Ethers for superactivation of mammalian dendritic cells

Abstract

The present disclosure relates to ether lipid (ETL) compounds, such as ether phospholipid (ETPL) compounds, and their use in the superactivation of mammalian dendritic cells, such as human or canine dendritic cells. The present disclosure also relates to compositions comprising an ETL, such as an ETPL, and one or more of a pathogen recognition receptor agonist, an antigen, and mammalian dendritic cells, as well as methods of making and using the compositions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 417,667, filed October 19, 2022, U.S. Provisional Patent Application No. 63 / 441,697, filed January 27, 2023, and U.S. Provisional Patent Application No. 63 / 451,885, filed March 13, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to ether lipid compounds, including ether phospholipid compounds, and their use in the hyperactivation of mammalian dendritic cells, such as human or canine dendritic cells. The present disclosure also relates to compositions comprising ether lipid compounds, such as ether phospholipid compounds, and one or more of a pathogen recognition receptor agonist, an antigen, and human or canine dendritic cells, as well as methods of making and using the compositions. [Background technology]

[0003] Typically, maturation of dendritic cells (DCs) with vaccine adjuvants such as Toll-like receptor agonists does not lead to the secretion of IL-1β. In some situations, such as inflammasome activation, IL-1β secretion occurs, but at the cost of DC death by a lytic process of cell death called pyroptosis (Evavold et al., J Mol Biol, 430(2):217-237, 2018). However, when DCs are matured using pathogen-associated molecular pattern (PAMP)-containing molecules, lipopolysaccharide (LPS), and damage-associated molecular pattern (DAMP)-containing molecules, such as PGPC (1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine), they produce and secrete IL-1β without pyroptosis, resulting in viable DCs characterized as superactivated (Zanoni et al., Science, 352(6290):1232-1236, 2016). Indeed, in a mouse model, superactivated DCs have been shown to have an improved ability to induce immune responses compared with cells activated with LPS alone (Zhivaki et al., Cell Rep, 33(7):108381, 2020). However, little is known about the stimulatory factors effective in superactivating human DCs.

[0004] Therefore, there is a need in the art for the identification of PAMPs and DAMPs suitable for the hyperactivation of human DCs. Furthermore, it is desirable to identify alternatives to the use of LPS and PGPC for the hyperactivation of mammalian DCs. In particular, LPS (endotoxin) is a potent PAMP, but its use in humans is contraindicated due to its potential to induce septic shock. Summary of the Invention

[0005] The present disclosure relates to ether lipid (ETL) compounds, such as ether phospholipid (ETPL) compounds, and their use in the hyperactivation of mammalian dendritic cells, such as human or canine dendritic cells. The present disclosure also relates to compositions comprising an ETL, such as an ETPL, and one or more of a pathogen recognition receptor agonist, an antigen, and human or canine dendritic cells, as well as methods of making and using the compositions.

[0006] The present disclosure provides a compound of Formula (I), Formula (II), Formula (III), Formula (III-A), Formula (III-A-1), Formula (III-A-2), Formula (III-B), Formula (III-B-1), Formula (III-B-2), Formula (IV), Formula (IV-A), Formula (IV-A-1), Formula (IV-A-2), Formula (IV-B), Formula (IV-B-1), Formula (IV-B-2), Formula (IV-C), Formula (IV-D), Formula (IV-E), Formula (IV-F), Formula (A), Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, or Compound 16, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof, as disclosed herein. In some embodiments, the ETL or ETPL is isolated.

[0007] The present disclosure relates to ether lipid (ETL) compounds of formula (I), formula (II), formula (III), formula (III-A), formula (III-A-1), formula (III-A-2), formula (III-B), formula (III-B-1), formula (III-B-2), formula (IV), formula (IV-A), formula (IV-A-1), formula (IV-A-2), formula (IV-B), formula (IV-B-1), formula (IV-B-2), formula (IV-C), formula (IV-D), formula (IV-E), formula (IV-F), formula (A), or provides a composition comprising an ether phospholipid (ETPL) compound, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, or Compound 16, or a protonated or deprotonated form thereof, if possible, or a pharmaceutically acceptable salt thereof, wherein the composition further comprises one or more of a TLR agonist, an antigen, and / or a dendritic cell. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist. In some embodiments, the ETL or ETPL is isolated.

[0008] The present disclosure relates to ether lipids of formula (I), formula (II), formula (III), formula (III-A), formula (III-A-1), formula (III-A-2), formula (III-B), formula (III-B-1), formula (III-B-2), formula (IV), formula (IV-A), formula (IV-A-1), formula (IV-A-2), formula (IV-B), formula (IV-B-1), formula (IV-B-2), formula (IV-C), formula (IV-D), formula (IV-E), formula (IV-F), formula (A), Compositions are provided comprising an ether phospholipid (ETL) compound or ether phospholipid (ETPL) compound, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, or Compound 16, or a protonated or deprotonated form thereof, if 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 ETL or ETPL is isolated.

[0009] The present disclosure relates to the ethers of formula (I), formula (II), formula (III), formula (III-A), formula (III-A-1), formula (III-A-2), formula (III-B), formula (III-B-1), formula (III-B-2), formula (IV), formula (IV-A), formula (IV-A-1), formula (IV-A-2), formula (IV-B), formula (IV-B-1), formula (IV-B-2), formula (IV-C), formula (IV-D), formula (IV-E), formula (IV-F), formula (A),

[0010] Compositions are provided comprising an ether phospholipid (ETL) compound or an ether phospholipid (ETPL) compound, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, or Compound 16, or a protonated or deprotonated form thereof, if 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 ETL or ETPL is isolated.

[0010] The present disclosure relates to the ethers of formula (I), formula (II), formula (III), formula (III-A), formula (III-A-1), formula (III-A-2), formula (III-B), formula (III-B-1), formula (III-B-2), formula (IV), formula (IV-A), formula (IV-A-1), formula (IV-A-2), formula (IV-B), formula (IV-B-1), formula (IV-B-2), formula (IV-C), formula (IV-D), formula (IV-E), formula (IV-F), formula (A), Compositions are provided comprising a lipid (ETL) compound or ether phospholipid (ETPL) compound, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, or Compound 16, or a protonated or deprotonated form thereof, if 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 ETL or ETPL is isolated.

[0011] The present disclosure provides a compound of Formula (I), Formula (II), Formula (III), Formula (III-A), Formula (III-A-1), Formula (III-A-2), Formula (III-B), Formula (III-B-1), Formula (III-B-2), Formula (IV), Formula (IV-A), Formula (IV-A-1), Formula (IV-A-2), Formula (IV-B), Formula (IV-B-1), Formula (IV-B-2), Formula (IV-C), Formula (IV-D), Formula (IV-E), Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, or Compound 13, or a protonated or deprotonated form thereof, if possible, or a pharmaceutically acceptable salt thereof, as disclosed herein. In some embodiments, the ETL or ETPL is isolated.

[0012] The present disclosure relates to an ether lipid (ETL) compound or ether lipid compound of formula (I), formula (II), formula (III), formula (III-A), formula (III-A-1), formula (III-A-2), formula (III-B), formula (III-B-1), formula (III-B-2), formula (IV), formula (IV-A), formula (IV-A-1), formula (IV-A-2), formula (IV-B), formula (IV-B-1), formula (IV-B-2), formula (IV-C), formula (IV-D), formula (IV-E), as disclosed herein. Compositions are provided comprising a phospholipid (ETPL) compound, Compound 1, Compound 2, Compound 3, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, or Compound 13, or a protonated or deprotonated form thereof, if possible, or a pharmaceutically acceptable salt thereof, wherein the composition further comprises one or more of a TLR agonist, an antigen, and / or a dendritic cell. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist. In some embodiments, the ETL or ETPL is isolated.

[0013] The present disclosure relates to ether lipids (ETLs) of formula (I), formula (II), formula (III), formula (III-A), formula (III-A-1), formula (III-A-2), formula (III-B), formula (III-B-1), formula (III-B-2), formula (IV), formula (IV-A), formula (IV-A-1), formula (IV-A-2), formula (IV-B), formula (IV-B-1), formula (IV-B-2), formula (IV-C), formula (IV-D), and formula (IV-E) disclosed herein. Compositions are provided comprising a compound or ether phospholipid (ETPL) compound, Compound 1, Compound 2, Compound 3, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, or Compound 13, or a protonated or deprotonated form thereof, if 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 ETL or ETPL is isolated.

[0014] The present disclosure relates to ether lipids (E) of formula (I), formula (II), formula (III), formula (III-A), formula (III-A-1), formula (III-A-2), formula (III-B), formula (III-B-1), formula (III-B-2), formula (IV), formula (IV-A), formula (IV-A-1), formula (IV-A-2), formula (IV-B), formula (IV-B-1), formula (IV-B-2), formula (IV-C), formula (IV-D), formula (IV-E),

[0010] Compositions are provided comprising an ether phospholipid (ETPL) compound or ether phospholipid (ETPL) compound, Compound 1, Compound 2, Compound 3, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, or Compound 13, or a protonated or deprotonated form thereof, if 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 ETL or ETPL is isolated.

[0015] The present disclosure relates to ether lipids (ETs) of formula (I), formula (II), formula (III), formula (III-A), formula (III-A-1), formula (III-A-2), formula (III-B), formula (III-B-1), formula (III-B-2), formula (IV), formula (IV-A), formula (IV-A-1), formula (IV-A-2), formula (IV-B), formula (IV-B-1), formula (IV-B-2), formula (IV-C), formula (IV-D), formula (IV-E), L) A composition comprising an ether phospholipid (ETPL) compound, Compound 1, Compound 2, Compound 3, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, or Compound 13, or a protonated or deprotonated form thereof, if possible, or a pharmaceutically acceptable salt thereof, wherein the composition further comprises a dendritic cell. 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 ETL or ETPL is isolated.

[0016] The present disclosure provides ether lipid (ETL) compounds, wherein the lipid alkyl chain is a C13-C24 n-alkyl chain or a C13-C22 n-alkyl chain. In some embodiments, the n-alkyl chain is a C18-C22 n-alkyl chain, a C21-C24 n-alkyl chain, or a C22 n-alkyl chain. In some embodiments, the disclosure provides compositions comprising an ether lipid compound, wherein the lipid alkyl chain is a C13-C24 n-alkyl chain, a C13-C22 n-alkyl chain, a C18-C22 n-alkyl chain, a C21-C24 n-alkyl chain, or a C22 n-alkyl chain, and the composition further comprises one or more of a TLR agonist, an antigen, and / or a dendritic cell. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist.

[0017] The present disclosure provides a composition comprising an isolated ether lipid (ETL) and a TLR7 / 8 agonist, wherein the lipid alkyl chain is a C13-C24 n-alkyl chain or a C13-C22 n-alkyl chain. In some embodiments, the n-alkyl chain is a C18-C22 n-alkyl chain, a C21-C24 n-alkyl chain, or a C22 n-alkyl chain. In some embodiments, the composition further comprises an antigen and / or dendritic cells. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist.

[0018] The present disclosure provides ether phospholipid (ETPL) compounds, wherein the lipid alkyl chain is a C13-C24 n-alkyl chain or a C13-C22 n-alkyl chain. In some embodiments, the n-alkyl chain is a C18-C22 n-alkyl chain, a C21-C24 n-alkyl chain, or a C22 n-alkyl chain. In some embodiments, the disclosure provides compositions comprising an ether phospholipid compound, wherein the lipid alkyl chain is a C13-C24 n-alkyl chain, a C13-C22 n-alkyl chain, a C18-C22 n-alkyl chain, a C21-C24 n-alkyl chain, or a C22 n-alkyl chain, and the composition further comprises one or more of a TLR agonist, an antigen, and / or a dendritic cell. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist.

[0019] The present disclosure provides a composition comprising an isolated ether phospholipid (ETPL) and a TLR agonist, wherein the lipid alkyl chain is a C13-C24 n-alkyl chain or a C13-C22 n-alkyl chain. In some embodiments, the n-alkyl chain is a C18-C22 n-alkyl chain, a C21-C24 n-alkyl chain, or a C22 n-alkyl chain. In some embodiments, the composition further comprises an antigen and / or dendritic cells. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist.

[0020] In some aspects, the present disclosure provides an ether lipid (ETL) compound having an n-alkyl chain, wherein the n-alkyl chain is a C21-C24 n-alkyl chain. In some embodiments, the present disclosure provides a composition comprising an ether lipid (ETL) compound having an n-alkyl chain, wherein the n-alkyl chain is a C21-C24 n-alkyl chain, and an antigen. In some embodiments, the composition further comprises dendritic cells and / or a TLR agonist. In some embodiments, the composition further comprises dendritic cells and / or a TLR7 / 8 agonist.

[0021] In some aspects, the present disclosure provides a composition comprising an isolated ether lipid (ETL) having an n-alkyl chain and an antigen, wherein the n-alkyl chain is a C21-C24 n-alkyl chain. In some embodiments, the composition further comprises dendritic cells and / or a TLR agonist. In some embodiments, the composition further comprises dendritic cells and / or a TLR7 / 8 agonist.

[0022] In some aspects, the present disclosure provides an ether phospholipid (ETPL) compound having an n-alkyl chain, wherein the n-alkyl chain is a C21-C24 n-alkyl chain. In some embodiments, the present disclosure provides a composition comprising an ether phospholipid (ETPL) compound having an n-alkyl chain, wherein the n-alkyl chain is a C21-C24 n-alkyl chain, and an antigen. In some embodiments, the composition further comprises dendritic cells and / or a TLR agonist. In some embodiments, the composition further comprises dendritic cells and / or a TLR7 / 8 agonist.

[0023] In some aspects, the present disclosure provides a composition comprising an isolated ether phospholipid (ETPL) having an n-alkyl chain and an antigen, wherein the n-alkyl chain is a C21-C24 n-alkyl chain. In some embodiments, the composition further comprises dendritic cells and / or a TLR agonist. In some embodiments, the composition further comprises dendritic cells and / or a TLR7 / 8 agonist.

[0024] In some aspects, the present disclosure provides a composition comprising an ether lipid (ETL) having an n-alkyl chain, wherein the n-alkyl chain is a C21-C24 n-alkyl chain, and a dendritic cell. In some embodiments, the composition further comprises a TLR agonist and / or an antigen. In some embodiments, the composition further comprises a TLR7 / 8 agonist and / or an antigen.

[0025] In some aspects, the present disclosure provides a composition comprising an isolated ether lipid (ETL) having an n-alkyl chain, wherein the n-alkyl chain is a C21-C24 n-alkyl chain, and a dendritic cell. In some embodiments, the composition further comprises a TLR agonist and / or an antigen. In some embodiments, the composition further comprises a TLR7 / 8 agonist and / or an antigen.

[0026] In some aspects, the present disclosure provides a composition comprising an ether phospholipid (ETPL) having an n-alkyl chain, wherein the n-alkyl chain is a C21-C24 n-alkyl chain, and a dendritic cell. In some embodiments, the composition further comprises a TLR agonist and / or an antigen. In some embodiments, the composition further comprises a TLR7 / 8 agonist and / or an antigen.

[0027] In some aspects, the present disclosure provides a composition comprising an isolated ether phospholipid (ETPL) having an n-alkyl chain, wherein the n-alkyl chain is a C21-C24 n-alkyl chain, and a dendritic cell. In some embodiments, the composition further comprises a TLR agonist and / or an antigen. In some embodiments, the composition further comprises a TLR7 / 8 agonist and / or an antigen.

[0028] In some aspects, the present disclosure provides an ether lipid (ETL) compound having an n-alkyl chain, wherein the n-alkyl chain is a C16-C20 n-alkyl chain. In some embodiments, the present disclosure provides a composition comprising an ether lipid (ETL) compound having an n-alkyl chain (wherein the n-alkyl chain is a C16-C20 n-alkyl chain) and an antigen. In some embodiments, the composition further comprises dendritic cells and / or a TLR agonist. In some embodiments, the composition further comprises dendritic cells and / or a TLR7 / 8 agonist.

[0029] In some aspects, the present disclosure provides a composition comprising an isolated ether lipid (ETL) having an n-alkyl chain and an antigen, wherein the n-alkyl chain is a C16-C20 n-alkyl chain. In some embodiments, the composition further comprises dendritic cells and / or a TLR agonist. In some embodiments, the composition further comprises dendritic cells and / or a TLR7 / 8 agonist.

[0030] In some aspects, the present disclosure provides an ether phospholipid (ETPL) compound having an n-alkyl chain, wherein the n-alkyl chain is a C16-C20 n-alkyl chain. In some embodiments, the present disclosure provides a composition comprising an ether phospholipid (ETPL) compound having an n-alkyl chain, wherein the n-alkyl chain is a C16-C20 n-alkyl chain, and an antigen. In some embodiments, the composition further comprises dendritic cells and / or a TLR agonist. In some embodiments, the composition further comprises dendritic cells and / or a TLR7 / 8 agonist.

[0031] In some aspects, the present disclosure provides a composition comprising an isolated ether phospholipid (ETPL) having an n-alkyl chain and an antigen, wherein the n-alkyl chain is a C16-C20 n-alkyl chain. In some embodiments, the composition further comprises dendritic cells and / or a TLR agonist. In some embodiments, the composition further comprises dendritic cells and / or a TLR7 / 8 agonist.

[0032] In some aspects, the present disclosure provides a composition comprising an ether lipid (ETL) having an n-alkyl chain, wherein the n-alkyl chain is a C16-C20 n-alkyl chain, and a dendritic cell. In some embodiments, the composition further comprises a TLR agonist and / or an antigen. In some embodiments, the composition further comprises a TLR7 / 8 agonist and / or an antigen.

[0033] In some aspects, the present disclosure provides a composition comprising an isolated ether lipid (ETL) having an n-alkyl chain, wherein the n-alkyl chain is a C16-C20 n-alkyl chain, and a dendritic cell. In some embodiments, the composition further comprises a TLR agonist and / or an antigen. In some embodiments, the composition further comprises a TLR7 / 8 agonist and / or an antigen.

[0034] In some aspects, the present disclosure provides a composition comprising an ether phospholipid (ETPL) having an n-alkyl chain, wherein the n-alkyl chain is a C16-C20 n-alkyl chain, and a dendritic cell. In some embodiments, the composition further comprises a TLR agonist and / or an antigen. In some embodiments, the composition further comprises a TLR7 / 8 agonist and / or an antigen.

[0035] In some aspects, the present disclosure provides a composition comprising an isolated ether phospholipid (ETPL) having an n-alkyl chain, wherein the n-alkyl chain is a C16-C20 n-alkyl chain, and a dendritic cell. In some embodiments, the composition further comprises a TLR agonist and / or an antigen. In some embodiments, the composition further comprises a TLR7 / 8 agonist and / or an antigen.

[0036] In some embodiments of the preceding aspects, the antigen is present in a biological sample obtained from the individual. In some embodiments, the biological sample comprises biopsy tissue. In some embodiments, the biological sample comprises cells. In other embodiments, the biological sample does not comprise cells. In some embodiments, the biological sample comprises pus from an abscess. In some embodiments, the antigen comprises a 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, the microbial antigen comprising 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.

[0037] In some embodiments, the composition does not include liposomes. In some embodiments, the composition does not include LPS or MPLA. In some embodiments, the composition does not include oxPAPC or oxPAPC species. In some embodiments, the composition does not include HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC, and / or PGPC. In some embodiments, the composition does not include lysophosphatidylcholine (LPC). In some embodiments, the composition does not include 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

[0038] In some embodiments, the composition further comprises an adjuvant, wherein the adjuvant comprises an aluminum salt adjuvant, a squalene-in-water emulsion, a saponin, or a combination thereof.

[0039] In some embodiments, the present disclosure provides a pharmaceutical formulation comprising the composition of any of the preceding aspects and a pharmaceutically acceptable excipient.

[0040] In an additional aspect, the present disclosure provides a method for producing superactivated dendritic cells, the method comprising contacting dendritic cells with a composition comprising an effective amount of an isolated ether lipid (ETL) comprising a C13-C22 n-alkyl chain or a C13-C24 n-alkyl chain and a TLR agonist to produce the superactivated dendritic cells, wherein the superactivated dendritic cells secrete IL-1 beta 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 a pharmaceutical formulation comprising the composition of any one of the preceding embodiments. In some aspects, the disclosure provides a pharmaceutical formulation comprising a plurality of superactivated dendritic cells produced by the preceding embodiments and a pharmaceutically acceptable excipient. In some embodiments, the plurality is at least 10 3 , 10 4 , 105 , 10 6 , 10 7 or 10 8 In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist.

[0041] In an additional aspect, the present disclosure provides a method for producing superactivated dendritic cells, the method comprising contacting dendritic cells with a composition comprising an effective amount of an isolated ether phospholipid (ETPL) comprising a C13-C22 n-alkyl chain or a C13-C24 n-alkyl chain and a TLR agonist to produce the superactivated dendritic cells, wherein the superactivated dendritic cells secrete IL-1 beta without undergoing pyroptosis. In some embodiments, dendritic cells are contacted ex vivo with the composition or pharmaceutical formulation of any one of the preceding embodiments. In other embodiments, dendritic cells are contacted in vivo with a pharmaceutical formulation comprising the composition of any one of the preceding embodiments. In some aspects, the disclosure provides a pharmaceutical formulation comprising a plurality of superactivated dendritic cells produced by the preceding embodiments and a pharmaceutically acceptable excipient. In some embodiments, the plurality is at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 or 10 8 In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist.

[0042] In an additional aspect, the present disclosure provides a composition comprising an isolated ether lipid (ETL) having an n-alkyl chain and a pathogen recognition receptor (PRR) agonist, wherein the n-alkyl chain is a C13-C22 n-alkyl chain or a C13-C24 n-alkyl chain. In some embodiments, the PRR agonist is an agonist of a toll-like receptor (TLR), a NOD-like receptor (NLR), a RIG-I-like receptor (RLR), or a C-type lectin receptor (CLR). In some embodiments, the PRR agonist is an agonist of a cytoplasmic DNA sensor (CDS) or a stimulator of IFN genes (STING). In some embodiments, the PRR agonist comprises a TLR7 / 8 agonist. In some embodiments, the composition further comprises an antigen and / or dendritic cells.

[0043] In an additional aspect, the present disclosure provides a composition comprising an isolated ether phospholipid (ETPL) having an n-alkyl chain and a pathogen recognition receptor (PRR) agonist, wherein the n-alkyl chain is a C13-C22 n-alkyl chain or a C13-C24 n-alkyl chain. In some embodiments, the PRR agonist is an agonist of a toll-like receptor (TLR), a NOD-like receptor (NLR), a RIG-I-like receptor (RLR), or a C-type lectin receptor (CLR). In some embodiments, the PRR agonist is an agonist of a cytoplasmic DNA sensor (CDS) or a stimulator of IFN genes (STING). In some embodiments, the PRR agonist comprises a TLR7 / 8 agonist. In some embodiments, the composition further comprises an antigen and / or dendritic cells.

[0044] In some embodiments of the preceding aspect, the n-alkyl chain of the ether lipid (ETL) is a C21-C24 n-alkyl chain. In some embodiments, the n-alkyl chain of the ETL is a C22 n-alkyl chain.

[0045] In some embodiments of the preceding aspect, the n-alkyl chain of the ether phospholipid (ETPL) is a C21-C24 n-alkyl chain. In some embodiments, the n-alkyl chain of the ETPL is a C22 n-alkyl chain.

[0046] In some embodiments of the preceding aspect, the ETPL comprises 1-docosyl-sn-glycerol-3-phosphocholine (DGPC). In some embodiments of the preceding aspect, the ETPL comprises 1-docosyl-sn-glycerol-3-phosphate (DGP).

[0047] In some embodiments of the preceding aspects, the TLR agonist is a small molecule with a molecular weight of 900 daltons or less. In some embodiments of the preceding aspects, the TLR7 / 8 agonist is a small molecule with a molecular weight of 900 daltons or less. In some embodiments, the TLR7 / 8 agonist comprises an imidazoquinoline compound. In some embodiments, the TLR7 / 8 agonist comprises resiquimod (R848). In some embodiments, the ETPL comprises DGPC and the TLR7 / 8 agonist comprises resiquimod (R848). In some embodiments, the ETPL comprises DGP and the TLR7 / 8 agonist comprises resiquimod (R848).

[0048] The present disclosure further provides a composition for the superactivation of human dendritic cells, comprising an ether lipid (ETL) compound having an n-alkyl chain and a pathogen recognition receptor (PRR) agonist, wherein the n-alkyl chain is a C22 n-alkyl chain, and the composition is effective for achieving a higher level of dendritic cell superactivation than a comparative composition comprising a comparative compound instead of ETL. The present disclosure further provides a composition for the superactivation of human dendritic cells, comprising an isolated ether lipid (ETL) compound having an n-alkyl chain and a pathogen recognition receptor (PRR) agonist, wherein the n-alkyl chain is a C22 n-alkyl chain, and the composition is effective for achieving a higher level of dendritic cell superactivation than a comparative composition comprising a comparative compound instead of ETL. In some embodiments, the superactivation occurs in vitro or ex vivo. In other embodiments, the superactivation occurs in vivo. In some embodiments, a high level of dendritic cell superactivation comprises inducing IL-1 beta secretion from human dendritic cells in vitro at a level at least two-fold, three-fold, or four-fold higher when contacted with a composition comprising an ETL and a PRR agonist than when contacted with a comparative composition comprising a comparative compound and a PRR agonist, where the PRR agonist is LPS. In some embodiments, the concentrations of ETL and the comparative compound are the same, optionally ranging from about 10 μM to about 80 μM, and LPS is present at a concentration of 1 μg / ml in both the composition and the comparative composition. In some embodiments, a high level of dendritic cell superactivation comprises a lipid activity index for IL-1 beta secretion from human dendritic cells for the composition comprising an ETL and a PRR agonist that is at least four-fold, five-fold, or six-fold higher in activity units than for the comparative composition comprising a comparative compound and a PRR agonist. In some embodiments, the comparative compound is PGPC. In some embodiments, the comparator compound is 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

[0049] The present disclosure further provides a composition for the superactivation of human dendritic cells, comprising an ether phospholipid (ETPL) compound having an n-alkyl chain and a pathogen recognition receptor (PRR) agonist, wherein the n-alkyl chain is a C22 n-alkyl chain, and the composition is effective for achieving a higher level of dendritic cell superactivation than a comparative composition comprising a comparative compound instead of the ETPL. The present disclosure further provides a composition for the superactivation of human dendritic cells, comprising an isolated ether phospholipid (ETPL) compound having an n-alkyl chain and a pathogen recognition receptor (PRR) agonist, wherein the n-alkyl chain is a C22 n-alkyl chain, and the composition is effective for achieving a higher level of dendritic cell superactivation than a comparative composition comprising a comparative compound instead of the ETPL. In some embodiments, the superactivation occurs in vitro or ex vivo. In other embodiments, the superactivation occurs in vivo. In some embodiments, a high level of dendritic cell superactivation comprises inducing IL-1beta secretion from human dendritic cells in vitro at a level at least two-fold, three-fold, or four-fold higher when contacted with a composition comprising an ETPL and a PRR agonist than when contacted with a comparative composition comprising a comparative compound and a PRR agonist, where the PRR agonist is LPS. In some embodiments, the concentrations of ETPL and the comparative compound are the same, optionally ranging from about 10 μM to about 80 μM, and LPS is present at a concentration of 1 μg / ml in both the composition and the comparative composition. In some embodiments, a high level of dendritic cell superactivation comprises a lipid activity index for IL-1beta secretion from human dendritic cells for the composition comprising an ETPL and a PRR agonist that is at least four-fold, five-fold, or six-fold higher in activity units than for the comparative composition comprising a comparative compound and a PRR agonist. In some embodiments, the comparative compound is PGPC. In some embodiments, the comparator compound is 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

[0050] Ether lipid (ETL) compounds, such as isolated ether lipid compounds, and ether phospholipid (ETPL) compounds, such as isolated ether phospholipid compounds, can be administered in the form of micelles.

[0051] Ether lipid (ETL) compounds, such as isolated ether lipid compounds, and ether phospholipid (ETPL) compounds, such as isolated ether phospholipid compounds, can be administered in the form of lipid nanoparticles (LNPs).

[0052] In some embodiments of the present disclosure, the LNPs of the composition are enriched in particles having a lipid bilayer (liposomes) compared to particles having a single lipid layer (micelles). Specifically, in some embodiments, the LNPs comprise liposomes and are largely or substantially free of 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.

[0053] In some embodiments, the present disclosure provides lipid nanoparticles comprising an ETL or ETPL compound and at least one additional lipid, and their use in hyperactivating mammalian dendritic cells. The present disclosure also relates to compositions comprising an ETL or ETPL compound and at least one additional lipid, further comprising one or more of a pathogen recognition receptor agonist, an antigen, and mammalian dendritic cells, as well as methods of making and using the compositions.

[0054] In some embodiments, the present disclosure provides a composition comprising an ETL or ETPL compound and a TLR agonist, such as a TLR7 / 8 agonist, wherein the ETL or ETPL compound is a compound represented by Formula (I), Formula (II), Formula (III), Formula (III-A), Formula (III-A-1), Formula (III-A-2), Formula (III-B), Formula (III-B-1), Formula (III-B-2), Formula (IV), Formula (IV-A), Formula (IV-A-1), Formula (IV-A-2), Formula (IV-B), Formula (IV-B-1), Formula (IV-B-2 ... The composition is a compound of Formula (IV-B-2), Formula (IV-C), Formula (IV-D), Formula (IV-E), Formula (IV-F), Formula (A), Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, or Compound 16, or a protonated or deprotonated form thereof, if possible, or a pharmaceutically acceptable salt thereof, wherein the ETL or ETPL and at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a PEGylated lipid, a structured lipid, and a mixture thereof. In some embodiments, the composition further comprises an antigen and / or dendritic cells.

[0055] In some embodiments, the present disclosure provides a composition comprising an ETL or ETPL compound and an antigen, wherein the ETL or ETPL compound is a compound represented by Formula (I), Formula (II), Formula (III), Formula (III-A), Formula (III-A-1), Formula (III-A-2), Formula (III-B), Formula (III-B-1), Formula (III-B-2), Formula (IV), Formula (IV-A), Formula (IV-A-1), Formula (IV-A-2), Formula (IV-B), Formula (IV-B-1), Formula (IV-B-2), Formula (IV-B-1), Formula (IV-B-2), Formula (IV-B-3), Formula (IV-B-4), Formula (IV-B-5), Formula (IV-B-6), Formula (IV-B-7), Formula (IV-B-8), Formula (IV-B-9), Formula (IV-B-10), Formula (IV-B-11), Formula (IV-B-12), Formula (IV-B-13), Formula (IV-B-14), Formula (IV-B-15), Formula (IV-B-16), Formula (IV-B-17), Formula (IV-B-18), Formula (IV-B-19), Formula (IV-B-21), Formula (IV-B-22), Formula (IV-B-19), Formula (IV-B-23), Formula (IV-B-24), Formula (IV-B-25), Formula (IV-B-26), Formula (IV-B-27), Formula (IV-B-28), Formula (IV-B-29 ... The composition is a compound of Formula (IV-C), Formula (IV-D), Formula (IV-E), Formula (IV-F), Formula (A), Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, or Compound 16, or a protonated or deprotonated form thereof, if possible, or a pharmaceutically acceptable salt thereof, wherein the ETL or ETPL and at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structured lipid, and a mixture thereof. In some embodiments, the composition further comprises a TLR agonist, such as a TLR7 / 8 agonist, and / or dendritic cells.

[0056] In some embodiments, the present disclosure provides a composition comprising an ETL or ETPL compound and a dendritic cell, wherein the ETL or ETPL compound is a compound represented by Formula (I), Formula (II), Formula (III), Formula (III-A), Formula (III-A-1), Formula (III-A-2), Formula (III-B), Formula (III-B-1), Formula (III-B-2), Formula (IV), Formula (IV-A), Formula (IV-A-1), Formula (IV-A-2), Formula (IV-B), Formula (IV-B-1), Formula (IV-B-2), as disclosed herein. , Formula (IV-C), Formula (IV-D), Formula (IV-E), Formula (IV-F), a compound of Formula (A), Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, or Compound 16, or a protonated or deprotonated form thereof, if possible, or a pharmaceutically acceptable salt thereof, wherein the ETL or ETPL and at least one additional lipid are part of a lipid nanoparticle (LNP). In additional embodiments, the ETL or ETPL compound is isolated. In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structured lipid, and a mixture thereof. In some embodiments, the composition further comprises a TLR agonist, such as a TLR7 / 8 agonist, and / or an antigen.

[0057] In some embodiments, the present disclosure provides a composition comprising an ETL or ETPL compound and a TLR agonist, such as a TLR7 / 8 agonist, wherein the ETL or ETPL compound is a compound represented by Formula (I), Formula (II), Formula (III), Formula (III-A), Formula (III-A-1), Formula (III-A-2), Formula (III-B), Formula (III-B-1), Formula (III-B-2), Formula (IV), Formula (IV-A), Formula (IV-A-1), Formula (IV-A-2), Formula (IV-B), Formula ( IV-B-1), a compound of Formula (IV-B-2), Formula (IV-C), Formula (IV-D), Formula (IV-E), Compound 1, Compound 2, Compound 3, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, or Compound 13, or a protonated or deprotonated form thereof, if possible, or a pharmaceutically acceptable salt thereof, wherein the ETL or ETPL and at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a PEGylated lipid, a structured lipid, and a mixture thereof. In some embodiments, the composition further comprises an antigen and / or dendritic cells.

[0058] In some embodiments, the present disclosure provides a composition comprising an ETL or ETPL compound and an antigen, wherein the ETL or ETPL compound is a compound represented by Formula (I), Formula (II), Formula (III), Formula (III-A), Formula (III-A-1), Formula (III-A-2), Formula (III-B), Formula (III-B-1), Formula (III-B-2), Formula (IV), Formula (IV-A), Formula (IV-A-1), Formula (IV-A-2), Formula (IV-B), Formula (IV-B-1), Formula ( IV-B-2), a compound of Formula (IV-C), Formula (IV-D), Formula (IV-E), Compound 1, Compound 2, Compound 3, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, or Compound 13, or a protonated or deprotonated form thereof, if possible, or a pharmaceutically acceptable salt thereof, wherein the ETL or ETPL and at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structured lipid, and a mixture thereof. In some embodiments, the composition further comprises a TLR agonist, such as a TLR7 / 8 agonist, and / or dendritic cells.

[0059] In some embodiments, the present disclosure provides a composition comprising an ETL or ETPL compound and a dendritic cell, wherein the ETL or ETPL compound is a compound represented by Formula (I), Formula (II), Formula (III), Formula (III-A), Formula (III-A-1), Formula (III-A-2), Formula (III-B), Formula (III-B-1), Formula (III-B-2), Formula (IV), Formula (IV-A), Formula (IV-A-1), Formula (IV-A-2), Formula (IV-B), Formula (IV-B-1), Formula (IV-B-2 ... (IV-B-2), a compound of Formula (IV-C), Formula (IV-D), Formula (IV-E), Compound 1, Compound 2, Compound 3, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, or Compound 13, or a protonated or deprotonated form thereof, if possible, or a pharmaceutically acceptable salt thereof, wherein the ETL or ETPL and at least one additional lipid are part of a lipid nanoparticle (LNP). In additional embodiments, the ETL or ETPL compound is isolated. In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structured lipid, and a mixture thereof. In some embodiments, the composition further comprises a TLR agonist, such as a TLR7 / 8 agonist, and / or an antigen.

[0060] In some embodiments of the preceding aspects, the antigen is present in a biological sample obtained from the individual. In some embodiments, the biological sample comprises biopsy tissue. In some embodiments, the biological sample comprises cells. In other embodiments, the biological sample does not comprise cells. In some embodiments, the biological sample comprises pus from an abscess. In some embodiments, the antigen comprises a 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, the microbial antigen comprising 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.

