M C 15 -C 17 n-alkyl.

21. The composition of claim 18, wherein the compound is miltefosine (MILT): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

22. A composition comprising an isolated compound of formula (III): where R P C 16 -C 20 n-alkyl or a protonated form thereof or a pharmaceutically acceptable salt thereof; and antigen.

23. The composition of claim 22, wherein R P C 17 -C 19 n-alkyl.

24. The composition of claim 22, wherein the compound is Perifosine (PERIF): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

25. The composition of any one of claims 14-24, further comprising a TLR7 / 8 agonist.

26. The composition of any one of claims 14-25, further comprising dendritic cells.

27. A composition comprising an isolated compound of formula (I): where R E1 C 16 -C 20 n-alkyl and R E2 is C1-C4 alkyl, or a protonated form thereof or a pharmaceutically acceptable salt thereof; and Dendritic cells.

28. The composition of claim 27, wherein R E1 C 17 -C 19 n-alkyl.

29. The composition of claim 27 or claim 15, wherein R E2 It is a C1-C2 alkyl group.

30. The composition of claim 27, wherein the compound is edelfosine (EDEL): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

31. A composition comprising an isolated compound of formula (II): where R M C 14 -C 20 n-alkyl or a protonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and Dendritic cells.

32. The composition of claim 31, wherein R M C 14 -C 18 n-alkyl.

33. The composition of claim 31, wherein R M C 15 -C 17 n-alkyl.

34. The composition of claim 31, wherein the compound is miltefosine (MILT): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

35. A composition comprising an isolated compound of formula (III): where R P C 16 -C 20 n-alkyl or a protonated form thereof or a pharmaceutically acceptable salt thereof; and Dendritic cells.

36. The composition of claim 35, wherein R P C 17 -C 19 n-alkyl.

37. The composition of claim 35, wherein the compound is Perifosine (PERIF): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

38. The composition of any one of claims 27-37, further comprising a TLR7 / 8 agonist.

39. The composition of any one of claims 27-38, further comprising an antigen.

40. The composition of any one of claims 1-39, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 Daltons or less.

41. The composition of claim 40, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

42. The composition of claim 41, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

43. The composition of claim 40 or claim 41, wherein the TLR7 / 8 agonist does not inhibit NLR family pyrin-containing domain 3 (NLRP3).

44. The composition of any one of claims 1-43, wherein the antigen is present in a biological sample obtained from an individual.

45. The composition of claim 44, wherein the biological sample comprises a biopsy.

46. ​​The composition of claim 44, wherein the biological sample comprises cells.

47. The composition of claim 44, wherein the biological sample does not comprise cells.

48. The composition of claim 44, wherein the biological sample comprises pus from an abscess.

49. The composition of any one of claims 1-48, wherein the antigen comprises a protein antigen.

50. The composition of any one of claims 1-49, wherein the antigen comprises a tumor antigen.

51. The composition of claim 50, wherein the tumor antigen comprises a synthetic or recombinant neoantigen.

52. The composition of claim 50 or claim 51, wherein the tumor antigen comprises a tumor cell lysate.

53. The composition of any one of claims 1-49, wherein the antigen comprises a microbial antigen, and the microbial antigen comprises one or more of a viral antigen, a bacterial antigen, a protozoan antigen, and a fungal antigen.

54. The composition of claim 53, wherein the microbial antigen comprises a purified or recombinant surface protein.

55. The composition of claim 53, wherein the microbial antigen comprises an inactivated whole virus.

56. The composition of any one of claims 1-55, wherein the composition does not comprise liposomes.

57. The composition of any one of claims 1-56, wherein the composition does not comprise LPS or MPLA.

58. The composition of any one of claims 1-57, wherein the composition does not comprise oxPAPC or oxPAPC species, optionally wherein the composition does not comprise HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC and / or PGPC.

