Over-activated ether lipids for mammalian dendritic cells

Abstract

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

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority benefits of U.S. Provisional Patent Application No. 63 / 417,667, filed on October 19, 2022; U.S. Provisional Patent Application No. 63 / 441,697, filed on January 27, 2023; and U.S. Provisional Patent Application No. 63 / 451,885, filed on March 13, 2023. The entire contents of those applications are hereby incorporated by reference herein. Technical field

[0003] The present disclosure relates to ether lipid compounds, including ether phospholipid compounds, and their use in over - activating mammalian dendritic cells, such as human dendritic cells 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 the following: pathogen - recognition receptor agonists, antigens, and human or canine dendritic cells, as well as the production methods and uses of such compositions. Background art

[0004] Generally, the maturation of dendritic cells (DCs) by vaccine adjuvants, such as Toll - like receptor agonists, does not result in IL - 1β secretion. In situations such as inflammasome activation, IL - 1β secretion does occur, but at the cost of the death of DCs due to a lytic cell death process called pyroptosis (Evavold et al., J Mol Biol, 430(2):217 - 237, 2018). However, when using molecules containing pathogen - associated molecular patterns (PAMPs), lipopolysaccharide (LPS), and molecules containing damage - associated molecular patterns (DAMPs), such as PGPC (1 - palmitoyl - 2 - glutaryl - sn - glycero - 3 - phosphocholine), to mature DCs, they produce and secrete IL - 1β without pyroptosis occurring, and these live DCs are characterized as over - activated (Zanoni et al., Science, 352(6290):1232 - 1236, 2016). In fact, in a mouse model, over - activated DCs have shown an improved ability to induce an immune response compared to cells activated with LPS alone (Zhivaki et al., Cell Rep, 33(7):108381, 2020). However, little is known about stimuli that are effective for the over - activation of human DCs.

[0005] Therefore, there is a need in the art to identify PAMPs and DAMPs suitable for the over - activation of human DCs. In addition, there is a need to identify alternatives to using LPS and PGPC for over - activating mammalian DCs. Specifically, while LPS (endotoxin) is an effective PAMP, it is prohibited for use in humans because it can cause septic shock. SUMMARY OF THE INVENTION

[0006] The present disclosure relates to ether lipid (ETL) compounds, such as ether phospholipid (ETPL) compounds, and their use in over - activating mammalian dendritic cells, such as human dendritic cells or canine dendritic cells. The present disclosure also relates to compositions comprising an ETL, such as an ETPL, and one or more of the following: pathogen recognition receptor agonists, antigens, and mammalian dendritic cells, as well as methods of production and uses of said compositions.

[0007] The present disclosure provides 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) as disclosed herein; 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 their protonated or de - protonated forms (where possible) or their pharmaceutically acceptable salts. In some embodiments, the ETL or ETPL is isolated.

[0008] The present disclosure also provides compositions comprising an ether lipid (ETL) or ether phospholipid (ETPL) 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; or their protonated or de - protonated forms (where possible) or their pharmaceutically acceptable salts; 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.

[0009] The present disclosure also provides a composition comprising an ether lipid (ETL) or ether phospholipid (ETPL) 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) as disclosed herein, 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 (where possible) or a pharmaceutically acceptable salt thereof; wherein the composition further comprises a TLR agonist. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist. In some embodiments, the composition further comprises an antigen. In some embodiments, the composition further comprises dendritic cells. In some embodiments, the composition further comprises an antigen and dendritic cells. In some embodiments, the ETL or ETPL is isolated.

[0010] The present disclosure also provides a composition comprising an ether lipid (ETL) or ether phospholipid (ETPL) 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) as disclosed herein, 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 (where possible) or a pharmaceutically acceptable salt thereof; wherein the composition further comprises an antigen. In some embodiments, the composition further comprises a TLR agonist. In some embodiments, the composition further comprises dendritic cells. In some embodiments, the composition further comprises a TLR agonist and dendritic cells. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist. In some embodiments, the ETL or ETPL is isolated.

[0011] The present disclosure also provides a composition comprising an ether lipid (ETL) or ether phospholipid (ETPL) 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) as disclosed herein, 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 (where possible) or a pharmaceutically acceptable salt thereof; wherein the composition further comprises dendritic cells. In some embodiments, the composition further comprises an antigen. In some embodiments, the composition further comprises a TLR agonist. In some embodiments, the composition further comprises an antigen and a TLR agonist. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist. In some embodiments, the ETL or ETPL is isolated.

[0012] 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) as disclosed herein, 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 (where possible) or a pharmaceutically acceptable salt thereof. In some embodiments, the ETL or ETPL is isolated.

[0013] The present disclosure also provides a composition comprising an ether lipid (ETL) or ether phospholipid (ETPL) 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, 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 (where 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.

[0014] The present disclosure also provides a composition comprising an ether lipid (ETL) or ether phospholipid (ETPL) 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, 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 (where possible) or a pharmaceutically acceptable salt thereof; wherein the composition further comprises a TLR agonist. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist. In some embodiments, the composition further comprises an antigen. In some embodiments, the composition further comprises a dendritic cell. In some embodiments, the composition further comprises an antigen and a dendritic cell. In some embodiments, the ETL or ETPL is isolated.

[0015] The present disclosure also provides a composition comprising an ether lipid (ETL) or ether phospholipid (ETPL) 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, 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 (where possible) or a pharmaceutically acceptable salt thereof; wherein the composition further comprises an antigen. In some embodiments, the composition further comprises a TLR agonist. In some embodiments, the composition further comprises dendritic cells. In some embodiments, the composition further comprises a TLR agonist and dendritic cells. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist. In some embodiments, the ETL or ETPL is isolated.

[0016] The present disclosure also provides a composition comprising an ether lipid (ETL) or ether phospholipid (ETPL) 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, 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 (where possible) or a pharmaceutically acceptable salt thereof; wherein the composition further comprises dendritic cells. In some embodiments, the composition further comprises an antigen. In some embodiments, the composition further comprises a TLR agonist. In some embodiments, the composition further comprises an antigen and a TLR agonist. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist. In some embodiments, the ETL or ETPL is isolated.

[0017] The present disclosure provides ether lipid (ETL) compounds, wherein the lipid alkyl chain is a C13-C24 normal alkyl chain or a C13-C22 normal alkyl chain. In some embodiments, the normal alkyl chain is a C18-C22 normal alkyl chain, a C21-C24 normal alkyl chain, or a C22 normal alkyl chain. In some embodiments, the present disclosure provides a composition comprising an ether lipid compound, wherein the lipid alkyl chain is a C13-C24 normal alkyl chain, a C13-C22 normal alkyl chain, a C18-C22 normal alkyl chain, a C21-C24 normal alkyl chain, or a C22 normal alkyl chain, and 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.

[0018] 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 normal alkyl chain or a C13-C22 normal alkyl chain. In some embodiments, the normal alkyl chain is a C18-C22 normal alkyl chain, a C21-C24 normal alkyl chain, or a C22 normal alkyl chain. In some embodiments, the composition further comprises an antigen and / or a dendritic cell. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist.

[0019] The present disclosure provides ether phospholipid (ETPL) compounds, wherein the lipid alkyl chain is a C13-C24 normal alkyl chain or a C13-C22 normal alkyl chain. In some embodiments, the normal alkyl chain is a C18-C22 normal alkyl chain, a C21-C24 normal alkyl chain, or a C22 normal alkyl chain. In some embodiments, the present disclosure provides a composition comprising an ether phospholipid compound, wherein the lipid alkyl chain is a C13-C24 normal alkyl chain, a C13-C22 normal alkyl chain, a C18-C22 normal alkyl chain, a C21-C24 normal alkyl chain, or a C22 normal alkyl chain, and 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.

[0020] 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 normal alkyl chain or a C13-C22 normal alkyl chain. In some embodiments, the normal alkyl chain is a C18-C22 normal alkyl chain, a C21-C24 normal alkyl chain, or a C22 normal alkyl chain. In some embodiments, the composition further comprises an antigen and / or a dendritic cell. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist.

[0021] In some aspects, the present disclosure provides ether lipid (ETL) compounds having a normal alkyl chain, wherein the normal alkyl chain is a C21-C24 normal alkyl chain. In some embodiments, the present disclosure provides a composition comprising an ether lipid (ETL) compound having a normal alkyl chain, wherein the normal alkyl chain is a C21-C24 normal 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.

[0022] In some aspects, the present disclosure provides a composition comprising a separated ether lipid (ETL) having a normal alkyl chain and an antigen, wherein the normal alkyl chain is a C21-C24 normal 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.

[0023] In some aspects, the present disclosure provides ether phospholipid (ETPL) compounds having a normal alkyl chain, wherein the normal alkyl chain is a C21-C24 normal alkyl chain. In some embodiments, the present disclosure provides a composition comprising an ether phospholipid (ETPL) compound having a normal alkyl chain, wherein the normal alkyl chain is a C21-C24 normal 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.

[0024] In some aspects, the present disclosure provides a composition comprising a separated ether phospholipid (ETPL) having a normal alkyl chain and an antigen, wherein the normal alkyl chain is a C21-C24 normal 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.

[0025] In some aspects, the present disclosure provides a composition comprising an ether lipid (ETL) having a normal alkyl chain and dendritic cells, wherein the normal alkyl chain is a C21-C24 normal alkyl chain. 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 a separated ether lipid (ETL) having a normal alkyl chain and dendritic cells, wherein the normal alkyl chain is a C21-C24 normal alkyl chain. 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 ether phospholipid (ETPL) having a normal alkyl chain and dendritic cells, wherein the normal alkyl chain is a C21-C24 normal alkyl chain. 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 a composition comprising an isolated ether phospholipid (ETPL) having a normal alkyl chain and dendritic cells, wherein the normal alkyl chain is a C21-C24 normal alkyl chain. 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.

[0029] In some aspects, the present disclosure provides an ether lipid (ETL) compound having a normal alkyl chain, wherein the normal alkyl chain is a C16-C20 normal alkyl chain. In some embodiments, the present disclosure provides a composition comprising an ether lipid (ETL) compound having a normal alkyl chain, wherein the normal alkyl chain is a C16-C20 normal 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.

[0030] In some aspects, the present disclosure provides a composition comprising an isolated ether lipid (ETL) having a normal alkyl chain and an antigen, wherein the normal alkyl chain is a C16-C20 normal 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.

[0031] In some aspects, the present disclosure provides an ether phospholipid (ETPL) compound having a normal alkyl chain, wherein the normal alkyl chain is a C16-C20 normal alkyl chain. In some embodiments, the present disclosure provides a composition comprising an ether phospholipid (ETPL) compound having a normal alkyl chain, wherein the normal alkyl chain is a C16-C20 normal 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.

[0032] In some aspects, the present disclosure provides a composition comprising an isolated ether phospholipid (ETPL) having a normal alkyl chain and an antigen, wherein the normal alkyl chain is a C16-C20 normal 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.

[0033] In some aspects, the present disclosure provides a composition comprising an ether lipid (ETL) having a normal alkyl chain and dendritic cells, wherein the normal alkyl chain is a C16-C20 normal alkyl chain. 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 isolated ether lipid (ETL) having a normal alkyl chain and dendritic cells, wherein the normal alkyl chain is a C16-C20 normal alkyl chain. 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 ether phospholipid (ETPL) having a normal alkyl chain and dendritic cells, wherein the normal alkyl chain is a C16-C20 normal alkyl chain. 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 aspects, the present disclosure provides a composition comprising an isolated ether phospholipid (ETPL) having a normal alkyl chain and dendritic cells, wherein the normal alkyl chain is a C16-C20 normal alkyl chain. 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.

[0037] In some embodiments of the foregoing aspects, the antigen is present in a biological sample obtained from an individual. In some embodiments, the biological sample comprises a biopsy tissue. In some embodiments, the biological sample comprises cells. In other embodiments, the biological sample does not comprise cells. In some embodiments, the biological sample comprises pus from an abscess. In some embodiments, the antigen comprises a protein antigen. In some embodiments, the antigen comprises a tumor antigen. In some embodiments, the tumor antigen comprises a synthetic or recombinant neoantigen. In some embodiments, the tumor antigen comprises a tumor cell lysate. In some embodiments, the antigen comprises a microbial antigen, and the microbial antigen comprises one or more of a viral antigen, a bacterial antigen, a protozoal antigen, and a fungal antigen. In some embodiments, the microbial antigen comprises a purified or recombinant surface protein. In some embodiments, the microbial antigen comprises an inactivated whole virus.

[0038] In some embodiments, the composition does not contain liposomes. In some embodiments, the composition does not contain LPS or MPLA. In some embodiments, the composition does not contain oxPAPC or oxPAPC species. In some embodiments, the composition does not contain HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC, and / or PGPC. In some embodiments, the composition does not contain lysophosphatidylcholine (LPC). In some embodiments, the composition does not contain 1-dodecanoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

[0039] In some embodiments, the composition further comprises an adjuvant, wherein the adjuvant comprises an aluminum salt adjuvant, a squalene-in-water emulsion, saponin, or a combination thereof.

[0040] In some embodiments, the present disclosure provides a pharmaceutical formulation comprising the composition of any of the foregoing aspects and a pharmaceutically acceptable excipient.

[0041] In other aspects, the present disclosure provides a method for generating over-activated dendritic cells, the method comprising contacting dendritic cells with a composition comprising an effective amount of a separated ether lipid (ETL) having a C13-C22 n-alkyl chain or a C13-C24 n-alkyl chain and a TLR agonist to generate over-activated dendritic cells, wherein the over-activated dendritic cells secrete IL-1β without undergoing pyroptosis. In some embodiments, the dendritic cells are contacted ex vivo with the composition or pharmaceutical formulation of any of the foregoing embodiments. In other embodiments, the dendritic cells are contacted in vivo with a pharmaceutical formulation comprising the composition of any of the foregoing embodiments. In some aspects, the present disclosure provides a pharmaceutical formulation comprising a plurality of over-activated dendritic cells generated by the foregoing embodiments and a pharmaceutically acceptable excipient. In some embodiments, the plurality comprises at least 10 3 、10 4 、10 5 、10 6 、10 7 or 10 8 over-activated DCs. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist.

[0042] In other aspects, the present disclosure provides a method for generating hyperactivated dendritic cells, the method comprising contacting dendritic cells with a composition comprising an effective amount of a separated ether phospholipid (ETPL) having a C13-C22 n-alkyl chain or a C13-C24 n-alkyl chain and a TLR agonist to generate hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing pyroptosis. In some embodiments, the dendritic cells are contacted ex vivo with the composition or pharmaceutical formulation of any of the foregoing embodiments. In other embodiments, the dendritic cells are contacted in vivo with a pharmaceutical formulation comprising the composition of any of the foregoing embodiments. In some aspects, the present disclosure provides a pharmaceutical formulation comprising a plurality of hyperactivated dendritic cells generated by the foregoing embodiments and a pharmaceutically acceptable excipient. In some embodiments, the plurality comprises at least 10 3 、10 4 、10 5 、10 6 、10 7 or 10 8 hyperactivated DCs. In some embodiments, the TLR agonist comprises a TLR7 / 8 agonist.

[0043] In other aspects, the present disclosure provides a composition comprising a separated ether lipid (ETL) having a 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 cytosolic 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 other aspects, the present disclosure provides a composition comprising a separated ether phospholipid (ETPL) having a 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 cytosolic 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.

[0045] In some embodiments of the foregoing 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.

[0046] In some embodiments of the foregoing 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.

[0047] In some embodiments of the foregoing aspect, the ETPL comprises 1-dodecyl-sn-glycero-3-phosphocholine (DGPC). In some embodiments of the foregoing aspect, the ETPL comprises 1-dodecyl-sn-glycero-3-phosphate (DGP).

[0048] In some embodiments of the foregoing aspect, the TLR agonist is a small molecule having a molecular weight of 900 daltons or less. In some embodiments of the foregoing aspect, the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 daltons or less. In some embodiments, the TLR7 / 8 agonist comprises an imidazoquinoline compound. In some embodiments, the TLR7 / 8 agonist comprises resiquimod (R848). In some embodiments, the 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).

[0049] The present disclosure further provides a composition for over-activating human dendritic cells, the composition comprising an ether lipid (ETL) compound having a normal alkyl chain and a pathogen recognition receptor (PRR) agonist, wherein the normal alkyl chain is a C22 normal alkyl chain, and wherein the composition is effective in achieving a higher level of dendritic cell over-activation compared to a comparative composition comprising a comparative compound instead of the ETL. The present disclosure further provides a composition for over-activating human dendritic cells, the composition comprising an isolated ether lipid (ETL) compound having a normal alkyl chain and a pathogen recognition receptor (PRR) agonist, wherein the normal alkyl chain is a C22 normal alkyl chain, and wherein the composition is effective in achieving a higher level of dendritic cell over-activation compared to a comparative composition comprising a comparative compound instead of the ETL. In some embodiments, the over-activation occurs in vitro or ex vivo. In other embodiments, the over-activation occurs in vivo. In some embodiments, the higher level of dendritic cell over-activation comprises at least 2, 3, or 4 times higher levels of in vitro induced IL-1β secretion from human dendritic cells when contacted with a composition comprising the ETL and the PRR agonist compared to when contacted with a comparative composition comprising the comparative compound and the PRR agonist, wherein the PRR agonist is LPS. In some embodiments, the concentration of the ETL and the concentration of the comparative compound are the same concentration, optionally in the range of about 10 μM to about 80 μM, and LPS is present at a concentration of 1 μg / ml in both the composition and the comparative composition. In some embodiments, the higher level of dendritic cell over-activation comprises at least 4, 5, or 6 times higher lipid activity index for IL-1β secretion from human dendritic cells by a composition comprising the ETL and the PRR agonist compared to a comparative composition comprising the comparative compound and the PRR agonist, in terms of activity units. In some embodiments, the comparative compound is PGPC. In some embodiments, the comparative compound is 1-docosanoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

[0050] The present disclosure further provides a composition for over - activating human dendritic cells, the composition comprising an ether phospholipid (ETPL) compound having a normal alkyl chain and a pathogen - recognition receptor (PRR) agonist, wherein the normal alkyl chain is a C22 normal alkyl chain, and wherein the composition effectively achieves a higher level of dendritic cell over - activation compared to a comparative composition comprising a comparative compound rather than ETPL. The present disclosure further provides a composition for over - activating human dendritic cells, the composition comprising an isolated ether phospholipid (ETPL) compound having a normal alkyl chain and a pathogen - recognition receptor (PRR) agonist, wherein the normal alkyl chain is a C22 normal alkyl chain, and wherein the composition effectively achieves a higher level of dendritic cell over - activation compared to a comparative composition comprising a comparative compound rather than ETPL. In some embodiments, the over - activation occurs in vitro or ex vivo. In other embodiments, the over - activation occurs in vivo. In some embodiments, the higher level of dendritic cell over - activation includes at least 2, 3, or 4 - fold higher levels of in vitro - induced IL - 1β secretion from human dendritic cells when contacting with a composition comprising ETPL and a PRR agonist compared to when contacting with a comparative composition comprising a comparative compound and a PRR agonist, wherein the PRR agonist is LPS. In some embodiments, the concentration of ETPL and the concentration of the comparative compound are the same concentration, optionally in the range of about 10 μM to about 80 μM, and LPS is present at a concentration of 1 μg / ml in both the composition and the comparative composition. In some embodiments, the higher level of dendritic cell over - activation includes at least 4, 5, or 6 - fold higher lipid activity indices in terms of activity units for IL - 1β secretion from human dendritic cells for a composition comprising ETPL and a PRR agonist compared to a comparative composition comprising a comparative compound and a PRR agonist. In some embodiments, the comparative compound is PGPC. In some embodiments, the comparative compound is 1 - docosanoyl - 2 - hydroxy - sn - glycero - 3 - phosphocholine [LPC(22:0)].

[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 micelle form.

[0052] 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 lipid nanoparticle (LNP) form.

[0053] In some embodiments of the present disclosure, relative to particles having a single lipid layer (micelles), the LNPs of the composition are enriched in particles having a lipid bilayer (liposomes). Specifically, in some embodiments, the LNPs comprise liposomes and comprise very few micelles to substantially no micelles. In some embodiments, the LNPs comprise liposomes and less than about 10% of the lipid particles present are micelles. In some embodiments, the LNPs comprise liposomes and less than about 5% of the lipid particles present are micelles. In some embodiments, the LNPs comprise liposomes and less than about 1% of the lipid particles present are micelles.

[0054] In some embodiments, the present disclosure provides lipid nanoparticles comprising an ETL or ETPL compound and at least one other lipid, and uses thereof in over-activating mammalian dendritic cells. The present disclosure also relates to a composition comprising an ETL or ETPL compound and at least one other lipid, wherein the composition further comprises one or more of a pathogen recognition receptor agonist, an antigen, and mammalian dendritic cells, and methods of production and uses of the composition.

[0055] In some aspects, 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 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) as disclosed herein; 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 (where possible) or a pharmaceutically acceptable salt thereof; and the ETL or ETPL and at least one other lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one other lipid is selected from the group consisting of ionizable lipids, cationic lipids, another phospholipid, a polyethylene glycolylated lipid, a structural lipid, and mixtures thereof. In some embodiments, the composition further comprises an antigen and / or dendritic cells.

[0056] In some aspects, the present disclosure provides a composition comprising an ETL or ETPL compound and an antigen, 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), formula (IV-F), formula (A) as disclosed herein; 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 (where possible) or a pharmaceutically acceptable salt thereof; and the ETL or ETPL and at least one other lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one other lipid is selected from the group consisting of: ionizable lipids, cationic lipids, another phospholipid, a polyethylene glycolylated lipid, a structural lipid, and mixtures thereof. In some embodiments, the composition further comprises a TLR agonist, such as a TLR7 / 8 agonist, and / or a dendritic cell.

[0057] In some aspects, the present disclosure provides a composition comprising an ETL or ETPL compound and dendritic cells, 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), formula (IV-F), formula (A) as disclosed herein; 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 (where possible) or a pharmaceutically acceptable salt thereof; and the ETL or ETPL and at least one other 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 other lipid is selected from the group consisting of: ionizable lipids, cationic lipids, another phospholipid, a polyethylene glycolylated lipid, a structural lipid, and mixtures thereof. In some embodiments, the composition further comprises a TLR agonist, such as a TLR7 / 8 agonist, and / or an antigen.

[0058] In some aspects, 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 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, 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 its protonated or deprotonated form (where possible) or its pharmaceutically acceptable salt; and the ETL or ETPL and at least one other lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one other lipid is selected from the group consisting of: ionizable lipids, cationic lipids, another phospholipid, polyethylene glycolated lipids, structural lipids, and mixtures thereof. In some embodiments, the composition further comprises an antigen and / or dendritic cells.

[0059] In some aspects, the present disclosure provides a composition comprising an ETL or ETPL compound and an antigen, 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) as disclosed herein, 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 its protonated or deprotonated form (where possible) or its pharmaceutically acceptable salt; and the ETL or ETPL and at least one other lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one other lipid is selected from the group consisting of: ionizable lipids, cationic lipids, another phospholipid, polyethylene glycolated lipids, structural lipids, and mixtures thereof. In some embodiments, the composition further comprises a TLR agonist, such as a TLR7 / 8 agonist, and / or dendritic cells.

[0060] In some aspects, the present disclosure provides a composition comprising an ETL or ETPL compound and dendritic cells, 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) as disclosed herein, 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 (where possible) or a pharmaceutically acceptable salt thereof; and the ETL or ETPL and at least one other 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 other lipid is selected from the group consisting of: ionizable lipids, cationic lipids, another phospholipid, a polyethylene glycolylated lipid, a structural lipid, and mixtures thereof. In some embodiments, the composition further comprises a TLR agonist, such as a TLR7 / 8 agonist, and / or an antigen.

[0061] In some embodiments of the foregoing aspects, the antigen is present in a biological sample obtained from an individual. In some embodiments, the biological sample comprises a biopsy tissue. In some embodiments, the biological sample comprises cells. In other embodiments, the biological sample does not comprise cells. In some embodiments, the biological sample comprises pus from an abscess. In some embodiments, the antigen comprises a protein antigen. In some embodiments, the antigen comprises a tumor antigen. In some embodiments, the tumor antigen comprises a synthetic or recombinant neoantigen. In some embodiments, the tumor antigen comprises a tumor cell lysate. In some embodiments, the antigen comprises a microbial antigen, and the microbial antigen comprises one or more of a viral antigen, a bacterial antigen, a protozoal antigen, and a fungal antigen. In some embodiments, the microbial antigen comprises a purified or recombinant surface protein. In some embodiments, the microbial antigen comprises an inactivated whole virus.

[0062] 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 separated 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 (P188) and / or Pluronic P123 (P123).

[0063] 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.

[0064] In some embodiments, the present disclosure provides a pharmaceutical preparation comprising a composition of any one of the aforementioned aspects and a pharmaceutically acceptable excipient. In some embodiments, the preparation does not include a surfactant (e.g., poloxamer). In some embodiments, the preparation does not include poloxamer 407 (KP407), poloxamer 188 (KP188) and / or pluronic P123 (P123).

[0065] In other aspects, the present disclosure provides a method for producing hyperactivated dendritic cells, the method comprising contacting dendritic cells with an effective amount of a composition or pharmaceutical preparation as in any one of the preceding embodiments to produce hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing pyroptosis, and ETL or ETPL and at least one other lipid are part of a lipid nanoparticle (LNP). In some embodiments, at least one other lipid is selected from the group consisting of: an ionizable lipid, a cationic lipid, another phospholipid, a pegylated lipid, a structural lipid, and a mixture thereof. In some embodiments, dendritic cells are contacted with a composition or pharmaceutical preparation of any one of the preceding embodiments ex vivo. In other embodiments, dendritic cells are contacted with a pharmaceutical preparation comprising a composition of any one of the preceding embodiments in vivo. In some aspects, the present disclosure provides a pharmaceutical preparation comprising a plurality of hyperactivated dendritic cells produced by the preceding embodiments and a pharmaceutically acceptable excipient. In some embodiments, the plurality comprises at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 or 10 8 Overactivated DC.