[0061] In some embodiments, the composition does not include LPS or MPLA. In some embodiments, the composition does not include oxPAPC or species of oxPAPC. In some embodiments, the composition does not include HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC, and / or PGPC. In some embodiments, the composition does not include isolated mRNA. In some embodiments, the composition does not include a surfactant (e.g., poloxamer). In some embodiments, the composition does not include poloxamer 407 (KP407), poloxamer 188 (KP188), and / or Pluronic P123 (P123).

[0062] In some embodiments, the composition further comprises an adjuvant, wherein the adjuvant comprises an aluminum salt adjuvant, a squalene-in-water emulsion, a saponin, or a combination thereof.

[0063] 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 does not include a surfactant (e.g., a poloxamer). In some embodiments, the formulation does not include poloxamer 407 (KP407), poloxamer 188 (KP188), and / or Pluronic P123 (P123).

[0064] In another aspect, the present disclosure provides a method for producing superactivated dendritic cells, comprising contacting dendritic cells with an effective amount of the composition or pharmaceutical formulation of any of the preceding embodiments to produce superactivated dendritic cells, wherein the superactivated dendritic cells secrete IL-1 beta without undergoing pyroptosis, and wherein the ETL or ETPL and at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a PEGylated lipid, a structured lipid, and mixtures thereof. In some embodiments, dendritic cells are contacted ex vivo with the composition or pharmaceutical formulation of any one of the preceding embodiments. In other embodiments, dendritic cells are contacted in vivo with a pharmaceutical formulation comprising the composition of any one of the preceding embodiments. In some aspects, the disclosure provides a pharmaceutical formulation comprising a plurality of superactivated dendritic cells produced by the preceding embodiments and a pharmaceutically acceptable excipient. In some embodiments, the plurality is at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 or 10 8 Contains superactivated DCs.

[0065] In other aspects, the present disclosure provides an ETL or ETPL (wherein the ETL compound or ETPL compound is a compound represented by Formula (I), Formula (II), Formula (III), Formula (III-A), Formula (III-A-1), Formula (III-A-2), Formula (III-B), Formula (III-B-1), Formula (III-B-2), Formula (IV), Formula (IV-A), Formula (IV-A-1), Formula (IV-A-2), Formula (IV-B), Formula (IV-B-1), Formula (IV-B-2), Formula (IV-C), Formula (IV-D), Formula (IV-E), Formula (IV-F), Formula (A),

[0010] Compositions are provided that include Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, or Compound 16, or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof, at least one additional lipid, and a pathogen recognition receptor (PRR) agonist, wherein the ETL or ETPL and the at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structured lipid, and a mixture thereof. In some embodiments, the PRR agonist is an agonist of a toll-like receptor (TLR), a NOD-like receptor (NLR), a RIG-I-like receptor (RLR), or a C-type lectin receptor (CLR). In some embodiments, the PRR agonist is an agonist of a cytoplasmic DNA sensor (CDS) or a stimulator of IFN genes (STING). In some embodiments, the PRR agonist comprises a TLR7 / 8 agonist. In some embodiments, the composition further comprises an antigen and / or dendritic cells.

[0066] In some embodiments of the preceding aspects, the TLR7 / 8 agonist is a small molecule with a molecular weight of 900 daltons or less. In some embodiments, the TLR7 / 8 agonist comprises an imidazoquinoline compound. In some embodiments, the TLR7 / 8 agonist comprises resiquimod (R848).

[0067] The present disclosure further provides a composition for the hyperactivation of human dendritic cells, comprising an ETL or ETPL (wherein the ETL compound or ETPL compound is a compound represented by formula (I), formula (II), formula (III), formula (III-A), formula (III-A-1), formula (III-A-2), formula (III-B), formula (III-B-1), formula (III-B-2), formula (IV), formula (IV-A), formula (IV-A-1), formula (IV-A-2), formula (IV-B), formula (IV-B-1), formula (IV-B-2), formula (IV-C), formula (IV-D), formula (IV-E), formula (IV-F), formula (A), Provided are compositions comprising Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, or Compound 16, or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof, at least one additional lipid, and a pathogen recognition receptor (PRR) agonist, wherein the composition is effective in achieving a higher level of dendritic cell superactivation than a comparative composition comprising the comparative compound instead of an ETL or ETPL. In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structured lipid, and a mixture thereof. In some embodiments, the superactivation occurs in vitro or ex vivo. In other embodiments, the superactivation occurs in vivo. In some embodiments, high levels of dendritic cell superactivation include inducing IL-1beta secretion from human dendritic cells in vitro at a level at least two-fold, three-fold, or four-fold higher when contacted with a composition comprising an ETL or ETPL and a PRR agonist than when contacted with a comparative composition comprising a comparative compound and a PRR agonist, where the PRR agonist is LPS. In some embodiments, the concentrations of ETL or ETPL and the comparative compound are the same, optionally in the range of about 10 μM to about 80 μM, and LPS is present at a concentration of 1 μg / ml in both the composition and the comparative composition.In some embodiments, a high level of dendritic cell superactivation includes a lipid activity index for IL-1beta secretion from human dendritic cells for a composition comprising an ETL or ETPL and a PRR agonist that is at least 4-fold, 5-fold, or 6-fold higher activity units than a comparative composition comprising a reference compound and a PRR agonist. In some embodiments, the reference compound is PGPC.

[0068] In other aspects, the present disclosure provides an ETL or ETPL (wherein the ETL compound or ETPL compound is a compound of Formula (I), Formula (II), Formula (III), Formula (III-A), Formula (III-A-1), Formula (III-A-2), Formula (III-B), Formula (III-B-1), Formula (III-B-2), Formula (IV), Formula (IV-A), Formula (IV-A-1), Formula (IV-A-2), Formula (IV-B), Formula (IV-B-1), Formula (IV-B-2), Formula (IV-C), Formula (IV-D), Formula (IV-E), or a compound of Formula (IV-F), as disclosed herein.

[0013] Compositions are provided that include Compound 1, Compound 2, Compound 3, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, or Compound 13, or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof; at least one additional lipid; and a pathogen recognition receptor (PRR) agonist, wherein the ETL or ETPL and the at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structured lipid, and a mixture thereof. In some embodiments, the PRR agonist is an agonist of a toll-like receptor (TLR), a NOD-like receptor (NLR), a RIG-I-like receptor (RLR), or a C-type lectin receptor (CLR). In some embodiments, the PRR agonist is an agonist of a cytoplasmic DNA sensor (CDS) or a stimulator of IFN genes (STING). In some embodiments, the PRR agonist comprises a TLR7 / 8 agonist. In some embodiments, the composition further comprises an antigen and / or dendritic cells.

[0069] In some embodiments of the preceding aspects, the TLR7 / 8 agonist is a small molecule with a molecular weight of 900 daltons or less. In some embodiments, the TLR7 / 8 agonist comprises an imidazoquinoline compound. In some embodiments, the TLR7 / 8 agonist comprises resiquimod (R848).

[0070] The present disclosure further provides a composition for the hyperactivation of human dendritic cells, comprising an ETL or ETPL (wherein the ETL or ETPL compound is a compound of formula (I), formula (II), formula (III), formula (III-A), formula (III-A-1), formula (III-A-2), formula (III-B), formula (III-B-1), formula (III-B-2), formula (IV), formula (IV-A), formula (IV-A-1), formula (IV-A-2), formula (IV-B), formula (IV-B-1), formula (IV-B-2), formula (IV-C), formula (IV-D), formula (IV-E), or a compound of formula (IV-F). The present invention provides a composition comprising Compound 1, Compound 2, Compound 3, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, or Compound 13, or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof, at least one additional lipid, and a pathogen recognition receptor (PRR) agonist, wherein the composition is effective in achieving a higher level of dendritic cell superactivation than a comparative composition comprising the comparative compound instead of an ETL or ETPL. In some embodiments, the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structured lipid, and a mixture thereof. In some embodiments, the superactivation occurs in vitro or ex vivo. In other embodiments, the superactivation occurs in vivo. In some embodiments, high levels of dendritic cell superactivation include inducing IL-1beta secretion from human dendritic cells in vitro at a level at least two-fold, three-fold, or four-fold higher when contacted with a composition comprising an ETL or ETPL and a PRR agonist than when contacted with a comparative composition comprising a comparative compound and a PRR agonist, where the PRR agonist is LPS. In some embodiments, the concentrations of ETL or ETPL and the comparative compound are the same, optionally in the range of about 10 μM to about 80 μM, and LPS is present at a concentration of 1 μg / ml in both the composition and the comparative composition.In some embodiments, a high level of dendritic cell superactivation includes a lipid activity index for IL-1beta secretion from human dendritic cells for a composition comprising an ETL or ETPL and a PRR agonist that is at least 4-fold, 5-fold, or 6-fold higher activity units than a comparative composition comprising a reference compound and a PRR agonist. In some embodiments, the reference compound is PGPC.

[0071] In any of the embodiments disclosed herein, the etherlipid may be in the form of a pharmaceutically acceptable salt.

[0072] In any of the embodiments disclosed herein, the ether phospholipid may be in the form of a pharmaceutically acceptable salt.

[0073] In any of the embodiments disclosed herein, when a compound of Formula (I), Formula (II), Formula (III), Formula (III-A), Formula (III-A-1), Formula (III-A-2), Formula (III-B), Formula (III-B-1), Formula (III-B-2), Formula (IV), Formula (IV-A), Formula (IV-A-1), Formula (IV-A-2), Formula (IV-B), Formula (IV-B-1), Formula (IV-B-2), Formula (IV-C), Formula (IV-D), Formula (IV-E), Formula (IV-F), Formula (A), Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, or Compound 16 is disclosed in an embodiment, the present disclosure also encompasses the use of a compound of any other formula or other specific compound in place of that embodiment.

[0074] The disclosure of methods that include administering the compounds and compositions of the present disclosure to a subject (e.g., a subject in need thereof) also relates to the use of the compounds and compositions for treating or preventing a disease or disorder, or treating a subject having a disease or disorder, and the use of the compounds and compositions in the manufacture of a medicament for treating or preventing a disease or disorder, or treating a subject having a disease or disorder.

[0075] In any of the embodiments comprising an antigen disclosed herein, the antigen may comprise one or more viral antigens. In some embodiments, the one or more viral antigens comprise one or both of an influenza A antigen and an influenza B antigen. In some embodiments, one or both of the influenza A antigen and the influenza B antigen comprise one or both of a hemagglutinin and a nucleoprotein. In some embodiments, the viral antigen comprises an inactivated virion, and optionally the inactivated virion comprises an inactivated split virion. In some embodiments comprising antigens of both influenza A and influenza B, the antigen is an antigen of influenza A virus H1N1, influenza A virus H3N2, Victoria lineage influenza B virus, or Yamagata lineage influenza B virus.

[0076] Data shown in bar graphs in the following figures are presented as means, with error bars representing standard deviation (SD). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and ns=not significant. [Brief explanation of the drawings]

[0077] [Figure 1] A shows cell viability under the indicated test conditions. B shows IL-1β secretion by human monocyte-derived dendritic cells (moDCs) under the indicated test conditions. [Figure 2A] Cell viability under the indicated test conditions is shown. [Figure 2B] IL-1β secretion by human moDCs under the test conditions indicated is shown. [Figure 3] A shows cell viability under the indicated test conditions. B shows IL-1β secretion by human moDCs under the indicated test conditions. [Figure 4]A shows IL-1β secretion under the indicated test conditions, B shows cell viability under the indicated test conditions, and C shows TNFα secretion by human moDCs under the indicated test conditions. [Figure 5] 1 shows dendritic cell migration from the skin to the draining lymph nodes under the test conditions shown. [Figure 6] Survival rates of LLC1 tumor-bearing mice immunized with PBS or whole tumor lysate in the presence of PAMPs and DAMPs are shown. [Figure 7] IFNγ-secreting cells in the draining lymph nodes of immunized mice are shown. [Figure 8] IL-1β secretion by human moDCs treated with 22:0 Lyso PC, DPD (compound 9), compound 10, or vehicle, with and without R848, is shown. [Figure 9] The viability of cells treated with 22:0 Lyso PC, DPD (compound 9), compound 10, or vehicle, with and without R848, is shown. [Figure 10] IL-6 secretion by human moDCs treated with 22:0 Lyso PC, Compound 9 (DPD), Compound 2 (DGP), Compound 7, Compound 8, or vehicle without R848, with R848, and with R848 and MCC950 is shown. [Figure 11] IL-1β secretion by human moDCs treated with 22:0 Lyso PC, Compound 9 (DPD), Compound 2 (DGP), Compound 7, Compound 8, or vehicle without R848, with R848, and with R848 and MCC950 is shown. [Figure 12] Shown is the viability of cells treated with 22:0 Lyso PC, Compound 9 (DPD), Compound 2 (DGP), Compound 7, Compound 8, or vehicle without R848, with R848, and with R848 and MCC950. [Figure 13] IL-6 secretion by human moDCs treated with Compound 11, Compound 12, or vehicle without R848, with R848, and with R848 and MCC950 is shown. [Figure 14]IL-1β secretion by human moDCs treated with Compound 11, Compound 12, or vehicle without R848, with R848, and with R848 and MCC950 is shown. [Figure 15] Shown is the viability of cells treated with Compound 11, Compound 12, or vehicle without R848, with R848, and with R848 and MCC950. [Figure 16] Figure 1 shows IL-6 secretion by human moDCs treated with compounds 1, 4, 6, 11, 12, 13, 14, 15, 16, 2, 22:0 LPC, or vehicle without R848, with R848, and with R848 and MCC950. The compound concentration tested was 41.25 micromolar. [Figure 17] Figure 1 shows IL-1β secretion by human moDCs treated with compounds 1, 4, 6, 11, 12, 13, 14, 15, 16, 2, 22:0 LPC, or vehicle without R848, with R848, and with R848 and MCC950. The compound concentration tested was 41.25 micromolar. [Figure 18] Cell viability is shown for cells treated with compounds 1, 4, 6, 11, 12, 13, 14, 15, 16, 2, 22:0 LPC, or vehicle without R848, with R848, and with R848 and MCC950. [Figure 19] Figure 1 shows cell viability of cells treated with Compound 1, 2, 22:0 LPC, or vehicle without R848, with R848, and with R848 and MCC950. The compound concentration tested was 20.6 micromolar. [Figure 20] Figure 1 shows IL-1β secretion by human moDCs treated with Compound 1, 2, 22:0 LPC, or vehicle without R848, with R848, and with R848 and MCC950. The compound concentration tested was 20.6 micromolar. [Figure 21]IL-1β secretion by human moDCs under the indicated test conditions is shown. moDCs in each plot were derived from a different healthy donor (HD), and each symbol represents a value obtained from biological replicates. Ordinary two-way ANOVA was performed, followed by Tukey's multiple comparisons with a single pooled variance. [Figure 22] Cell viability was determined by measuring lactate dehydrogenase (LDF) release after treatment of human moDCs under the indicated test conditions. Each symbol represents the mean value of biological triplicates of moDCs derived from a given healthy donor (HD93, HD94, HD95, and HD96). Dashed lines indicate the acceptable range of cell viability. [Figure 23] The number of viable CD11c+CD209+ cells present in a fixed volume obtained from each sample, as determined by flow cytometry, is shown. Each symbol type is unique to each healthy donor. Statistical testing used repeated measures one-way ANOVA, followed by Tukey's comparison of individual variances. [Figure 24] A shows the percentage of viable CD11c+CD209+ cells expressing CD83. B shows the mean fluorescence intensity (MFI) of CD83 staining of viable CD11c+CD209+ cells. Each symbol type is unique to each donor. Statistical testing used repeated measures one-way ANOVA followed by Tukey's comparison of individual variances. [Figure 25] A shows the percentage of viable CD11c+CD209+ cells expressing CD86. B shows the MFI of CD86 staining of viable CD11c+CD209+ cells. Each symbol shape is unique to each donor. Statistical testing used repeated measures one-way ANOVA followed by Tukey's comparison of individual variances. [Figure 26] A shows the percentage of viable CD11c+CD209+ cells expressing CD40. B shows the MFI of CD40 staining of viable CD11c+CD209+ cells. Each symbol shape is unique to each donor. Statistical testing used repeated measures one-way ANOVA followed by Tukey's comparison of individual variances. [Figure 27] A shows the percentage of viable CD11c+CD209+ cells expressing MHC class I (HLA-ABC). B shows the MFI of MHC class I staining of viable CD11c+CD209+ cells. Each symbol shape is unique to each donor. Statistical testing used repeated measures one-way ANOVA followed by Tukey's comparison of individual variances. [Figure 28] A shows the percentage of viable CD11c+CD209+ cells expressing MHC class II (HLA-DR). B shows the MFI of MHC class II staining of viable CD11c+CD209+ cells. Each symbol shape is unique to each donor. Statistical testing used repeated measures one-way ANOVA followed by Tukey's comparison of individual variances. [Figure 29] A shows the percentage of viable CD11c+CD209+ cells expressing CCR7. B shows the MFI of CCR7 staining of viable CD11c+CD209+ cells. Each symbol shape is unique to each donor. Statistical testing used repeated measures one-way ANOVA followed by Tukey's comparison of individual variances. [Figure 30] Figure 1 shows the concentration of IL-1β present in cell culture supernatants after 24 hours of treatment of moDCs under the indicated conditions. Graphs show data from individual human donor samples, with each symbol representing a value obtained from a biological replicate. Statistical comparisons were performed using a standard two-way ANOVA followed by Tukey's multiple comparison test with a single pooled variance. [Figure 31] Panel A shows cell viability determined by measuring LDH activity in cell culture supernatants of moDCs treated for 24 hours under the indicated conditions. Panel B shows cell viability determined by measuring ATP-induced luminescence signal using CellTiter-Glo 2.0 reagent after lysis of moDCs for 24 hours under the indicated conditions. The x-axis labels apply to both panels. Each symbol in the graph represents a biological replicate of the donor. The dashed lines indicate the acceptable range of cell viability. [Figure 32A]Figure 1 shows NF-kB-dependent gene expression by human moDCs after treatment under the indicated conditions. The concentration of IL-6 present in cell culture supernatants after 24 hours of moDC treatment is shown. Each symbol represents a biological replicate. Statistical comparisons were performed using a conventional two-way ANOVA followed by Tukey's multiple comparison test with a single pooled variance. [Figure 32B] Figure 1 shows NF-kB-dependent gene expression by human moDCs after treatment under the indicated conditions. The concentration of IL-10 present in cell culture supernatants after 24 hours of moDC treatment is shown. Each symbol represents a biological replicate. Statistical comparisons were performed using a conventional two-way ANOVA followed by Tukey's multiple comparison test with a single pooled variance. [Figure 32C] Figure 1 shows NF-kB-dependent gene expression by human moDCs after treatment under the indicated conditions. The concentration of IL12p70 present in cell culture supernatants after 24 hours of moDC treatment is shown. Each symbol represents a biological replicate. Statistical comparisons were performed using a conventional two-way ANOVA followed by Tukey's multiple comparison test with a single pooled variance. [Figure 33] A-B show IRF-dependent gene expression by human moDCs after treatment under the indicated conditions. A shows the concentration of IP-10 present in the cell culture supernatant after 24 hours of treatment of moDCs. B shows the concentration of IFNα2. Each symbol represents a biological replicate. For statistical comparisons, a regular two-way ANOVA was performed, followed by Tukey's multiple comparison test with single pooled variance. [Figure 34-1]Figures A-B show the migration of human moDCs derived from three different donors (HD87, HD92, and HD93) after treatment with the indicated stimulatory factors. Briefly, cells were plated in the top chamber of a 5-µm-pore transwell. Medium containing the indicated concentrations of CCL19 was added to the bottom chamber, and the cells were incubated overnight. moDC migration was quantified by counting cells in the bottom chamber. Each symbol represents a biological replicate. Statistical comparisons were performed using a conventional two-way ANOVA followed by Tukey's multiple comparison test with single pooled variances. [Figure 34-2] (C) Migration of human moDCs derived from three different donors (HD87, HD92, and HD93) after treatment with the indicated stimulatory factors. Briefly, cells were plated in the upper chamber of a 5-μm-pore transwell. Medium containing the indicated concentrations of CCL19 was added to the bottom chamber, and cells were incubated overnight. moDC migration was quantified by counting cells in the bottom chamber. Each symbol represents a biological replicate. Statistical comparisons were performed using a conventional two-way ANOVA followed by Tukey's multiple comparison test with single pooled variances. [Figure 35A] Figure 1 shows the effect of human moDC hyperactivation on T cells. The concentration of IL-6 present in cell culture supernatants after 2 days of treatment of cocultures of moDCs and memory CD4+ T cells with the indicated stimulators is shown. Each column represents the mean value, and data points represent values ​​from biological replicates. A regular two-way ANOVA was performed, followed by Tukey's multiple comparisons with a single pooled variance. [Figure 35B] Figure 1 shows the effect of human moDC hyperactivation on T cells. The concentration of IL-6 present in cell culture supernatants after 2 days of treatment of CD4+ T cells with the indicated stimulators is shown. IL-6 was measured from cell culture supernatants using a Lumit immunoassay. Each column represents the mean value, and data points represent values ​​from biological replicates. A regular two-way ANOVA was performed, followed by Tukey's multiple comparisons with a single pooled variance. [Figure 36A]Figure 1 shows that stimulation of human moDCs with R848 and DGP mediates hyperactivation in cocultures. The concentration of IL-1β present in cell culture supernatants after 2 days of treatment of cocultures of moDCs and memory CD4+ T cells with the indicated stimulators is shown. IL-1β was measured from cell culture supernatants using a Lumit immunoassay. Each column represents the mean value, and data points represent values ​​from biological replicates. A regular two-way ANOVA was performed, followed by Tukey's multiple comparisons with a single pooled variance. [Figure 36B] Figure 1 shows that stimulation of human moDCs with R848 and DGP mediates hyperactivation in cocultures. Cell viability is shown after 2 days of treatment of cocultures of moDCs and memory CD4+ T cells with the indicated stimulators. Each column represents the mean value, and data points represent biological replicates. Ordinary two-way ANOVA was performed, followed by Tukey's multiple comparisons with single pooled variance. [Figure 37A] Figure 1 shows that human moDC stimulated with R848 and DGP induces Th1 responses. The concentration of IFNγ present in cell culture supernatants after 2 days of treatment of cocultures of moDC and memory CD4+ T cells (with anti-CD3) with the indicated stimulators is shown. IFNγ was measured using a Lumit immunoassay. Each column represents the mean value, and data points represent values ​​from biological replicates. A regular two-way ANOVA was performed, followed by Tukey's multiple comparisons with single pooled variances. [Figure 37B] Figure 1 shows that human moDCs stimulated with R848 and DGP induce Th1 responses. IFNγ levels in cell culture supernatants after 2 days of treatment of moDCs alone, moDCs and CD4+ T cells, and CD4+ T cells alone with 2.85 μM R848, 82.5 μM DGP, and 0.1 ng / mL anti-CD3 are shown. IFNγ was measured using a Lumit immunoassay. Each column represents the mean value, and data points represent biological replicates. A two-way ordinary ANOVA was performed, followed by Tukey's multiple comparisons with a single pooled variance. [Figure 37C]Figure 1 shows that human moDCs stimulated with R848 and DGP induce Th1 responses. The concentration of IFNγ present in cell culture supernatants after 2 days of treatment of cocultures of moDCs and memory CD4+ T cells with the indicated stimulators (without anti-CD3) is shown. IFNγ was measured using a Lumit immunoassay. Each column represents the mean value, and data points represent values ​​from biological replicates. A regular two-way ANOVA was performed, followed by Tukey's multiple comparisons with single pooled variances. [Figure 38A] Figure 1 shows that minimal Th2 cytokines are induced by human moDCs stimulated with R848 and DGP. The concentration of IL-4 present in cell culture supernatants after 2 days of treatment of cocultures of moDCs and memory CD4+ T cells with the indicated stimulators is shown. Cytokines were measured using a Lumit immunoassay. Each column represents the mean value, and data points represent values ​​from biological replicates. A regular two-way ANOVA was performed, followed by Tukey's multiple comparisons with single pooled variances. [Figure 38B] Figure 1 shows that minimal Th2 cytokines are induced by human moDCs stimulated with R848 and DGP. The concentration of IL-5 present in cell culture supernatants after 2 days of treatment of cocultures of moDCs and memory CD4+ T cells with the indicated stimulators is shown. Cytokines were measured using a Lumit immunoassay. Each column represents the mean value, and data points represent values ​​from biological replicates. A regular two-way ANOVA was performed, followed by Tukey's multiple comparisons with single pooled variances. [Figure 38C] Figure 1 shows that minimal Th2 cytokines are induced by human moDCs stimulated with R848 and DGP. The concentration of IL-13 present in cell culture supernatants after 2 days of treatment of cocultures of moDCs and memory CD4+ T cells with the indicated stimulators is shown. Cytokines were measured using a Lumit immunoassay. Each column represents the mean value, and data points represent values ​​from biological replicates. A regular two-way ANOVA was performed, followed by Tukey's multiple comparisons with single pooled variances. [Figure 39A] Figure 1 shows that human moDCs stimulated with R848 and DGP induce Th17 responses. The concentration of IL-17A present in cell culture supernatants after 2 days of treatment of cocultures of moDCs and memory CD4+ T cells with the indicated stimulators is shown. Cytokines were measured using a Lumit immunoassay. Each column represents the mean value, and data points represent values ​​from biological replicates. Ordinary two-way ANOVA was performed, followed by Tukey's multiple comparisons with single pooled variances. [Figure 39B] Figure 1 shows that human moDCs stimulated with R848 and DGP induce Th17 responses. The concentration of IL-17F present in cell culture supernatants after 2 days of treatment of cocultures of moDCs and memory CD4+ T cells with the indicated stimulators is shown. Cytokines were measured using a Lumit immunoassay. Each column represents the mean value, and data points represent values ​​from biological replicates. Ordinary two-way ANOVA was performed, followed by Tukey's multiple comparisons with single pooled variances. [Figure 39C] Figure 1 shows that human moDCs stimulated with R848 and DGP induce Th17 responses. The concentrations of IL-IL-22 present in cell culture supernatants after 2 days of treatment of cocultures of moDCs and memory CD4+ T cells with the indicated stimulators are shown. Cytokines were measured using a Lumit immunoassay. Each column represents the mean value, and data points represent values ​​from biological replicates. Ordinary two-way ANOVA was performed, followed by Tukey's multiple comparisons with single pooled variances. [Figure 39D]Figure 1 shows that human moDCs stimulated with R848 and DGP induce Th17 responses. The concentrations of IL-17A present in cell culture supernatants after 2 days of treatment of moDCs alone, moDCs and CD4+ T cells, and CD4+ T cells alone with 2.85 μM R848, 41.3 μM DGP, and 0.1 ng / mL anti-CD3 are shown. Cytokines were measured using a Lumit immunoassay. Each column represents the mean value, and data points represent biological replicates. Ordinary two-way ANOVA was performed, followed by Tukey's multiple comparisons with a single pool of variances. [Figure 39E] Figure 1 shows that human moDCs stimulated with R848 and DGP induce Th17 responses. The concentrations of IL-17F present in cell culture supernatants after 2 days of treatment of moDCs alone, moDCs and CD4+ T cells, and CD4+ T cells alone with 2.85 μM R848, 41.3 μM DGP, and 0.1 ng / mL anti-CD3 are shown. Cytokines were measured using a Lumit immunoassay. Each column represents the mean value, and data points represent biological replicates. Ordinary two-way ANOVA was performed, followed by Tukey's multiple comparisons with a single pooled variance. [Figure 39F] Figure 1 shows that human moDCs stimulated with R848 and DGP induce Th17 responses. The concentration of IL-22 present in cell culture supernatants after 2 days of treatment of moDCs alone, moDCs and CD4+ T cells, and CD4+ T cells alone with 2.85 μM R848, 41.3 μM DGP, and 0.1 ng / mL anti-CD3 is shown. Cytokines were measured using a Lumit immunoassay. Each column represents the mean value, and data points represent biological replicates. Ordinary two-way ANOVA was performed, followed by Tukey's multiple comparisons with a single pooled variance. [Figure 40] We show that R848 in combination with DGP (compound 2) enhances antigen-specific reactivation of CD8+ T cells. Briefly, the concentration of IFNγ present in the cell culture supernatant of CD8+ T cells cocultured with pretreated Flt3L-DCs for 96 hours was quantified as an index of T cell activation. [Figure 41] We demonstrate a process for reducing the size of DGP (Compound 2) DP (formulation) using jet mill micronization of DGP DS (drug substance) and homogenization of DGP DP, which increases DC hyperactivation in vitro and in vivo. In initial size reduction studies, sonication was used instead of homogenization. [Figure 42] Micronization, sonication, and a combination of the two are shown to reduce the size of DGP DP. [Figure 43] 1 shows that micronization and / or sonication of DGP DP increases IL-1β secretion by human moDCs when treated with R848 and DGP DP compared to unmodified DGP DP. [Figure 44] This shows that micronization and / or sonication of DGP DP increases the expression of CCR7 on DCs migrating to draining lymph nodes 4 hours after administration of R848 and DGP DP, compared to unmodified DGP DP. [Figure 45] A shows the frequency of SIINFEKL+ CD8+ T cells in the blood of immunized mice, and B shows their absolute numbers. Data from five mice per group are shown, and each symbol represents one mouse. [Figure 46] A shows the frequency of SIINFEKL+ CD8+ T cells in the draining lymph nodes of immunized mice, and B shows their absolute numbers. Data from 4-5 mice per group are shown, and each symbol represents one mouse. [Figure 47] The frequency of OVA-specific IFNγ-secreting T cells in the draining lymph nodes of immunized mice is shown. IFNγ-secreting cells were measured by ELISPOT assay after culturing cells in the presence or absence of OVA peptidomimetic for 18 hours. Data from 4-5 mice per group are shown, and each symbol represents one mouse. [Figure 48A] 1 shows the structures of cationic and ionizable lipids suitable for use in lipid nanoparticles (LNPs) of the present disclosure. [Figure 48B]The structures of other types of lipids suitable for use in the LNPs of the present disclosure are shown. See also Hou et al., Nature Review Materials, 6:1078-1094, 2021, which is incorporated herein by reference. [Figure 49] Heatmaps depicting normalized concentrations of cytokines and chemokines detected at 2, 24, and 48 hours post-injection are shown. Abbreviations: MCP1 = monocyte chemoattractant protein 1, MIP-1α = macrophage inflammatory protein 1 alpha, MIP-1β = macrophage inflammatory protein 1 beta, Rantes = normal T-cell expressed and secreted activation regulator, Eotaxin = eosinophil chemotaxin, MDC = macrophage-derived chemokine, KC = keratinocyte-derived chemokine, IP-10 = interferon-inducible protein 10, IFNα = interferon alpha. , IFNβ = interferon beta, TNFα = tumor necrosis factor alpha, IL-6 = interleukin 6, IL-10 = interleukin 10, IL-12p40 = interleukin 12 subunit P40, IL-12p70 = interleukin 12 subunit P70, IL-23 = interleukin 23, IL-27 = interleukin 27, TSLP = thymic stromal lymphopoietin, MIG = monokine induced by gamma. [Figure 50] A–D are graphs showing the absolute numbers of monocytes (A), moDCs (B), macrophages (C), and cDCs (D) in the dLN at 4 and 48 hours after injection. There were five mice per group, and each symbol represents one mouse. [Figure 51] A–D are graphs showing the absolute numbers of monocytes (A), moDCs (B), macrophages (C), and cDCs (D) in the spleen at 4 and 48 hours after injection. There were five mice per group; each symbol represents one mouse. Some samples were excluded due to low cell viability after dissociation. [Figure 52] A to D are graphs showing the MFI of CD69 expression on the surface of monocytes (A), moDCs (B), macrophages (C), and cDCs (D) in the dLN at 4 or 48 hours after injection. [Figure 53]A to D are graphs showing the MFI of CD69 expression on the surface of (A), moDCs (B), macrophages (C), and cDCs (D) in the spleen at 4 and 48 hours after injection. [Figure 54] AB are graphs showing the MFI of CCR7 expression on the surface of DCs in the dLN (A) and spleen (B) at 4 and 48 hours after injection. [Figure 55] Spleen weights of immunized mice at the endpoint are shown, with each symbol representing one mouse, 7 mice per group. [Figure 56] Panel A shows the frequency of IFNγ SFCs in each sample and restimulation condition. Panel B shows the frequency of Afluria-specific SFCs in each mouse after subtracting the background from the unstimulated condition. Statistical significance was determined by Student's t-test. Each symbol represents one mouse, with 4-7 mice per group. [Figure 57] Panel A shows the IFNγ concentration determined for each sample and restimulation condition. Panel B shows the Afluria-specific IFNγ secretion for each mouse after background subtraction from the unstimulated condition. Statistical significance was determined by Student's t-test. Each symbol represents one mouse, with 4-7 mice per group. [Figure 58] Panel A shows the frequency of IL-5 SFCs in each sample and restimulation condition. Panel B shows the frequency of Afluria-specific SFCs in each mouse after subtracting the background from the unstimulated condition. Statistical significance was determined by Student's t-test. Each symbol represents one mouse, n = 4–7 per group. [Figure 59] Panel A shows the IL-5 concentration determined for each sample and restimulation condition. Panel B shows the Afluria-specific IL-5 secretion for each mouse after background subtraction from the unstimulated condition. Statistical significance was determined by Student's t-test. Each symbol represents one mouse, n = 4–7 per group. [Figure 60]A shows the ratio of IFNγ SFC to IL-5 SFC for each sample. B shows the ratio of IFNγ concentration to IL-5 concentration for each sample. Each symbol represents one mouse, with 6-7 mice per group. [Figure 61] Geometric mean titers (GMT) of antigen-specific antibodies in a hemagglutinin inhibition (HAI) assay performed using Afluria vaccine as the viral antigen are shown. [Figure 62] Panel A shows antigen-specific IgG in the serum of immunized mice, detected by ELISA using Afluria vaccine as the coating antigen. Panel B shows the affinity of antigen-specific IgG in the serum of immunized mice, detected by ELISA using Afluria vaccine as the coating antigen. Statistical significance was determined by Student's t-test. Each symbol represents one mouse, with 4 to 7 mice per group. [Figure 63A] The frequency of DC is shown. [Figure 63B] The frequency of CD8+ TCM is shown. [Figure 63C] The frequency of CD4+ TCM is shown. [Figure 63D] The frequency of CD8+ TEM is shown. [Figure 63E] The frequency of CD4+ TEM is shown. [Figure 63F] The frequency of GCB is shown. [Figure 63G] The frequency of TFH cells is shown. Each symbol represents one mouse, with 4 to 7 mice per group. [Figure 64] A-D show the hyperactivation of canine PBCM. A shows the relative viability measured by ATP content in each condition compared to R848 alone. B shows IL-1β in cell culture supernatants after 48 hours of stimulation with the indicated treatments. C shows IL-6 in cell culture supernatants after 48 hours of stimulation with the indicated treatments. D shows IFNγ secretion in cell culture supernatants after 48 hours of stimulation with the indicated treatments. Each symbol represents one canine donor, n = 4 donors. Statistical significance was determined by one-way analysis of variance followed by Dunnett's multiple comparison test. [Figure 65] A shows the clinical scores in immunized mice after challenge with live influenza (PR8) virus. B shows the weight changes in immunized mice after challenge with live influenza (PR8) virus. C shows the survival rate in immunized mice after challenge with live influenza (PR8) virus. [Figure 66] A shows the influenza (PR8) virus load in bronchoalveolar lavage (BAL) fluid of immunized mice 5 days after challenge. B shows the influenza virus hemagglutinin (HA) antigen concentration in bronchoalveolar lavage (BAL) fluid of immunized mice 5 days after challenge. [Figure 67] A) Anti-hemagglutinin (HA) IgG antibody titers in the serum of immunized mice before influenza virus challenge. B) Anti-nucleoprotein (NP) IgG antibody titers in the serum of immunized mice before influenza virus challenge. [Figure 68] A shows the percentage of influenza nucleoprotein-specific CD8+ T cells in the blood of immunized mice before influenza virus challenge. B shows the absolute number of influenza nucleoprotein-specific CD8+ T cells in the blood of immunized mice before influenza virus challenge. Statistical significance was determined by one-way ANOVA with Tukey's post-hoc analysis. DETAILED DESCRIPTION OF THE INVENTION

[0078] The present disclosure relates to ether lipid (ETL) compounds, such as ether phospholipid (ETPL) compounds, and their use in the hyperactivation of human dendritic cells. The present disclosure also relates to compositions comprising an ETL, such as an ETPL, and one or more of a pathogen recognition receptor agonist, an antigen, and human dendritic cells, as well as methods of making and using 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.