59. The composition of claim 58, wherein the composition does not comprise lysophosphatidylcholine (LPC), optionally wherein the composition does not comprise 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

60. The composition of any one of claims 1-59, further comprising an adjuvant, wherein the adjuvant comprises an aluminum salt adjuvant, a squalene-in-water emulsion, a saponin, or a combination thereof.

61. A pharmaceutical formulation comprising the composition of any one of claims 1-60 and a pharmaceutically acceptable excipient.

62. A method for producing hyperactivated dendritic cells, the method comprising contacting the dendritic cells with a composition comprising an effective amount of an isolated compound of formula (I) or a protonated form or a pharmaceutically acceptable salt thereof and a TLR7 / 8 agonist for producing hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing pyroptosis.

63. The method of claim 62, wherein R in formula (I) E1 C 17 -C 19 n-alkyl.

64. The method of claim 62 or claim 63, wherein R in formula (I) E2 It is a C1-C2 alkyl group.

65. The method of claim 62, wherein the compound of formula (I) is edelfosine (EDEL): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

66. A method for producing hyperactivated dendritic cells, the method comprising contacting the dendritic cells with a composition comprising an effective amount of an isolated compound of formula (II) or a protonated form or a pharmaceutically acceptable salt thereof and a TLR7 / 8 agonist for producing hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing pyroptosis.

67. The method of claim 66, wherein R in formula (II) M C 14 -C 18 n-alkyl.

68. The method of claim 66, wherein R in formula (II) M C 15 -C 17 n-alkyl.

69. The method of claim 66, wherein the compound of formula (II) is miltefosine (MILT): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

70. A method for producing hyperactivated dendritic cells, the method comprising contacting the dendritic cells with a composition comprising an effective amount of an isolated compound of formula (III) or a protonated form or a pharmaceutically acceptable salt thereof and a TLR7 / 8 agonist for producing hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing pyroptosis.

71. The method of claim 70, wherein R of formula (III) P C 17 -C 19 n-alkyl.

72. The method of claim 70, wherein the compound of formula (III) is perifosine (PERIF): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

73. The method of any one of claims 62-72, wherein the dendritic cells are contacted ex vivo with the composition of any one of claims 1-60 or the formulation of claim 61.

74. The method of any one of claims 62-72, wherein the dendritic cells are contacted with the formulation of claim 61 in vivo.

75. A pharmaceutical preparation comprising at least 10 3 , 10 4 , 10 5 or 10 6 A hyperactivated dendritic cell produced by the method of claim 73, and a pharmaceutically acceptable excipient.

76. A method of stimulating an immune response to an antigen, comprising administering to an individual in need thereof an effective amount of the formulation of claim 73 to stimulate an immune response to the antigen.

77. A method of treating cancer comprising administering to a subject in need thereof an effective amount of the formulation of claim 61 to treat the cancer.

78. A method for inhibiting abnormal cell proliferation, comprising administering to an individual in need thereof an effective amount of the formulation of claim 61 to inhibit abnormal cell proliferation.

79. A method of treating an infectious disease comprising administering to an individual in need thereof an effective amount of the formulation of claim 61 to treat the infectious disease.

80. Use of the formulation of claim 61 for inducing an immune response against an antigen in an individual in need thereof.

81. Use of the formulation of claim 61 for inducing an anti-tumor immune response in an individual in need thereof, wherein the individual is or was a tumor carrier.

82. Use of the formulation of claim 61 for inducing an anti-microbial immune response in an individual in need thereof, wherein the individual is infected with or has not been exposed to the microorganism.

83. The composition, formulation, method or use of any one of claims 44-82, wherein the individual is a mammalian subject.

84. The composition, formulation, method or use of any one of claims 44-82, wherein the individual is a human subject.

85. A method of preparing an immunogenic composition, the method comprising: a) depleting a cell suspension prepared from a tumor to obtain a suspension enriched in tumor cells; b) lysing cells from the tumor cell-rich suspension to obtain a tumor cell lysate; and c) contacting the tumor cell lysate with the isolated compound of formula (I) or a protonated form or a pharmaceutically acceptable salt thereof and a toll-like receptor 7 / 8 (TLR7 / 8) agonist to obtain the immunogenic composition.

