Lipids for hyperactivation of mammalian dendritic cells

Abstract

The present disclosure relates to compounds of the various formulas disclosed herein, including Formula (I) and Formula (II), and uses thereof in hyperactivating mammalian dendritic cells, such as human dendritic cells or canine dendritic cells. The present disclosure also relates to compositions comprising a compound of the various formulas disclosed herein, including Formula (I) and Formula (II), and one or more of a pathogen recognition receptor agonist, an antigen, and mammalian dendritic cells, as well as methods for production and use of the compositions.

Description

Attorney Docket No.: 16553-20010.40 LIPIDS FOR HYPERACTIVATION OF MAMMALIAN DENDRITIC CELLS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority benefit of U.S. Provisional Patent Application No. 63 / 634,738, filed April 16, 2024. The contents of that application are hereby incorporated by reference herein in their entirety. FIELD

[0002] The present disclosure relates to lipid compounds, including phospholipid compounds, and uses thereof in hyperactivating mammalian dendritic cells, such as human dendritic cells or canine dendritic cells. The present disclosure also relates to compositions comprising a lipid compound, such as a phospholipid compound, and one or more of a pathogen recognition receptor agonist, an antigen, and human or canine dendritic cells, as well as methods for production and use of the compositions. BACKGROUND

[0003] Typically, dendritic cell (DC) maturation by vaccine adjuvants such as Toll-like receptor agonists does not lead to IL-1beta secretion. In circumstances such as inflammasome activation, IL-1beta secretion does occur but at the cost of DC death by a lytic process of cell death termed pyroptosis (Evavold et al., J Mol Biol, 430(2):217-237, 2018). However, when DCs are matured using the pathogen-associated molecular pattern (PAMP)- containing molecule, lipopolysaccharide (LPS) and the damage-associated molecular pattern (DAMP)-containing molecule such as PGPC (1-palmitoyl-2-glutaryl-sn-glycero-3- phosphocholine) they produce and secrete IL-1beta without pyroptosing, characterizing these viable DCs as hyperactive (Zanoni et al., Science, 352(6290):1232-1236, 2016). In fact, in mouse models, hyperactivated DCs have demonstrated an improved ability to induce an immune response compared to cells activated using LPS alone (Zhivaki et al., Cell Rep, 33(7):108381, 2020). However, little is known about stimuli effective for hyperactivation of human DCs.

[0004] As such, the identification of PAMPs and DAMPs suitable for hyperactivation of human DCs is needed in the art. Additionally, the identification of alternatives to the use of LPS and PGPC for hyperactivation of mammalian DCs is desirable. In particular, while LPS 1sf-6685008Attorney Docket No.: 16553-20010.40 (endotoxin) is a potent PAMP, it is contraindicated for use in humans as it can lead to septic shock. BRIEF SUMMARY

[0005] The current application provides lipid compounds and uses thereof in hyperactivating mammalian dendritic cells.

[0006] Provided herein are compounds of Formula (I):

[0008] A1is independently O, S, -NRA-, or -CH2-;

[0009] A2and A3are independently O, S, -NRA-, -CH2-, -NRA-C(=G)-O-, -NRA-C(=G)- NRA-,-O-C(=G)-NRA-, -O-C(=G)-O-, -O-C(=S)-O-, -S- or -S-S- ;

[0010] wherein each RAis independently H, C1-C4alkyl, benzyl, C2-C4alkenyl, C2-C4alkynyl, C3-C10cycloalkyl, C3-C10cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA;

[0011] R2is H, C1-C4alkyl, , C2-C4alkenyl, C2-C4alkynyl, C3-C10cycloalkyl, C3-C10cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA;;

[0012] R3is C10-C30n-alkyl;

[0013] where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N- (CH2)P-, where RNis -CH3or -CH2CH3and p is an integer from 1 to 6 inclusive; 2sf-6685008Attorney Docket No.: 16553-20010.40

[0014] each R5is independently C1-C4alkyl;

[0015] R6is O or S;

[0016] or a protonated or deprotonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and all stereoisomers thereof;

[0017] wherein

[0018] i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or

[0019] ii) G is S or NH; or

[0020] iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or - CH2CH3 and p is an integer from 1 to 6 inclusive; or

[0021] iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or

[0022] v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or

[0023] vi) R6is S.

[0024] The present disclosure provides compounds of Formula (I), or a protonated or deprotonated form thereof where possible, or a pharmaceutically acceptable salt thereof. In some embodiments, the compounds of Formula (I), or a protonated or deprotonated form thereof where possible, or a pharmaceutically acceptable salt thereof, are isolated.

[0025] The present disclosure also provides compositions comprising a compound of Formula (I), or a protonated or deprotonated form thereof where possible, or a pharmaceutically acceptable salt thereof; wherein the composition further comprises one or more of a TLR agonist, an antigen, and / or dendritic cells. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist. In some embodiments, the compound of Formula (I), or a protonated or deprotonated form thereof where possible, or a pharmaceutically acceptable salt thereof is isolated.

[0026] The present disclosure also provides compositions comprising a compound of Formula (I), or a protonated or deprotonated form thereof where possible, or a pharmaceutically acceptable salt thereof; wherein the composition further comprises a TLR agonist. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist. In some embodiments, the composition further comprises an antigen. In some embodiments, the composition further comprises dendritic cells. In some embodiments, the composition further comprises an antigen and dendritic cells. In some embodiments, the compound of Formula (I) is isolated. 3sf-6685008Attorney Docket No.: 16553-20010.40

[0027] The present disclosure also provides compositions comprising a compound of Formula (I), or a protonated or deprotonated form thereof where possible, or a pharmaceutically acceptable salt thereof; wherein the composition further comprises an antigen. In some embodiments, the composition further comprises a TLR agonist. In some embodiments, the composition further comprises dendritic cells. In some embodiments, the composition further comprises a TLR agonist and dendritic cells. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist. In some embodiments, the compound of Formula (I) is isolated.

[0028] The present disclosure also provides compositions comprising a compound of Formula (I), or a protonated or deprotonated form thereof where possible, or a pharmaceutically acceptable salt thereof; wherein the composition further comprises dendritic cells. In some embodiments, the composition further comprises an antigen. In some embodiments, the composition further comprises a TLR agonist. In some embodiments, the composition further comprises an antigen and a TLR agonist. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist. In some embodiments, the compound of Formula (I) is isolated.

[0029] In some embodiments of the preceding aspects, the antigen is present in a biological sample obtained from an individual. In some embodiments, the biological sample comprises biopsy tissue. In some embodiments, the biological sample comprises cells. In other embodiments, the biological sample does not comprise cells. In some embodiments, the biological sample comprises pus from an abscess. In some embodiments, the antigen comprises a proteinaceous 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.

[0030] 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 a species of 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 4sf-6685008Attorney Docket No.: 16553-20010.40 (LPC). In some embodiments, the composition does not comprise 1-behenoyl-2-hydroxy-sn- glycero-3-phosphocholine [LPC(22:0)]. In some embodiments, the composition does not comprise isolated mRNA. In some embodiments, the composition does not comprise a surfactant (e.g., a poloxamer). In some embodiments, the composition does not comprise Poloxamer 407 (KP407), Poloxamer 188 (KP188), and / or Pluronic P123 (P123).

[0031] 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 combinations thereof.

[0032] In some embodiments, the present disclosure provides a pharmaceutical formulation comprising the composition of any of the preceding aspects and a pharmaceutically acceptable excipient.

[0033] In additional aspects, the present disclosure provides a method for production of hyperactivated dendritic cells, the method comprising contacting the dendritic cells with a composition comprising an effective amount of a compound of Formula (I) and a TLR agonist for production of hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1beta without undergoing pyroptosis. In some embodiments, the dendritic cells are contacted ex vivo with the composition or pharmaceutical formulation of any one of the preceding embodiments. In other embodiments, the dendritic cells are contacted in vivo with the pharmaceutical formulation comprising the composition of any one of the preceding embodiments. In some aspects, the present disclosure provides a pharmaceutical formulation comprising a plurality of the hyperactivated dendritic cells produced by the preceding embodiments, and a pharmaceutically acceptable excipient. In some embodiments, the plurality comprises at least 103, 104, 105, 106, 107or 108hyperactivated DCs. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist.

[0034] In some embodiments of the preceding aspects, the TLR agonist is a small molecule with a molecule weight of 900 daltons or less. In some embodiments of the preceding aspects, the TLR7 / 8 agonist is a small molecule with a molecule weight of 900 daltons or less. In some embodiments, the TLR7 / 8 agonist comprises an imidazoquinoline compound. In some embodiments, the TLR7 / 8 agonist comprises resiquimod (R848).

[0035] The present disclosure further provides compositions for hyperactivation of human dendritic cells, comprising a compound of Formula (I) and a pathogen recognition receptor (PRR) agonist, wherein the composition is effective for achieving a higher level of 5sf-6685008Attorney Docket No.: 16553-20010.40 dendritic cell hyperactivation than a comparator composition comprising a comparator compound in place of the compound of Formula (I). In some embodiments, the hyperactivation occurs in vitro or ex vivo. In other embodiments, the hyperactivation occurs in vivo. In some embodiments, the comparator compound is 1-palmitoyl-2-glutaryl-sn- glycero-3-phosphocholine (PGPC). In some embodiments, the comparator compound is 1- behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

[0036] A compound of Formula (I) can be administered in the form of micelles.

[0037] A compound of Formula (I) can be administered in the form of lipid nanoparticles (LNPs). In some embodiments of the present disclosure, the LNPs of the compositions are enriched in particles with lipid bilayers (liposomes) relative to particles with a single lipid layer (micelle). Specifically, in some embodiments, the LNPs comprise liposomes, and little to substantially no micelles. In some embodiments, the LNPs comprise liposomes, and less than about 10% of the lipid particles present are micelles. In some embodiments, the LNPs comprise liposomes, and less than about 5% of the lipid particles present are micelles. In some embodiments, the LNPs comprise liposomes, and less than about 1% of the lipid particles present are micelles.

[0038] In some embodiments, the present disclosure provides lipid nanoparticles comprising a compound of Formula (I) and at least one further lipid, and uses thereof in hyperactivating mammalian dendritic cells. The present disclosure also relates to compositions comprising a compound of Formula (I) and at least one further lipid, wherein the compositions further comprise one or more of a pathogen recognition receptor agonist, an antigen, and mammalian dendritic cells, as well as methods for production and use of the compositions.

[0039] 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 combinations thereof.

[0040] In some embodiments, the present disclosure provides a pharmaceutical formulation comprising the composition of any of the preceding aspects and a pharmaceutically acceptable excipient. In some embodiments, the formulation comprises a surfactant (e.g., a poloxamer). In some embodiments, the formulation comprises Poloxamer 407 (KP407), Poloxamer 188 (KP188), and / or Pluronic P123 (P123).

[0041] In some embodiments, the present disclosure provides a pharmaceutical formulation comprising the composition of any of the preceding aspects and a 6sf-6685008Attorney Docket No.: 16553-20010.40 pharmaceutically acceptable excipient. In some embodiments, the formulation does not comprise a surfactant (e.g., a poloxamer). In some embodiments, the formulation does not comprise Poloxamer 407 (KP407), Poloxamer 188 (KP188), and / or Pluronic P123 (P123).

[0042] In other aspects, the present disclosure provides a method for production of hyperactivated dendritic cells, the method comprising contacting the dendritic cells with an effective amount of the composition or pharmaceutical formulation of any of the preceding embodiments for production of hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1beta without undergoing pyroptosis, and the compound of Formula (I) and the at least one further lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one further lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, a further phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof. In some embodiments, the dendritic cells are contacted ex vivo with the composition or pharmaceutical formulation of any one of the preceding embodiments. In other embodiments, the dendritic cells are contacted in vivo with the pharmaceutical formulation comprising the composition of any one of the preceding embodiments. In some aspects, the present disclosure provides a pharmaceutical formulation comprising a plurality of the hyperactivated dendritic cells produced by the preceding embodiments, and a pharmaceutically acceptable excipient. In some embodiments, the plurality comprises at least 103, 104, 105, 106, 107or 108hyperactivated DCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG. 1A shows structures of cationic and ionizable lipids suitable for use in the lipid nanoparticles (LNPs) of the present disclosure. FIG. 1B shows structures of other types of lipids suitable for use in LNPs of the present disclosure. See also, Hou et al., Nature Review Materials, 6:1078-1094, 2021, which is incorporated herein by reference. DETAILED DESCRIPTION

[0044] The present disclosure relates to compounds of Formula (I), and uses thereof in hyperactivating human dendritic cells. The present disclosure also relates to compositions comprising a compound of Formula (I), and one or more of a pathogen recognition receptor agonist, an antigen, and human dendritic cells, as well as methods for production and use of 7sf-6685008Attorney Docket No.: 16553-20010.40 the compositions. In further embodiments, the dendritic cells are non-human dendritic cells, with the proviso that the dendritic cells are not rodent dendritic cells. General Techniques and Definitions

[0045] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0046] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless indicated otherwise. For example, “an” excipient includes one or more excipients.

[0047] The phrase “comprising” as used herein is open-ended, indicating that such embodiments may include additional elements. In contrast, the phrase “consisting of” is closed, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase “consisting essentially of” is partially closed, indicating that such embodiments may further comprise elements that do not materially change the basic characteristics of such embodiments.

[0048] The term “about” as used herein in reference to a value, encompasses from 90% to 110% of that value (e.g., a molecular weight of about 900 daltons, refers to a molecular weight of from 810 daltons to 990 daltons).

[0049] An “effective amount” or a “sufficient amount” of a substance is that amount sufficient to effect beneficial or desired results, including clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For instance, in the context of administering an immunogenic composition, an effective amount contains sufficient antigen, and one or both of a compound of Formula (I) and a PRR agonist, to stimulate an immune response against the antigen (e.g., antigen-reactive antibody and / or cellular immune response).

[0050] The terms “individual” and “subject” refer to mammals. “Mammals” include, but are not limited to, humans, non-human primates (e.g., monkeys), farm animals, sport animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats). In some embodiments, the subject is a human patient, such as a human patient suffering from cancer and / or an infectious disease. 8sf-6685008Attorney Docket No.: 16553-20010.40

[0051] The term “dose” as used herein in reference to an immunogenic composition refers to a measured portion of the immunogenic composition taken by (administered to or received by) a subject at any one time.

[0052] The terms “isolated” and “purified” as used herein refers to a material that is removed from at least one component with which it is otherwise associated during production of the material (e.g., removed from its original environment). As an example, when used in reference to a compound of Formula (I), the compound is at least 90%, 95%, 96%, 97%, 98% or 99% pure 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 cell that produced the protein. As a further example, when used in reference to a synthesized compound, an isolated compound or a purified compound has been removed from the reaction mixture in which it was synthesized.

[0053] The terms “pharmaceutical formulation” and “pharmaceutical composition” refer to preparations that are in such form as to permit the biological activity of the active ingredient to be effective, and that contain no additional components that are unacceptably toxic to an individual to which the formulation or composition would be administered. Such formulations or compositions are intended to be sterile.

[0054] “Excipients” as used herein include pharmaceutically acceptable excipients, carriers, vehicles or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable excipient is an aqueous pH buffered solution.

[0055] The term “antigen” refers to a substance that is recognized and bound specifically by an antibody or by a T cell antigen receptor. Antigens can include peptides, polypeptides, proteins, glycoproteins, polysaccharides, complex carbohydrates, sugars, gangliosides, lipids and phospholipids; portions thereof and combinations thereof. Antigens when present in the compositions of the present disclosure can be synthetic or isolated from nature. Antigens suitable for administration in the methods of the present disclosure include any molecule capable of eliciting 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 is rendered immunogenic when conjugated with a generally larger immunogenic molecule (carrier). 9sf-6685008Attorney Docket No.: 16553-20010.40

[0056] “Polypeptide antigens” can include purified native peptides, synthetic peptides, recombinant peptides, crude peptide extracts, or peptides in a partially purified or unpurified active state (such as peptides that are part of attenuated or inactivated viruses, microorganisms or cells), or fragments of such peptides. Polypeptide antigens are preferably at least eight amino acid residues in length.

[0057] The term “agonist” is used in the broadest sense and includes any molecule that activates signaling through a receptor. In some embodiments, the agonist binds to the receptor. For instance, a TLR8 agonist binds to a TLR8 receptor and activates a TLR8- signaling pathway.

[0058] “Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groups. Cxalkyl refers to an alkyl group having x number of carbon atoms. Cx-Cyalkyl or Cx-yalkyl refers to an alkyl group having between x number and y number of carbon atoms, inclusive. An “n- alkyl” group refers to a straight-chain, i.e. linear, alkyl group.

[0059] “Alkylene” refers to divalent saturated aliphatic hydrocarbyl groups.

[0060] “Alkenyl” refers to monovalent hydrocarbyl groups having at least one double bond (>C=C<). Cx alkenyl refers to an alkenyl group having x number of carbon atoms. Cx- Cyalkenyl or Cx-yalkenyl refers to an alkenyl group having between x number and y number of carbon atoms, inclusive.

[0061] “Cycloalkyl” as used herein refers to and includes, unless otherwise stated, saturated cyclic univalent hydrocarbon structures, having the number of carbon atoms designated (i.e., C3-C10means three to ten carbon atoms). Cycloalkyl can consist of one ring, such as cyclohexyl, or multiple rings, such as adamantyl. A cycloalkyl comprising more than one ring may be fused, spiro or bridged, or combinations thereof. Particular cycloalkyl groups are those having from 3 to 12 annular carbon atoms. A preferred cycloalkyl is a cyclic hydrocarbon having from 3 to 8 annular carbon atoms (a "C3-C8 cycloalkyl"), having 3 to 6 annular carbon atoms (a “C3-C6 cycloalkyl”), or having from 3 to 4 annular carbon atoms (a "C3-C4 cycloalkyl"). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, and the like.

[0062] “Cycloalkenyl” refers to and includes, unless otherwise stated, an unsaturated cyclic non-aromatic univalent hydrocarbon structure, having at least one site of olefinic unsaturation (i.e., having at least one moiety of the formula C=C) and having the number of carbon atoms designated (i.e., C3-C10 means three to ten carbon atoms). Cycloalkenyl can consist of one ring, such as cyclohexenyl, or multiple rings, such as norbornenyl. A preferred 10sf-6685008Attorney Docket No.: 16553-20010.40 cycloalkenyl is an unsaturated cyclic hydrocarbon having from 3 to 8 annular carbon atoms (a “C3-C8 cycloalkenyl”). Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, norbornenyl, and the like.

[0063] “Heterocycle”, “heterocyclic”, or “heterocyclyl” as used herein refers to a saturated or an unsaturated non-aromatic cyclic group having a single ring or multiple condensed rings, and, unless otherwise specified, having from 1 to 14 annular carbon atoms and from 1 to 6 annular heteroatoms, such as nitrogen, sulfur or oxygen, and the like

[0064] “Stimulation” of a response or parameter includes eliciting and / or enhancing that response or parameter when compared to otherwise same conditions except for a parameter of interest, or alternatively, as compared to another condition (e.g., increase in TLR-signaling in the presence of a TLR agonist as compared to the absence of the TLR agonist). For example, “stimulation” of an immune response means an increase in the response. Depending upon the parameter measured, the increase may be from 2-fold to 2,000-fold, or from 5-fold to 500-fold or over, or from 2, 5, 10, 50, or 100-fold to 500, 1,000, 2,000, 5,000, or 10,000- fold.