[0079] General Techniques and Definitions 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 those in the art.

[0080] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless otherwise indicated. For example, "an excipient" includes one or more excipients.

[0081] As used herein, the phrase "comprising" is open-ended and indicates that such embodiment may include additional elements. In contrast, the phrase "consisting of" is a limitation and indicates that such embodiment does not include additional elements (except for trace impurities). The phrase "consisting essentially of" is a partial limitation and indicates that such embodiment may include further elements that do not materially alter the basic characteristics of such embodiment.

[0082] The term "about" as used herein in reference to a value includes 90% to 110% of that value (eg, a molecular weight of about 900 daltons refers to a molecular weight of 810 daltons to 990 daltons).

[0083] An "effective amount" or "sufficient amount" of a substance is an amount sufficient to produce a beneficial or desired result, including a clinical result, and therefore, "effective amount" will depend on the context in which it is applied. For example, with respect to administration of an immunogenic composition, an effective amount would contain sufficient antigen to stimulate an immune response to the antigen (e.g., antigen-reactive antibodies and / or a cellular immune response), and one or both of an ether lipid (ETL) compound, such as an ether phospholipid (ETPL) compound, and a PRR agonist.

[0084] The terms "individual" and "subject" refer to a mammal. "Mammal" includes, but is not limited to, humans, non-human primates (e.g., monkeys), farm animals, sport animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats). In some embodiments, the subject is a human patient, e.g., a human patient suffering from cancer and / or an infectious disease.

[0085] The term "dose" as used herein with respect to an immunogenic composition refers to the measured portion of the immunogenic composition that is taken (administered or ingested) by a subject at one time.

[0086] As used herein, the terms "isolated" and "purified" refer to material that has been removed from at least one component that normally coexists with the material during its production (e.g., removed from its original environment). By way of example, when used with reference to an ETL, such as an ETPL, an isolated ETL or ETPL is at least 90%, 95%, 96%, 97%, 98%, or 99% pure as measured by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), or gas chromatography (GC). By way of further example, when used with reference to a recombinant protein, an isolated protein refers to a protein that has been removed from the culture medium of a host cell in which the protein was produced. By way of further example, when used with reference to a synthesized compound, an isolated or purified compound has been removed from the reaction mixture in which the compound was synthesized.

[0087] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the individual to whom the formulation or composition is administered. Such formulations or compositions are intended to be sterile.

[0088] As used herein, "excipient" includes a pharmaceutically acceptable excipient, carrier, vehicle, or stabilizer that is nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. Often, the physiologically acceptable excipient is an aqueous pH buffered solution.

[0089] The term "antigen" refers to a substance that is specifically recognized and bound by an antibody or T cell antigen receptor. Antigens can include peptides, polypeptides, proteins, glycoproteins, polysaccharides, glycoconjugates, 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 generally becomes immunogenic when conjugated to a larger immunogenic molecule (carrier).

[0090] "Polypeptide antigens" can include purified natural peptides, synthetic peptides, recombinant peptides, crude peptide extracts, or partially purified or non-purified active peptides (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 8 amino acid residues in length.

[0091] The term "agonist" is used in the broadest sense and includes any molecule that activates receptor-mediated signaling. In some embodiments, an agonist binds to a receptor. For example, a TLR8 agonist binds to the TLR8 receptor and activates the TLR8 signaling pathway.

[0092] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group. x Alkyl refers to an alkyl group having x carbon atoms. x -Cy Alkyl or C x-y Alkyl refers to an alkyl group having from x to y carbon atoms (inclusive). An "n-alkyl" group refers to a straight-chain, ie, linear, alkyl group.

[0093] "Alkylene" refers to a divalent saturated aliphatic hydrocarbyl group.

[0094] "Alkenyl" refers to a monovalent hydrocarbyl group having at least one double bond (>C=C<). x Alkenyl refers to an alkenyl group having x carbon atoms. x -C y Alkenyl or C x-y Alkenyl refers to an alkenyl group having from x to y (inclusive) carbon atoms.

[0095] "Stimulation" of a response or parameter includes eliciting and / or enhancing that response or parameter when compared to conditions that are otherwise the same except for the parameter of interest, or when compared to another condition (e.g., increased TLR signaling in the presence of a TLR agonist compared to the absence of the TLR agonist). For example, "stimulation" of an immune response refers to an increase in the response. Depending on the parameter being measured, the increase can be 2-fold to 2,000-fold, or 5-fold to 500-fold or more, or 2-, 5-, 10-, 50-, or 100-fold to 500-, 1,000-, 2,000-, 5,000-, or 10,000-fold.

[0096] Conversely, "inhibition" of a response or parameter includes a decrease and / or suppression of the response or parameter when compared to conditions that are otherwise the same except for the parameter of interest, or when compared to another condition (e.g., a decrease in abnormal cell proliferation after administration of a composition comprising an ETL compound, such as an ETPL compound, and one or more of a pathogen recognition receptor agonist, an antigen, and human dendritic cells, compared to administration of a placebo composition or no treatment). For example, "inhibition" of an immune response refers to a decrease in the response. Depending on the parameter being measured, the decrease can be 2-fold to 2,000-fold, or 5-fold to 500-fold or more, or 2, 5, 10, 50, or 100-fold to 500, 1,000, 2,000, 5,000, or 10,000-fold.

[0097] The relative terms "higher" and "lower" refer to a measurable increase or decrease in a response or parameter, respectively, when compared to conditions that are otherwise the same except for the parameter of interest, or when compared to another condition. For example, a "higher level of DC hyperactivation" refers to a level of DC hyperactivation resulting from a treatment condition (including an ETL compound, such as an ETPL compound of the present disclosure) that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold higher than the level of DC hyperactivation resulting from a control condition (e.g., without ETL or ETPL, PGPC, oxPAPC, etc.). Similarly, a "lower level of DC hyperactivation" refers to a level of DC hyperactivation resulting from a treatment condition (including an ETL compound, such as an ETPL compound of the present disclosure) that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold lower than the level of DC hyperactivation resulting from a control condition (e.g., without ETL or ETPL, PGPC, oxPAPC, etc.). In some embodiments, the control condition includes a comparison compound in place of the ETL compound of the treatment condition, which may be an ETPL compound.

[0098] As used herein, the term "immunization" refers to the process of mounting a mammalian subject's response to an antigen, thereby improving the subject's ability to resist or overcome infection and / or disease.

[0099] As used herein, the term "vaccination" refers to the introduction of a vaccine into a mammalian subject.

[0100] "Adjuvant" refers to a substance that, when added to a composition containing an antigen, enhances or augments the immune response to the antigen in a mammalian recipient upon exposure.

[0101] The terms "treating" or "treatment" of a disease refer to carrying out a protocol, which may include administering one or more therapeutic agents to an individual (human or otherwise), to obtain a beneficial or desired result for the individual, including a clinical result. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more signs or symptoms of the disease, reduction in the extent of the disease, a stable (i.e., not worsening) disease state, prevention of disease spread, delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging survival as compared to the expected survival of an individual not receiving treatment. Furthermore, "treating" and "treatment" can be performed by administration of a single dose of one or more therapeutic agents or by administration of a series of doses of one or more therapeutic agents. "Treating" or "treatment" specifically includes protocols that do not require complete alleviation of signs or symptoms, do not require a cure, and have only a palliative effect on the individual. To "ameliorate" a disease or disorder means to lessen the severity and / or undesirable clinical symptoms of the disease or disorder and / or slow the time course of progression of the disease or disorder compared to the expected untreated outcome.

[0102] The compounds described herein can be administered in any pharmaceutically acceptable form, for example, in the form of a pharmaceutically acceptable salt, or in the form of a free base or free acid, provided that such form is pharmaceutically acceptable. The compounds described herein, or their pharmaceutically acceptable salts, can be administered in a pharmaceutically acceptable carrier or excipient. As used herein, "pharmaceutically acceptable" or "pharmacologically acceptable" means that the material is not biologically or otherwise undesirable; for example, the material can be incorporated into a pharmaceutical composition administered to a patient without causing significant undesirable biological effects or adversely interacting with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicology and manufacturing testing and / or are listed in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration. A "pharmaceutically acceptable salt" is a salt that retains at least some of the biological activity of the free (non-salt) compound and can be administered to an individual as a drug or pharmaceutical. Such salts include, for example, (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with organic acids such as acetic acid, oxalic acid, propionic acid, succinic acid, maleic acid, tartaric acid, and the like; and (2) salts formed when an acidic proton present in the parent compound 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 that can be used to prepare salts include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. Pharmaceutically acceptable salts can be prepared in situ during the manufacturing process, or by separately reacting a purified compound of the invention in its organic acid or organic base form with a suitable organic or inorganic base or acid, respectively, and isolating the salt thus formed during subsequent purification.

[0103] I. Ether Lipid (ETL) Compounds and Ether Phospholipid (ETPL) Compounds "Ether lipid" (ETL) or "ether lipid molecule" refers to a glycerol molecule having a hydrocarbyl group on one of the hydroxyl groups of the glycerol. The remaining hydroxyl group may be unsubstituted (free hydroxyl group) or substituted. The hydrocarbyl group may be an aliphatic hydrocarbyl group, such as an alkyl group, for example, an n-alkyl group. The alkyl group or n-alkyl group in any of the compounds disclosed herein is preferably unsubstituted, i.e., consists of only carbon and hydrogen atoms.

[0104] "Ether phospholipid" (ETPL) or "ether phospholipid molecule" refers to a specific type of ether lipid, a glycerol molecule having a phosphate group on a hydroxyl of the glycerol and a hydrocarbyl group on one of the other two hydroxyl groups of the glycerol. The remaining hydroxyl group may be unsubstituted (a free hydroxyl group) or substituted. The hydrocarbyl group may be an aliphatic hydrocarbyl group, such as an alkyl group, e.g., an n-alkyl group. The alkyl or n-alkyl group in any of the compounds disclosed herein is preferably unsubstituted, i.e., composed only of carbon and hydrogen atoms.

[0105] The present disclosure provides ether lipids, e.g., ether phospholipids. The present disclosure provides isolated ether lipids, e.g., isolated ether phospholipids.

[0106] In some embodiments, provided herein are ether lipid compounds of formula (I), such as isolated ether lipids (ETLs) having alkyl chains of formula (I): [ka]

[0107] (In the formula, R 1 is H or [ka] and

[0108] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5,

[0109] R 3 is C 13- C 24 n-alkyl,

[0110] where R 4 is H or (CH3)3N + -(CH2)2-,

[0111] Each R 5 are independently C1-C4 alkyl,

[0112] or a protonated or deprotonated form thereof, or a salt thereof, such as a pharmaceutically acceptable salt thereof, and all stereoisomers thereof.

[0113] In some embodiments, provided herein are ether lipid compounds of formula (II), such as isolated ether lipids (ETLs) having alkyl chains of formula (II): [ka]

[0114] (In the formula, R 1 is H or [ka] and

[0115] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R5 )2, or -CH2-C6H5,

[0116] R 3 is C 13- C 24 n-alkyl,

[0117] where R 4 is H or (CH3)3N + -(CH2)2-,

[0118] Each R 5 are independently C1-C4 alkyl,

[0119] or a protonated or deprotonated form thereof, or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0120] In some embodiments, provided herein are ether lipid compounds of formula (III), such as isolated ether lipids (ETLs) having alkyl chains of formula (III): [ka]

[0121] (In the formula, R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5,

[0122] R 3 is C 13- C 24 n-alkyl,

[0123] Each R 5 are independently C1-C4 alkyl,

[0124] or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0125] In some embodiments, provided herein are ether lipid compounds of formula (III-A), such as isolated ether lipids (ETLs) having alkyl chains of formula (III-A): [ka]

[0126] (In the formula, R 2 are -(C=O)-NH2, -(C=O)-NH(R 5 ), or -(C=O)-N(R 5 )2,

[0127] R 3 is C 13- C 24 n-alkyl,

[0128] Each R 5 are independently C1-C4 alkyl,

[0129] or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some embodiments, R 2 is —(C═O)—NH. In some embodiments, R 2 is —(C═O)—NH—CH. In some embodiments, R 2 is —(C═O)—N(CH). In some embodiments, R 3 is C 21 -C 24 In some embodiments, R 2 is -(C=O)-NH2, and R 3 is C 21 -C 24 In some embodiments, R 2 is -(C=O)-NH-CH3, and R 3 is C 21 -C 24 In some embodiments, R 2 is -(C=O)-N(CH3)2, and R 3 is C 21 -C24 In some embodiments, R 2 is -(C=O)-NH2, and R 3 is C 22 In some embodiments, R 2 is -(C=O)-NH-CH3, and R 3 is C 22 In some embodiments, R 2 is -(C=O)-N(CH3)2, and R 3 is C 22 It is n-alkyl.

[0130] In some embodiments, provided herein are ether lipid compounds of formula (III-A-1), such as isolated ether lipids (ETLs) having alkyl chains of formula (III-A-1): [ka]

[0131] (In the formula, R 2 are -(C=O)-NH2, -(C=O)-NH(R 5 ), or -(C=O)-N(R 5 )2,

[0132] R 3 is C 21- C 24 n-alkyl,

[0133] Each R 5 are independently C1-C4 alkyl,

[0134] or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some embodiments, R 2 is —(C═O)—NH. In some embodiments, R 2 is —(C═O)—NH—CH. In some embodiments, R 2 is —(C═O)—N(CH). In some embodiments, R 3is C 22 In some embodiments, R 2 is -(C=O)-NH2, and R 3 is C 22 In some embodiments, R 2 is -(C=O)-NH-CH3, and R 3 is C 22 In some embodiments, R 2 is -(C=O)-N(CH3)2, and R 3 is C 22 It is n-alkyl.

[0135] In some embodiments, provided herein are ether lipid compounds of formula (III-A-2), such as isolated ether lipids (ETLs) having alkyl chains of formula (III-A-2): [ka]

[0136] (In the formula, R 2 are -(C=O)-NH2, -(C=O)-NH(R 5 ), or -(C=O)-N(R 5 )2,

[0137] R 3 is C 16- C 20 n-alkyl,

[0138] Each R 5 are independently C1-C4 alkyl,

[0139] or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some embodiments, R 2 is —(C═O)—NH. In some embodiments, R 2 is —(C═O)—NH—CH. In some embodiments, R 2 is —(C═O)—N(CH). In some embodiments, R3 is C 18 In some embodiments, R 2 is -(C=O)-NH2, and R 3 is C 18 In some embodiments, R 2 is -(C=O)-NH-CH3, and R 3 is C 18 In some embodiments, R 2 is -(C=O)-N(CH3)2, and R 3 is C 18 It is n-alkyl.

[0140] In some embodiments, provided herein are ether lipid compounds of formula (III-B), such as isolated ether lipids (ETLs) having alkyl chains of formula (III-B): [ka]

[0141] (In the formula, R 3 is C 13- C 24 n-alkyl), or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some embodiments, R 3 is C 21 -C 24 In some embodiments, R 3 is C 22 It is n-alkyl.

[0142] In some embodiments, provided herein are ether lipid compounds of formula (III-B-1), such as isolated ether lipids (ETLs) having alkyl chains of formula (III-B-1): [ka]

[0143] (In the formula, R 3 is C 21- C24 n-alkyl), or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some embodiments, R 3 is C 22 It is n-alkyl.

[0144] In some embodiments, provided herein are ether lipid compounds of formula (III-B-2), such as isolated ether lipids (ETLs) having alkyl chains of formula (III-B-2): [ka]

[0145] (In the formula, R 3 is C 16- C 20 n-alkyl), or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some embodiments, R 3 is C 18 It is n-alkyl.

[0146] In some embodiments, provided herein are ether lipid compounds of formula (IV), such as isolated ether phospholipids (ETPLs) having alkyl chains of formula (IV): [ka]

[0147] (In the formula, R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5,

[0148] R 3 is C 13- C 24 n-alkyl,

[0149] R 4 is H or (CH3)3N + -(CH2)2-,

[0150] Each R 5 are independently C1-C4 alkyl,

[0151] or a protonated or deprotonated form thereof, or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0152] In some embodiments, provided herein are ether lipid compounds of formula (IV-A), such as isolated ether phospholipids (ETPLs) having alkyl chains of formula (IV-A): [ka]

[0153] (In the formula, R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5,

[0154] R 3 is C 13- C 24 n-alkyl,

[0155] Each R 5 are independently C1-C4 alkyl,

[0156] or a protonated or deprotonated form thereof, or a salt thereof, such as a pharmaceutically acceptable salt thereof. 2 is H. In some embodiments, R 2 is —(C═O)—NH. In some embodiments, R 2 is —(C═O)—NH—CH. In some embodiments, R 2 is —(C═O)—N(CH). In some embodiments, R 3 is C 21 -C 24In some embodiments, R 2 is H and R 3 is C 21 -C 24 In some embodiments, R 2 is -(C=O)-NH2, and R 3 is C 21 -C 24 In some embodiments, R 2 is -(C=O)-NH-CH3, and R 3 is C 21 -C 24 In some embodiments, R 2 is -(C=O)-N(CH3)2, and R 3 is C 21 -C 24 In some embodiments, R 2 is H and R 3 is C 22 In some embodiments, R 2 is -(C=O)-NH2, and R 3 is C 22 In some embodiments, R 2 is -(C=O)-NH-CH3, and R 3 is C 22 In some embodiments, R 2 is -(C=O)-N(CH3)2, and R 3 is C 22 It is n-alkyl.

[0157] In some embodiments, provided herein are ether lipid compounds of formula (IV-A-1), such as isolated ether phospholipids (ETPLs) having alkyl chains of formula (IV-A-1): [ka]

[0158] (In the formula, R 2is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5,

[0159] R 3 is C 21- C 24 n-alkyl,

[0160] Each R 5 are independently C1-C4 alkyl,

[0161] or a protonated or deprotonated form thereof, or a salt thereof, such as a pharmaceutically acceptable salt thereof. 2 is H. In some embodiments, R 2 is —(C═O)—NH. In some embodiments, R 2 is —(C═O)—NH—CH. In some embodiments, R 2 is —(C═O)—N(CH). In some embodiments, R 3 is C 22 In some embodiments, R 2 is H and R 3 is C 22 In some embodiments, R 2 is -(C=O)-NH2, and R 3 is C 22 In some embodiments, R 2 is -(C=O)-NH-CH3, and R 3 is C 22 In some embodiments, R 2 is -(C=O)-N(CH3)2, and R 3 is C 22 It is n-alkyl.

[0162] In some embodiments, provided herein are ether lipid compounds of formula (IV-A-2), such as isolated ether phospholipids (ETPLs) having alkyl chains of formula (IV-A-2): [ka]

[0163] (In the formula, R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5,

[0164] R 3 is C 16- C 20 n-alkyl,

[0165] Each R 5 are independently C1-C4 alkyl,

[0166] or a protonated or deprotonated form thereof, or a salt thereof, such as a pharmaceutically acceptable salt thereof. 2 is H. In some embodiments, R 2 is —(C═O)—NH. In some embodiments, R 2 is —(C═O)—NH—CH. In some embodiments, R 2 is —(C═O)—N(CH). In some embodiments, R 3 is C 18 In some embodiments, R 2 is H and R 3 is C 18 In some embodiments, R 2 is -(C=O)-NH2, and R 3 is C 18 In some embodiments, R 2 is -(C=O)-NH-CH3, and R3 is C 18 In some embodiments, R 2 is -(C=O)-N(CH3)2, and R 3 is C 18 It is n-alkyl.

[0167] In some embodiments, provided herein are ether lipid compounds of formula (IV-B), such as isolated ether phospholipids (ETPLs) having alkyl chains of formula (IV-B): [ka]

[0168] (In the formula, R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5,

[0169] R 3 is C 13- C 24 n-alkyl,

[0170] Each R 5 are independently C1-C4 alkyl,

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

[0172] In some embodiments, provided herein are ether lipid compounds of formula (IV-B-1), such as isolated ether phospholipids (ETPLs) having alkyl chains of formula (IV-B-1): [ka]

[0173] (In the formula, R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5,

[0174] R 3 is C 21- C 24 n-alkyl,

[0175] Each R 5 are independently C1-C4 alkyl,

[0176] or a protonated form thereof, or a salt thereof, such as a pharmaceutically acceptable salt thereof. 2 is H. In some embodiments, R 2 is —(C═O)—NH. In some embodiments, R 2 is —(C═O)—NH—CH. In some embodiments, R 2 is —(C═O)—N(CH). In some embodiments, R 3 is C 22 In some embodiments, R 2 is H and R 3 is C 22 In some embodiments, R 2 is -(C=O)-NH2, and R 3 is C 22 In some embodiments, R 2 is -(C=O)-NH-CH3, and R 3 is C 22 In some embodiments, R 2 is -(C=O)-N(CH3)2, and R 3 is C 22 It is n-alkyl.

[0177] In some embodiments, provided herein are ether lipid compounds of formula (IV-B-2), such as isolated ether phospholipids (ETPLs) having alkyl chains of formula (IV-B-2): [ka]

[0178] (In the formula, R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5,

[0179] R 3 is C 16- C 20 n-alkyl,

[0180] Each R 5 are independently C1-C4 alkyl,

[0181] or a protonated form thereof, or a salt thereof, such as a pharmaceutically acceptable salt thereof. 2 is H. In some embodiments, R 2 is —(C═O)—NH. In some embodiments, R 2 is —(C═O)—NH—CH. In some embodiments, R 2 is —(C═O)—N(CH). In some embodiments, R 3 is C 18 In some embodiments, R 2 is H and R 3 is C 18 In some embodiments, R 2 is -(C=O)-NH2, and R 3 is C 18 In some embodiments, R 2 is -(C=O)-NH-CH3, and R 3 is C 18 In some embodiments, R 2 is -(C=O)-N(CH3)2, and R 3 is C 18It is n-alkyl.

[0182] In some embodiments, provided herein are ether lipid compounds of formula (IV-C), such as isolated ether phospholipids (ETPLs) having alkyl chains of formula (IV-C): [ka]

[0183] (In the formula, R 3 is C 13- C 24 n-alkyl,

[0184] R 4 is H or (CH3)3N + -(CH2)2-),

[0185] or a protonated or deprotonated form thereof, or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0186] In some embodiments, provided herein are ether lipid compounds of formula (IV-D), such as isolated ether phospholipids (ETPLs) having alkyl chains of formula (IV-D): [ka]

[0187] (In the formula, R 3 is C 13- C 24 n-alkyl),

[0188] or a protonated form thereof, or a salt thereof, such as a pharmaceutically acceptable salt thereof. 3 is C 16 -C 20 In some embodiments, R 3 is C 21 -C 24 In some embodiments, R3 is C 22 It is n-alkyl.

[0189] In some embodiments, provided herein are ether lipid compounds of formula (IV-E), such as isolated ether phospholipids (ETPLs) having alkyl chains of formula (IV-E): [ka]

[0190] (In the formula, R 3 is C 13- C 24 n-alkyl),

[0191] or a protonated or deprotonated form thereof, or a salt thereof, such as a pharmaceutically acceptable salt thereof. 3 is C 21 -C 24 It is n-alkyl.

[0192] In some embodiments, provided herein are ether lipid compounds of formula (IV-F), such as isolated ether phospholipids (ETPLs) having alkyl chains of formula (IV-F): [ka]

[0193] (In the formula, R 2 is H, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, and R 3 is C 21- C 24 n-alkyl, and each R 5 are independently C1-C4 alkyl), or a protonated or deprotonated form thereof, or a salt thereof. In some embodiments, R 2 is H. In some embodiments, R 2is —(C═O)—NH. In some embodiments, R 2 is -(C=O)-NH(R 5 In some embodiments, R 2 is -(C=O)-N(R 5 )2. In some embodiments, R 3 is C 21 In some embodiments, R 3 is unsubstituted. In some embodiments, R 5 is —CH3. Formula (IV-F) is a combination of certain compounds of formula (IV-A) and formula (IV-E).

[0194] In some embodiments, provided herein are ether lipid compounds of formula (A), such as isolated ether lipids (ETLs) having alkyl chains of formula (I): [ka]

[0195] (In the formula, R 1 is H or [ka] and

[0196] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5,

[0197] R 3 is C 10- C 30 n-alkyl,

[0198] where R 4 is H or (CH3)3N + -(CH2)2-,

[0199] Each R5 are independently C1-C4 alkyl,

[0200] or a protonated or deprotonated form thereof, or a salt thereof, such as a pharmaceutically acceptable salt thereof, and all stereoisomers thereof.

[0201] In some embodiments, the ether phospholipid (ETPL) having an n-alkyl chain is a compound of formula (IV), wherein R 4 is (CH3)3N + -(CH2)2-, and R 2 is H and R 3 is C 22 n-alkyl, and the compound is 1-docosyl-sn-glycerol-3-phosphocholine (DGPC): [ka] or a protonated form thereof, or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0202] In some embodiments, the isolated ether phospholipid (ETPL) having an n-alkyl chain is a compound of formula (IV), wherein R 4 is (CH3)3N + -(CH2)2-, and R 2 is H and R 3 is C 22 n-alkyl, and the compound is 1-docosyl-sn-glycerol-3-phosphocholine (DGPC): [ka] or a protonated form thereof, or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0203] In some embodiments, the ether phospholipid (ETPL) having an n-alkyl chain is a compound of formula (IV), wherein R 4 is H and R 2 is H and R 3 is C 22n-alkyl, and the compound is 1-docosyl-sn-glycerol-3-phosphate (DGP): [ka] or a protonated or deprotonated form thereof, or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0204] In some embodiments, the isolated ether phospholipid (ETPL) having an n-alkyl chain is a compound of formula (IV), wherein R 4 is H and R 2 is H and R 3 is C 22 n-alkyl, and the compound is 1-docosyl-sn-glycerol-3-phosphate (DGP): [ka] or a protonated or deprotonated form thereof, or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0205] The ether lipid (ETL) and ether phospholipid (ETPL) compounds of the present disclosure have alkyl chains, where the n-alkyl chain is a C13-C22 n-alkyl chain or a C13-C24 n-alkyl chain. In some embodiments, the n-alkyl chain is a C18-C22 n-alkyl chain or a C21-C24 n-alkyl chain. In some embodiments, the n-alkyl chain is a C16-C20 n-alkyl chain. In some embodiments, the n-alkyl chain is a C21-C24 n-alkyl chain. In some preferred embodiments, the n-alkyl chain is a C22 n-alkyl chain. The structures of exemplary ETPL and ETL compounds of the present disclosure are set forth below in Tables I and II, respectively. The structures set forth in Tables I and II may alternatively be protonated or deprotonated forms of the structures set forth in Tables I and II, i.e., protonated is defined below as O -where deprotonation refers to the removal of a proton from any or all of the phosphate OH groups, and / or may be a salt of the structure shown in Table I and Table II, e.g., a pharmaceutically acceptable salt thereof. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2]

[0206] II. Pathogen Recognition Receptor Agonists The compositions and methods of the present disclosure may further include 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 comprises a cytoplasmic DNA sensor (CDS) or a stimulator of IFN genes (STING). In some embodiments, the PRR agonist comprises a TLR7 / 8 agonist.

[0207] A. TLR agonists and TLR7 / 8 agonists As used herein, the term "TLR agonist" refers to an agonist of at least one TLR. As used herein, the term "TLR7 / 8 agonist" refers to an agonist of TLR7 and / or TLR8. In one embodiment, the TLR7 / 8 agonist is a TLR7 agonist. In a further embodiment, the TLR7 / 8 agonist is a TLR8 agonist. In a further embodiment, the TLR7 / 8 agonist is an agonist of both TLR7 and TLR8. The TLR7 / 8 agonists of the present disclosure are suitable for superactivating human dendritic cells in the presence of LPC.

[0208] In some embodiments, the TLR agonist is a small molecule. In some embodiments, the TLR7 / 8 agonist is a small molecule. In some embodiments, the TLR7 / 8 agonist is a small molecule, or a salt thereof, with a molecular weight of 900 daltons or less. That is, small molecule TLR7 / 8 agonists are not macromolecules, such as recombinant proteins or synthetic oligonucleotides, that can be regulated by the USFDA's Center for Biologics Evaluation and Research. Rather, small molecule TLR7 / 8 agonists can be regulated by the FDA's Center for Drug Evaluation and Research. In some embodiments, the small molecule has a molecular weight of about 90 to about 900 daltons. In some embodiments, the TLR7 / 8 agonist comprises an imidazoquinoline compound. In some preferred embodiments, the TLR7 / 8 agonist comprises resiquimod (R848).

[0209] B. Other PRR agonists In some embodiments, the pathogen recognition receptor (PRR) agonist includes a toll-like receptor (TLR) agonist, with the proviso that the TLR agonist does not include a TLR7 / 8 agonist. In some embodiments, the TLR agonist includes an agonist of one or more of TLR2, TLR3, TLR4, TLR5, TLR9, and TLR13. In some embodiments, the PRR agonist is a TLR2 / 6 agonist, e.g., Pam2CSK4. In other embodiments, the TLR agonist is a TLR4 agonist, e.g., monophosphoryl lipid A (MPLA). However, in preferred embodiments, the TLR agonist is not an agonist of TLR2, TLR4, and / or TLR9. For example, in preferred embodiments, the TLR9 agonist is not a TLR4 ligand, such as LPS (endotoxin).

[0210] In additional embodiments, the PRR agonist comprises a NOD-like receptor (NLR) agonist. In further embodiments, the PRR agonist comprises a RIG-I-like receptor (RLR) agonist. In additional embodiments, the PRR agonist comprises a C-type lectin receptor (CLR) agonist. In still further embodiments, the PRR agonist comprises a CDS agonist or a STING agonist.

[0211] III. Antigen The compositions and methods of the present disclosure may further include an antigen. In some embodiments, the antigen comprises a proteinaceous antigen. The terms "polypeptide" and "protein" are used interchangeably herein to refer to a proteinaceous antigen comprising a peptide chain at least 8 amino acids in length. In some embodiments, the proteinaceous antigen is 8 to 1800 amino acids in length, 9 to 1000 amino acids in length, 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.

[0212] In some embodiments, the antigen is a tumor antigen comprising the amino acid sequence of at least one full-length protein or a fragment thereof. In some embodiments, the tumor antigen comprises an amino acid sequence derived from an oncoprotein or a fragment thereof. In some embodiments, the mammalian antigen is a neoantigen or is encoded by a gene comprising a mutation relative to a gene present in normal cells from the mammalian subject. Neoantigens are believed to be particularly useful in enabling T cells to distinguish between cancer cells and non-cancerous cells (see, e.g., Schumacher and Schreiber, Science, 348:69-74, 2015). In other embodiments, the tumor antigen comprises a viral antigen, e.g., an antigen of a cancer-causing virus.

[0213] In some embodiments, the tumor antigen is a fusion protein comprising two or more polypeptides, each polypeptide comprising an amino acid sequence derived from a different tumor antigen or non-contiguous amino acid sequences derived from the same tumor antigen. In some of these embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, each polypeptide comprising a non-contiguous amino acid sequence derived from the same tumor antigen.

[0214] In some embodiments, the antigen is a microbial antigen. In some embodiments, the microbial antigen comprises a viral antigen, a bacterial antigen, a protozoan antigen, a fungal antigen, or a combination thereof. In some embodiments, the microbial antigen comprises a surface protein or other antigenic subunit of a microorganism. In other embodiments, the microbial antigen comprises an inactivated or attenuated microorganism. For example, the microbial antigen may comprise an inactivated virus, e.g., a chemically or genetically inactivated virus. Alternatively, the microbial antigen may comprise a virus-like particle.

[0215] In some embodiments, the antigen may be present in a biological sample obtained from an individual, e.g., a human patient. For example, the antigen may include cancer cells. In a further aspect, the antigen may include microbially infected cells, e.g., virally infected cells.

[0216] IV. Dendritic cells The compositions and methods of the present disclosure may further include dendritic cells (DCs), which are antigen-presenting cells believed to bridge the innate and adaptive immune systems of mammals. In a preferred embodiment, the DCs are subset 1 classical DCs (cDC1s, formerly called myeloid DC1s), as opposed to plasmacytoid DCs (pDCs).

[0217] In some embodiments, the DCs are hyperactivated DCs that express high levels of CD40 and IL-12p70. As used herein, the term "hyperactivated dendritic cells" refers to a cellular state in which DCs can secrete IL-1β while maintaining cell viability (e.g., without undergoing pyroptosis). Thus, hyperactivated dendritic cells can stimulate robust T cell immunity (Figure 1), which clearly combines the advantages of activated and pyroptotic dendritic cells (Zhivaki et al., Cell Reports, 33 (7), 2020, 108381).

[0218] V. Pharmaceutical Preparations Some compositions of the present disclosure are pharmaceutical formulations comprising a pharmaceutically acceptable excipient and an ETL compound, such as an ETPL compound. Some compositions of the present disclosure are pharmaceutical formulations comprising a pharmaceutically acceptable excipient and an ETL compound or a lipid nanoparticle (LNP) comprising an ETPL compound and at least one additional lipid. In some embodiments, the pharmaceutical formulation further comprises a PRR agonist, dendritic cells, an antigen, an adjuvant, or any combination thereof. The pharmaceutical formulations of the present disclosure may be in the form of a solution or suspension. Alternatively, the pharmaceutical formulation may be a dehydrated solid (e.g., a freeze-dried or spray-dried solid). The pharmaceutical formulations of the present disclosure are preferably sterile and preferably essentially endotoxin-free. The term "pharmaceutical formulation" is used interchangeably herein with the terms "medicine" and "drug." In some embodiments, the pharmaceutical formulation comprises various components in specific ratios based on the intended purpose of the formulation. In some embodiments, the pharmaceutical formulation comprises an ETL compound, such as an ETPL compound, and a non-ionic surfactant. In some embodiments, the nonionic surfactant comprises an ethylene oxide-propylene oxide copolymer (i.e., a poloxamer), such as poloxamer-407 (CAS Registry Number 977057-91-2).

[0219] A. Excipients Pharmaceutically acceptable excipients of the present disclosure include, for example, solvents, buffers, tonicity adjusters, bulking agents, and preservatives (see, e.g., Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, pharmaceutical formulations may include excipients that function as one or more of a solvent, buffer, tonicity adjuster, and bulking agent (e.g., sodium chloride in saline can function as both an aqueous vehicle and an isotonicity adjuster). Pharmaceutically acceptable excipients of the present disclosure also include detergents, wetting agents, thickeners, emulsifiers, foaming agents, and dispersing agents, as well as surfactants.

[0220] Many of the lipids disclosed herein are poorly soluble in water.Surfactants can be used to solubilize lipids in aqueous formulations.A wide variety of surfactants are available and can be classified into anionic surfactants, nonionic surfactants, cationic surfactants, and zwitterionic surfactants.

[0221] Some examples of nonionic surfactants include poloxamers, which have 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 aforementioned values ​​of a and b may vary slightly).

[0222] Other nonionic 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), which is obtained by reacting 40 moles of ethylene oxide with 1 mole of hydrogenated castor oil.

[0223] In some embodiments, the pharmaceutical formulation comprises an aqueous vehicle as a solvent. Suitable vehicles include, for example, sterile water, saline, phosphate-buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic.

[0224] The pharmaceutical formulation may contain a buffering agent. The buffering agent controls the pH and prevents degradation of the active agent during processing, storage, and optional reconstitution. Suitable buffering agents include salts, including, for example, acetate, citrate, phosphate, or sulfate. Other suitable buffers include amino acids, such as arginine, glycine, histidine, and lysine. The buffering agent may further contain hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the composition in the range of 6 to 9. In some embodiments, the pH is greater than 6, 7, or 8 (lower limit). In some embodiments, the pH is less than 9, 8, or 7 (upper limit). That is, the pH is within the range of about 6 to 9, with the lower limit being less than the upper limit.