86. The method of claim 85, wherein R in formula (I) E1 C 17 -C 19 n-alkyl.

87. The method of claim 85 or claim 86, wherein R in formula (I) E2 It is a C1-C2 alkyl group.

88. The method of claim 85, wherein the compound of formula (I) is edelfosine (EDEL): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

89. A method of preparing an immunogenic composition, the method comprising: a) depleting a cell suspension prepared from a tumor for leukocytes to obtain a tumor cell-enriched suspension; b) lysing cells from the tumor cell-rich suspension to obtain a tumor cell lysate; and c) contacting the tumor cell lysate with the isolated compound of formula (II) or a protonated form or a pharmaceutically acceptable salt thereof and a toll-like receptor 7 / 8 (TLR7 / 8) agonist to obtain the immunogenic composition.

90. The method of claim 89, wherein R in formula (II) M C 14 -C 18 n-alkyl.

91. The method of claim 89, wherein R in formula (II) M C 15 -C 17 n-alkyl.

92. The method of claim 89, wherein the compound of formula (II) is miltefosine (MILT): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

93. A method of preparing an immunogenic composition, the method comprising: a) depleting a cell suspension prepared from a tumor for leukocytes to obtain a tumor cell-enriched suspension; b) lysing cells from the tumor cell-rich suspension to obtain a tumor cell lysate; and c) contacting the tumor cell lysate with the isolated compound of formula (III) or a protonated form or a pharmaceutically acceptable salt thereof and a toll-like receptor 7 / 8 (TLR7 / 8) agonist to obtain the immunogenic composition.

94. The method of claim 93, wherein R of formula (III) P C 17 -C 19 n-alkyl.

95. The method of claim 93, wherein the compound of formula (III) is perifosine (PERIF): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

96. The method of any one of claims 85-95, wherein in step a) the leukocytes are depleted by negative selection using an anti-CD45 antibody.

97. The method of any one of claims 85-96, wherein in step b) the cells are lysed by one or more freeze-thaw cycles.

98. The method of any one of claims 85-97, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 Daltons or less.

99. The method of claim 98, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

100. The method of claim 99, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

101. The method of claim 98 or claim 99, wherein the TLR7 / 8 agonist does not inhibit NLR family pyrin-containing domain 3 (NLRP3).

102. The method of any one of claims 85-101, further comprising obtaining a sample from a tumor of a mammalian subject suffering from cancer prior to step a) and preparing a cell suspension from the sample.

103. An immunogenic composition prepared by the method of any one of claims 85-102.

104. A method of eliciting an anti-cancer immune response, the method comprising: Administering an effective amount of the immunogenic composition of claim 103 to a mammalian subject having cancer.

105. The method of claim 104, wherein the anti-cancer immune response comprises a cellular immune response.

106. The method of claim 105, wherein the anti-cancer immune response comprises cancer antigen-induced IL-1β secretion and / or activation of CD8+ T lymphocytes.

107. The method of any one of claims 104-106, wherein the cancer is a non-hematological cancer.

108. The method of claim 107, wherein the non-hematological cancer is a carcinoma, a sarcoma, or a melanoma.

109. The method of any one of claims 104-106, wherein the cancer is lymphoma.

110. A method of treating cancer, the method comprising: a) preparing an immunogenic composition comprising a tumor cell lysate, a compound of formula (I) or a protonated form thereof or a pharmaceutically acceptable salt thereof, and a toll-like receptor 7 / 8 (TLR7 / 8) agonist, wherein the tumor cell lysate is or has been prepared from a tumor sample obtained from a mammalian subject suffering from cancer; and b) administering an effective amount of the immunogenic composition to the subject.