[0065] Conversely, “inhibition” of a response or parameter includes reducing and / or repressing that response or parameter when compared to otherwise same conditions except for a parameter of interest, or alternatively, as compared to another condition (e.g., decrease in abnormal cell proliferation after administration of a composition comprising a compound of Formula (I), and one or more of a pathogen recognition receptor agonist, an antigen, and human dendritic cells, as compared to the administration of a placebo composition or no treatment). For example, “inhibition” of an immune response means a decrease in the response. Depending upon the parameter measured, the decrease may be from 2-fold to 2,000-fold, or from 5-fold to 500-fold or over, or from 2, 5, 10, 50, or 100-fold to 500, 1,000, 2,000, 5,000, or 10,000-fold.

[0066] The relative terms “higher” and “lower” refer to a measurable increase or decrease, respectively, in a response or parameter when compared to otherwise same conditions except for a parameter of interest, or alternatively, as compared to another condition. For instance, a “higher level of DC hyperactivation” refers to a level of DC hyperactivation as a consequence of a treatment condition (comprising a compound of Formula (I) of the present disclosure) that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold above a level of DC hyperactivation as a consequence of a control condition (e.g., no a compound of Formula (I), PGPC, oxPAPC, etc.). Likewise, a “lower level of DC hyperactivation” refers to 11sf-6685008Attorney Docket No.: 16553-20010.40 a level of DC hyperactivation as a consequence of a treatment condition (comprising a compound of Formula (I) of the present disclosure) that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10- fold below a level of DC hyperactivation as a consequence of a control condition (e.g., no a compound of Formula (I), PGPC, oxPAPC, etc.). In some embodiments, the control condition comprises a comparator compound in the place of a compound of Formula (I) of the treatment condition.

[0067] As used herein the term “immunization” refers to a process that increases a mammalian subject’s reaction to antigen and therefore improves its ability to resist or overcome infection and / or resist disease.

[0068] The term “vaccination” as used herein refers to the introduction of vaccine into a body of a mammalian subject.

[0069] “Adjuvant” refers to a substance which, when added to a composition comprising an antigen, enhances or potentiates an immune response to the antigen in the mammalian recipient upon exposure.

[0070] The terms “treating” or “treatment” of a disease refer to executing a protocol, which may include administering one or more therapeutic agents to an individual (human or otherwise), in an effort to obtain beneficial or desired results in the individual, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more signs or symptoms of a disease, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total). “Treatment” also can mean prolonging survival as compared to expected survival of an individual not receiving treatment. Further, “treating” and “treatment” may occur by administration of one dose of a therapeutic agent or therapeutic agents, or may occur upon administration of a series of doses of a therapeutic agent or therapeutic agents. “Treating” or “treatment” does not require complete alleviation of signs or symptoms, and does not require a cure, and specifically includes protocols that have only a palliative effect on the individual. “Palliating” a disease or disorder means that the extent and / or undesirable clinical manifestations of the disease or disorder are lessened and / or time course of progression of the disease or disorder is slowed, as compared to the expected untreated outcome.

[0071] The compounds described herein can be administered in any pharmaceutically acceptable form, such as in the form of a pharmaceutically acceptable salt, or in free base or 12sf-6685008Attorney Docket No.: 16553-20010.40 free acid form if said form is pharmaceutically acceptable. The compounds described herein, or pharmaceutically acceptable salts thereof, can be administered in pharmaceutically acceptable carriers or excipients. As used herein, by “pharmaceutically acceptable” or “pharmacologically acceptable” is meant a material that is not biologically or otherwise undesirable, e.g. , the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and / or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration. “Pharmaceutically acceptable salts” are those salts which retain at least some of the biological activity of the free (non-salt) compound and which can be administered as drugs or pharmaceuticals to an individual. Such salts, for example, include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, oxalic acid, propionic acid, succinic acid, maleic acid, tartaric acid and the like; (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g. , an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine and the like. Acceptable inorganic bases which can be used to prepared salts include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. Pharmaceutically acceptable salts can be prepared in situ in the manufacturing process, or by separately reacting a purified compound of the invention in its free acid or base form with a suitable organic or inorganic base or acid, respectively, and isolating the salt thus formed during subsequent purification. I. Compounds of Formula (I) and Formula (II)

[0072] Provided herein are compounds of Formula (I), such as isolated compounds of Formula (I): 13sf-6685008Attorney Docket No.: 16553-20010.40

[0074] A1is independently O, S, -NRA-, or -CH2-;

[0075] A2and A3are independently O, S, -NRA-, -CH2-, -NRA-C(=G)-O-, -NRA-C(=G)- NRA-,-O-C(=G)-NRA-, -O-C(=G)-O-, -O-C(=S)-O-, -S- or -S-S- ;

[0076] wherein each RAis independently H, C1-C4 alkyl, benzyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C10cycloalkyl, C3-C10cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA;

[0077] R2is H, C1-C4 alkyl, , C2-C4 alkenyl, C2-C4 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA;;

[0078] R3is C10-C30n-alkyl;

[0079] where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N- (CH2)P-, where RNis -CH3 or -CH2CH3 and p is an integer from 1 to 6 inclusive;

[0080] each R5is independently C1-C4alkyl;

[0081] R6is O or S; 14sf-6685008Attorney Docket No.: 16553-20010.40

[0082] or a protonated or deprotonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and all stereoisomers thereof;

[0083] wherein:

[0084] i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or

[0085] ii) G is S or NH; or

[0086] iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or - CH2CH3and p is an integer from 1 to 6 inclusive; or

[0087] iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or

[0088] v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or

[0089] vi) R6is S.

[0090] Also provided herein are compounds of Formula (II), such as isolated compounds of Formula (II):

[0092] A1and A2are independently O, S, -NRA-, or -CH2-, A3is independently O, S, - NRA-, -CH2-, or -NH-C(=O)-O- , and RAis H or C1-C4alkyl; 15sf-6685008Attorney Docket No.: 16553-20010.40

[0093] R2is H, C1-C4alkyl, -(C=G)-NH2, -(C=G)-NH(R5) , -(C=G)-N(R5)2, or -CH2- C6H5 , wherein G is O, S, or NH;

[0094] R3is C10-C30 n-alkyl;

[0095] where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N- (CH2)P-, where RNis -CH3 or -CH2CH3 and p is an integer from 1 to 6 inclusive;

[0096] each R5is independently C1-C4 alkyl;

[0097] R6is O or S;

[0098] or a protonated or deprotonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and all stereoisomers thereof;

[0099] wherein:

[0100] i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or

[0101] ii) G is S or NH; or

[0102] iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or - CH2CH3and p is an integer from 1 to 6 inclusive; or

[0103] iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or

[0104] v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or

[0105] vi) R6is S. II. Pathogen Recognition Receptor Agonists

[0106] Compositions and methods of the present disclosure may further comprise a pathogen recognition receptor (PRR) agonist. In some embodiments, the PRR agonist comprises 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 other embodiments, the PRR agonist 16sf-6685008Attorney Docket No.: 16553-20010.40 comprises a cytosolic DNA sensor (CDS) or a stimulator of IFN genes (STING). In some embodiments, the PRR agonist comprises a TLR7 / 8 agonist. A. TLR Agonists and TLR7 / 8 Agonists

[0107] The term “TLR agonist” as used herein refers to an agonist of at least one 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 a further aspect, the TLR7 / 8 agonist is a TLR8 agonist. In a further aspect, the TLR7 / 8 agonist is an agonist of both TLR7 and TLR8. TLR7 / 8 agonists of the present disclosure are suitable for hyperactivating human dendritic cells in the presence of LPC.

[0108] 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 with a molecule weight of 900 daltons or less, or a salt thereof. That is, the small molecule TLR7 / 8 agonist is not a large molecule like a recombinant protein or a synthetic oligonucleotide, which is regulatable by the U.S. FDA’s Center for Biologics Evaluation and Research. Rather the small molecule TLR7 / 8 agonist is regulatable by the FDA’s Center for Drug Evaluation and Research. In some embodiments, the small molecule has a molecule weight of from about 90 to about 900 daltons. In some embodiments, the TLR7 / 8 agonist comprises an imidazoquinoline compound. In some preferred embodiments, the TLR7 / 8 agonist comprises resiquimod (R848). B. Other PRR Agonists

[0109] In some aspects, the pathogen recognition receptor (PRR) agonist comprises a toll-like receptor (TLR) agonist with the proviso that the TLR agonist does not comprise a TLR7 / 8 agonist. In some embodiments, the TLR agonist comprises 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 instance, in preferred embodiments, the TLR9 agonist is not a TLR4 ligand such as LPS (endotoxin).

[0110] In additional aspects, the PRR agonist comprises a NOD-like receptor (NLR) agonist. In further aspects, the PRR agonist comprises a RIG-I-like receptor (RLR) agonist. In additional aspects, the PRR agonist comprises a C-type lectin receptor (CLR) agonist. In still further aspects, the PRR agonist comprises a CDS agonist or a STING agonist. 17sf-6685008Attorney Docket No.: 16553-20010.40 III. Antigens

[0111] Compositions and methods of the present disclosure may further comprise an antigen. In some embodiments, the antigen comprises a proteinaceous antigen. The terms “polypeptide” and “protein” are used interchangeably herein to refer to proteinaceous antigens that comprise peptide chains that are at least 8 amino acids in length. In some embodiments, the proteinaceous antigen is from 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 such as by phosphorylation, hydroxylation, sulfonation, palmitoylation, and / or glycosylation.

[0112] In some embodiments, the antigen is a tumor antigen that comprises the amino acid sequence of at least one full length protein or fragment thereof. In some embodiments, the tumor antigen comprises an amino acid sequence or fragment thereof from an oncoprotein. In some embodiments, the mammalian antigen is a neoantigen or encoded by a gene comprising a mutation relative to the gene present in normal cells from a mammalian subject. Neoantigens are thought to be particularly useful in enabling T cells to distinguish between cancer cells and non-cancer cells (see, e.g., Schumacher and Schreiber, Science, 348:69-74, 2015). In other embodiments, the tumor antigen comprises a viral antigen, such as an antigen of a cancer-causing virus.

[0113] 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 non-contiguous amino acid sequences from the same tumor antigen. In some of these embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, wherein each polypeptide comprises non-contiguous amino acid sequences from the same tumor antigen.

[0114] In some embodiments, the antigen is a microbial antigen. In some embodiments, the microbial antigen comprises a viral antigen, a bacterial antigen, a protozoan antigen, a fungal antigen, or combinations thereof. In some embodiments, the microbial antigen comprises a surface protein or other antigenic subunit of a microbe. In other embodiments, the microbial antigen comprises an inactivated or attenuated microbe. For instance, the microbial antigen may comprise an inactivated virus, such as a chemically or genetically- inactivated virus. Alternatively, the microbial antigen may comprise a virus-like particle. 18sf-6685008Attorney Docket No.: 16553-20010.40

[0115] In some embodiments, the antigen may be present in a biological sample obtained from an individual, such as a human patient. For instance, the antigen may comprise cancer cells. In a further aspect, the antigen may comprise microbially-infected cells, such as virally- infected cells. IV. Dendritic Cells

[0116] Compositions and methods of the present disclosure may further comprise dendritic cells (DCs), which are antigen presenting cells that are thought to bridge the innate and adaptive immune systems of mammals. In preferred embodiments, the DCs are subset-1 conventional DCs (cDC1s, previously referred to as myeloid DC1s), as opposed to plasmacytoid DCs (pDCs).

[0117] In some embodiments, the DCs are hyperactive DCs that express high levels of CD40 and IL-12p70. As used herein, the term “hyperactive dendritic cells” refer to a cell state in which DCs are able to secrete IL-1β while maintaining cellular viability (e.g., without undergoing pyroptosis). In this way, hyperactivated dendritic cells are able to stimulate robust T cell immunity (FIG. 1), which apparently combines the benefits of activated and pyroptotic dendritic cells (Zhivaki et al., Cell Reports, 33 (7), 2020, 108381). V. Pharmaceutical Formulations

[0118] Some compositions of the present disclosure are pharmaceutical formulations comprising a pharmaceutically acceptable excipient, and a compound of Formula (I). Some compositions of the present disclosure are pharmaceutical formulations comprising a pharmaceutically acceptable excipient, and a lipid nanoparticle (LNP) comprising a compound of Formula (I) and at least one further lipid. In some embodiments, the pharmaceutical formulations further comprise a PRR agonist, a dendritic cell, an antigen, an adjuvant, or any combination thereof. Pharmaceutical formulations of the present disclosure may be in the form of a solution or a suspension. Alternatively, the pharmaceutical formulations may be a dehydrated solid (e.g., freeze dried or spray dried solid). The pharmaceutical formulations of the present disclosure are preferably sterile, and preferably essentially endotoxin-free. The term “pharmaceutical formulations” is used interchangeably herein with the terms “medicinal product” and “medicament”. In some embodiments, the pharmaceutical formation comprises specific ratios of the various components based on the intended purpose of the formulation. In some embodiments, the pharmaceutical formulations 19sf-6685008Attorney Docket No.: 16553-20010.40 comprise a compound of Formula (I) and non-ionic surfactant. In some embodiments, the non-ionic surfactant comprises an ethylene oxide-propylene oxide copolymer (that is, a poloxamer), such as Poloxamer-407 (CAS Registry No. 977057-91-2). A. Excipients

[0119] Pharmaceutically acceptable excipients of the present disclosure include for instance, solvents, buffering agents, tonicity adjusting agents, bulking agents, and preservatives (See, e.g., Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the pharmaceutical formulations may comprise an excipient that functions as one or more of a solvent, a buffering agent, a tonicity adjusting agent, and a bulking agent (e.g., sodium chloride in saline may serve as both an aqueous vehicle and a tonicity adjusting agent). Pharmaceutically acceptable excipients of the present disclosure also include detergents, wetting agents, thickening agents, emulsifiers, foaming agents, and dispersants, as well as surfactants.

[0120] Many of the lipids disclosed herein are sparingly soluble in water. Surfactants can be used to solubilize the lipids in aqueous formulations. A wide variety of surfactants are available, which can be classified as anionic surfactants, non-ionic surfactants, cationic surfactants, and zwitterionic surfactants.

[0121] Some examples of non-ionic surfactants include poloxamers, which are triblock copolymers of ethylene oxide and propylene oxide of the general formula: HO-[CH2CH2-O-]a-[CH2CH(CH3)-O-]b-[CH2-CH2-O-]a-H, where a is typically about 2 to 130 and b is typically about 15 to 67. Some poloxamers are sold under the trade name Pluronic® (PLURONIC is a registered trademark of BASF SE, Ludwigshafen, Germany). Examples of poloxamers are Poloxamer 407 (KP407; a = 101, b = 56); Poloxamer 188 (KP188; a = 80, b = 27); Pluronic® P84 (P-84; a = 19, b = 39); and Pluronic® P123 (P-123; a = 20, b =70) (the foregoing values for a and b can be subject to slight variation).

[0122] Other non-ionic surfactants include the Cremophor® series (CREMAPHOR is a registered trademark of BASF SE, Ludwigshafen, Germany). Cremophor® surfactants include Cremophor® EL (K EL), a mixture of polyoxyethylated triglycerides produced by reacting castor oil with ethylene oxide in a molar ratio of approximately 1:35, and Cremophor® RH40 (also known as Kolliphor® RH40; KOLLIPHOR is a registered trademark of BASF SE), obtained by reacting 40 moles of ethylene oxide with 1 mole of hydrogenated castor oil. 20sf-6685008Attorney Docket No.: 16553-20010.40

[0123] In some embodiments, the pharmaceutical formulations comprise an aqueous vehicle as a solvent. Suitable vehicles include for instance sterile water, saline solution, phosphate buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic.

[0124] The pharmaceutical formulations may comprise a buffering agent. Buffering agents control pH to inhibit degradation of the active agent during processing, storage and optionally reconstitution. Suitable buffers include for instance salts comprising acetate, citrate, phosphate or sulfate. Other suitable buffers include for instance amino acids such as arginine, glycine, histidine, and lysine. The buffering agent may further comprise hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the composition within a 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 in the range of from about 6 to 9 in which the lower limit is less than the upper limit.

[0125] The pharmaceutical compositions may comprise a tonicity adjusting agent. Suitable tonicity adjusting agents include for instance dextrose, glycerol, sodium chloride, glycerin and mannitol.

[0126] The pharmaceutical formulations may comprise a bulking agent. Bulking agents are particularly useful when the pharmaceutical composition is to be lyophilized before administration. In some embodiments, the bulking agent is a protectant that aids in the stabilization and prevention of degradation of the active agents during freeze or spray drying and / or during storage. Suitable bulking agents are sugars (mono-, di- and polysaccharides) such as sucrose, lactose, trehalose, mannitol, sorbital, glucose and raffinose.

[0127] The pharmaceutical formulations may comprise a preservative. Suitable preservatives include for instance antioxidants and antimicrobial agents. However, in preferred embodiments, the pharmaceutical formulation is prepared under sterile conditions and is in a single use container, and thus does not necessitate inclusion of a preservative. Methods for preparing sterile, pharmaceutically acceptable compositions include steam sterilization, dry-heat sterilization, gas sterilization, ionizing radiation, or sterile filtration. Sterile pharmaceutical formulations are compounded or manufactured according to pharmaceutical-grade sterilization standards (United States Pharmacopeia Chapters 797, 1072, and 1211; California Business & Professions Code 4127.7; 16 California Code of Regulations 1751, 21 Code of Federal Regulations 211) known to those of skill in the art. 21sf-6685008Attorney Docket No.: 16553-20010.40

[0128] In some embodiments, the pharmaceutical formulation is a homogenous solution. In some embodiments, the homogenous solution is supplied in a pre-filled syringe. In some embodiments, the pharmaceutical formulation is supplied as a suspension. In some embodiments, the suspension is refrigerated. In some embodiments, the suspension is frozen. In some embodiments, methods provided herein further comprise the step of warming the refrigerated suspension to room temperature and / or agitating the suspension to ensure that the active ingredient(s) are dissolved and / or evenly distributed in solution prior to administration. In some embodiments, methods provided herein further comprise the step of thawing the frozen suspension and warming to room temperature and / or agitating the suspension to ensure that the active ingredient(s) are dissolved and / or evenly distributed in solution prior to administration. In some embodiments, the suspension is diluted prior to administration. In some embodiments, the suspension is supplied as a pre-filled syringe. In some embodiments, the suspension comprises a pharmaceutically acceptable excipient, e.g., surfactant, glycerol, non-ionic surfactant, buffer, glycol, salt, or any combination thereof.

[0129] 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). B. Adjuvants

[0130] Pharmaceutically acceptable adjuvants of the present disclosure include for instance, an aluminum salt adjuvant, a squalene-in-water emulsion, a saponin, or combinations thereof. In some embodiments, the adjuvant is an aluminum salt adjuvant selected from the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, and combinations thereof. In other embodiments, the adjuvant is a squalene-in-water emulsion such as MF59 or AS03. In other embodiments, the adjuvant is a saponin, such as Quil A or QS-21, as in AS01 or AS02. C. Kits

[0131] Also provided herein are kits comprising at least one pharmaceutical formulation described herein. In some embodiments, the kit comprises a lyophilized or freeze-dried pharmaceutical formulation (e.g., one unit dose in a vial) disclosed herein and a solution for dissolving, diluting, and / or reconstituting the lyophilized pharmaceutical composition. In some embodiments, the solution for reconstituting or dilution is supplied as a pre-filled syringe. In some embodiments, the kit comprises a frozen suspension of a pharmaceutical 22sf-6685008Attorney Docket No.: 16553-20010.40 formulation (e.g., one unit dose in a vial). In some embodiments, the kit includes a buffer that helps to prevent aggregation upon reconstituting the pharmaceutical composition disclosed herein. In some embodiments, the pharmaceutical composition is provided in a pre-filled syringe. In some embodiments, a kit comprises a dual-chamber syringe or container wherein one of the chambers contains a buffer for dissolving or diluting the pharmaceutical composition. In some embodiments, the kit comprises a syringe for injection. In some embodiments, the reconstituted solution is filtered before administration. In some embodiments, the kit comprises a filter or a filter syringe for filtering the reconstituted pharmaceutical composition before administration. In some embodiments, the kit further comprises instructions for use, e.g., instructions for hyperactivating cells. D. Particle Size of Drug Product

[0132] The particle size of the drug particles can affect the uptake of drug by cells. Particle size can be controlled by milling of the drug substance, such as DGP (Compound 2) by techniques well-known in the pharmaceutical arts. Dry milling techniques that can be used include, but are not limited to, jet milling, hammer milling, and pin milling. Wet milling techniques that can be used include, but are not limited to, rotor-stator milling, colloid milling, and media milling. Milling of the drug substance can be performed before further steps in the method, such as combining the drug substance with solutions, buffers, and / or other components to form a suspension.