[0066] In other aspects, the present disclosure provides a composition 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), formula (IV-F), formula (A) as disclosed herein, 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 (where possible) or a pharmaceutically acceptable salt thereof; at least one other lipid and a pathogen recognition receptor (PRR) agonist, and the ETL or ETPL and at least one other lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one other lipid is selected from the group consisting of: ionizable lipids, cationic lipids, another phospholipid, pegylated lipids, structural lipids, and mixtures thereof. In some embodiments, the PRR agonist is an agonist of a toll-like receptor (TLR), NOD-like receptor (NLR), RIG-I-like receptor (RLR), or C-type lectin receptor (CLR). In some embodiments, the PRR agonist is an agonist of a cytosolic DNA sensor (CDS) or a stimulator of interferon 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.

[0067] In some embodiments of the foregoing aspect, the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 daltons or less. In some embodiments, the TLR7 / 8 agonist comprises an imidazoquinoline compound. In some embodiments, the TLR7 / 8 agonist comprises resiquimod (R848).

[0068] The present disclosure further provides a composition for hyperactivating human dendritic cells, the composition comprising 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), formula (IV-F), formula (A) as disclosed herein, 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 its protonated or deprotonated form (where possible) or its pharmaceutically acceptable salt; at least one other lipid and a pathogen recognition receptor (PRR) agonist, wherein the composition is effective to achieve a higher level of dendritic cell hyperactivation compared to a comparative composition comprising a comparative compound rather than ETL or ETPL. In some embodiments, the at least one other lipid is selected from the group consisting of: ionizable lipids, cationic lipids, another phospholipid, polyethylene glycolated lipids, structured lipids, and mixtures thereof. In some embodiments, the hyperactivation occurs in vitro or ex vivo. In other embodiments, the hyperactivation occurs in vivo. In some embodiments, the higher level of dendritic cell hyperactivation includes a level of IL-1β secretion from human dendritic cells that is at least 2, 3, or 4 times higher when contacted with a composition comprising ETL or ETPL and a PRR agonist compared to when contacted with a comparative composition comprising a comparative compound and a PRR agonist, wherein the PRR agonist is LPS. In some embodiments, the concentration of ETL or ETPL and the concentration of the comparative compound are the same concentration, optionally in the range of about 10 μM to about 80 μM, and LPS is present at a concentration of 1 μg / ml in both the composition and the comparative composition. In some embodiments, the higher level of dendritic cell hyperactivation includes a lipid activity index for IL-1β secretion from human dendritic cells that is at least 4, 5, or 6 times higher for a composition comprising ETL or ETPL and a PRR agonist compared to a comparative composition comprising a comparative compound and a PRR agonist. In some embodiments, the comparative compound is PGPC.

[0069] In other aspects, the present disclosure provides a composition 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) as disclosed herein, 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 its protonated or deprotonated form (where possible) or its pharmaceutically acceptable salt; at least one other lipid and a pathogen recognition receptor (PRR) agonist, and the ETL or ETPL and at least one other lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one other lipid is selected from the group consisting of: ionizable lipids, cationic lipids, another phospholipid, polyethylene glycolated lipids, structural lipids, and mixtures thereof. In some embodiments, the PRR agonist is an agonist of a toll-like receptor (TLR), NOD-like receptor (NLR), RIG-I-like receptor (RLR), or C-type lectin receptor (CLR). In some embodiments, the PRR agonist is an agonist of a cytosolic DNA sensor (CDS) or a stimulator of interferon 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.

[0070] In some embodiments of the foregoing aspects, the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 daltons or less. In some embodiments, the TLR7 / 8 agonist comprises an imidazoquinoline compound. In some embodiments, the TLR7 / 8 agonist comprises resiquimod (R848).

[0071] The present disclosure further provides a composition for over-activating human dendritic cells, the composition comprising 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) as disclosed herein, 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 its protonated or deprotonated form (where possible) or its pharmaceutically acceptable salt; at least one other lipid and a pathogen recognition receptor (PRR) agonist, wherein the composition is effective in achieving a higher level of dendritic cell over-activation compared to a comparative composition comprising a comparative compound rather than ETL or ETPL. In some embodiments, the at least one other lipid is selected from the group consisting of: ionizable lipids, cationic lipids, another phospholipid, pegylated lipids, structured lipids, and mixtures thereof. In some embodiments, the over-activation occurs in vitro or ex vivo. In other embodiments, the over-activation occurs in vivo. In some embodiments, the higher level of dendritic cell over-activation includes at least 2, 3, or 4 times higher levels of in vitro-induced secretion of IL-1β from human dendritic cells upon contact with a composition comprising ETL or ETPL and a PRR agonist compared to contact with a comparative composition comprising a comparative compound and a PRR agonist, wherein the PRR agonist is LPS. In some embodiments, the concentration of ETL or ETPL and the concentration of the comparative compound are the same concentration, optionally in the range of about 10 μM to about 80 μM, and LPS is present at a concentration of 1 μg / ml in both the composition and the comparative composition. In some embodiments, the higher level of dendritic cell over-activation includes a lipid activity index for the secretion of IL-1β from human dendritic cells that is at least 4, 5, or 6 times higher in units of activity for a composition comprising ETL or ETPL and a PRR agonist compared to a comparative composition comprising a comparative compound and a PRR agonist. In some embodiments, the comparative compound is PGPC.

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

[0073] In any of the embodiments disclosed herein, the ether phospholipid may be in the form of a pharmaceutically acceptable salt.

[0074] In any of the embodiments disclosed herein, whenever 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 disclosure also encompasses the alternative use in that embodiment of a compound of any other formula or other specific compounds.

[0075] The disclosure of methods that include administering the compounds and compositions of the disclosure to a subject (e.g., a subject in need) is also related 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.

[0076] In any of the embodiments disclosed herein that include an antigen, the antigen can include one or more viral antigens. In some embodiments, the one or more viral antigens include 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 include one or both of hemagglutinin and nucleoprotein. In some embodiments, the viral antigen includes inactivated virus particles, optionally wherein the inactivated virus particles include inactivated split virus particles. In some embodiments that include both an influenza A antigen and an influenza B antigen, the antigen is an H1N1 influenza A virus, an H3N2 influenza A virus, a Victoria lineage influenza B virus, and a Yamagata lineage influenza B virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] The data presented in the bar graphs in the figures below are shown as mean values with error bars representing the standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns = not significant.

[0078] Figure 1A showing cell viability and Figure 1BShows the IL-1β secretion of human monocyte-derived dendritic cells (moDCs) under specified test conditions.

[0079] Figure 2A Shows cell viability and Figure 2B Shows the IL-1β secretion of human moDCs under specified test conditions.

[0080] Figure 3A Shows cell viability and Figure 3B Shows the IL-1β secretion of human moDCs under specified test conditions.

[0081] Figure 4A Shows IL-1β secretion, Figure 4B Shows cell viability, and Figure 4C Shows the TNFα secretion of human moDCs under specified test conditions.

[0082] Figure 5 Shows the migration of dendritic cells from the skin to the draining lymph nodes under specified test conditions.

[0083] Figure 6 Shows the survival rate of mice bearing LLC1 tumors immunized with PBS or whole tumor lysate in the presence of PAMP and DAMP.

[0084] Figure 7 Shows IFNγ-secreting cells in the draining lymph nodes of immunized mice.

[0085] Figure 8 Shows the IL-1β secretion of human moDCs treated with 22:0Lyso PC, DPD (Compound 9), Compound 10, or vehicle with and without R848.

[0086] Figure 9 Shows the viability of cells treated with 22:0Lyso PC, DPD (Compound 9), Compound 10, or vehicle with and without R848.

[0087] Figure 10 Shows the IL-6 secretion of human moDCs treated with 22:0LysoPC, Compound 9 (DPD), Compound 2 (DGP), Compound 7, Compound 8, or vehicle without R848, with R848, and with R848 and MCC950.

[0088] Figure 11IL-1β secretion of human moDCs treated with 22:0 LysoPC, Compound 9 (DPD), Compound 2 (DGP), Compound 7, Compound 8, or vehicle without R848, with R848, and with R848 and MCC950 is shown.

[0089] Figure 12 Cell viability of cells treated with 22:0 LysoPC, Compound 9 (DPD), Compound 2 (DGP), Compound 7, Compound 8, or vehicle without R848, with R848, and with R848 and MCC950 is shown.

[0090] Figure 13 IL-6 secretion of human moDCs treated with Compound 11, Compound 12, or vehicle without R848, with R848, and with R848 and MCC950 is shown.

[0091] Figure 14 IL-1β secretion of human moDCs treated with Compound 11, Compound 12, or vehicle without R848, with R848, and with R848 and MCC950 is shown.

[0092] Figure 15 Cell viability of cells treated with Compound 11, Compound 12, or vehicle without R848, with R848, and with R848 and MCC950 is shown.

[0093] Figure 16 IL-6 secretion of human moDCs treated with Compound 1, 4, 6, 11, 12, 13, 14, 15, 16, 2, 22:0 LPC, or vehicle without R848, with R848, and with R848 and MCC950 is shown. The tested compound concentration is 41.25 μM.

[0094] Figure 17 IL-1β secretion of human moDCs treated with Compound 1, 4, 6, 11, 12, 13, 14, 15, 16, 2, 22:0 LPC, or vehicle without R848, with R848, and with R848 and MCC950 is shown. The tested compound concentration is 41.25 μM.

[0095] Figure 18 Cell viability of cells treated with Compound 1, 4, 6, 11, 12, 13, 14, 15, 16, 2, 22:0 LPC, or vehicle without R848, with R848, and with R848 and MCC950 is shown.

[0096] Figure 19 Shows the cell viability of cells treated with Compound 1, 2, 22:0 LPC, or vehicle in the absence of R848, in the presence of R848, and in the presence of R848 and MCC950. The tested compound concentration was 20.6 μM.

[0097] Figure 20 Shows the IL-1β secretion of human moDCs treated with Compound 1, 2, 22:0 LPC, or vehicle in the absence of R848, in the presence of R848, and in the presence of R848 and MCC950. The tested compound concentration was 20.6 μM.

[0098] Figure 21 Shows the IL-1β secretion of human moDCs under the specified test conditions. The moDCs in each figure were from different healthy donors (HDs), and the symbols represent values obtained from biological replicate samples. Ordinary two-way ANOVA was performed, followed by Tukey's multiple comparisons using a pooled variance.

[0099] Figure 22 Shows the cell viability as determined by measuring the release of lactate dehydrogenase (LDF) after treating human moDCs under the specified test conditions. The symbols represent the mean of biological triplicates of moDCs from given healthy donors (HD93, HD94, HD95, and HD96). The dashed line indicates the acceptable range of cell viability.

[0100] Figure 23 Shows the number of live CD11c+CD209+ cells present in a fixed volume obtained from each sample as determined by flow cytometry. The symbol shape for each healthy donor is unique. Statistical tests were completed using one-way ANOVA with repeated measures, followed by Tukey's comparisons using individual variances.

[0101] Figure 24A Shows the percentage of live CD11c+CD209+ cells expressing CD83, and Figure 24B Shows the mean fluorescence intensity (MFI) of CD83 staining of live CD11c+CD209+ cells. The symbol shape for each donor is unique. Statistical tests were completed using one-way ANOVA with repeated measures, followed by Tukey's comparisons using individual variances.

[0102] Figure 25A Shows the percentage of live CD11c+CD209+ cells expressing CD86, and Figure 25BShows the MFI of CD86 staining of viable CD11c+CD209+ cells. The symbol shapes for each donor are unique. Statistical tests were completed using one-way ANOVA with repeated measures, followed by Tukey's comparison using individual variances.

[0103] Figure 26A Shows the percentage of viable CD11c+CD209+ cells expressing CD40, and Figure 26B Shows the MFI of CD40 staining of viable CD11c+CD209+ cells. The symbol shapes for each donor are unique. Statistical tests were completed using one-way ANOVA with repeated measures, followed by Tukey's comparison using individual variances.

[0104] Figure 27A Shows the percentage of viable CD11c+CD209+ cells expressing MHC class I (HLA-ABC), and Figure 27B Shows the MFI of MHC class I staining of viable CD11c+CD209+ cells. The symbol shapes for each donor are unique. Statistical tests were completed using one-way ANOVA with repeated measures, followed by Tukey's comparison using individual variances.

[0105] Figure 28A Shows the percentage of viable CD11c+CD209+ cells expressing MHC class II (HLA-DR), and Figure 28B Shows the MFI of MHC class II staining of viable CD11c+CD209+ cells. The symbol shapes for each donor are unique. Statistical tests were completed using one-way ANOVA with repeated measures, followed by Tukey's comparison using individual variances.

[0106] Figure 29A Shows the percentage of viable CD11c+CD209+ cells expressing CCR7, and Figure 29B Shows the MFI of CCR7 staining of viable CD11c+CD209+ cells. The symbol shapes for each donor are unique. Statistical tests were completed using one-way ANOVA with repeated measures, followed by Tukey's comparison using individual variances.

[0107] Figure 30 Shows the concentration of IL-1β present in the cell culture supernatant after treating moDC for 24 hours under the specified conditions. The graph shows data from individual human donor samples, and the symbols represent values obtained from biological replicate samples. For statistical comparison, ordinary two-way ANOVA was performed, followed by Tukey's multiple comparison test using a single pooled variance.

[0108] Figure 31AShows the cell viability as determined by measuring the LDH activity of the cell culture supernatant of moDCs treated for 24 hours under specified conditions. Figure 31B Shows the cell viability as determined by measuring the luminescence signal induced by ATP using CellTiter-Glo 2.0 reagent after lysing moDCs for 24 hours under specified conditions. The x-axis label applies to both groups. The symbols in the figure represent biological replicate samples from donors. The dashed line indicates the acceptable range of cell viability.

[0109] Figure 32A - 32C Shows the NF-kB-dependent gene expression of human moDCs after treatment under specified conditions. Figure 32A Shows the concentration of IL-6, Figure 32B Shows the concentration of IL-10, and Figure 32C Shows the concentration of IL12p70 present in the cell culture supernatant after 24 hours of moDC treatment. The symbols represent biological replicate samples. For statistical comparison, ordinary two-way ANOVA was performed, followed by Tukey's multiple comparison test using a single pooled variance.

[0110] Figure 33A - 33B Shows the IRF-dependent gene expression of human moDCs after treatment under specified conditions. Figure 33A Shows the concentration of IP-10, and Figure 33B Shows the concentration of IFNα2 present in the cell culture supernatant after 24 hours of moDC treatment. The symbols represent biological replicate samples. For statistical comparison, ordinary two-way ANOVA was performed, followed by Tukey's multiple comparison test using a single pooled variance.

[0111] Figure 34A - 34C Shows the migration of human moDCs derived from three different donors (HD87, HD92, and HD93) after treatment with the specified stimuli. Briefly, cells were seeded in the upper chamber of a 5 μm pore transwell. Medium containing the specified concentration of CCL19 was added to the lower chamber, and the cells were incubated overnight. moDC migration was quantified by counting the cells in the lower chamber. The symbols represent biological replicate samples. For statistical comparison, ordinary two-way ANOVA was performed, followed by Tukey's multiple comparison test using a single pooled variance.

[0112] Figure 35A - 35B Shows the effect of human moDC overactivation on T cells. Figure 35A Shows the concentration of IL-6 present in the cell culture supernatant after 2 days of co-culturing moDCs and memory CD4+ T cells treated with the specified stimuli. Figure 35BShows the concentration of IL-6 present in the cell culture supernatant after treating CD4+ T cells with the specified stimuli for 2 days. IL-6 was measured from the cell culture supernatant using a Lumit immunoassay. Columns represent the mean, and data points represent the values of biological replicate samples. Ordinary two-way ANOVA was performed, followed by Tukey's multiple comparisons using a single pooled variance.

[0113] Figure 36A - 36B Shows the overactivation in co-cultures mediated by human moDCs stimulated with R848 and DGP. Figure 36A Shows the concentration of IL-1β present in the cell culture supernatant after treating a co-culture of moDCs and memory CD4+ T cells with the specified stimuli for 2 days. IL-1β was measured from the cell culture supernatant using a Lumit immunoassay. Figure 36B Shows the cell viability after treating a co-culture of moDCs and memory CD4+ T cells with the specified stimuli for 2 days. Columns represent the mean, and data points represent the values of biological replicate samples. Ordinary two-way ANOVA was performed, followed by Tukey's multiple comparisons using a single pooled variance.

[0114] Figure 37A - 37C Shows that a Th1 response is induced by human moDCs stimulated with R848 and DGP. Figure 37A Shows the concentration of IFNγ present in the cell culture supernatant after treating a co-culture of moDCs and memory CD4+ T cells with the specified stimuli (using anti-CD3) for 2 days. Figure 37B Shows the IFNγ present in the cell culture supernatant after treating individual moDCs, moDCs and CD4+ T cells, and individual CD4+ T cells with 2.85 μM R848, 82.5 μM DGP, and 0.1 ng / mL anti-CD3 for 2 days. Figure 37C Shows the concentration of IFNγ present in the cell culture supernatant after treating a co-culture of moDCs and memory CD4+ T cells with the specified stimuli (without using anti-CD3) for 2 days. IFNγ was measured using a Lumit immunoassay. Columns represent the mean, and data points represent the values of biological replicate samples. Ordinary two-way ANOVA was performed, followed by Tukey's multiple comparisons using a single pooled variance.

[0115] Figure 38A - 38C Shows that human moDCs stimulated with R848 and DGP induce the lowest amount of Th2 cytokines. Figure 38A Shows the concentration of IL-4, Figure 38B Shows the concentration of IL-5, and Figure 38CShows the concentration of IL-13 present in the cell culture supernatant 2 days after treating a co-culture of moDCs and memory CD4+ T cells with the specified stimuli. Cytokines were measured using a Lumit immunoassay. Columns represent the mean, and data points represent the values of biological replicate samples. Ordinary two-way ANOVA was performed, followed by Tukey's multiple comparisons using a single pooled variance.

[0116] Figure 39A - 39F Shows that the Th17 response is induced by human moDCs stimulated with R848 and DGP. Figure 39A Shows the concentration of IL-17A, Figure 39B shows the concentration of IL-17F, and Figure 39C shows the concentration of IL-IL-22 present in the cell culture supernatant 2 days after treating a co-culture of moDCs and memory CD4+ T cells with the specified stimuli. Figure 39D Shows the concentration of IL-17A, Figure 39E shows the concentration of IL-17F, and Figure 39F Shows the concentration of IL-22 present in the cell culture supernatant 2 days after treating individual moDCs, moDCs and CD4+ T cells, and individual CD4+ T cells with 2.85 μM R848, 41.3 μM DGP, and 0.1 ng / mL anti-CD3. Cytokines were measured using a Lumit immunoassay. Columns represent the mean, and data points represent the values of biological replicate samples. Ordinary two-way ANOVA was performed, followed by Tukey's multiple comparisons using a single pooled variance.

[0117] Figure 40 Shows that the combination of R848 and DGP (Compound 2) enhances the antigen-specific reactivation of CD8+ T cells. Briefly, the concentration of IFNγ present in the cell culture supernatant of CD8+ T cells co-cultured with pre-treated Flt3L-DCs for 96 hours was quantified as a measure of T cell activation.

[0118] Figure 41 Shows a method for reducing the size of DGP (Compound 2) DP (drug product) by jet milling micronization of DGP DS (drug substance) and homogenization of DGP DP, which method increases DC overactivation in vitro and in vivo. In the initial size reduction studies, sonication was used in place of homogenization.

[0119] Figure 42 Shows that micronization, sonication, and the combination of both reduce the size of DGP DP.

[0120] Figure 43Shows that micronization and / or sonication of DGP DP increases IL-1β secretion of human moDCs when treated with R848 and DGP DP compared to unmodified DGP DP.

[0121] Figure 44 Shows that micronization and / or sonication of DGP DP increases CCR7 expression on DCs migrating to the draining lymph nodes 4 hours after administration of R848 and DGP DP compared to unmodified DGP DP.

[0122] Figure 45A Shows the frequency, and Figure 45B Shows the absolute number of SIINFEKL + CD8 + T cells in the blood of immunized mice. Data from a group of 5 mice are shown, and each symbol represents one mouse.

[0123] Figure 46A Shows the frequency, and Figure 46B Shows the absolute number of SIINFEKL + CD8 + T cells in the draining lymph nodes of immunized mice. Data from a group of 4 - 5 mice are shown, and each symbol represents one mouse.

[0124] Figure 47 Shows the frequency of OVA - specific, IFNγ - secreting T cells in the draining lymph nodes of immunized mice. Cells were cultured for 18 hours in the presence or absence of OVA peptivator, and IFNγ - secreting cells were measured by ELISPOT assay. Data from a group of 4 - 5 mice are shown, and each symbol represents one mouse.

[0125] Figure 48A Shows the structures of cationic lipids and ionizable lipids of lipid nanoparticles (LNPs) applicable to the present disclosure. Figure 48B Shows the structures of other types of lipids of LNPs suitable for the present disclosure. See also Hou et al., Nature Review Materials, 6:1078 - 1094, 2021, which is incorporated herein by reference.

[0126] Figure 49A heatmap showing the normalized concentrations of cytokines and chemokines detected 2 hours, 24 hours, and 48 hours after injection. Abbreviations: MCP1 = monocyte chemoattractant protein 1, MIP-1α = macrophage inflammatory protein-1α, MIP-1β = macrophage inflammatory protein-1β, Rantes = regulated on activation, normal T cell expressed and secreted, Eotaxin = eosinophil chemotactic factor, MDC = macrophage-derived chemokine, KC = keratinocyte-derived chemokine, IP-10 = interferon-inducible protein 10, IFNα = interferon α, IFNβ = interferon β, TNFα = tumor necrosis factor α, 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 interferon.

[0127] Figure 50A - 50D Graph showing the absolute numbers of monocytes ( Figure 50A ), moDCs ( Figure 50B ), macrophages ( Figure 50C ), and cDCs ( Figure 50D ) in the dLNs at 4 hours and 48 hours after injection. There were five mice per group and each symbol represents one mouse.

[0128] Figure 51A - 51D Graph showing the absolute numbers of monocytes ( Figure 51A ), moDCs ( Figure 51B ), macrophages ( Figure 51C ), and cDCs ( Figure 51D ) in the spleen at 4 hours and 48 hours after injection. There were five mice per group and each symbol represents one mouse. Some samples were excluded due to low cell viability after dissociation.

[0129] Figure 52A - 52D Graph showing the mean fluorescence intensity (MFI) of CD69 expression on the surface of monocytes ( Figure 52A ), moDCs ( Figure 52B ), macrophages ( Figure 52C ), and cDCs ( Figure 52D ) in the dLNs at 4 hours or 48 hours after injection.

[0130] Figure 53A - 53D Graph showing the mean fluorescence intensity (MFI) of CD69 expression on the surface of monocytes ( Figure 53A ), moDCs ( Figure 53B ), macrophages ( Figure 53C ), and cDCs ( Figure 53D ) in the spleen at 4 hours and 48 hours after injection.

[0131] Figure 54A - 54B Graph showing the MFI of CCR7 expression on the surface of DCs in the dLNs ( Figure 54A ) and spleen ( Figure 54B ) at 4 hours and 48 hours post-injection.

[0132] Figure 55 Shows the spleen weights of immunized mice at the end point. Each symbol represents one mouse, with 7 mice per group.

[0133] Figure 56A Shows the frequencies of IFNγ SFCs for each sample and restimulation condition. Figure 56B Shows the frequencies of Afluria-specific SFCs for 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.

[0134] Figure 57A Shows the IFNγ concentrations determined for each sample and restimulation condition. Figure 57B Shows the Afluria-specific IFNγ secretion for 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.

[0135] Figure 58A Shows the frequencies of IL-5 SFCs for each sample and restimulation condition. Figure 58B Shows the frequencies of Afluria-specific SFCs for 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 mice / group.

[0136] Figure 59A Shows the IL-5 concentrations measured for each sample and restimulation condition. Figure 59B Shows the Afluria-specific IL-5 secretion for 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 mice / group.

[0137] Figure 60A Shows the ratio of IFNγ SFC to IL-5 SFC for each sample. Figure 60B Shows the ratio of IFNγ to IL-5 concentration for each sample. Each symbol represents one mouse, with 6 - 7 mice per group.

[0138] Figure 61 Shows the geometric mean titer (GMT) of antigen-specific antibodies in a hemagglutination inhibition (HAI) assay performed with the Afluria vaccine as the viral antigen.

[0139] Figure 62AShows antigen - specific IgG in the sera of immunized mice detected by ELISA using the Afluria vaccine as the coated antigen. Figure 62B Shows the avidity of antigen - specific IgG in the sera of immunized mice detected by ELISA using the Afluria vaccine as the coated antigen. Statistical significance was determined by Student's t - test. Each symbol represents one mouse, with 4 - 7 mice per group.

[0140] Figure 63A - 63G Shows DC( Figure 63A ), CD8+ TCM( Figure 63B ), CD4+ TCM( Figure 63C ), CD8+ TEM( Figure 63D ), CD4+ TEM( Figure 63E ), GC B( Figure 63F ), and TFH cell( Figure 63G ) frequencies. Each symbol represents one mouse, with 4 - 7 mice per group.

[0141] Figure 64A - 64D Shows the overactivation of canine PBCM. Figure 64A Shows the relative viability measured by ATP content under each condition compared to R848 alone. Figure 64B Shows IL - 1β, Figure 64C Shows IL - 6, and Figure 64D Shows IFNγ secretion in the cell - culture supernatants after stimulation with the indicated treatments for 48 hours. Each symbol represents one canine donor, n = 4 donors. Statistical significance was determined by one - way ANOVA followed by Dunnett's multiple - comparison test.

[0142] Figure 65A Shows the clinical score, Figure 65B Shows the body - weight change, and Figure 65C Shows the survival rate of immunized mice after challenge with live influenza (PR8) virus.