[0225] The pharmaceutical composition may comprise an isotonicity adjusting agent. Suitable isotonicity adjusting agents include, for example, dextrose, glycerol, sodium chloride, glycerin, and mannitol.

[0226] The pharmaceutical formulation may contain a bulking agent. Bulking agents are particularly useful when the pharmaceutical composition is lyophilized before administration. In some embodiments, the bulking agent is a protective agent that helps stabilize and prevent degradation of the active agent during lyophilization or spray drying and / or storage. Suitable bulking agents are sugars (monosaccharides, disaccharides, and polysaccharides) such as sucrose, lactose, trehalose, mannitol, sorbital, glucose, and raffinose.

[0227] Pharmaceutical preparations may contain preservatives. Suitable preservatives include, for example, antioxidants and antibacterial agents. However, in a preferred embodiment, the pharmaceutical preparations are prepared under aseptic conditions and are contained in single-use containers, so preservatives are not necessary. Methods for preparing pharmaceutically acceptable sterile compositions include steam sterilization, dry heat sterilization, gas sterilization, ionizing radiation, or sterile filtration. Sterile pharmaceutical preparations are compounded or manufactured in accordance with pharmaceutical-grade sterilization standards known to those skilled in the art (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).

[0228] In some embodiments, the pharmaceutical formulation is a homogeneous solution. In some embodiments, the homogeneous solution is provided in the form of a pre-filled syringe. In some embodiments, the pharmaceutical formulation is provided as a suspension. In some embodiments, the suspension is refrigerated. In some embodiments, the suspension is frozen. In some embodiments, the methods provided herein further comprise warming the refrigerated suspension to room temperature and / or stirring the suspension prior to administration to ensure that the active ingredient(s) are dissolved and / or uniformly dispersed in the solution. In some embodiments, the methods provided herein further comprise thawing the frozen suspension and warming it to room temperature and / or stirring the suspension prior to administration to ensure that the active ingredient(s) are dissolved and / or uniformly dispersed in the solution. In some embodiments, the suspension is diluted prior to administration. In some embodiments, the suspension is provided as a pre-filled syringe. In some embodiments, the suspension comprises a pharmaceutically acceptable excipient, such as a surfactant, glycerol, a non-ionic surfactant, a buffer, a glycol, a salt, or any combination thereof.

[0229] The pharmaceutical formulations of the present disclosure are suitable for parenteral administration, i.e., they are not intended for enteral administration (e.g., not by oral, gastric, or rectal administration).

[0230] B. Adjuvants Pharmaceutically acceptable adjuvants of the present disclosure include, for example, aluminum salt adjuvants, squalene-in-water emulsions, saponins, or combinations thereof. In some embodiments, the adjuvant is an aluminum salt adjuvant selected from the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, and combinations thereof. In other embodiments, the adjuvant is a squalene-in-water emulsion such as MF59 or AS03. In other embodiments, the adjuvant is a saponin such as Quil A or QS-21, such as AS01 or AS02.

[0231] C. Kit 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 disclosed herein (e.g., a single unit dose in a vial) and a solution for dissolving, diluting, and / or reconstituting the lyophilized pharmaceutical composition. In some embodiments, the reconstitution or dilution solution is provided as a pre-filled syringe. In some embodiments, the kit comprises a frozen suspension of the pharmaceutical formulation (e.g., a single unit dose in a vial). In some embodiments, the kit comprises a buffer that helps prevent aggregation when reconstituting the pharmaceutical composition disclosed herein. In some embodiments, the pharmaceutical composition is provided in a pre-filled syringe. In some embodiments, the kit comprises a dual-chamber syringe or container, one chamber of which contains a buffer for dissolving or diluting the pharmaceutical composition. In some embodiments, the kit comprises an injection syringe. In some embodiments, the reconstituted solution is filtered before administration. In some embodiments, the kit comprises a filter or filter syringe for filtering the reconstituted pharmaceutical composition before administration. In some embodiments, the kit further comprises instructions for use, eg, instructions for superactivating cells.

[0232] D. Particle size of the formulation The particle size of drug particles can affect cellular uptake of the drug. Particle size can be controlled by milling a drug substance, such as DGP (Compound 2), using techniques well known in the pharmaceutical arts. Dry milling techniques that can be used include, but are not limited to, jet mills, hammer mills, and pin mills. Wet milling techniques that can be used include, but are not limited to, rotor-stator mills, colloid mills, and media mills. Milling of a drug substance can be performed prior to further steps in the method, such as combining the drug substance with a solution, buffer, and / or other ingredients to form a suspension.

[0233] The drug substance can be combined with a solution or buffer, such as phosphate buffered saline and a poloxamer (e.g., poloxamer 407 or poloxamer 188), and formulated. Additional procedures, including sonication and homogenization, can also be used to reduce particle size in the formulation.

[0234] In embodiments, about 50% of the particles in the formulation have a diameter of less than about 40 microns (D 50 <40 microns). In embodiments, about 50% of the particles in the formulation have a diameter of less than about 30 microns (D 50 <30 microns). In embodiments, about 50% of the particles in the formulation have a diameter of less than about 20 microns to about 40 microns (D 50 <20 microns to 40 microns). In embodiments, about 50% of the particles in the formulation have a diameter of about 20 microns to less than about 30 microns (D 50 <20 microns to 30 microns). In embodiments, about 50% of the particles in the formulation have a diameter of less than about 20 microns (D 50 <20 microns). In embodiments, about 50% of the particles in the formulation have a diameter of about 10 microns to less than about 30 microns (D 50 <10 microns to 30 microns). In embodiments, about 50% of the particles in the formulation have a diameter of less than about 10 microns (D 50 <10 microns). In embodiments, about 50% of the particles in the formulation have a diameter of about 10 microns to less than about 20 microns (D 50 <10 microns to 20 microns). In embodiments, about 50% of the particles in the formulation have a diameter of about 5 microns to less than about 20 microns (D 50 <5 microns to 20 microns). In embodiments, about 50% of the particles in the formulation have a diameter of less than about 5 microns (D 50 <5 microns).

[0235] In embodiments, about 50% of the particles in the formulation have a diameter of less than about 40 microns (D 90 <40 microns). In embodiments, about 50% of the particles in the formulation have a diameter of less than about 30 microns (D90 <30 microns). In embodiments, about 50% of the particles in the formulation have a diameter of less than about 20 microns to about 40 microns (D 90 <20 microns to 40 microns). In embodiments, about 50% of the particles in the formulation have a diameter of about 20 microns to less than about 30 microns (D 90 <20 microns to 30 microns). In embodiments, about 50% of the particles in the formulation have a diameter of less than about 20 microns (D 90 <20 microns). In embodiments, about 50% of the particles in the formulation have a diameter of about 10 microns to less than about 30 microns (D 90 <10 microns to 30 microns). In embodiments, about 50% of the particles in the formulation have a diameter of less than about 10 microns (D 90 <10 microns). In embodiments, about 50% of the particles in the formulation have a diameter of about 10 microns to less than about 20 microns (D 90 <10 microns to 20 microns). In embodiments, about 50% of the particles in the formulation have a diameter of about 5 microns to less than about 20 microns (D 90 <5 microns to 20 microns). In embodiments, about 50% of the particles in the formulation have a diameter of less than about 5 microns (D 90 <5 microns).

[0236] The particles of the formulations described herein comprise one or more of: i) a surfactant, e.g., a non-ionic surfactant such as a poloxamer or Pluronic, e.g., poloxamer 407, poloxamer 188, Pluronic 84, or Pluronic 123; a humectant such as P407, P188, or polysorbate 80; or a thickener such as carboxymethylcellulose; and ii) a soluble polymeric polymer of Formula (I), Formula (II), Formula (III), Formula (III-A), Formula (III-A-1), Formula (III-A-2), Formula (III-B), Formula (III-B-1), Formula (III-B-2), Formula (IV ... ), Formula (IV-A), Formula (IV-A-1), Formula (IV-A-2), Formula (IV-B), Formula (IV-B-1), Formula (IV-B-2), Formula (IV-C), Formula (IV-D), Formula (IV-E), Formula (IV-F), Formula (A), ether lipid (ETL) compounds or ether phospholipid (ETPL) compounds, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, or Compound 16, or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof. The particles may have a size or size range as indicated above.

[0237] VI. Preparation method In some aspects, the present disclosure relates to methods for preparing superactivated dendritic cells and methods for preparing immunogenic compositions suitable for in vitro, ex vivo, or in vivo superactivation of dendritic cells.

[0238] In one aspect, the present disclosure provides a method for producing superactivated dendritic cells (DCs), comprising contacting dendritic cells with an effective amount of an isolated ether lipid (ETL) having an n-alkyl chain (e.g., isolated ether phospholipid (ETPL)) and a PRR agonist to produce superactivated dendritic cells, wherein the superactivated dendritic cells secrete IL-1 beta without undergoing pyroptosis. In some embodiments, the DCs are isolated; in other embodiments, the DCs are present in a biological sample obtained from a mammalian subject, such as a human patient. In some embodiments, the DCs are monocyte-derived DCs, preferably cDC1s.

[0239] In a further aspect, the present disclosure provides a method for producing an immunogenic composition, comprising combining an antigen with an effective amount of an isolated ether lipid (ETL) having an n-alkyl chain (such as an isolated ether phospholipid (ETPL)) and a PRR agonist to produce the immunogenic composition. In some embodiments, the antigen comprises a proteinaceous antigen 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.

[0240] In certain embodiments, the present disclosure provides a method for producing 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 an isolated ether lipid (ETL) having an n-alkyl chain (such as an isolated ether phospholipid (ETPL)) and a PRR agonist to obtain the immunogenic composition.

[0241] In some embodiments, leukocytes are depleted from a tumor cell-enriched cell suspension by contacting the tumor cell suspension with a leukocyte-specific antibody. In some embodiments, leukocytes are depleted by contacting the tumor cell-enriched suspension with an anti-CD45 antibody. In some embodiments, cells are lysed by a physical disruption-based cell lysis method, such as, but not limited to, mechanical lysis, liquid homogenization, sonication, freeze-thawing, or manual crushing. In some preferred embodiments, cells are lysed by one or more freeze-thaw cycles.

[0242] In some embodiments of the aforementioned method, the alkyl chain of the ETL (e.g., ETPL) is a C13-C22 n-alkyl chain or a C13-C24 n-alkyl chain. In some embodiments, the alkyl chain of the ETL (e.g., ETPL) is a C18-C22 n-alkyl chain or a C18-C24 n-alkyl chain. In some preferred embodiments, the alkyl chain of the ETL (e.g., ETPL) is a C22 n-alkyl chain. In some preferred embodiments, the ETPL is DGPC. In some preferred embodiments, the ETPL is DGP. In some embodiments, 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, and a particularly preferred embodiment is resiquimod (R848).

[0243] VII. More Lipids The compositions and methods of the present disclosure include at least one additional lipid, wherein the LPC and the at least one additional lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one additional lipid comprises an ionizable lipid, a cationic lipid, an additional phospholipid, a PEGylated lipid, a structured lipid, or a mixture thereof. In some embodiments, the LNP comprises a first phospholipid (lysophosphatidylcholine having a single C13-C24 acyl chain [LPC:C13-C24]), an ionizable lipid, a second phospholipid, a PEGylated lipid, and a structured lipid. The structures of additional lipids suitable for use in the compositions and methods of the present disclosure are shown in Figures 48A and 48B (reproduced from Hou et al., Nature Review Materials, 6:1078-1094, 2021).

[0244] In some embodiments, the at least one additional lipid comprises one or both of an additional phospholipid and a structural lipid, optionally wherein the additional phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and the structural lipid comprises cholesterol. In some embodiments, the at least one additional lipid comprises, or further comprises, a pegylated lipid, optionally wherein the pegylated lipid comprises polyethylene glycol [PEG] 2000 dimyristoylglycerol [DMG]. In some embodiments, the at least one additional lipid comprises, or further comprises, an ionizable lipid, optionally wherein the ionizable lipid comprises (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA) or an analog or derivative thereof. In some embodiments, the at least one additional lipid comprises at least one lipid from the following list (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 )-heptadeca-8-salt-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)dipyropiolide nate;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-1H-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethan-1-aminium bromide;C12-200: 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecahydrocarbonyl)amino) 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)butyl DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOSPA: 2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate; DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane; DOTMA: 1,2-di-O-octadecenyl-3-trimethylammonium-propane; DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine; ePC: ethylphosphatidylcholine;FTT5: hexa(octan-3-yl) 9,9',9'',9''',9'''',9''''-((((benzene-1,3,5-tricarbonyl)iris(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,12 Z,12'Z,12''Z,12''Z)-tetrakis(octadeca-9,12-dienoate); PEG2000-DMG: 1,2-dimyristoyl rac-glycero-3-methoxypolyethylene glycol-2000; TT3: N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide, shown in Figures 48A and 48B.

[0245] VIII. Antigen-encoding mRNA The compositions and methods of the present disclosure include mRNA encoding an antigen or are suitable for use with formulations that otherwise include mRNA encoding an antigen. In some embodiments, the antigen is a proteinaceous antigen. The terms "polypeptide" and "protein" are used interchangeably herein to refer to an antigen comprising a peptide chain that is at least 8 amino acids in length. In some embodiments, the antigen is 8 to 1800 amino acids in length, 9 to 1000 amino acids in length, 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.

[0246] In some embodiments, the antigen is a tumor antigen comprising the amino acid sequence of at least one full-length protein or a fragment thereof. In some embodiments, the tumor antigen comprises an amino acid sequence derived from an oncoprotein or a fragment thereof. In some embodiments, the mammalian antigen is a neoantigen or is encoded by a gene comprising a mutation relative to a gene present in normal cells from the mammalian subject. Neoantigens are believed to be particularly useful in enabling T cells to distinguish between cancer cells and non-cancerous cells (see, e.g., Schumacher and Schreiber, Science, 348:69-74, 2015). In other embodiments, the tumor antigen comprises a viral antigen, e.g., an antigen of a cancer-causing virus.

[0247] In some embodiments, the tumor antigen is a fusion protein comprising two or more polypeptides, each polypeptide comprising an amino acid sequence derived from a different tumor antigen or non-contiguous amino acid sequences derived from the same tumor antigen. In some of these embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, each polypeptide comprising a non-contiguous amino acid sequence derived from the same tumor antigen.

[0248] In some embodiments, the antigen is a microbial antigen. In some embodiments, the microbial antigen comprises a viral antigen, a bacterial antigen, a protozoan antigen, a fungal antigen, or a combination thereof. In some embodiments, the microbial antigen comprises a surface protein or other antigenic subunit of a microorganism.

[0249] 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, hi some preferred embodiments, the mRNA comprises one or both of a 5' cap structure and a polyA tail.

[0250] IX. Lipid-Based Delivery Vehicles The compositions and methods of the present disclosure include lipid-based delivery vehicles for antigen-encoding mRNA. 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).

[0251] In some embodiments, the LNP is an ether lipid (ETL) compound of Formula (I), Formula (II), Formula (III), Formula (III-A), Formula (III-A-1), Formula (III-A-2), Formula (III-B), Formula (III-B-1), Formula (III-B-2), Formula (IV), Formula (IV-A), Formula (IV-A-1), Formula (IV-A-2), Formula (IV-B), Formula (IV-B-1), Formula (IV-B-2), Formula (IV-C), Formula (IV-D), Formula (IV-E), Formula (IV-F), Formula (A), or The present invention relates to an ether phospholipid (ETPL) compound, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, or Compound 16, or a protonated or deprotonated form thereof, if 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 structured lipid, and mixtures thereof. In some embodiments, the ether lipid (ETL) or ether phospholipid (ETPL) is isolated. In some embodiments, at least one lipid comprises an ionizable lipid. In some embodiments, at least one lipid comprises a cationic lipid. In some embodiments, at least one lipid comprises a second phospholipid. In some embodiments, at least one lipid comprises a PEGylated lipid. In some embodiments, at least one lipid comprises a structured lipid. In some embodiments, the at least one lipid comprises an ionizable lipid, a second phospholipid, a pegylated lipid, and a structured lipid.

[0252] In some embodiments, the LNP comprises an ether lipid (ETL) compound or ether lipid of Formula (I), Formula (II), Formula (III), Formula (III-A), Formula (III-A-1), Formula (III-A-2), Formula (III-B), Formula (III-B-1), Formula (III-B-2), Formula (IV), Formula (IV-A), Formula (IV-A-1), Formula (IV-A-2), Formula (IV-B), Formula (IV-B-1), Formula (IV-B-2), Formula (IV-C), Formula (IV-D), Formula (IV-E), as disclosed herein. The present invention relates to an ether lipid (ETPL) compound, Compound 1, Compound 2, Compound 3, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, or Compound 13, or a protonated or deprotonated form thereof, if 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 structured lipid, and mixtures thereof. In some embodiments, the ether lipid (ETL) or ether phospholipid (ETPL) is isolated. In some embodiments, at least one lipid comprises an ionizable lipid. In some embodiments, at least one lipid comprises a cationic lipid. In some embodiments, at least one lipid comprises a second phospholipid. In some embodiments, at least one lipid comprises a PEGylated lipid. In some embodiments, at least one lipid comprises a structured lipid. In some embodiments, the at least one lipid comprises an ionizable lipid, a second phospholipid, a pegylated lipid, and a structured lipid.

[0253] In some embodiments, the lipid component of the RNA-Lipoplex comprises one or more lipids. In some preferred embodiments, the one or more lipids comprise a first lipid and a second lipid, and the first lipid is different from the second lipid. In some embodiments, the first lipid is a cationic lipid, and the second lipid is a neutral lipid or an anionic lipid.

[0254] The structures of lipids suitable for use in the lipid-based mRNA delivery vehicles of the present disclosure are shown in Figures 48A and 48B (reproduced from Hou et al., Nature Review Materials, 6:1078-1094, 2021).

[0255] X.How to use In some embodiments, the present disclosure relates to a method of using any one of the compositions or formulations described herein, comprising an ETL compound, such as an ETPL compound. In some embodiments, the composition or formulation further comprises a PRR agonist, dendritic cells, an antigen, an adjuvant, or any combination thereof. The method of use is suitable for multiple uses, including stimulating an immune response. In some embodiments, the method of use includes treating cancer. In some embodiments, the method of use includes inhibiting abnormal cell proliferation. In some embodiments, the method of use includes treating an infectious disease. The method comprises administering an effective amount of a formulation or composition described herein to an individual in need thereof to achieve a particular result. The individual is a mammalian subject, e.g., a human patient. In other embodiments, the individual is a non-human patient. In some embodiments, the individual is a canine patient. That is, in some embodiments, the method of use includes clinical use, and in other embodiments, the method of use includes preclinical and / or veterinary use. For preclinical use, the mammalian subject may be a non-human primate (e.g., a monkey or ape) or a rodent (e.g., a mouse or rat). For veterinary use, the mammalian subject can be a farm animal (eg, a cow), a sport animal (eg, a horse), or a pet (eg, a companion animal such as a dog or cat).

[0256] A. Stimulation of the immune response Briefly, the present disclosure provides methods for 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 result from eliciting a new immune response (e.g., as a result of a primary vaccination regimen) or augmenting an existing immune response (e.g., as a result of a booster vaccination regimen). In some embodiments, stimulating an immune response includes one or more of the following: stimulating cytokine production, stimulating B lymphocyte proliferation, stimulating interferon pathway-related gene expression, stimulating chemotactic factor-related gene expression, and stimulating dendritic cell (DC) maturation. Methods for measuring the stimulation of an immune response are known in the art.

[0257] For example, the present disclosure provides methods for eliciting an antigen-specific immune response in an individual by administering to the individual a composition or formulation described herein in an amount sufficient to elicit an antigen-specific immune response in the individual. In preferred embodiments, the composition or formulation comprises an antigen. In some embodiments, the composition or formulation is administered to an individual's tissue that contains the antigen. The immune response may include one or both of an antigen-specific antibody response and an antigen-specific cytotoxic T lymphocyte (CTL) response. "Inducing" an antigen-specific antibody response means increasing the titer of an antigen-specific antibody above a threshold, such as a baseline titer or serum protective level, before administration. "Inducing" an antigen-specific CTL response means increasing the frequency of antigen-specific CTLs found in peripheral blood above the baseline frequency before administration.

[0258] Analysis of immune responses (both qualitative and quantitative) can be performed by any method known in the art, including, but not limited to, measuring antigen-specific antibody production (including measurement of specific antibody subclasses), activation of specific lymphocyte populations such as B cells and helper T cells, production of cytokines such as IFN-alpha, IFN-gamma, IL-6, IL-12, and / or histamine release. Methods for measuring antigen-specific antibody responses include enzyme-linked immunosorbent assay (ELISA). Activation of specific lymphocyte populations can be measured by proliferation assays and fluorescence-activated cell sorting (FACS). Cytokine production can also be measured by ELISA. In some embodiments, the method of stimulating an immune response includes stimulating interleukin-1 beta (IL-1β), interferon-gamma (IFN-γ), and / or tumor necrosis factor-alpha (TNF-α) secretion by monocyte-derived dendritic cells or peripheral blood mononuclear cells. In some embodiments, the method of stimulating an immune response comprises stimulating secretion of one or more of IFN-γ, IL-17a, IL-17f, and IL-22 by memory CD4+ T cells. In some embodiments, the method of stimulating an immune response comprises increasing Th1 differentiation of naive CD4+ T cells. In some preferred embodiments, at least 50%, 55%, 60%, 65%, 70%, or 75% of cells contacted with a composition of the present disclosure remain viable 40 to 56 hours (or about 48 hours) after contact.

[0259] In some embodiments, the method is suitable for stimulating an anti-tumor immune response. In other embodiments, the method is suitable for stimulating an anti-microbial immune response. In some embodiments, the anti-microbial response is an anti-bacterial immune response. In some embodiments, the anti-microbial response is an anti-fungal immune response. In some embodiments, the anti-microbial response is an anti-viral immune response. In some embodiments, the anti-microbial response is an anti-protozoan immune response.

[0260] B. Treating or preventing a disease The present disclosure further provides a method of treating or preventing a disease in an individual, comprising administering to the individual a composition or formulation described herein in an amount sufficient to treat or prevent the disease in the individual. In some embodiments, the disease is cancer. In some embodiments, the disease is abnormal cell proliferation. In other embodiments, the disease is an infectious disease.

[0261] In one aspect, the method can include administering a crude product containing an ETL compound, such as an ETPL compound, to a subject in need thereof. In some embodiments, the composition further comprises a PRR agonist, an antigen, an adjuvant, or any combination thereof. In a further aspect, the method includes adoptive cell therapy and includes administering a composition comprising dendritic cells, such as superactivated dendritic cells, and an ETL compound (such as an ETPL compound) to a subject in need thereof. In some embodiments, the composition further comprises a PRR agonist, an antigen, an adjuvant, or any combination thereof.

[0262] In some embodiments, the method includes treating cancer in an individual or otherwise treating a mammalian subject with cancer. In some embodiments, the method includes: a) preparing an immunogenic composition comprising a tumor cell lysate, an isolated ether lipid (ETL) having an n-alkyl chain (e.g., an isolated ether phospholipid (ETPL)), 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 was prepared from a tumor sample obtained from a subject with cancer, and the alkyl chain is a C13-C22 n-alkyl chain or a C13-C24 n-alkyl chain; and b) administering an effective amount of the immunogenic composition to the subject. In some embodiments, the cancer is a hematological cancer, e.g., lymphoma, leukemia, or myeloma. In other embodiments, the cancer is a non-hematological cancer, e.g., sarcoma, carcinoma, or melanoma. In some embodiments, the cancer is malignant.

[0263] In some embodiments, the methods include inhibiting abnormal cell growth in an individual. "Abnormal cell growth" refers to the growth of a benign or malignant tumor. A malignant tumor can be a metastatic tumor.

[0264] In some embodiments, the method includes treating or preventing an infectious disease in an individual. In some embodiments, the infectious disease is caused by a viral infection. In other embodiments, the infectious disease is caused by a bacterial infection. In further embodiments, the infectious disease is caused by a fungal infection. In still further embodiments, the infectious disease is caused by a protozoan infection. Of particular interest are infectious diseases caused by zoonotic pathogens that infect humans as well as other animals, such as mammals or birds. In some embodiments, zoonotic pathogens infect humans via an intermediate species (vector).

[0265] Enumeration of Embodiments The embodiments listed below are representative of some aspects of the present disclosure, and the features of each embodiment may be combined with any of the other embodiments where appropriate and practical.

[0266] Embodiment 1. An isolated ether lipid (ETL) of formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated form thereof, or a pharmaceutically acceptable salt thereof; A composition comprising a TLR7 / 8 agonist.

[0267] Embodiment 2.R 3 But C 18 -C 22 n-Alkyl or C 21 -C 24 The composition of embodiment 1, wherein the alkyl is n-alkyl.

[0268] Embodiment 3. The composition of embodiment 1 or embodiment 2, further comprising an antigen.

[0269] Embodiment 4. The composition of any one of embodiments 1 to 3, further comprising dendritic cells.

[0270] Embodiment 5. An isolated ether lipid (ETL) of formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4 is H or (CH3)3N +-(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated form thereof, or a pharmaceutically acceptable salt thereof; and an antigen.

[0271] Embodiment 6 The composition of embodiment 5, further comprising dendritic cells.

[0272] Embodiment 7. The composition of embodiment 5 or embodiment 6, further comprising a TLR7 / 8 agonist.

[0273] Embodiment 8. An isolated ether lipid (ETL) of formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated form thereof, or a pharmaceutically acceptable salt thereof; and a dendritic cell.

[0274] Embodiment 9. The composition of embodiment 8, further comprising a TLR7 / 8 agonist.

[0275] Embodiment 10. The composition of embodiment 8 or embodiment 9, further comprising an antigen.

[0276] Embodiment 11.R 3 But C 22 The composition of any one of embodiments 1 to 10, wherein the alkyl group is n-alkyl.

[0277] Embodiment 12. The composition of any one of embodiments 1-11, wherein the ETL is an ether phospholipid (ETPL) comprising 1-docosyl-sn-glycerol-3-phosphocholine (DGPC), or a pharmaceutically acceptable salt thereof.

[0278] Embodiment 13. The composition of any one of embodiments 1-11, wherein the ETL is an ETPL comprising 1-docosyl-sn-glycerol-3-phosphate (DGP), or a pharmaceutically acceptable salt thereof.

[0279] Embodiment 14. The composition of any one of embodiments 1 to 13, wherein the TLR7 / 8 agonist is a small molecule with a molecular weight of 900 daltons or less.

[0280] Embodiment 15. The composition of embodiment 14, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

[0281] Embodiment 16. The composition of embodiment 15, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

[0282] Embodiment 17. The composition of embodiment 14 or embodiment 15, wherein said TLR7 / 8 agonist does not inhibit NLR family pyrin domain-containing 3 (NLRP3).

[0283] Embodiment 18. The composition of embodiment 13, wherein the ETPL comprises one or both of DGPC and DGP, and the TLR7 / 8 agonist comprises resiquimod (R848).

[0284] Embodiment 19. The composition of any one of embodiments 1 to 18, wherein the antigen is present in a biological sample obtained from an individual.

[0285] Embodiment 20. The composition of embodiment 19, wherein the biological sample comprises biopsy tissue.

[0286] Embodiment 21. The composition of embodiment 19, wherein the biological sample comprises cells.

[0287] Embodiment 22. The composition of embodiment 19, wherein the biological sample does not contain cells.

[0288] Embodiment 23 The composition of embodiment 19, wherein the biological sample comprises pus from an abscess.

[0289] Embodiment 24. The composition of any one of embodiments 1 to 23, wherein the antigen comprises a proteinaceous antigen.

[0290] Embodiment 25. The composition of embodiment 24, wherein the antigen comprises a tumor antigen.

[0291] Embodiment 26. The composition of embodiment 25, wherein the tumor antigen comprises a synthetic or recombinant neoantigen.

[0292] Embodiment 27. The composition of embodiment 26, wherein the tumor antigen comprises a tumor cell lysate.

[0293] Embodiment 28. The composition of embodiment 24, wherein the antigen comprises a microbial antigen, the microbial antigen comprising one or more of a viral antigen, a bacterial antigen, a protozoan antigen, and a fungal antigen.

[0294] Embodiment 29. The composition of embodiment 28, wherein the microbial antigen comprises a purified or recombinant surface protein.

[0295] Embodiment 30. The composition of embodiment 28, wherein the microbial antigen comprises an inactivated whole virus.

[0296] Embodiment 31. The composition of any one of embodiments 1 to 30, wherein the composition does not contain liposomes.

[0297] Embodiment 32. The composition of any one of embodiments 1 to 31, wherein the composition does not contain LPS or MPLA.

[0298] Embodiment 33. The composition of any one of embodiments 1 to 32, wherein the composition does not comprise oxPAPC or species of oxPAPC, and optionally the composition does not comprise HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC, and / or PGPC.

[0299] Embodiment 34. The composition of embodiment 33, wherein the composition does not contain lysophosphatidylcholine (LPC), and optionally, the composition does not contain 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

[0300] Embodiment 35. The composition of any one of embodiments 1 to 34, further comprising an adjuvant, wherein the adjuvant comprises an aluminum salt adjuvant, a squalene-in-water emulsion, a saponin, or a combination thereof.

[0301] Embodiment 36. A pharmaceutical formulation comprising the composition of any one of embodiments 1 to 35 and a pharmaceutically acceptable excipient.

[0302] Embodiment 37. A method for the production of superactivated dendritic cells, comprising administering to said dendritic cells an effective amount of an isolated ether lipid (ETL) of formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated form thereof, or a pharmaceutically acceptable salt thereof, and The method comprises contacting a composition comprising a TLR7 / 8 agonist with a subject to produce superactivated dendritic cells, wherein the superactivated dendritic cells secrete IL-1 beta without undergoing pyroptosis.

[0303] Embodiment 38. The method of embodiment 37, wherein the dendritic cells are contacted ex vivo with the composition of any one of embodiments 1 to 35 or the formulation of embodiment 36.

[0304] Embodiment 39 The method of embodiment 37, wherein the dendritic cells are contacted in vivo with the formulation of embodiment 36.

[0305] Embodiment 40. At least 10 produced by the method of embodiment 38 3 , 10 4 , 10 5 or 10 6 A pharmaceutical preparation comprising the superactivated dendritic cells and a pharmaceutically acceptable excipient.

[0306] Embodiment 41. A method of stimulating an immune response to an antigen, comprising administering to an individual in need thereof an effective amount of the formulation of embodiment 36 to stimulate the immune response to the antigen.

[0307] Embodiment 42. A method of treating cancer, comprising administering an effective amount of the formulation of embodiment 36 to an individual in need thereof to treat the cancer.

[0308] Embodiment 43. A method of inhibiting abnormal cell growth, comprising administering to an individual in need thereof an effective amount of the formulation of embodiment 36 to inhibit abnormal cell growth.

[0309] Embodiment 44. A method of treating an infection, comprising administering an effective amount of the formulation of embodiment 36 to an individual in need thereof to treat the infection.

[0310] Embodiment 45. Use of the formulation of embodiment 36 for inducing an immune response against said antigen in an individual in need thereof.

[0311] Embodiment 46. Use of the formulation of embodiment 36 for inducing an anti-tumor immune response in an individual in need thereof, wherein the individual has or has had a tumor.

[0312] Embodiment 47. Use of the formulation of embodiment 36 for inducing an antimicrobial immune response in an individual in need thereof, wherein the individual is infected with or has not been exposed to the microorganism.

[0313] Embodiment 48. A composition, formulation, method or use according to any one of embodiments 19 to 47, wherein the individual is a mammalian subject.

[0314] Embodiment 49. A composition, formulation, method or use according to any one of embodiments 19 to 47, wherein the individual is a human subject.

[0315] Embodiment 50. A method for preparing an immunogenic composition, 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) administering the tumor cell lysate to an isolated ether lipid (ETL) of formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated form thereof, or a pharmaceutically acceptable salt thereof, and and contacting the antibody with a toll-like receptor 7 / 8 (TLR7 / 8) agonist to obtain the immunogenic composition.

[0316] Embodiment 51. The method of embodiment 50, wherein the leukocytes are depleted in step a) by negative selection using an anti-CD45 antibody.

[0317] Embodiment 52. The method of embodiment 50 or embodiment 51, wherein the cells are lysed in step b) by one or more freeze-thaw cycles.

[0318] Embodiment 53. R3 is C18 -C 22 Alkyl or C 18 -C 24 The method of any one of embodiments 50-52, wherein the alkyl is alkyl.

[0319] Embodiment 54. The method of embodiment 53, wherein the ETL comprises one or both of DGPC and DGP, or pharmaceutically acceptable salts thereof.

[0320] Embodiment 55. The method of any one of embodiments 50-54, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 daltons or less.

[0321] Embodiment 56 The method of embodiment 55, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

[0322] Embodiment 57. The method of embodiment 56, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

[0323] Embodiment 58. The method of embodiment 55 or embodiment 56, wherein said TLR7 / 8 agonist does not inhibit NLR family pyrin domain-containing 3 (NLRP3).

[0324] Embodiment 59. The method of embodiment 54, wherein the ETL comprises one or both of DGPC and DGP, or pharmaceutically acceptable salts thereof, and the TLR7 / 8 agonist comprises resiquimod (R848).

[0325] Embodiment 60. The method of any one of embodiments 50 to 59, further comprising, prior to step a), obtaining a sample derived from the tumor from a mammalian subject having cancer and preparing a suspension of the cells from the sample.

[0326] Embodiment 61. An immunogenic composition prepared by the method of any one of embodiments 50 to 60.

[0327] Embodiment 62. A method for inducing an anti-cancer immune response, comprising administering to a mammalian subject having cancer an effective amount of the immunogenic composition of embodiment 61.

[0328] Embodiment 63. The method of embodiment 62, wherein the anti-cancer immune response comprises a cellular immune response.

[0329] Embodiment 64. The method of embodiment 63, wherein the anti-cancer immune response comprises cancer antigen-induced IL-1 beta secretion and / or activation of CD8+ T lymphocytes.

[0330] Embodiment 65. The method of any one of embodiments 62 to 64, wherein the cancer is a non-hematological cancer.

[0331] Embodiment 66. The method of embodiment 65, wherein the non-hematological cancer is carcinoma, sarcoma, or melanoma.

[0332] Embodiment 67. The method of any one of embodiments 62 to 64, wherein the cancer is lymphoma.

[0333] Embodiment 68. A method of treating cancer, comprising: a) administering to a subject a tumor cell lysate an isolated ether lipid (ETL) of formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated form thereof, or a pharmaceutically acceptable salt thereof, and The method comprises: a) preparing an immunogenic composition comprising a toll-like receptor 7 / 8 (TLR7 / 8) agonist, wherein the tumor cell lysate is a tumor sample obtained from the mammalian subject having cancer or is prepared from the tumor cell lysate; and b) administering to the subject an effective amount of the immunogenic composition.

[0334] Embodiment 69.R 3 But C 18 -C 22 Alkyl chain or C 18 -C 24 69. The method of any one of embodiments 62-68, wherein the alkyl chain is an alkyl chain.

[0335] Embodiment 70. The method of embodiment 68, wherein the ETL comprises one or both of DGPC and DGP, or pharmaceutically acceptable salts thereof.

[0336] Embodiment 71. The method of any one of embodiments 62 to 70, wherein the TLR7 / 8 agonist is a small molecule with a molecular weight of 900 daltons or less.

[0337] Embodiment 72. The method of embodiment 71, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

[0338] Embodiment 73. The method of embodiment 72, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

[0339] Embodiment 74. The method of embodiment 70, wherein the ETPL comprises DGPC or a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist comprises resiquimod (R848).