[0133] The drug substance can be combined with solutions or buffers, such as phosphate- buffered saline and a poloxamer (for example, poloxamer 407 or poloxamer 188), to give a drug product. Further procedures can be used to reduce particle size in the drug product, including sonication and homogenization.

[0134] In embodiments, about 50% of the particles in the drug product have a diameter less than about 40 microns (D50 < 40 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than about 30 microns (D50 < 30 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than between about 20 microns and about 40 microns (D50 < 20 microns to 40 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than between about 20 microns and about 30 microns (D50< 20 microns to 30 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than about 20 microns (D50 < 20 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than between about 10 microns and about 30 microns (D50 < 10 23sf-6685008Attorney Docket No.: 16553-20010.40 microns to 30 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than between about 10 microns (D50 < 10 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than between about 10 microns and about 20 microns (D50< 10 microns to 20 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than between about 5 microns and about 20 microns (D50 < 5 microns to 20 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than between about 5 microns (D50< 5 microns).

[0135] In embodiments, about 50% of the particles in the drug product have a diameter less than about 40 microns (D90 < 40 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than about 30 microns (D90< 30 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than between about 20 microns and about 40 microns (D90 < 20 microns to 40 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than between about 20 microns and about 30 microns (D90< 20 microns to 30 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than about 20 microns (D90 < 20 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than between about 10 microns and about 30 microns (D90< 10 microns to 30 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than between about 10 microns (D90 < 10 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than between about 10 microns and about 20 microns (D90< 10 microns to 20 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than between about 5 microns and about 20 microns (D90 < 5 microns to 20 microns). In embodiments, about 50% of the particles in the drug product have a diameter less than between about 5 microns (D90< 5 microns).

[0136] The particles of the drug product as described herein can comprise i) one or more of a surfactant, such as a non-ionic surfactant, such as a poloxamer or a Pluronic, such as Poloxamer 407, Poloxamer 188, Pluronic 84, or Pluronic 123; a wetting agent, such as P407, P188, or polysorbate 80; or a thickening agent, such as carboxymethyl cellulose; and ii) a compound of Formula (I) as disclosed herein; or a protonated or deprotonated form thereof where possible, or a pharmaceutically acceptable salt thereof. The particles can have a size or size range as indicated above. 24sf-6685008Attorney Docket No.: 16553-20010.40 VI. Methods for Production

[0137] The present disclosure relates, in some aspects, 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.

[0138] In one aspect, the present disclosure provides a method for production of hyperactivated dendritic cells (DCs), the method comprising contacting dendritic cells with effective amounts of a compound of Formula (I) and a PRR agonist for production of hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1beta without undergoing pyroptosis. In some embodiments, the DCs are isolated, while in other embodiments, the DCs are present within a biological sample obtained from a mammalian subject, such as a human patient. In some embodiments, the DCs are monocyte-derived DCs, preferably cDC1s.

[0139] In a further aspect, the present disclosure provides a method for production of an immunogenic composition, the method comprising combining an antigen with effective amounts of a compound of Formula (I) and a PRR agonist for production of an immunogenic composition. In some embodiments, the antigen comprises a proteinaceous antigen that is present in or purified from a biological sample obtained from a mammalian subject. In some embodiments, the proteinaceous 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.

[0140] In specific embodiments, the present disclosure provide a method for production of an immunogenic composition, the method comprising: a) preparing a suspension of cells from a tumor to obtain a tumor cell suspension; b) lysing cells from the tumor cell suspension to obtain a tumor cell lysate; and c) contacting the tumor cell lysate with a compound of Formula (I) and a PRR agonist to obtain the immunogenic composition. In some embodiments, leukocytes are depleted from the tumor cell suspension by contacting the suspension with an antibody specific to leukocytes to obtain a tumor cell-enriched suspension (e.g., negative selection). In some embodiments, the leukocytes are depleted by contacting the tumor cell-enriched suspension with an anti-CD45 antibody. In some embodiments, the cells are lysed by a physical disruption-based cell lysis method, such as, but not limited to, mechanical lysis, liquid 25sf-6685008Attorney Docket No.: 16553-20010.40 homogenization, sonication, freeze-thaw, or manual grinding. In some preferred embodiments, the cells are lysed by one or more freeze-thaw cycles.

[0141] In specific embodiments, the present disclosure provide a method for production of an immunogenic composition, the method comprising: a) lysing cells from a tumor cell suspension to obtain a tumor cell lysate; and b) contacting the tumor cell lysate with a compound of Formula (I) and a PRR agonist to obtain the immunogenic composition. In some embodiments, leukocytes are depleted from the tumor cell suspension by contacting the suspension with an antibody specific to leukocytes to obtain a tumor cell-enriched suspension (e.g., negative selection). In some embodiments, the leukocytes are depleted by contacting the tumor cell-enriched suspension with an anti-CD45 antibody. In some embodiments, the cells are lysed by a physical disruption-based cell lysis method, such as, but not limited to, mechanical lysis, liquid homogenization, sonication, freeze-thaw, or manual grinding. In some preferred embodiments, the cells are lysed by one or more freeze-thaw cycles.

[0142] In some embodiments of the afore-mentioned methods, the PRR agonist is a TLR agonist. In some embodiments, the PRR agonist is a TLR7 / 8 agonist. In some preferred embodiments, the TLR7 / 8 agonist is an imidazoquinoline compound, which in particularly preferred embodiments is resiquimod (R848). VII. Further Lipids

[0143] Compositions and methods of the present disclosure can comprise at least one further lipid, wherein the LPC and the at least one further lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one further lipid comprises an ionizable lipid, a cationic lipid, a further phospholipid, a pegylated lipid, a structural lipid, or a mixture thereof. In some embodiments, the LNP comprises a first phospholipid (lysophosphatidylcholine with a single C13-C24 acyl chain [LPC:C13-C24]), an ionizable lipid, a second phospholipid, a pegylated lipid, and a structural lipid. Structures of further lipids suitable for use in the compositions and methods of the present disclosure are shown in FIG. 1A and FIG. 1B (reproduced from Hou et al., Nature Review Materials, 6:1078-1094, 2021).

[0144] In some embodiments, the at least one further lipid comprises one or both of a further phospholipid and a structural lipid, optionally wherein the further phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and the structural lipid comprises cholesterol. In some embodiments, the at least one further lipid comprises or 26sf-6685008Attorney Docket No.: 16553-20010.40 further comprises a pegylated lipid, optionally wherein the pegylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG]. In some embodiments, at least one further lipid comprises or further comprises an ionizable lipid, optionally wherein the ionizable lipid comprises (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA) or analogs or derivatives thereof. In some embodiments, the at least one further lipid comprises at least one lipid from the following list (disclosed in Hou et al., Nature Review Materials 6, 1078–1094 (2021)); these lipids include 306Oi10, tetrakis(8- methylnonyl) 3,3′,3″,3‴-(((methylazanediyl) bis(propane-3,1 diyl))bis(azanetriyl))tetrapropionate; 9A1P9, decyl (2-(dioctylammonio)ethyl) phosphate;A2- Iso5-2DC18, ethyl 5,5- di((Z)- heptadec-8- en-1- yl)-1-(3-(pyrrolidin-1- yl)propyl)-2,5- dihydro-1H- imidazole-2- carboxylate; ALC-0315, ((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-ethyl-6- methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H- cyclopenta[a]phenanthren-3- ol; BAME-O16B,bis(2-(dodecyldisulfanyl)ethyl) 3,3′-((3- methyl-9- oxo-10- oxa-13,14- dithia-3,6- diazahexacosyl)azanediyl)dipropionate; BHEM- Cholesterol, 2-(((((3S,8S,9S,10R,13R,14S,17R)-10,13- dimethyl-17-((R)-6- methylheptan-2- yl)-2,3,4,7 ,8,9,10,11,12,13,14,15,16,17- tetradecahydro-1Hcyclopenta[a]phenanthren-3- yl)oxy)carbonyl)amino)- N,N- bis(2- hydroxyethyl)- Nmethylethan-1- aminium 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)- heptatriaconta-6,9,28,31- tetraen-19- yl 4-(dimethylamino)butanoate; DOPE, 1,2- dioleoyl- sn- glycero-3- phosphoethanolamine; DOSPA, 2,3-dioleyloxy- N-[2- (sperminecarboxamido)ethyl]- N,N- dimethyl-1- propanaminiumtrifluoroacetate; DOTAP, 1,2- dioleoyl-3- trimethylammonium- propane; DOTMA,1,2- di- O- octadecenyl-3- trimethylammonium- propane; DSPC, 1,2- distearoyl- snglycero-3- phosphocholine; ePC, ethylphosphatidylcholine; FTT5, hexa(octan-3- yl)9,9′,9″,9‴,9″″,9‴″- ((((benzene-1,3,5- tricarbonyl)yris(azanediyl)) tris (propane-3,1- diyl))tris(azanetriyl))hexanonanoate; Lipid H (SM-102), heptadecan-9- yl 8-((2- hydroxyethyl)(6- oxo-6- (undecyloxy)hexyl)amino) octanoate; OF- Deg- Lin, (((3,6- dioxopiperazine-2,5- diyl)bis(butane-4, 1- diyl))bis(azanetriyl))tetrakis(ethane-2,1- diyl) (9Z,9′Z,9″Z,9‴Z,12Z,12′Z,12″Z,12‴Z)- tetrakis 27sf-6685008Attorney Docket No.: 16553-20010.40 (octadeca-9,12- dienoate); PEG2000- DMG, 1,2-dimyristoylrac-glycero-3- methoxypolyethylene glycol-2000; TT3, N1,N3,N5- tris(3-(didodecylamino)propyl)benzene- 1,3,5- tricarboxamide, and are illustrated in FIG. 1A and FIG. 1B. VIII. mRNA Encoding An Antigen

[0145] Compositions and methods of the present disclosure comprise an mRNA encoding an antigen or are otherwise suitable for use with a formulation comprising an mRNA encoding an antigen. In some embodiments, the antigen is a proteinaceous antigen. The terms “polypeptide” and “protein” are used interchangeably herein in reference to antigens that comprise peptide chains that are at least 8 amino acids in length. In some embodiments, the antigen is from 8 to 1800 amino acids, 9 to 1000 amino acids, or 10 to 100 amino acids in length. The polypeptide may be post-translationally modified such as by phosphorylation, hydroxylation, sulfonation, palmitoylation, and / or glycosylation.

[0146] In some embodiments, the antigen is a tumor antigen that comprises the amino acid sequence of at least one full length protein or fragment thereof. In some embodiments, the tumor antigen comprises an amino acid sequence or fragment thereof from an oncoprotein. In some embodiments, the mammalian antigen is a neoantigen or encoded by a gene comprising a mutation relative to the gene present in normal cells from a mammalian subject. Neoantigens are thought to be particularly useful in enabling T cells to distinguish between cancer cells and non-cancer cells (see, e.g., Schumacher and Schreiber, Science, 348:69-74, 2015). In other embodiments, the tumor antigen comprises a viral antigen, such as an antigen of a cancer-causing virus.

[0147] 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 non-contiguous amino acid sequences from the same tumor antigen. In some of these embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, wherein each polypeptide comprises non-contiguous amino acid sequences from the same tumor antigen.

[0148] In some embodiments, the antigen is a microbial antigen. In some embodiments, the microbial antigen comprises a viral antigen, a bacterial antigen, a protozoan antigen, a fungal antigen, or combinations thereof. In some embodiments, the microbial antigen comprises a surface protein or other antigenic subunit of a microbe. 28sf-6685008Attorney Docket No.: 16553-20010.40

[0149] In some preferred embodiments, the mRNA comprises a 5’ untranslated region (5’UTR) at the 5’ end of the coding region and a 3’ untranslated region (3’UTR) at the 3’ end of the coding region. In some preferred embodiments, the mRNA comprises one or both of a 5’ cap structure and a polyA tail. IX. Lipid-Based Delivery Vehicles

[0150] Compositions and methods of the present disclosure comprise a lipid-based delivery vehicle for the mRNA encoding an antigen. In some embodiments, the vehicle is a lipid nanoparticle (LNP). In other embodiments, the vehicle is a lipid that forms a complex with the mRNA (RNA-Lipoplex).

[0151] In some embodiments, the LNP comprises a compound of Formula (I), or a protonated or deprotonated form thereof where possible, or a pharmaceutically acceptable salt thereof; and at least one lipid selected from the group consisting of an ionizable lipid, a cationic lipid, a second phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof. In some embodiments, the compound of Formula (I) is isolated. In some embodiments, the at least one lipid comprises an ionizable lipid. In some embodiments, the at least one lipid comprises a cationic lipid. In some embodiments, the at least one lipid comprises a second phospholipid. In some embodiments, the at least one lipid comprises a pegylated lipid. In some embodiments, the at least one lipid comprises a structural lipid. In some embodiments, the at least one lipid comprise an ionizable lipid, a second phospholipid, a pegylated lipid, and a structural lipid.

[0152] In some embodiments, the LNP comprises a compound of Formula (I) as disclosed herein; or a protonated or deprotonated form thereof where possible, or a pharmaceutically acceptable salt thereof; and at least one lipid selected from the group consisting of an ionizable lipid, a cationic lipid, a second phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof. In some embodiments, the compound of Formula (I) is isolated. In some embodiments, the at least one lipid comprises an ionizable lipid. In some embodiments, the at least one lipid comprises a cationic lipid. In some embodiments, the at least one lipid comprises a second phospholipid. In some embodiments, the at least one lipid comprises a pegylated lipid. In some embodiments, the at least one lipid comprises a structural lipid. In some embodiments, the at least one lipid comprise an ionizable lipid, a second phospholipid, a pegylated lipid, and a structural lipid. 29sf-6685008Attorney Docket No.: 16553-20010.40

[0153] In some embodiments, the lipid component of RNA-Lipoplex comprises one or more lipids. In some preferred embodiments, the one or more lipids comprise a first lipid and a second lipid, wherein the first lipid is distinct from the second lipid. In some embodiments, the first lipid is a cationic lipid and the second lipid is a neutral or anionic lipid.

[0154] Structure of lipids suitable for use in the lipid-based mRNA delivery vehicles of the present disclosure are shown in FIG. 1A and FIG. 1B (reproduced from Hou et al., Nature Review Materials, 6:1078-1094, 2021). X. Methods of Use

[0155] In some aspects, the present disclosure relates to methods of use of any one of the compositions or formulations described herein, which comprise a compound of Formula (I). In some embodiments, the compositions or formulations further comprise a PRR agonist, a dendritic cell, an antigen, an adjuvant, or any combination thereof. The methods of use are suitable for a plurality of uses involving stimulating an immune response. In some embodiments, the methods of use comprise methods of treating cancer. In some embodiments, the methods of use comprise methods of inhibiting abnormal cell proliferation. In some embodiments, the methods of use comprise methods of treating an infectious disease. The methods comprise administering an effective amount of a formulation or a composition described herein to an individual in need thereof to achieve a specific outcome. The individual is a mammalian subject, such as a human patient. In other embodiments, the individual is a non-human patient. In some embodiments, the individual is a canine patient. That is in some embodiments, the methods of use involve clinical uses, while in other embodiments the methods of use involve pre-clinical and / or veterinary uses. For preclinical uses, the mammalian subject may be a non-human primate (e.g., monkey or ape) or a rodent (e.g., mouse or rat). For veterinary uses the mammalian subject may be a farm animal (e.g., cow), a sport animal (e.g., horse), or a pet (e.g., companion animal such as a dog or cat). A. Stimulation of an Immune Response

[0156] In brief, the present disclosure provides methods of stimulating an immune response in an individual, comprising administering to the individual a composition or formulation described herein in an amount sufficient to stimulate an immune response in the individual. “Stimulating” an immune response (used interchangeably with “eliciting” an immune response), means increasing the immune response, which can arise from eliciting a de novo immune response (e.g., as a consequence of an initial vaccination regimen) or 30sf-6685008Attorney Docket No.: 16553-20010.40 enhancing an existing immune response (e.g., as a consequence of a booster vaccination regimen). In some embodiments, stimulating an immune response comprises one or more of the group consisting of: stimulating cytokine production; stimulating B lymphocyte proliferation; stimulating interferon pathway-associated gene expression; stimulating chemoattractant-associated gene expression; and stimulating dendritic cell DC maturation. Methods for measuring stimulation of an immune response are known in the art.

[0157] For instance, the present disclosure provides methods of inducing an antigen- specific immune response in an individual by administering to the individual a composition or formulation described herein in an amount sufficient to induce an antigen-specific immune response in the individual. In preferred embodiments, the composition or formulation comprises the antigen. In some embodiments, the composition or formulation is administered to a tissue of the individual comprising the antigen. The immune response may comprise one or both of an antigen-specific antibody response and an antigen-specific cytotoxic T lymphocyte (CTL) response. “Inducing” an antigen-specific antibody response means increasing titer of the antigen-specific antibodies above a threshold level such as a pre- administration baseline titer or a seroprotective level. “Inducing” an antigen-specific CTL response means increasing frequency of antigen-specific CTL found in peripheral blood above a pre-administration baseline frequency.

[0158] Analysis (both qualitative and quantitative) of the immune response can be by any method known in the art, including, but not limited to, measuring antigen-specific antibody production (including measuring specific antibody subclasses), activation of specific populations of lymphocytes such as B cells and helper T cells, production of cytokines such as IFN-alpha, IFN-gamma, IL-6, IL-12 and / or release of histamine. Methods for measuring antigen-specific antibody responses include enzyme-linked immunosorbent assay (ELISA). Activation of specific populations of lymphocytes can be measured by proliferation assays, and with fluorescence-activated cell sorting (FACS). Production of cytokines can also be measured by ELISA. In some embodiments, methods of stimulating an immune response comprise stimulation of interleukin-1beta (IL-1β) secretion, interferon-gamma (IFN-γ) secretion, and / or tumor necrosis factor-alpha (TNF-α) secretion by monocyte-derived dendritic cells or peripheral blood mononuclear cells. In some embodiments, methods of stimulating an immune response comprise stimulation of secretion of one or more of IFN-γ, IL-17a, IL-17f, and IL-22 by memory CD4+ T cells. In some embodiments, methods of stimulating an immune response comprise increasing Th1 differentation of naïve CD4+ T 31sf-6685008Attorney Docket No.: 16553-20010.40 cells. In some preferred embodiments, at least 50%, 55%, 60%, 65%, 70% or 75% of the cells contacted with a composition of the present disclosure remain viable at 40-56 hours (or about 48 hours) post-contact.

[0159] 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- microbe immune response. In some embodiments, the anti-microbe response is an anti- bacterial immune response. In some embodiments, the anti-microbe response is an anti- fungal immune response. In some embodiments, the anti-microbe response is, an anti-viral immune response. In some embodiments, the anti-microbe response is an anti-protozoan immune response. B. Treating or Preventing Disease

[0160] The present disclosure further provides methods of treating or preventing a disease in an individual, comprising administering to the individual a composition or formulation described herein in an amount sufficient to treat or prevent a 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.