[0143] Figure 66A Shows the influenza (PR8) virus load, and Figure 66B Shows the concentration of influenza virus hemagglutinin (HA) antigen in the bronchoalveolar lavage (BAL) fluid of immunized mice on day 5 after challenge.

[0144] Figure 67A Shows the titer of anti - hemagglutinin (HA) IgG, and Figure 67B Shows the anti - nucleoprotein (NP) IgG antibody in the sera of immunized mice before influenza virus challenge.

[0145] Figure 68A Shows the percentage of influenza - nucleoprotein - specific CD8+ T cells in the blood of immunized mice before influenza virus challenge, andFigure 68B Shows the absolute number of said cells. Statistical significance was determined by one-way ANOVA with Tukey's post hoc analysis. Detailed implementation mode

[0146] The present disclosure relates to ether lipid (ETL) compounds, such as ether phospholipid (ETPL) compounds, and their use in over-activated human dendritic cells. The present disclosure also relates to compositions comprising an ETL, such as an ETPL, and one or more of the following: pathogen recognition receptor agonists, antigens, and mammalian dendritic cells, as well as methods for producing and using said compositions. In other embodiments, the dendritic cells are non-human dendritic cells, provided that the dendritic cells are not rodent dendritic cells.

[0147] General techniques and definitions

[0148] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.

[0149] Unless otherwise indicated, the singular forms "a", "an", and "the" as used herein and in the appended claims include plural references. For example, "an" excipient includes one or more excipients.

[0150] As used herein, the phrase "comprising" is open-ended, indicating that such embodiments may include other elements. In contrast, the phrase "consisting of" is closed-ended, indicating that such embodiments do not include other elements (except for trace impurities). The phrase "consisting essentially of" is partially closed-ended, indicating that such embodiments may also contain elements that do not substantially alter the basic characteristics of such embodiments.

[0151] As used herein, the term "about" in reference to a numerical value encompasses 90% to 110% of said value (e.g., a molecular weight of about 900 daltons refers to a molecular weight of 810 daltons to 990 daltons).

[0152] An "effective amount" or "sufficient amount" of a substance is an amount sufficient to achieve a beneficial or desired result, including a clinical result, and thus the "effective amount" depends on the context in which it is applied. For example, in the context of administering an immunogenic composition, an effective amount contains a sufficient amount of antigen, as well as one or both of an ether lipid (ETL) compound such as an ether phospholipid (ETPL) compound and a PRR agonist, to stimulate an immune response against the antigen (e.g., antigen-reactive antibodies and / or a cellular immune response).

[0153] 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, such as a human patient suffering from cancer and / or an infectious disease.

[0154] As used herein with reference to an immunogenic composition, the term "dose" refers to the measured portion of an immunogenic composition taken (administered to or received by a subject) by a subject at any one time.

[0155] The terms "isolated" and "purified" as used herein refer to a material that has been removed (e.g., removed from its original environment) from at least one component with which it was associated during the production of the material. As an example, when used in reference to an ETL (such as an ETPL), the purity of the isolated ETL or ETPL is at least 90%, 95%, 96%, 97%, 98%, or 99% as determined by thin layer chromatography (TLC), high pressure liquid chromatography (HPLC), or gas chromatography (GC). As another example, when used in reference to a recombinant protein, an isolated protein refers to a protein that has been removed from the culture medium of the host cell that produced the protein. As another example, when used in reference to a synthetic compound, an isolated compound or a purified compound has been removed from the reaction mixture in which it was synthesized.

[0156] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a formulation that is in a form that permits the bioactivity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to the individual to whom the formulation or composition will be administered. Such a formulation or composition is intended to be sterile.

[0157] As used herein, "excipient" includes a pharmaceutically acceptable excipient, carrier, vehicle, or stabilizer that is non-toxic to cells or mammals exposed to it at the dosages and concentrations used. A commonly physiologically acceptable excipient is an aqueous pH buffer solution.

[0158] The term "antigen" refers to a substance that is specifically recognized and bound by an antibody or by a T cell antigen receptor. Antigens can include peptides, polypeptides, proteins, glycoproteins, polysaccharides, complex carbohydrates, sugars, gangliosides, lipids, and phospholipids; portions thereof, and combinations thereof. When present in the compositions of the present disclosure, antigens 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 chemical compound that is not immunogenic by itself but becomes immunogenic when conjugated to a generally larger immunogenic molecule (carrier).

[0159] "Polypeptide antigen" can include purified natural peptides, synthetic peptides, recombinant peptides, crude peptide extracts, or peptides in a partially purified or unpurified active state (such as peptides that are part of an attenuated or inactivated virus, microorganism, or cell), or fragments of such peptides. The length of the polypeptide antigen is preferably at least eight amino acid residues.

[0160] The term "agonist" is used in the broadest sense and includes any molecule that activates signal transduction via a receptor. In some embodiments, the agonist binds to the receptor. For example, a TLR8 agonist binds to the TLR8 receptor and activates the TLR8 signal transduction pathway.

[0161] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbon group. C x Alkyl refers to an alkyl group having x number of carbon atoms. C x -C y Alkyl or C x-y Alkyl refers to an alkyl group having between x number and y number (including the endpoints) of carbon atoms. "Normal alkyl" refers to a straight-chain, i.e., linear, alkyl group.

[0162] "Alkylene" refers to a divalent saturated aliphatic hydrocarbon group.

[0163] "Alkenyl" refers to a monovalent hydrocarbon group having at least one double bond (>C=C<). C x Alkenyl refers to an alkenyl group having x number of carbon atoms. C x -C y Alkenyl or C x-y Alkenyl refers to an alkenyl group having between x number and y number (including the endpoints) of carbon atoms.

[0164] "Stimulation" of a response or parameter includes initiating and / or promoting the response or parameter when compared to other identical conditions except for the parameter of interest, or alternatively, when compared to another condition (e.g., TLR signaling increases in the presence of a TLR agonist compared to the absence of a TLR agonist). By way of example, "stimulation" of an immune response means 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.

[0165] Conversely, "inhibition" of a response or parameter includes reducing and / or suppressing the response or parameter when compared to other identical conditions except for the parameter of interest, or alternatively, when compared to another condition (e.g., abnormal cell proliferation decreases 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). By way of example, "inhibition" of an immune response means 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.

[0166] The relative terms "higher" and "lower" mean that a response or parameter is measurably increased or decreased, respectively, when compared to other identical conditions except for the parameter of interest, or alternatively, when compared to another condition. By way of example, "higher levels of DC hyperactivation" means the level of DC hyperactivation caused by a treatment condition (comprising an ETL compound of the present disclosure such as an ETPL compound) that is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold higher than the level of DC hyperactivation caused by a control condition (e.g., no ETL or ETPL, PGPC, oxPAPC, etc.). Similarly, "lower levels of DC hyperactivation" means the level of DC hyperactivation caused by a treatment condition (comprising an ETL compound of the present disclosure such as an ETPL compound) that is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold lower than the level of DC hyperactivation caused by a control condition (e.g., no ETL or ETPL, PGPC, oxPAPC, etc.). In some embodiments, the control condition includes an ETL compound that is a comparative compound replacement treatment condition, which can be an ETPL compound.

[0167] As used herein, the term "immunization" refers to a method of enhancing the response of a mammalian subject to an antigen and thus improving its ability to resist or overcome infection and / or disease.

[0168] As used herein, the term "vaccination" refers to the introduction of a vaccine into a mammalian subject.

[0169] "Adjuvant" refers to a substance that, when added to a composition containing an antigen after exposure, enhances or potentiates the immune response to the antigen in a mammalian recipient.

[0170] The term "treating" or "treatment" of a disease means implementing a regimen that can include administering one or more therapeutic agents to an individual (human or other individual) to seek to obtain a beneficial or desired result in the individual, including a clinical result. Beneficial or desired clinical results include, but are not limited to, alleviation or improvement of one or more signs or symptoms of the disease, reduction in the severity of the disease, stabilization of the disease state (i.e., not worsening), prevention of the spread of the disease, delay or slowing of the progression of the disease, improvement or palliation of the disease state, and remission (partial or complete). "Treatment" can also mean an extension of survival compared to the expected survival of an individual not receiving treatment. Additionally, "treating" and "treatment" can be effected by administering a single dose of one or more therapeutic agents, or can be effected after administering a series of doses of one or more therapeutic agents. Additionally, "treating" or "treatment" does not require complete alleviation of signs and symptoms and does not require a cure, and specifically includes regimens that only have a palliative effect on an individual. "Palliating" a disease or disorder means a reduction in the degree and / or undesired clinical manifestations of the disease or disorder and / or a slowing of the course of progression of the disease or disorder compared to the expected untreated outcome.

[0171] The compounds described herein can be administered in any pharmaceutically acceptable form, such as in the form of a pharmaceutically acceptable salt, or in the free base or free acid form (if 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 a substance that is not biologically or otherwise undesirable, e.g., the substance can be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a harmful manner with any other components of the composition containing it. Pharmaceutically acceptable carriers or excipients preferably meet the required standards of toxicological and manufacturing tests and / or are included in the Inactive Ingredient Guide established by the U.S. Food and Drug Administration. "Pharmaceutically acceptable salts" are those salts that retain at least some of the biological activity of the free (non-salt) compound and can be administered as a drug or medicine to an individual. Such salts include, for example: (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or acid addition salts formed with organic acids such as acetic acid, oxalic acid, propionic acid, succinic acid, maleic acid, tartaric acid, etc.; (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 metal ion, or an aluminum ion) or coordinated with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, etc. Acceptable inorganic bases that can be used to prepare salts include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, etc. Pharmaceutically acceptable salts can be prepared in situ during the manufacturing process or by reacting the purified compound of the present invention in its free acid or base form with a suitable organic or inorganic base or acid, respectively, and isolating the salt so formed during subsequent purification.

[0172] I. Ether Lipids (ETL) and Ether Phospholipids (ETPL) Compounds

[0173] "Ether lipid" (ETL) or "ether lipid molecule" refers to a glycerol molecule having a hydrocarbon group on one of the hydroxyl groups of glycerol. The remaining hydroxyl group may be unsubstituted (free hydroxyl group) or may be substituted. The hydrocarbon group may be an aliphatic hydrocarbon group such as an alkyl group, such as a normal alkyl group. The alkyl or normal alkyl group in any of the compounds disclosed herein is preferably unsubstituted, i.e., it consists only of carbon atoms and hydrogen atoms.

[0174] “Ether phospholipid” (ETPL) or “ether phospholipid molecule” is a specific type of ether lipid and refers to a glycerol molecule having a phosphate ester group on a hydroxyl group of glycerol and a hydrocarbon group on one of the other two hydroxyl groups of glycerol. The remaining hydroxyl group may be unsubstituted (free hydroxyl group) or may be substituted. The hydrocarbon group may be an aliphatic hydrocarbon group, such as an alkyl group, such as a normal alkyl group. The alkyl group or normal alkyl group in any of the compounds disclosed herein is preferably unsubstituted, i.e., it consists only of carbon atoms and hydrogen atoms.

[0175] The present disclosure provides ether lipids, such as ether phospholipids. The present disclosure provides isolated ether lipids, such as isolated ether phospholipids.

[0176] In some embodiments, the present disclosure provides an ether lipid compound of formula (I), such as an isolated ether lipid (ETL) having an alkyl chain of formula (I):

[0177]

[0178] wherein R 1 is H or

[0179] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0180] R 3 is C 13- C 24 normal alkyl;

[0181] wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0182] each R 5 is independently C1-C4 alkyl;

[0183] or its protonated or deprotonated form; or its salt, such as its pharmaceutically acceptable salt; and all its stereoisomers.

[0184] In some embodiments, the present disclosure provides an ether lipid compound of formula (II), such as an isolated ether lipid (ETL) having an alkyl chain of formula (II):

[0185]

[0186] wherein R 1 is H or

[0187] R 2is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0188] R 3 is C 13- C 24 n-alkyl;

[0189] wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0190] each R 5 is independently C1-C4 alkyl;

[0191] or its protonated or deprotonated form; or its salt, such as its pharmaceutically acceptable salt.

[0192] In some embodiments, the present disclosure provides an ether lipid compound of formula (III), such as an isolated ether lipid (ETL) of formula (III) having an alkyl chain:

[0193]

[0194] wherein R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0195] R 3 is C 13- C 24 n-alkyl; and

[0196] each R 5 is independently C1-C4 alkyl;

[0197] or its salt, such as its pharmaceutically acceptable salt.

[0198] In some embodiments, the present disclosure provides an ether lipid compound of formula (III-A), such as an isolated ether lipid (ETL) of formula (III-A) having an alkyl chain:

[0199]

[0200] wherein R 2 is -(C=O)-NH2, -(C=O)-NH(R 5 ), or -(C=O)-N(R 5 )2;

[0201] R 3 is C 13- C 24 is a n-alkyl; and

[0202] each R 5 is independently a C1-C4 alkyl;

[0203] or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some embodiments, R 2 is -(C=O)-NH2. In some embodiments, R 2 is -(C=O)-NH-CH3. In some embodiments, R 2 is -(C=O)-N(CH3)2. In some embodiments, R 3 is C 21 -C 24 is a n-alkyl. In some embodiments, R 2 is -(C=O)-NH2 and R 3 is C 21 -C 24 is a n-alkyl. In some embodiments, R 2 is -(C=O)-NH-CH3 and R 3 is C 21 -C 24 is a n-alkyl. In some embodiments, R 2 is -(C=O)-N(CH3)2 and R 3 is C 21 -C 24 is a n-alkyl. In some embodiments, R 2 is -(C=O)-NH2 and R 3 is C 22 is a n-alkyl. In some embodiments, R 2 is -(C=O)-NH-CH3 and R 3 is C 22 is a n-alkyl. In some embodiments, R 2 is -(C=O)-N(CH3)2 and R 3 is C 22 is a n-alkyl.

[0204] In some embodiments, provided herein are ether lipid compounds of formula (III-A-1), such as isolated ether lipids (ETLs) having an alkyl chain of formula (III-A-1):

[0205]

[0206] wherein R 2 is -(C=O)-NH2, -(C=O)-NH(R 5) or -(C═O)-N(R 5 )2;

[0207] R 3 is C 21- C 24 n-alkyl; and

[0208] each R 5 is independently a C1-C4 alkyl;

[0209] or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some embodiments, R 2 is -(C═O)-NH2. In some embodiments, R 2 is -(C═O)-NH-CH3. In some embodiments, R 2 is -(C═O)-N(CH3)2. In some embodiments, R 3 is C 22 n-alkyl. In some embodiments, R 2 is -(C═O)-NH2 and R 3 is C 22 n-alkyl. In some embodiments, R 2 is -(C═O)-NH-CH3 and R 3 is C 22 n-alkyl. In some embodiments, R 2 is -(C═O)-N(CH3)2 and R 3 is C 22 n-alkyl.

[0210] In some embodiments, provided herein are ether lipid compounds of formula (III-A-2), such as isolated ether lipids (ETLs) of formula (III-A-2) having an alkyl chain:

[0211]

[0212] wherein R 2 is -(C═O)-NH2, -(C═O)-NH(R 5 ) or -(C═O)-N(R 5 )2;

[0213] R 3 is C 16- C 20 n-alkyl; and

[0214] each R 5 is independently a C1-C4 alkyl;

[0215] or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some embodiments, R 2is -(C=O)-NH2. In some embodiments, R 2 is -(C=O)-NH-CH3. In some embodiments, R 2 is -(C=O)-N(CH3)2. In some embodiments, R 3 is C 18 n-alkyl. In some embodiments, R 2 is -(C=O)-NH2 and R 3 is C 18 n-alkyl. In some embodiments, R 2 is -(C=O)-NH-CH3 and R 3 is C 18 n-alkyl. In some embodiments, R 2 is -(C=O)-N(CH3)2 and R 3 is C 18 n-alkyl.

[0216] In some embodiments, the present disclosure provides ether lipid compounds of formula (III-B), such as isolated ether lipids (ETLs) of formula (III-B) having an alkyl chain:

[0217]

[0218] wherein 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 n-alkyl. In some embodiments, R 3 is C 22 n-alkyl.

[0219] In some embodiments, the present disclosure provides ether lipid compounds of formula (III-B-1), such as isolated ether lipids (ETLs) of formula (III-B-1) having an alkyl chain:

[0220]

[0221] wherein R 3 is C 21- C 24 n-alkyl; or a salt thereof, such as a pharmaceutically acceptable salt thereof. In some embodiments, R 3 is C 22 n-alkyl.

[0222] In some embodiments, the present disclosure provides ether lipid compounds of formula (III-B-2), such as isolated ether lipids (ETLs) of formula (III-B-2) having an alkyl chain:

[0223]

[0224] wherein 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 n-alkyl.

[0225] In some embodiments, provided herein are ether lipid compounds of formula (IV), such as isolated ether phospholipids (ETPLs) of formula (IV) having an alkyl chain:

[0226]

[0227] wherein R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0228] R 3 is C 13- C 24 n-alkyl;

[0229] R 4 is H or (CH3)3N + -(CH2)2-; and

[0230] each R 5 is independently C1-C4 alkyl;

[0231] or its protonated or deprotonated form; or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0232] In some embodiments, provided herein are ether lipid compounds of formula (IV-A), such as isolated ether phospholipids (ETPLs) of formula (IV-A) having an alkyl chain:

[0233]

[0234] wherein R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0235] R 3 is C 13- C 24 n-alkyl; and

[0236] Each R 5 is independently a C1-C4 alkyl group;

[0237] or its protonated or deprotonated form; or its salt, such as its pharmaceutically acceptable salt. In some embodiments, R 2 is H. In some embodiments, R 2 is -(C=O)-NH2. In some embodiments, R 2 is -(C=O)-NH-CH3. In some embodiments, R 2 is -(C=O)-N(CH3)2. In some embodiments, R 3 is C 21 -C 24 -n-alkyl. In some embodiments, R 2 is H and R 3 is C 21 -C 24 -n-alkyl. In some embodiments, R 2 is -(C=O)-NH2 and R 3 is C 21 -C 24 -n-alkyl. In some embodiments, R 2 is -(C=O)-NH-CH3 and R 3 is C 21 -C 24 -n-alkyl. In some embodiments, R 2 is -(C=O)-N(CH3)2 and R 3 is C 21 -C 24 -n-alkyl. In some embodiments, R 2 is H and R 3 is C 22 -n-alkyl. In some embodiments, R 2 is -(C=O)-NH2 and R 3 is C 22 -n-alkyl. In some embodiments, R 2 is -(C=O)-NH-CH3 and R 3 is C 22 -n-alkyl. In some embodiments, R 2 is -(C=O)-N(CH3)2 and R 3 is C 22 -n-alkyl.

[0238] In some embodiments, provided herein are ether lipid compounds of formula (IV-A-1), such as isolated ether phospholipids (ETPLs) of formula (IV-A-1) having an alkyl chain:

[0239]

[0240] wherein R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 2 or -CH2-C6H5;

[0241] R 3 is C 21- C 24 n-alkyl; and

[0242] each R 5 is independently C1-C4 alkyl;

[0243] or its protonated or deprotonated form; or its salt, such as its pharmaceutically acceptable salt. In some embodiments, R 2 is H. In some embodiments, R 2 is -(C=O)-NH2. In some embodiments, R 2 is -(C=O)-NH-CH3. In some embodiments, R 2 is -(C=O)-N(CH3)2. In some embodiments, R 3 is C 22 n-alkyl. In some embodiments, R 2 is H and R 3 is C 22 n-alkyl. In some embodiments, R 2 is -(C=O)-NH2 and R 3 is C 22 n-alkyl. In some embodiments, R 2 is -(C=O)-NH-CH3 and R 3 is C 22 n-alkyl. In some embodiments, R 2 is -(C=O)-N(CH3)2 and R 3 is C 22 n-alkyl.

[0244] In some embodiments, provided herein are ether lipid compounds of formula (IV-A-2), such as isolated ether phospholipids (ETPL) having an alkyl chain of formula (IV-A-2):

[0245]

[0246] wherein R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R5 ) 2 or -CH2-C6H5;

[0247] R 3 is C 16- C 20 n-alkyl; and

[0248] each R 5 is independently a C1-C4 alkyl;

[0249] or its protonated or deprotonated form; or its salt, such as its pharmaceutically acceptable salt. In some embodiments, R 2 is H. In some embodiments, R 2 is -(C=O)-NH2. In some embodiments, R 2 is -(C=O)-NH-CH3. In some embodiments, R 2 is -(C=O)-N(CH3)2. In some embodiments, R 3 is C 18 n-alkyl. In some embodiments, R 2 is H and R 3 is C 18 n-alkyl. In some embodiments, R 2 is -(C=O)-NH2 and R 3 is C 18 n-alkyl. In some embodiments, R 2 is -(C=O)-NH-CH3 and R 3 is C 18 n-alkyl. In some embodiments, R 2 is -(C=O)-N(CH3)2 and R 3 is C 18 n-alkyl.

[0250] In some embodiments, provided herein are ether lipid compounds of formula (IV-B), such as isolated ether phospholipids (ETPL) of formula (IV-B) having an alkyl chain:

[0251]

[0252] wherein R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0253] R 3 is C 13- C 24 n-alkyl; and

[0254] Each R 5 is independently a C1-C4 alkyl group;

[0255] or its protonated form; or its salt, such as its pharmaceutically acceptable salt.

[0256] In some embodiments, provided herein are ether lipid compounds of formula (IV-B-1), such as isolated ether phospholipids (ETPLs) of formula (IV-B-1) having an alkyl chain:

[0257]

[0258] wherein R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0259] R 3 is C 21- C 24 n-alkyl; and

[0260] each R 5 is independently a C1-C4 alkyl group;

[0261] or its protonated form; or its salt, such as its pharmaceutically acceptable salt. In some embodiments, R 2 is H. In some embodiments, R 2 is -(C=O)-NH2. In some embodiments, R 2 is -(C=O)-NH-CH3. In some embodiments, R 2 is -(C=O)-N(CH3)2. In some embodiments, R 3 is C 22 n-alkyl. In some embodiments, R 2 is H and R 3 is C 22 n-alkyl. In some embodiments, R 2 is -(C=O)-NH2 and R 3 is C 22 n-alkyl. In some embodiments, R 2 is -(C=O)-NH-CH3 and R 3 is C 22 n-alkyl. In some embodiments, R 2 is -(C=O)-N(CH3)2 and R 3 is C 22 n-alkyl.

[0262] In some embodiments, provided herein are ether lipid compounds of formula (IV-B-2), such as isolated ether phospholipids (ETPLs) of formula (IV-B-2) having an alkyl chain:

[0263]

[0264] wherein R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0265] R 3 is C 16- C 20 n-alkyl; and

[0266] each R 5 is independently C1-C4 alkyl;

[0267] or its protonated form; or its salt, such as its pharmaceutically acceptable salt. In some embodiments, R 2 is H. In some embodiments, R 2 is -(C=O)-NH2. In some embodiments, R 2 is -(C=O)-NH-CH3. In some embodiments, R 2 is -(C=O)-N(CH3)2. In some embodiments, R 3 is C 18 n-alkyl. In some embodiments, R 2 is H and R 3 is C 18 n-alkyl. In some embodiments, R 2 is -(C=O)-NH2 and R 3 is C 18 n-alkyl. In some embodiments, R 2 is -(C=O)-NH-CH3 and R 3 is C 18 n-alkyl. In some embodiments, R 2 is -(C=O)-N(CH3)2 and R 3 is C 18 n-alkyl.

[0268] In some embodiments, provided herein are ether lipid compounds of formula (IV-C), such as isolated ether phospholipids (ETPLs) of formula (IV-C) having an alkyl chain:

[0269]

[0270] wherein R3 is C 13- C 24 a normal alkyl group; and

[0271] R 4 is H or (CH3)3N + -(CH2)2-;

[0272] or its protonated or deprotonated form; or its salt, such as its pharmaceutically acceptable salt.

[0273] In some embodiments, the present disclosure provides ether lipid compounds of formula (IV-D), such as isolated ether phospholipids (ETPL) having an alkyl chain of formula (IV-D):

[0274]

[0275] wherein R 3 is C 13- C 24 a normal alkyl group;

[0276] or its protonated form; or its salt, such as its pharmaceutically acceptable salt. In some embodiments, R 3 is C 16 -C 20 a normal alkyl group. In some embodiments, R 3 is C 21 -C 24 a normal alkyl group. In some embodiments, R 3 is C 22 a normal alkyl group.

[0277] In some embodiments, the present disclosure provides ether lipid compounds of formula (IV-E), such as isolated ether phospholipids (ETPL) having an alkyl chain of formula (IV-E):

[0278]

[0279] wherein R 3 is C 13- C 24 a normal alkyl group;

[0280] or its protonated or deprotonated form; or its salt, such as its pharmaceutically acceptable salt. In some embodiments, R 3 is C 21 -C 24 a normal alkyl group.

[0281] In some embodiments, the present disclosure provides ether lipid compounds of formula (IV-F), such as isolated ether phospholipids (ETPL) having an alkyl chain of formula (IV-F):

[0282]

[0283] wherein R 2 is H, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 2), or -CH2-C6H5; R 3 is C 21- C 24 n-alkyl; and each R 5 is independently a C1-C4 alkyl; or its protonated or deprotonated form; or its salt. In some embodiments, R 2 is H. In some embodiments, R 2 is -(C=O)-NH2. 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 n-alkyl. 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).

[0284] In some embodiments, provided herein are ether lipid compounds of formula (A), such as isolated ether lipids (ETLs) of formula (I) having an alkyl chain:

[0285]

[0286] wherein R 1 is H or

[0287] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 2), or -CH2-C6H5;

[0288] R 3 is C 10- C 30 n-alkyl;

[0289] wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0290] each R 5 is independently a C1-C4 alkyl;

[0291] or its protonated or deprotonated form; or its salts, such as its pharmaceutically acceptable salts; and all of its stereoisomers.