[0340] Embodiment 75. The method of embodiment 70, wherein the ETPL comprises DGP or a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist comprises resiquimod (R848).

[0341] Embodiment 76. The method of any one of claims 68 to 75, further comprising administering to the subject an effective amount of an additional therapeutic agent.

[0342] Embodiment 77. The method of embodiment 76, wherein the additional therapeutic agent comprises one or more of the group consisting of an immune checkpoint inhibitor, an anti-tumor agent, and radiation therapy.

[0343] Embodiment 78. An isolated ether lipid (ETL) of formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated form thereof, or a pharmaceutically acceptable salt thereof; and a pathogen recognition receptor (PRR) agonist.

[0344] Embodiment 79. The composition of embodiment 78, 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).

[0345] Embodiment 80. The composition of embodiment 78, wherein the PRR agonist is an agonist of a cytoplasmic DNA sensor (CDS) or a stimulator of IFN genes (STING).

[0346] Embodiment 81. The composition of embodiment 78, wherein the PRR agonist comprises one or more of R848, TL8-506, LPS, Pam2CSK4, and ODN2336.

[0347] Embodiment 82. The composition of any one of embodiments 78 to 81, further comprising an antigen.

[0348] Embodiment 83. The composition of any one of embodiments 78 to 82, further comprising dendritic cells.

[0349] Embodiment 84. A pharmaceutical formulation comprising the composition of any one of embodiments 78 to 83 and a pharmaceutically acceptable excipient.

[0350] Embodiment 85. An isolated ether lipid (ETL) of formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C24 n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated form thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient.

[0351] Embodiment 86. A pharmaceutical formulation of embodiment 84 or embodiment 85, wherein the alkyl chain is a C22 n-alkyl chain.

[0352] Embodiment 87. The pharmaceutical formulation of embodiment 86, wherein the ETL comprises one or both of DGPC and DGP, or pharmaceutically acceptable salts thereof.

[0353] Embodiment 88. A composition for the hyperactivation of human dendritic cells, comprising an isolated ether lipid (ETL) of formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated form thereof, or a pharmaceutically acceptable salt thereof; and a pathogen recognition receptor (PRR) agonist, wherein the alkyl chain is a C22 n-alkyl chain, and the composition is effective in achieving a higher level of dendritic cell hyperactivation than a comparative composition comprising PGPC instead of the ETL.

[0354] Embodiment 89.R 3 But C 22 The composition of embodiment 88, wherein the alkyl is n-alkyl.

[0355] Embodiment 90. The composition of embodiment 88 or embodiment 89, wherein the high level of dendritic cell hyperactivation comprises inducing at least two-fold, three-fold, or four-fold higher levels of IL-1 beta secretion from the human dendritic cells in vitro when contacted with the composition comprising the ETL and the PRR agonist than when contacted with the comparative composition comprising the PGPC and the PRR agonist, and the PRR agonist is LPS.

[0356] Embodiment 91. The composition of embodiment 90, wherein the concentration of the ETL and the concentration of the PGPC are the same and range 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 comparative composition.

[0357] Embodiment 92. The composition of embodiment 90, wherein the high level of dendritic cell hyperactivation comprises a lipid activity index for IL-1 beta secretion from human dendritic cells for the composition comprising the ETL and the PRR agonist that is at least 4-fold, 5-fold, or 6-fold higher activity units than the comparative composition comprising the PGPC and the PRR agonist.

[0358] Embodiment 93. A composition, formulation, method or use according to any one of embodiments 19 to 47, wherein the individual is a human subject.

[0359] Embodiment 94. A composition, formulation, method or use according to any one of embodiments 19 to 47, wherein the individual is a canine subject.

[0360] Embodiment 95. A composition, formulation, method or use according to any one of embodiments 60 to 92, wherein the mammalian subject is a human patient.

[0361] Embodiment 96. The composition, formulation, method or use of any one of embodiments 60 to 92, wherein the mammalian subject is a non-human patient.

[0362] Embodiment 97. A composition, formulation, method or use according to any one of embodiments 60 to 92, wherein the mammalian subject is a canine patient.

[0363] Embodiment 98. A composition, formulation, method or use according to any one of embodiments 1 to 93 or 95, wherein the dendritic cells are human dendritic cells.

[0364] Embodiment 99. The composition, formulation, method or use of any one of embodiments 1 to 48, 50 to 87 or 97, wherein the dendritic cells are canine dendritic cells.

[0365] Embodiment 100. The composition, method or use of embodiment 98 or embodiment 99, wherein the dendritic cells are present in a composition comprising peripheral blood mononuclear cells (PBMCs).

[0366] Embodiment 101. The composition, method or use of any one of embodiments 37 to 49 or embodiments 98 to 99, wherein the superactivated dendritic cells secrete one or both of IFNγ and TNFα.

[0367] Embodiment 102. A composition, formulation, method or use according to any one of embodiments 1 to 101, comprising a surfactant.

[0368] Embodiment 103. A composition, formulation, method or use according to embodiment 102, wherein the surfactant comprises a non-ionic surfactant.

[0369] Embodiment 104. A composition, formulation, method or use according to embodiment 103, wherein the nonionic surfactant comprises an ethylene oxide-propylene oxide copolymer.

[0370] Embodiment 105. The composition, formulation, method or use of embodiment 103, wherein the nonionic surfactant comprises one or more of poloxamer 407, poloxamer 188, and P123.

[0371] Embodiment 106. The composition, formulation, method or use of embodiment 103, wherein the nonionic surfactant comprises poloxamer 407.

[0372] Embodiment 107. A composition, formulation, method or use according to any one of embodiments 103 to 106, wherein i) the ETL is dissolved in alcohol to form an ETL alcohol solution, ii) the ETL alcohol solution 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 ETL and the non-ionic surfactant.

[0373] Embodiment 108. The composition, formulation, method or use of any one of embodiments 103 to 107, wherein the nonionic surfactant is present in an amount of about 2.5% to 25% (w / w), optionally about 5% to 20% (w / w), optionally about 15% (w / w).

[0374] Embodiment 109. The composition, formulation, method or use of any one of embodiments 103 to 108, wherein the ETL and the non-ionic surfactant are present in particles having a diameter of about 1000 to 15,000 nanometers, optionally about 5000 nanometers.

[0375] Embodiment A1. An isolated ether lipid (ETL) of formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof; and a TLR agonist.

[0376] Embodiment A2. The composition of embodiment A1, wherein the TLR agonist comprises a TLR7 / 8 agonist.

[0377] Embodiment A3.R 3 But C 18 -C 22 n-Alkyl or C 21 -C 24 The composition of embodiment A1 or embodiment A2, wherein the alkyl is n-alkyl.

[0378] Embodiment A4.R 3 But C 16 -C 20 The composition of any one of embodiments A1 through A3, wherein the alkyl is n-alkyl.

[0379] Embodiment A5. The composition of any one of embodiments A1-A4, further comprising an antigen.

[0380] Embodiment A6. The composition of any one of embodiments A1-A5, further comprising dendritic cells.

[0381] Embodiment A7. An isolated ether lipid (ETL) of formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof; and an antigen.

[0382] Embodiment A8. The composition of embodiment A7, further comprising dendritic cells.

[0383] Embodiment A9. The composition of embodiment A7 or embodiment A8, further comprising a TLR agonist.

[0384] Embodiment A10. The composition of embodiment A9, wherein the TLR agonist comprises a TLR7 / 8 agonist.

[0385] Embodiment A11. An isolated ether lipid (ETL) of formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof; and a dendritic cell.

[0386] Embodiment A12. The composition of embodiment A11, further comprising a TLR agonist.

[0387] Embodiment A13. The composition of embodiment A12, wherein the TLR agonist comprises a TLR7 / 8 agonist.

[0388] Embodiment A14. The composition of any one of embodiments A11-A13, further comprising an antigen.

[0389] Embodiment A15.R 3 But C 22 The composition of any one of embodiments A1 through A14, wherein the alkyl is n-alkyl.

[0390] Embodiment A16. The composition of any one of embodiments A1-A15, wherein the ETL is an ether phospholipid (ETPL) comprising 1-docosyl-sn-glycerol-3-phosphocholine (DGPC), or a pharmaceutically acceptable salt thereof.

[0391] Embodiment A17. The composition of any one of embodiments A1-A15, wherein the ETL is an ETPL comprising 1-docosyl-sn-glycerol-3-phosphate (DGP), or a pharmaceutically acceptable salt thereof.

[0392] Embodiment A18. The composition of any one of embodiments A1-A17, wherein the TLR agonist is a small molecule having a molecular weight of 900 daltons or less.

[0393] Embodiment A19. The composition of any one of embodiments A1-A18, wherein the TLR agonist comprises a TLR7 / 8 agonist.

[0394] Embodiment A20. The composition of embodiment A19, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

[0395] Embodiment A21. The composition of embodiment A19, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

[0396] Embodiment A22. The composition of any one of embodiments A18-A20, wherein said TLR7 / 8 agonist does not inhibit NLR family pyrin domain-containing 3 (NLRP3).

[0397] Embodiment A23. The composition of any one of Embodiments A1 to A14, wherein the ETPL comprises one or both of DGPC and DGP, and the TLR7 / 8 agonist comprises resiquimod (R848).

[0398] Embodiment A24. The composition of any one of embodiments A1-A23, wherein the antigen is present in a biological sample obtained from an individual.

[0399] Embodiment A25. The composition of embodiment A24, wherein the biological sample comprises biopsy tissue.

[0400] Embodiment A26. The composition of embodiment A24, wherein the biological sample comprises cells.

[0401] Embodiment A27. The composition of embodiment A24, wherein the biological sample does not contain cells.

[0402] Embodiment A28. The composition of embodiment A24, wherein the biological sample comprises pus from an abscess.

[0403] Embodiment A29. The composition of any one of embodiments A1-A28, wherein the antigen comprises a proteinaceous antigen.

[0404] Embodiment A30. The composition of embodiment A29, wherein the antigen comprises a tumor antigen.

[0405] Embodiment A31. The composition of embodiment A30, wherein the tumor antigen comprises a synthetic or recombinant neoantigen.

[0406] Embodiment A32. The composition of embodiment A30, wherein the tumor antigen comprises a tumor cell lysate.

[0407] Embodiment A33. The composition of embodiment A29, wherein the antigen comprises a microbial antigen, the microbial antigen comprising one or more of a viral antigen, a bacterial antigen, a protozoan antigen, and a fungal antigen.

[0408] Embodiment A34. The composition of embodiment A33, wherein the microbial antigen comprises a purified or recombinant surface protein.

[0409] Embodiment A35. The composition of embodiment A33, wherein the microbial antigen comprises an inactivated whole virus.

[0410] Embodiment A36. The composition of any one of embodiments A1-A35, wherein the composition does not comprise liposomes.

[0411] Embodiment A37. The composition of any one of embodiments A1-A36, wherein the composition does not include LPS or MPLA.

[0412] Embodiment A38. The composition of any one of embodiments A1 to A37, wherein the composition does not comprise oxPAPC or species of oxPAPC, and optionally the composition does not comprise HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC, and / or PGPC.

[0413] Embodiment A39. The composition of any one of embodiments A1-A38, wherein the composition does not comprise lysophosphatidylcholine (LPC), and optionally the composition does not comprise 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

[0414] Embodiment A40. The composition of any one of Embodiments A1-A39 further comprising an adjuvant, wherein the adjuvant comprises an aluminum salt adjuvant, a squalene-in-water emulsion, a saponin, or a combination thereof.

[0415] Embodiment A41. A pharmaceutical formulation comprising the composition of any one of embodiments A1-A40 and a pharmaceutically acceptable excipient.

[0416] Embodiment A42. A method for the production of superactivated dendritic cells, comprising administering to said dendritic cells an effective amount of an isolated ether lipid (ETL) of formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof, and The method comprises contacting a composition comprising a TLR7 / 8 agonist with a subject to produce superactivated dendritic cells, wherein the superactivated dendritic cells secrete IL-1 beta without undergoing pyroptosis.

[0417] Embodiment A43. The method of embodiment A42, wherein the dendritic cells are contacted ex vivo with the composition of any one of embodiments A1-A40 or the formulation of embodiment A41.

[0418] Embodiment A44. The method of embodiment A42, wherein the dendritic cells are contacted in vivo with the formulation of embodiment A41.

[0419] Embodiment A45. At least 10 3 , 10 4 , 10 5 or 10 6 A pharmaceutical preparation comprising the superactivated dendritic cells and a pharmaceutically acceptable excipient.

[0420] Embodiment A46. A method of stimulating an immune response to an antigen, comprising administering to an individual in need thereof an effective amount of the formulation of embodiment A41 to stimulate the immune response to the antigen.

[0421] Embodiment A47. A method of treating cancer comprising administering to an individual in need thereof an effective amount of the formulation of embodiment A41 so as to treat said cancer.

[0422] Embodiment A48. A method of inhibiting abnormal cell growth comprising administering to an individual in need thereof an effective amount of the formulation of embodiment A41 to inhibit abnormal cell growth.

[0423] Embodiment A49. A method of treating an infection comprising administering to an individual in need thereof an effective amount of the formulation of embodiment A41 so as to treat the infection.

[0424] Embodiment A50. Use of the formulation of embodiment A41 for inducing an immune response against said antigen in an individual in need thereof.

[0425] Embodiment A51. Use of the formulation of embodiment A41 for inducing an anti-tumor immune response in an individual in need thereof, wherein the individual has or has had a tumor.

[0426] Embodiment A52. Use of the formulation of embodiment A41 for inducing an antimicrobial immune response in an individual in need thereof, wherein the individual is infected with or has not been exposed to the microorganism.

[0427] Embodiment A53. The composition, formulation, method or use of any one of embodiments A24 to A52, wherein the individual is a mammalian subject.

[0428] Embodiment A54. The composition, formulation, method or use of any one of embodiments A24 to A52, wherein the individual is a human subject.

[0429] Embodiment A55. A method of preparing an immunogenic composition 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) administering to the tumor cell lysate an isolated ether lipid (ETL) of Formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof, and and contacting the antibody with a toll-like receptor (TLR) agonist to obtain the immunogenic composition.

[0430] Embodiment A56. The method of embodiment A55, wherein the TLR agonist comprises a TLR7 / 8 agonist.

[0431] Embodiment A57. The method of embodiment A55 or embodiment A56, wherein the leukocytes are depleted in step a) by negative selection using an anti-CD45 antibody.

[0432] Embodiment A58. The method of any one of embodiments A55 to A57, wherein the cells are lysed in step b) by one or more freeze-thaw cycles.

[0433] Embodiment A59.R 3 But C 18 -C 22 Alkyl or C 18 -C 24 The method of any one of embodiments A55 to A58 wherein is alkyl.

[0434] Embodiment A60.R 3 But C 16 -C 20 The method of any one of embodiments A55 to A58 wherein is alkyl.

[0435] Embodiment A61.R 3 But C 21 -C 24 The method of any one of embodiments A55 to A58 wherein is alkyl.

[0436] Embodiment A62. The method of embodiment A59 or embodiment A61, wherein the ETL comprises one or both of DGPC and DGP, or pharmaceutically acceptable salts thereof.

[0437] Embodiment A63. The method of any one of embodiments A55-A62, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 daltons or less.

[0438] Embodiment A64. The method of embodiment A63, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

[0439] Embodiment A65. The method of embodiment A64, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

[0440] Embodiment A66. The method of any one of embodiments A63 to A65, wherein said TLR7 / 8 agonist does not inhibit NLR family pyrin domain-containing 3 (NLRP3).

[0441] Embodiment A67. The method of embodiment A62, wherein the ETL comprises one or both of DGPC and DGP, or pharmaceutically acceptable salts thereof, and the TLR7 / 8 agonist comprises resiquimod (R848).

[0442] Embodiment A68. The method of any one of embodiments A55 to A67, further comprising, prior to step a), obtaining a sample derived from the tumor from a mammalian subject having cancer and preparing a suspension of the cells from the sample.

[0443] Embodiment A69. An immunogenic composition prepared by the method of any one of embodiments A55 to A68.

[0444] Embodiment A70. A method of inducing an anti-cancer immune response comprising administering to a mammalian subject having cancer an effective amount of the immunogenic composition of embodiment A69.

[0445] Embodiment A71. The method of embodiment A70, wherein the anti-cancer immune response comprises a cellular immune response.

[0446] Embodiment A72. The method of embodiment A63, wherein the anti-cancer immune response comprises cancer antigen-induced IL-1beta secretion and / or activation of CD8+ T lymphocytes.

[0447] Embodiment A73. The method of any one of embodiments A62-A64, wherein the cancer is a non-hematological cancer.

[0448] Embodiment A74. The method of embodiment A65, wherein the non-hematologic cancer is carcinoma, sarcoma, or melanoma.

[0449] Embodiment A75. The method of any one of embodiments A70-A74, wherein the cancer is lymphoma.

[0450] Embodiment A76. A method of treating cancer comprising: a) administering to a subject a tumor cell lysate an isolated ether lipid (ETL) of formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof, and The method comprises: a) preparing an immunogenic composition comprising a toll-like receptor (TLR) agonist, wherein the tumor cell lysate is or has been prepared from a tumor sample obtained from the mammalian subject having cancer; and b) administering to the subject an effective amount of the immunogenic composition.

[0451] Embodiment A77. The method of embodiment A76, wherein the TLR agonist comprises a TLR7 / 8 agonist.

[0452] Embodiment A78.R 3 But C 18 -C22 Alkyl chain or C 18 -C 24 The method of any one of embodiments A70 to A77, wherein the alkyl chain is an alkyl chain.

[0453] Embodiment A79.R 3 But C 16 -C 20 The method of any one of embodiments A70 to A77 wherein is alkyl.

[0454] Embodiment A80.R 3 But C 21 -C 24 The method of any one of embodiments A70 to A77 wherein is alkyl.

[0455] Embodiment A81 The method of embodiment A76 or embodiment A77, wherein the ETL comprises one or both of DGPC and DGP, or pharmaceutically acceptable salts thereof.

[0456] Embodiment A82. The method of any one of embodiments A70-A81, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 daltons or less.

[0457] Embodiment A83 The method of embodiment A82 wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

[0458] Embodiment A84. The method of embodiment A83, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

[0459] Embodiment A85. The method of embodiment A81 wherein the ETPL comprises DGPC or a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist comprises resiquimod (R848).

[0460] Embodiment A86. The method of embodiment A81 wherein the ETPL comprises DGP or a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist comprises resiquimod (R848).

[0461] Embodiment A87. The method of any one of claims 68-75, further comprising administering to the subject an effective amount of an additional therapeutic agent.

[0462] Embodiment A88. The method of embodiment A76, wherein the additional therapeutic agent comprises one or more of the group consisting of an immune checkpoint inhibitor, an anti-tumor agent, and radiation therapy.

[0463] Embodiment A89. An isolated ether lipid (ETL) of Formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof; and a pathogen recognition receptor (PRR) agonist.

[0464] Embodiment A90. The composition of embodiment A89, wherein said 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).

[0465] Embodiment A91. The composition of embodiment A89, wherein the PRR agonist is an agonist of a cytoplasmic DNA sensor (CDS) or a stimulator of IFN genes (STING).

[0466] Embodiment A92. The composition of embodiment A89, wherein the PRR agonist comprises one or more of R848, TL8-506, LPS, Pam2CSK4, and ODN2336.

[0467] Embodiment A93. The composition of any one of embodiments A89-A92, further comprising an antigen.

[0468] Embodiment A94. The composition of any one of embodiments A89-A93, further comprising dendritic cells.

[0469] Embodiment A95. A pharmaceutical formulation comprising the composition of any one of embodiments A89-A94 and a pharmaceutically acceptable excipient.

[0470] Embodiment A96. An isolated ether lipid (ETL) of Formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient.

[0471] Embodiment A97. The pharmaceutical formulation of embodiment A95 or embodiment A96, wherein the alkyl chain is a C22 n-alkyl chain.

[0472] Embodiment A98. The pharmaceutical formulation of embodiment A97, wherein the ETL comprises one or both of DGPC and DGP, or pharmaceutically acceptable salts thereof.

[0473] Embodiment A99. A composition for the hyperactivation of human dendritic cells comprising an isolated ether lipid (ETL) of formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof; and a pathogen recognition receptor (PRR) agonist, wherein the alkyl chain is a C22 n-alkyl chain, and the composition is effective in achieving a higher level of dendritic cell hyperactivation than a comparative composition comprising PGPC instead of the ETL.

[0474] Embodiment A100.R 3 But C 22 The composition of embodiment A99, wherein the alkyl is n-alkyl.

[0475] Embodiment A101. The composition of embodiment A99 or embodiment A100, wherein the high level of dendritic cell hyperactivation comprises inducing at least a two-fold, three-fold, or four-fold higher level of IL-1 beta secretion from the human dendritic cells in vitro when contacted with the composition comprising the ETL and the PRR agonist than when contacted with the comparative composition comprising the PGPC and the PRR agonist, and the PRR agonist is LPS.

[0476] Embodiment A102. The composition of embodiment A101, wherein the concentration of the ETL and the concentration of the PGPC are the same and range 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 comparative composition.

[0477] Embodiment A103. The composition of embodiment A101, wherein the high level of dendritic cell hyperactivation comprises a lipid activity index for IL-1beta secretion from the human dendritic cells for the composition comprising the ETL and the PRR agonist that is at least 4-fold, 5-fold, or 6-fold higher activity units than the comparative composition comprising the PGPC and the PRR agonist.

[0478] Embodiment A104. The composition, formulation, method or use of any one of embodiments A24 to A52, wherein the individual is a human subject.

[0479] Embodiment A105. The composition, formulation, method or use of any one of embodiments A24 to A52, wherein the individual is a canine subject.

[0480] Embodiment A106. The composition, formulation, method or use of any one of embodiments A68 to A103, wherein the mammalian subject is a human patient.

[0481] Embodiment A107. The composition, formulation, method or use of any one of embodiments A68 to A103, wherein the mammalian subject is a non-human patient.

[0482] Embodiment A108. The composition, formulation, method or use of any one of embodiments A68 to A103, wherein the mammalian subject is a canine patient.

[0483] Embodiment A109. The composition, formulation, method or use of any one of embodiments A1 to A104 or A106, wherein the dendritic cells are human dendritic cells.

[0484] Embodiment A110. The composition, formulation, method or use of any one of embodiments A1 to A53, A55 to A98 or A108, wherein the dendritic cells are canine dendritic cells.

[0485] Embodiment A111. A composition, method or use according to embodiment A109 or embodiment A110, wherein the dendritic cells are present in a composition comprising peripheral blood mononuclear cells (PBMCs).

[0486] Embodiment A112. The composition, method or use of any one of embodiments A42 to A54 or embodiments A109 to A110, wherein the superactivated dendritic cells secrete one or both of IFNγ and TNFα.

[0487] Embodiment A113. A composition, formulation, method or use according to any one of embodiments A1 to A112, comprising a surfactant.

[0488] Embodiment A114. A composition, formulation, method or use according to embodiment A113, wherein the surfactant comprises a non-ionic surfactant.

[0489] Embodiment A115. A composition, formulation, method or use according to embodiment A114, wherein the nonionic surfactant comprises an ethylene oxide-propylene oxide copolymer.

[0490] Embodiment A116. A composition, formulation, method or use according to embodiment A114, wherein the nonionic surfactant comprises one or more of poloxamer 407, poloxamer 188, and P123.

[0491] Embodiment A117. A composition, formulation, method or use according to embodiment A114, wherein the nonionic surfactant comprises poloxamer 407.

[0492] Embodiment A118. The composition, formulation, method or use of any one of embodiments A114 to A117, wherein i) the ETL is dissolved in alcohol to form an ETL alcoholic solution, ii) the ETL alcoholic solution 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 ETL and the non-ionic surfactant.

[0493] Embodiment A119. The composition, formulation, method or use of any one of embodiments A104 to A118, wherein the nonionic surfactant is present in an amount of about 2.5% to 25% (w / w), optionally about 5% to 20% (w / w), optionally about 15% (w / w).

[0494] Embodiment A120. The composition, formulation, method, or use of any one of embodiments A104 to A119, wherein the ETL and the non-ionic surfactant are present in particles having a diameter of about 1000 to 15,000 nanometers, optionally about 5000 nanometers.

[0495] Embodiment A121. An isolated ether lipid (ETL) of formula (I): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4 is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated or deprotonated form thereof, or a salt thereof.

[0496] Embodiment A122. The isolated etherlipid is a compound of Formula (II): [ka] (In the formula, R 1 is H or [ka] and R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, where R 4is H or (CH3)3N + -(CH2)2-, Each R 5 are independently C1-C4 alkyl, or a protonated or deprotonated form thereof, or a salt thereof.

[0497] Embodiment A123. The isolated etherlipid is a compound of formula (III): [ka] (In the formula, R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, Each R 5 are independently C1-C4 alkyl, An isolated ether lipid according to embodiment A121, which is: or a salt thereof.

[0498] Embodiment A124. The isolated ether lipid is an isolated ether phospholipid (ETPL) compound of Formula (IV): [ka] (In the formula, R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, R 4 is H or (CH3)3N + -(CH2)2-, Each R5 are independently C1-C4 alkyl, or a protonated or deprotonated form thereof, or a salt thereof.

[0499] Embodiment A125. The isolated ether lipid is an isolated ether phospholipid (ETPL) compound of Formula (IV-A): [ka] (In the formula, R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, Each R 5 are independently C1-C4 alkyl, or a protonated or deprotonated form thereof, or a salt thereof.

[0500] Embodiment A126. The isolated ether lipid is an isolated ether phospholipid (ETPL) compound of Formula (IV-B): [ka] (In the formula, R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2, or -CH2-C6H5, R 3 is C 13- C 24 n-alkyl, Each R 5 are independently C1-C4 alkyl, or a protonated form thereof, or a salt thereof.

[0501] Embodiment A127. The isolated ether lipid is an isolated ether phospholipid (ETPL) compound of Formula (IV-C): [ka] (In the formula, R 3 is C 13- C 24 n-alkyl, R 4 is H or (CH3)3N + -(CH2)2-), or a protonated or deprotonated form thereof, or a salt thereof.

[0502] Embodiment A128. A compound of Formula 2: [ka] or a protonated form thereof, or a pharmaceutically acceptable salt thereof.

[0503] Embodiment A129. The compound of embodiment A128, wherein the compound is isolated.

[0504] Embodiment A130. Isolated Compound 1 of Formula 1: [ka] or a protonated form thereof, or a pharmaceutically acceptable salt thereof.

[0505] Embodiment A131. A compound of Formula (III-A-1): [ka] (In the formula, R 2 are -(C=O)-NH2, -(C=O)-NH(R 5), or -(C=O)-N(R 5 )2, R 3 is C 21- C 24 n-alkyl, Each R 5 are independently C1-C4 alkyl, or a pharmaceutically acceptable salt thereof.

[0506] Embodiment A132.R 2 A compound according to embodiment A131, wherein is -(C=O)-NH2.

[0507] Embodiment A133.R 2 A compound according to embodiment A131, wherein is -(C=O)-NH-CH3.

[0508] Embodiment A134.R 2 A compound according to embodiment A131, wherein is -(C=O)-N(CH3)2.

[0509] Embodiment A135.R 3 C 22 The compound according to any one of embodiments A131 to A134, which is n-alkyl.

[0510] Embodiment A136. Compound 7 of Formula 7: [ka] or a pharmaceutically acceptable salt thereof.

[0511] Embodiment A137. The compound of embodiment A136, wherein the compound is isolated.

[0512] Embodiment A138. Compound 8 of Formula 8: [ka] or a pharmaceutically acceptable salt thereof.

[0513] Embodiment A139. The compound of embodiment A138, wherein the compound is isolated.

[0514] Embodiment A140. A composition comprising a compound according to any one of embodiments A121-A139 and a pharmaceutically acceptable excipient.

[0515] Embodiment A141. The composition of embodiment A140, wherein the pharmaceutically acceptable excipient comprises phosphate buffered saline.

[0516] Embodiment A142. The composition of embodiment A140, wherein the pharmaceutically acceptable excipient comprises an aqueous solution of poloxamer 407.

[0517] Embodiment A143. The composition of embodiment A140, wherein the pharmaceutically acceptable excipients comprise phosphate buffered saline and poloxamer 407.

[0518] Embodiment A144. The composition of any one of embodiments A140-A143, wherein the composition is sterile.

[0519] Embodiment A145. An article of manufacture comprising a container enclosing a liquid formulation of a compound of any one of embodiments A121-A139 and a pharmaceutically acceptable excipient.

[0520] Embodiment A146. The product of embodiment A145, wherein the container is a syringe.

[0521] Embodiment A147. The product of embodiment A146, wherein the syringe is further housed within an injection device.

[0522] Embodiment A148. The product of embodiment A147, wherein the injection device is an auto-injector.

[0523] Embodiment A149. An isolated ether lipid (ETL) or ether phospholipid (ETPL), Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, Compound 16, Compound 17, Compound 18, Compound 19, Compound 20, Compound 21, Compound 22, Compound 23, Compound 24, Compound 25, Compound 26, Compound 27, Compound 28, Compound 29, Compound 30, Compound 31, Compound 32, Compound 33, Compound 34, Compound 35, Compound 36, Compound 37, Compound 38, Compound 39, Compound 40, Compound 41, Compound 42, Compound 43, Compound 44, Compound 45, Compound 46, Compound 47, Compound 48, Compound 49, Compound 50, Compound 51, Compound 52, Compound 53, Compound 54, Compound 55, Compound 56, Compound 57, Compound 58, Compound 59, Compound 60, Compound 61, Compound 62, Compound 63, Compound 64, Compound 65, Compound 66, Compound 67, Compound 68, Compound 69, Compound 69, Compound 69, Compound 69, Compound 69, Compound 69, Compound 69, Compound 70, Compound 71, Compound 72, Compound 73, Compound 74, Compound 75, Compound 76, Compound 77, Compound 78, Compound 79, Compound 79, Compound 79, Compound 79, Compound 79, Compound 79, Compound 79, Compound 79, Compound 71, Compound 72, Compound 73, Compound 74, Compound 75, Compound 76, Compound 77 1. A composition comprising Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, or Compound 13, or a protonated or deprotonated form thereof, if possible, or a pharmaceutically acceptable salt thereof, and at least one additional lipid, wherein the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structured lipid, and mixtures thereof.

[0524] Embodiment A150. The composition of embodiment A149, wherein the ETL or the ETPL and the at least one additional lipid are part of a lipid nanoparticle (LNP).

[0525] Embodiment A151. The composition of embodiment A149 or embodiment A150, further comprising an antigen.

[0526] Embodiment A152. The composition of any one of embodiments A149-A151, further comprising dendritic cells.

[0527] Embodiment A153. A composition according to any one of embodiments A149 to A152, further comprising a TLR agonist.

[0528] Embodiment A154. A composition according to any one of embodiments A149 to A152, further comprising a TLR7 / 8 agonist.

[0529] Embodiment A155. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (II), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0530] Embodiment A156. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (III), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0531] Embodiment A157. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (III-A), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0532] Embodiment A158. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (III-A-1), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0533] Embodiment A159. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (III-A-2), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0534] Embodiment A160. The composition of any one of embodiments A149-A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (III-B), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0535] Embodiment A161. The composition of any one of embodiments A149-A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (III-B-1), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0536] Embodiment A162. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (III-B-2), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0537] Embodiment A163. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0538] Embodiment A164. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV-A), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0539] Embodiment A165. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV-A-1), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0540] Embodiment A166. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV-A-2), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0541] Embodiment A167. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV-B), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0542] Embodiment A168. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV-B-1), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0543] Embodiment A169. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV-B-2), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0544] Embodiment A170. The composition of any one of embodiments A149-A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV-C), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0545] Embodiment A171. The composition of any one of embodiments A149-A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV-D), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0546] Embodiment A172. The composition of any one of embodiments A149-A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV-E), or, if possible, a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0547] Embodiment A173. The composition of any one of embodiments A149-A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is Compound 1, or, if possible, its protonated or deprotonated form, or a pharmaceutically acceptable salt thereof.

[0548] Embodiment A174. The composition of any one of embodiments A149-A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is Compound 2, or possibly a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0549] Embodiment A175. The composition of any one of embodiments A149-A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 3, or possibly a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0550] Embodiment A176. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 4, or possibly a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0551] Embodiment A177. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 5, or possibly a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0552] Embodiment A178. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 6, or possibly a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0553] Embodiment A179. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 7, or possibly a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0554] Embodiment A180. The composition of any one of embodiments A149-A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 8, or possibly a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0555] Embodiment A181. The composition of any one of embodiments A149-A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 9, or possibly a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0556] Embodiment A182. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 10, or possibly a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0557] Embodiment A183. The composition of any one of embodiments A149-A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 11, or possibly its protonated or deprotonated form, or a pharmaceutically acceptable salt thereof.

[0558] Embodiment A184. The composition of any one of embodiments A149 to A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 12, or possibly a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0559] Embodiment A185. The composition of any one of embodiments A149-A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 13, or possibly a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof.

[0560] Synthesis scheme The following synthetic schemes illustrate general synthetic procedures, which may be used as described or further modified or combined to prepare the compounds disclosed herein. Furthermore, the chemical reactions of the synthetic examples provided herein can be readily adapted to prepare the compounds disclosed herein. For example, the synthesis of non-exemplary compounds disclosed herein can be carried out without difficulty by modifications known to those skilled in the art, such as the use of alternative protecting groups, the utilization of other suitable reagents known in the art other than those described, or routine modification of reaction conditions. It will also be recognized that other reactions disclosed herein or known in the art can be applied to prepare other compounds disclosed herein.

[0561] Scheme 1: Synthesis of compounds of formula IV-D [ka] Compounds of formula (IV-D) can be readily prepared according to Scheme 1. Starting material SC-1-1, (R)-2,3-dihydroxypropyl(2-(trimethylammonio)ethyl)phosphate, is commercially available (CAS number 28319-77-9; suppliers include Ambeed, Arlington Heights, Illinois, United States). BuSnO (2.89 g, 0.0116 mol) was used to form intermediate SC-1-2, followed by R 3 -Br(wherein, R 3 is C 13 -C 24 n-alkyl) to give compounds of formula (IV-D).

[0562] Scheme 2: Synthesis of compounds of formula III-B [ka] Compounds of formula (III-B) can be readily prepared according to Scheme 2. Alcohol SC-2-1 (wherein R 3 is C 13 -C 24 (III-B) is reacted with (S)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl 4-methylbenzenesulfonate SC-2-2 (commercially available from Sigma-Aldrich, Saint Louis, Missouri, United States) to form intermediate SC-2-3. The dioxolane ring is opened with acetic acid to give compound of formula (III-B).

[0563] Scheme 3: Compounds of formula III (R 2 Synthesis of (benzyl) [ka] Scheme 3 illustrates the synthesis of a compound of formula (III) 2 is benzyl), and compounds of formula (IV-A) 2 = benzyl). Starting from the compound of formula (III-B) prepared in Scheme 2, the terminal hydroxy group is protected, for example with a TBDPSCl group. Benzyl bromide is then added to the unprotected 2-hydroxy group. Deprotection of the terminal hydroxy group gives the compound of formula (III) (where R 2 is benzyl and R 3 is C 13 -C 24 n-alkyl) is obtained.

[0564] Scheme 4: Compounds of formula (IV-A) (R 2 Synthesis of (benzyl) [ka] As shown in Scheme 4, a compound of formula (IV-A) (wherein R 2 is benzyl), by reacting a compound of formula (III) 2 is benzyl) with tetrabenzyl pyrophosphate (tetrabenzyl diphosphate) to give a compound of formula (III) 2 is benzyl), and then removing the benzyl group from the phosphate group to give a compound of formula (IV-A), 2 is benzyl and R 3 is C 13 -C 24 n-alkyl) is obtained.

[0565] Scheme 5: Synthesis of compounds of formula (IV-E) [ka] Compounds of formula (IV-E) can be prepared starting from intermediate SC-4-8 of Scheme 4 and removing all benzyl groups, for example, using catalytic hydrogenation as shown in Scheme 5, where R 3 is C 13 -C 24 n-alkyl).