[0161] In one aspect, the methods may comprise administering a composition a compound of Formula (I) to a subject in need thereof. In some embodiments, the compositions further comprise a PRR agonist, an antigen, an adjuvant, or any combination thereof. In a further aspect, the methods involve adoptive cell therapy, and comprise administering a composition comprising a dendritic cell, such as a hyperactivated dendritic cell, and a compound of Formula (I) to a subject in need thereof. In some embodiments, the compositions further comprise a PRR agonist, an antigen, an adjuvant, or any combination thereof.

[0162] In some embodiments, the methods involve treating cancer in an individual or otherwise treating a mammalian subject with cancer. In some embodiments, the methods comprise: a) preparing an immunogenic composition comprising a tumor cell lysate, an isolated compound of Formula (I), 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 sample of a tumor obtained from the subject with cancer; and b) administering to the subject an effective amount of the immunogenic composition. In some embodiments, the cancer is a hematologic cancer, such as a lymphoma, a leukemia, or a myeloma. In other embodiments, 32sf-6685008Attorney Docket No.: 16553-20010.40 the cancer is a non-hematologic cancer, such as a sarcoma, a carcinoma, or a melanoma. In some embodiments, the cancer is malignant.

[0163] In some embodiments, the methods involve inhibiting abnormal cell proliferation in an individual. “Abnormal cell proliferation” refers to proliferation of a benign tumor or a malignant tumor. The malignant tumor may be a metastatic tumor.

[0164] In some embodiments, the methods involve 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 still further embodiments, the infectious disease is caused by a protozoal infection. Of particular importance are infectious diseases caused by zoonotic pathogens that infect humans as well as other animals such as mammals or birds. In some embodiments, the zoonotic pathogen is transmitted to humans via an intermediate species (vector). SYNTHETIC SCHEMES

[0165] The following synthetic schemes describe general synthetic procedures, which can be used as described or further modified or combined to prepare the compounds disclosed herein. For example, the synthesis of non-exemplified compounds disclosed herein can be successfully performed by modifications known to the skilled artisan, such as by using alternative protecting groups, by utilizing other suitable reagents known in the art other than those described, or by making routine modifications of reaction conditions. Other reactions disclosed herein or known in the art will be recognized as being applicable for preparing other compounds disclosed herein. 33sf-6685008Attorney Docket No.: 16553-20010.40 Scheme A1

[0166] (i) HBTU, DIEA / CH2Cl2; (ii) Acid (e.g., HCl in tetrahydrofuran); (iii) dimethoxytrityl chloride (DMTr-Cl), 4-(dimethylamino)pyridine (DMAP) / pyridine; (iv) benzoyl chloride, diisopropylethylamine (DIEA) / CH2Cl2; (v) Acid (e.g., dichloroacetic acid or trichloroacetic acid in CH2Cl2); (vi) phosphitylation; (vii) phosphoramidite coupling and oxidation with water, iodine, and pyridine (Y = O) / sulfurization with 3- (dimethylaminomethylidene)amino-3H-1,2,4-dithiazole-3-thione (DDTT) (Y = S); (viii) ammonia or ammonia and methylamine and (ix) (a) phosphorous oxychloride (POCl3), DIEA and (b) aqueous ammonia or aqueous ammonia and methylamine. The R or S isomer of 300, 300a, 301, and 301a, and the racemic mixtures of 300, 300a, 301, and 301a, are prepared from the corresponding stereochemically pure isomer of compound 12 or a racemic mixture of compound 12, respectively. 300a has an additional stereo center at the phosphorothioate phosphorus. When prepared from a single stereoisomer of compound 12, two diastereomers of 300a will be produced. When prepared from a racemic mixture of compound 12, all four stereoisomers of 300a will be produced. 34sf-6685008Attorney Docket No.: 16553-20010.40 Scheme A2

[0167] (i) N,N′-Disuccinimidyl carbonate (DSC), DIEA / CH2Cl2; (ii) Acid (e.g., HCl in tetrahydrofuran); (iii) DMTr-Cl, DMAP / pyridine; (iv) benzoyl chloride, DIEA / CH2Cl2; (v) Acid; (vi) phosphitylation; (vii) phosphoramidite coupling and oxidation (Y =. O) / Sulfurization (Y = S); (viii) ammonia or ammonia and methylamine and (ix) (a) phosphorous oxychloride (POCl3), DIEA and (b) aqueous ammonia or aqueous ammonia and methylamine. The R or S isomer of 302, 302a, 303, and 303a, and the racemic mixture are prepared from the corresponding stereochemically pure isomer of compound 19 or a racemic mixture of compound 19, respectively. 302a has an additional stereo center at the phosphorothioate phosphorus. When prepared from a single stereoisomer of compound 19, two diastereomers of 302a will be produced. When prepared from a racemic mixture of compound 19, all four stereoisomers of 302a will be produced. 35sf-6685008Attorney Docket No.: 16553-20010.40 Scheme A3Y = S 304 Y = O304a Y = S

[0168] (i) N,N′-Disuccinimidyl carbonate (DSC), DIEA / CH2Cl2; (ii) Acid; (iii) DMTr-Cl, DMAP / pyridine; (iv) benzoyl chloride, DIEA / CH2Cl2; (v) Acid; (vi) phosphitylation; (vii) phosphoramidite coupling and oxidation (Y = O) or sulfurization. (Y = S); (viii) ammonia or ammonia and methylamine and (ix) (a) phosphorous oxychloride (POCl3), DIEA and (b) aqueous ammonia or aqueous ammonia and methylamine. The R or S isomer of 304, 304a, 305, and 305a, and the racemic mixture are prepared from corresponding stereochemically pure isomer of compound 12 or a racemic mixture of compound 12, respectively. 304a has an additional stereo center at the phosphorothioate phosphorus. When prepared from a single stereoisomer of compound 12, two diastereomers of 304a will be produced. When prepared from a racemic mixture of compound 19, all four stereoisomers of 304a will be produced. Scheme A4 36sf-6685008Attorney Docket No.: 16553-20010.40

[0169] (i) DSC, DIEA / CH2Cl2; (ii) Acid; (iii) DMTr-Cl, DMAP / pyridine; (iv) t-butyldimethylsilyl chloride (TBDMS-Cl), imidazole / CH2Cl2; (v) Acid; (vi) phosphitylation; (vii) phosphoramidite coupling and oxidation Y = O) or sulfurization. (Y = S); (viii) ammonia or ammonia and methylamine and (ix) (a) phosphorous oxychloride (POCl3), DIEA, (b) aqueous ammonia or aqueous ammonia and methylamine and (c) triethylamine trihydrofluoride. The R or S isomer of 306, 306a, 307, and 307a, and the racemic mixture are prepared from the corresponding stereochemically pure isomers of compound 19 or a racemic mixture of compound 19, respectively. 306a has an additional stereo center at the phosphorothioate phosphorus. When prepared from a single stereoisomer of compound 19, two diastereomers of 306a will be produced. When prepared from a racemic mixture of compound 19, all four stereoisomers of 306a will be produced. 37sf-6685008Attorney Docket No.: 16553-20010.40 Scheme A5

[0170] (i) DSC, DIEA / CH2Cl2; (ii) Acid; (iii) DMTr-Cl, DMAP / pyridine; (iv) benzoyl chloride, DIEA / CH2Cl2; (v) Acid; (vi) phosphitylation; (vii) phosphoramidite coupling and oxidation (Y = O) or sulfurization (Y = S); (viii) ammonia or ammonia and methylamine and (ix) (a) phosphorous oxychloride (POCl3), DIEA and (b) aqueous ammonia or aqueous ammonia and methylamine. The R or S isomer of 47, 48, and 48a, and the racemic mixture are prepared from the corresponding stereochemically pure isomer of compound 12 or a racemic mixture of compound 30, respectively. 47a has an additional stereo center at the phosphorothioate phosphorus. When prepared from a single stereoisomer of compound 12, two diastereomers of 47a will be produced. When prepared from a racemic mixture of compound 12, all four stereoisomers of 47a will be produced 38sf-6685008Attorney Docket No.: 16553-20010.40 Scheme A6

[0171] (i) Benzoic anhydride, DIEA / CH2Cl2; (ii) Acetic anhydride, DIEA / CH2Cl2; (iii) DSC, DIEA, methylamine; (iv) DSC, DIEA, ammonia; (v) DSC, DIEA, dimethylamine; (vi) 1,1′-thiocarbonyldiimidazole, DIEA, dimethylamine; (vii) 1,1′-thiocarbonyldiimidazole, DIEA, ammonia; (viii). 1,1′-thiocarbonyldiimidazole, DIEA, methylamine; (ix) DSC, DIEA, methanol; (x) DSC, DIEA, ethanol; (xi) DSC, DIEA, isopropanol; (xii) DSC, DIEA, benzyl alcohol. 39sf-6685008Attorney Docket No.: 16553-20010.40 Scheme. A7

[0172] (i) Trityl chloride, DMAP, pyridine; (ii) C18 or C22 alkyl bromide, base Scheme A8

[0173] (i) Acid; (ii) (a) POCl3, base and (b) aqueous base 40sf-6685008Attorney Docket No.: 16553-20010.40 Scheme A9

[0174] (i) (a) Phosphitylation of 126a-137b; (ii) the formed phosphoramidite is coupled to choline in the presence 5-(Ethylthio)-1H-tetrazole (ETT) followed by oxidation (Y = O) or sulfurization (Y = S) to yield compounds 150a-161b. 41sf-6685008Attorney Docket No.: 16553-20010.40 Scheme A10

[0175] (i) Bis(2-cyanoethyl) N,N-diisopropylphosphoramidite, ETT / dichloromethane; (ii) a. oxidation or sulfurization and (b) aqueous base and (iii). Hydrogenation. 42sf-6685008Attorney Docket No.: 16553-20010.40 Scheme A11209a Y = O 208a Y = O 209b Y = S 208b Y = S

[0176] (i) Lithium aluminium hydride (LAH) / tetrahydrofuran (THF); (ii) benzyl chloroformate, diisopropylethylamine / CH2Cl2; (iii) Acid (e.g., HCl in THF); (iv) hydrogenation on Pd-C in methanol or methanol / THF (v) dimethoxytrityl chloride (DMTr- Cl), 4-(dimethylamino)pyridine (DMAP) / pyridine; (vi) benzoyl chloride, 43sf-6685008Attorney Docket No.: 16553-20010.40 diisopropylethylamine (DIEA) / CH2Cl2; (vii) Acid (e.g., dichloroacetic acid or trichloroacetic acid in CH2Cl2); (viii) phosphitylation; (ix) phosphoramidite coupling and oxidation with water, iodine, and pyridine (Y = O) / sulfurization with 3- (dimethylaminomethylidene)amino-3H-1,2,4-dithiazole-3-thione (DDTT) (Y = S); (x) aqueous ammonia or aqueous ammonia and methylamine and (xi) (a) phosphorous oxychloride (POCl3), DIEA and (b) aqueous ammonia or aqueous ammonia and methylamine. The R or S isomer and the racemic mixtures of 209a, 209b, 211 and 212, are prepared from the corresponding stereochemically pure or racemic precursor compound 13, respectively. Compounds 209b has an additional stereo center at the phosphorothioate phosphorus. When prepared from a single stereoisomer of compound 13, two diastereomers of compounds 209b will be produced. When prepared from a racemic mixture of compound 13, all four stereoisomers of 209b will be produced. EXAMPLES

[0177] Abbreviations: BM (bone marrow); BMDC (bone marrow-derived dendritic cell); CDS (cytosolic DNA sensor); CLR (C-type lectin receptor); DAMP (damage-associated molecular pattern); DC (dendritic cell); DGPC (1-docosyl-sn-glycerol-3-phosphocholine); DGP (1-docosyl-sn-glycerol-3-phosphate); dLN (draining lymph node); ETL (ether lipid); 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-gamma); IL-1b / IL1-beta / IL-1β (Interleukin-1beta); KOdiA-PC (1-(Palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine); KOOA-PC (1- palmitoyl-(5-keto-8-oxo-6-octenoyl)-sn-glycero-3-phosphocholine); KP407 (poloxamer 407); LPC / Lyso PC (lysophosphatidylcholine); Lyso PC(22:0) (1-behenoyl-2-hydroxy-sn-glycero- 3-phosphocholine); LPS (lipopolysaccharide); MFI (mean fluorescence intensity); moDC (monocyte-derived dendritic cell); MPLA (monophosphoryl lipid A); NLR (NOD-like receptor); oxPAPC (oxidized 1-palmitoyl-2-arachidonyl-sn- glycero-3-phosphorylcholine); PAMP (pathogen-associated molecular pattern); PBMCs (peripheral blood mononuclear cells); PGPC (1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine); POVPC (1-palmitoyl-2- (5'-oxo-valeroyl)-sn-glycero-3-phosphocholine); PRR (pathogen recognition receptor); RLR 44sf-6685008Attorney Docket No.: 16553-20010.40 (RIG-I-like receptor); R848 (resiquimod); STING (stimulator of IFN genes); TNFα (tumor necrosis factor-alpha); TLR (toll-like receptor); and WTL (whole tumor lysate). Example P-1: Preparation of Lipid Nanoparticles Comprising a Compound of Formula (I)

[0178] This example describes preparation of lipid nanoparticles (LNPs) loaded with a a compound of Formula (I) in a microfluidic process. Materials and Methods

[0179] LNPs are synthesized using the NanoAssemblr® Ignite™ microfluidic instrument (Precision Nanosystems, Vancouver, BC, Canada). A kit containing GenVoy-ILM™ ionizable lipid mix (Precision Nanosystems, Vancouver, BC, Canada) is used to produce LNPs. The kit without mRNA is used to build empty LNP vehicles, and LNPs loaded with a compound of Formula (I) are generated by adding the appropriate compound of Formula (I) to a molar ratio of 10% of the total LNP content. LNPs are also produced using individual components (without a kit) to determine if loading a compound of Formula (I) into LNPs can be intentionally varied. Lipids are first dissolved in ethanol and then combined following the molarity percentages shown in Table III. Lipids in ethanol are combined with PBS, pH 7.4 at a 1:3 volumetric ratio. The NanoAssemblr® Ignite™ microfluidic instrument is programmed with a flow rate of 12 mL / min, a start waste of 0.35 mL, and an end waste of 0.05 mL. LNPs are washed in PBS, pH 7.4 to remove residual ethanol, and then are concentrated using Amicon 10K MWCO centrifugal filters by spinning at 2000xg for 30 min. Table III. LNP Formulations^^Percent molarity of components of different LNP formulations. LNP 2 and LNP 3 formulations share the same vehicle (LNP Vehicle 2).

[0180] Loading of a compound of Formula (I) into LNPs is assessed using HPLC. LNPs in PBS are frozen at -80°C, then lyophilized and stored at -20°C until they are quantified. LNPs are reconstituted in ethanol, and then mixed with water to dissolve the PBS. A seven 45sf-6685008Attorney Docket No.: 16553-20010.40 point standard curve of a compound of Formula (I) is prepared in ethanol with water and PBS is added to match sample preparation. Standards and samples are filtered through a 0.45 um filter prior to running on the HPLC. HPLC quantification is performed using an Agilent 1260 Infinity II HPLC equipped with a 1260 Infinity II Evaporative Light Scattering Detector (ELSD). A Luna 5µm NH2100Å, 150X4.6 mm LC Column (Phenomenex, Torrance, CA) with a column temperature of 30°C is used to detect samples. Two eluents are used: A, 100% water; and B, 100% acetonitrile. An initial mobile phase composed of 5% / 95% A / B is used to load the column, with a gradient reaching 24% / 76% A / B after 2.5 min. A more shallow gradient is used from 2.5 to 6 min, with A / B slowly reaching 25% / 75% during that time frame. A post time of 3 min is used to return the gradient to starting conditions prior to the next sample run. The flow rate is set to 1 mL / min, and the injection volume is 5 µL for samples and standards. The ELSD uses an evaporator temperature of 80°C, a nebulizer temperature of 30°C, and a nitrogen gas flow rate of 0.9 standard liters / min. Agilent CDS 2.6 software is used for HPLC instrument control, data acquisition, and processing.

[0181] The size of the LNPs is assessed using dynamic light scattering (DLS) on the NanoBrook Omni particle size and zeta potential analyzer (Brookhaven Instruments Corp., Holtsville, NY). Four measurements are made for each sample for 120 seconds each, with the first measurement made for each sample excluded from downstream analyses as time needed for sample equilibration. 46sf-6685008Attorney Docket No.: 16553-20010.40 BIOLOGICAL EXAMPLES Introduction

[0182] Dendritic cell (DC) hyperactivation is a cellular state defined by its ability to secrete IL-1β while remaining viable. IL-1βeta is an important cytokine in the induction of T cell responses. IL-1β is synthesized within a cell in a pro-form that is then cleaved by an activated inflammasome. Dendritic cell (DC) hyperactivation is a cellular state defined by its ability to secrete IL-1β while remaining viable. IL-1β is an important cytokine in the induction of T cell responses. IL-1β is synthesized within a cell in a pro-form that is then cleaved by an activated inflammasome. The mature (active) form of the cytokine is then released via pores formed as a result of inflammasome activation. Previously, inflammasome activation and pore formation was thought to result in pyroptotic cell death. However, hyperactivated DCs remain alive while secreting IL-1β. Additionally, hyperactive DCs have enhanced migratory capacity. As a result, these antigen presenting cells can traffic to lymph nodes where they can signal to other immune cell types and initiate adaptive immune responses. DC hyperactivation leads to enhanced T cell responses with an especially durable memory population (Zhivaki et al., Cell Reports, 33 (7), 2020, 108381). The enhanced T cell responses can be harnessed to treat diseases such as cancer.

[0183] Previous work identified oxidized lipids and lyso phosphatidylcholine (PC) lipids as chemical entities that induce DC hyperactivation. In particular, the compound 22:0 lyso PC (1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine) has been identified as a potent hyperactivating lipid. The inventors sought to develop additional lipids with increased stability and / or hyperactivating potency. Example B-1: Hyperactivation of Human moDCs Materials and Methods

[0184] Human monocyte-derived DC generation. Human monocytes are isolated from Leukopaks purchased from Miltenyi using the StraightFrom Leukopak CD14 microbead kit (Miltenyi). Isolations are completed following manufacturer’s instructions. Monocytes are then aliquoted and frozen in fetal bovine serum containing 10% dimethyl sulfoxide. For studies with monocyte-derived dendritic cell (moDC) cultures, monocytes are thawed and cultured in RPMI medium containing 10% FBS, 50 units / mL penicillin, 50 mg / mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 mM beta-mercaptoethanol, 10mM HEPES, and Gibco MEM non-essential amino acids (R10 media). To differentiate 47sf-6685008Attorney Docket No.: 16553-20010.40 monocytes into moDCs, recombinant human GM-CSF (50 ng / mL) and IL-4 (25 ng / mL) are added to R10 media. Cells are cultured for 6 days with GM-CSF and IL-4, with an additional cell feeding with R10 containing GM-CSF and IL-4 on day 3.

[0185] Lipid preparation. Lipid stocks are formulated at 650µg / mL lipid in 0.5 or 4% Kolliphor P407 (KP407) in PBS. Lipids are prepared from lyophilized stocks by mixing with a cold solution of KP407 at 1000rpm for 1 hour at RT. A 10X PBS solution is then added and the lipids are mixed at RT for an additional 30 min to make the 0.5 or 4% KP407 stock solution isotonic. Lipid stocks are then further diluted in PBS to treat cells.

[0186] Hyperactivation of human moDCs. Six days after differentiation, moDC are collected and counted. Cells are plated into 96-well flat-bottom plates at 1x105cells / well. Cells are treated with or without 1 µg / mL R848 (final) and with or without a lipid of the present disclosure (or a vehicle control). In some experiments, MCC950, an inhibitor of the NLRP3 inflammasome, is added at a final concentration of 10uM. In some experiments, nigericin, which causes NLRP3 inflammasome activation and subsequent pyroptosis, is used as a positive control at 20uM. Cells and stimuli totaled a final volume of 200uL / well.