[0292] In some embodiments, the ether phospholipid (ETPL) having a normal alkyl chain is a compound of formula (IV), wherein R 4 is (CH3)3N + -(CH2)2-; R 2 is H; R 3 is C 22 normal alkyl, and the compound is 1-docosyl-sn-glycero-3-phosphocholine (DGPC):

[0293]

[0294] or its protonated form; or its salts, such as its pharmaceutically acceptable salts.

[0295] In some embodiments, the isolated ether phospholipid (ETPL) having a normal alkyl chain is a compound of formula (IV), wherein R 4 is (CH3)3N + -(CH2)2-; R 2 is H; R 3 is C 22 normal alkyl, and the compound is 1-docosyl-sn-glycero-3-phosphocholine (DGPC):

[0296]

[0297] or its protonated form; or its salts, such as its pharmaceutically acceptable salts.

[0298] In some embodiments, the ether phospholipid (ETPL) having a normal alkyl chain is a compound of formula (IV), wherein R 4 is H; R 2 is H; R 3 is C 22 normal alkyl, and the compound is 1-docosyl-sn-glycero-3-phosphate (DGP):

[0299]

[0300] or its protonated or deprotonated form; or its salts, such as its pharmaceutically acceptable salts.

[0301] In some embodiments, the isolated ether phospholipid (ETPL) having a normal alkyl chain is a compound of formula (IV), wherein R 4 is H; R 2 is H; R 3 is C22 a normal alkyl group, and the compound is 1-docosyl-sn-glycero-3-phosphate (DGP):

[0302]

[0303] or its protonated or deprotonated form; or its salt, such as its pharmaceutically acceptable salt.

[0304] The ether lipid (ETL) and ether phospholipid (ETPL) compounds of the present disclosure have an alkyl chain, wherein the normal alkyl chain is a C13-C22 normal alkyl chain or a C13-C24 normal alkyl chain. In some embodiments, the normal alkyl chain is a C18-C22 normal alkyl chain or a C21-C24 normal alkyl chain. In some embodiments, the normal alkyl chain is a C16-C20 normal alkyl chain. In some embodiments, the normal alkyl chain is a C21-C24 normal alkyl chain. In some preferred embodiments, the normal alkyl chain is a C22 normal alkyl chain. The structures of exemplary ETPL and ETL compounds of the present disclosure are shown in Table I and Table II below. The structures shown in Table I and Table II may alternatively be the protonated or deprotonated forms of the structures shown in Table I and Table II, i.e., wherein protonation indicates a proton on any or all of the phosphate oxygen depicted below as O - and deprotonation indicates the removal of a proton from any or all of the phosphate OH groups; and / or may be a salt of the structures shown in Table I and Table II, such as its pharmaceutically acceptable salt.

[0305] Table I. Ether Phospholipids

[0306]

[0307]

[0308]

[0309] Table II. Ether Lipids

[0310]

[0311]

[0312] II. Pathogen Recognition Receptor Agonists

[0313] The compositions and methods of the present disclosure may also comprise pathogen recognition receptor (PRR) agonists. In some embodiments, the PRR agonist comprises an agonist of a toll-like receptor (TLR), NOD-like receptor (NLR), RIG-I-like receptor (RLR), or C-type lectin receptor (CLR). In other embodiments, the PRR agonist comprises a cytoplasmic DNA sensor (CDS) or stimulator of IFN genes (STING). In some embodiments, the PRR agonist comprises a TLR7 / 8 agonist.

[0314] A. TLR agonists and TLR7 / 8 agonists

[0315] 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 aspect, the TLR7 / 8 agonist is a TLR7 agonist. In another aspect, the TLR7 / 8 agonist is a TLR8 agonist. In another aspect, the TLR7 / 8 agonist is an agonist of both TLR7 and TLR8. The TLR7 / 8 agonists of the present disclosure are suitable for over-activating human dendritic cells in the presence of LPC.

[0316] In some aspects, the TLR agonist is a small molecule. In some aspects, the TLR7 / 8 agonist is a small molecule. In some embodiments, the TLR7 / 8 agonist is a small molecule or a salt thereof having a molecular weight of 900 daltons or less. That is, the small molecule TLR7 / 8 agonist is not a macromolecule such as a recombinant protein or a synthetic oligonucleotide, which may be regulated by the Center for Biologics Evaluation and Research of the US FDA. Instead, the small molecule TLR7 / 8 agonist may be regulated by the Center for Drug Evaluation and Research of the FDA. In some embodiments, the small molecule has a molecular weight of about 90 to about 900 daltons. In some embodiments, the TLR7 / 8 agonist comprises an imidazoquinoline compound. In some preferred embodiments, the TLR7 / 8 agonist comprises resiquimod (R848).

[0317] B. Other PRR agonists

[0318] In some aspects, the pathogen recognition receptor (PRR) agonist comprises a toll-like receptor (TLR) agonist, with the proviso that the TLR agonist does not comprise a TLR7 / 8 agonist. In some embodiments, the TLR agonist comprises an agonist of one or more of TLR2, TLR3, TLR4, TLR5, TLR9, and TLR13. In some embodiments, the PRR agonist is a TLR2 / 6 agonist, such as Pam2CSK4. In other embodiments, the TLR agonist is a TLR4 agonist, such as monophosphoryl lipid A (MPLA). However, in a preferred embodiment, the TLR agonist is not an agonist of TLR2, TLR4, and / or TLR9. For example, in a preferred embodiment, the TLR9 agonist is not a TLR4 ligand, such as LPS (endotoxin).

[0319] In other aspects, the PRR agonist comprises a NOD-like receptor (NLR) agonist. In other aspects, the PRR agonist comprises a RIG-I-like receptor (RLR) agonist. In additional aspects, the PRR agonist comprises a C-type lectin receptor (CLR) agonist. In other aspects, the PRR agonist comprises a CDS agonist or a STING agonist.

[0320] III. Antigen

[0321] The compositions and methods of the present disclosure may also comprise an antigen. In some embodiments, the antigen comprises a protein antigen. The terms "polypeptide" and "protein" are used interchangeably herein to refer to a protein antigen comprising a peptide chain of at least 8 amino acids in length. In some embodiments, the protein antigen is 8 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.

[0322] 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 the amino acid sequence of 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 the gene present in normal cells of a mammalian subject. Neoantigens are thought to be particularly useful for enabling T cells to distinguish cancer cells from non-cancer cells (see, for example, Schumacher and Schreiber, Science, 348:69-74, 2015). In other embodiments, the tumor antigen comprises a viral antigen, such as an antigen of an oncogenic virus.

[0323] In some embodiments, the tumor antigen is a fusion protein comprising two or more polypeptides, wherein each polypeptide comprises an amino acid sequence from a different tumor antigen or non - contiguous amino acid sequences from the same tumor antigen. In some of these embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, wherein each polypeptide comprises non - contiguous amino acid sequences from the same tumor antigen.

[0324] In some embodiments, the antigen is a microbial antigen. In some embodiments, the microbial antigen comprises a viral antigen, a bacterial antigen, a protozoal antigen, a fungal antigen, or a combination thereof. In some embodiments, the microbial antigen comprises a surface protein or other antigenic subunit of the microorganism. In other embodiments, the microbial antigen comprises an inactivated or attenuated microorganism. For example, the microbial antigen can comprise an inactivated virus, such as a chemically or genetically inactivated virus. Alternatively, the microbial antigen can comprise virus - like particles.

[0325] In some embodiments, the antigen can be present in a biological sample obtained from an individual, such as a human patient. For example, the antigen can comprise cancer cells. In another aspect, the antigen can comprise microbially infected cells, such as virus - infected cells.

[0326] IV. Dendritic Cells

[0327] The compositions and methods of the present disclosure can also comprise dendritic cells (DCs), which are antigen - presenting cells that are thought to bridge the innate and adaptive immune systems of mammals. In preferred embodiments, the DCs are subset 1 conventional DCs (cDC1, previously called myeloid DC1), as opposed to plasmacytoid DCs (pDCs).

[0328] In some embodiments, the DCs are hyperactivated DCs that express high levels of CD40 and IL - 12p70. As used herein, the term "hyperactivated dendritic cell" refers to a cell state in which the DC is able to secrete IL - 1β while maintaining cell viability (e.g., not undergoing pyroptosis). In this way, hyperactivated dendritic cells are able to stimulate a robust T - cell immunity (Figure 1), which apparently combines the benefits of activated dendritic cells and pyroptotic dendritic cells (Zhivaki et al., Cell Reports, 33(7), 2020, 108381).

[0329] V. Pharmaceutical Formulations

[0330] Some compositions of the present disclosure are pharmaceutical formulations that comprise a pharmaceutically acceptable excipient and an ETL compound, such as an ETPL compound. Some compositions of the present disclosure are pharmaceutical formulations that comprise a pharmaceutically acceptable excipient and lipid nanoparticles (LNPs) that comprise an ETL or ETPL compound and at least one other lipid. In some embodiments, the pharmaceutical formulation further comprises a PRR agonist, a dendritic cell, an antigen, an adjuvant, or any combination thereof. The pharmaceutical formulations of the present disclosure may be in the form of a solution or a suspension. Alternatively, the pharmaceutical formulation may be a dehydrated solid (e.g., a lyophilized or spray-dried solid). The pharmaceutical formulations of the present disclosure are preferably sterile and are preferably substantially endotoxin-free. The term "pharmaceutical formulation" may be used interchangeably with the terms "pharmaceutical product" and "drug" herein. In some embodiments, depending on the intended purpose of the formulation, the pharmaceutical formulation comprises specific ratios of the various components. In some embodiments, the pharmaceutical formulation comprises an ETL compound, such as an ETPL compound and a nonionic 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).

[0331] A. Excipient

[0332] The pharmaceutically acceptable excipients of the present disclosure include, for example, solvents, buffers, tonicity regulators, bulking agents, and preservatives (see, e.g., Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the pharmaceutical formulation may comprise an excipient that serves as one or more of a solvent, a buffer, a tonicity regulator, and a bulking agent (e.g., sodium chloride in physiological saline may serve as both an aqueous vehicle and a tonicity regulator). The pharmaceutically acceptable excipients of the present disclosure also include detergents, wetting agents, thickening agents, emulsifying agents, foaming agents, and dispersing agents, as well as surfactants.

[0333] Many of the lipids disclosed herein are water-soluble. Surfactants can be used to solubilize the lipids in an aqueous formulation. A variety of surfactants are available and can be classified as anionic surfactants, nonionic surfactants, cationic surfactants, and zwitterionic surfactants.

[0334] Some examples of nonionic surfactants include poloxamers, which are triblock copolymers of ethylene oxide and propylene oxide of the following general formula: HO-[CH2CH2-O-] a -[CH2CH(CH3)-O-] b -[CH2-CH2-O-] a -H, where a is generally from about 2 to 130 and b is generally from about 15 to 67. Some poloxamers are available under the trade name For sale (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); P84 (P-84; a = 19, b = 39); and P123 (P-123; a = 20, b = 70) (the foregoing values of a and b may vary slightly).

[0335] Other nonionic surfactants include CREMAPHOR series (CREMAPHOR is a registered trademark of BASF SE, Ludwigshafen, Germany). Surfactants include EL (KEL), which is a mixture of polyoxyethylated triglycerides produced by reacting castor oil with ethylene oxide in a molar ratio of approximately 1:35, and RH40 (also known as 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.

[0336] In some embodiments, the pharmaceutical formulation comprises an aqueous medium as a solvent. Suitable media include, for example, sterile water, saline solution, phosphate buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic.

[0337] The pharmaceutical formulation may comprise a buffer. The buffer controls the pH to inhibit degradation of the active agent during processing, storage, and optional reconstitution. Suitable buffers include, for example, salts containing acetate, citrate, phosphate, or sulfate. Other suitable buffers include, for example, amino acids such as arginine, glycine, histidine, and lysine. The buffer may also comprise hydrochloric acid or sodium hydroxide. In some embodiments, the buffer maintains the pH of the composition within the range of 6 to 9. In some embodiments, the pH is greater than (lower limit) 6, 7, or 8. In some embodiments, the pH is less than (upper limit) 9, 8, or 7. That is, the pH is in the range of about 6 to 9, where the lower limit is less than the upper limit.

[0338] The pharmaceutical composition may comprise a tonicity regulator. Suitable tonicity regulators include, for example, dextrose, glycerol, sodium chloride, glycerin, and mannitol.

[0339] The pharmaceutical preparation may contain bulking agents. Bulking agents are particularly suitable when the pharmaceutical composition is lyophilized before administration. In some embodiments, the bulking agent is a protective agent that helps to stabilize and prevent degradation of the active agent during freeze-drying or spray-drying and / or during storage. Suitable bulking agents are sugars (monosaccharides, disaccharides, and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose, and raffinose.

[0340] The pharmaceutical preparation may contain preservatives. Suitable preservatives include, for example, antioxidants and antimicrobial agents. However, in preferred embodiments, the pharmaceutical preparation is prepared under aseptic conditions and in single-dose containers and thus does not need to include preservatives. Methods for preparing sterile pharmaceutically acceptable compositions include steam sterilization, dry heat sterilization, gas sterilization, ionizing radiation, or sterile filtration. Sterile pharmaceutical preparations are compounded or manufactured according to pharmaceutical grade sterilization standards known to those skilled in the art (Chapter 797, 1072, and 1211 of the United States Pharmacopeia; California Business & Professions Code 4127.7; 16 California Code of Regulations 1751, 21 Code of Federal Regulations 211).

[0341] In some embodiments, the pharmaceutical preparation is a homogeneous solution. In some embodiments, the homogeneous solution is provided in a pre-filled syringe. In some embodiments, the pharmaceutical preparation is provided in the form of a suspension. In some embodiments, the suspension is refrigerated. In some embodiments, the suspension is frozen. In some embodiments, the methods provided herein further include the step of warming the refrigerated suspension to room temperature and / or stirring the suspension to ensure that the active ingredient is dissolved and / or uniformly distributed in the solution before administration. In some embodiments, the methods provided herein further include the step of thawing the frozen suspension and warming it to room temperature and / or stirring the suspension to ensure that the active ingredient is dissolved and / or uniformly distributed in the solution before administration. In some embodiments, the suspension is diluted before administration. In some embodiments, the suspension is provided in the form of a pre-filled syringe. In some embodiments, the suspension contains pharmaceutically acceptable excipients, such as surfactants, glycerol, nonionic surfactants, buffers, diols, salts, or any combination thereof.

[0342] The pharmaceutical preparations of the present disclosure are suitable for parenteral administration. That is, the pharmaceutical preparations of the present disclosure are not intended for enteral administration (e.g., not by oral, gastric, or rectal administration).

[0343] B. Adjuvants

[0344] The 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 hydroxyaluminum sulfate, aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, and combinations thereof. In other embodiments, the adjuvant is a squalene-in-water emulsion, such as MF59 or AS03. In other embodiments, the adjuvant is a saponin, such as Quil A or QS-21, as in AS01 or AS02.

[0345] C. Kit

[0346] Also provided herein are kits that contain at least one of the pharmaceutical formulations described herein. In some embodiments, the kit contains a lyophilized or freeze-dried pharmaceutical formulation (e.g., one unit dose in a vial) disclosed herein and a solution for dissolving, diluting, and / or reconstituting the lyophilized pharmaceutical composition. In some embodiments, the solution for reconstitution or dilution is provided in a pre-filled syringe. In some embodiments, the kit contains a frozen suspension of the pharmaceutical formulation (e.g., one unit dose in a vial). In some embodiments, the kit includes a buffer that helps prevent aggregation during reconstitution of the pharmaceutical compositions disclosed herein. In some embodiments, the pharmaceutical composition is provided in a pre-filled syringe. In some embodiments, the kit contains a dual-chamber syringe or container, where one chamber contains a buffer for dissolving or diluting the pharmaceutical composition. In some embodiments, the kit contains an injection syringe. In some embodiments, the reconstitution solution is filtered before administration. In some embodiments, the kit contains a filter or filter syringe for filtering the reconstituted pharmaceutical composition before administration. In some embodiments, the kit further contains instructions for use, such as instructions for over-activated cells.

[0347] D. Particle Size of Pharmaceutical Products

[0348] The particle size of drug particles can affect the uptake of drugs by cells. The particle size can be controlled by grinding the drug substance, such as DGP (Compound 2), by techniques well known in the pharmaceutical art. Dry grinding techniques that can be used include, but are not limited to, jet milling, hammer milling, and needle milling. Wet grinding techniques that can be used include, but are not limited to, rotor-stator milling, colloid milling, and media milling. The grinding of the drug substance can be carried out at other steps of the method, such as before mixing the drug substance with a solution, buffer, and / or other components to form a suspension.

[0349] The drug substance can be combined with a solution or buffer, such as phosphate buffered saline and poloxamer (e.g., poloxamer 407 or poloxamer 188), to obtain a pharmaceutical product. Further procedures can be used to reduce the particle size in the pharmaceutical product, including sonication and homogenization.

[0350] In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 40 micrometers (D 50 <40 micrometers). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 30 micrometers (D 50 <30 micrometers). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 20 micrometers to about 40 micrometers (D 50 <20 micrometers to 40 micrometers). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 20 micrometers to about 30 micrometers (D 50 <20 micrometers to 30 micrometers). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 20 micrometers (D 50 <20 micrometers). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 10 micrometers to about 30 micrometers (D 50 <10 micrometers to 30 micrometers). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 10 micrometers (D 50 <10 micrometers). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 10 micrometers to about 20 micrometers (D 50 <10 micrometers to 20 micrometers). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 5 micrometers to about 20 micrometers (D 50 <5 micrometers to 20 micrometers). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 5 micrometers (D 50 <5 micrometers).

[0351] In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 40 micrometers (D 90 <40 micrometers). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 30 micrometers (D 90 <30 micrometers). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 20 micrometers to about 40 micrometers (D 90 <20 micrometers to 40 micrometers). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 20 micrometers to about 30 micrometers (D 90 <20 micrometers to 30 micrometers). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 20 micrometers (D 90 <20 micrometers). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 10 micrometers to about 30 micrometers (D 90 <10 micrometers to 30 micrometers). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 10 micrometers (D 90(< 10 microns). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 10 microns to about 20 microns (D 90 <10 microns to 20 microns). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 5 microns to about 20 microns (D 90 <5 microns to 20 microns). In an embodiment, about 50% of the particles in the pharmaceutical product have a diameter less than about 5 microns (D 90 <5 microns).

[0352] The particles of the pharmaceutical product as described herein may comprise i) one or more of the following: surfactants, such as nonionic surfactants, such as poloxamers or pluronics, such as poloxamer 407, poloxamer 188, pluronic 84 or pluronic 123; wetting agents, such as P407, P188 or polysorbate 80; or thickening agents, such as carboxymethyl cellulose; and ii) ether lipid (ETL) 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 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) as disclosed herein; or their protonated or deprotonated forms (where possible) or their pharmaceutically acceptable salts. The particles may have the size or size range as indicated above.

[0353] VI. Production methods

[0354] In some aspects, the present disclosure relates to methods for preparing over-activated dendritic cells and methods for preparing immunogenic compositions. The immunogenic compositions are suitable for over-activating dendritic cells in vitro, ex vivo or in vivo.

[0355] In one aspect, the present disclosure provides a method for generating hyperactivated dendritic cells (DCs), the method comprising contacting dendritic cells with an effective amount of a separated ether lipid (ETL) having a positive alkyl chain, such as separated ether phospholipid (ETPL), and a PRR agonist to generate hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing pyroptosis. In some embodiments, the DCs are isolated, while in other embodiments, the DCs are present within a biological sample obtained from a mammalian subject, such as a human patient. In some embodiments, the DCs are monocyte-derived DCs, preferably cDC1s.

[0356] In another aspect, the present disclosure provides a method for generating an immunogenic composition, the method comprising combining an antigen with an effective amount of a separated ether lipid (ETL) having a positive alkyl chain, such as separated ether phospholipid (ETPL), and a PRR agonist to generate an immunogenic composition. In some embodiments, the antigen comprises a protein antigen present in or purified from a biological sample obtained from a mammalian subject. In some embodiments, the protein antigen is a synthetic or recombinant protein. In some preferred embodiments, the antigen is a tumor antigen. In some preferred embodiments, the antigen is a microbial antigen.

[0357] In a specific embodiment, the present disclosure provides a method for generating an immunogenic composition, the method comprising:

[0358] a) clearing leukocytes from a cell suspension prepared from a tumor to obtain a tumor cell-rich suspension;

[0359] b) lysing the cells from the tumor cell-rich suspension to obtain a tumor cell lysate; and

[0360] c) contacting the tumor cell lysate with a separated ether lipid (ETL) having a positive alkyl chain, such as separated ether phospholipid (ETPL), and a PRR agonist to obtain an immunogenic composition. In some embodiments, leukocytes are cleared from the tumor cell-rich cell suspension by contacting the tumor cell-rich suspension with an antibody specific for leukocytes. In some embodiments, leukocytes are cleared by contacting the tumor cell-rich suspension with an anti-CD45 antibody. In some embodiments, the cells are lysed by a cell lysis method based on physical disruption, such as but not limited to mechanical lysis, liquid homogenization, sonication, freeze-thaw, or manual grinding. In some preferred embodiments, the cells are lysed by one or more freeze-thaw cycles.

[0361] In some embodiments of the foregoing method, the alkyl chain of the ETL (such as ETPL) is a C13-C22 n-alkyl chain or a C13-C24 n-alkyl chain. In some embodiments, the alkyl chain of the ETL (such as 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 (such as ETPL) is a C22 n-alkyl chain. In some preferred embodiments, ETPL is DGPC. In some preferred embodiments, 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, which is resiquimod (R848) in a particularly preferred embodiment.

[0362] VII. Other Lipids

[0363] The compositions and methods of the present disclosure include at least one other lipid, wherein the LPC and at least one other lipid are part of a lipid nanoparticle (LNP). In some embodiments, the at least one other lipid comprises an ionizable lipid, a cationic lipid, another phospholipid, a PEGylated lipid, a structural 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 structural lipid. The structures of other lipids suitable for the compositions and methods of the present disclosure are shown in Figure 48A and Figure 48B (reproduced from Hou et al., Nature Review Materials, 6:1078-1094, 2021).

[0364] In some embodiments, at least one other lipid comprises one or both of another phospholipid and a structural lipid, optionally wherein the other phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and the structural lipid comprises cholesterol. In some embodiments, at least one other lipid comprises or further comprises a PEGylated lipid, optionally wherein the PEGylated lipid comprises polyethylene glycol [PEG]2000 dimyristoyl glycerol [DMG]. In some embodiments, at least one other lipid comprises or further comprises an ionizable lipid, optionally wherein the ionizable lipid comprises (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA) or an analogue or derivative thereof. In some embodiments, at least one other lipid comprises at least one lipid from the following list (disclosed in Hou et al., Nature Review Materials 6, 1078-1094 (2021)); these lipids include 306Oi10, 3,3',3”,3”'-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrakis(8-methylnonyl) tetrapropionate; 9A1P9, decyl (2-(dioctylammonio)ethyl) phosphate; A2-Iso5-2DC18, ethyl 5,5-bis((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate; ALC-0315, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate); ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide; 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-(dodecyldithio)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazapentacosanyl)azanediyl)dipropionate; BHEM-cholesterol, 2-(((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-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-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol); cKK-E12, 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione; DC-cholesterol, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol; DLin-MC3-DMA, 4-(dimethylamino)butanoic acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl ester; 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, hexakis(octan-3-yl) 9,9',9”,9”',9””,9””'-((((benzene-1,3,5-tricarbonyl)tris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanononanoate; Lipid H (SM-102), 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octadecan-9-yl octanoate; OF-Deg-Lin, (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl) (9Z,9'Z,9”Z,9”'Z,12Z,12'Z,12”Z,12”'Z)-tetrakis(octadeca-9,12-dienoate); PEG2000-DMG, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000; TT3, N1,N3,N5-tris(3-(dilauryamino)propyl)benzene-1,3,5-tricarboxamide, and are shown in; Figure 48A and Figure 48B in.

[0365] VIII. mRNA encoding an antigen

[0366] The compositions and methods of the present disclosure comprise mRNA encoding an antigen or are otherwise suitable for use with a formulation comprising mRNA encoding an antigen. In some embodiments, the antigen is a protein antigen. With reference to an antigen comprising a peptide chain of at least 8 amino acids in length, the terms “polypeptide” and “protein” are used interchangeably herein. 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.

[0367] 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 the amino acid sequence of 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 the gene present in normal cells from a mammalian subject. Neoantigens are thought to be particularly suitable for enabling T cells to distinguish cancer cells from non-cancer cells (see, e.g., Schumacher and Schreiber, Science, 348:69-74, 2015). In other embodiments, the tumor antigen comprises a viral antigen, such as an antigen of an oncogenic virus.

[0368] In some embodiments, the tumor antigen is a fusion protein comprising two or more polypeptides, wherein each polypeptide comprises the amino acid sequence of a different tumor antigen or non-consecutive amino acid sequences from the same tumor antigen. In some of these embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, wherein each polypeptide comprises non-consecutive amino acid sequences from the same tumor antigen.

[0369] In some embodiments, the antigen is a microbial antigen. In some embodiments, the microbial antigen comprises a viral antigen, a bacterial antigen, a protozoal antigen, a fungal antigen, or a combination thereof. In some embodiments, the microbial antigen comprises a surface protein of the microbe or other antigenic subunit.

[0370] In some preferred embodiments, the mRNA comprises a 5' untranslated region (5'UTR) at the 5' end of the coding region and comprises a 3' untranslated region (3'UTR) at the 3' end of the coding region. In some preferred embodiments, the mRNA comprises one or both of a 5' cap structure and a polyA tail.

[0371] IX. Lipid-Based Delivery Vehicles

[0372] The compositions and methods of the present disclosure include lipid-based delivery vehicles for mRNA encoding an antigen. In some embodiments, the vehicle is a lipid nanoparticle (LNP). In other embodiments, the vehicle is a lipid that forms a complex with the mRNA (RNA-lipid complex).