[0566] Scheme 6: Synthesis of compounds of formula (III-A) [ka] Compounds of formula (III-A) can be prepared via compounds of formula (III-B) as intermediates, as shown in Scheme 6. In Scheme 6, R 2 are -(C=O)-NH2, -(C=O)-NH(R 5 ), or -(C=O)-N(R 5 )2, and each R 5 are independently C1-C4 alkyl, and R 3 is C 13 -C 24 It is n-alkyl.

[0567] Scheme 7: Synthesis of compounds of formula (IV-A) [ka] Compounds of formula (IV-A) can be prepared by the reaction of compounds of formula (III-A) (wherein R 2 are -(C=O)-NH2, -(C=O)-NH(R 5 ), or -(C=O)-N(R 5 )2, and each R 5 are independently C1-C4 alkyl, and R 3 is C 13 -C 24 It can be prepared starting from n-alkyl.

[0568] Scheme 8: Synthesis of compounds of formula (III-A), formula (IV-A), formula (IV-B), and formula (IV-C) [ka] [ka] Compounds of formula (III-A), formula (IV-A), and formula (IV-B) can be prepared as shown in Scheme 8, where R 3 is C 13 -C 24 n-alkyl, and each R 11 are independently selected from H or methyl). The synthesis of carbamates is shown. To synthesize compounds of formula (IV-B) having an alkyl or benzyl group at the 2-hydroxy position of the glycerol moiety, an alkylating or benzylating agent, such as an alkyl bromide or benzyl bromide, is used instead of di(pyridin-2-yl)carbonate, reagent SC-8-5. To synthesize compounds of formula (IV-C) having a free hydroxy group at the 2-hydroxy position of the glycerol moiety, a protecting group can be placed on the hydroxy group and removed at the end of the synthesis (e.g., the benzyl group can be protected with benzyl bromide instead of reagent SC-8-5, followed by catalytic hydrogenation). [Example]

[0569] Abbreviations: BM (bone marrow); BMDC (bone marrow-derived dendritic cells); CDS (cytoplasmic 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); 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-1 beta); KOdiA-PC (1-(palmitoyl)-2-(5-keto-6-octenedioyl)phosphatidylcholine); KOOA-PC (1-palmitoyl-(5-keto-8-oxo-6-octenoyl)-sn-glycero-3-phosphocholine); 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 cells); MPLA (monophosphoryl lipid A); NLR (NOD-like receptor); oxPAPC (oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine); PAMP (pathogen-associated molecular pattern); PBMC (peripheral blood mononuclear cells); PG PC (1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine); POVPC (1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine); PRR (pathogen recognition receptor); RLR (RIG-I-like receptor); R848 (resiquimod); STING (stimulator of IFN genes); TNFα (tumor necrosis factor alpha); TLR (toll-like receptor); and WTL (whole tumor lysate).

[0570] Synthesis Example Example S-1: Synthesis of Compound 1 [ka] To a stirred solution of (R)-2,3-dihydroxypropyl(2-(trimethylammonio)ethyl)phosphate 1-1 (2 g, 0.0077 mol) in IPA (150 mL), BuSnO (2.89 g, 0.0116 mol) was added, and the reaction mixture was heated at 100 °C for 16 h. The progress of the reaction was monitored by TLC. The crude reaction mixture was evaporated by rotary evaporation to give the crude material, which was used in the next step without analysis.

[0571] To a stirred solution of crude intermediate 1-2 (2 g, 0.0040 mol) in IPA (150 mL), KOtBu (0.672 g, 0.006 mol) and 1-bromodocosane (1-3) (1.86 g, 0.0048 mol) were added at 0 °C, and the reaction mixture was stirred at room temperature for 16 h. The reaction progress was monitored by TLC. The crude reaction mixture was evaporated by rotary evaporation to give the crude material. The resulting crude material was purified by column chromatography (silica gel basified with NH4OH) using 30% MeOH and 10% NH4OH in DCM as the eluent. The impure material was repurified by Combiflash chromatography (ELSD) (12 g column) using 40% MeOH and 10% NH4OH in DCM as the eluent to give compound 1 (75 mg, 0.13 mmol, 3.3%) as a white solid. HRMS: 566.3606;HPLC-ELSD: 97.13%; 1 H NMR (CDCl3) 400 MHz) δ ppm 4.28-4.27 (m, 1H), 3.92-3.85 (m, 3H), 3.64-3.61 (m, 3H), 3.46-3.43 (m, 3H), 3.24 (bs, 9 H), 1.59-1.52 (m, 2H), 1.42-1.24, (m, 40H), 0.89 (t, J= 6.4Hz, 3H).

[0572] Example S-2: Synthesis of Compound 9, Compound 2, and Compound 10 [ka] Synthesis of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (2-3) To a stirred solution of 1-docosanol 2-1 (10 g, 0.0306 mol) and (S)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl 4-methylbenzenesulfonate 2-2 (7.77 g, 0.0367 mol) in toluene (150 mL), KOtBu (6.86 g, 0.0612 mol) was added at 0 °C and heated to 110 °C for 16 h. The reaction progress was monitored by TLC. Upon completion of the reaction, the reaction mixture was concentrated in vacuo to give the crude material. The resulting crude material was purified by MPLC flash column chromatography using 10% EtOAc in hexane as the eluent to give (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (2-3, 15 g, 0.0340 mmol, 38%) as an off-white solid. HPLC (ELSD): 97.39%, 1 H NMR (CDCl3, 400 MHz): δ ppm 4.29-4.23 (m, 1H), 4.07-4.04 (m, 1H), 3.74-3.71 (m, 1H), 3.53-3.39 (m, 3H), 1.57-1.51 (m, 1H), 1.42 (s, 3H), 1.37 (m, 3H), 1.31-1.14 (m, 40H), 0.89-0.82 (m, 3H).

[0573] Synthesis of compound 9 A solution of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane 2-3 (15 g, 0.034 mol) in acetic acid:HO (volume ratio 10:1) was heated to 60 °C, and the reaction mixture was stirred for 16 h. The progress of the reaction was monitored by TLC analysis. Upon completion of the reaction, the reaction mixture was concentrated in vacuo to give the crude material. The resulting crude material was washed with n-hexane and dried in vacuo to give (S)-3-(docosyloxy)propane-1,2-diol (9.00 g, 66%) as an off-white solid. HPLC (ELSD): 86.61%, 1H NMR (CDCl 3, 400 MHz): δ ppm 3.86 (brs, 1H), 3.71-3.67 (m, 2H), 3.53-3.44 (m, 4H), 2.62 (brs, 1H), 2.19 (brs, 1H), 1.61-1.54 (m, 2H), 1.31-1.14 (m, 38H), 0.89-0.82 (m, 3H).

[0574] The crude material (1.00 g, 2.49 mmol) was purified by dissolving in 20% EtOAc:hexane (200 mL) and stirring for 30 minutes. After 30 minutes, the solid was filtered and dried under vacuum to give compound 9 (600 mg, 1.49 mmol, 60%) as an off-white solid. HPLC (ELSD): 98.33%, 1 H NMR (CDCl 3, 400 MHz): δ ppm 3.86-3.85 (m, 1H), 3.70-3.66 (m, 2H), 3.53-3.44 (m, 4H), 2.63-2.62 (m, 1H), 2.19 (brs, 1H), 1.62-1.53 ​​(m, 2H), 1.31-1.14 (m, 38H), 0.89-0.82 (m, 3H).

[0575] Synthesis of (R)-1-((tert-butyldiphenylsilyl)oxy)-3-(docosyloxy)propan-2-ol (2-4): To a stirred solution of crude (S)-3-(docosyloxy)propane-1,2-diol (compound 9) (6 g, 0.0149 mol) in DCM (150 mL) was added imidazole (2.54 g, 0.0374 mol) and TBDPSCl (4.7 mL, 0.0179 mol) at 0 °C, and the reaction mixture was stirred at room temperature for 4 h. The reaction progress was monitored by TLC analysis. Upon completion of the reaction, the reaction mixture was diluted with water, and the product was extracted into DCM (2 × 200 mL). The combined organic layers were dried over NaSO and concentrated in vacuo to give the crude material. The resulting crude material was purified by MPLC flash column chromatography (12 g Clariscep C-series) using 10% EtOAc in hexane as the eluent to give (R)-1-((tert-butyldiphenylsilyl)oxy)-3-(docosyloxy)propan-2-ol (2-4) (5.00 g, 52%) as an off-white solid. 1 H NMR (CDCl3, 400 MHz): δ ppm 7.67-7.65 (m, 5H), 7.42-7.36 (m, 5H), 3.89-3.71 (m, 1H), 3.70-3.53 (m, 2H), 3.52-3.41 (m, 4H), 1.61 (brs, 1H), 1.56-1.53 ​​(m, 2H), 1.31-1.14 (m, 38H), 1.22 (s, 9H), 0.89-0.86 (m, 3H).

[0576] Synthesis of (R)-(2-(benzyloxy)-3-(docosyloxy)propoxy)(tert-butyl)diphenylsilane (2-5): To a stirred solution of (R)-1-((tert-butyldiphenylsilyl)oxy)-3-(docosyloxy)propan-2-ol (2-4) (3 g, 0.0046 mol) in THF (50 mL) was added NaH (0.45 g, 0.0938 mol) at 0 °C and stirred for 20 min. After 20 min, BnBr (0.7 mL, 0.0056 mol) was added at room temperature, and the reaction mixture was stirred at room temperature for 4 h. The progress of the reaction was monitored by TLC analysis. Upon completion of the reaction, the reaction mixture was quenched with ice, and the product was extracted into EtOAc (100 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo to give the crude material. The resulting crude material was purified by MPLC flash column chromatography using 10% EtOAc in hexane as the eluent to give (R)-(2-(benzyloxy)-3-(docosyloxy)propoxy)(tert-butyl)diphenylsilane (2-5) (2.2 g, 65%) as a pale yellow gum. 1 H NMR (CDCl3, 400 MHz): δ ppm 7.61-7.58 (m, 5H), 7.34-7.26 (m, 10H), 4.60 (s, 2H), 3.69-3.68 (m, 2H) 3.63-3.33 (m, 5H), 1.51-1.45 (m, 2H), 1.31-1.14 (m, 38H), 1.01 (s, 9H), 0.82-0.77 (m, 3H).

[0577] Synthesis of (S)-2-(benzyloxy)-3-(docosyloxy)propan-1-ol (2-6): To a stirred solution of (R)-(2-(benzyloxy)-3-(docosyloxy)propoxy)(tert-butyl)diphenylsilane (2-5, 2.9 g, 0.003 mol) in THF (30 mL) was added TBAF (8 mL, 0.007 mol) at 0 °C, and the reaction mixture was stirred at room temperature for 4 h. The reaction progress was monitored by TLC analysis. Upon completion of the reaction, the reaction mixture was quenched with ice, and the product was extracted into EtOAc (100 mL). The combined organic layers were dried over NaSO and concentrated in vacuo to give the crude material. The resulting crude material was purified by MPLC flash column chromatography using 10% EtOAc in hexane as the eluent to give (S)-2-(benzyloxy)-3-(docosyloxy)propan-1-ol (2-6, 2.0 g, 98%) as an off-white solid. 1 H NMR (DMSO-d6, 400 MHz, 1 H NMR (CDCl3): δ ppm 7.35-7.28 (m, 5H), 4.73-4.61 (m, 2H), 3.68-3.65 (m, 1H) 3.61-3.42 (m, 7H), 2.19 (brs, 1H), 1.62-1.54 (m, 2H), 1.31-1.14 (m, 38H), 0.92-0.82 (m, 3H).

[0578] Synthesis of (R)-dibenzyl(2-(benzyloxy)-3-(docosyloxy)propyl)phosphate (2-8): To a stirred solution of (S)-2-(benzyloxy)-3-(docosyloxy)propan-1-ol (2-6, 600 mg, 1.224 mmol) in THF (20 mL) was added KOtBu (205 mg, 1.836 mmol) and tetrabenzyl diphosphate 2-7 (790 mg, 1.469 mmol) at 0 °C and stirred at room temperature for 2 h. The reaction was monitored for completion by TLC. The reaction progress was monitored by TLC analysis. Upon completion of the reaction, the reaction mixture was quenched using ice-containing NH4Cl, and the product was extracted into EtOAc (100 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo to give the crude material. The resulting crude material was purified by MPLC flash column chromatography using 20% ​​EtOAc in hexane as the eluent to give (R)-dibenzyl(2-(benzyloxy)-3-(docosyloxy)propyl)phosphate (2-8, 450 mg, 49%) as an off-white solid. HPLC (ELSD): 90.68%, 1 H NMR (CDCl3, 400 MHz): δ ppm 7.34-7.26 (m, 15H), 5.04-5.02 (m, 4H) 4.63-4.62 (m, 2H), 4.17-4.07 (m, 2H) 3.72-3.70 (m, 1H), 3.48-3.36 (m, 4H), 1.54-1.51 (m, 2H), 1.31-1.14 (m, 38H), 0.90-0.86 (m, 3H).

[0579] Synthesis of compound 2: To a stirred solution of (R)-dibenzyl(2-(benzyloxy)-3-(docosyloxy)propyl)phosphate 2-8 (450 mg, 0.599 mmol) in MeOH (50 mL) was added Pd(OH) (45 mg), and the reaction mixture was stirred at room temperature under H balloon pressure (40 psi) for 4 hours. The reaction progress was monitored by TLC. Upon completion of the reaction, the reaction mixture was filtered through a Celite bed, and the filtrate was concentrated in vacuo to give the crude material. The crude material was washed with diethyl ether, filtered, and dried in vacuo to give compound 2 (145 mg, 0.27 mmol, 50%) as an off-white solid. HPLC (ELSD): 99.14%, HRMS (M+1): 481.3962; 1 H NMR (CDCl3, 400 MHz): δ ppm 3.80-3.73 (m, 3H), 3.40-3.29 (m, 4H), 1.50-1.40 (m, 2H), 1.31-1.21 (m, 40 H), 0.87-0.84 (m, 3H).

[0580] Synthesis of compound 10 To a stirred solution of (R)-dibenzyl(2-(benzyloxy)-3-(docosyloxy)propyl)phosphate 2-8 (500 mg, 0.666 mmol) in dioxane (5 mL) was added HCl (4.0 M) in dioxane (20 mL) at 0° C., and the reaction mixture was stirred at room temperature for 48 hours. The reaction progress was monitored by TLC. Upon completion of the reaction, the reaction mixture was concentrated in vacuo, and the residue was diluted with water. The resulting solid was filtered and dried in vacuo to give impure material. The resulting impure material was further purified by stirring in ACN (100 mL) for 30 minutes, filtered, and the solid was dried in vacuo to give compound 10 (120 mg, 0.17 mmol, 31%) as an off-white solid. HPLC (ELSD): 99.13%, HRMS (M+1): 571.4449; 1H NMR (CDCl3, 400 MHz): δ ppm 7.28- 7.13 (m, 5H), 4.64-4.56 (m, 2H), 3.99-3.95 (m, 2H), 3.72 (bs, 1H), 3.51-3.22 (m, 4H), 1.48-1.43 (m, 2H), 1.30-1.21 (m, 40H), 0.80-0.87 (m, 3H).

[0581] Alternative synthesis of compound 9 and compound 2 [ka] Synthesis of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (2A-3) To a stirred solution of 1-bromodocosane (2A-2) (16.96 g, 128.3664 mmol) in toluene at 0 °C, potassium tert-butoxide (28.8 g, 256.7328 mmol) and (R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (2A-1) (16.96 g, 128.3664 mmol) were added. The reaction mixture became a thick mass. The reaction mixture was stirred at room temperature for 1 hour, and then the reaction mixture was heated to 110 °C for 16 hours. The completion of the reaction was monitored by TLC. Upon completion of the reaction, ether was added to the reaction mixture and stirred for 10 minutes. A brine solution was added to the reaction mixture, and it was extracted with ether. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to give the crude product (60 g) as a brown solid. H NMR confirmed the reaction. 1 H NMR (CDCl3) 400 MHz δ ppm 5.01-4.91 (m, 1H), 4.29-4.22 (m, 1H), 4.06-4.04 (m, 1H), 3.74-3.72 (m, 1H), 3.53-3.39 (m, 3H), 1.59-1.53 ​​(m, 2H), 1.45-1.18 (brm, 44H), 0.86 (t, J=13.6 Hz, 3H). 1 1 H NMR showed the desired product along with an impurity. 1H NMR values ​​were assigned based on the product peaks in the next step.

[0582] Synthesis of compound 9 To a stirred solution of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (2A-3) (60 g, 136.1315 mmol) in MeOH (500 mL), concentrated HCl (125 mL) was added and heated to 70 °C for 16 h. Completion of the reaction was monitored by TLC. The reaction mixture was diluted with water, filtered, and the filtered solid was stirred again with water and filtered to give a solid. The solid was stirred with hexane and filtered to give the product containing water. Acetonitrile was added to the product and distilled three times to remove water to give compound 9 (30 g, 55% over two steps) as an off-white solid. 1 The result was confirmed by H NMR (CDCl3) 400 MHz. 1 H NMR (CDCl3) 400 MHz δ ppm 3.86 (bs, 1H), 3.71-3.66 (m, 2H), 3.53-3.44 (m, 4H), 2.59 (bs, 1H), 2.15 (bs, 1H), 1.58-1.54 (m, 2H), 1.38-1.18 (bs, 38H), 0.88 (t, J=6.4 Hz, 3H).

[0583] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (2A-4) To a stirred solution of compound 9 ((S)-3-(docosyloxy)propane-1,2-diol (20.0 g, 49.913 mmol)) in pyridine (100.0 mL) at 0 °C, trityl chloride (13.91 g, 49.913 mmol) was added at 0 °C and heated to 120 °C in a sealed tube for 16 h. The starting materials, pyridine, and trityl chloride were anhydrous because moisture inhibits the reaction. The completion of the reaction was monitored by TLC. Upon completion of the reaction, the reaction mixture was evaporated under reduced pressure to give the crude product. The crude product was purified by Combiflash using 5% EtOAc in hexane as the eluent. After evaporation of the fractions, the product was washed with n-pentane (500.0 mL), stirred for 1 h, filtered, and dried to give the desired compound as a white solid contaminated with trityl impurity. The desired product 2A-4 (19.4 g, 60%) was obtained as a white solid and was characterized and confirmed by 1H NMR. 1 H NMR (400 MHz, CDCl3): δ =7.25-7.43 (m, 15 H), 3.94 (m, 1 H), 3.42-3.52 (m, 4H), 3.18 (m, 2H), 1.22-1.48 (m, 38 H), 0.87 (m, 3H).

[0584] Synthesis of (R)-((3-(docosyloxy)-2-((4-methoxybenzyl)oxy)propoxy)methanetriyl)tribenzene (2A-5) To a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (2A-4) (10.00 g, 15.55 mmol) in DMF (150 mL) was added NaH (1.55 g, 38.88 mmol) at 0 °C, and the reaction mixture was stirred for 20 min. After 20 min, p-methoxybenzyl chloride (3.14 mL, 23.32 mmol) was added dropwise, and the reaction mixture was stirred for 16 h. Completion of the reaction was monitored by TLC. The reaction mixture was quenched with ice-cold water (100 mL) and extracted with EtOAc (2 × 150 mL). The organic layer was dried over NaSO and evaporated in vacuo to give the crude material. The resulting crude material was purified by Combiflash chromatography (40 g column) using 0.5% EtOAc and 1% triethylamine in hexane as the eluent to give the title compound 2A-5 as a colorless liquid (12 g, impure; the trityl impurity was not separated at this stage). 1 Confirmed by 1 H NMR. 1 H NMR (400 MHz, CDCl3): δ = 7.45-6.84 (m, 19H), 4.59-4.57(m, 2H), 3.81-3.80 (m, 3H), 3.73-3.70 (m, 1 H), 3.55-3.53 (m, 2H), 3.40-3.36(m, 2H), 3.20-3.19 (m, 2H), 1.52-1.49(m, 2H), 1.30-1.20 (m, 38H), 0.89-0.84 (m, 3H).

[0585] Synthesis of (S)-3-(docosyloxy)-2-((4-methoxybenzyl)oxy)propan-1-ol (2A-6) To a stirred solution of (R)-((3-(docosyloxy)-2-((4-methoxybenzyl)oxy)propoxy)methanetriyl)tribenzene (2A-5) (12.00 g, 15.72 mmol)) in DCM:MeOH (120 mL) was added camphorsulfonic acid (3.65 g, 15.72 mmol) at 0 °C, and the reaction mixture was stirred at room temperature for 2 h. Completion of the reaction was monitored by TLC. The reaction mixture was diluted with DCM (200 mL) and washed with water (2 × 150 mL). The organic layer was dried over NaSO and evaporated under vacuum to give the crude material. The resulting crude material was purified by Combiflash chromatography (40 g column) using 20% ​​EtOAc and 1% triethylamine in hexane as the eluent to give the title compound 2A-6 (4.1 g, 50%) as a white solid. 1 Confirmed by 1 H NMR. 1 H NMR (400 MHz, CDCl3): δ =7.28 (d, J=8.8Hz, 2H), 6.88 (d, J=8.4Hz, 2H), 4.66-4.53(m, 2H), 3.73 (s, 3H), 3.72-3.66 (m, 1 H), 3.65-3.62 (m, 2H), 3.57-3.51(m, 2H), 3.45-3.42 (m, 2H), 2.16-2.13(m, 1H), 1.57-1.54(m, 2H), 1.31-1.19 (m, 38 H), 0.89-0.86 (m, 3H).

[0586] Synthesis of (R)-3-(docosyloxy)-2-((4-methoxybenzyl)oxy)propyl dihydrogen phosphate (2A-8) To a stirred solution of distilled POCl3 (0.8 mL, 8.5567 mmol) in THF (7 mL) at -20 °C (salt-ice mixture), a solution of (S)-3-(docosyloxy)-2-((4-methoxybenzyl)oxy)propan-1-ol (2A-6) (1 g, 1.9199 mmol) and anhydrous Et3N (6 mL, 43.0477 mmol) in anhydrous THF (7 mL) was added dropwise over 10 min and stirred at -20 °C for an additional 20 min. The reaction mixture was initially off-white for the first 20 min but slowly turned pale yellow. The completion of the reaction was monitored by TLC. The reaction mixture was slowly quenched with 10% aqueous NaHCO3 (5 mL) and stirred at the same temperature for 30 min. The ice bath temperature reached -10 °C, and then the reaction mixture was acidified with 6 N HCl and extracted with DCM (60 mL). The DCM layer was washed with 10% NaHCO3 solution (2 x 30 mL), and the aqueous layer was separated, acidified with concentrated HCl, and extracted with ethyl acetate and DCM. The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure at 40 °C to give the title compound 2A-8 as an off-white solid (0.6 g, 52%). 1H NMR and 31P NMR confirmed the identity.

[0587] 1 H NMR (CD3OD) 400 MHz VT 50℃ δ ppm 7.29 (d, J=8.4 Hz, 2H), 6.87 (d, J=8.4 Hz, 2H), 4.65-4.57 (m, 2H), 4.05-3.98 (m, 1H), 3.79-3.76 (bs, 4H), 3.55-3.48 (m, 2H), 3.43 (t, J=6.8 Hz, 2H), 1.58-1.51 (m, 2H), 1.33-1.21 (bs, 38H), 0.89 (t, J=6.4 Hz, 3H) 31P NMR (CDCl3) 400 MHz) δ ppm 0.719 (t). 1 H NMR and 31 P NMR was recorded in CD3OD and CD3OD + CDCl3 at 50-60 °C due to the low solubility of the compounds. HPLC: tRet 7.705 min (99.56%) HPLC method conditions: Column: LUNA HILIC (250*4.6) mm, 5 μm, 200A Mobile phase-A: 10 Mm ammonium acetate aqueous solution; Mobile phase-B: ACN 100% Method-T / %B: -0 / 10, 2 / 10, 6 / 100, 13 / 100, 14 / 10, 15 / 10 Flow rate: 1.0 ml / min Column temperature: 30 °C Diluent: THF

[0588] Synthesis of (R)-3-(docosyloxy)-2-hydroxypropyl dihydrogen phosphate (compound 2) To a stirred suspension of (R)-3-(docosyloxy)-2-((4-methoxybenzyl)oxy)propyl dihydrogen phosphate) (2A-8) (200 mg, 0.3328 mmol) in acetonitrile (10 mL), 4 M HCl in 1,4-dioxane (0.2 mL) was added at room temperature, and the reaction mixture was stirred in a sealed tube at 40° C. for 16 hours. Acetonitrile was added to the reaction mixture, and the solvent was removed with a dropper. This process was repeated three times, and then the solvent was removed under reduced pressure at 40° C. to give compound 2 (140 mg, 87.5%) as an off-white solid. H NMR and P NMR confirmed the identity.

[0589] 1 H NMR (CD3OD) 400 MHz VT 50℃ δ ppm 4.02-3.89 (m, 3H), 3.19-3.45 (m, 4H), 1.61-1.54 (m, 2H), 1.37-1.21 (bs, 38H), 0.89 (t, J=6.8 Hz, 3H). 31P NMR (CD3OD) 400 MHz VT 50℃ δ ppm 0.76 (t) Note: 1 H NMR, 31 PNMR was recorded in CD3OD and CD3OD + CDCl3 at 50-60 °C due to the low solubility of the compounds. HPLC: tRet 7.746 min (98.43%) HPLC method conditions: Column: LUNA HILIC (250*4.6) mm, 5 μm, 200A Mobile phase-A: 10 Mm ammonium acetate aqueous solution; Mobile phase-B: ACN 100% Method-T / %B: -0 / 10, 2 / 10, 6 / 100, 13 / 100, 14 / 10, 15 / 10 Flow rate: 1.0 ml / min Column temperature: 30 °C Diluent: ACN + H2O.

[0590] Example S-3: Synthesis of Compound 7 [ka] Synthesis of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (3-3): To a stirred solution of 1-bromodocosane (3-1) (16.96 g, 128.3664 mmol) in toluene at 0 °C, potassium tert-butoxide (28.8 g, 256.7328 mmol) and (R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (3-2) (50 g, 128.3664 mmol) were added, and the reaction mixture became a thick mass, so stirring was stopped. The reaction mixture was stirred at room temperature for 1 hour, and then the reaction mixture was heated to 110 °C for 16 hours. Completion of the reaction was monitored by TLC. Upon completion of the reaction, diethyl ether was added to the reaction mixture and stirred for 10 minutes. Brine solution was added to the reaction mixture, which was then extracted with diethyl ether. The organic layer was dried over anhydrous NaSO and concentrated under reduced pressure to give (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (3-3) as a brown solid (57 g, crude). HPLC (ELSD): 88.67%, 1 H NMR (CDCl3, 400 MHz): δ ppm 4.29-4.23 (m, 1H), 4.06-4.04 (m, 1H), 3.74-3.71 (m, 1H), 3.53-3.41 (m, 3H), 1.57-1.51 (m, 2 H), 1.45 (s, 3H), 1.38 (m, 3H), 1.31-1.14 (m, 40H), 0.89-0.86 (m, 3H).

[0591] Synthesis of compound 9: To a stirred solution of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (3-3) (57 g, 129.3250 mmol) in MeOH (500 mL) was added concentrated HCl (125 mL) and heated to 70 °C for 16 h. Completion of the reaction was monitored by TLC. The reaction mixture was diluted with water, filtered, and the filtered solid was stirred again with water and filtered to give a solid. The solid was stirred with hexane and filtered to give pure material. The resulting material contained water, so acetonitrile was added to the product and co-distilled three times (to remove traces of water) to give compound 9 as an off-white solid (28 g, 54% over two steps). HPLC (ELSD): 99.70%, 1 H NMR (CDCl 3, 400 MHz): δ ppm 3.88-3.83 (m, 1H), 3.71-3.67 (m, 2H), 3.53-3.44 (m, 4H), 2.59-2.58 (m, 1H), 2.15 (t, J= 5.6 Hz, 1H), 1.58-1.54 (m, 2H), 1.31-1.28 (m, 40 H), 0.88 (t, J= 6.8 Hz, 3H).

[0592] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (3-4): To a stirred solution of (S)-3-propoxypropane-1,2-diol (3-3) (25 g, 0.0625 mol) in pyridine (150 mL), trityl chloride (15.6 g, 0.0562 mol) was added at 0 °C, and the reaction mixture was stirred at 120 °C for 16 hours. Completion of the reaction was monitored by TLC. Upon completion of the reaction, the reaction mixture was concentrated in vacuo to give a crude material. The resulting crude material was purified by MPLC flash column chromatography using 10% EtOAc in hexane as the eluent to give (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (3-4) as an off-white solid (25 g, 62%). 1H NMR (CDCl3, 400 MHz): δ ppm 7.44 -7.21 (m, 15 H), 3.97-3.93 (m, 1H), 3.52-3.41 (m, 4H), 3.22 -3.15 (m, 2H), 2.42 (d, J=3.6 Hz, 1H), 1.57-1.52 (m, 2H), 1.31-1.28 (m, 40H), 0.87 (t, J= 6.4 Hz, 3H).

[0593] Synthesis of (R)-4-nitrophenyl(1-propoxy-3-(trityloxy)propan-2-yl)carbonate (3-5): To a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (3-4) (1.2 g, 1.866 mmol) in THF (30 mL) at room temperature, EtN (0.52 mL, 3.732 mmol) was added, followed by 4-nitrophenyl chloroformate (0.56 g, 2.7993 mmol). The reaction mixture was heated to 80 °C in a sealed tube for 16 h. TLC showed the formation of product along with starting material, so additional EtN (1.3 mL, 9.33 mmol) and 4-nitrophenyl chloroformate (1.88 g, 9.33 mmol) were added and heated to 80 °C in a sealed tube for 16 h. Completion of the reaction was monitored by TLC. The reaction mixture was concentrated in vacuo. The residue was dissolved in EtOAc and washed with brine solution, and the combined organic layers were dried over anhydrous NaSO and concentrated in vacuo to give crude material. The resulting crude material was purified by combi-flash column chromatography using 30% EtOAc in hexane as the eluent to give (R)-4-nitrophenyl (1-propoxy-3-(trityloxy)propan-2-yl) carbonate (5) as an off-white solid (1.2 g, impure). HPLC (ELSD): 99.89%, 1 H NMR (CDCl 3,400 MHz): δ ppm 8.28 -8.25 (m, 2 H), 7.45 -7.23 (m, 17 H ), 5.17 -5.14 (m, 1H), 3.70-3.63 (m, 2H), 3.45-3.34 (m, 4H), 1.56-1.53 ​​(m, 2H), 1.31-1.28 (m, 40 H), 0.87 (t, J= 6.8 Hz, 3H).

[0594] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylcarbamate (3-6): A stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-yl(4-nitrophenyl)carbonate (3-5) (1.2 g, 1.4849 mmol) in THF at 0 °C was purged with ammonia gas and stirred at room temperature in a sealed tube for 16 hours. Completion of the reaction was monitored by TLC. The reaction mixture was concentrated in vacuo to give the crude material. The resulting crude material was purified by combiflash column chromatography using 5% EtOAc in hexane to remove the 4-nitrophenol impurity, followed by elution with 30% EtOAc in hexane as the eluent to give (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylcarbamate (3-6) as a colorless waxy solid (0.75 g, 73.62%). 1 H NMR (CDCl 3, 400 MHz): δ ppm 7.45 -7.23 (m, 15 H ), 5.09 -5.04 (m, 1H), 4.66 (brs, 2H), 3.65-3.60 (m, 2H), 3.44-3.37 (m, 2H), 3.35-3.23 (m, 2H) 1.50-1.47 (m, 2H), 1.31-1.28 (m, 40 H), 0.87 (t, J= 6.4 Hz, 3H).

[0595] Synthesis of (S)-1-(docosyloxy)-3-hydroxypropan-2-ylcarbamate (compound 7): To a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylcarbamate (3-6) (0.75 g, 1.0932 mmol) in MeOH and DCM (1:1, 10 mL), CSA (0.05 g, 0.21864 mmol) was added, and the reaction mixture was stirred at room temperature for 16 hours. Completion of the reaction was monitored by TLC. The reaction mixture was filtered, and the solid was washed with ether and dried under vacuum to give (S)-1-(docosyloxy)-3-hydroxypropan-2-ylcarbamate (compound 7) as an off-white solid (0.33 g, 68.03%). HPLC (ELSD): 99.85%, HRMS: 444.4623; 1 H NMR (CDCl 3, 400 MHz): δ ppm 4.90-4.85 (m, 1H), 4.72 (brs, 2H), 3.84 (t, J= 5.6 Hz, 3H), 3.66-3.64 (m, 2H), 3.49-3.42 (m, 2H), 2.45( t, J= 6.4 Hz, 1H), 1.55-1.53 ​​(m, 2H), 1.31-1.28 (m, 40 H), 0.88 (t, J= 6.4 Hz, 3H).

[0596] Example S-4: Synthesis of Compound 8 [ka] Synthesis of (R)-4-nitrophenyl(1-propoxy-3-(trityloxy)propan-2-yl)carbonate (4-2): To a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (4-1) (2.5 g, 0.0038 mol) in THF (30 mL) at room temperature, EtN (1.1 mL, 0.0076 mol) was added, followed by 4-nitrophenyl chloroformate (1.17 g, 0.0058 mol) and stirred at 80 °C for 16 h. Completion of the reaction was monitored by TLC. The reaction mixture was concentrated in vacuo. The residue was dissolved in EtOAc and washed with brine solution, and the combined organic layers were dried over anhydrous NaSO and concentrated in vacuo to give crude material. The resulting crude material was purified by combiflash column chromatography using 20% ​​EtOAc in hexane as the eluent to give (R)-4-nitrophenyl (1-propoxy-3-(trityloxy)propan-2-yl)carbonate (4-2) as an off-white solid (3 g, impure). 1 H NMR (CDCl 3, 400 MHz): δ ppm 8.27 (d, J=9.2 Hz, 2 H), 7.45 -7.23 (m, 17 H ), 5.17 -5.14 (m, 1H), 3.70-3.63 (m, 2H), 3.45-3.34 (m, 4H), 1.56-1.53 (m, 2H), 1.31-1.28 (m, 40 H), 0.87 (t, J= 6.8 Hz, 3H).

[0597] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylmethylcarbamate (4-3): To a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-yl(4-nitrophenyl)carbonate (4-2) (3.5 g, 0.0043 mmol) in THF (40 mL) at 0 °C, a solution of methylamine in THF (5 mL) was added, and the reaction mixture was stirred at room temperature for 16 h. Completion of the reaction was monitored by TLC. The reaction mixture was concentrated in vacuo to give the crude material. The resulting crude material was purified by combiflash column chromatography using 20% ​​EtOAc in hexane as the eluent to give (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylmethylcarbamate (4-3) as an off-white solid (3 g, impure). 1 H NMR (CDCl 3, 400 MHz): δ ppm 7.43 -7.20 (m, 15 H ), 6.82 (brs, 1H) 5.09 -5.04 (m, 1H), 4.72-4.71 (m, 1H), 3.67-3.58 (m, 2H), 3.44-3.37 (m, 2H), 3.27-3.22 (m, 2H), 2.80 (d, J= 4.8 Hz, 3H), 1.50-1.47 (m, 2H), 1.31-1.28 (m, 40 H), 0.87 (t, J= 6.4 Hz, 3H).