[0187] After an overnight incubation, cells and culture supernatant are used for downstream readouts. One hundred and fifty microliters of cell supernatant are collected. Viability is measured using the CellTiter-Glo assay (Promega), which measures ATP content from cells. Fifty microliters of CellTiter-Glo reagent are added to 50uL of cells. Luminescence is quantified on a SpectraMax m5e plate reader using an integration time of 500 milliseconds. Viability data are set relative to control conditions where cells are treated with R848 only. To measure IL-1β secretion, the human IL-1β Lumit kit (Promega) is used to assay cell culture supernatant. Culture supernatant samples are incubated with enzyme- linked antibodies in a 384-well plate for 1 hour before addition of luminescent substrate. Samples are measured for luminescence with an integration time of 500 milliseconds. IL-1β concentrations of samples is determined by interpolation from a standard curve using 4- parameter logistic regression analysis. Studies are performed on several different human donor samples, and each biological condition is tested in triplicate. 48sf-6685008Attorney Docket No.: 16553-20010.40 Results

[0188] Preferred lipids of the present disclosure are expected to cause minimal cell toxicity, which is defined herein as greater than 75% cell viability, alone or in combination with R848, relative to the treatment with R848 alone. In addition, treatment of human moDCs with lipids of the present disclosure in combination with R848, but not in the absence of R848, is expected to induce secretion of IL-1β. Example B-2: Hyperactivation of Murine FLT3L-DCs Materials and Methods

[0189] Murine bone marrow-derived FLT3L-DC generation. Leg femur and tibia are removed from mice, cut with scissors, and flushed into sterile tubes. Bone marrow suspension is treated with ACK for 1 minute, then passed through a 40um cell strainer. Cells are counted and resuspended in media consisting of complete IMDM containing 10% FBS, penicillin and streptomycin, and supplements of L-glutamine and sodium pyruvate (I10). Cells are then plated at 8x106bone marrow cells / well in a P12 plate. Recombinant mouse FLT3L (Miltenyi) is added to cultures at 200ng / mL. Differentiated cells are used for subsequent assays on day 8. The efficiency of differentiation is monitored by flow cytometry using BD Symphony A3, and CD11c+MHC-II+cells are routinely above 80% of living cells. For each experiment, 5 mice are used to collect BM and to generate DCs.

[0190] Lipid preparation. Lipid stocks are formulated at 650µg / mL lipid in 4% Kolliphor P407 (KP407). Lipids are prepared from lyophilized stocks by mixing with a cold solution of KP407 at 1000rpm for 1 hour at RT. A 10X PBS solution is then added and the lipids are mixed at RT for an additional 30 min to make the 4% KP407 stock solution isotonic. Lipid stocks are then further diluted in PBS to treat cells.

[0191] Hyperactivation of murine bone marrow-derived FLT3L-DCs for cytokine assays. BMDCs are harvested on day 8 post differentiation, washed with PBS, and re-plated in FLT3L-containing I10 at a concentration of 1x105cells / mL. Cells are primed with or without 1µg / mL R848 (final) then treated with or without a lipid of the present disclosure (or a vehicle control). Forty-eight hours post stimulation, supernatants are collected for cytokine measurement. Viability is measured using the CellTiter-Glo assay (Promega), which measures ATP content from cells. Fifty microliters of CellTiter-Glo reagent are added to 50uL of cells. Luminescence is quantified on a SpectraMax m5e plate reader using an integration time of 500 milliseconds. Viability data is set relative to control conditions where 49sf-6685008Attorney Docket No.: 16553-20010.40 cells were treated with R848. IL-1β and TNFα cytokine secretion is measured using sandwich ELISAs (Invitrogen).

[0192] Hyperactivation of murine bone marrow-derived FLT3L-DCs for migration assays. BMDCs are harvested on day 8 post differentiation, washed with PBS, and re-plated in FLT3L-containing I10 at a concentration of 1x106cells / mL. For DC hyperactivation, 500 µl of R848 is added at a final concentration of 1µg / mL, and 500 µl of a compound of Formula (I) prepared in 4% KP407 is diluted to a final concentration of 41uM. Cells are incubated for 24 hours at 37°C on a tube rotator. Twenty-four hours post-stimulation, cells are washed with PBS and stained with CFSE (1:1000) for 30 min at 37°C in the dark. DCs are then counted and 1.10x106cells are injected subcutaneously (SC) in 100ul per mouse. 24 hours post-injection, the skin draining lymph nodes (dLN) are dissected. A single cell suspension is prepared, and cells are stained in PBS with Live Dead Fixable dye (ThermoFisher) for 20 min at 4°C. Cells are then washed again and stained for 20 min at 4°C in MACS buffer (PBS with 1% FCS and 2 mM EDTA) containing the following fluorescently conjugated antibodies: anti-CD11c and anti-I-A / I-E (MHC-II). To determine the absolute number of CD11c+MHC-IIhighamong living cells, countBright counting beads (ThermoFisher) are used, following the manufacturer’s protocol. Data are acquired on a BD FACS Symphony (Becton-Dickenson). Data are analyzed using FlowJo software (Tree Star). At least five mice are used for each experimental group.

[0193] DC migration to the skin dLN following chemical injection. To assess the migration of DCs from the skin to the skin draining lymph node (dLN), mice are subcutaneously injected with 100ug or 50ug or 20ug or 10ug or 1 ug of R848 in combination with 65ug or 50ug or 20ug of a compound of Formula (I) that is prepared in KP407 at 4.0% final. 24 hours post-injection, the skin draining lymph nodes (dLN) are dissected. A single cell suspension iss prepared, and cells are stained in PBS with Live Dead Fixable dye (ThermoFisher) for 20 min at 4°C. Cells are then washed again and stained for 20 min at 4°C in MACS buffer (PBS with 1% FCS and 2 mM EDTA) containing the following fluorescently conjugated antibodies: anti-CD11c and anti-I-A / I-E (MHC-II). To determine the absolute number of CD11c+MHC-IIhighamong living cells, countBright counting beads (ThermoFisher) are used, following the manufacturer’s protocol. Data are acquired on a BD FACS Symphony (Becton-Dickenson). Data are analyzed using FlowJo software (Tree Star). Five mice are used for each experimental group. 50sf-6685008Attorney Docket No.: 16553-20010.40

[0194] Ex vivo whole tumor lysate preparation. Syngeneic whole tumor lysates (WTL) are prepared from tumors explants of unimmunized tumor-bearing mice. Briefly, tumors from unimmunized mice bearing a tumor 10-12 mm of size are mechanically disaggregated using gentle MACS dissociator (Miltenyi Biotec) and enzymatically digested using the Tumor Dissociation Kit (Miltenyi Biotec) following the manufacturer’s protocol. After digestion, tumor cell suspensions are washed with PBS and passed through 70um and then 30um filters. Tumor cells are then counted and resuspended at 2x107cells / ml then lysed by 3-4 cycles of freeze-thawing. The lysed cells are further disrupted by repeatedly passing the material through 18G, then 21G, and finally 25G needles. Lysate is filtered again through 70um and 30um cell strainers and stored in aliquots at -80°C until use. Protein quantification in the lysates is performed using the BCA assay. WTL are used for immunotherapy at a concentration equivalent to 50ug per mouse.

[0195] Immunotherapy. C57BL / 6J mice are injected SC with 50,000 cells on the right upper back. Seven days later, mice are either injected SC with PBS or immunized distally from the tumor injection site with 50ug of WTL derived from syngeneic tumors in combination with the hyperactivating stimuli R848100ug / mouse + 22:0 lyso PC (65ug / mouse), R848100ug / mouse + DGP (65ug / mouse) or R848 (100ug / mouse) + a compound of Formula (I) (65ug / mouse). Mice receive 4 boost injections with same doses of WTL+ hyperactivating stimuli on days 11, 14, 18, and 21 post-tumor inoculation. Results

[0196] Murine DCs treated with R848 alone or in combination with the vehicle control KP407, or DCs treated with PBS and KP407, are not expected to induce IL-1β secretion. In contrast, DC treated with R848 + a compound of Formula (I) are expected to induce high levels of IL-1β secretion from viable cells. In addition, TNFα secretion, which is indicative of NF-kB activation by R848, is expected to be comparable when DCs are treated with R848 alone or R848 in combination with a compound of Formula (I). As such, lipids of the present disclosure are expected to induce a state of DC hyperactivation.

[0197] Moreover, R848 in combination with lipids of the present disclosure is expected to enhance DC migration from the skin to the dLN. Also, R848 in combination with lipids of the present disclosure are expected to induce potent anti-tumor responses in mice, when administered with a whole tumor lysate. 51sf-6685008Attorney Docket No.: 16553-20010.40 Example B-3: Hyperactivation to Induce T Cell Responses In Vivo Materials and Methods

[0198] Vaccine formulation. A lipid of the present disclosure is prepared from powder stock by resuspension in a cold solution of 4% KP407 at 650µg / mL. The mixture is stirred for 1 hour at RT using a magnetic stir bar at 1000rpm. After 1 hour, 10X PBS is added to make an isotonic solution and stirred at RT for another 30 min. The lipid is then combined with additional components depending on the treatment group. VacciGrade™ clinical grade R848 (InvivoGen) is prepared by dissolving in PBS at a stock of 10mg / mL for further combination depending on treatment group. For antigen, chicken ovalbumin (OVA) (InvivoGen) and SARS-COV-2 Spike (R&D Systems) protein are dissolved in PBS at 10mg / mL and 1mg / mL, respectively, to create stock solutions.

[0199] Immunization. Naïve C57BL / 6J mice are grouped into four different immunization conditions. As negative controls, mice received PBS injections or antigen only (200ug OVA and 5ug spike). A third group received 10ug of R848 in addition to the antigens. Finally, the fourth group received antigens, 10ug R848, and 65ug of a compound of Formula (I). Vaccinations are injected subcutaneously in the dorsal flank at 200uL / mouse. The same immunization treatments are repeated twice at one-week intervals. Seven days after the final immunizations (day 21 from start of study), mice are euthanized and draining lymph nodes are collected for downstream analyses. Organs are kept in MACS tissue storage solution (Miltenyi) at 4°C until processed. Four mice are allotted to each treatment group, but draining lymph nodes in the PBS treatment group are pooled.

[0200] ELISPOT analysis. Spleen dissociation kits (Miltenyi) are used to dissociate draining lymph nodes according to manufacturer’s protocol. Cells are counted and plated at 200,000 cells / well. IFNγ ELISPOT kits (R&D Systems) are used according to manufacturer protocol to study IFNγ T cells responses. To restimulate cells, 10µg / mL of OVA or Spike peptivators are added to cell cultures (Miltenyi). Restimulations are done in triplicate, and responses for each mouse in the study are averaged. Data graphed are the means from each mouse. 52sf-6685008Attorney Docket No.: 16553-20010.40 Results

[0201] Hyperactivating stimuli act on dendritic cells to potentiate T-cell responses to antigens. When immune cells are restimulated with peptide libraries derived from OVA or Spike proteins, most treatment groups are expected to have minimal increases in numbers of IFNγ spot-forming cells as compared to unstimulated cells. In contrast, mice immunized with antigen in combination with R848 and a compound of Formula (I) are expected to greater numbers of IFNγ spot-forming cells as compared to unstimulated cells. Example B-4: Hyperactivation of Human moDCs with Lipid Nanoparticles (LNPs)

[0202] This example describes the hyperactivation of human monocyte-derived dendritic cells (moDCs) with a TLR7 / 8 agonist in combination LNPs loaded with a lipid of the present disclosure. Materials and Methods

[0203] Human monocytes are isolated from Leukopaks purchased from Miltenyi Inc. (San Jose, CA) using the StraightFrom Leukopak CD14 microbead kit according to the manufacturer’s instructions. Monocytes are then aliquoted and frozen in fetal bovine serum containing 10% dimethyl sulfoxide. For studies with monocyte-derived dendritic cell (moDC) cultures, monocytes are thawed and cultured in RPMI medium containing 10% FBS, 50 units / mL penicillin, 50 mg / mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 mM beta-mercaptoethanol, 10mM HEPES, and Gibco MEM non-essential amino acids (R10 media). To differentiate monocytes into moDCs, recombinant human GM-CSF (50 ng / mL) and IL-4 (25 ng / mL) are added to R10 media. Cells are cultured for 6 days with GM- CSF and IL-4, with an additional cell feeding with R10 media containing GM-CSF and IL-4 on day 3.

[0204] Six days after differentiation, moDC are collected and counted. Cells are plated into 96-well flat-bottom plates at 1x105cells / well. Cells are treated with or without 1 µg / mL R848 (final) and with or without a compound of Formula (I) (or vehicle control). Hyperactivity induced by LNPs is measured using two assays. The CellTiter-Glo assay (Promega) detects ATP as a measure of cell viability. The IL-1β Lumit assay (Promega) measures IL-1β cytokine amount present in the moDC cell culture supernatant. Experimental conditions are tested in triplicate and the mean result from one donor is plotted. Example B-5: Hyperactivated moDCs Improve Th1 and Th17 Cytokine Responses 53sf-6685008Attorney Docket No.: 16553-20010.40 Materials and Methods

[0205] Monocyte Isolation, Storage, and Differentiation into moDC. Human leukapheresis blood products are freshly obtained from AllCells via same day or overnight shipping at 4°C. Miltenyi StraightFrom Leukopak CD14 MicroBead isolation kits are used according to manufacturer instructions to isolate CD14+ monocytes. Briefly, leukopaks ranging in quantity from 5x109to 1x1010total nucleated cells are evenly aliquoted into 50mL conical tubes. Cells are incubated with CD14 microbeads and then loaded on the MultiMACS using the program “Possel2”. After washing out the negative population, CD14+ cells are eluted from the columns and counted. An aliquot of monocytes is used to stain for purity of cells to ensure that isolated live cells are >80% CD11c+CD14+ by flow cytometry. Cells are centrifuged at 400xg for 5 min and resuspended at 1.25x107cells / mL in freezing media (FBS containing 10% DMSO) and aliquoted at 5mL per cryogenic freezing vial. Vials are placed in CoolCell freezing containers and placed in a -80°C freezer and stored no longer than 1 week. Frozen cells are transferred for long-term storage to the vapor phase of a liquid nitrogen freezer until use.

[0206] To differentiate monocytes into moDC, vials of cells are taken out of the liquid nitrogen freezer and quickly thawed in a 37°C water bath. Cells are diluted ten-fold in warmed R10++ media containing 50 units / mL benzonase. R10++ media is composed of RPMI 1640 media containing 10% fetal bovine serum, 50 units / mL penicillin, 50 mg / mL streptomycin, 2mM L-glutamine, 1mM sodium pyruvate, 50mM beta-mercaptoethanol, 10mM HEPES, and 1% Gibco MEM non-essential amino acids. After gentle mixing by inversion of tubes, cells are incubated at 37°C for 10 min. Cells are pelleted at 400xg for 5 min and supernatant is aspirated. Cells are resuspended in R10++ media and counted on the MoxiGo by diluting an aliquot in PBS containing a final concentration of 2.5µg / mL propidium iodide. Cells are plated in T75 culture flasks at 3-6x107cells per flask in a total of 20mL R10++ media containing a final concentration of 50ng / mL GM-CSF and 25ng / mL IL- 4. Three days after the start of culture, an additional 10mL R10++ containing 50ng / mL GM- CSF and 25ng / mL IL-4 is added to each flask.

[0207] Six days after start of differentiation, R10++ media containing cells are placed into 50mL conical tubes. Flasks are washed with an additional 10mL PBS and also collected. Cells are centrifuged at 400xg for 5 min and supernatant is aspirated. Cells are resuspended in R10++ media and counted on the MoxiGo by diluting an aliquot in PBS containing a final 54sf-6685008Attorney Docket No.: 16553-20010.40 concentration of 2.5µg / mL propidium iodide. Cells are then adjusted to 1x106cells / mL in R10++ media.

[0208] Quality Control Staining of moDC Differentiation. An aliquot of cells is stained to ensure monocyte differentiation into moDC. Cells are plated into a 96-well V-bottom plate at 1x105cells / well. Cells are also plated for fluorescence minus one (FMO) staining controls. Cells are washed twice with PBS by centrifuging at 400xg for 4 min and removing supernatant. Live / Dead NIR viability dye is diluted 1000-fold in PBS and added to samples at 100µL / well. For the control FMO sample not receiving live / dead staining, PBS is used to resuspend pelleted cells. Cells are incubated for 10 min at 4°C. Fc block is diluted in FACS buffer 100-fold and added to wells at 100µL / well. Cells are incubated at 4°C for another 10 min. Cells are then pelleted at 400xg for 4 min and supernatant is discarded. Cells are then stained with antibody staining cocktail containing CD11c (FITC), CD209 (APC), and SIRPa (PE-Cy7) at 200-fold dilutions. Antibodies are diluted in a buffer mixture composed of FACS buffer and Brilliant Buffer Stain (mixed at 1:1 volume ratio). For FMO controls, staining mixtures are prepared with one target antibody removed to define negative cell populations. Cells are incubated at 4°C for 15 min in antibody cocktails. After incubation, cells are washed twice with FACS buffer by topping up wells to 200µL / well, centrifuging at 400xg for 4 min, and discarding supernatant. When cells are analyzed by flow cytometry immediately, they are resuspended in 150µL / well FACS buffer. When cells are not analyzed immediately, cells are fixed using a solution of 4% PFA in PBS by resuspending cells at 100µL / well. Cells are incubated in the dark at RT for 20 min. The fixation buffer is diluted with 100µL / well FACS buffer, and cells are pelleted at 400xg for 4 min. Supernatant is collected and disposed of as hazardous waste. Cells are washed with 200µL / well FACS buffer and centrifuged at 400xg for 4 min. Supernatant is discarded and cells are resuspended in a final volume of 150µL / well FACS buffer. Fixed cells are stored at 4°C protected from light until flow cytometry analysis. From each sample, 75µL is acquired.

[0209] LDH Release Assay. Lactate dehydrogenase (LDH) reagents are stored in -20°C and allowed to return to RT before starting the assay. About 45 min prior to supernatant collection, 20µL / well of 10X lysis buffer from LDH release assay kit is added to LDH maximum control wells. Lysis is allowed to occur for 45 min in the 37°C incubator before plates are spun at 400xg for 4 min. During the incubation, the Substrate Mix is resuspended in 11.4mL of UltraPure Distilled Water, and 600µL of Assay Buffer Stock Solution is added to it to make the Reaction Mixture. After vortexing, Reaction Mixture is kept at RT in the 55sf-6685008Attorney Docket No.: 16553-20010.40 dark until use. Freshly collected cell culture is used for the LDH release assay. 25µL of sample supernatant is added to the wells of a 96-well flat bottom ELISA plate, and an additional 25µL of kit Reaction Mixture is added to wells. Samples are gently tapped to mix and left to incubate at RT protected from light for 30 min. 25µL of kit Stop Solution is added to each well and samples are gently tapped to mix them. Absorbance is read on the Spectramax M3 plate reader at 490nm and 680nm.

[0210] To analyze data, 680nm absorbance readings are subtracted from 490nm values. Values from blank media control samples are used to remove background signal. Samples are divided by the mean of the LDH maximum samples (lysed cells controls) and multiplied by 100 to obtain percent cytotoxicity for a sample. The percent cytotoxicity value is subtracted from 100 to obtain the percent viability.