[0373] In some embodiments, the LNP comprises an ether lipid (ETL) or ether phospholipid (ETPL) 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) as disclosed herein, 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 (where possible) or a pharmaceutically acceptable salt thereof; and at least one lipid selected from the group consisting of: an ionizable lipid, a cationic lipid, a second phospholipid, a polyethylene glycolylated lipid, a structural 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 polyethylene glycolylated lipid. In some embodiments, at least one lipid comprises a structural lipid. In some embodiments, at least one lipid comprises an ionizable lipid, a second phospholipid, a polyethylene glycolylated lipid, and a structural lipid.

[0374] In some embodiments, the LNP comprises an ether lipid (ETL) or ether phospholipid (ETPL) 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, 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 (where possible) or a pharmaceutically acceptable salt thereof; and at least one lipid selected from the group consisting of: an ionizable lipid, a cationic lipid, a second phospholipid, a pegylated lipid, a structural lipid, and mixtures thereof. In some embodiments, the 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 structural lipid. In some embodiments, at least one lipid comprises an ionizable lipid, a second phospholipid, a pegylated lipid, and a structural lipid.

[0375] In some embodiments, the lipid component of the RNA-lipid complex comprises one or more lipids. In some preferred embodiments, the one or more lipids comprise a first lipid and a second lipid, wherein the first lipid is different from the second lipid. In some embodiments, the first lipid is a cationic lipid and the second lipid is a neutral or anionic lipid.

[0376] The structures of lipids suitable for the lipid-based mRNA delivery vehicles of the present disclosure are shown in Figure 48A and Figure 48B (reproduced from Hou et al., Nature Review Materials, 6:1078-1094, 2021).

[0377] X. Methods of Use

[0378] In some aspects, the present disclosure relates to methods of using any of the compositions or formulations described herein, the formulations comprising an ETL compound, such as an ETPL compound. In some embodiments, the composition or formulation further comprises a PRR agonist, a dendritic cell, an antigen, an adjuvant, or any combination thereof. The methods of use are suitable for a variety of uses involving stimulating an immune response. In some embodiments, the methods of use include a method of treating cancer. In some embodiments, the methods of use include a method of inhibiting abnormal cell proliferation. In some embodiments, the methods of use include a method of treating an infectious disease. The methods include administering to an individual in need thereof an effective amount of the formulation or composition described herein to achieve a particular result. The individual is a mammalian subject, such as a human patient. In other embodiments, the individual is a non-human patient. In some embodiments, the individual is a canine patient. That is, in some embodiments, the methods of use relate to clinical use, while in other embodiments, the methods of use relate to preclinical and / or veterinary use. For preclinical use, the mammalian subject can be a non-human primate (e.g., a monkey or an ape) or a rodent (e.g., a mouse or a rat). For veterinary use, the mammalian subject can be a farm animal (e.g., a cow), a sport animal (e.g., a horse), or a pet (e.g., a companion animal, such as a dog or a cat).

[0379] A. Stimulation of the immune response

[0380] Briefly, the present disclosure provides a method of stimulating an immune response in an individual, the method comprising administering to the individual an amount of the composition or formulation described herein sufficient to stimulate an immune response in the individual. "Stimulating" an immune response (which can be used interchangeably with "eliciting" an immune response) means increasing the immune response, which can result from eliciting a de novo immune response (e.g., caused by a primary vaccination regimen) or promoting an existing immune response (e.g., caused by a booster vaccination regimen). In some embodiments, stimulating the immune response comprises one or more of the group consisting of: stimulating cytokine production; stimulating B lymphocyte proliferation; stimulating gene expression related to the interferon pathway; stimulating gene expression related to chemokines; and stimulating dendritic cell DC maturation. Methods for measuring the stimulation of an immune response are known in the art.

[0381] For example, the present disclosure provides methods of inducing an antigen - specific immune response in an individual, the method comprising administering to the individual a composition or formulation described herein in an amount sufficient to induce an antigen - specific immune response in the individual. In a preferred embodiment, the composition or formulation comprises an antigen. In some embodiments, the composition or formulation is administered to a tissue of an individual that contains the antigen. The immune response can include one or both of an antigen - specific antibody response and an antigen - specific cytotoxic T lymphocyte (CTL) response. "Inducing" an antigen - specific antibody response means increasing the titer of antigen - specific antibodies to a level higher than a threshold level, such as the baseline titer or the serum protective level before administration. "Inducing" an antigen - specific CTL response means increasing the frequency of antigen - specific CTLs found in peripheral blood to a level higher than the baseline frequency before administration.

[0382] Analysis (both qualitative and quantitative) of the immune response can be carried out by any method known in the art, including but not limited to measuring antigen - specific antibody production (including measuring specific antibody subclasses), activation of specific lymphocyte populations such as B cells and helper T cells, production of cytokines (such as IFN - α, IFN - γ, IL - 6, IL - 12) and / or release of histamine. Methods for measuring antigen - specific antibody responses include enzyme - linked immunosorbent assay (ELISA). Activation of specific lymphocyte populations can be measured by proliferation assays and fluorescence - activated cell sorting (FACS). Production of cytokines can also be measured by ELISA. In some embodiments, the method of stimulating an immune response includes stimulating interleukin - 1β (IL - 1β) secretion, interferon - γ (IFN - γ) secretion, and / or tumor necrosis factor - α (TNF - α) secretion by monocyte - derived dendritic cells or peripheral blood mononuclear cells. In some embodiments, the method of stimulating an immune response includes stimulating memory CD4+ T cells to secrete one or more of IFN - γ, IL - 17a, IL - 17f, and IL - 22. In some embodiments, the method of stimulating an immune response includes increasing the Th1 differentiation of naive CD4+ T cells. In some preferred embodiments, at least 50%, 55%, 60%, 65%, 70%, or 75% of the cells in contact with the composition of the present disclosure remain viable 40 to 56 hours (or about 48 hours) after contact.

[0383] 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 immune response is an anti - bacterial immune response. In some embodiments, the anti - microbial response is an anti - fungal immune response. In some embodiments, the anti - microbial response is an anti - viral immune response. In some embodiments, the anti - microbial response is an anti - protozoal immune response.

[0384] B. Treating or preventing diseases

[0385] The present disclosure further provides a method of treating or preventing a disease in an individual, the method 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.

[0386] In one aspect, the method may comprise administering to a subject in need thereof a composition comprising an ETL compound, such as an ETPL compound. In some embodiments, the composition further comprises a PRR agonist, an antigen, an adjuvant, or any combination thereof. In another aspect, the method involves adoptive cell therapy and comprises administering to a subject in need thereof a composition comprising dendritic cells (such as over-activated dendritic cells) and an ETL compound (such as an ETPL compound). In some embodiments, the composition further comprises a PRR agonist, an antigen, an adjuvant, or any combination thereof.

[0387] In some embodiments, the method involves treating cancer in an individual or otherwise treating a mammalian subject having cancer. In some embodiments, the method comprises: a) preparing an immunogenic composition comprising a tumor cell lysate, an isolated ether lipid (ETL) having a normal alkyl chain (such as 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 prepared from or has been prepared from a tumor sample obtained from a subject having cancer, and the alkyl chain is a C13-C22 normal alkyl chain or a C13-C24 normal alkyl chain; and b) administering to the subject an effective amount of the immunogenic composition. In some embodiments, the cancer is a hematological cancer, such as lymphoma, leukemia, or myeloma. In other embodiments, the cancer is a non-hematological cancer, such as sarcoma, carcinoma, or melanoma. In some embodiments, the cancer is malignant.

[0388] In some embodiments, the method involves inhibiting abnormal cell proliferation in an individual. "Abnormal cell proliferation" refers to the proliferation of a benign or malignant tumor. The malignant tumor may be a metastatic tumor.

[0389] In some embodiments, the method involves 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 other embodiments, the infectious disease is caused by a fungal infection. In still other embodiments, the infectious disease is caused by a protozoan infection. Of particular importance are infectious diseases caused by zoonotic pathogens that infect humans as well as other animals such as mammals or birds. In some embodiments, the zoonotic pathogen is transmitted to humans via an intermediate species (vector).

[0390] The recited embodiments

[0391] The following recited embodiments represent aspects of the present disclosure. Where appropriate and feasible, the features of each embodiment may be combined with any other embodiment.

[0392] Embodiment 1. A composition comprising an isolated ether lipid (ETL) of formula (I):

[0393]

[0394] Wherein:

[0395] R 1 is H or

[0396] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0397] R 3 is C 13- C 24 n-alkyl;

[0398] Wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0399] Each R 5 is independently C1-C4 alkyl;

[0400] or its protonated form; or a pharmaceutically acceptable salt thereof; and

[0401] A TLR7 / 8 agonist.

[0402] Embodiment 2. The composition according to embodiment 1, wherein R 3 is C 18 -C 22 n-alkyl or C 21-C 24 n-alkyl

[0403] Embodiment 3. The composition according to Embodiment 1 or Embodiment 2, further comprising an antigen.

[0404] Embodiment 4. The composition according to any one of Embodiments 1-3, further comprising dendritic cells.

[0405] Embodiment 5. A composition comprising a separated ether lipid (ETL) of formula (I):

[0406]

[0407] wherein:

[0408] R 1 is H or

[0409] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0410] R 3 is C 13- C 24 n-alkyl;

[0411] where R 4 is H or (CH3)3N + -(CH2)2-; and

[0412] each R 5 is independently C1-C4 alkyl;

[0413] or its protonated form; or its pharmaceutically acceptable salt; and

[0414] an antigen.

[0415] Embodiment 6. The composition according to Embodiment 5, further comprising dendritic cells.

[0416] Embodiment 7. The composition according to Embodiment 5 or Embodiment 6, further comprising a TLR7 / 8 agonist.

[0417] Embodiment 8. A composition comprising a separated ether lipid (ETL) of formula (I):

[0418]

[0419] wherein:

[0420] R 1is H or

[0421] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0422] R 3 is C 13- C 24 n-alkyl;

[0423] wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0424] each R 5 is independently C1-C4 alkyl;

[0425] or its protonated form; or its pharmaceutically acceptable salt; and

[0426] dendritic cells.

[0427] Embodiment 9. The composition according to Embodiment 8, further comprising a TLR7 / 8 agonist.

[0428] Embodiment 10. The composition according to Embodiment 8 or Embodiment 9, further comprising an antigen.

[0429] Embodiment 11. The composition according to any one of Embodiments 1-10, wherein R 3 is C 22 n-alkyl.

[0430] Embodiment 12. The composition according to any one of Embodiments 1-11, wherein the ETL is an ether phospholipid (ETPL) comprising 1-didodecyl-sn-glycero-3-phosphocholine (DGPC) or its pharmaceutically acceptable salt.

[0431] Embodiment 13. The composition according to any one of Embodiments 1-11, wherein the ETL is an ETPL comprising 1-didodecyl-sn-glycero-3-phosphate (DGP) or its pharmaceutically acceptable salt.

[0432] Embodiment 14. The composition according to any one of Embodiments 1-13, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 daltons or less.

[0433] Embodiment 15. The composition according to Embodiment 14, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

[0434] Embodiment 16. The composition according to embodiment 15, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

[0435] Embodiment 17. The composition according to embodiment 14 or embodiment 15, wherein the TLR7 / 8 agonist does not inhibit NLR family pyrin domain containing 3 (NLRP3).

[0436] Embodiment 18. The composition according to embodiment 13, wherein the ETPL comprises one or both of DGPC and DGP, and the TLR7 / 8 agonist comprises resiquimod (R848).

[0437] Embodiment 19. The composition according to any one of embodiments 1-18, wherein the antigen is present in a biological sample obtained from an individual.

[0438] Embodiment 20. The composition according to embodiment 19, wherein the biological sample comprises biopsy tissue.

[0439] Embodiment 21. The composition according to embodiment 19, wherein the biological sample comprises cells.

[0440] Embodiment 22. The composition according to embodiment 19, wherein the biological sample does not comprise cells.

[0441] Embodiment 23. The composition according to embodiment 19, wherein the biological sample comprises pus from an abscess.

[0442] Embodiment 24. The composition according to any one of embodiments 1-23, wherein the antigen comprises a protein antigen.

[0443] Embodiment 25. The composition according to embodiment 24, wherein the antigen comprises a tumor antigen.

[0444] Embodiment 26. The composition according to embodiment 25, wherein the tumor antigen comprises a synthetic or recombinant neoantigen.

[0445] Embodiment 27. The composition according to embodiment 26, wherein the tumor antigen comprises a tumor cell lysate.

[0446] Embodiment 28. The composition according to embodiment 24, wherein the antigen comprises a microbial antigen, and the microbial antigen comprises one or more of a viral antigen, a bacterial antigen, a protozoal antigen, and a fungal antigen.

[0447] Embodiment 29. The composition according to embodiment 28, wherein the microbial antigen comprises a purified or recombinant surface protein.

[0448] Embodiment 30. The composition according to embodiment 28, wherein the microbial antigen comprises an inactivated whole virus.

[0449] Embodiment 31. The composition according to any one of embodiments 1-30, wherein the composition does not comprise liposomes.

[0450] Embodiment 32. The composition according to any one of embodiments 1-31, wherein the composition does not comprise LPS or MPLA.

[0451] Embodiment 33. The composition according to any one of embodiments 1-32, wherein the composition does not comprise oxPAPC or oxPAPC species, optionally wherein the composition does not comprise HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC and / or PGPC.

[0452] Embodiment 34. The composition according to embodiment 33, wherein the composition does not comprise lysophosphatidylcholine (LPC), optionally wherein the composition does not comprise 1-dodecanoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

[0453] Embodiment 35. The composition according to any one of embodiments 1-34, further comprising an adjuvant, wherein the adjuvant comprises an aluminum salt adjuvant, a water-in-squalene emulsion, saponin or a combination thereof.

[0454] Embodiment 36. A pharmaceutical formulation comprising the composition according to any one of embodiments 1-35 and a pharmaceutically acceptable excipient.

[0455] Embodiment 37. A method for generating hyperactivated dendritic cells, the method comprising contacting the dendritic cells with a composition comprising an effective amount of an isolated ether lipid (ETL) of formula (I):

[0456]

[0457] Wherein:

[0458] R 1 is H or

[0459] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2

[0460] or -CH2-C6H5;

[0461] R 3 is C 13- C 24 n-alkyl;

[0462] wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0463] each R 5 is independently a C1-C4 alkyl group;

[0464] or its protonated form; or a pharmaceutically acceptable salt thereof; and

[0465] a TLR7 / 8 agonist to produce hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing pyroptosis.

[0466] Embodiment 38. The method according to embodiment 37, wherein the dendritic cells are contacted ex vivo with the composition according to any one of embodiments 1-35 or the formulation according to embodiment 36.

[0467] Embodiment 39. The method according to embodiment 37, wherein the dendritic cells are contacted in vivo with the formulation according to embodiment 36.

[0468] Embodiment 40. A pharmaceutical formulation comprising at least 10 3 、10 4 、10 5 or 10 6 hyperactivated dendritic cells produced by the method according to embodiment 38 and a pharmaceutically acceptable excipient.

[0469] Embodiment 41. A method of stimulating an immune response against an antigen, the method comprising administering to an individual in need thereof an effective amount of the formulation according to embodiment 36 to stimulate an immune response against the antigen.

[0470] Embodiment 42. A method of treating cancer, the method comprising administering to an individual in need thereof an effective amount of the formulation according to embodiment 36 to treat the cancer.

[0471] Embodiment 43. A method of inhibiting abnormal cell proliferation, the method comprising administering to an individual in need thereof an effective amount of the formulation according to embodiment 36 to inhibit abnormal cell proliferation.

[0472] Embodiment 44. A method for treating an infectious disease, the method comprising administering to an individual in need thereof an effective amount of the preparation according to Embodiment 36 to treat the infectious disease.

[0473] Embodiment 45. Use of the preparation according to Embodiment 36 for inducing an immune response against the antigen in an individual in need thereof.

[0474] Embodiment 46. Use of the preparation according to Embodiment 36 for inducing an anti-tumor immune response in an individual in need thereof, wherein the individual has or has had a tumor.

[0475] Embodiment 47. Use of the preparation according to Embodiment 36 for inducing an anti-microbial immune response in an individual in need thereof, wherein the individual is infected with the microbe or has not been exposed to the microbe.

[0476] Embodiment 48. The composition, preparation, method or use according to any one of Embodiments 19-47, wherein the individual is a mammalian subject.

[0477] Embodiment 49. The composition, preparation, method or use according to any one of Embodiments 19-47, wherein the individual is a human subject.

[0478] Embodiment 50. A method for preparing an immunogenic composition, the method comprising:

[0479] a) removing white blood cells from a cell suspension prepared from a tumor to obtain a tumor cell-rich suspension;

[0480] b) lysing the cells from the tumor cell-rich suspension to obtain a tumor cell lysate; and

[0481] c) combining the tumor cell lysate with a separated ether lipid (ETL) of formula (I):

[0482]

[0483] wherein:

[0484] R 1 is H or

[0485] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0486] R 3 is C 13- C24 n-alkyl;

[0487] wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0488] each R 5 is independently a C1-C4 alkyl;

[0489] or its protonated form; or a pharmaceutically acceptable salt thereof; and

[0490] contacted with a toll-like receptor 7 / 8 (TLR7 / 8) agonist to obtain the immunogenic composition.

[0491] Embodiment 51. The method according to embodiment 50, wherein in step a), leukocytes are depleted by negative selection using an anti-CD45 antibody.

[0492] Embodiment 52. The method according to embodiment 50 or embodiment 51, wherein in step b), the cells are lysed by one or more freeze-thaw cycles.

[0493] Embodiment 53. The method according to any one of embodiments 50-52, wherein R3 is C 18 -C 22 alkyl or C 18 -C 24 alkyl.

[0494] Embodiment 54. The method according to embodiment 53, wherein the ETL comprises one or both of DGPC and DGP, or a pharmaceutically acceptable salt thereof.

[0495] Embodiment 55. The method according to any one of embodiments 50-54, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 daltons or less.

[0496] Embodiment 56. The method according to embodiment 55, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

[0497] Embodiment 57. The method according to embodiment 56, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

[0498] Embodiment 58. The method according to embodiment 55 or embodiment 56, wherein the TLR7 / 8 agonist does not inhibit NLR family pyrin domain containing 3 (NLRP3).

[0499] Embodiment 59. The method according to embodiment 54, 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).

[0500] Embodiment 60. The method according to any one of embodiments 50-59, the method further comprising obtaining a sample from a tumor of a mammalian subject suffering from cancer and preparing a cell suspension from the sample before step a).

[0501] Embodiment 61. An immunogenic composition prepared by the method according to any one of embodiments 50-60.

[0502] Embodiment 62. A method of eliciting an anti-cancer immune response, the method comprising administering to a mammalian subject suffering from cancer an effective amount of the immunogenic composition according to embodiment 61.

[0503] Embodiment 63. The method according to embodiment 62, wherein the anti-cancer immune response comprises a cellular immune response.

[0504] Embodiment 64. The method according to embodiment 63, wherein the anti-cancer immune response comprises cancer antigen-induced IL-1β secretion and / or activation of CD8+ T lymphocytes.

[0505] Embodiment 65. The method according to any one of embodiments 62-64, wherein the cancer is a non-blood cancer.

[0506] Embodiment 66. The method according to embodiment 65, wherein the non-blood cancer is a carcinoma, a sarcoma or a melanoma.

[0507] Embodiment 67. The method according to any one of embodiments 62-64, wherein the cancer is lymphoma.

[0508] Embodiment 68. A method of treating cancer, the method comprising:

[0509] a) preparing an immunogenic composition, the immunogenic composition comprising a tumor cell lysate, an isolated ether lipid (ETL) of formula (I):

[0510]

[0511] wherein:

[0512] R 1 is H or

[0513] R 2is H, a C1-C4 alkyl group, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0514] R 3 is a C 13- C 24 n-alkyl group;

[0515] wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0516] each R 5 is independently a C1-C4 alkyl group;

[0517] or its protonated form; or its pharmaceutically acceptable salt; and

[0518] a toll-like receptor 7 / 8 (TLR7 / 8) agonist, wherein the tumor cell lysate is prepared from or has been prepared from a tumor sample obtained from a mammalian subject having cancer; and

[0519] b) administering to the subject an effective amount of the immunogenic composition.

[0520] Embodiment 69. The method according to any one of Embodiments 62-68, wherein R 3 is a C 18 -C 22 alkyl chain or a C 18 -C 24 alkyl chain.

[0521] Embodiment 70. The method according to Embodiment 68, wherein the ETL comprises one or both of DGPC and DGP, or a pharmaceutically acceptable salt thereof.

[0522] Embodiment 71. The method according to any one of Embodiments 62-70, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 daltons or less.

[0523] Embodiment 72. The method according to Embodiment 71, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

[0524] Embodiment 73. The method according to Embodiment 72, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

[0525] Embodiment 74. The method according to embodiment 70, wherein the ETPL comprises DGPC or a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist comprises resiquimod (R848).

[0526] Embodiment 75. The method according to embodiment 70, wherein the ETPL comprises DGP or a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist comprises resiquimod (R848).

[0527] Embodiment 76. The method according to any one of claims 68 - 75, the method further comprising administering to the subject an effective amount of an additional therapeutic agent.

[0528] Embodiment 77. The method according to embodiment 76, wherein the additional therapeutic agent comprises one or more of the group consisting of: immune checkpoint inhibitors, anti - neoplastic agents, and radiotherapy.

[0529] Embodiment 78. A composition comprising a separated ether lipid (ETL) of formula (I):

[0530]

[0531] Wherein:

[0532] R 1 is H or

[0533] R 2 is H, C1 - C4 alkyl, -(C = O)-NH2, -(C = O)-NH(R 5 ), -(C = O)-N(R 5 )2 or -CH2 - C6H5;

[0534] R 3 is C 13- C 24 n - alkyl;

[0535] Wherein R 4 is H or (CH3)3N + -(CH2)2 -; and

[0536] each R 5 is independently C1 - C4 alkyl;

[0537] or its protonated form; or a pharmaceutically acceptable salt thereof; and

[0538] a pathogen recognition receptor (PRR) agonist.

[0539] Embodiment 79. The composition according to embodiment 78, wherein the PRR agonist is an agonist of toll-like receptor (TLR), NOD-like receptor (NLR), RIG-I-like receptor (RLR), or C-type lectin receptor (CLR).

[0540] Embodiment 80. The composition according to embodiment 78, wherein the PRR agonist is an agonist of cytosolic DNA sensor (CDS) or stimulator of IFN genes (STING).

[0541] Embodiment 81. The composition according to embodiment 78, wherein the PRR agonist comprises one or more of R848, TL8-506, LPS, Pam2CSK4, and ODN 2336.

[0542] Embodiment 82. The composition according to any one of embodiments 78-81, further comprising an antigen.

[0543] Embodiment 83. The composition according to any one of embodiments 78-82, further comprising dendritic cells.

[0544] Embodiment 84. A pharmaceutical preparation comprising the composition according to any one of embodiments 78-83 and a pharmaceutically acceptable excipient.

[0545] Embodiment 85. A pharmaceutical preparation comprising an isolated ether lipid (ETL) of formula (I):

[0546]

[0547] Wherein:

[0548] R 1 is H or

[0549] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0550] R 3 is C 13- C 24 n-alkyl;

[0551] Wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0552] Each R 5 is independently C1-C4 alkyl;

[0553] or its protonated form; or a pharmaceutically acceptable salt thereof; and

[0554] a pharmaceutically acceptable excipient.

[0555] Embodiment 86. The pharmaceutical preparation according to Embodiment 84 or Embodiment 85, wherein the alkyl chain is a C22 normal alkyl chain.

[0556] Embodiment 87. The pharmaceutical preparation according to Embodiment 86, wherein the ETL comprises one or both of DGPC and DGP or a pharmaceutically acceptable salt thereof.

[0557] Embodiment 88. A composition for overactivation of human dendritic cells, the composition comprising an isolated ether lipid (ETL) of formula (I):

[0558]

[0559] wherein:

[0560] R 1 is H or

[0561] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0562] R 3 is C 13- C 24 normal alkyl;

[0563] wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0564] each R 5 is independently C1-C4 alkyl;

[0565] or its protonated form; or a pharmaceutically acceptable salt thereof; and

[0566] a pathogen recognition receptor (PRR) agonist, wherein the alkyl chain is a C22 normal alkyl chain, and wherein the composition effectively achieves a higher level of dendritic cell overactivation compared to a comparative composition comprising PGPC instead of ETL.

[0567] Embodiment 89. The composition according to Embodiment 88, wherein R 3 is C 22 normal alkyl.

[0568] Embodiment 90. The composition according to Embodiment 88 or Embodiment 89, wherein the higher level of dendritic cell over-activation comprises in vitro inducing a level of IL-1β secretion from human dendritic cells that is at least 2, 3, or 4 times higher when contacting with a composition comprising ETL and a PRR agonist compared to when contacting with a comparative composition comprising PGPC and a PRR agonist, wherein the PRR agonist is LPS.

[0569] Embodiment 91. The composition according to Embodiment 90, wherein the concentration of the ETL and the concentration of the PGPC are the same concentration within the range of about 10 μM to about 80 μM, and LPS is present in both the composition and the comparative composition at a concentration of 1 μg / ml.

[0570] Embodiment 92. The composition according to Embodiment 90, wherein the higher level of dendritic cell over-activation comprises a lipid activity index for IL-1β secretion from human dendritic cells that is at least 4, 5, or 6 times higher in a composition comprising ETL and a PRR agonist compared to a comparative composition comprising PGPC and a PRR agonist, in terms of activity units.

[0571] Embodiment 93. The composition, formulation, method, or use according to any one of Embodiments 19 - 47, wherein the individual is a human subject.

[0572] Embodiment 94. The composition, formulation, method, or use according to any one of Embodiments 19 - 47, wherein the individual is a canine subject.

[0573] Embodiment 95. The composition, formulation, method, or use according to any one of Embodiments 60 - 92, wherein the mammalian subject is a human patient.