111. The method of claim 110, wherein R in formula (I) E1 C 17 -C 19 n-alkyl.

112. The method of claim 110 or claim 111, wherein R in formula (I) E2 It is a C1-C2 alkyl group.

113. The method of claim 110, wherein the compound of formula (I) is edelfosine (EDEL): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

114. A method of treating cancer, the method comprising: a) preparing an immunogenic composition comprising a tumor cell lysate, a compound of formula (II) or a protonated form thereof or a pharmaceutically acceptable salt thereof, and a toll-like receptor 7 / 8 (TLR7 / 8) agonist, wherein the tumor cell lysate is or has been prepared from a tumor sample obtained from a mammalian subject suffering from cancer; and b) administering an effective amount of the immunogenic composition to the subject.

115. The method of claim 114, wherein R in formula (II) M C 14 -C 18 n-alkyl.

116. The method of claim 114, wherein R in formula (II) M C 15 -C 17 n-alkyl.

117. The method of claim 114, wherein the compound of formula (II) is miltefosine (MILT): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

118. A method of treating cancer, the method comprising: a) preparing an immunogenic composition comprising a tumor cell lysate, a compound of formula (III) or a protonated form thereof or a pharmaceutically acceptable salt thereof, and a toll-like receptor 7 / 8 (TLR7 / 8) agonist, wherein the tumor cell lysate is or has been prepared from a tumor sample obtained from a mammalian subject suffering from cancer; and b) administering an effective amount of the immunogenic composition to the subject.

119. The method of claim 118, wherein R of formula (III) P C 17 -C 19 n-alkyl.

120. The method of claim 118, wherein the compound of formula (III) is perifosine (PERIF): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

121. The method of any one of claims 104-120, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 Daltons or less.

122. The method of claim 121, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

123. The method of claim 122, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

124. The method of any one of claims 110-123, further comprising administering to the subject an effective amount of an additional therapeutic agent.

125. The method of claim 124, wherein the additional therapeutic agent comprises one or more of the group consisting of an immune checkpoint inhibitor, an anti-tumor agent, and radiation therapy.

126. A composition comprising a compound of formula (I) or a protonated form or a pharmaceutically acceptable salt thereof, and a pathogen recognition receptor (PRR) agonist.

127. The composition of claim 126, wherein R in formula (I) E1 C 17 -C 19 n-alkyl.

128. The composition of claim 126 or claim 127, wherein R in formula (I) E2 It is a C1-C2 alkyl group.

129. The composition of claim 126, wherein the compound is edelfosine (EDEL): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

130. A composition comprising a compound of formula (II) or a protonated form or a pharmaceutically acceptable salt thereof, and a pathogen recognition receptor (PRR) agonist.

131. The composition of claim 130, wherein R in formula (II) M C 14 -C 18 n-alkyl.

132. The composition of claim 130, wherein R in formula (II) M C 15 -C 17 n-alkyl.

133. The composition of claim 130, wherein the compound is miltefosine (MILT): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

134. A composition comprising a compound of formula (III) or a protonated form or a pharmaceutically acceptable salt thereof, and a pathogen recognition receptor (PRR) agonist.

135. The composition of claim 134, wherein R in formula (III) P C 17 -C 19 n-alkyl.

136. The composition of claim 134, wherein the compound is Perifosine (PERIF): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

137. The composition of any of claims 126-136, 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).

138. The composition of any of claims 126-136, wherein the PRR agonist is an agonist of the cytoplasmic DNA sensor (CDS) or stimulator of IFN genes (STING).

139. The composition of any of claims 126-136, wherein the PRR agonist comprises one or more of R848, TL8-506, LPS, Pam2CSK4, and ODN 2336.

140. The composition of any one of claims 126-139, further comprising an antigen.

141. The composition of any one of claims 126-140, further comprising dendritic cells.

142. A pharmaceutical formulation comprising the composition of any one of claims 126-141 and a pharmaceutically acceptable excipient.