[0211] Memory CD4+ T Cell Isolation. The CD14- fraction of the leukapheresis product are collected and used for further isolation of memory CD4+ T cells. First, approximately 5x109total nucleated cells or less of the blood product are pelleted at 200xg for 10 min. The cells are resuspended in two 50mL conical tubes each containing 25mL in volume. Cells are then incubated with microbeads targeting red blood cells and granulocytes at 4°C for 15 min. Cells are loaded on equilibrated MultiMACS columns using the “Deplete” program. Columns are washed twice with separation buffer and the flow-through of peripheral blood mononuclear cells (PBMCs) are collected and counted. The Memory CD4+ T Cell Isolation Kit from Miltenyi is then used to isolate memory CD4+ T cells from the PBMCs. After following the manufacturer’s protocol for antibody and microbead incubations, cells are loaded onto equilibrated MultiMACS columns using the “Deplete” program. After 2 column washes using separation buffer, the negative fraction is collected and counted. An aliquot is stained to confirm the purity of the memory CD4 T cell population. Cells are centrifuged at 400xg for 5 min and resuspended at 1.25x107cells / mL in freezing media (FBS containing 10% DMSO) and aliquoted at 3mL per cryogenic freezing vial. Vials are placed in CoolCell freezing containers and placed in a -80°C freezer for no longer than 1 week. Frozen cells are transferred for long-term storage to the vapor phase of a liquid nitrogen freezer until use.

[0212] Treatments of Cells For Analysis of Cytokine Secretion. Human moDCs are plated at 5x104cells / well in 96-well U-bottom tissue culture plates in R10++ media by adding 50µL of cells per well. To each well, 50µL of memory CD4 T cells at 2.5x106cells / mL is added. For control conditions where only moDC or only CD4 T cells are plated, 56sf-6685008Attorney Docket No.: 16553-20010.40 the missing cells are replaced with an equivalent volume of R10++ media. After adding all treatments, cell cultures have a total volume of 200µL / well.

[0213] Lyophilized R848 stocks are prepared as described above and frozen at -80°C for storage. An aliquot of frozen R848 is thawed and further diluted to 22.8µM in R10++ (8µg / mL R848). A portion of diluted R848 is combined with MCC950 to reach a concentration of 80µM MCC950. Another portion of diluted R848 is combined with anti-IL- 1β blocking antibody to reach a concentration of 80µg / mL anti-IL-1β antibody. Cell cultures receive 25µL of these mixes depending on desired treatment. For wells not receiving R848 treatment, an equivalent volume of R10++ media is added to wells. For these three chemicals, final concentrations in the wells are 2.85µM R848, 10µM MCC950, and 10µg / mL anti-IL- 1β.

[0214] A 4.44% solution of KP407 dissolved in sterile, distilled water is prepared at a temperature of 4°C. Lipid from powder is prepared by adding KP407 solution to a compound of Formula (I). Using a magnetic stir bar, the solution is agitated at 1000rpm for 1 hour at RT. Sterile 10X PBS is added to the solution resulting in a 1X PBS concentration. The mixture is then stirred at 1000rpm for another 30 min at RT. The final stock concentration of the compound of Formula (I) is 650µg / mL in 4% KP407 in 1X PBS. The stock solution is then further diluted in PBS to achieve an 8X concentration of the compound of Formula (I) from the final target of 41.3µM. The solution is then added to cell cultures at 25µL / well. For wells not receiving a compound of Formula (I), 4% KP407 in 1X PBS is diluted in PBS and added to cells as a vehicle control. Volume of vehicle control matches the volume carrying a compound of Formula (I) in the study.

[0215] To activate memory CD4+ T cells, an anti-CD3 antibody is used to induce T cell receptor signaling. Anti-CD3 antibody is diluted in R10++ media to 0.4ng / mL and added to wells at 50µL / well. The final concentration in the wells after addition is 0.1ng / mL. For control conditions not receiving anti-CD3 antibody, an equivalent volume of R10++ media is added to wells.

[0216] Plates are sealed with Breathe-Easy Membranes plate sealers and placed in a 37°C, 5% CO2incubator for two days. For study readouts, plates are centrifuged at 400xg for 4 min and cell culture supernatant is collected in 96-well U-bottom plates.

[0217] CellTiter-Glo 2.0 Cell Viability Assay. To measure cell viability, CellTiter-Glo 2.0 Cell Viability Assay kit from Promega is used according to the manufacturer’s instructions. Briefly, frozen reagent is thawed at 4°C and aliquoted. Aliquots are stored at 57sf-6685008Attorney Docket No.: 16553-20010.40 4°C in the dark. After removing 150µL of the cell culture volume for use in other assays, cells are lysed with 50µL / well CellTiter-Glo reagent by pipetting up and down. After incubating in the dark at RT for 10 min, samples are transferred to white opaque 96-well plates. Luminescence is measured for 500ms / well using a Spectramax M3 plate reader. Luminescence values are normalized to the mean values of control samples and multiplied 100-fold to report values as percentage viability.

[0218] Lumit IL-6, IL-1β, IFNγ Immunoassays. To determine the concentrations of IL-6, IL-1β, and IFNγ secreted into the cell culture supernatant, Lumit Immunoassays are used. Conceptually, two antibodies are used to bind to the target cytokine in the sample, and each antibody is tethered to a subunit of an enzyme. Binding of the two antibodies to the target cytokine enables the subunits to be in close enough proximity to bind and function. A substrate for the enzyme is then added to create a luminescent signal that indicates the cytokine concentration in the supernatant.

[0219] The IL-6 and IFNγ kit standards are prepared in cell culture medium (R10++) starting at 25,000pg / mL with two-fold dilutions to 24.4pg / mL. The IL-1β standard is prepared by diluting in R10++ medium to 40,000pg / mL. The top standard is then serially diluted two-fold to 39pg / mL. Blank controls are included for all standards. Sample supernatants are diluted 5-fold in R10++ for the IL-6 and IFNγ assay. Sample supernatants are used undiluted for the IL-1β immunoassay.

[0220] To prepare antibodies for each assay, recommended protocols from the manufacturer are followed to dilute antibodies in R10++ media to reach a 2-fold concentrate. Antibodies are combined with sample supernatant or standards (12.5µL of each) by pipetting into white opaque 384-well plates. Samples are then incubated at 37°C for 1 hour. Samples are then allowed to cool to RT in the dark while substrate is prepared. For every 3,040µL of Detection Buffer B used, 175µL of Detection Substrate B is added. Substrate is mixed by briefly vortexing and added at 6.25µL / well. On the Spectramax M3 plate reader, sample luminescence is recorded at 500ms / well. Prism software is used to construct a standard curve using 4-parameter logistics analysis. Interpolated results are corrected for sample dilution.

[0221] LEGENDplex Assay. To detect multiple cytokines of interest (IL-4, IL-5, IL-13, IL-17A, IL-17F, and IL-22), the predefined LEGENDplex Human Th Cytokine Panel from Biolegend is utilized. Conceptually, each targeted cytokine has antibodies bound to a unique bead population defined by bead size and APC fluorescence signal. When incubated with sample supernatant, cytokines are bound by the antibodies. Biotinylated secondary antibodies 58sf-6685008Attorney Docket No.: 16553-20010.40 are then added to bind the cytokines of interest, and streptavidin-bound PE is then incubated with samples. Using flow cytometry, each bead population specific for a cytokine can be defined based on bead size and APC signal intensity. The PE fluorescence signal on beads then indicates the amount of cytokine present in the sample.

[0222] To run the assay, samples are diluted 2-fold in Assay Buffer. Standards are prepared according to manufacturer instructions. Lyophilized standard is reconstituted in 250µL Assay Buffer. After a 10 min incubation at RT, this top standard concentration is serially diluted 4-fold to make an 8-point standard curve with the last point set as blank buffer. In each well of a V-bottom plate, 25µL Assay Buffer is added with 25µL of sample or standard. Beads are vortexed thoroughly and added at 25µL / well. Samples are incubated in the dark at RT for 2 hours on an orbital shaker set at 200-300rpm. The beads are then pelleted at 250xg for 5 min and resuspended in 200µL / well 1X wash buffer. After pelleting beads again, samples are resuspended in 25µL / well Detection Antibodies. Samples are incubated for 1 hour at RT in the dark on the orbital shaker. Without washing, 25µL / well of streptavidin-PE is added to samples and incubated at RT in the dark for 30 min on the orbital shaker. After the incubation, beads are washed again and resuspended in 150µL / well wash buffer. Samples are analyzed the same day using the Novocyte Quanteon flow cytometer.

[0223] Raw data is exported as FCS files and uploaded to the LEGENDplex Data Analysis Software Suite from Qognit for analysis. Five parameter logistic curves are constructed from the standards, and sample results are interpolated using the cloud software. Sample dilutions are incorporated into the analysis, and correct bead population gating is verified on all samples. Results are exported from the cloud software as a Microsoft Excel file.

[0224] Data Analysis. Data are analyzed and graphed using Microsoft Excel and GraphPad Prism software. Data are reported in graphs as mean with ranges indicating SDs. Graphs depict data from individual donors, and data points represent biological replicates.

[0225] For statistical analyses, ordinary two-way ANOVA is conducted, followed by Tukey’s multiple comparisons test with a single pooled variance. Statistical testing is completed using Prism software. Example B-6: Incorporation of a Lipid of Formula (I) into Lipid Nanoparticles in the Presence or Absence of mRNA Encoding an Antigen to Improve Vaccine Response 59sf-6685008Attorney Docket No.: 16553-20010.40

[0226] This example describes the preparation and testing of lipid nanoparticles (LNPs) containing a lipid of the present disclosure, loaded with or without mRNA encoding an antigen. The lipid-containing LNPs are suitable for hyperactivating mammalian dendritic cells in combination with a small molecule pathogen-associated molecular pattern (PAMP) (e.g., R848). Hyperactivation of DCs induces pro-inflammatory cytokines and adds to their cytokine repertoire IL-1β, a critical cytokine for memory T cell formation. The aims of these experiments are to: 1) test whether the compound of Formula (I) can effectively hyperactivate mammalian dendritic cells when delivered via an LNP, and 2) determine if this DC hyperactivation will enhance de novo T cell activation and memory T cell reactivation in mice. Materials and Methods

[0227] LNP Production. LNPs are prepared by combining the following components with or without mRNA: (8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid), 1-octylnonyl ester (SM102) or other ionizable lipid, 1,2-distearoyl-sn-glycero-3- phosphocholine (DPSC) or other structural lipid, cholesterol, and 1,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) (or other PEG / stealth lipid), in the presence or absence of a a compound of Formula (I). Table 6-1 (with mRNA) and Table 6-2 (without mRNA) detail exemplary molar percentages of each component for exemplary LNP formulations.

[0228] LNPs are synthesized using the NanoAssemblr Ignite instrument (Precision Nanosystems). Lipids are first dissolved in ethanol and then combined to final concentrations as shown in Table 6-1 and Table 6-2. Lipids in ethanol are combined with sodium citrate buffer (pH 4) with or without mRNA at a 1:3 volumetric ratio, at a flow rate of 12 mL / min. mRNA (e.g., OVA mRNA) is added into sodium citrate buffer for loading into LNPs. After synthesis, LNPs are washed using 10 volumes of PBS, pH 7.4 to remove residual ethanol, and then concentrated using Amicon 10K MWCO centrifugal filters. 60sf-6685008Attorney Docket No.: 16553-20010.40 Table 6-1. Percent Molarity of Lipids in LNP Formulations Containing mRNATable 6-2. Percent Molarity of Lipids in LNP Formulations Lacking mRNA

[0229] LNP Characterization. Loading of the compound of Formula (I) into LNPs is assessed using HPLC. LNPs are dissolved in ethanol to release lipids prior to quantification. Standards prepared with known quantities of lipid are filtered through a 0.45 um filter. HPLC quantification is performed using an Agilent 1260 Infinity II HPLC equipped with a 1260 Infinity II Evaporative Light Scattering Detector. Column chemistry, mobile phases, and gradients depend on the structure of the lipid that is being detected.

[0230] Loading of mRNA into LNPs is quantified using a RiboGreen assay (ThermoFisher) following the manufacturer’s protocol. Samples are diluted to fall within the range of the standard curve. LNPs are lysed using Triton X-100 to assess encapsulation of mRNA into LNPs. Both total mRNA and encapsulated mRNA are quantified. The size and charge of the LNPs is assessed using dynamic light scattering (DLS) and zeta potential on the NanoBrook Omni (Brookhaven).

[0231] Human moDC Production and Hyperactivation. Human monocytes are isolated from Leukopaks purchased from Miltenyi using the StraightFrom Leukopak CD14 microbead kit (Miltenyi) according to the manufacturer’s instructions. Monocytes are then aliquoted and frozen in fetal bovine serum containing 10% dimethyl sulfoxide. To differentiate monocyte- derived dendritic cell (moDC) cultures, monocytes are thawed and cultured in RPMI medium containing 10% FBS, 50 units / mL penicillin, 50 mg / mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 mM beta-mercaptoethanol (R10 media). To differentiate 61sf-6685008Attorney Docket No.: 16553-20010.40 monocytes into moDCs, recombinant human GM-CSF (50 ng / mL) and IL-4 (25 ng / mL) are added to R10 media. Cells are cultured for 6 days with GM-CSF and IL-4, with an additional cell feeding with R10 containing GM-CSF and IL-4 on day 3.

[0232] After differentiation, moDCs are plated into a 96-well flat-bottom plates at 1x105cells / well. Cells are treated with or without 1 μg / mL R848 (final), with or without LNPs loaded with a compound of Formula (I) (or vehicle control) in the presence or absence of mRNA. Hyperactivation induced by LNPs is measured after 24-48 hrs in culture with the LNPs. DC hyperactivation in response to the compounds of Formula (I) in LNPs is assessed as IL-1β secretion by live cells. Cell Viability is assessed using the LDH CyQuant Kit (Invitrogen) following manufacturer’s instructions. IL-1β and IL-6 Lumit assays (Promega) are used to measure IL-1β and IL-6 present in moDC cell culture supernatants. In addition to IL-1β secretion, expression of moDC activation markers is quantified using flow cytometry. moDCs collected after 24-48 hrs in culture with LNPs, are stained with Live / Dead stain to identify live cells, followed by staining with antibodies specific for CD11c, CD40, CD86, HLA-DR, and HLA-ABC. Live cells are selected for analysis, and then CD11c+cells are assessed for antigen presentation was assessed using antibodies specific for HLA-DR and HLA-ABC to determine if hyperactivation interferes with antigen presentation. Activation is assessed by staining for CD40 and CD86 to determine if hyperactivation increases expression of activation markers.

[0233] Immunization. C57BL / 6J mice are immunized subcutaneously with LNPs as detailed in Table 6-3. OVA mRNA-loaded LNPs are used to deliver transcripts for production of OVA antigen in vivo, in combination with LNPs containing either a compound of Formula (I) and R848 delivered via LNP or exogenously. The OVA mRNA dose is fixed at 1-5 µg / mouse, while the compound of Formula (I) is dosed at 50-100 µg / mouse, and the R848 is dosed at 10-50 µg / mouse (Table 6-3). Mice are given a primary immunization on Day 0, with a boost immunization with the same doses on Day 7. For assessment of short- term effector responses, on Day 14, blood and secondary lymphoid organs are collected. For assessment of long-term memory responses, on Day 40, blood and secondary lymphoid organs are collected. Blood is collected for measurement of antibody and T-cell responses. Serum is collected from the blood using serum separation tubes, while blood for cellular analysis is collected using K2EDTA tubes. After blood collection, mice are euthanized, and the draining lymph nodes and spleen are collected and processed into single cell suspensions. 62sf-6685008Attorney Docket No.: 16553-20010.40 Table 6-3. Immunization Groups

[0234] Assessment of Effector and Memory Immune Responses. OVA-specific T cells in the blood and draining lymph node of mice are assessed 14 and 40 days post primary immunization. CD8+ T cells specific for an MHC-I-restricted T cell epitope of ovalbumin (OVA) are quantified in the blood and dLN by SIINFEKL-tetramer staining. Briefly, red blood cells are lysed using a RBC lysis buffer, with lysis completed twice to remove all RBCs in the blood. Cells are washed, then stained for viability (Live / Dead), SIINFEKL- tetramer binding (MBL), and CD3, CD4, and CD8 expression. Cells are fixed with 4% paraformaldehyde, and counting beads are added to permit total cell counts to be determined. Data are collected using a BD FACS Symphony and analyzed using Flowjo (BD).

[0235] The frequency of T effector and T memory cells are assessed by flow cytometry, in the blood and draining lymph node of mice receiving OVA LNP immunization. Briefly, cell suspensions are stained with CD3, CD4, CD8, CD62L and CD44 antibodies to measure the frequency of T effector cells (CD44LowCD62Lneg) and T memory cells (CD44highCD62L+) using a BD FACS Symphony instrument and data is analyzed using Flowjo (BD).

[0236] OVA-specific antibodies in serum of mice receiving OVA LNP immunization are assessed 14 and 40 days post primary immunization. OVA-specific total IgG, IgG1, and IgG2b are assessed by ELISA. Briefly, ELISA plates are coated with 10 µg / mL Endofit Ovalbumin (InvivoGen) overnight, then washed and blocked with 2% bovine serum albumin. 63sf-6685008Attorney Docket No.: 16553-20010.40 Plates are washed again, and then serum is added to the plates at a 1:500 dilution, followed by 1:5 dilutions for a total of 7 serum dilutions. Samples are washed, then incubated with detection antibodies specific for IgG, IgG1, or IgG2b, which are conjugated to HRP (Southern Biotech), to detect total, Th2-skewed, and Th1-skewed OVA-specific antibodies, respectively. Plates are washed, then incubated with TMB, and stop solution is added once color development is complete.

[0237] OVA-specific T cell responses are determined from assessment of secondary lymphoid organs. Post-immunization, draining lymph nodes and spleens are collected from the mice at early (Day 14) and late (Day 40) time points. Harvested lymph nodes and spleens are dissociated into single cell suspensions, which are used in ELISPOT assays. ELISPOT is used to detect IFNγ and IL-5 secretion by T cells, which are indicative of Th1 and Th2 responses, respectively. Cells from draining lymph nodes and spleens are plated in RPMI medium containing 10% FBS, 100 units / mL penicillin-streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 mM beta-mercaptoethanol, 10 mM HEPES, and 1X Gibco MEM non-essential amino acids (R10 media). Cells are plated at 200,000 cells / well in a 96 well ELISPOT plate and restimulated with 10µg / mL OVA peptivator or 1µg / mL OVA peptide antigens. As controls, additional plated cells are left unstimulated or stimulated with irrelevant antigens (not used in vaccination). Completion of the assay results in spots that can be visually quantified where cytokines were secreted by T cells, as a method to quantify the number of T cells responding to restimulation. Example B-7: Assessment of Effect of R848 + Lipid Adjuvant on Immunogenicity of Inactivated Influenza A Virus Vaccine

[0238] PR8 is a live virus that is propagated in specific pathogen-free chicken embryonated eggs. This strain was isolated in 1934 from a human patient in Puerto Rico and was deposited by the Centers for Disease Control and Prevention. The PR8 influenza virus strain is an attenuated virus, and is unable to replicate in humans, as a result of over 100 passages in each of mice, ferrets and embryonated chicken eggs. According to the ATCC, there have been no reports of laboratory-acquired infections with strain A / PR / 8 / 34. Material and Methods

[0239] PR8 Virus. Influenza A virus (H1N1), also called PR8 (ATCC VR-95PQ) is a high-titer, purified, live virus suspended in 1X PBS + 0.1% sodium azide. This product was prepared from ATCC VR-95 via purification through sucrose gradient centrifugation and is 64sf-6685008Attorney Docket No.: 16553-20010.40 devoid of cellular debris and contaminants. Influenza PR8 strain has been used for the past 30 years to produce inactivated influenza vaccines. Virus inactivation was done by heating the live PR8 virus at 56 degrees for 45 min. Inactivated virus was titrated for HA content and used for immunization.