[0574] Embodiment 96. The composition, formulation, method, or use according to any one of Embodiments 60 - 92, wherein the mammalian subject is a non-human patient.

[0575] Embodiment 97. The composition, formulation, method, or use according to any one of Embodiments 60 - 92, wherein the mammalian subject is a canine patient.

[0576] Embodiment 98. The composition, formulation, method, or use according to any one of Embodiments 1 - 93 or 95, wherein the dendritic cells are human dendritic cells.

[0577] Embodiment 99. The composition, formulation, method, or use according to any one of Embodiments 1 - 48, 50 - 87, or 97, wherein the dendritic cells are canine dendritic cells.

[0578] Embodiment 100. The composition, method or use according to Embodiment 98 or Embodiment 99, wherein the dendritic cells are present in a composition comprising peripheral blood mononuclear cells (PBMC).

[0579] Embodiment 101. The composition, method or use according to any one of Embodiments 37-49 or Embodiments 98-99, wherein the over-activated dendritic cells secrete one or both of IFNγ and TNFα.

[0580] Embodiment 102. The composition, formulation, method or use according to any one of Embodiments 1-101, which comprises a surfactant.

[0581] Embodiment 103. The composition, formulation, method or use according to Embodiment 102, wherein the surfactant comprises a non-ionic surfactant.

[0582] Embodiment 104. The composition, formulation, method or use according to Embodiment 103, wherein the non-ionic surfactant comprises an ethylene oxide-propylene oxide copolymer.

[0583] Embodiment 105. The composition, formulation, method or use according to Embodiment 103, wherein the non-ionic surfactant comprises one or more of poloxamer 407, poloxamer 188 and P123.

[0584] Embodiment 106. The composition, formulation, method or use according to Embodiment 103, wherein the non-ionic surfactant comprises poloxamer 407.

[0585] Embodiment 107. The composition, formulation, method or use according to any one of Embodiments 103-106, wherein i) the ETL is dissolved in an 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.

[0586] Embodiment 108. The composition, formulation, method or use according to any one of Embodiments 103-107, 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).

[0587] Embodiment 109. The composition, formulation, method or use according to any one of Embodiments 103-108, wherein the ETL and the non-ionic surfactant are present in particles having a diameter of about 1000 to 15,000 nanometers, optionally a diameter of about 5000 nanometers.

[0588] Embodiment A1. A composition comprising a separated ether lipid (ETL) of formula (I):

[0589]

[0590] Wherein:

[0591] R 1 is H or

[0592] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0593] R 3 is C 13- C 24 n-alkyl;

[0594] Wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0595] each R 5 is independently C1-C4 alkyl;

[0596] or its protonated or deprotonated form; or its pharmaceutically acceptable salt; and

[0597] a TLR agonist.

[0598] Embodiment A2. The composition according to Embodiment A1, wherein the TLR agonist comprises a TLR7 / 8 agonist.

[0599] Embodiment A3. The composition according to Embodiment A1 or Embodiment A2, wherein R 3 is C 18 -C 22 n-alkyl or C 21 -C 24 n-alkyl.

[0600] Embodiment A4. The composition according to any one of Embodiments A1-A3, wherein R 3 is C 16 -C 20 n-alkyl.

[0601] Embodiment A5. The composition according to any one of Embodiments A1 - A4, further comprising an antigen.

[0602] Embodiment A6. The composition according to any one of Embodiments A1 - A5, further comprising dendritic cells.

[0603] Embodiment A7. A composition comprising an isolated ether lipid (ETL) of formula (I):

[0604]

[0605] Wherein:

[0606] R 1 is H or

[0607] R 2 is H, C1 - C4 alkyl, -(C = O)-NH2, -(C = O)-NH(R 5 ), -(C = O)-N(R 5 )2 or -CH2 - C6H5;

[0608] R 3 is C 13- C 24 n - alkyl;

[0609] Where R 4 is H or (CH3)3N + -(CH2)2 -; and

[0610] Each R 5 is independently C1 - C4 alkyl;

[0611] or its protonated or de - protonated form; or its pharmaceutically acceptable salt; and

[0612] an antigen.

[0613] Embodiment A8. The composition according to Embodiment A7, further comprising dendritic cells.

[0614] Embodiment A9. The composition according to Embodiment A7 or Embodiment A8, further comprising a TLR agonist.

[0615] Embodiment A10. The composition according to Embodiment A9, wherein the TLR agonist comprises a TLR7 / 8 agonist.

[0616] Embodiment A11. A composition comprising an isolated ether lipid (ETL) of formula (I):

[0617]

[0618] Wherein:

[0619] R 1 is H or

[0620] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0621] R 3 is C 13- C 24 n-alkyl;

[0622] Wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0623] each R 5 is independently C1-C4 alkyl;

[0624] or its protonated or deprotonated form; or its pharmaceutically acceptable salt; and

[0625] dendritic cells.

[0626] Embodiment A12. The composition according to Embodiment A11, further comprising a TLR agonist.

[0627] Embodiment A13. The composition according to Embodiment A12, wherein the TLR agonist comprises a TLR7 / 8 agonist.

[0628] Embodiment A14. The composition according to any one of Embodiments A11-A13, further comprising an antigen.

[0629] Embodiment A15. The composition according to any one of Embodiments A1-A14, wherein R 3 is C 22 n-alkyl.

[0630] Embodiment A16. The composition according to any one of Embodiments A1-A15, wherein the ETL is an ether phospholipid (ETPL) comprising 1-didodecyl-sn-glycero-3-phosphocholine (DGPC) or its pharmaceutically acceptable salt.

[0631] Embodiment A17. The composition according to any one of Embodiments A1-A15, wherein the ETL is an ETPL comprising 1-didodecyl-sn-glycero-3-phosphate (DGP) or its pharmaceutically acceptable salt.

[0632] Embodiment A18. The composition according to any one of embodiments A1 - A17, wherein the TLR agonist is a small molecule having a molecular weight of 900 daltons or less.

[0633] Embodiment A19. The composition according to any one of embodiments A1 - A18, wherein the TLR agonist comprises a TLR7 / 8 agonist.

[0634] Embodiment A20. The composition according to embodiment A19, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

[0635] Embodiment A21. The composition according to embodiment A19, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

[0636] Embodiment A22. The composition according to any one of embodiments A18 - A20, wherein the TLR7 / 8 agonist does not inhibit NLR family pyrin domain containing 3 (NLRP3).

[0637] Embodiment A23. The composition according to any one of embodiments A1 - A14, wherein the ETPL comprises one or both of DGPC and DGP, and the TLR7 / 8 agonist comprises resiquimod (R848).

[0638] Embodiment A24. The composition according to any one of embodiments A1 - A23, wherein the antigen is present in a biological sample obtained from an individual.

[0639] Embodiment A25. The composition according to embodiment A24, wherein the biological sample comprises biopsy tissue.

[0640] Embodiment A26. The composition according to embodiment A24, wherein the biological sample comprises cells.

[0641] Embodiment A27. The composition according to embodiment A24, wherein the biological sample does not comprise cells.

[0642] Embodiment A28. The composition according to embodiment A24, wherein the biological sample comprises pus from an abscess.

[0643] Embodiment A29. The composition according to any one of embodiments A1 - A28, wherein the antigen comprises a protein antigen.

[0644] Embodiment A30. The composition according to embodiment A29, wherein the antigen comprises a tumor antigen.

[0645] Embodiment A31. The composition as described in Embodiment A30, wherein the tumor antigen comprises a synthetic or recombinant neoantigen.

[0646] Embodiment A32. The composition as described in Embodiment A30, wherein the tumor antigen comprises a tumor cell lysate.

[0647] Embodiment A33. The composition as described in Embodiment A29, wherein the antigen comprises a microbial antigen, and the microbial antigen comprises one or more of a viral antigen, a bacterial antigen, a protozoal antigen, and a fungal antigen.

[0648] Embodiment A34. The composition as described in Embodiment A33, wherein the microbial antigen comprises a purified or recombinant surface protein.

[0649] Embodiment A35. The composition as described in Embodiment A33, wherein the microbial antigen comprises an inactivated whole virus.

[0650] Embodiment A36. The composition as described in any one of Embodiments A1 - A35, wherein the composition does not contain liposomes.

[0651] Embodiment A37. The composition as described in any one of Embodiments A1 - A36, wherein the composition does not contain LPS or MPLA.

[0652] Embodiment A38. The composition as described in any one of Embodiments A1 - A37, wherein the composition does not contain oxPAPC or oxPAPC species, optionally wherein the composition does not contain HOdiA - PC, KOdiA - PC, HOOA - PC, KOOA - PC, and / or PGPC.

[0653] Embodiment A39. The composition as described in any one of Embodiments A1 - A38, wherein the composition does not contain lysophosphatidylcholine (LPC), optionally wherein the composition does not contain 1 - dodecanoyl - 2 - hydroxy - sn - glycero - 3 - phosphocholine [LPC(22:0)].

[0654] Embodiment A40. The composition as described in any one of Embodiments A1 - A39, which further comprises an adjuvant, wherein the adjuvant comprises an aluminum salt adjuvant, a water - in - squalene emulsion, saponin, or a combination thereof.

[0655] Embodiment A41. A pharmaceutical formulation comprising the composition as described in any one of Embodiments A1 - A40 and a pharmaceutically acceptable excipient.

[0656] Embodiment A42. A method for generating hyperactivated dendritic cells, the method comprising contacting the dendritic cells with a composition comprising an effective amount of an isolated ether lipid (ETL) of formula (I):

[0657]

[0658] Wherein:

[0659] R 1 is H or

[0660] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0661] R 3 is C 13- C 24 n-alkyl;

[0662] Where R 4 is H or (CH3)3N + -(CH2)2-; and

[0663] each R 5 is independently C1-C4 alkyl;

[0664] or its protonated or deprotonated form; or its pharmaceutically acceptable salt; and

[0665] a TLR7 / 8 agonist, to generate hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing pyroptosis.

[0666] Embodiment A43. The method according to embodiment A42, wherein the dendritic cells are contacted ex vivo with the composition according to any one of embodiments A1-A40 or the formulation according to embodiment A41.

[0667] Embodiment A44. The method according to embodiment A42, wherein the dendritic cells are contacted in vivo with the formulation according to embodiment A41.

[0668] Embodiment A45. A pharmaceutical formulation comprising at least 10 3 、10 4 、10 5 or 10 6 hyperactivated dendritic cells generated by the method according to embodiment A43 and a pharmaceutically acceptable excipient.

[0669] Embodiment A46. A method of stimulating an immune response against an antigen, the method comprising administering to an individual in need thereof an effective amount of the preparation of Embodiment A41 to stimulate an immune response against the antigen.

[0670] Embodiment A47. A method of treating cancer, the method comprising administering to an individual in need thereof an effective amount of the preparation of Embodiment A41 to treat the cancer.

[0671] Embodiment A48. A method of inhibiting abnormal cell proliferation, the method comprising administering to an individual in need thereof an effective amount of the preparation of Embodiment A41 to inhibit abnormal cell proliferation.

[0672] Embodiment A49. A method of treating an infectious disease, the method comprising administering to an individual in need thereof an effective amount of the preparation of Embodiment A41 to treat the infectious disease.

[0673] Embodiment A50. Use of the preparation of Embodiment A41 for inducing an immune response against the antigen in an individual in need thereof.

[0674] Embodiment A51. Use of the preparation 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.

[0675] Embodiment A52. Use of the preparation of Embodiment A41 for inducing an anti-microbial immune response in an individual in need thereof, wherein the individual is infected with the microbe or has not been exposed to the microbe.

[0676] Embodiment A53. The composition, preparation, method or use according to any one of Embodiments A24 - A52, wherein the individual is a mammalian subject.

[0677] Embodiment A54. The composition, preparation, method or use according to any one of Embodiments A24 - A52, wherein the individual is a human subject.

[0678] Embodiment A55. A method of preparing an immunogenic composition, the method comprising:

[0679] a) removing white blood cells from a cell suspension prepared from a tumor to obtain a tumor cell-rich suspension;

[0680] b) lysing the cells from the tumor cell-rich suspension to obtain a tumor cell lysate; and

[0681] c) combining the tumor cell lysate with a separated ether lipid (ETL) of formula (I):

[0682]

[0683] Wherein:

[0684] R 1 is H or

[0685] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0686] R 3 is C 13- C 24 n-alkyl;

[0687] Wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0688] each R 5 is independently C1-C4 alkyl;

[0689] or its protonated or deprotonated form; or its pharmaceutically acceptable salt; and

[0690] contacted with a toll-like receptor (TLR) agonist to obtain the immunogenic composition.

[0691] Embodiment A56. The method according to embodiment A55, wherein the TLR agonist comprises a TLR7 / 8 agonist.

[0692] Embodiment A57. The method according to embodiment A55 or embodiment A56, wherein in step a), leukocytes are depleted by negative selection using an anti-CD45 antibody.

[0693] Embodiment A58. The method according to any one of embodiments A55-A57, wherein in step b), cells are lysed by one or more freeze-thaw cycles.

[0694] Embodiment A59. The method according to any one of embodiments A55-A58, wherein R 3 is C 18 -C 22 alkyl or C 18 -C 24 alkyl.

[0695] Embodiment A60. The method according to any one of embodiments A55-A58, wherein R 3 is C 16 -C20 Alkyl group.

[0696] Embodiment A61. The method according to any one of embodiments A55 - A58, wherein R 3 is C 21 -C 24 alkyl group.

[0697] Embodiment A62. The method according to embodiment A59 or embodiment A61, wherein the ETL comprises one or both of DGPC and DGP or a pharmaceutically acceptable salt thereof.

[0698] Embodiment A63. The method according to any one of embodiments A55 - A62, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 daltons or less.

[0699] Embodiment A64. The method according to embodiment A63, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

[0700] Embodiment A65. The method according to embodiment A64, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

[0701] Embodiment A66. The method according to any one of embodiments A63 - A65, wherein the TLR7 / 8 agonist does not inhibit NLR family pyrin domain containing 3 (NLRP3).

[0702] Embodiment A67. The method according to embodiment A62, 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).

[0703] Embodiment A68. The method according to any one of embodiments A55 - A67, the method further comprising obtaining a sample from a tumor of a mammalian subject having cancer and preparing a cell suspension from the sample before step a).

[0704] Embodiment A69. An immunogenic composition prepared by the method according to any one of embodiments A55 - A68.

[0705] Embodiment A70. A method of eliciting an anti - cancer immune response, the method comprising:

[0706] administering to a mammalian subject having cancer an effective amount of the immunogenic composition according to embodiment A69.

[0707] Embodiment A71. The method according to embodiment A70, wherein the anti-cancer immune response comprises a cellular immune response.

[0708] Embodiment A72. The method according to embodiment A63, wherein the anti-cancer immune response comprises cancer antigen-induced IL-1β secretion and / or activation of CD8+ T lymphocytes.

[0709] Embodiment A73. The method according to any one of embodiments A62 - A64, wherein the cancer is a non-blood cancer.

[0710] Embodiment A74. The method according to embodiment A65, wherein the non-blood cancer is a carcinoma, sarcoma or melanoma.

[0711] Embodiment A75. The method according to any one of embodiments A70 - A74, wherein the cancer is lymphoma.

[0712] Embodiment A76. A method for treating cancer, the method comprising:

[0713] a) preparing an immunogenic composition comprising a tumor cell lysate, an isolated ether lipid (ETL) of formula (I):

[0714]

[0715] wherein:

[0716] R 1 is H or

[0717] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0718] R 3 is C 13- C 24 n-alkyl;

[0719] wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0720] each R 5 is independently C1-C4 alkyl;

[0721] or its protonated or deprotonated form; or its pharmaceutically acceptable salt; and

[0722] Toll-like receptor (TLR) agonist, wherein the tumor cell lysate is or has been prepared from a tumor sample obtained from a mammalian subject suffering from cancer; and

[0723] b) administering to the subject an effective amount of the immunogenic composition.

[0724] Embodiment A77. The method according to embodiment A76, wherein the TLR agonist comprises a TLR7 / 8 agonist.

[0725] Embodiment A78. The method according to any one of embodiments A70 - A77, wherein R 3 is C 18 -C 22 alkyl chain or C 18 -C 24 alkyl chain.

[0726] Embodiment A79. The method according to any one of embodiments A70 - A77, wherein R 3 is C 16 -C 20 alkyl.

[0727] Embodiment A80. The method according to any one of embodiments A70 - A77, wherein R 3 is C 21 -C 24 alkyl.

[0728] Embodiment A81. The method according to embodiment A76 or embodiment A77, wherein the ETL comprises one or both of DGPC and DGP or a pharmaceutically acceptable salt thereof.

[0729] Embodiment A82. The method according to any one of embodiments A70 - A81, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 daltons or less.

[0730] Embodiment A83. The method according to embodiment A82, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

[0731] Embodiment A84. The method according to embodiment A83, wherein the TLR7 / 8 agonist comprises resiquimod (R848).

[0732] Embodiment A85. The method according to embodiment A81, wherein the ETPL comprises DGPC or a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist comprises resiquimod (R848).

[0733] Embodiment A86. The method according to Embodiment A81, wherein the ETPL comprises DGP or a pharmaceutically acceptable salt thereof, and the TLR7 / 8 agonist comprises resiquimod (R848).

[0734] Embodiment A87. The method according to any one of Technical Solutions 68-75, the method further comprising administering to the subject an effective amount of an additional therapeutic agent.

[0735] Embodiment A88. The method according to Embodiment A76, wherein the additional therapeutic agent comprises one or more of the group consisting of: immune checkpoint inhibitors, anti-neoplastic agents, and radiotherapy.

[0736] Embodiment A89. A composition comprising a separated ether lipid (ETL) of formula (I):

[0737]

[0738] Wherein:

[0739] R 1 is H or

[0740] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0741] R 3 is C 13- C 24 n-alkyl;

[0742] Wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0743] Each R 5 is independently C1-C4 alkyl;

[0744] or its protonated or deprotonated form; or a pharmaceutically acceptable salt thereof; and

[0745] A pathogen recognition receptor (PRR) agonist.

[0746] Embodiment A90. The composition according to Embodiment A89, wherein the PRR agonist is an agonist of toll-like receptor (TLR), NOD-like receptor (NLR), RIG-I-like receptor (RLR), or C-type lectin receptor (CLR).

[0747] Embodiment A91. The composition according to Embodiment A89, wherein the PRR agonist is an agonist of cytosolic DNA sensors (CDS) or a stimulator of IFN genes (STING).

[0748] Embodiment A92. The composition according to Embodiment A89, wherein the PRR agonist comprises one or more of R848, TL8-506, LPS, Pam2CSK4, and ODN 2336.

[0749] Embodiment A93. The composition according to any one of Embodiments A89-A92, further comprising an antigen.

[0750] Embodiment A94. The composition according to any one of Embodiments A89-A93, further comprising dendritic cells.

[0751] Embodiment A95. A pharmaceutical preparation comprising the composition according to any one of Embodiments A89-A94 and a pharmaceutically acceptable excipient.

[0752] Embodiment A96. A pharmaceutical preparation comprising an isolated ether lipid (ETL) of formula (I):

[0753]

[0754] Wherein:

[0755] R 1 is H or

[0756] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0757] R 3 is C 13- C 24 n-alkyl;

[0758] Wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0759] each R 5 is independently C1-C4 alkyl;

[0760] or its protonated or deprotonated form; or its pharmaceutically acceptable salt; and

[0761] a pharmaceutically acceptable excipient.

[0762] Pharmaceutical preparation according to embodiment A95 or embodiment A96, wherein the alkyl chain is a C22 normal alkyl chain.

[0763] Embodiment A98. The pharmaceutical preparation according to embodiment A97, wherein the ETL comprises one or both of DGPC and DGP or a pharmaceutically acceptable salt thereof.

[0764] Embodiment A99. A composition for the overactivation of human dendritic cells, the composition comprising an isolated ether lipid (ETL) of formula (I):

[0765]

[0766] Wherein:

[0767] R 1 is H or

[0768] R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0769] R 3 is C 13- C 24 normal alkyl;

[0770] wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0771] each R 5 is independently C1-C4 alkyl;

[0772] or its protonated or deprotonated form; or a pharmaceutically acceptable salt thereof; and

[0773] a pathogen recognition receptor (PRR) agonist, wherein the alkyl chain is a C22 normal alkyl chain, and wherein the composition effectively achieves a higher level of dendritic cell overactivation compared to a comparative composition comprising PGPC instead of ETL.

[0774] Embodiment A100. The composition according to embodiment A99, wherein R 3 is C 22 normal alkyl.

[0775] Embodiment A101. The composition as described in Embodiment A99 or Embodiment A100, wherein the higher level of dendritic cell overactivation comprises at least 2, 3, or 4 times higher levels of in vitro induced secretion of IL-1β from human dendritic cells when contacting with a composition comprising ETL and a PRR agonist compared to when contacting with a comparative composition comprising PGPC and a PRR agonist, wherein the PRR agonist is LPS.

[0776] Embodiment A102. The composition as described in Embodiment A101, wherein the concentration of the ETL and the concentration of the PGPC are the same concentration within the range of about 10 μM to about 80 μM, and the LPS is present at a concentration of 1 μg / ml in both the composition and the comparative composition.

[0777] Embodiment A103. The composition as described in Embodiment A101, wherein the higher level of dendritic cell overactivation comprises at least 4, 5, or 6 times higher lipid activity index of IL-1β secreted from human dendritic cells by a composition comprising ETL and a PRR agonist compared to a comparative composition comprising PGPC and a PRR agonist, in terms of activity units.

[0778] Embodiment A104. The composition, formulation, method, or use as described in any one of Embodiments A24 - A52, wherein the individual is a human subject.

[0779] Embodiment A105. The composition, formulation, method, or use as described in any one of Embodiments A24 - A52, wherein the individual is a canine subject.

[0780] Embodiment A106. The composition, formulation, method, or use as described in any one of Embodiments A68 - A103, wherein the mammalian subject is a human patient.

[0781] Embodiment A107. The composition, formulation, method, or use as described in any one of Embodiments A68 - A103, wherein the mammalian subject is a non - human patient.

[0782] Embodiment A108. The composition, formulation, method, or use as described in any one of Embodiments A68 - A103, wherein the mammalian subject is a canine patient.

[0783] Embodiment A109. The composition, formulation, method, or use as described in any one of Embodiments A1 - A104 or A106, wherein the dendritic cells are human dendritic cells.

[0784] Embodiment A110. A composition, preparation, method or use according to any one of Embodiments A1 - A53, A55 - A98 or A108, wherein the dendritic cells are canine dendritic cells.

[0785] 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 (PBMC).

[0786] Embodiment A112. A composition, method or use according to any one of Embodiments A42 - A54 or Embodiments A109 - A110, wherein the overactivated dendritic cells secrete one or both of IFNγ and TNFα.

[0787] Embodiment A113. A composition, preparation, method or use according to any one of Embodiments A1 - A112, which comprises a surfactant.

[0788] Embodiment A114. A composition, preparation, method or use according to Embodiment A113, wherein the surfactant comprises a nonionic surfactant.

[0789] Embodiment A115. A composition, preparation, method or use according to Embodiment A114, wherein the nonionic surfactant comprises an ethylene oxide - propylene oxide copolymer.

[0790] Embodiment A116. A composition, preparation, method or use according to Embodiment A114, wherein the nonionic surfactant comprises one or more of poloxamer 407, poloxamer 188 and P123.

[0791] Embodiment A117. A composition, preparation, method or use according to Embodiment A114, wherein the nonionic surfactant comprises poloxamer 407.

[0792] Embodiment A118. A composition, preparation, method or use according to any one of Embodiments A114 - A117, wherein i) the ETL is dissolved in an alcohol to form an ETL alcohol solution; ii) the ETL alcohol solution is mixed with a nonionic surfactant to form a mixture; and iii) the alcohol is evaporated from the mixture to form particles comprising the ETL and the nonionic surfactant.

[0793] Embodiment A119. The composition, formulation, method or use according to any one of embodiments A104 - A118, 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).

[0794] Embodiment A120. The composition, formulation, method or use according to any one of embodiments A104 - A119, wherein the ETL and the non-ionic surfactant are present in particles having a diameter of about 1000 to 15,000 nanometers, optionally a diameter of about 5000 nanometers.

[0795] Embodiment A121. An isolated ether lipid (ETL) of formula (I):

[0796]

[0797] Formula (I)

[0798] wherein R 1 is H or

[0799] R 2 is H, C1 - C4 alkyl, -(C = O)-NH2, -(C = O)-NH(R 5 ), -(C = O)-N(R 5 )2 or -CH2 - C6H5;

[0800] R 3 is C 13- C 24 n-alkyl;

[0801] wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0802] each R 5 is independently C1 - C4 alkyl;

[0803] or its protonated or deprotonated form; or its salt.

[0804] Embodiment A122. The isolated ether lipid according to embodiment A121, wherein the isolated ether lipid is a compound of formula (II):

[0805]

[0806] wherein R 1 is H or

[0807] R 2is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0808] R 3 is C 13- C 24 n-alkyl;

[0809] wherein R 4 is H or (CH3)3N + -(CH2)2-; and

[0810] each R 5 is independently C1-C4 alkyl;

[0811] or its protonated or deprotonated form; or its salt.

[0812] Embodiment A123. The separated ether lipid as described in Embodiment A121, wherein the separated ether lipid is a compound of formula (III):

[0813]

[0814] wherein R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0815] R 3 is C 13- C 24 n-alkyl; and

[0816] each R 5 is independently C1-C4 alkyl;

[0817] or its salt.

[0818] Embodiment A124. The separated ether lipid as described in Embodiment A121, wherein the separated ether lipid is a separated ether phospholipid (ETPL) compound of formula (IV):

[0819]

[0820] wherein R 2 is H, C1-C4 alkyl, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0821] R3 is C 13- C 24 is a normal alkyl group;

[0822] R 4 is H or (CH3)3N + -(CH2)2-; and

[0823] each R 5 is independently a C1-C4 alkyl group;

[0824] or its protonated or deprotonated form; or its salt.