[0598] Synthesis of (S)-1-(docosyloxy)-3-hydroxypropan-2-ylmethylcarbamate (compound 8): To a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylmethylcarbamate (4-3) (3 g, 0.00429 mmol) in DCM; MeOH (40 mL) was added CSA (0.9 g, 0.00429 mol) at 0 °C, and the reaction mixture was stirred at room temperature for 1 h. Completion of the reaction was monitored by TLC. The residue was dissolved in EtOAc and washed with brine solution, and the combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude material. The resulting crude material was purified by combiflash column chromatography using 30% EtOAc in hexane as the eluent to give (S)-1-(docosyloxy)-3-hydroxypropan-2-ylmethylcarbamate compound 8 as an off-white solid. HPLC (ELSD): 99.88%, 1 H NMR (CDCl 3, 400 MHz): δ ppm 4.90 (brs, 1H), 4.77 (brs, 1H), 3.83 (brs, 2H), 3.64-3.63 (m, 2H), 3.49-3.44 (m, 2H), 2.82 (d, J= 5.2 Hz, 3H), 2.56 (brs, 1H), 1.59 (brs, 2H), 1.31-1.28 (m, 40H), 0.90 (t, J= 6.4 Hz, 3H).

[0599] Example S-5: Synthesis of Compound 12, Compound 11, and Compound 13 [ka] Synthesis of (R)-2,2-dimethyl-4-((octadecyloxy)methyl)-1,3-dioxolane (5-3): To a stirred solution of 1-bromooctadecane (5-2, 25.22 g, 75.66 mmol, 1.0 equiv.) in toluene at 0 °C, potassium tertiary butoxide (16.97 g, 151.32 mmol, 2.0 equiv.) and (R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (5-1, 10.0 g, 75.66 mmol, 1.0 equiv.) were added. The thick mass of the reaction mixture was stirred at room temperature for 1 h and then heated to 110 °C for 16 h. The reaction was monitored for completion by TLC. Upon completion of the reaction, diethyl ether (500 mL) was added to the reaction mixture and stirred for 10 min. Brine solution (500 mL) was added to the reaction mixture and extracted with ether. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude product 5-3 (29 g) as a brown semi-solid. crude 1 Confirmed by 1 H NMR.

[0600] 1 H NMR (400 MHz, CDCl3): δ =4.23-4.29 (m, 1 H), 4.23-4.29 (m, 1 H), 4.04-4.06 (m, 1H), 3.72-3.74 (m, 1H), 3.41-3.53 (m, 3H), 1.55-1.57 (m, 2H), 1.38 (m, 30H), 0.86-0.89(m, 3H).

[0601] Synthesis of ((S)-3-(octadecyloxy)propane-1,2-diol) (Compound 12) To a stirred solution of (R)-2,2-dimethyl-4-((octadecyloxy)methyl)-1,3-dioxolane 5-3 (14.0 g, 37.695 mmol, 1.0 equiv) in methanol (140 mL) was added concentrated HCl (41.5 mL), stirred, and heated to 70 °C for 16 h. The reaction was monitored for completion by TLC. All solvent was evaporated, and then acetonitrile (100.0 mL) was added to the crude reaction mixture and stirred for 2 h. An off-white, mobile solid precipitated, which was filtered and dried. The crude powder was triturated with n-pentane (50.0 mL), filtered, and dried to give compound 12 as an off-white solid (7.4 g, 58% over two steps). 1 The product was confirmed by H NMR (CDCl3) 400 MHz, CDCl3 + D2O, and HRMS. 1 H NMR (400 MHz, CDCl3): δ =3.84-3.88 (m, 1 H), 3.71-3.72 (m, 2 H), 3.44-3.54 (m, 3H), 2.57-2.58 (d, j=4.8 HZ, 1H), 2.12-2.15 (m, 1H), 1.22-1.4 (m, 32 H), 0.86-0.89 (t, 3H). HPLC: 99.79%. HRMS: 344.59 (matched), dimer mass: (689.13) matched.

[0602] Synthesis of (R)-1-(octadecyloxy)-3-(trityloxy)propan-2-ol (5-4) To a stirred solution of (S)-3-(octadecyloxy)propane-1,2-diol compound 12 (1.3 g, 3.772 mmol, 1.0 equiv.) in pyridine (5.0 mL) at 0 °C, trityl chloride (1.05 g, 3.772 mmol) was added and heated to 120 °C in a sealed tube for 16 h. The starting materials, pyridine, and trityl chloride were anhydrous because moisture inhibits the reaction. The reaction completion was monitored by TLC. The reaction mixture was evaporated under reduced pressure, and the crude product was purified by CombiFlash using 5% ethyl acetate in hexane as the eluent. The fractions were collected and concentrated. Finally, the compound was triturated with n-pentane (25.0 mL), filtered, and dried to give the title compound 5-4 (1.3 g, 62%) as an off-white solid containing a trace of the deprotected trityl alcohol impurity. 1 Confirmed by H NMR (CDCl3) 400 MHz.

[0603] 1 H NMR (400 MHz, CDCl3): δ =7.18-7.50 (m, 15 H), 3.93-3.97 (m, 1 H), 3.42-3.52 (m, 4H), 3.16-3.22 (m, 2H), 1.50-1.56 (m, 2H), 1.22-1.38 (m, 30H), 0.86-0.93 (t, 3H).

[0604] Synthesis of (R)-1-propoxy-3-(trityloxy)propan-2-ylpyridin-2-yl carbonate (5-6) To a stirred solution of (R)-1-(octadecyloxy)-3-(trityloxy)propan-2-ol (5-4) (1.0 g, 1.703 mmol, 1.0 equiv.) in dry THF (10.0 mL), triethylamine (1.42 mL, 10.218 mmol, 6.0 equiv.) was added followed by di(pyridin-2-yl)carbonate 5-5 (0.736 g, 3.407 mmol, 2.0 equiv.) at room temperature, and the reaction mixture was then stirred at 80° C. for 16 hours in a sealed tube. The completion of the reaction was monitored by TLC. Intermediate 5-6 was unstable and was used directly in the next step without workup. The formation of intermediate 5-6 was confirmed by HPLC.1 H NMR (CDCl3) 400 MHz: δ = 7.28-7.48 (m, 20 H), 3.2-3.8 (m, 4 H), 1.28-1.38 (m, 32 H).

[0605] Synthesis of (R)-1-(octadecyloxy)-3-(trityloxy)propan-2-ylmethylcarbamate (5-7) To a stirred solution of intermediate (R)-1-propoxy-3-(trityloxy)propan-2-ylpyridin-2-yl carbonate 5-6 (1.0 g, 1.446 mmol, 1.0 equiv) in dry THF (20.0 mL) was added 7% methylamine in THF (10.0 mL) at 0 °C. The reaction mixture was stirred in a sealed tube at 80 °C for 16 h. Completion of the reaction was monitored by TLC. The reaction mixture was evaporated under reduced pressure. The crude product was dissolved in ethyl acetate (50.0 mL) and extracted with water (25.0 mL × 2). The organic layer was dried over anhydrous sodium sulfate and concentrated to give the title compound 5-7 (1.2 g, crude) as an amber semi-solid. The crude product was 1 It was characterized by H NMR (CDCl3, 400 MHz) and used in the next step without further purification.

[0606] 1 H NMR (400 MHz, CDCl3): δ =7.26-7.43 (m, 15 H), 5.079 (m, 1 H), 4.66 (m, 1H), 2.81 (m, 3H), 1.25-1.55 (m, 32 H), 1.22-1.38 (m, 30 H), 0.88 (t, 3H).

[0607] Synthesis of (S)-1-hydroxy-3-(octadecyloxy)propan-2-ylmethylcarbamate (compound 11) To a stirred solution of (R)-1-(octadecyloxy)-3-(trityloxy)propan-2-ylmethylcarbamate 5-7 (1.2 g, 1.863 mmol, 1.0 equiv.) in anhydrous DCM:anhydrous MeOH (1:1) (10.0 mL:10.0 mL) solvent mixture at 0 °C, DL-10-camphorsulfonic acid (CSA; 0.433 g, 1.863 mmol, 1.1 equiv.) was added, and the reaction mixture was stirred at room temperature for 5 h. Completion of the reaction was monitored by TLC. The reaction mixture was diluted with DCM (100.0 mL) and extracted with water (50.0 mL × 2). The organic layer was dried over anhydrous sodium sulfate and concentrated to give the crude compound. The crude product was triturated with n-pentane (20.0 mL) to give compound 11 (420.0 mg, 56.0%) as an off-white solid. The structure of the product was confirmed by 1H NMR (400 MHz) (CDCl3, CDCl3 + D2O) and HRMS.

[0608] 1 H NMR (400 MHz, CDCl3): δ = 4.487-4.89 (m, 1H), 4.76 (m, 1H), 3.80-3.83 (m, 2H), 3.59-3.66 (m, 2H), 3.42-3.48 (m, 2 H), 2.80-2.82 (d, 3H), 2.54-2.57 (m, 1H), 1.52-1.57 (m, 2H), 1.20-1.4 (m, 32H), 0.86-0.89 (t, 3H).HRMS:401.43(match), HPLC:99.84%.

[0609] Synthesis of (R)-1-((bis(benzyloxy)phosphoryl)oxy)-3-(octadecyloxy)propan-2-ylmethylcarbamate (5-8) To a stirred solution of (S)-1-hydroxy-3-(octadecyloxy)propan-2-ylmethylcarbamate compound 11 (1.0 g, 2.489 mmol, 1.0 equiv.) in anhydrous THF (80.0 mL) at 0 °C, potassium tert-butoxide (0.279 g, 2.489 mmol, 2.0 equiv.) was added and stirred for 5 min. Tetrabenzyl diphosphate (1.34 g, 2.489 mmol, 1.0 equiv.) was added, and the reaction mixture was stirred at room temperature for 3 h. Completion of the reaction was monitored by TLC. The reaction mixture was diluted with ethyl acetate (50.0 mL) and extracted with water (50.0 mL × 2). The organic layer was dried over anhydrous sodium sulfate and concentrated to give 2.0 g of crude compound. The crude compound was purified by preparative HPLC to give compound 5-8 (750.0 mg, 45%) as a white solid. 1 H NMR, 31 Confirmed by P NMR.

[0610] 1 H NMR (400 MHz, CDCl3): δ = 4.46 (m, 1H), 4.71 (m, 1H), 4.18-4.27 (m, 2H), 2.66-2.70 (m, 2H), 1.48-1.51 (m, 2H), 1.29 (m, 30H), 0.86-0.89 (t, 3H). 31 P NMR:match

[0611] Synthesis of (R)-1-(octadecyloxy)-3-(phosphonooxy)propan-2-ylmethylcarbamate (compound 13) To a stirred solution of (R)-1-((bis(benzyloxy)phosphoryl)oxy)-3-(octadecyloxy)propan-2-ylmethylcarbamate 5-8 (400.0 mg, 0.604 mmol, 1.0 equiv.) in MeOH, 20% Pd(OH)2 / C (100.0 mg) was added and hydrogenated at 40 PSI for 4 hours. The reaction was monitored for completion by TLC. The reaction mixture was filtered through Celite, washed with 250 mL of methanol, and the filtrate was passed through a micron filter (NYL 0.45 um) and concentrated to give the crude product. The crude compound was triturated with n-pentane and dried under high vacuum to give compound 13 (350 mg, 99%) as a white solid. H NMR, P NMR, and HRMS confirmed the identity of the compound.

[0612] 1 H NMR (400 MHz, CDOD): δ = 4.35-4.40 (m, 1H), 4.14-4.18 (m, 1H), 4.05-4.08 (m, 1H), 3.47-3.55 (m, 2H), 3.37-3.39 (m, 2 H), 2.59-2.64 (d, 3H), 1.42-1.47 (m, 2H), 1.20-1.30 (m, 30H), 0.78-0.81 (t, 3H).HRMS: 482.01 (match), HPLC: 99.87%.

[0613] Example S-6: Synthesis of Compound 6 [ka] Synthesis of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (6-3) To a stirred solution of 1-bromodocosane (6-2) (16.96 g, 128.3664 mmol) in toluene at 0 °C, potassium tert-butoxide (28.8 g, 256.7328 mmol) and (R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (6-1) (16.96 g, 128.3664 mmol) were added. The reaction mixture became a thick mass. The reaction mixture was stirred at room temperature for 1 hour, and then the reaction mixture was heated to 110 °C for 16 hours. The completion of the reaction was monitored by TLC. Upon completion of the reaction, ether was added to the reaction mixture and stirred for 10 minutes. A brine solution was added to the reaction mixture, and it was extracted with ether. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to give the crude product (60 g) as a brown solid. H NMR confirmed the reaction. 1 H NMR (CDCl3) 400 MHz δ ppm 5.01-4.91 (m, 1H), 4.29-4.22 (m, 1H), 4.06-4.04 (m, 1H), 3.74-3.72 (m, 1H), 3.53-3.39 (m, 3H), 1.59-1.53 ​​(m, 2H), 1.45-1.18 (brm, 44H), 0.86 (t, J=13.6 Hz, 3H). 1 H NMR shows the desired product along with impurities, 1 H NMR values ​​were assigned based on the product peaks in the next step.

[0614] Synthesis of compound 9 To a stirred solution of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (6-3) (60 g, 136.1315 mmol) in MeOH (500 mL) was added concentrated HCl (125 mL) and heated to 70 °C for 16 h. Completion of the reaction was monitored by TLC. The reaction mixture was diluted with water, filtered, and the filtered solid was stirred again with water and filtered to give a solid. The solid was stirred with hexane and filtered to give the product. The product contained water. Acetonitrile was added to the product and distilled three times to remove water to give compound 9 (30 g, 55% over two steps) as an off-white solid. 1The result was confirmed by H NMR (CDCl3) 400 MHz. 1 H NMR (CDCl3) 400 MHz δ ppm 3.86 (bs, 1H), 3.71-3.66 (m, 2H), 3.53-3.44 (m, 4H), 2.59 (bs, 1H), 2.15 (bs, 1H), 1.58-1.54 (m, 2H), 1.38-1.18 (bs, 38H), 0.88 (t, J=6.4 Hz, 3H).

[0615] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (6-4) To a stirred solution of compound 9 ((S)-3-(docosyloxy)propane-1,2-diol (20.0 g, 49.913 mmol)) in pyridine (100.0 mL) at 0 °C, trityl chloride (13.91 g, 49.913 mmol) was added at 0 °C and heated to 120 °C in a sealed tube for 16 h. The reaction was monitored for completion by TLC. Upon completion, the reaction mixture was evaporated under reduced pressure to give the crude product. The crude product was purified by CombiFlash using 5% EtOAc in hexane as the eluent. After evaporation of the fractions, the product was washed with n-pentane (500.0 mL), stirred for 1 h, filtered, and dried to give the desired compound as a white solid contaminated with trityl impurity. The desired product 6-4 (19.4 g, 60%) was obtained as a white solid and characterized by H NMR, which confirmed its identity. 1 H NMR (400 MHz, CDCl3): δ =7.25-7.43 (m, 15 H), 3.94 (m, 1 H), 3.42-3.52 (m, 4H), 3.18 (m, 2H), 1.22-1.48 (m, 38 H), 0.87 (m, 3H).

[0616] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylpyridin-2-yl carbonate (6-6) To a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (5 g, 7.7759 mmol) in THF at room temperature, EtN (4.3 mL, 31.1036 mmol) was added, followed by di(pyridin-2-yl)carbonate (3.36 g, 15.5518 mmol), and the mixture was stirred at 80 °C in a sealed tube for 16 h. The starting materials, pyridine, and trityl chloride were anhydrous because moisture inhibits the reaction. The reaction was monitored for completion by TLC. Due to the instability of the resulting reaction mixture, this crude mixture of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylpyridin-2-yl carbonate 6-6 (5 g, crude, light brown reaction mixture) was used in the next step without workup.

[0617] 1 H NMR (CDCl3) 400 MHz) δ ppm 7.80-7.76 (m, 1H), 7.47-7.27 (m, 16H), 6.57 (d, J=9.2 Hz, 1H), 6.30-6.27 (m, 1H), 5.14-5.12 (m, 1H), 3.75-3.33 (m, 6H), 1.52-1.49 (m, 2H), 1.4-1.2 (bs, 38H), 0.87 (t, J=6.4 Hz, 3H). Crude 1 1 H NMR showed the desired product along with an impurity.

[0618] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylmethylcarbamate (6-7) To a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylpicolinate (6-6) (5 g, 6.6835 mmol) in THF (50 mL) was added 7% methylamine in THF (10 mL) at 0 °C, stirred, and heated at 80 °C in a sealed tube for 16 h. The reaction was monitored for completion by TLC. The reaction mixture was evaporated under reduced pressure to give the crude product. The crude product was dissolved in ethyl acetate (200 mL), washed with water (250 mL × 2), extracted, and separated. The organic layer was dried over anhydrous sodium sulfate and concentrated to give (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylmethylcarbamate 6-7 (6 g, crude) as a light brown solid, which was characterized by H NMR. 1 H NMR (CDCl3) 400 MHz) δ ppm 7.43-7.42 (m, 15H), 5.08-5.06 (m, 1H), 3.65-3.21 (m, 6H), 2.8 (d, J=4.4 Hz, 3H), 1.50-1.43 (m, 2H), 1.31-1.27 (brs, 38H), 0.87 (t, J=6.4 Hz, 3H).

[0619] Synthesis of (S)-1-(docosyloxy)-3-hydroxypropan-2-ylmethylcarbamate (compound 8) To a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylmethylcarbamate (6-7) (6 g, 8.5773 mmol) in a 1:1 DCM:MeOH (60 mL:60 mL) solvent mixture at 0 °C, DL-10-camphorsulfonic acid (1.99 g, 8.5773 mmol) was added. The whole reaction mixture was stirred at room temperature for 2 hours. The completion of the reaction was monitored by TLC. The reaction mixture was diluted with DCM (200 mL), washed with water (50.0 mL × 2), extracted, separated, and the organic layer was dried over anhydrous sodium sulfate and concentrated to give the crude compound. The crude product was washed with n-pentane (100 mL) and stirred for 15 minutes, resulting in the precipitation of an off-white solid. This was filtered and dried to give compound 8 (1.5 g) as an off-white solid. 1H NMR (CDCl3) 400 MHz) δ ppm 4.90-4.87 (m, 1H), 4.80 (bs, 1H), 3.85-3.81 (m, 2H), 3.64-3.61 (m, 2H), 3.47-3.43 (m, 2H), 2.81 (d, J=5.2 Hz, 3H), 2.62 (t, J=6 Hz, 1H), 1.63-1.52 (m, 2H), 1.4-1.2 (brs, 38H), 0.88 (t, J=6.4 Hz, 3H).

[0620] Synthesis of (R)-1-((bis(benzyloxy)phosphoryl)oxy)-3-(docosyloxy)propan-2-ylmethylcarbamate (6-10) To a stirred solution of compound 8 ((S)-1-(docosyloxy)-3-hydroxypropan-2-ylmethylcarbamate (0.5 g, 1.092 mmol)) in anhydrous THF (100 mL) at 0 °C, potassium tert-butoxide (0.245 g, 2.1846 mmol) was added. The mixture was stirred for 10 min, tetrabenzyl diphosphate (1.17 g, 2.1846 mmol) was added, and the reaction mixture was stirred at 0 °C for 3 h. TLC showed the formation of product along with starting material. Additional potassium tert-butoxide (0.12 g, 1.0923 mmol) and tetrabenzyl diphosphate (0.6 g, 1.0923 mmol) were added and the mixture was stirred at 0 °C for an additional 3 h. Completion of the reaction was monitored by TLC. The reaction mixture was quenched with ice water and extracted with ethyl acetate (2 × 100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated to give 1.3 g of crude compound, which was purified by chiral preparative HPLC to separate peak 1 and peak 2. Peak 1 (as a pale pink liquid) was obtained as the title compound 6-10 (130 mg), confirmed by H NMR and P NMR.

[0621] 1H NMR (CDCl3) 400 MHz) δ ppm 7.36-7.3 (bs, 10H), 5.1-5.0 (m, 4H), 4.69 (bs, 1H), 4.30-4.27 (m, 1H), 4.19-4.13 (m, 1H), 355-3.51 (m, 2H), 3.41-3.36 (m, 2H), 2.67 (bs, 3H), 1.52-1.47 (m, 2H), 1.33-1.24 (bs, 38H), 0.88 (t, J=6.4 Hz, 3H. 31P NMR (CDCl3) 400 MHz) δ ppm -1.913 (bs), HRMS: (M+1) =718.4727. HPLC:t Ret 11.017 min (99.35%) HPLC method conditions: Column: Kinetex EVO, C18 (150*4.6) mm, 5 μm, 100A Mobile phase-A: 0.1% formic acid aqueous solution; Mobile phase-B: ACN 100% Method-T / %B: -0 / 60, 2 / 60, 6 / 100, 16 / 100, 17 / 60, 18 / 60 Flow rate: 1.5 ml / min Column temperature: 30 °C Diluent: THF

[0622] Synthesis of (R)-1-(docosyloxy)-3-(phosphonooxy)propan-2-ylmethylcarbamate: Compound 6 To a stirred solution of (R)-1-((bis(benzyloxy)phosphoryl)oxy)-3-(docosyloxy)propan-2-ylmethylcarbamate 6-10 (130 mg, 0.181 mmol) in EtOAc, 20% Pd(OH)2 / C (50 mg) was added and hydrogenated at 40 PSI for 2 hours. The completion of the reaction was monitored by TLC. The reaction mixture was diluted with 10% MeOH in DCM and filtered through Celite. The filtrate was concentrated under reduced pressure to give the crude product. The crude product was washed with pentane and dried to give compound 6 (50 mg) as an off-white solid. 1H NMR, 31P NMR confirmed the reaction.

[0623] 1H NMR (CD3OD) 400 MHz VT 50℃ δ ppm 4.38-4.36 (bs, 1H), 4.19-4.16 (m, 1H), 4.08-4.04 (m, 1H), 3.56-3.5 (m, 2H), 3.38 (t, J=6.4 Hz, 2H), 2.60 (s, 3H), 1.49-1.42 (m, 2H), 1.3-1.11 (bs, 38H). 31P NMR (CDCl3) 400 MHz) δ ppm 0.276 (bs) HPLC: tRet 7.616 min (99.58%) HPLC method conditions: Column: LUNA HILIC (250*4.6) mm, 5 μm, 200A Mobile phase-A: 10 Mm ammonium acetate aqueous solution; Mobile phase-B: ACN 100% Method-T / %B: -0 / 10, 2 / 10, 6 / 100, 13 / 100, 14 / 10, 15 / 10 Flow rate: 1.0 ml / min Column temperature: 30 °C Diluent: THF.

[0624] Example S-7: Synthesis of Compound 4: [ka] Synthesis of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (7-3): To a stirred solution of 1-bromodocosane (16.96 g, 128.3664 mmol) in toluene at 0 °C, potassium tert-butoxide (28.8 g, 256.7328 mmol) and (R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (16.96 g, 128.3664 mmol) were added. The reaction mixture became a thick mass, so stirring was stopped. The reaction mixture was stirred at room temperature for 1 hour and then heated to 110 °C for 16 hours. Completion of the reaction was monitored by TLC. Upon completion of the reaction, ether was added to the reaction mixture and stirred for 10 minutes. Brine solution was added to the reaction mixture and extracted with ether. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to give crude product 3 (60 g) as a brown solid. H NMR confirmed the reaction. 1H NMR (CDCl) 400 MHz δ ppm 5.01-4.91 (m, 1H), 4.29-4.22 (m, 1H), 4.06-4.04 (m, 1H), 3.74-3.72 (m, 1H), 3.53-3.39 (m, 3H), 1.59-1.53 ​​(m, 2H), 1.45-1.18 (m, 40H), 0.86 (t, J = 13.6 Hz, 3H). Crude H NMR showed the desired product along with impurities; H NMR values ​​were assigned based on the product peaks in the next step.

[0625] Synthesis of compound 9 To a stirred solution of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (60 g, 136.1315 mmol) in MeOH (500 mL) was added concentrated HCl (125 mL) and heated to 70° C. for 16 h. Completion of the reaction was monitored by TLC. The reaction mixture was diluted with water, filtered, and the filtered solid was stirred again with water and filtered to give a solid. The solid was stirred with hexane and filtered to give the product. Since the product contained water, acetonitrile was added to the product and distilled three times to remove water, giving compound 9 as an off-white solid (30 g, 55% over two steps). 1 H NMR (CDCl3) 400 MHz) confirmed: 1 H NMR (CDCl3) 400 MHz δ ppm 3.86 (bs, 1H), 3.71-3.66 (m, 2H), 3.53-3.44 (m, 4H), 2.59 (bs, 1H), 2.15 (bs, 1H), 1.58-1.54 (m, 2H), 1.38-1.18 (bs, 40H), 0.88 (t, J=6.4 Hz, 3H).

[0626] Synthesis of R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (7-4) To a stirred solution of (S)-3-(docosyloxy)propane-1,2-diol (20.0 g, 49.913 mmol) in pyridine (100.0 mL) at 0 °C, trityl chloride (13.91 g, 49.913 mmol) was added at 0 °C and heated to 120 °C in a sealed tube for 16 h. (Note that the starting materials, pyridine, and trityl chloride must be anhydrous. If the reaction mixture contains water, the reaction will not proceed.) Completion of the reaction was monitored by TLC. Upon completion, the reaction mixture was evaporated under reduced pressure to give the crude product. The crude product was purified by Combiflash chromatography using 5% EtOAc in hexane as the eluent. After evaporation of the fractions, the product was washed with n-pentane (500 mL), stirred for 1 h, filtered, and dried to give the desired compound as a white solid slightly contaminated with trityl chloride. The desired product 7-4 (19.4 g, 60%) was obtained as a white solid and characterized by H NMR, confirming: 1 H NMR (400 MHz, CDCl): δ = 7.25-7.43 (m, 15 H), 3.94 (m, 1 H), 3.42-3.52 (m, 4 H), 3.18 (m, 2 H), 1.58-1.54 (m, 2 H), 1.22-1.48 (m, 38 H), 0.87 (m, 3 H). Crude H NMR showed the desired product along with impurities. H NMR values ​​were assigned based on the product peaks in the next step.

[0627] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylpyridin-2-yl carbonate (7-6): To a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (5 g, 7.7759 mmol) in THF at room temperature, EtN (4.3 mL, 31.1036 mmol) was added, followed by di(pyridin-2-yl)carbonate (3.36 g, 15.5518 mmol) and stirred in a sealed tube at 80 °C for 16 h. The reaction was monitored for completion by TLC. Due to the instability of the resulting reaction mixture, the crude mixture was carried on to the next step without workup to give (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylpyridin-2-yl carbonate 7-6 (5 g, crude) as a light brown reaction mixture. 1 H NMR (CDCl) 400 MHz) δ ppm 7.80-7.76 (m, 1H), 7.47-7.27 (m, 16H), 6.57 (d, J = 9.2 Hz, 1H), 6.30-6.27 (m, 1H), 5.14-5.12 (m, 1H), 3.75-3.33 (m, 6H), 1.52-1.49 (m, 2H), 1.4-1.2 (d, 38H), 0.87 (t, J = 6.4 Hz, 3H). Crude H NMR showed the desired product along with impurities. H NMR values ​​were assigned based on the product peaks in the next step.

[0628] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylmethylcarbamate (7-7) To the crude compound of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylpyridin-2-yl carbonate in THF (100 ml) was added 2M methylamine in THF (50 mL) and heated to 80° C. for 16 hours. Completion of the reaction was monitored by TLC. The reaction mixture was evaporated under reduced pressure to give the crude product. The crude product was dissolved in EtOAc (300 mL) and washed with water (2×200 mL), and the organic layer was dried over anhydrous NaSO and concentrated under reduced pressure to give the product. The crude compound was carried on directly to the next step (9 g, crude) and characterized by H NMR: 1H NMR (CDCl3) 400 MHz) δ ppm 7.43-7.18 (m, 15H), 5.08-5.06 (m, 1H), 4.74-4.72 (m, 1H), 3.65-3.60 (m, 2H), 3.43-3.36 (m, 2H), 3.27-3.21 (m, 2H), 2.80 (d, J=4.8Hz, 3H), 1.50-1.47 (m, 2H), 1.28 (brs, 40H), 0.87 (t, J=6.4 Hz, 3H).

[0629] Synthesis of (S)-1-(docosyloxy)-3-hydroxypropan-2-ylmethylcarbamate (7-8) To a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylmethylcarbamate (9.0 g, 12.865 mmol) in 100 mL of MeOH and DCM (1:1) was added camphorsulfonic acid (2.98 g, 12.865 mmol) at 0° C. The reaction mixture was stirred at room temperature for 2 hours. The completion of the reaction was monitored by TLC. The reaction mixture was diluted with DCM (300.0 mL) and washed with water (200 mL×2), and then the organic layer was dried over anhydrous sodium sulfate and concentrated to give the crude compound. The crude was washed with pentane to give the pure title compound (2.8 g, pure) as an off-white solid, which was characterized by H NMR: 1 H NMR (CDCl3) 400 MHz) δ ppm 5.02 (bs, 1H), 4.89-4.87 (m, 1H), 3.80-3.77 (m, 2H), 3.63-3.61 (m, 2H), 3.47-3.43 (m, 2H), 3.01-2.98 (m, 1H), 2.79 (d, J=4.8Hz, 3H), 1.57-1.52 (m, 2H), 1.28 (s, 40H), 0.88 (t, J=6.4 Hz, 3H).

[0630] Synthesis of (R)-1-(docosyloxy)-3-((2-oxido-1,3,2-dioxaphospholan-2-yl)oxy)propan-2-ylmethylcarbamate (7-9) To a stirred solution of (S)-1-(docosyloxy)-3-hydroxypropan-2-ylmethylcarbamate (0.3 g, 0.655 mmol) and EtN (0.27 mL, 1.967 mmol) in THF (5 mL) was added 2-chloro-1,3,2-dioxaphospholane-2-oxide (0.18 mL, 1.967 mmol) at 0 °C, and the reaction mixture was stirred for 1 h. Completion of the reaction was monitored by TLC. The reaction mixture was filtered, and the filtrate was concentrated in vacuo. The resulting crude compound (0.3 g) was characterized by H NMR and used in the next step. 1 H NMR (CDCl3) 400 MHz) δ ppm 4.98-4.96 (m, 1H), 4.46-4.43 (m, 2H), 4.28-4.25 (m, 4H), 3.74-3.71 (m, 2H), 3.55-3.44(m, 2H), 2.69(s, 3H), 1.55-1.52 (m, 2H), 1.30 (bs, 40H), 0.90-0.87 (m, 3H).

[0631] Synthesis of (R)-3-(docosyloxy)-2-((methylcarbamoyl)oxy)propyl(2-(trimethylammonio)ethyl)phosphate: Compound 4 To a stirred solution of (R)-1-(docosyloxy)-3-((2-oxido-1,3,2-dioxaphospholan-2-yl)oxy)propan-2-ylmethylcarbamate (0.3 g, 0.532 mmol) in ACN (15 mL) was added 2 M trimethylamine solution (3 mL) at 0° C., and the reaction mixture was stirred at 65° C. for 16 h. The completion of the reaction was monitored by TLC. The reaction mixture was cooled to 0° C., and then the precipitated solid was filtered and dried under vacuum to give the crude material. The crude compound was washed with water (15 mL) and acetonitrile (30 mL) to give the title compound (0.23 g, pure) as an off-white solid. 1 Characterized by H NMR: 1H NMR (CDCl3) 400 MHz) δ ppm 4.97-4.95 (m, 1H), 4.27-4.25 (m, 2H),4.01-3.96 (m, 2H), 3.63-3.58 (m, 4H), 3.48-3.40(m, 2H), 3.22 (s, 9H), 2.69(s, 3H), 1.55-1.53 ​​(m, 2H), 1.28 (bs, 40H), 0.90 (t, J = 5.6Hz, 3H).31P NMR (CD3OD, 162 MHz): 0.17 ppm.HRMS = 622.6053.

[0632] Example S-8: Synthesis of Compound 14, Compound 15, and Compound 16 [ka] Synthesis of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (8-3): To a stirred solution of 1-bromodocosane (16.96 g, 128.3664 mmol) in toluene at 0 °C, potassium tert-butoxide (28.8 g, 256.7328 mmol) and (R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (16.96 g, 128.3664 mmol) were added. The reaction mixture became a thick mass, so stirring was stopped and the reaction mixture was allowed to warm to room temperature and stirred at room temperature for 1 hour. The reaction mixture was then heated to 110 °C for 16 hours. Completion of the reaction was monitored by TLC. Upon completion of the reaction, ether was added to the reaction mixture and stirred for 10 minutes. Brine solution was added to the reaction mixture and extracted with ether. The organic layer was dried over anhydrous NaSO and concentrated under reduced pressure to give the crude product (60 g) as a brown solid. H NMR confirmed: 1H NMR (CDCl) 400 MHz δ ppm 5.01-4.91 (m, 1H), 4.29-4.22 (m, 1H), 4.06-4.04 (m, 1H), 3.74-3.72 (m, 1H), 3.53-3.39 (m, 3H), 1.59-1.53 ​​(m, 2H), 1.45-1.18 (m, 44H), 0.86 (t, J = 13.6 Hz, 3H). Crude H NMR showed the desired product along with impurities; H NMR values ​​were assigned based on the product peaks in the next step.

[0633] Synthesis of compound 9: To a stirred solution of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (60 g, 136.1315 mmol) in MeOH (500 mL) was added concentrated HCl (125 mL) and heated to 70° C. for 16 h. Completion of the reaction was monitored by TLC. The reaction mixture was diluted with water, filtered, and the filtered solid was stirred again with water and filtered to give a solid. The solid was stirred with hexane and filtered to give the product. Since the product contained water, acetonitrile was added to the product and evaporated three times to remove water to give compound 9 (30 g, 55% over two steps) as an off-white solid. 1 The result was confirmed by H NMR (CDCl3) 400 MHz. 1 H NMR (CDCl3) 400 MHz δ ppm 3.86 (bs, 1H), 3.71-3.66 (m, 2H), 3.53-3.44 (m, 4H), 2.59 (bs, 1H), 2.15 (bs, 1H), 1.58-1.54 (m, 2H), 1.38-1.18 (bs, 38H), 0.88 (t, J=6.4 Hz, 3H).

[0634] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (8-4): To a stirred solution of (S)-3-(docosyloxy)propane-1,2-diol (20.0 g, 49.913 mmol) in pyridine (100.0 mL) at 0 °C, trityl chloride (13.91 g, 49.913 mmol) was added at 0 °C and heated to 120 °C in a sealed tube for 16 h. (Note that in this step, the starting material, pyridine, and trityl chloride must be anhydrous. If the reaction mixture contains water, the reaction will not proceed.) The completion of the reaction was monitored by TLC. Upon completion, the reaction mixture was evaporated under reduced pressure. The crude product was purified by Combiflash chromatography using 5% EtOAc in hexane as the eluent. After evaporation of the fractions, the material was washed with n-pentane (500 mL), stirred for 1 h, filtered, and dried to give the desired compound as a white solid contaminated with trityl chloride. The desired product 8-4 (19.4 g, 60%) was obtained as a white solid and was confirmed by 1H NMR. 1 H NMR (400 MHz, CDCl3): δ =7.25-7.43 (m, 15 H), 3.94 (m, 1 H), 3.42-3.52 (m, 4H), 3.18 (m, 2H), 1.22-1.48 (m, 38 H), 0.87 (m, 3H).

[0635] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylpyridin-2-yl carbonate (8-6): To a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (5 g, 7.7759 mmol) in THF at room temperature, EtN (4.3 mL, 31.1036 mmol) was added, followed by di(pyridin-2-yl)carbonate (3.36 g, 15.5518 mmol) and stirred in a sealed tube at 80 °C for 16 h. The reaction was monitored for completion by TLC. Due to the instability of the resulting reaction mixture, this crude mixture was used in the next step without workup to give (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylpyridin-2-yl carbonate 8-6 (5 g, crude) as a light brown reaction mixture. 1H NMR (CDCl) 400 MHz) δ ppm 7.80-7.76 (m, 1H), 7.47-7.27 (m, 16H), 6.57 (d, J = 9.2 Hz, 1H), 6.30-6.27 (m, 1H), 5.14-5.12 (m, 1H), 3.75-3.33 (m, 6H), 1.52-1.49 (m, 2H), 1.4-1.2 (d, 38H), 0.87 (t, J = 6.4 Hz, 3H). H NMR of the crude compound showed the desired product along with impurities, and H NMR values ​​were assigned based on the product peaks in the next step.