[0240] Immunization. Ten BALB / c mice per group are immunized subcutaneously on D- 14 with either PBS or inactivated virus alone or inactivated virus in combination with R848 + a compound of Formula (I), or inactivated virus plus AddaVax™ squalene-oil-in-water adjuvant (InvivoGen), which has similar formulation to the MF59® adjuvant of Novartis. Mice are then challenged on day 0 with 1000 PFU of live PR8 virus (ATCC) injected intranasal (25uL in one nostril) on D0. Details for each group are listed in Table 7-1. Table 7-1. Immunization Groups

[0241] Clinical Score Assessment. Out of the 10 mice per group: 5 mice per group are assessed for body weight changes throughout the study, as well as mortality and morbidity using a murine scoring system as shown in Table 7-2. Four areas of focus for clinical score are established: appearance, mobility, breathing and eyes. Each mouse is assigned a clinical score that would be the mean of the scores in all four areas. Body weight and clinical scores are assessed daily starting on D0 and until D10. Table 7-2. Murine Behavioral and Clinical Score65sf-6685008Attorney Docket No.: 16553-20010.40

[0242] Blood Collection. Blood is collected from all mice on D-1 (one day before challenge). Briefly, BALB / c mice are placed under Isoflurane for approximately 10 min for anesthesia. Using a 21G needle, mice are gently poked through the skin to the submandibular space to induce bleeding. Five-Six drops are collected in a mini collect K2EDTA blood collection tube. Blood samples are transported back to Corner Therapeutics lab at RT and are allowed to warm up to RT for at least 15 min. To process the blood, 1 mL of RBC lysis buffer is added to 150µl of whole blood into each well of 96 deep-well plate. Samples are then mixed with multi-channel pipette and incubated at RT for 20 min. 600µl of PBS is then added to all wells and samples are centrifuged at 600xg for 5 min. This step is repeated twice, then pellets are resuspended in 200µl of PBS and transferred to 96-well V bottom plate for tetramer staining.

[0243] Tetramer Staining. 200µL of processed blood samples (as described above) are plated into 96-well V bottom plates. Cells are spun at 400xg for 4 min then washed with PBS at least once before cell staining. Cells are then resuspended in 100ul PBS containing Live / Dead Aqua (1:1000) and incubated for 20 min at 4oC. Cells are then washed and resuspend in 100ul of FACS buffer containing Fc block (1:100) for 10 min, then washed again with 100ul FACS buffer. For tetramer staining, cells are resuspended in 100µl of FACS buffer containing H-2Kd Influenza NP Tetramer-TYQRTRALV-PE and H-2Kd Influenza NP Tetramer-TYQRTRALV-APC (1:20) and incubated at 37oC for 2 hours. For surface markers staining, cells are washed with 100µl FACS buffer and spun for 4 min at 400xg. The cell pellet was stained with anti-mouse CD3, anti-mouse CD4 and anti-mouse CD8 antibodies for 20 min at 4oC. Cells are then washed and resuspended in 100µl of 4% PFA to fix the cells for 20 min at RT. After fixation, cells are washed twice with FACS buffer and kept in 4oC overnight in 150µl FACS buffer. Prior to sample acquisition on the Symphony A3, Countbright counting beads are added to the samples to allow the measurement of absolute number of cells.

[0244] Antibody Responses. HA-specific antibodies and NP-specific antibodies in the serum of mice receiving immunization are assessed on D-1 (one day before challenge with live PR8 virus). NP and HA-specific total IgG are assessed using ELISA. Briefly, ELISA 66sf-6685008Attorney Docket No.: 16553-20010.40 plates are coated with 1ug / mL HA or NP recombinant proteins overnight, then washed and blocked with 2% bovine serum albumin. Plates are washed again, and then serum is added to the plates at a 1:500 dilution, followed by 1:5 dilutions completed for a total of 7 serum dilutions tested. Samples are washed, then incubated with detection antibody specific for IgG conjugated to HRP (Southern Biotech). Plates are washed, then incubated with TMB, and stop solution is added once color development is complete.

[0245] BAL Collection. The bronchoalveolar lavage fluid (BAL) is collected from the lungs for viral load testing by inserting a catheter into the trachea of mice. During the procedure, 3 washes with PBS is performed by injecting 1mL of PBS each time to wash the airways and retrieve the fluid sample. The collected BAL from each mouse is centrifuged for 5 min at 4oC before freezing at -80oC.

[0246] Plaque Assay. The viral load in the BAL is measured by plaque assay. For the plaque assay, 3.5x105MDCK cells are seeded into wells of two 6-well plates per BAL. The cells are cultured in MDCK media (EMEM+ 10%FBS + Pen-Strep) for 16hrs. The wells are washed once with 3ml 1X PBS and once with 3ml of infection media (IMDM + Pen-Strep+ 0.2% BSA + 1mg / ml of TPCK-trypsin). Ten-fold dilutions of BAL (up to 10-6dilution) in infection media are prepared and MDCK cells are infected with 500ml of each virus dilution in duplicate. MDCK cells are infected for 1hr at 37oC. After infection, inoculum is removed and 3ml of 0.3% agarose prepared in infection media is layered on top of the cells. The cells are incubated at 37oC for 72hrs, followed by addition of 4% paraformaldehyde to fix the cells. One hour after fixation, the agarose layer is gently removed without disrupting the monolayer. Then, 2ml of 2% crystal violet are added to stain the live cells for 10 min at RT. The plates are washed under running tap water to remove the crystal violet stain. Plates are allowed to dry overnight. The plaques are counted and based on the dilution of the virus the plaque forming units per ml is calculated.

[0247] HA ELISA. For quantitative determination of Influenza Hemagglutinin (HA) levels in the BAL, Influenza A H1N1 (A / Puerto Rico / 8 / 1934) Hemagglutinin / HA ELISA Pair Set (Sino Biologics) is used for a sandwich ELISA.

[0248] Data Analysis. In in vivo studies, n refers to the number of animals per condition. Graphical data are shown as mean values with error bars indicating the SD of 5 mice / group. Each symbol represents one mouse. All experiments are analyzed using Prism 7 (GraphPad Software). Statistical differences are calculated by one-way ANOVA with Tukey post-hoc 67sf-6685008Attorney Docket No.: 16553-20010.40 test. P values of < 0.05 (*), < 0.01 (**) or < 0.001 (***); < 0.0001 (****) indicate significant differences between groups.

[0249] Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the examples herein should not be construed as limiting the scope of the present disclosure. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. 68sf-6685008

Claims

Attorney Docket No.: 16553-20010.40 CLAIMS We claim:

1. A composition comprising a compound of Formula (I):A1is independently O, S, -NRA-, or -CH2-; A2and A3are independently O, S, -NRA-, -CH2-, -NRA-C(=G)-O-, -NRA-C(=G)-NRA-,- O-C(=G)-NRA-, -O-C(=G)-O-, -O-C(=S)-O-, -S- or -S-S- ; wherein each RAis independently H, C1-C4 alkyl, benzyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA; R2is H, C1-C4alkyl, , C2-C4alkenyl, C2-C4alkynyl, C3-C10cycloalkyl, C3-C10cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA;; R3is C10-C30 n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3or -CH2CH3and p is an integer from 1 to 6 inclusive;sf-6685008Attorney Docket No.: 16553-20010.40 each R5is independently C1-C4alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and all stereoisomers thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3or - CH2CH3 and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; and a TLR agonist; or a composition comprising a compound of Formula (II):A1and A2are independently O, S, -NRA-, or -CH2-, A3is independently O, S, -NRA-, -CH2-, or -NH-C(=O)-O- , and RAis H or C1-C4alkyl;sf-6685008Attorney Docket No.: 16553-20010.40 R2is H, C1-C4alkyl, -(C=G)-NH2, -(C=G)-NH(R5) , -(C=G)-N(R5)2, or -CH2-C6H5, wherein G is O, S, or NH; R3is C10-C30 n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3or -CH2CH3and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a pharmaceutically acceptable salt thereof; or a stereoisomer thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3or - CH2CH3and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; and a TLR agonist.

2. The composition of claim 1, wherein the TLR agonist comprises a TLR7 / 8 agonist.

3. The composition of claim 1 or claim 2, wherein R3is C18-C22 n-alkyl or C21-C24 n- alkyl.

4. The composition of any one of claims 1-3, wherein R3is C16-C20n-alkyl.

5. The composition of any one of claims 1-4, further comprising an antigen.

6. The composition of any one of claims 1-5, further comprising dendritic cells.sf-6685008Attorney Docket No.: 16553-20010.40 7. A composition comprising a compound of Formula (I):A1is independently O, S, -NRA-, or -CH2-; A2and A3are independently O, S, -NRA-, -CH2-, -NRA-C(=G)-O-, -NRA-C(=G)-NRA-,- O-C(=G)-NRA-, -O-C(=G)-O-, -O-C(=S)-O-, -S- or -S-S- ; wherein each RAis independently H, C1-C4alkyl, benzyl, C2-C4alkenyl, C2-C4alkynyl, C3-C10cycloalkyl, C3-C10cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA; R2is H, C1-C4 alkyl, , C2-C4 alkenyl, C2-C4 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA;; R3is C10-C30 n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or -CH2CH3 and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4 alkyl; R6is O or S;sf-6685008Attorney Docket No.: 16553-20010.40 or a protonated or deprotonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and all stereoisomers thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3or - CH2CH3and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; and an antigen or a composition comprising a compound of Formula (II):sf-6685008Attorney Docket No.: 16553-20010.40 R3is C10-C30n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or -CH2CH3 and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4 alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a pharmaceutically acceptable salt thereof; or a stereoisomer thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or - CH2CH3 and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; and an antigen.

8. The composition of claim 7, further comprising dendritic cells.

9. The composition of claim 7 or claim 8, further comprising a TLR agonist.

10. The composition of claim 9, wherein the TLR agonist comprises a TLR7 / 8 agonist.

11. The composition of claim 10, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

12. The composition of claim 10, wherein the TLR7 / 8 agonist comprises resiquimod (R848).sf-6685008Attorney Docket No.: 16553-20010.40 13. A composition comprising a compound of Formula (I):A1is independently O, S, -NRA-, or -CH2-; A2and A3are independently O, S, -NRA-, -CH2-, -NRA-C(=G)-O-, -NRA-C(=G)-NRA-,- O-C(=G)-NRA-, -O-C(=G)-O-, -O-C(=S)-O-, -S- or -S-S- ; wherein each RAis independently H, C1-C4alkyl, benzyl, C2-C4alkenyl, C2-C4alkynyl, C3-C10cycloalkyl, C3-C10cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA; R2is H, C1-C4 alkyl, , C2-C4 alkenyl, C2-C4 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA;; R3is C10-C30 n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or -CH2CH3 and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4 alkyl; R6is O or S;sf-6685008Attorney Docket No.: 16553-20010.40 or a protonated or deprotonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and all stereoisomers thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3or - CH2CH3and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; and dendritic cells; or a composition comprising a compound of Formula (II):sf-6685008Attorney Docket No.: 16553-20010.40 R3is C10-C30n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or -CH2CH3 and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4 alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a pharmaceutically acceptable salt thereof; or a stereoisomer thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or - CH2CH3 and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; and dendritic cells.

14. The composition of claim 13, further comprising a TLR agonist.

15. The composition of claim 14, wherein the TLR agonist comprises a TLR7 / 8 agonist.

16. The composition of any one of claims 13-15, further comprising an antigen.

17. A composition of any one of claims 13-16, wherein R3is C22n-alkyl.

18. The composition of any one of claims 1-17, wherein the TLR agonist is a small molecule with a molecule weight of 900 daltons or less. sf-6685008Attorney Docket No.: 16553-20010.40 19. The composition of any one of claims 1-18, wherein the TLR agonist comprises a TLR7 / 8 agonist.

20. The composition of claim 19, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

21. The composition of claim 19, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

22. The composition of any one of claims 1-21, wherein the TLR7 / 8 agonist does not inhibit NLR family pyrin domain containing 3 (NLRP3).

23. The composition of any one of claims 1-22, wherein the antigen is present in a biological sample obtained from an individual.

24. The composition of claim 23, wherein the biological sample comprises biopsy tissue.

25. The composition of claim 23, wherein the biological sample comprises cells.

26. The composition of claim 23, wherein the biological sample does not comprise cells.

27. The composition of claim 23, wherein the biological sample comprises pus from an abscess.

28. The composition of any one of claims 1-27, wherein the antigen comprises a proteinaceous antigen.

29. The composition of claim 28, wherein the antigen comprises a tumor antigen.sf-6685008Attorney Docket No.: 16553-20010.40 30. The composition of claim 29, wherein the tumor antigen comprises a synthetic or recombinant neoantigen.

31. The composition of claim 29, wherein the tumor antigen comprises a tumor cell lysate.

32. The composition of claim 28, 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.

33. The composition of claim 32, wherein the microbial antigen comprises a purified or recombinant surface protein.

34. The composition of claim 32, wherein the microbial antigen comprises an inactivated, whole virus.

35. The composition of any one of claims 1-34, wherein the composition does not comprise liposomes.

36. The composition of any one of claims 1-34, wherein the composition does not comprise LPS or MPLA.

37. The composition of any one of claims 1-36, wherein the composition does not comprise oxPAPC or a species of oxPAPC, optionally wherein the composition does not comprise HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC, and / or PGPC.

38. The composition of any one of claims 1-37, 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)] .sf-6685008Attorney Docket No.: 16553-20010.40 39. The composition of any one of claims 1-38, further comprising an adjuvant, wherein the adjuvant comprises an aluminum salt adjuvant, a squalene-in-water emulsion, a saponin, or combinations thereof.

40. A pharmaceutical formulation comprising the composition of any one of claims 1- 39 and a pharmaceutically acceptable excipient.

41. A method for production of hyperactivated dendritic cells, the method comprising contacting the dendritic cells with a composition comprising effective amounts of a composition comprising a compound of Formula (I):A1is independently O, S, -NRA-, or -CH2-; A2and A3are independently O, S, -NRA-, -CH2-, -NRA-C(=G)-O-, -NRA-C(=G)- NRA-,-O-C(=G)-NRA-, -O-C(=G)-O-, -O-C(=S)-O-, -S- or -S-S- ; wherein each RAis independently H, C1-C4 alkyl, benzyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA; R2is H, C1-C4alkyl, , C2-C4alkenyl, C2-C4alkynyl, C3-C10cycloalkyl, C3-C10cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atomsf-6685008Attorney Docket No.: 16553-20010.40 selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA;; R3is C10-C30 n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N- (CH2)P-, where RNis -CH3or -CH2CH3and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and all stereoisomers thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3or - CH2CH3and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; and a TLR7 / 8 agonist for production of hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1beta without undergoing pyroptosis; or a method for production of hyperactivated dendritic cells, the method comprising contacting the dendritic cells with a composition comprising effective amounts of a composition comprising a compound of Formula (II):sf-6685008Attorney Docket No.: 16553-20010.40A1and A2are independently O, S, -NRA-, or -CH2-, A3is independently O, S, -NRA-, -CH2-, or -NH-C(=O)-O- , and RAis H or C1-C4alkyl; R2is H, C1-C4 alkyl, -(C=G)-NH2, -(C=G)-NH(R5) , -(C=G)-N(R5)2 , or -CH2-C6H5 , wherein G is O, S, or NH; R3is C10-C30 n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3or -CH2CH3and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a pharmaceutically acceptable salt thereof; or a stereoisomer thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3or - CH2CH3and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; and a TLR7 / 8 agonist for production of hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1beta without undergoing pyroptosis.

42. The method of claim 41, wherein the dendritic cells are contacted ex vivo with the composition of any one of claims 1-39 or the formulation of claim 40.sf-6685008Attorney Docket No.: 16553-20010.40 43. The method of claim 41, wherein the dendritic cells are contacted in vivo with the formulation of claim 40.

44. A pharmaceutical formulation comprising at least 103, 104, 105or 106of the hyperactivated dendritic cells produced by the method of any one of claims 41-43, and a pharmaceutically acceptable excipient.

45. A method of stimulating an immune response against an antigen, comprising administering an effective amount of the formulation of claim 40 to an individual in need thereof to stimulate the immune response against the antigen.

46. A method of treating cancer, comprising administering an effective amount of the formulation of claim 40 to an individual in need thereof to treat the cancer.

47. A method of inhibiting abnormal cell proliferation, comprising administering an effective amount of the formulation of claim 40 to an individual in need thereof to inhibit abnormal cell proliferation.

48. A method of treating an infectious disease, comprising administering an effective amount of the formulation of claim 40 to an individual in need thereof to treat the infectious disease.

49. Use of the formulation of claim 40 for inducing an immune response against the antigen in an individual in need thereof.

50. Use of the formulation of claim 40 for inducing an anti-tumor immune response in an individual in need thereof, wherein the individual is or was tumor-bearing.

51. Use of the formulation of claim 40 for inducing an anti-microbe immune response in an individual in need thereof, wherein the individual is infected with the microbe or has not been exposed to the microbe.sf-6685008Attorney Docket No.: 16553-20010.40 52. The composition, formulation, method or use of any one of claims 23-51, wherein the individual is a mammalian subject.

53. The composition, formulation, method or use of any one of claims 23-52, wherein the individual is a human subject.

54. A method of preparing an immunogenic composition, the method comprising: a) depleting leukocytes from a suspension of cells prepared from a tumor to obtain a tumor cell-enriched suspension; b) lysing cells from the tumor cell-enriched suspension to obtain a tumor cell lysate; and c) contacting the tumor cell lysate with a composition comprising a compound of Formula (I):A1is independently O, S, -NRA-, or -CH2-; A2and A3are independently O, S, -NRA-, -CH2-, -NRA-C(=G)-O-, -NRA-C(=G)- NRA-,-O-C(=G)-NRA-, -O-C(=G)-O-, -O-C(=S)-O-, -S- or -S-S- ; wherein each RAis independently H, C1-C4 alkyl, benzyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C10cycloalkyl, C3-C10cycloalkenyl, or a three- to ten-membered heterocyclicsf-6685008Attorney Docket No.: 16553-20010.40 ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA; R2is H, C1-C4 alkyl, , C2-C4 alkenyl, C2-C4 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA;; R3is C10-C30n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N- (CH2)P-, where RNis -CH3 or -CH2CH3 and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4 alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and all stereoisomers thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or - CH2CH3 and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; and a toll-like receptor (TLR) agonist to obtain the immunogenic composition; or a method of preparing an immunogenic composition, the method comprising: a) depleting leukocytes from a suspension of cells prepared from a tumor to obtain a tumor cell- enriched suspension; b) lysing cells from the tumor cell-enriched suspension to obtain a tumor cell lysate; and c) contacting the tumor cell lysate with a composition comprising a compound of Formula (II):sf-6685008Attorney Docket No.: 16553-20010.40A1and A2are independently O, S, -NRA-, or -CH2-, A3is independently O, S, -NRA-, -CH2-, or -NH-C(=O)-O- , and RAis H or C1-C4 alkyl; R2is H, C1-C4 alkyl, -(C=G)-NH2, -(C=G)-NH(R5) , -(C=G)-N(R5)2 , or -CH2-C6H5 , wherein G is O, S, or NH; R3is C10-C30 n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3or -CH2CH3and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4 alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a pharmaceutically acceptable salt thereof; or a stereoisomer thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or - CH2CH3 and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S;sf-6685008Attorney Docket No.: 16553-20010.40 and a toll-like receptor (TLR) agonist to obtain the immunogenic composition.

55. The method of claim 54, wherein the TLR agonist comprises a TLR7 / 8 agonist.

56. The method of claim 54 or claim 55, wherein the leukocytes are depleted in step a) by negative selection using an anti-CD45 antibody.