143. A pharmaceutical formulation comprising an isolated compound of formula (I) or a protonated form or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

144. The composition of claim 143, wherein R in formula (I) E1 C 17 -C 19 n-alkyl.

145. The composition of claim 143 or claim 144, wherein R in formula (I) E2 It is a C1-C2 alkyl group.

146. The composition of claim 143, wherein the compound is edelfosine (EDEL): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

147. A pharmaceutical formulation comprising an isolated compound of formula (II) or a protonated form or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

148. The formulation of claim 147, wherein R in formula (II) M C 14 -C 18 n-alkyl.

149. The formulation of claim 147, wherein R in formula (II) M C 15 -C 17 n-alkyl.

150. The formulation of claim 147, wherein the compound is miltefosine (MILT): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

151. A pharmaceutical formulation comprising an isolated compound of formula (III) or a protonated form or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

152. The composition of claim 151, wherein R in formula (III) P C 17 -C 19 n-alkyl.

153. The composition of claim 151, wherein the compound is Perifosine (PERIF): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

154. A composition for hyperactivating human dendritic cells, comprising a compound of formula (I) or a protonated form or a pharmaceutically acceptable salt thereof, and a pathogen recognition receptor (PRR) agonist, wherein the composition is effective in achieving a higher level of dendritic cell hyperactivation than a comparative composition comprising PGPC instead of the compound of formula (I).

155. The composition of claim 154, wherein R in formula (I) E1 C 17 -C 19 n-alkyl.

156. The composition of claim 154 or claim 155, wherein R in formula (I) E2 It is a C1-C2 alkyl group.

157. The composition of claim 154, wherein the compound is edelfosine (EDEL): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

158. A composition for hyperactivating human dendritic cells, comprising a compound of formula (II) or a protonated form or a pharmaceutically acceptable salt thereof, and a pathogen recognition receptor (PRR) agonist, wherein the composition is effective in achieving a higher level of dendritic cell hyperactivation than a comparative composition comprising PGPC instead of the compound of formula (II).

159. The composition of claim 158, wherein R in formula (II) M C 14 -C 18 n-alkyl.

160. The composition of claim 158, wherein R in formula (II) M C 15 -C 17 n-alkyl.

161. The composition of claim 158, wherein the compound is miltefosine (MILT): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

162. A composition for superactivating human dendritic cells, comprising a compound of formula (III) or a protonated form or a pharmaceutically acceptable salt thereof, and a pathogen recognition receptor (PRR) agonist, wherein the composition is effective in achieving a higher level of dendritic cell superactivation than a comparative composition comprising PGPC instead of the compound of formula (III).

163. The composition of claim 162, wherein R in formula (III) P C 17 -C 19 n-alkyl.

164. The composition of claim 162, wherein the compound is Perifosine (PERIF): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

165. The composition of any of claims 154-164, wherein the higher level of dendritic cell hyperactivation comprises inducing in vitro secretion of IL-1β from human dendritic cells when contacted with a composition comprising the compound and a PRR agonist at a level that is at least 2, 3, or 4 times higher than when contacted with a comparative composition comprising PGPC and a PRR agonist, wherein the PRR agonist is LPS.

166. The composition of claim 165, wherein the concentration of the compound and the concentration of the PGPC are the same concentration within the range of about 10 μM to about 80 μM, and the LPS is present in the composition and the comparative composition at a concentration of 1 g / ml.

167. The composition of claim 165, wherein the higher level of dendritic cell hyperactivation comprises a lipid activity index for IL-1β secretion from human dendritic cells for a composition comprising the compound and the PRR agonist that is at least 4, 5, or 6 times greater in activity units than the lipid activity index of a comparative composition comprising the PGPC and the PRR agonist.

168. The composition, formulation, method or use of any one of claims 44-82, wherein the individual is a canine subject.

169. The composition, formulation, method or use of any one of claims 44-82 or 102-167, wherein the mammalian subject is a human patient.

170. The composition, formulation, method or use of any one of claims 44-82 or 102-167, wherein the mammalian subject is a non-human patient.

171. The composition, formulation, method or use of any one of claims 44-82 or 102-167, wherein the mammalian subject is a canine patient.

172. The composition, formulation, method or use of any one of claims 1-167 or 169, wherein the dendritic cells are human dendritic cells.