[0825] Embodiment A125. The separated ether lipid as described in Embodiment A121, wherein the separated ether lipid is a separated ether phospholipid (ETPL) compound of formula (IV-A):

[0826]

[0827] wherein R 2 is H, a C1-C4 alkyl group, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0828] R 3 is C 13- C 24 is a normal alkyl group; and

[0829] each R 5 is independently a C1-C4 alkyl group;

[0830] or its protonated or deprotonated form; or its salt.

[0831] Embodiment A126. The separated ether lipid as described in Embodiment A121, wherein the separated ether lipid is a separated ether phospholipid (ETPL) compound of formula (IV-B):

[0832]

[0833] wherein R 2 is H, a C1-C4 alkyl group, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5;

[0834] R 3 is C 13- C 24 is a normal alkyl group; and

[0835] each R 5Independently a C1-C4 alkyl group;

[0836] or its protonated form; or its salt.

[0837] Embodiment A127. The separated ether lipid as described in Embodiment A121, wherein the separated ether lipid is a separated ether phospholipid (ETPL) compound of formula (IV-C):

[0838]

[0839] wherein R 3 is a C 13- C 24 n-alkyl group; and

[0840] R 4 is H or (CH3)3N + -(CH2)2-;

[0841] or its protonated or deprotonated form; or its salt.

[0842] Embodiment A128. A compound of formula 2:

[0843]

[0844] or its protonated form; or its pharmaceutically acceptable salt.

[0845] Embodiment A129. The compound as described in Embodiment A128, wherein the compound is separated.

[0846] Embodiment A130. A separated compound 1 of formula 1:

[0847]

[0848] or its protonated form; or its pharmaceutically acceptable salt.

[0849] Embodiment A131. A compound of formula (III-A-1):

[0850]

[0851] wherein:

[0852] R 2 is -(C=O)-NH2, -(C=O)-NH(R 5 ) or -(C=O)-N(R 5 )2;

[0853] R 3 is a C 21- C 24 n-alkyl group; and

[0854] Each R 5 is independently a C1-C4 alkyl group;

[0855] or a pharmaceutically acceptable salt thereof.

[0856] Embodiment A132. A compound as described in Embodiment A131, wherein R 2 is -(C=O)-NH2.

[0857] Embodiment A133. A compound as described in Embodiment A131, wherein R 2 is -(C=O)-NH-CH3.

[0858] Embodiment A134. A compound as described in Embodiment A131, wherein R2 is -(C=O)-N(CH3)2.

[0859] Embodiment A135. A compound as described in any one of Embodiments A131-A134, wherein R 3 is C 22 n-alkyl.

[0860] Embodiment A136. A compound of formula 7:

[0861]

[0862] or a pharmaceutically acceptable salt thereof.

[0863] Embodiment A137. A compound as in Embodiment A136, wherein the compound is isolated.

[0864] Embodiment A138. A compound of formula 8:

[0865]

[0866] or a pharmaceutically acceptable salt thereof.

[0867] Embodiment A139. A compound as in Embodiment A138, wherein the compound is isolated.

[0868] Embodiment A140. A composition comprising a compound as described in any one of Embodiments A121-A139 and a pharmaceutically acceptable excipient.

[0869] Embodiment A141. A composition as in Embodiment A140, wherein the pharmaceutically acceptable excipient comprises phosphate buffered saline.

[0870] Embodiment A142. A composition as in Embodiment A140, wherein the pharmaceutically acceptable excipient comprises an aqueous solution of poloxamer 407.

[0871] Embodiment A143. The composition according to Embodiment A140, wherein the pharmaceutically acceptable excipient comprises phosphate buffered saline and poloxamer 407.

[0872] Embodiment A144. The composition according to any one of Embodiments A140 - A143, wherein the composition is sterile.

[0873] Embodiment A145. An article comprising a container encapsulating a liquid formulation of a compound according to any one of Embodiments A121 - A139 and a pharmaceutically acceptable excipient.

[0874] Embodiment A146. The article according to Embodiment A145, wherein the container is a syringe.

[0875] Embodiment A147. The article according to Embodiment A146, wherein the syringe is further contained within an injection device.

[0876] Embodiment A148. The article according to Embodiment A147, wherein the injection device is an auto - injector.

[0877] Embodiment A149. A composition comprising a separated ether lipid (ETL) or ether phospholipid (ETPL) 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, 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 its protonated or de - protonated form (where possible) or its pharmaceutically acceptable salt; and at least one other lipid,

[0878] wherein the at least one other lipid is selected from the group consisting of: ionizable lipids, cationic lipids, another phospholipid, polyethylene glycolated lipids, structured lipids, and mixtures thereof.

[0879] Embodiment A150. The composition according to Embodiment A149, wherein the ETL or ETPL and the at least one other lipid are part of a lipid nanoparticle (LNP).

[0880] Embodiment A151. The composition as described in Embodiment A149 or Embodiment A150, further comprising an antigen.

[0881] Embodiment A152. The composition as described in any one of Embodiments A149 - A151, further comprising dendritic cells.

[0882] Embodiment A153. The composition as described in any one of Embodiments A149 - A152, further comprising a TLR agonist.

[0883] Embodiment A154. The composition as described in any one of Embodiments A149 - A152, further comprising a TLR7 / 8 agonist.

[0884] Embodiment A155. The composition as described in any one of Embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (II) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0885] Embodiment A156. The composition as described in any one of Embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (III) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0886] Embodiment A157. The composition as described in any one of Embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (III - A) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0887] Embodiment A158. The composition as described in any one of Embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (III - A - 1) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0888] Embodiment A159. The composition as described in any one of Embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (III - A - 2) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0889] Embodiment A160. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (III - B) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0890] Embodiment A161. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (III - B - 1) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0891] Embodiment A162. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (III - B - 2) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0892] Embodiment A163. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0893] Embodiment A164. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV - A) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0894] Embodiment A165. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV - A - 1) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0895] Embodiment A166. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV - A - 2) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0896] Embodiment A167. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV - B) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0897] Embodiment A168. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV - B - 1) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0898] Embodiment A169. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV - B - 2) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0899] Embodiment A170. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV - C) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0900] Embodiment A171. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV - D) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0901] Embodiment A172. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is a compound of formula (IV - E) or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0902] Embodiment A173. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is Compound 1 or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0903] Embodiment A174. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is Compound 2 or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0904] Embodiment A175. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is Compound 3 or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0905] Embodiment A176. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 4 or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0906] Embodiment A177. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 5 or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0907] Embodiment A178. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 6 or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0908] Embodiment A179. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 7 or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0909] Embodiment A180. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 8 or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0910] Embodiment A181. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 9 or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0911] Embodiment A182. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 10 or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0912] Embodiment A183. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 11 or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0913] Embodiment A184. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 12 or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0914] Embodiment A185. The composition according to any one of embodiments A149 - A154, wherein the ether lipid (ETL) or ether phospholipid (ETPL) is compound 13 or its protonated or deprotonated form (where possible), or a pharmaceutically acceptable salt thereof.

[0915] Synthesis process

[0916] The following synthesis process describes a general synthesis procedure, which can be used as described or further modified or combined to prepare the compounds disclosed herein. Additionally, the chemical reactions in the synthesis 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 successfully carried out by modifications known to those skilled in the art, such as by using alternative protecting groups, by utilizing other suitable reagents known in the art in addition to those described reagents, or by making routine modifications to the reaction conditions. Other reactions disclosed herein or known in the art will be recognized as suitable for preparing other compounds disclosed herein.

[0917] Process 1: Synthesis of compound of formula IV - D

[0918]

[0919] The compound of formula (IV - D) can be readily prepared according to Process 1. The 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). Bu2SnO (2.89 g, 0.0116 mol) can be used to form intermediate SC - 1 - 2, which is then reacted with R 3 -Br, where R 3 is C 13 -C 24 n - alkyl to produce the compound of formula (IV - D).

[0920] Process 2: Synthesis of compound of formula III - B

[0921]

[0922] The compound of formula (III - B) can be prepared according to Process 2. Alcohol SC - 2 - 1 (where R3 is C 13 -C 24 The n-alkyl) reacts 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. Opening the dioxolane ring with acetic acid gives the compound of formula (III-B).

[0923] Scheme 3: Synthesis of the compound of formula III, R 2 = benzyl

[0924]

[0925] Scheme 3 outlines the synthesis of the following: the compound of formula (III), where R 2 is benzyl, and the compound of formula (IV-A), where R 2 = benzyl. Starting with the compound of formula (III-B) prepared in Scheme 2, the terminal hydroxyl group is protected, for example, with a TBDPSCl group. Then benzyl bromide is added to the unprotected 2-hydroxy group. The terminal hydroxyl group is deprotected to give the compound of formula (III), where R 2 is benzyl and R 3 is C 13 -C 24 n-alkyl.

[0926] Scheme 4: Synthesis of the compound of formula (IV-A), R 2 = benzyl

[0927]

[0928] As shown in Scheme 4, a phosphate group can be added to the compound of formula (III), where R 2 is benzyl, to prepare the compound of formula (IV-A), where R 2 is benzyl, which is achieved by reacting the compound of formula (III) (where R 2 is benzyl) with tetraphenylmethyl pyrophosphate (diphosphoric acid tetraphenylmethyl ester), and then removing the benzyl group from the phosphate ester to obtain the compound of formula (IV-A), where R 2 is benzyl and R 3 is C 13 -C 24 n-alkyl.

[0929] Scheme 5: Synthesis of the compound of formula (IV-E)

[0930]

[0931] The compound of formula (IV-E) can be prepared starting from the SC-4-8 intermediate in Scheme 4 and removing all benzyl groups, for example, by catalytic hydrogenation as shown in Scheme 5, where R 3 is C 13 -C 24 n-alkyl.

[0932] Scheme 6: Synthesis of the compound of formula (III-A)

[0933]

[0934] The compound of formula (III-A) can be prepared as shown in Scheme 6, via the compound of formula (III-B) as an intermediate. In Scheme 6, R 2 is -(C=O)-NH2, -(C=O)-NH(R 5 ) or -(C=O)-N(R 5 )2; each R 5 is independently C1-C4 alkyl; and R 3 is C 13 -C 24 n-alkyl.

[0935] Scheme 7: Synthesis of the compound of formula (IV-A)

[0936]

[0937] The compound of formula (IV-A) can be prepared starting from the compound of formula (III-A) as shown in Scheme 7, where R 2 is -(C=O)-NH2, -(C=O)-NH(R 5 ) or -(C=O)-N(R 5 )2; each R 5 is independently C1-C4 alkyl; and R 3 is C 13 -C 24 n-alkyl.

[0938] Scheme 8: Synthesis of the compounds of formula (III-A), formula (IV-A), formula (IV-B) and formula (IV-C)

[0939]

[0940] The 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 11Independently selected from H or methyl. The synthesis of carbamates is shown. To synthesize a compound of formula (IV-B) having an alkyl or benzyl group at the 2-hydroxy position in the glycerol moiety, an alkylating agent or benzylating agent such as alkyl bromide or benzyl bromide is used in place of reagent SC-8-5, bis(pyridin-2-yl) carbonate. To synthesize a compound of formula (IV-C) having a free hydroxy group at the 2-hydroxy position in the glycerol moiety, a protecting group can be placed on the hydroxy group and removed at the end of the synthesis (such as benzyl, by adding benzyl bromide in place of reagent SC-8-5 and removing it by catalytic hydrogenation).

[0941] Example

[0942] Abbreviations: BM (bone marrow); BMDC (bone marrow-derived dendritic cell); CDS (cytosolic DNA sensor); CLR (C-type lectin receptor); DAMP (damage-associated molecular pattern); DC (dendritic cell); DGPC (1-dodecyl-sn-glycero-3-phosphocholine); DGP (1-dodecyl-sn-glycero-3-phosphate); dLN (draining lymph node); HOdiA-PC (1-palmitoyl-2-(5-hydroxy-8-oxo-6-octenedioyl)-sn-glycero-3-phosphocholine); HOOA-PC (1-palmitoyl-2-(5-hydroxy-8-oxooct-6-enoyl)-sn-glycero-3-phosphocholine); IFNγ (interferon-γ); IL-1b / IL1-β / IL-1β (interleukin-1β); KOdiA-PC (1-(palmitoyl)-2-(5-oxo-6-octenedioyl) phosphatidylcholine); KOOA-PC (1-palmitoyl-(5-oxo-8-oxo-6-octenoyl)-sn-glycero-3-phosphocholine); KP407 (poloxamer 407); LPC / Lyso PC (lysophosphatidylcholine); Lyso PC(22:0) (1-dodecanoyl-2-hydroxy-sn-glycero-3-phosphocholine); LPS (lipopolysaccharide); MFI (mean fluorescence intensity); moDC (monocyte-derived dendritic cell); MPLA (monophosphoryl lipid A); NLR (NOD-like receptor); oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine); PAMP (pathogen-associated molecular pattern); PBMC (peripheral blood mononuclear cell); PGPC (1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine); POVPC (1-palmitoyl-2-(5'-oxo-valeryl)-sn-glycero-3-phosphocholine); PRR (pathogen recognition receptor); RLR (RIG-I-like receptor); R848 (resiquimod); STING (stimulator of IFN genes); TNFα (tumor necrosis factor-α); TLR (toll-like receptor); and WTL (whole tumor lysate).

[0943] Synthesis Examples

[0944] Example S-1: Synthesis of Compound 1

[0945]

[0946] To a stirred solution of (R)-2,3-dihydroxypropyl (2-(trimethylammonio)ethyl) phosphate 1-1 (2 g, 0.0077 mol) in IPA (150 mL) was added Bu2SnO (2.89 g, 0.0116 mol) 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 on a rotary evaporator to afford the crude material. The crude material obtained was used in the next step without analysis.

[0947] To a stirred solution of crude intermediate 1-2 (2 g, 0.0040 mol) in IPA (150 mL) at 0 °C was added KOtBu (0.672 g, 0.006 mol) and 1-bromodocosane (1-3) (1.86 g, 0.0048 mol) and the reaction mixture was stirred at room temperature for 16 h. The progress of the reaction was monitored by TLC. The crude reaction mixture was evaporated on a rotary evaporator to afford the crude material. The crude material obtained was purified by column chromatography (silica gel alkalized with NH4OH) using 30% MeOH / DCM and 10% NH4OH as eluents. The impure material was repurified by Combiflash chromatography (ELSD); 12 g column, using 40% MeOH / DCM and 10% NH4OH as eluents to give compound 1 (75 mg, 0.13 mmol, 3.3%) as a white solid. HRMS: 566.3606; HPLC-ELSD: 97.13%; 1 1H 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, 9H), 1.59 - 1.52 (m, 2H), 1.42 - 1.24, (m, 40H), 0.89 (t, J = 6.4 Hz, 3H).

[0948] Example S-2: Synthesis of Compound 9, Compound 2 and Compound 10

[0949]

[0950] Synthesis of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (2-3)

[0951] At 0 °C, KOtBu (6.86 g, 0.0612 mol) was added 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), and the mixture was heated to 110 °C for 16 h. The reaction progress was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated in vacuo to give the crude material. The obtained crude material was purified by MPLC flash column chromatography using 10% EtOAc / 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 1H 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).

[0952] Synthesis of Compound 9

[0953] A solution of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane 2-3 (15 g, 0.034 mol) in acetic acid:H2O (10:1 v / v) was heated to 60 °C, and the reaction mixture was stirred for 16 h. The reaction progress was monitored by TLC analysis. After completion of the reaction, the reaction mixture was concentrated in vacuo to give the crude material. The obtained 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%, 1 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).

[0954] The crude material (1.00 g, 2.49 mmol) was purified by dissolving in 20% EtOAc; hexane (200 mL) and stirred for 30 minutes. After 30 minutes, the solid was filtered and dried in vacuo 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).

[0955] Synthesis of (R)-1-((tert-butyldiphenylsilyl)oxy)-3-(docosyloxy)propan-2-ol (2-4):

[0956] To a stirred solution of crude (S)-3-(docosyloxy)propane-1,2-diol (compound 9) (6 g, 0.0149 mol) in DCM (150 mL) at 0 °C was added imidazole (2.54 g 0.0374 mol), TBDPSCl (4.7 mL, 0.0179 mol), and the reaction mixture was stirred at room temperature for 4 hours. The progress of the reaction was monitored by TLC analysis. After completion of the reaction, the reaction mixture was diluted with water and the product was extracted with DCM (2 x 200 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo to afford the crude material. The crude material obtained was purified by MPLC flash column chromatography; 12 g Clariscep C-series, using 10% EtOAc / 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).

[0957] Synthesis of (R)-(2-(Benzyloxy)-3-(docosyloxy)propoxy)(tert-butyl)diphenylsilane (2-5):

[0958] 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) at 0 °C was added NaH (0.45 g, 0.0938 mol) and the mixture was stirred for 20 minutes. After 20 minutes, BnBr (0.7 mL, 0.0056 mol) was added at room temperature and the reaction mixture was stirred at room temperature for 4 hours. The progress of the reaction was monitored by TLC analysis. After 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 obtained crude material was purified by MPLC flash column chromatography using 10% EtOAc / 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 1H 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).

[0959] Synthesis of (S)-2-(Benzyloxy)-3-(docosyloxy)propan-1-ol (2-6):

[0960] 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) at 0 °C was added TBAF (8 mL, 0.007 mol) and the reaction mixture was stirred at room temperature for 4 hours. The progress of the reaction was monitored by TLC analysis. After 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 obtained crude material was purified by MPLC flash column chromatography using 10% EtOAc / 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. 11H NMR (DMSO-d6, 400 MHz, 1 1H 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).

[0961] (R)-Dibenzyl (2-(benzyloxy)-3-(docosyloxy)propyl) phosphate (2 - 8) synthesis:

[0962] To a stirred solution of (S)-2-(benzyloxy)-3-(docosyloxy)propan-1-ol (2 - 6, 600 mg, 1.224 mmol) in THF (20 mL) at 0 °C was added KOtBu (205 mg, 1.836 mmol) and tetrabenzyl diphosphate 2 - 7 (790 mg, 1.469 mmol) and the mixture was stirred at room temperature for 2 h. The completion of the reaction was monitored by TLC. The progress of the reaction was monitored by TLC analysis. After completion of the reaction, the reaction mixture was quenched with NH4Cl and 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 obtained crude material was purified by MPLC flash column chromatography using 20% EtOAc / 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 1H 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).

[0963] Synthesis of compound 2:

[0964] To a stirred solution of (R)-diphenylmethyl (2-(benzyloxy)-3-(docosyloxy)propyl) phosphate 2-8 (450 mg, 0.599 mmol) in MeOH (50 mL) was added Pd(OH)2 (45 mg) and the reaction mixture was stirred at room temperature under H2 balloon pressure (40 psi) for 4 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was filtered through a bed of Celite and the filtrate was concentrated in vacuo to afford 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, 40H), 0.87 - 0.84 (m, 3H).

[0965] Synthesis of Compound 10

[0966] To a stirred solution of (R)-diphenylmethyl (2-(benzyloxy)-3-(docosyloxy)propyl) phosphate 2-8 (500 mg, 0.666 mmol) in dioxane (5 mL) at 0 °C was added HCl in dioxane (4.0 M) (20 mL) and the reaction mixture was stirred at room temperature for 48 h. The progress of the reaction was monitored by TLC. After completion, the mixture was concentrated in vacuo and the residue was diluted with water. The resulting solid was filtered and dried in vacuo to afford the impure material. The impure material obtained was further purified by stirring in ACN (100 mL) for 30 min, filtering and drying the solid 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; 1 H 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).

[0967] Alternative Syntheses of Compound 9 and Compound 2

[0968]

[0969] (R)-4-((Docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (2A-3) Synthesis

[0970] 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 to a stirred solution of 1-bromodocosane (2A-2) (16.96 g, 128.3664 mmol) in toluene. The reaction mixture became a viscous substance. The reaction mixture was stirred at room temperature for 1 h and then heated to 110 °C for 16 h. Completion of the reaction was monitored by TLC. After completion of the reaction, diethyl ether was added to the reaction mixture and stirred for 10 min. An aqueous brine solution was added to the reaction mixture and extracted with diethyl 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. Confirmed by 1 1H NMR. 1 1H 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 1H NMR showed the desired product and impurities; 1 1H NMR values were assigned based on the product peaks in the next step.

[0971] Synthesis of Compound 9

[0972] Concentrated HCl (125 mL) was added 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) and heated to 70 °C for 16 h. Completion of the reaction was monitored by TLC. The reaction mixture was diluted with water and filtered, and the filtered solid was stirred with water again and filtered to obtain a solid. The solid was stirred with hexane and filtered to obtain the product containing water. Acetonitrile was added to the product and distilled three times to remove the water, giving Compound 9 (30 g, 55%, over two steps) as an off-white solid. Confirmed by 1 1H NMR (CDCl3) 400 MHz). 11H 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).

[0973] Synthesis of (R)-1-(Docosyloxy)-3-(trityloxy)propan-2-ol (2A-4)

[0974] At 0 °C, trityl chloride (13.91 g, 49.913 mmol) was added 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 and the mixture was heated to 120 °C in a sealed tube for 16 h. The starting material, pyridine, and trityl chloride were anhydrous because moisture hindered the reaction. The completion of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was evaporated under reduced pressure to give the crude material. The crude product was purified by combi flash using 5% EtOAc / 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 a white solid as the desired compound contaminated with trityl impurities. The desired product 2A-4 (19.4 g, 60%) was obtained as a white solid and characterized and confirmed by 1H NMR. 1 1H NMR (400 MHz, CDCl3): δ = 7.25 - 7.43 (m, 15H), 3.94 (m, 1H), 3.42 - 3.52 (m, 4H), 3.18 (m, 2H), 1.22 - 1.48 (m, 38H), 0.87 (m, 3H).

[0975] Synthesis of (R)-(((3-(Docosyloxy)-2-((4-methoxybenzyl)oxy)propoxy)methanetriyl)tris)benzene (2A-5)

[0976] At 0 °C, NaH (1.55 g, 38.88 mmol) was added 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), and the reaction mixture was stirred for 20 minutes. After 20 minutes, p-methoxybenzyl chloride (3.14 mL, 23.32 mmol) was added dropwise, and the reaction mixture was stirred for 16 hours. Completion of the reaction was monitored by TLC. The reaction mixture was quenched with ice-cold water (100 mL) and extracted with EtOAc (2 x 150 mL). The organic layer was dried over Na2SO4 and evaporated in vacuo to give the crude material. The crude material obtained was purified by combi-flash chromatography (40 g column) using 0.5% EtOAc and 1% triethylamine / hexane as eluent to give the title compound 2A-5 (12 g, impure; the trityl impurity was not separated at this stage) as a colorless liquid. Confirmed by 1 1H NMR. 1 1H 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, 1H), 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).

[0977] Synthesis of (S)-3-(docosyloxy)-2-((4-methoxybenzyl)oxy)propan-1-ol (2A-6)

[0978] At 0 °C, camphorsulfonic acid (3.65 g, 15.72 mmol) was added to a stirred solution of (R)-((3-(docosyloxy)-2-((4-methoxybenzyl)oxy)propoxy)methanetriyl)trisbenzene (2A-5) (12.00 g, 15.72 mmol) in DCM:MeOH (120 mL), and the reaction mixture was stirred at room temperature for 2 hours. Completion of the reaction was monitored by TLC. The reaction mixture was diluted with DCM (200 mL) and washed with water (2 x 150 mL). The organic layer was dried over Na2SO4 and evaporated in vacuo to give the crude material. The crude material obtained was purified by Combi-flash chromatography (40 g column) using 20% EtOAc and 1% triethylamine / hexane as eluent to give the title compound 2A-6 (4.1 g, 50%) as a white solid, confirmed by 1 1H NMR. 11H NMR (400 MHz, CDCl3): δ = 7.28 (d, J = 8.8 Hz, 2H), 6.88 (d, J = 8.4 Hz, 2H), 4.66 - 4.53 (m, 2H), 3.73 (s, 3H), 3.72 - 3.66 (m, 1H), 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, 38H), 0.89 - 0.86 (m, 3H).

[0979] Synthesis of (R)-3-(Docosyloxy)-2-((4-methoxybenzyl)oxy)propyl dihydrogen phosphate (2A-8)

[0980] 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 a 10-minute period to a stirred solution of distilled POCl3 (0.8 mL, 8.5567 mmol) in THF (7 mL) (salt-ice mixture) at -20 °C and stirred for an additional 20 minutes at -20 °C. Initially, the reaction mixture was grayish-white within the first 20 minutes and slowly turned light yellow. The completion of the reaction was monitored by TLC. The reaction mixture was quenched slowly with 10% aqueous NaHCO3 (5 mL) and stirred for 30 minutes at the same temperature. The temperature of the ice bath was allowed to reach -10 °C, then the reaction mixture was acidified with 6N HCl and extracted with DCM (60 mL). The DCM layer was washed with 10% NaHCO3 solution (2 x 30 mL), the aqueous layer was separated and 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 (0.6 g, 52%) as a grayish-white solid. Confirmed by 1H NMR, 31P NMR.

[0981] 11H NMR (CD3OD) 400 MHz VT, 50 °C 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). Due to the low solubility of the compound, 1H NMR and 31P NMR were recorded in CD3OD and CD3OD + CDCl3 at 50 °C - 60 °C. 1 1H NMR and 31 31P NMR.

[0982] HPLC: tRet 7.705 min (99.56%) HPLC method conditions: Column: LUNA HILIC (250 * 4.6) mm, 5 μm, 200 Å, Mobile phase - A: (aqueous solution) containing 10 mM ammonium acetate; 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

[0983] (R)-3-(Docosyloxy)-2-hydroxypropyl dihydrogen phosphate (Compound 2) synthesis

[0984] 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) at room temperature was added 1,4 - dioxane (0.2 mL) containing 4 M HCl, and the reaction mixture was stirred in a sealed tube at 40 °C for 16 h. Acetonitrile was added to the reaction mixture, and the solvent was removed with a dropper, and this process was repeated three times, then the solvent was removed under reduced pressure at 40 °C to give Compound 2 (140 mg, 87.5%) as an off - white solid. Confirmed by 1H NMR, 31P NMR.