57. The method of any one of claims 54-56, wherein the cells are lysed in step b) by one or more freeze-thaw cycles.

58. The method of any one of claims 54-57, wherein R3in Formula (I) or Formula (II) is C18-C22alkyl or C18-C24alkyl.

59. The method of any one of claims 54-57, wherein R3in Formula (I) or Formula (II) is C16-C20 alkyl.

60. The method of any one of claims 54-57, wherein R3is C21-C24 alkyl.

61. The method of any one of claims 54-60, wherein the TLR7 / 8 agonist is a small molecule with a molecule weight of 900 daltons or less.

62. The method of claim 61, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

63. The method of claim 61, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

64. The method of any one of claims 54-63, wherein the TLR7 / 8 agonist does not inhibit NLR family pyrin domain containing 3 (NLRP3).sf-6685008Attorney Docket No.: 16553-20010.40 65. The method of any one of claims 54-64, further comprising before step a) obtaining a sample from the tumor from a mammalian subject with cancer and preparing the suspension of cells from the sample.

66. An immunogenic composition prepared by the method of any one of claims 54- 65.

67. A method of eliciting an anti-cancer immune response, the method comprising: administering to a mammalian subject with cancer an effective amount of the immunogenic composition of claim 66.

68. The method of claim 67, wherein the anti-cancer immune response comprises cellular immune response.

69. The method of claim 67, wherein the anti-cancer immune response comprises cancer antigen-induced IL-1beta secretion and / or activation of CD8+ T lymphocytes.

70. The method of any one of claims 67-69, wherein the cancer is a non-hematologic cancer.

71. The method of claim 70, wherein the non-hematologic cancer is a carcinoma, a sarcoma, or a melanoma.

72. The method of any one of claims 67-69, wherein the cancer is a lymphoma.

73. A method of treating cancer, the method comprising: a) preparing an immunogenic composition comprising a tumor cell lysate, a composition comprising a compound of Formula (I):sf-6685008Attorney Docket No.: 16553-20010.40A1is independently O, S, -NRA-, or -CH2-; A2and A3are independently O, S, -NRA-, -CH2-, -NRA-C(=G)-O-, -NRA-C(=G)- NRA-,-O-C(=G)-NRA-, -O-C(=G)-O-, -O-C(=S)-O-, -S- or -S-S- ; wherein each RAis independently H, C1-C4alkyl, benzyl, C2-C4alkenyl, C2-C4alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA; R2is H, C1-C4 alkyl, , C2-C4 alkenyl, C2-C4 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA;; R3is C10-C30 n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N- (CH2)P-, where RNis -CH3 or -CH2CH3 and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4 alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and all stereoisomers thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; orsf-6685008Attorney Docket No.: 16553-20010.40 ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or - CH2CH3 and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; and a toll-like receptor (TLR) agonist, wherein the tumor cell lysate is or has been prepared from a sample of a tumor obtained from the mammalian subject with cancer; and b) administering to the subject an effective amount of the immunogenic composition; or a method of treating cancer, the method comprising: a) preparing an immunogenic composition comprising a tumor cell lysate, a composition comprising a compound of Formula (II):A1and A2are independently O, S, -NRA-, or -CH2-, A3is independently O, S, -NRA-, -CH2-, or -NH-C(=O)-O- , and RAis H or C1-C4alkyl;sf-6685008Attorney Docket No.: 16553-20010.40 R2is H, C1-C4alkyl, -(C=G)-NH2, -(C=G)-NH(R5) , -(C=G)-N(R5)2, or -CH2-C6H5, wherein G is O, S, or NH; R3is C10-C30 n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3or -CH2CH3and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a pharmaceutically acceptable salt thereof; or a stereoisomer thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3or - CH2CH3and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; and a toll-like receptor (TLR) agonist, wherein the tumor cell lysate is or has been prepared from a sample of a tumor obtained from the mammalian subject with cancer; and b) administering to the subject an effective amount of the immunogenic composition.

74. The method of claim 73, wherein the TLR agonist comprises a TLR7 / 8 agonist.

75. The method of claim 73 or claim 74, wherein R3in Formula (I) or Formula (II) is a C18-C22alkyl chain or a C18-C24alkyl chain.

76. The method of claim 73 or claim 74, wherein R3in Formula (I) or Formula (II) is C16-C20 alkyl.sf-6685008Attorney Docket No.: 16553-20010.40 77. The method of claim 73 or claim 74, wherein R3is C21-C24alkyl.

78. The method of any one of claims 73-77, wherein the TLR7 / 8 agonist is a small molecule with a molecule weight of 900 daltons or less.

79. The method of claim 78, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

80. The method of claim 79, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

81. The method of any one of claims 73-80, further comprising administering to the subject an effective amount of an additional therapeutic agent.

82. The method of claim 81, wherein the additional therapeutic agent comprises one or more of the group consisting of an immune checkpoint inhibitor, an antineoplastic agent, and radiation therapy.

83. A composition comprising a compound of Formula (I):sf-6685008Attorney Docket No.: 16553-20010.40 A1is independently O, S, -NRA-, or -CH2-; A2and A3are independently O, S, -NRA-, -CH2-, -NRA-C(=G)-O-, -NRA-C(=G)- NRA-,-O-C(=G)-NRA-, -O-C(=G)-O-, -O-C(=S)-O-, -S- or -S-S- ; wherein each RAis independently H, C1-C4 alkyl, benzyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C10cycloalkyl, C3-C10cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA; R2is H, C1-C4alkyl, , C2-C4alkenyl, C2-C4alkynyl, C3-C10cycloalkyl, C3-C10cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA;; R3is C10-C30 n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N- (CH2)P-, where RNis -CH3or -CH2CH3and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4 alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and all stereoisomers thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or - CH2CH3 and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; and a pathogen recognition receptor (PRR) agonist or a composition comprising a compound of Formula (II):sf-6685008Attorney Docket No.: 16553-20010.40A1and A2are independently O, S, -NRA-, or -CH2-, A3is independently O, S, -NRA-, -CH2-, or -NH-C(=O)-O- , and RAis H or C1-C4 alkyl; R2is H, C1-C4 alkyl, -(C=G)-NH2, -(C=G)-NH(R5) , -(C=G)-N(R5)2 , or -CH2-C6H5 , wherein G is O, S, or NH; R3is C10-C30n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or -CH2CH3 and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4 alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a pharmaceutically acceptable salt thereof; or a stereoisomer thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or - CH2CH3and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; orsf-6685008Attorney Docket No.: 16553-20010.40 v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; and a pathogen recognition receptor (PRR) agonist.

84. The composition of claim 83, 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).

85. The composition of claim 83, wherein the PRR agonist is an agonist of a cytosolic DNA sensor (CDS) or a stimulator of IFN genes (STING).

86. The composition of claim 83, wherein the PRR agonist comprises one or more of R848, TL8-506, LPS, Pam2CSK4, and ODN 2336.

87. The composition of any one of claims 83-86, further comprising an antigen.

88. The composition of any one of claims 83-87, further comprising dendritic cells.

89. A pharmaceutical formulation comprising the composition of any one of claims 83-88 and a pharmaceutically acceptable excipient.

90. A pharmaceutical formulation comprising a compound of Formula (I):sf-6685008Attorney Docket No.: 16553-20010.40A1is independently O, S, -NRA-, or -CH2-; A2and A3are independently O, S, -NRA-, -CH2-, -NRA-C(=G)-O-, -NRA-C(=G)- NRA-,-O-C(=G)-NRA-, -O-C(=G)-O-, -O-C(=S)-O-, -S- or -S-S- ; wherein each RAis independently H, C1-C4 alkyl, benzyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C10cycloalkyl, C3-C10cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA; R2is H, C1-C4 alkyl, , C2-C4 alkenyl, C2-C4 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA;; R3is C10-C30n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N- (CH2)P-, where RNis -CH3 or -CH2CH3 and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4 alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and all stereoisomers thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or - CH2CH3 and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; orsf-6685008Attorney Docket No.: 16553-20010.40 vi) R6is S; and a pharmaceutically acceptable excipient; or a pharmaceutical formulation comprising a composition comprising a compound of Formula (II):A1and A2are independently O, S, -NRA-, or -CH2-, A3is independently O, S, -NRA-, -CH2-, or -NH-C(=O)-O- , and RAis H or C1-C4alkyl; R2is H, C1-C4 alkyl, -(C=G)-NH2, -(C=G)-NH(R5) , -(C=G)-N(R5)2 , or -CH2-C6H5 , wherein G is O, S, or NH; R3is C10-C30 n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3or -CH2CH3and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a pharmaceutically acceptable salt thereof; or a stereoisomer thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; orsf-6685008Attorney Docket No.: 16553-20010.40 iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3or - CH2CH3 and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; and a pharmaceutically acceptable excipient.

91. The pharmaceutical formulation of claim 90, wherein R3is C22 n-alkyl.

92. A composition for hyperactivation of human dendritic cells, comprising a composition comprising a compound of Formula (I):A1is independently O, S, -NRA-, or -CH2-; A2and A3are independently O, S, -NRA-, -CH2-, -NRA-C(=G)-O-, -NRA-C(=G)- NRA-,-O-C(=G)-NRA-, -O-C(=G)-O-, -O-C(=S)-O-, -S- or -S-S- ; wherein each RAis independently H, C1-C4alkyl, benzyl, C2-C4alkenyl, C2-C4alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, or a three- to ten-membered heterocyclicsf-6685008Attorney Docket No.: 16553-20010.40 ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA; R2is H, C1-C4 alkyl, , C2-C4 alkenyl, C2-C4 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA;; R3is C10-C30n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N- (CH2)P-, where RNis -CH3 or -CH2CH3 and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4 alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and all stereoisomers thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or - CH2CH3 and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; and a pathogen recognition receptor (PRR) agonist, wherein the alkyl chain is a C22 n-alkyl chain, and wherein the composition is effective for achieving a higher level of dendritic cell hyperactivation than a comparator composition comprising PGPC in place of the compound of Formula (I); or a composition for hyperactivation of human dendritic cells, comprising a composition comprising a compound of Formula (II):sf-6685008Attorney Docket No.: 16553-20010.40A1and A2are independently O, S, -NRA-, or -CH2-, A3is independently O, S, -NRA-, -CH2-, or -NH-C(=O)-O- , and RAis H or C1-C4 alkyl; R2is H, C1-C4 alkyl, -(C=G)-NH2, -(C=G)-NH(R5) , -(C=G)-N(R5)2 , or -CH2-C6H5 , wherein G is O, S, or NH; R3is C10-C30n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or -CH2CH3 and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4 alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a pharmaceutically acceptable salt thereof; or a stereoisomer thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or - CH2CH3and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; orsf-6685008Attorney Docket No.: 16553-20010.40 v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; and a pathogen recognition receptor (PRR) agonist, wherein the alkyl chain is a C22 n-alkyl chain, and wherein the composition is effective for achieving a higher level of dendritic cell hyperactivation than a comparator composition comprising PGPC in place of the compound of Formula (II).

93. The composition of claim 92, wherein R3is C22 n-alkyl.

94. The composition of claim 92 or claim 93, wherein the higher level of dendritic cell hyperactivation comprises induction of IL-1beta secretion from the human dendritic cells in vitro at a level that is at least 2, 3 or 4 fold higher when contacted with the composition comprising the compound of Formula (I) or Formula (II) and the PRR agonist than when contacted with the comparator composition comprising the PGPC and the PRR agonist, wherein the PRR agonist is LPS.

95. The composition of claim 94, wherein the concentration of the compound of Formula (I) or Formula (II) and the concentration of the PGPC are the same concentration in a range of from about 10 µM to about 80 µM, and the LPS is present at a concentration of 1 µg / ml in both the composition and the comparator composition.

96. The composition of claim 94 or claim 95, wherein the higher level of dendritic cell hyperactivation comprises a lipid activity index for IL-1beta secretion from the human dendritic cells for the composition comprising the compound of Formula (I) or Formula (II) and the PRR agonist that is at least 4, 5 or 6 fold higher in activity units than that of the comparator composition comprising the PGPC and the PRR agonist.

97. The composition, formulation, method or use of any one of claims 6, 8-53, 88, or 89, wherein the dendritic cells are human dendritic cells.sf-6685008Attorney Docket No.: 16553-20010.40 98. The composition, formulation, method or use of any one of claims 6, 8-53, 88, or 89, wherein the dendritic cells are canine dendritic cells.

99. The composition, method or use of any one of claims 92-98, wherein the dendritic cells are present in a composition comprising peripheral blood mononuclear cells (PBMCs).

100. The composition, method or use of any one of claims 41-53 or 92-99, wherein the hyperactivated dendritic cells secrete one or both of IFNγ and TNFα.

101. The composition, formulation, method or use of any one of claims 1-100, further comprising a surfactant.

102. The composition, formulation, method or use of claim 101, wherein the surfactant comprises a non-ionic surfactant.

103. The composition, formulation, method or use of claim 102, wherein the non-ionic surfactant comprises an ethylene oxide-propylene oxide copolymer (a poloxamer).

104. The composition, formulation, method or use of claim 102, wherein the non-ionic surfactant comprises one or more of Poloxamer 407, Poloxamer 188, and P123.

105. The composition, formulation, method or use of claim 102, wherein the non-ionic surfactant comprises Poloxamer 407.

106. The composition, formulation, method or use of any one of claims 102-105, wherein i) the compound of Formula (I) or Formula (II) is dissolved in an alcohol to form an alcohol solution of the compound of Formula (I) or Formula (II); ii) the alcohol solution of the compound of Formula (I) or Formula (II) is mixed with the non-ionic surfactant to form a mixture; and iii) the alcohol is evaporated from the mixture to form particles comprising the compound of Formula (I) or Formula (II) and the non-ionic surfactant.sf-6685008Attorney Docket No.: 16553-20010.40 107. The composition, formulation, method or use of any one of claims 102-106, wherein the non-ionic 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).

108. The composition, formulation, method or use of any one of claims 102-107, wherein the compound of Formula (I) or Formula (II) and non-ionic surfactant are present in particles with a diameter of about 1000 to 15,000 nanometers, optionally with a diameter of about 5000 nanometers.

109. An isolated compound of Formula (I):A1is independently O, S, -NRA-, or -CH2-; A2and A3are independently O, S, -NRA-, -CH2-, -NRA-C(=G)-O-, -NRA-C(=G)- NRA-,-O-C(=G)-NRA-, -O-C(=G)-O-, -O-C(=S)-O-, -S- or -S-S- ; wherein each RAis independently H, C1-C4 alkyl, benzyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA;sf-6685008Attorney Docket No.: 16553-20010.40 R2is H, C1-C4alkyl, , C2-C4alkenyl, C2-C4alkynyl, C3-C10cycloalkyl, C3-C10cycloalkenyl, or a three- to ten-membered heterocyclic ring with at least one atom selected from N, O or S, wherein the cycloalkyl, cycloalkenyl, and heterocyclic ring are optionally substituted with =O, =S, or =NRA;; R3is C10-C30n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N- (CH2)P-, where RNis -CH3or -CH2CH3and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a salt thereof, such as a pharmaceutically acceptable salt thereof; and all stereoisomers thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or - CH2CH3 and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S; an isolated compound of Formula (II):sf-6685008Attorney Docket No.: 16553-20010.40A1and A2are independently O, S, -NRA-, or -CH2-, A3is independently O, S, -NRA-, -CH2-, or -NH-C(=O)-O- , and RAis H or C1-C4alkyl; R2is H, C1-C4alkyl, -(C=G)-NH2, -(C=G)-NH(R5) , -(C=G)-N(R5)2, or -CH2-C6H5, wherein G is O, S, or NH; R3is C10-C30 n-alkyl; where R4is H, (RN)3N+-(CH2)P- , (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3 or -CH2CH3 and p is an integer from 1 to 6 inclusive; each R5is independently C1-C4alkyl; R6is O or S; or a protonated or deprotonated form thereof; or a pharmaceutically acceptable salt thereof; or a stereoisomer thereof; wherein i) at least one of A1, A2, or A3is S, -NRA-, or -CH2-; or ii) G is S or NH; or iii) R4is (RN)2N-(CH2)P-; (RN)HN-(CH2)P- or H2N-(CH2)P-, where RNis -CH3or - CH2CH3and p is an integer from 1 to 6 inclusive; or iv) R4is (CH3CH2)3N+-(CH2)P- and p is an integer from 1 to 6 inclusive; or v) R4is (CH3)3N+-(CH2)- or (CH3)3N+-(CH2)Q-, where Q is an integer from 3 to 6 inclusive; or vi) R6is S.

110. A compound of claim 109, wherein said compound is isolated.

111. A composition comprising the compound of claim 109 or claim 110 and a pharmaceutically acceptable excipient.sf-6685008Attorney Docket No.: 16553-20010.40 112. The composition of claim 111, further comprising a surfactant.

113. The composition of claim 112, wherein the surfactant is selected from the group consisting of a non-ionic surfactant, a wetting agent, P407, P188, polysorbate 80, a thickening agent, and carboxymethyl cellulose.

114. The composition of any one of claims 111-113, comprising particles having a diameter less than between about 5 microns and about 20 microns (D50< 5 microns to 20 microns), where the particles comprise a lipid and a non-ionic surfactant.

115. The composition of any one of claims 111-114, wherein the pharmaceutically acceptable excipient comprises phosphate-buffered saline.

116. The composition of any one of claims 111-115, wherein the pharmaceutically acceptable excipient comprises an aqueous solution of an ethylene oxide-propylene oxide copolymer (a poloxamer), or further comprises an ethylene oxide-propylene oxide copolymer..

117. The composition of any one of claims 111-114, wherein the pharmaceutically acceptable excipient comprises phosphate-buffered saline and at least one of Poloxamer 407, Poloxamer 188, and P123.

118. The composition of any one of claims 111-117, wherein said composition is sterile.

119. An article of manufacture comprising a container enclosing a liquid formulation of the composition of any one of claims 111-118 and a pharmaceutically acceptable excipient.

120. The article of manufacture of claim 119, wherein the container is a syringe.

121. The article of manufacture of claim 120, wherein the syringe is further contained within an injection device.sf-6685008Attorney Docket No.: 16553-20010.40 122. The article of manufacture of claim 121, wherein the injection device is an auto- injector.

123. A composition comprising a compound of Formula (I) or Formula (II); or a protonated or deprotonated form thereof where possible, or a pharmaceutically acceptable salt thereof, or a stereoisomer tehreof; and at least one further lipid, wherein the at least one further lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, a further phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof.

124. The composition of claim 123, wherein the compound of Formula (I) or Formula (II) and the at least one further lipid are part of a lipid nanoparticle (LNP).

125. The composition of claim 123 or claim 124, further comprising an antigen.

126. The composition of any one of claims 123-125, further comprising dendritic cells.

127. The composition of any one of claims 123-126, further comprising a TLR agonist.

128. The composition of any one of claims 123-127, further comprising a TLR7 / 8 agonist.

129. The composition, method, or use of any one of claims 5-12, 16-40, 45-82, or 87-89, wherein the antigen comprises one or more viral antigens.

130. The composition, method, or use of claim 129, wherein the one or more viral antigens comprise one or both of influenza A and influenza B antigens.

131. The composition, method, or use of claim 130, wherein the one or both of influenza A and influenza B antigens comprise one or both of hemagglutinin and nucleoprotein.sf-6685008Attorney Docket No.: 16553-20010.40 132. The composition, method, or use of any one of claims 129-131, wherein the viral antigens comprise inactivated virions, optionally wherein the inactivated virions comprise inactivated, split virions.

133. The composition, method, or use of any one of claim 130-132, comprising both influenza A and influenza B antigens of an H1N1 influenza A virus, an H3N2 influenza A virus, a Victoria lineage influenza B virus, and a Yamagata lineage influenza B virus.sf-6685008

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