173. The composition, formulation, method or use of any one of claims 1-168 or 171, wherein the dendritic cells are canine dendritic cells.

174. The composition, method or use of claim 172 or claim 173, wherein the dendritic cells are present in a composition comprising peripheral blood mononuclear cells (PBMCs).

175. The composition, method or use of any one of claims 62-84, 168 or 169 or claim 91, wherein the hyperactivated dendritic cells secrete one or both of IFNγ and TNFα.

176. The composition, formulation, method or use of any one of claims 1-175, comprising a surfactant.

177. The composition, formulation, method or use of claim 176, wherein the surfactant comprises a nonionic surfactant.

178. The composition, formulation, method or use of claim 177, wherein the nonionic surfactant comprises an ethylene oxide-propylene oxide copolymer.

179. The composition, formulation, method or use of claim 177, wherein the nonionic surfactant comprises one or more of poloxamer 407, poloxamer 188 and P123.

180. The composition, formulation, method or use of claim 177, wherein the nonionic surfactant comprises poloxamer 407.

181. The composition, formulation, method or use of any one of claims 177 to 180, wherein i) the PLA is dissolved in alcohol to form a PLA alcohol solution; ii) the PLA alcohol solution is mixed with the nonionic surfactant to form a mixture; and iii) the alcohol is evaporated from the mixture to form particles comprising the PLA and the nonionic surfactant.

182. The composition, formulation, method or use of any of claims 177-181, wherein the nonionic surfactant is present in an amount of about 2.5% to 25% (w / w), optionally about 5% to 20% (w / w), optionally about 15% (w / w).

183. The composition, formulation, method or use of any one of claims 177-182, wherein the PLA and nonionic surfactant are present in particles having a diameter of about 1000 to 2000 nanometers, optionally about 1500 nanometers in diameter.

184. A composition comprising a compound of formula (I) or a protonated form or a pharmaceutically acceptable salt thereof; and at least one additional lipid, wherein the at least one additional lipid is selected from the group consisting of ionizable lipids, cationic lipids, additional phospholipids, PEGylated lipids, structured lipids, and mixtures thereof.

185. The composition of claim 184, wherein R in formula (I) E1 C 17 -C 19 n-alkyl.

186. The composition of claim 184 or claim 185, wherein R in formula (I) E2 It is a C1-C2 alkyl group.

187. The composition of claim 183, wherein the compound is edelfosine (EDEL): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

188. A composition comprising a compound of formula (II) or a protonated form or a pharmaceutically acceptable salt thereof; and at least one additional lipid, wherein the at least one additional lipid is selected from the group consisting of ionizable lipids, cationic lipids, additional phospholipids, PEGylated lipids, structured lipids, and mixtures thereof.

189. The composition of claim 188, wherein R in formula (II) M C 14 -C 18 n-alkyl.

190. The composition of claim 188, wherein R in formula (II) M C 15 -C 17 n-alkyl.

191. The composition of claim 188, wherein the compound is miltefosine (MILT): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

192. A composition comprising a compound of formula (III) or a protonated form or a pharmaceutically acceptable salt thereof; and at least one additional lipid, wherein the at least one additional lipid is selected from the group consisting of ionizable lipids, cationic lipids, additional phospholipids, PEGylated lipids, structured lipids, and mixtures thereof.

193. The composition of claim 192, wherein R in formula (III) P C 17 -C 19 n-alkyl.

194. The composition of claim 192, wherein the compound is Perifosine (PERIF): or a protonated form thereof or a pharmaceutically acceptable salt thereof.

195. The composition of any one of claims 183-193, wherein the compound and the at least one additional lipid are part of a lipid nanoparticle (LNP).

196. The composition of any one of claims 184-195, further comprising an antigen.

197. The composition of any one of claims 184-196, further comprising dendritic cells.

198. The composition of any one of claims 184-197, further comprising a TLR agonist.

199. The composition of any one of claims 184-197, further comprising a TLR7 / 8 agonist.

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