[0636] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-yldimethylcarbamate (8-7): To the crude product of 8-6(R)-1-(docosyloxy)-3-(trityloxy)propan-2-ylpyridin-2-yl carbonate in 50 ml of THF was added 7% dimethylamine in THF (40 mL) and heated to 80° C. for 16 hours. Completion of the reaction was monitored by TLC. The reaction mixture was evaporated under reduced pressure. The crude product was dissolved in 200 mL of EtOAc and washed with 2×50 mL of water. The organic layer was dried over anhydrous NaSO and concentrated under reduced pressure to give the product. The crude compound was purified by Combiflash chromatography. The product was eluted with 5% EtOAc / 1% triethylamine / hexane. The fractions containing the product were concentrated and thoroughly dried to give the title product 7 (6 g, pure) as an off-white solid, characterized by H NMR. 1 H NMR (CDCl3) 400 MHz) δ ppm 7.45-7.43 (m, 5H), 7.30-7.20 (m, 10H), 5.08-5.05 (m, 1H), 3.66-3.63 (m, 2H), 3.41-3.38 (m, 2H), 3.27-3.23 (m, 2H), 2.95-2.94 (bs, 6H), 1.49-1.46 (m, 2H), 1.31-1.23 (brs, 38H), 0.87 (t, J=6.4 Hz, 3H).

[0637] Synthesis of (S)-1-(docosyloxy)-3-hydroxypropan-2-yldimethylcarbamate (compound 14): To a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-yldimethylcarbamate (3.0 g, 4.2011 mmol) in 30 mL of MeOH and DCM (1:1) was added camphorsulfonic acid (1.46 g, 6.3017 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 4 h. Completion of the reaction was monitored by TLC. The reaction mixture was concentrated to dryness. The residue was dissolved in ethyl acetate (250 mL), washed with water (50 mL × 2), dried over sodium sulfate, and concentrated to dryness. The crude compound from the previous batch was mixed with this batch and purified by Combiflash chromatography. The product was eluted with 40% EtOAc in hexane. The product-containing fractions were concentrated and thoroughly dried to give the title compound 14 (3.2 g, pure) as an off-white foamy solid, characterized by H NMR. 1 H NMR (CDCl3) 400 MHz) δ ppm 4.87-4.85 (m, 1H), 3.83-3.87 (m, 2H),3.67-3.59 (m, 2H), 3.47-3.43 (m, 2H), 2.901 (bs, 6H), 2.88-2.87 (m, 1H), 1.57-1.52 (m, 2H), 1.28-1.21 (bs, 38H), 0.88 (t, J=6.4 Hz, 3H).HRMS (M+1) = 472.1716.

[0638] Synthesis of (R)-1-((diethoxyphosphoryl)oxy)-3-(docosyloxy)propan-2-yldimethylcarbamate (8-8) To a stirred solution of (S)-1-(docosyloxy)-3-hydroxypropan-2-yldimethylcarbamate compound 14 (1 g, 2.1196 mmol) in THF (10 mL) at 0 °C, DIPEA (1.84 mL, 10.5983 mmol) and DMAP (0.51 g, 4.2392 mmol) were added, followed by dropwise addition of diethyl chlorophosphate (1.52 mL, 10.5983 mmol) and stirring at room temperature for 24 h. TLC showed the formation of starting material along with the product. Further addition of DIPEA (0.92 mL, 5.299 mmol), DMAP (0.26 g, 2.1196 mmol), and diethyl chlorophosphate (0.76 mL, 5.299 mmol) was followed by stirring at room temperature for an additional 24 h. Completion of the reaction was monitored by TLC. The reaction mixture was diluted with ethyl acetate (100 mL), washed with water (2×50 mL), and the organic layer was separated, dried over sodium sulfate, and concentrated under reduced pressure to give the crude product. The crude product was purified by Combiflash chromatography. The product was eluted with 30% EtOAc in hexanes. The fractions containing the product were concentrated and dried thoroughly to give the title compound (1 g, pure) as a pale pink waxy solid, characterized by H NMR and P NMR: 1 H NMR (CDCl3) 400 MHz) δ ppm 5.03-4.89 (m, 1H), 4.23-4.09 (m, 6H), 3.59-3.57 (m, 2H), 2.87 (s, 6H), 1.58-1.50 (m, 2H), 1.33-1.15 (m, 44H), 0.88 (t, J = 6.4 Hz, 3H); 31P NMR showed a single peak at -0.437 in CDCl3.

[0639] Synthesis of (R)-1-(docosyloxy)-3-(phosphonooxy)propan-2-yldimethylcarbamate: Compound 15: To a stirred solution of 8-8(R)-1-((diethoxyphosphoryl)oxy)-3-(docosyloxy)propan-2-yldimethylcarbamate (1 g, 1.6451 mmol) in DCM (10 mL) at 0 °C, TMSBr (1.3 mL, 9.8708 mmol) was added, followed by N,O-bis(trimethylsilyl)acetamide (2.4 mL, 9.8708 mmol), and the mixture was stirred at room temperature for 4 h. The reaction progress was monitored by TLC. The reaction mixture was cooled to 0 °C, and 1 mL of methanol was added and stirred for 10 min. 1 mL of water was added to the reaction mixture, which was stirred at 0 °C for an additional 10 min. Saturated NaHCO3 solution (50 mL) was added to the reaction mixture, which was then washed three times with EtOAc (3 × 50 mL). The aqueous layer was slowly acidified with 6 N HCl solution at 0 °C and extracted with ether. The organic layer was dried over anhydrous NaSO and concentrated under reduced pressure to give a crude white gummy solid, which was stirred with acetonitrile and methanol and filtered to give the title compound (0.35 g, pure) as an off-white solid, characterized by H NMR. 1 H NMR (CD3OD) 400 MHz: δ ppm 4.97-4.90 (m, 1H), 4.14-4.08 (m, 2H), 3.60-3.59 (m, 2H), 3.49-3.43 (m, 2H), 3.45-3.43 (m, 2H), 2.92 (d, J = 16 Hz, 6H), 1.40-1.20 (m, 38H), 0.89 (t, J = 6.4 Hz, 3H). 31P NMR revealed a single peak at 1.312 for CD3OD. HRMS: (M+1)=551.044.

[0640] Synthesis of (R)-2-((dimethylcarbamoyl)oxy)-3-ethoxypropyl(2-(trimethylammonio)ethyl)phosphate--icosane (1 / 1) compound 16: [ka] Synthesis of (R)-1-(docosyloxy)-3-((2-oxido-1,3,2-dioxaphospholan-2-yl)oxy)propan-2-yldimethylcarbamate (8-9): To a stirred solution of (S)-1-(docosyloxy)-3-hydroxypropan-2-yldimethylcarbamate compound 14 (0.5 g, 1.0598 mmol) in THF at 0 °C, triethylamine (0.44 mL, 3.1795 mmol) was added, followed by 2-chloro-1,3,2-dioxaphospholane 2-oxide (0.3 mL, 3.1795 mmol), and the mixture was stirred at 0 °C for 4 h. The reaction mixture was monitored for completion by TLC. The reaction mixture was filtered to remove salts, and the filtrate was concentrated under reduced pressure to give 0.62 g of crude product. Due to the unstable nature of the product, the crude product was used in the next step without a...

Claims

1. Compound of formula (IV-F): 【Chemistry 1】 (In the formula, R ２ is H, -(C=O)-NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C ２１－ C ２４ is n-alkyl, Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a salt thereof.

2. R ２ The compound of claim 1 , wherein is H.

3. R ２ is -(C=O)-NH ２ 2. The compound of claim 1, wherein:

4. R ２ is -(C=O)-NH(R ５ 2. The compound of claim 1, wherein

5. R ２ is -(C=O)-N(R ５ ) ２ 2. The compound of claim 1, wherein:

6. R ３ But C ２１ The compound according to any one of claims 1 to 5, which is n-alkyl.

7. R ３ The compound of any one of claims 1 to 6, wherein is unsubstituted.

8. R ５ But -CH ３ The compound according to any one of claims 1 to 2 or 4 to 7,

9. A compound of the formula: 【Chemistry 2】 or a protonated or deprotonated form thereof, or a salt thereof.

10. A compound of the formula: 【Transformation 3】 or a protonated or deprotonated form thereof, or a salt thereof.

11. A compound of the formula: 【Chemistry 4】 or a protonated or deprotonated form thereof, or a salt thereof.

12. A compound of the formula: 【Transformation 5】 or a protonated or deprotonated form thereof, or a salt thereof.

13. The compound of any one of claims 1 to 12, wherein the compound is isolated.

14. A composition comprising a compound according to any one of claims 1 to 13 and 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 1 to 15, further comprising an antigen.

17. The composition of any one of claims 1 to 16, further comprising dendritic cells.

18. The composition of any one of claims 14 to 17, wherein the TLR agonist is a small molecule with a molecular weight of 900 daltons or less.

19. The composition of any one of claims 15 to 18, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

20. The composition of any one of claims 15 to 19, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

21. Isolated ether lipids (ETL) of formula (I): 【Transformation 6】 (In the formula, R １ is H or 【Transformation 7】 and R ２ is H, C １ -C ４ Alkyl, —(C═O)—NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C １３－ C ２４ is n-alkyl, Here, R ４ is H or (CH ３ ) ３ N ＋ - (CH ２ ) ２ - and Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof; and a TLR agonist.

22. 22. The composition of claim 21, wherein the TLR agonist comprises a TLR7 / 8 agonist.

23. R ３ But C １８ -C ２２ n-Alkyl or C ２１ -C ２４ The composition of claim 21 or claim 22, wherein the alkyl group is n-alkyl.

24. R ３ But C １６ -C ２０ The composition of any one of claims 21 to 23, wherein the alkyl group is n-alkyl.

25. The composition of any one of claims 21 to 24, further comprising an antigen.

26. The composition of any one of claims 21 to 25, further comprising dendritic cells.

27. Isolated ether lipids (ETL) of formula (I): 【Transformation 8】 (In the formula, R １ is H or 【Chemistry 9】 and R ２ is H, C １ -C ４ Alkyl, —(C═O)—NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C １３－ C ２４ is n-alkyl, Here, R ４ is H or (CH ３ ) ３ N ＋ - (CH ２ ) ２ - and Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof; and an antigen.

28. 28. The composition of claim 27, further comprising dendritic cells.

29. 29. The composition of claim 27 or claim 28, further comprising a TLR agonist.

30. 30. The composition of claim 29, wherein the TLR agonist comprises a TLR7 / 8 agonist.

31. Isolated ether lipids (ETL) of formula (I): 【Chemistry 10】 (In the formula, R １ is H or 【Chemistry 11】 and R ２ is H, C １ -C ４ Alkyl, —(C═O)—NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C １３－ C ２４ is n-alkyl, Here, R ４ is H or (CH ３ ) ３ N ＋ - (CH ２ ) ２ - and Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof; and a dendritic cell.

32. 32. The composition of claim 31, further comprising a TLR agonist.

33. 33. The composition of claim 32, wherein the TLR agonist comprises a TLR7 / 8 agonist.

34. The composition of any one of claims 31 to 33, further comprising an antigen.

35. R ３ But C ２２ The composition of any one of claims 21 to 34, wherein the alkyl group is n-alkyl.

36. 36. The composition of any one of claims 21 to 35, wherein the ETL is an ether phospholipid (ETPL) comprising 1-docosyl-sn-glycerol-3-phosphocholine (DGPC), or a pharmaceutically acceptable salt thereof.

37. The composition of any one of claims 21 to 35, wherein the ETL is an ETPL comprising 1-docosyl-sn-glycerol-3-phosphate (DGP) or a pharmaceutically acceptable salt thereof.

38. The composition of any one of claims 21 to 37, wherein the TLR agonist is a small molecule with a molecular weight of 900 daltons or less.

39. The composition of any one of claims 21 to 38, wherein the TLR agonist comprises a TLR7 / 8 agonist.

40. 40. The composition of claim 39, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

41. 40. The composition of claim 39, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

42. The composition of any one of claims 14 to 41, wherein the TLR7 / 8 agonist does not inhibit NLR family pyrin domain-containing 3 (NLRP3).

43. The composition of any one of claims 21 to 34, wherein the ETPL comprises one or both of DGPC and DGP, and the TLR7 / 8 agonist comprises resiquimod (R848).

44. The composition of any one of claims 14 to 43, wherein the antigen is present in a biological sample obtained from an individual.

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

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

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

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

49. The composition of any one of claims 16 to 48, wherein the antigen comprises a proteinaceous antigen.

50. 50. The composition of claim 49, wherein the antigen comprises a tumor antigen.

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

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

53. 50. The composition of claim 49, wherein the antigen comprises a microbial antigen, the microbial antigen comprising one or more of a viral antigen, a bacterial antigen, a protozoan antigen, and a fungal antigen.

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

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

56. The composition of any one of claims 14 to 55, wherein the composition does not contain liposomes.

57. The composition of any one of claims 14 to 56, wherein the composition does not contain LPS or MPLA.

58. 58. The composition of any one of claims 14-57, wherein the composition does not include oxPAPC or species of oxPAPC, and optionally the composition does not include HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC, and / or PGPC.

59. 59. The composition of any one of claims 14 to 58, wherein the composition does not comprise lysophosphatidylcholine (LPC), and optionally the composition does not comprise 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

60. 60. The composition of any one of claims 14 to 59, further comprising an adjuvant, wherein the adjuvant comprises an aluminum salt adjuvant, a squalene-in-water emulsion, a saponin, or a combination thereof.

61. The composition of any one of claims 14 to 60, wherein the n-alkyl group is unsubstituted.

62. A pharmaceutical formulation comprising the composition of any one of claims 14 to 61 and a pharmaceutically acceptable excipient.

63. 1. A method for the production of superactivated dendritic cells, comprising administering to said dendritic cells an effective amount of an isolated ether lipid (ETL) of the following formula: i) Formula (I): 【Chemistry 12】 (In the formula, R １ is H or 【Chemistry 13】 and R ２ is H, C １ -C ４ Alkyl, —(C═O)—NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C １３－ C ２４ is n-alkyl, Here, R ４ is H or (CH ３ ) ３ N ＋ - (CH ２ ) ２ - and Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof; or II) Formula (IV-F): 【Chemistry 14】 (In the formula, R ２ is H, -(C=O)-NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C ２１－ C ２４ is n-alkyl, Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a salt thereof, and The method comprises contacting a subject with a composition comprising a TLR7 / 8 agonist to produce superactivated dendritic cells, wherein the superactivated dendritic cells secrete IL-1 beta without undergoing pyroptosis.

64. The method of claim 63, wherein the dendritic cells are contacted ex vivo with the composition of any one of claims 14 to 61 or the formulation of claim 62.

65. 64. The method of claim 63, wherein the dendritic cells are contacted with the formulation of claim 62 in vivo.

66. 65. At least 10 produced by the method of claim 64 ３ , 10 ４ , 10 ５ or 10 ６ A pharmaceutical preparation comprising the superactivated dendritic cells and a pharmaceutically acceptable excipient.

67. 63. A method of stimulating an immune response to an antigen, comprising administering to an individual in need thereof an effective amount of the formulation of claim 62 to stimulate the immune response to the antigen.

68. 63. A method of treating cancer, comprising administering to an individual in need thereof an effective amount of the formulation of claim 62 to treat said cancer.

69. 63. A method of inhibiting abnormal cell proliferation, comprising administering to an individual in need thereof an effective amount of the formulation of claim 62 to inhibit abnormal cell proliferation.

70. 63. A method of treating an infectious disease, comprising administering an effective amount of the formulation of claim 62 to an individual in need thereof to treat the infectious disease.

71. 63. Use of the formulation of claim 62 for inducing an immune response against said antigen in an individual in need thereof.

72. 63. Use of the formulation of claim 62 for inducing an anti-tumor immune response in an individual in need thereof, wherein the individual has or has had a tumor.

73. 63. Use of the formulation of claim 62 for inducing an antimicrobial immune response in an individual in need thereof, wherein the individual is infected with or has not been exposed to the microorganism.

74. 74. The composition, formulation, method or use of any one of claims 44 to 73, wherein the individual is a mammalian subject.

75. 74. The composition, formulation, method or use of any one of claims 44 to 73, wherein the individual is a human subject.

76. 1. A method for preparing an immunogenic composition, 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) treating the tumor cell lysate with an isolated ether lipid (ETL) of the formula: i) Formula (I): 【Chemistry 15】 (In the formula, R １ is H or 【Chemistry 16】 and R ２ is H, C １ -C ４ Alkyl, —(C═O)—NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C １３－ C ２４ is n-alkyl, Here, R ４ is H or (CH ３ ) ３ N ＋ - (CH ２ ) ２ - and Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof; ii) Formula (IV-F): 【Chemistry 17】 (In the formula, R ２ is H, -(C=O)-NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C ２１－ C ２４ is n-alkyl, Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a salt thereof, and and contacting the antibody with a toll-like receptor (TLR) agonist to obtain the immunogenic composition.

77. 77. The method of claim 76, wherein the TLR agonist comprises a TLR7 / 8 agonist.

78. 78. The method of claim 76 or claim 77, wherein the leukocytes are depleted in step a) by negative selection using an anti-CD45 antibody.

79. 79. The method of any one of claims 76 to 78, wherein the cells are lysed in step b) by one or more freeze-thaw cycles.

80. R in formula (I) ３ But C １８ -C ２２ Alkyl or C １８ -C ２４ 80. The method of any one of claims 76 to 79, wherein the alkyl is alkyl.

81. R in formula (I) ３ But C １６ -C ２０ 80. The method of any one of claims 76 to 79, wherein the alkyl is alkyl.

82. R ３ But C ２１ -C ２４ 80. The method of any one of claims 76 to 79, wherein the alkyl is alkyl.

83. 80. The method of any one of claims 76 to 79, wherein the ETL comprises one or both of DGPC and DGP, or a pharmaceutically acceptable salt thereof.

84. 84. The method of any one of claims 76 to 83, wherein the TLR7 / 8 agonist is a small molecule with a molecular weight of 900 daltons or less.

85. 85. The method of claim 84, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

86. 86. The method of claim 85, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

87. 87. The method of any one of claims 84 to 86, wherein the TLR7 / 8 agonist does not inhibit NLR family pyrin domain-containing 3 (NLRP3).

88. 84. The method of claim 83, wherein the ETL comprises one or both of DGPC and DGP, or a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist comprises resiquimod (R848).

89. 89. The method of any one of claims 76 to 88, further comprising, prior to step a), obtaining a sample derived from said tumor from a mammalian subject with cancer and preparing a suspension of said cells from said sample.

90. An immunogenic composition prepared by the method of any one of claims 76 to 89.

91. 91. A method for inducing an anti-cancer immune response, comprising administering to a mammalian subject having cancer an effective amount of the immunogenic composition of claim 90.

92. 93. The method of claim 92, wherein the anti-cancer immune response comprises a cellular immune response.

93. 92. The method of claim 91, wherein the anti-cancer immune response comprises cancer antigen-induced IL-1 beta secretion and / or activation of CD8+ T lymphocytes.

94. 94. The method of any one of claims 91 to 93, wherein the cancer is a non-hematological cancer.

95. 95. The method of claim 94, wherein the non-hematological cancer is carcinoma, sarcoma, or melanoma.

96. 96. The method of any one of claims 91 to 95, wherein the cancer is lymphoma.

97. 1. A method of treating cancer, comprising: a) administering to a tumor cell lysate an isolated ether lipid (ETL) of the formula: i) Formula (I): [Chemistry 18] (In the formula, R １ is H or 【Chemistry 19】 and R ２ is H, C １ -C ４ Alkyl, —(C═O)—NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C １３－ C ２４ is n-alkyl, Here, R ４ is H or (CH ３ ) ３ N ＋ - (CH ２ ) ２ - and Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof; or ii) Formula (IV-F): 【Chemistry 20】 (In the formula, R ２ is H, -(C=O)-NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C ２１－ C ２４ is n-alkyl, Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a salt thereof, and The method comprises: preparing an immunogenic composition comprising a toll-like receptor (TLR) agonist, wherein the tumor cell lysate is or has been prepared from a tumor sample obtained from the mammalian subject having cancer; and b) administering to the subject an effective amount of the immunogenic composition.

98. 98. The method of claim 97, wherein the TLR agonist comprises a TLR7 / 8 agonist.

99. R in formula (I) ３ But C １８ -C ２２ Alkyl chain or C １８ -C ２４ 99. The method of claim 97 or claim 98, wherein the alkyl chain is an alkyl chain.

100. R in formula (I) ３ But C １６ -C ２０ 99. The method of claim 97 or claim 98, wherein the alkyl is alkyl.

101. R ３ But C ２１ -C ２４ 99. The method of claim 97 or claim 98, wherein the alkyl is alkyl.

102. 99. The method of claim 97 or claim 98, wherein the ETL comprises one or both of DGPC and DGP, or a pharmaceutically acceptable salt thereof.

103. The method of any one of claims 97 to 102, wherein the TLR7 / 8 agonist is a small molecule with a molecular weight of 900 daltons or less.

104. 104. The method of claim 103, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

105. 105. The method of claim 104, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

106. The method of any one of claims 97 to 105, wherein the ETL comprises DGPC or a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist comprises resiquimod (R848).

107. The method of any one of claims 97 to 105, wherein the ETL comprises DGP or a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist comprises resiquimod (R848).

108. 108. The method of any one of claims 97-107, further comprising administering to the subject an effective amount of an additional therapeutic agent.

109. 109. The method of claim 108, wherein the additional therapeutic agent comprises one or more of the group consisting of an immune checkpoint inhibitor, an anti-tumor agent, and radiation therapy.

110. Isolated ether lipids (ETLs) of the formula: i) Formula (I): 【Chemistry 21】 (In the formula, R １ is H or 【Chemistry 22】 and R ２ is H, C １ -C ４ Alkyl, —(C═O)—NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C １３－ C ２４ is n-alkyl, Here, R ４ is H or (CH ３ ) ３ N ＋ - (CH ２ ) ２ - and Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof; or ii) Formula (IV-F): 【Chemistry 23】 (In the formula, R ２ is H, -(C=O)-NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C ２１－ C ２４ is n-alkyl, Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a salt thereof; and a pathogen recognition receptor (PRR) agonist.

111. 111. The composition of claim 110, 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).

112. The composition of claim 110, wherein the PRR agonist is an agonist of a cytoplasmic DNA sensor (CDS) or a stimulator of IFN genes (STING).

113. 111. The composition of claim 110, wherein the PRR agonist comprises one or more of R848, TL8-506, LPS, Pam2CSK4, and ODN2336.

114. The composition of any one of claims 110 to 113, further comprising an antigen.

115. The composition of any one of claims 110 to 114, further comprising dendritic cells.

116. A pharmaceutical formulation comprising the composition of any one of claims 110 to 115 and a pharmaceutically acceptable excipient.

117. Isolated ether lipids (ETLs) of the formula: i) Formula (I): 【Chemistry 24】 (In the formula, R １ is H or 【Chemistry 25】 and R ２ is H, C １ -C ４ Alkyl, —(C═O)—NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C １３－ C ２４ is n-alkyl, Here, R ４ is H or (CH ３ ) ３ N ＋ - (CH ２ ) ２ - and Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof; or ii) Formula (IV-F): 【Chemistry 26】 (In the formula, R ２ is H, -(C=O)-NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C ２１－ C ２４ is n-alkyl, Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a salt thereof; and a pharmaceutically acceptable excipient.

118. R ３ But C ２２ 118. The pharmaceutical preparation of claim 117, wherein the alkyl is n-alkyl.

119. 119. The pharmaceutical formulation of claim 118, wherein the ETL comprises one or both of DGPC and DGP, or pharmaceutically acceptable salts thereof.

120. A composition for the hyperactivation of human dendritic cells, comprising an isolated ether lipid (ETL) of the formula: i) Formula (I): 【Chemistry 27】 (In the formula, R １ is H or 【Chemistry 28】 and R ２ is H, C １ -C ４ Alkyl, —(C═O)—NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C １３－ C ２４ is n-alkyl, Here, R ４ is H or (CH ３ ) ３ N ＋ - (CH ２ ) ２ - and Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a pharmaceutically acceptable salt thereof; or ii) Formula (IV-F): 【Chemistry 29】 (In the formula, R ２ is H, -(C=O)-NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C ２１－ C ２４ is n-alkyl, Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a salt thereof; and a pathogen recognition receptor (PRR) agonist, wherein the alkyl chain is a C22 n-alkyl chain, and the composition is effective in achieving a higher level of dendritic cell hyperactivation than a comparative composition comprising PGPC instead of the ETL.

121. R ３ But C ２２ The composition of claim 120, wherein the alkyl is n-alkyl.

122. The composition of claim 120 or claim 121, wherein the high level of dendritic cell hyperactivation comprises inducing IL-1 beta secretion from the human dendritic cells in vitro at a level that is at least 2-fold, 3-fold, or 4-fold higher when contacted with the composition comprising the ETL and the PRR agonist than when contacted with the comparative composition comprising the PGPC and the PRR agonist, and the PRR agonist is LPS.

123. The composition of claim 122, wherein the concentration of the ETL and the concentration of the PGPC are the same and range 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 comparative composition.

124. The composition of claim 122 or claim 123, wherein the high level of dendritic cell hyperactivation comprises a lipid activity index for IL-1 beta secretion from the human dendritic cells for the composition comprising the ETL and the PRR agonist that is at least 4, 5, or 6 times higher activity units than the comparative composition comprising the PGPC and the PRR agonist.

125. 74. The composition, formulation, method or use of any one of claims 44 to 73, wherein the individual is a human subject.

126. 74. The composition, formulation, method or use of any one of claims 44 to 73, wherein the individual is a canine subject.

127. 125. The composition, formulation, method or use of any one of claims 89 to 124, wherein the mammalian subject is a human patient.

128. 125. The composition, formulation, method or use of any one of claims 89 to 124, wherein the mammalian subject is a non-human patient.

129. 125. The composition, formulation, method or use of any one of claims 89 to 124, wherein the mammalian subject is a canine patient.

130. 128. The composition, formulation, method or use of any one of claims 14 to 125 or 127, wherein the dendritic cells are human dendritic cells.

131. 130. The composition, formulation, method or use of any one of claims 14 to 74, 76 to 119 or 129, wherein the dendritic cells are canine dendritic cells.

132. 132. The composition, method or use of claim 130 or claim 131, wherein the dendritic cells are present in a composition comprising peripheral blood mononuclear cells (PBMCs).

133. The composition, method or use of any one of claims 42 to 54 or 109 to 110, wherein the superactivated dendritic cells secrete one or both of IFNγ and TNFα.

134. 134. The composition, formulation, method or use of any one of claims 14 to 133, further comprising a surfactant.

135. 135. The composition, formulation, method or use of claim 134, wherein the surfactant comprises a non-ionic surfactant.

136. 136. The composition, formulation, method or use of claim 135, wherein the non-ionic surfactant comprises an ethylene oxide-propylene oxide copolymer (poloxamer).

137. 136. The composition, formulation, method or use of claim 135, wherein the non-ionic surfactant comprises one or more of poloxamer 407, poloxamer 188, and P123.

138. 136. The composition, formulation, method or use of claim 135, wherein the non-ionic surfactant comprises poloxamer 407.

139. 139. The composition, formulation, method or use of any one of claims 135 to 138, comprising: i) dissolving the ETL in alcohol to form an alcoholic ETL solution; ii) mixing the alcoholic ETL solution with the non-ionic surfactant to form a mixture; and iii) evaporating the alcohol from the mixture to form particles comprising the ETL and the non-ionic surfactant.

140. 140. The composition, formulation, method or use of any one of claims 135 to 139, 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).

141. 141. The composition, formulation, method or use of any one of claims 135-140, wherein the ETL and the non-ionic surfactant are present in particles having a diameter of about 1000 to 15,000 nanometers, optionally about 5000 nanometers.

142. Isolated ether lipids (ETL) of formula (I): 【Transformation 30】 (In the formula, R １ is H or 【Chemistry 31】 and R ２ is H, C １ -C ４ Alkyl, —(C═O)—NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C １３－ C ２４ is n-alkyl, Here, R ４ is H or (CH ３ ) ３ N ＋ - (CH ２ ) ２ - and Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a salt thereof.

143. The isolated ether lipid is a compound of formula (II): 【Chemistry 32】 (In the formula, R １ is H or 【Transformation 33】 and R ２ is H, C １ -C ４ Alkyl, —(C═O)—NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C １３－ C ２４ is n-alkyl, Here, R ４ is H or (CH ３ ) ３ N ＋ - (CH ２ ) ２ - and Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a salt thereof.

144. The isolated ether lipid is a compound of formula (III): 【Transformation 34】 (In the formula, R ２ is H, C １ -C ４ Alkyl, —(C═O)—NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C １３－ C ２４ is n-alkyl, Each R ５ are independently １ -C ４ alkyl), 143. The isolated ether lipid of claim 142, wherein the ether lipid is:

145. The isolated ether lipid is an isolated ether phospholipid (ETPL) compound of formula (IV): 【Chemistry 35】 (In the formula, R ２ is H, C １ -C ４ Alkyl, —(C═O)—NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C １３－ C ２４ is n-alkyl, R ４ is H or (CH ３ ) ３ N ＋ - (CH ２ ) ２ - and Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a salt thereof.

146. The isolated ether lipid is an isolated ether phospholipid (ETPL) compound of formula (IV-A): 【Transformation 36】 (In the formula, R ２ is H, C １ -C ４ Alkyl, —(C═O)—NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C １３－ C ２４ is n-alkyl, Each R ５ are independently １ -C ４ alkyl), or a protonated or deprotonated form thereof, or a salt thereof.

147. The isolated ether lipid is an isolated ether phospholipid (ETPL) compound of formula (IV-B): 【Chemistry 37】 (In the formula, R ２ is H, C １ -C ４ Alkyl, —(C═O)—NH ２ , -(C=O)-NH(R ５ ), -(C=O)-N(R ５ ) ２ , or -CH ２ -C ６ H ５ and R ３ is C １３－ C ２４ is n-alkyl, Each R ５ are independently １ -C ４ alkyl), 143. The isolated ether lipid of claim 142, wherein the ether lipid is a protonated form thereof, or a salt thereof.

148. The isolated ether lipid is an isolated ether phospholipid (ETPL) compound of formula (IV-C): 【Chemistry 38】 (In the formula, R ３ is C １３－ C ２４ is n-alkyl, R ４ is H or (CH ３ ) ３ N ＋ - (CH ２ ) ２ -), or a protonated or deprotonated form thereof, or a salt thereof.

149. Compound of Formula 2: 【Chemistry 39】 or a protonated form thereof, or a pharmaceutically acceptable salt thereof.

150. 150. The compound of claim 149, wherein the compound is isolated.

151. Isolated Compound 1 of Formula 1: 【Chemistry 40】 or a protonated form thereof, or a pharmaceutically acceptable salt thereof.

152. Compound of formula (III-A-1): 【Chemistry 41】 (In the formula, R ２ is -(C=O)-NH ２ , -(C=O)-NH(R ５ ), or —(C═O)—N(R ５ ) ２ and R ３ is C ２１－ C ２４ is n-alkyl, Each R ５ are independently １ -C ４ alkyl), or a pharmaceutically acceptable salt thereof.

153. R ２ is -(C=O)-NH ２ 153. The compound of claim 152, wherein:

154. R ２ is -(C=O)-NH-CH ３ 153. The compound of claim 152, wherein:

155. R ２ is -(C=O)-N(CH ３ ) ２ 153. The compound of claim 152, wherein:

156. R ３ is C ２２ 156. The compound of any one of claims 152 to 155, which is n-alkyl.

157. Compound 7 of Formula 7: 【Chemistry 42】 or a pharmaceutically acceptable salt thereof.

158. 158. The compound of claim 157, wherein the compound is isolated.

159. Compound 8 of Formula 8: 【Chemistry 43】 or a pharmaceutically acceptable salt thereof.

160. 160. The compound of claim 159, wherein the compound is isolated.

161. A compound of the formula: 【Chemistry 44-1】 【Chemistry 44-2】 【Chemistry 44-3】 or a protonated form thereof, or a salt thereof.

162. 162. The compound of claim 161, wherein the compound is isolated.

163. 163. A composition comprising a compound according to any one of claims 142 to 162 and a pharmaceutically acceptable excipient.

164. 164. The composition of claim 163, further comprising a surfactant.

165. 165. The composition of claim 164, wherein the surfactant is selected from the group consisting of nonionic surfactants, wetting agents, P407, P188, polysorbate 80, thickeners, and carboxymethylcellulose.

166. Diameter of about 5 microns to less than about 20 microns (D ５０ 166. The composition of any one of claims 163 to 165, comprising particles having a particle size of <5 microns to 20 microns, said particles comprising an ether lipid and a non-ionic surfactant.

167. 167. The composition of any one of claims 163 to 166, wherein the pharmaceutically acceptable excipient comprises phosphate buffered saline.

168. 168. The composition of any one of claims 163 to 167, wherein the pharmaceutically acceptable excipient comprises an aqueous solution of an ethylene oxide-propylene oxide copolymer (poloxamer), or further comprises an ethylene oxide-propylene oxide copolymer.

169. 166. The composition of any one of claims 163-165, wherein the pharmaceutically acceptable excipient comprises phosphate buffered saline and at least one of poloxamer 407, poloxamer 188, and P123.

170. 170. The composition of any one of claims 163 to 169, wherein the composition is sterile.

171. 171. An article of manufacture comprising a container enclosing a liquid formulation of a compound of any one of claims 142 to 170 and a pharmaceutically acceptable excipient.

172. 172. The product of claim 171, wherein the container is a syringe.

173. 173. The article of manufacture of claim 172, wherein the syringe is further housed within an injection device.

174. 174. The article of manufacture of claim 173, wherein the injection device is an auto-injector.

175. The present specification discloses an isolated ether lipid (ETL) or ether phospholipid (ETPL), Compound 1, Compound 2, and the like, which are represented by formula (I), formula (II), formula (III), formula (III-A), formula (III-A-1), formula (III-A-2), formula (III-B), formula (III-B-1), formula (III-B-2), formula (IV), formula (IV-A), formula (IV-A-1), formula (IV-A-2), formula (IV-B), formula (IV-B-1), formula (IV-B-2), formula (IV-C), formula (IV-D), formula (IV-E), formula (IV-F), and formula (A). , Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, or Compound 16, or a protonated or deprotonated form thereof, if possible, or a pharmaceutically acceptable salt thereof, and at least one additional lipid, wherein the at least one additional lipid is selected from the group consisting of an ionizable lipid, a cationic lipid, an additional phospholipid, a pegylated lipid, a structured lipid, and mixtures thereof.

176. The composition of claim 175, wherein the ETL or the ETPL and the at least one additional lipid are part of a lipid nanoparticle (LNP).

177. 177. The composition of claim 175 or claim 176, further comprising an antigen.

178. 178. The composition of any one of claims 175 to 177, further comprising dendritic cells.

179. 179. The composition of any one of claims 175 to 178, further comprising a TLR agonist.

180. 179. The composition of any one of claims 175 to 178, further comprising a TLR7 / 8 agonist.

181. 178. The composition, method, or use of any one of claims 16, 25, 27, 34, 67, 71, 114, or 177, wherein the antigen comprises one or more viral antigens.

182. 182. The composition, method, or use of claim 181, wherein the one or more viral antigens comprise one or both of an influenza A antigen and an influenza B antigen.

183. 183. The composition, method, or use of claim 182, wherein one or both of the influenza A antigen and the influenza B antigen comprises one or both of a hemagglutinin and a nucleoprotein.

184. 184. The composition, method or use of any one of claims 181 to 183, wherein the viral antigen comprises an inactivated virion, and optionally the inactivated virion comprises an inactivated split virion.

185. 185. The composition, method or use of any one of claims 182 to 184, comprising antigens of both influenza A and influenza B, influenza A virus H1N1, influenza A virus H3N2, influenza B virus of Victoria lineage, and influenza B virus of Yamagata lineage.

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