[0985] 1 1H NMR (CD3OD) 400 MHz VT, 50 °C 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).

[0986] 31P NMR (CD3OD) 400 MHz VT, 50 °C δ ppm 0.76 (t) Note: Due to the low solubility of the compound, it was recorded in CD3OD and CD3OD + CDCl3 at 50 °C - 60 °C 1 1H NMR, 31 31P NMR.

[0987] HPLC: tRet 7.746 min (98.43%), HPLC method conditions: Column: LUNA HILIC (250 * 4.6) mm, 5 μm, 200 A, Mobile phase - A: (aqueous solution) containing 10 mM ammonium acetate; 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.

[0988] Example S - 3: Synthesis of Compound 7

[0989]

[0990] (R)-4-((Docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (3 - 3) synthesis:

[0991] To a stirred solution of 1-bromodocosane (3 - 1) (16.96 g, 128.3664 mmol) in toluene at 0 °C was added 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). The reaction mixture became a viscous substance and stirring ceased. 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. After completion of the reaction, diethyl ether was added to the reaction mixture and stirred for 10 minutes. An aqueous brine solution was added to the reaction mixture and extracted with diethyl ether. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to give (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (3 - 3) (57 g, crude) as a brown solid. HPLC (ELSD): 88.67%, 1HNMR(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, 2H), 1.45 (s, 3H), 1.38 (m, 3H), 1.31 - 1.14 (m, 40H), 0.89 - 0.86 (m, 3H).

[0992] Synthesis of Compound 9:

[0993] 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 the mixture was heated to 70 °C for 16 h. The completion of the reaction was monitored by TLC. The reaction mixture was diluted with water and filtered, and the filtered solid was stirred with water again and filtered to obtain a solid. The solid was stirred with hexane and filtered to obtain the pure substance. The obtained substance contained water. Acetonitrile was added to the product and co-distilled three times (to remove trace amounts 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, 40H), 0.88 (t, J = 6.8 Hz, 3H).

[0994] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (3-4):

[0995] To a stirred solution of (S)-3-propoxypropane-1,2-diol (3-3) (25 g, 0.0625 mol) in pyridine (150 mL) at 0 °C was added trityl chloride (15.6 g, 0.0562 mol), and the reaction mixture was stirred at 120 °C for 16 h. The completion of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was concentrated in vacuo to give a crude material. The obtained crude material was purified by MPLC flash column chromatography using 10% EtOAc / hexane as the eluent to give (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (3-4) as an off-white solid (25 g, 62%). 11H NMR (CDCl3, 400 MHz): δ ppm 7.44 - 7.21 (m, 15H), 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).

[0996] Synthesis of (R)-4-nitrophenyl (1-propoxy-3-(trityloxy)propan-2-yl) carbonate (3-5):

[0997] 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 was added Et3N (0.52 mL, 3.732 mmol), 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 indicated the formation of starting material along with the product. Additional Et3N (1.3 mL, 9.33 mmol) and 4-nitrophenyl chloroformate (1.88 g, 9.33 mmol) were added and the reaction was 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. The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo to give the crude material. The crude material obtained was purified by combi-flash column chromatography using 30% EtOAc / hexane as the eluent to give (R)-4-nitrophenyl (1-propoxy-3-(trityloxy)propan-2-yl) carbonate (5) (1.2 g, impure) as an off-white solid. HPLC (ELSD): 99.89%, 1 1H NMR (CDCl 3, 400 MHz): δ ppm 8.28 - 8.25 (m, 2H), 7.45 - 7.23 (m, 17H), 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, 40H), 0.87 (t, J = 6.8 Hz, 3H).

[0998] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-yl carbamate (3-6):

[0999] 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 was purged with ammonia at 0 °C and stirred in a sealed tube at room temperature for 16 h. Completion of the reaction was monitored by TLC. The reaction mixture was concentrated in vacuo to afford the crude material. The obtained crude material was purified by combi-flash column chromatography using 5% EtOAc / hexane to remove 4-nitrophenol impurities and then eluted with 30% EtOAc / hexane as the eluent to give (R)-1-(docosyloxy)-3-(trityloxy)propan-2-yl carbamate (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, 15H), 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, 40H), 0.87 (t, J = 6.4 Hz, 3H).

[1000] Synthesis of (S)-1-(docosyloxy)-3-hydroxypropan-2-yl carbamate (Compound 7):

[1001] To a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-yl carbamate (3-6) (0.75 g, 1.0932 mmol) in MeOH and DCM (1:1, 10 mL) was added CSA (0.05 g, 0.21864 mmol) and the reaction mixture was stirred at room temperature for 16 h. Completion of the reaction was monitored by TLC. The reaction mixture was filtered, the solid was washed with diethyl ether and dried in vacuo to give (S)-1-(docosyloxy)-3-hydroxypropan-2-yl carbamate (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, 40H), 0.88 (t, J = 6.4 Hz, 3H).

[1002] Example S-4: Synthesis of Compound 8

[1003]

[1004] (Synthesis of (R)-4-nitrophenyl (1-propoxy-3-(trityloxy)propan-2-yl) carbonate (4-2):

[1005] 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 was added Et3N (1.1 mL, 0.0076 mol), followed by the addition of 4-nitrophenyl chloroformate (1.17 g, 0.0058 mol) and the mixture was stirred at 80 °C for 16 h. The 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. The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo to give the crude product. The obtained crude product was purified by combi-flash column chromatography using 20% EtOAc / hexane as the eluent to give (R)-4-nitrophenyl (1-propoxy-3-(trityloxy)propan-2-yl) carbonate (4-2) (3 g, impure) as an off-white solid. 1 H NMR(CDCl 3, 400 MHz): δ ppm 8.27 (d, J = 9.2 Hz, 2H), 7.45 - 7.23 (m, 17H), 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, 40H), 0.87 (t, J = 6.8 Hz, 3H).

[1006] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-yl methylcarbamate (4-3):

[1007] At 0 °C, a solution of methylamine in THF (5 mL) was added 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), and the reaction mixture was stirred at room temperature for 16 h. The completion of the reaction was monitored by TLC. The reaction mixture was concentrated in vacuo to afford the crude material. The obtained crude material was purified by combi-flash column chromatography using 20% EtOAc / hexane as the eluent to give (R)-1-(docosyloxy)-3-(trityloxy)propan-2-yl methylcarbamate (4-3) (3 g, impure) as an off-white solid. 1 H NMR(CDCl 3, 400 MHz): δ ppm 7.43 - 7.20 (m, 15H), 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, 40H), 0.87 (t, J = 6.4 Hz, 3H).

[1008] Synthesis of (S)-1-(docosyloxy)-3-hydroxypropan-2-yl methylcarbamate (Compound 8):

[1009] At 0 °C, CSA (0.9 g, 0.00429 mol) was added to a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-yl methylcarbamate (4-3) (3 g, 0.00429 mmol) in DCM; MeOH (40 mL), and the reaction mixture was stirred at room temperature for 1 h. The 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 in vacuo to afford the crude material. The obtained crude material was purified by combi-flash column chromatography using 30% EtOAc / hexane as the eluent to give (S)-1-(docosyloxy)-3-hydroxypropan-2-yl methylcarbamate Compound 8. 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).

[1010] Example S-5: Synthesis of Compound 12, Compound 11 and Compound 13

[1011]

[1012] (R)-2,2-Dimethyl-4-((octadecyloxy)methyl)-1,3-dioxolane (5-3) Synthesis:

[1013] At 0 °C, potassium tert-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 to a stirred solution of 1-bromooctadecane (5-2, 25.22 g, 75.66 mmol, 1.0 equiv) in toluene. The viscous material of the reaction mixture was stirred at room temperature for 1 h and heated to 110 °C for 16 h. The completion of the reaction was monitored by TLC. After completion of the reaction, diethyl ether (500.0 mL) was added to the reaction mixture and stirred for 10 min, and then an aqueous brine solution (500.0 mL) was added to the reaction mixture and extracted with diethyl ether. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product 5-3 (29 g) as a brown semi-solid. Confirmed by crude 1 1H NMR.

[1014] 1 1H NMR (400 MHz, CDCl3): δ = 4.23 - 4.29 (m, 1H), 4.23 - 4.29 (m, 1H), 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).

[1015] ((S)-3-(Octadecyloxy)propane-1,2-diol) (Compound 12) Synthesis

[1016] 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), and the mixture was stirred and heated to 70 °C for 16 h. The reaction completion was monitored by TLC. All solvents were evaporated, and then acetonitrile (100.0 mL) was added to the crude reaction mixture and stirred for 2 h. A grayish-white free-flowing solid precipitated, which was filtered and dried. This crude powder was wet-milled with n-pentane (50.0 mL), filtered, and dried to give Compound 12 as a grayish-white solid (7.4 g, 58%, over two steps). Confirmed by 1 1H NMR (CDCl3, 400 MHz), CDCl3 + D2O, and HRMS. 1 1H NMR (400 MHz, CDCl3): δ = 3.84 - 3.88 (m, 1H), 3.71 - 3.72 (m, 2H), 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, 32H), 0.86 - 0.89 (t, 3H). HPLC: 99.79%. HRMS: 344.59 (match), dimer mass: (689.13) match.

[1017] Synthesis of (R)-1-(octadecyloxy)-3-(trityloxy)propan-2-ol (5-4)

[1018] 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 was added trityl chloride (1.05 g, 3.772 mmol), and the mixture was heated to 120 °C in a sealed tube for 16 h. The starting material, pyridine, and trityl chloride were anhydrous as moisture hindered the reaction. The reaction completion was monitored by TLC. The reaction mixture was evaporated under reduced pressure and the crude product was purified by combi-flash using 5% ethyl acetate / hexane as the eluent. The fractions were collected and concentrated, and finally the compound was wet-milled with n-pentane (25.0 mL), filtered, and dried to give the title compound 5-4 as a grayish-white solid with trace amounts of deprotected trityl alcohol impurity (1.3 g, 62%). Confirmed by 1 1H NMR (CDCl3, 400 MHz)

[1019] 11H NMR (400 MHz, CDCl3): δ = 7.18 - 7.50 (m, 15H), 3.93 - 3.97 (m, 1H), 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).

[1020] Synthesis of (R)-1-propoxy-3-(trityloxy)propan-2-yl pyridin-2-yl carbonate (5-6)

[1021] At room temperature, triethylamine (1.42 mL, 10.218 mmol, 6.0 equiv) was added 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 anhydrous THF (10.0 mL), followed by addition of bis(pyridin-2-yl) carbonate 5-5 (0.736 g, 3.407 mmol, 2.0 equiv). The reaction mixture was then stirred in a sealed tube at 80 °C for 16 h. The completion of the reaction was monitored by TLC. Since the intermediate 5-6 is unstable, it was used directly in the next step without further treatment. The formation of intermediate 5-6 was confirmed by 1 1H NMR (CDCl3) 400 MHz: δ = 7.28 - 7.48 (m, 20H), 3.2 - 3.8 (m, 4H), 1.28 - 1.38 (m, 32H).

[1022] Synthesis of (R)-1-(octadecyloxy)-3-(trityloxy)propan-2-yl methylcarbamate (5-7)

[1023] At 0 °C, THF (10.0 mL) containing 7% methylamine was added to a stirred solution of intermediate (R)-1-propoxy-3-(trityloxy)propan-2-yl pyridin-2-yl carbonate 5-6 (1.0 g, 1.446 mmol, 1.0 equiv) in anhydrous THF (20.0 mL). The reaction mixture was stirred in a sealed tube at 80 °C for 16 h. The 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), extracted with water (25.0 mL x 2), and 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 characterized by 1 1H NMR (CDCl3, 400 MHz) and used without further purification in the next step.

[1024] 11H NMR (400 MHz, CDCl3): δ = 7.26 - 7.43 (m, 15H), 5.079 (m, 1H), 4.66 (m, 1H), 2.81 (m, 3H), 1.25 - 1.55 (m, 32H), 1.22 - 1.38 (m, 30H), 0.88 (t, 3H).

[1025] Synthesis of (S)-1-hydroxy-3-(octadecyloxy)propan-2-yl methylcarbamate (Compound 11)

[1026] At 0 °C, DL-10-camphorsulfonic acid (CSA; 0.433 g, 1.863 mmol, 1.1 eq) was added to a stirred solution of (R)-1-(octadecyloxy)-3-(trityloxy)propan-2-yl methylcarbamate 5-7 (1.2 g, 1.863 mmol, 1.0 eq) in a mixture of anhydrous DCM:anhydrous MeOH (1:1) 10.0 mL:10.0 mL, 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 x 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 product structure was confirmed by 1H NMR (400 MHz) in CDCl3, CDCl3 + D2O, and HRMS.

[1027] 1 1H 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, 2H), 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 (in agreement), HPLC: 99.84%,

[1028] Synthesis of (R)-1-((bis(benzyloxy)phosphoryl)oxy)-3-(octadecyloxy)propan-2-yl methylcarbamate (5-8)

[1029] At 0 °C, potassium tert-butoxide (0.279 g, 2.489 mmol, 2.0 equiv) was added to a stirred solution of methyl (S)-1-hydroxy-3-(octadecyloxy)propan-2-yl carbamate compound 11 (1.0 g, 2.489 mmol, 1.0 equiv) in anhydrous THF (80.0 mL) solvent, and the mixture was stirred for 5 minutes. Tetraphenylmethyl diphosphate (1.34 g, 2.489 mmol, 1.0 equiv) was added, and the reaction mixture was stirred at room temperature for 3 hours. The 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 x 2). The organic layer was dried over anhydrous sodium sulfate and concentrated to give 2.0 g of a crude compound. The crude compound was purified by preparative HPLC to give compound 5-8 (750.0 mg, 45%) as a white solid. Confirmed by 1 1H NMR, 31 31P NMR.

[1030] 1 1H 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 31P NMR: Consistent with

[1031] Synthesis of methyl (R)-1-(octadecyloxy)-3-(phosphonyloxy)propan-2-yl carbamate (Compound 13)

[1032] To a stirred solution of methyl (R)-1-((bis(benzyloxy)phosphoryl)oxy)-3-(octadecyloxy)propan-2-yl carbamate 5-8 (400.0 mg, 0.604 mmol, 1.0 equiv) in MeOH was added 20% Pd(OH)2 / C (100.0 mg) and hydrogenated at 40 PSI for 4 hours. The completion of the reaction was monitored by TLC. The reaction mixture was filtered through diatomaceous earth and washed with 250 mL of methanol, and the filtrate was passed through a micron filter NYL 0.45 um and concentrated to give a crude product. The crude compound was wet milled with n-pentane and dried in high vacuum to give compound 13 (350 mg, 99%) as a white solid. Confirmed by 1H NMR, 31P NMR and HRMS.

[1033] 11H 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, 2H), 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 (conformed), HPLC: 99.87%.

[1034] Example S-6: Synthesis of Compound 6

[1035]

[1036] (R)-4-((Docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (6-3) Synthesis

[1037] At 0 °C, potassium tert-butoxide (28.8 g, 256.7328 mmol) and (R)-(2,2-dimethyl-1,3-dioxol-4-yl)methanol (6-1) (16.96 g, 128.3664 mmol) were added to a stirred solution of 1-bromodocosane (6-2) (16.96 g, 128.3664 mmol) in toluene. The reaction mixture became a viscous substance. The reaction mixture was stirred at room temperature for 1 hour and then heated to 110 °C for 16 hours. The completion of the reaction was monitored by TLC. After the reaction was completed, diethyl ether was added to the reaction mixture and stirred for 10 minutes. An aqueous brine solution was added to the reaction mixture and extracted with diethyl ether. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a crude product (60 g) as a brown solid. Confirmed by 1H NMR. 1 1H 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 1H NMR showed the desired product and impurities; 1 1H NMR values were assigned based on the product peaks in the next step.

[1038] Synthesis of Compound 9

[1039] 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 the mixture was heated to 70 °C for 16 h. Completion of the reaction was monitored by TLC. The reaction mixture was diluted with water and filtered, and the filtered solid was stirred with water again 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 the water, giving compound 9 as an off-white solid (30 g, 55%, over two steps). Confirmed by 1 1H NMR (CDCl3, 400 MHz). 1 1H 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).

[1040] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (6-4)

[1041] 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 was added trityl chloride (13.91 g, 49.913 mmol) and the mixture was heated to 120 °C in a sealed tube for 16 h. Completion of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was evaporated under reduced pressure to give a crude product. The crude product was purified by combi-flash using 5% EtOAc / 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 a white solid as the desired compound contaminated with trityl impurities. The desired product 6-4 was obtained as a white solid (19.4 g, 60%) and characterized and confirmed by 1H NMR. 1 1H NMR (400 MHz, CDCl3): δ = 7.25 - 7.43 (m, 15H), 3.94 (m, 1H), 3.42 - 3.52 (m, 4H), 3.18 (m, 2H), 1.22 - 1.48 (m, 38H), 0.87 (m, 3H).

[1042] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propyl pyridin-2-yl carbonate (6-6)

[1043] At room temperature, Et3N (4.3 mL, 31.1036 mmol) was added to a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (5 g, 7.7759 mmol) in THF, followed by the addition of bis(pyridin-2-yl) carbonate (3.36 g, 15.5518 mmol), and the mixture was stirred in a sealed tube at 80 °C for 16 h. The starting materials, pyridine, and trityl chloride were anhydrous as moisture hindered the reaction. The completion of the reaction was monitored by TLC. Since the product reaction mixture was unstable, this crude mixture of (R)-1-(docosyloxy)-3-(trityloxy)propyl pyridin-2-yl carbonate 6-6 (5 g, crude material, light brown reaction mixture) was used in the next step without further purification.

[1044] 1 1H 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 1H NMR showed the desired product along with impurities.

[1045] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propyl methylcarbamate (6-7)

[1046] At 0 °C, THF solution containing 7% methylamine (10 mL) was added to a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propyl pyridin-2-yl carbonate (6-6) (5 g, 6.6835 mmol) in THF (50 mL), and the mixture was stirred and heated in a sealed tube at 80 °C for 16 h. The completion of the reaction was monitored by TLC. The reaction mixture was evaporated under reduced pressure to give a crude material. 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)propyl methylcarbamate 6-7 (6 g, crude product) as a light brown solid, which was characterized by 1H NMR. 11H 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).

[1047] Synthesis of (S)-1-(Docosyloxy)-3-hydroxypropan-2-yl Methylcarbamate (Compound 8)

[1048] To a stirred solution of (R)-1-(Docosyloxy)-3-(trityloxy)propan-2-yl Methylcarbamate (6 - 7) (6 g, 8.5773 mmol) in a mixture of DCM:MeOH (1:1) 60 mL:60 mL solvent at 0 °C was added DL-10-Camphorsulfonic acid (1.99 g, 8.5773 mmol). The whole 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 (50.0 mL x 2), extracted and separated, and the organic layer was dried over anhydrous sodium sulfate and concentrated to give the crude compound. The crude material was washed with n-Pentane (100 mL), stirred for 15 min, and a grayish-white solid was precipitated, which was filtered and dried to give Compound 8 (1.5 g) as a grayish-white solid. 1 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).

[1049] Synthesis of (R)-1-(((Bis(benzyloxy)phosphoryl)oxy)-3-(docosyloxy)propan-2-yl Methylcarbamate (6 - 10)

[1050] At 0 °C, potassium tert-butoxide (0.245 g, 2.1846 mmol) was added to a stirred solution of compound 8 methylcarbamic acid ((S)-1-(docosyloxy)-3-hydroxypropan-2-yl ester (0.5 g, 1.092 mmol)) in anhydrous THF (100 mL) solvent. The mixture was stirred for 10 minutes, diphenylmethyl diphosphate (1.17 g, 2.1846 mmol) was added, and the reaction mixture was stirred at 0 °C for 3 hours. TLC indicated the formation of starting material along with the product. Additional potassium tert-butoxide (0.12 g, 1.0923 mmol) and diphenylmethyl diphosphate (0.6 g, 1.0923 mmol) were added and the mixture was stirred at 0 °C for an additional 3 hours. Completion of the reaction was monitored by TLC. The reaction mixture was quenched with ice water and extracted with ethyl acetate (2 x 100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated to give 1.3 g of the crude compound which was purified by chiral preparative HPLC to separate peak-1 and peak-2. Peak-1 (as a light pink liquid) gave the title compound 6-10 (130 mg). Confirmed by 1H NMR, 31P NMR.

[1051] 1 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), 3.55 - 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).

[1052] 31P NMR (CDCl3) 400 MHz) δ ppm -1.913 (bs), HRMS: (M+1) = 718.4727.

[1053] 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: (aqueous solution) containing 0.1% formic acid; 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

[1054] (R)-1-(Docosyloxy)-3-(phosphonyloxy)propan-2-yl methylcarbamate: Synthesis of Compound 6

[1055] To a stirred solution of methylcarbamic acid (R)-1-((bis(benzyloxy)phosphoryl)oxy)-3-(docosyloxy)propan-2-yl ester 6-10 (130 mg, 0.181 mmol) in EtOAc was added 20% Pd(OH)2 / C (50 mg), and the mixture was hydrogenated at 40 PSI for 2 h. Completion of the reaction was monitored by TLC. The reaction mixture was diluted with 10% MeOH / 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. Confirmed by 1H NMR, 31P NMR.

[1056] 1 1H NMR (CD3OD) 400 MHz VT, 50 °C 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).

[1057] 31P NMR (CDCl3) 400 MHz) δ ppm 0.276 (bs)

[1058] HPLC: tRet 7.616 min (99.58%), HPLC method conditions: column: LUNA HILIC (250*4.6) mm, 5 μm, 200 A, mobile phase - A: (aqueous solution) containing 10 mM ammonium acetate; 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.

[1059] Example S-7: Synthesis of compound 4:

[1060]

[1061] Synthesis of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (7-3):

[1062] 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 to a stirred solution of 1-bromodocosane (16.96 g, 128.3664 mmol) in toluene. The reaction mixture became a viscous substance and stirring ceased. The reaction mixture was stirred at room temperature for 1 h and then heated to 110 °C for 16 h. The completion of the reaction was monitored by TLC. After completion of the reaction, diethyl ether was added to the reaction mixture and stirred for 10 min, an aqueous brine solution was added to the reaction mixture and extracted with diethyl ether. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to give the crude product 3 (60 g) as a brown solid. Confirmed by 1H NMR. 1 1H 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, 40H), 0.86 (t, J = 13.6 Hz, 3H). The crude 1H NMR shows the desired product along with impurities; the 1H NMR values are assigned based on the product peaks in the next step.

[1063] Synthesis of Compound 9

[1064] Concentrated HCl (125 mL) was added to a stirred solution of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (60 g, 136.1315 mmol) in MeOH (500 mL) and heated to 70 °C for 16 h. The completion of the reaction was monitored by TLC. The reaction mixture was diluted with water and filtered, the filtered solid was stirred with water again 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 the water, giving Compound 9 (30 g, 55%, over two steps) as an off-white solid. Confirmed by 1 1H NMR (CDCl3) 400 MHz): 1 1H 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).

[1065] (R)-1-(Docosyloxy)-3-(trityloxy)propan-2-ol (7-4) Synthesis

[1066] At 0 °C, trityl chloride (13.91 g, 49.913 mmol) was added 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 and the mixture was heated to 120 °C in a sealed tube for 16 h. (Note that the starting material, pyridine, and trityl chloride should be anhydrous; if the reaction mixture contains any moisture, no reaction will occur). The completion of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was evaporated under reduced pressure to give the crude material. The crude product was purified by combiflash chromatography using 5% EtOAc / 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 a white solid as the desired compound with some trityl chloride contamination. The desired product 7-4 (19.4 g, 60%) was obtained as a white solid and characterized and confirmed by 1H NMR: 1 1H NMR (400 MHz, CDCl3): δ = 7.25 - 7.43 (m, 15H), 3.94 (m, 1H), 3.42 - 3.52 (m, 4H), 3.18 (m, 2H), 1.58 - 1.54 (m, 2H), 1.22 - 1.48 (m, 38H), 0.87 (m, 3H). The crude 1H NMR showed the desired product along with impurities. The 1H NMR values were assigned based on the product peaks in the subsequent steps.

[1067] (R)-1-(Docosyloxy)-3-(trityloxy)propyl pyridin-2-yl carbonate (7-6) Synthesis:

[1068] At room temperature, Et3N (4.3 mL, 31.1036 mmol) was added to a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (5 g, 7.7759 mmol) in THF, followed by bis(pyridin-2-yl) carbonate (3.36 g, 15.5518 mmol), and the mixture was stirred in a sealed tube at 80 °C for 16 h. The completion of the reaction was monitored by TLC. Since the product reaction mixture was unstable, this crude mixture was used in the next step without further workup to give (R)-1-(docosyloxy)-3-(trityloxy)propyl pyridin-2-yl carbonate 7-6 (5 g, crude) as a light brown reaction mixture. 11H 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). The crude 1H NMR shows the desired product along with impurities. The 1H NMR values were assigned based on the product peaks in the subsequent steps.

[1069] Synthesis of (R)-1-(Docosyloxy)-3-(trityloxy)propan-2-yl methylcarbamate (7-7)

[1070] To a solution of the crude compound (R)-1-(Docosyloxy)-3-(trityloxy)propyl pyridin-2-yl carbonate (100 mL of THF) was added a solution of 2 M methylamine in THF (50 mL), and the mixture was heated to 80 °C for 16 h. The completion of the reaction was monitored by TLC. The reaction mixture was evaporated under reduced pressure to give a crude material. The crude product was dissolved in EtOAc (300 mL) and washed with water (2 x 200 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to give the product. The crude compound was used as such in the next step (9 g, crude) and characterized by 1H NMR: 1 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 = 4.8 Hz, 3H), 1.50 - 1.47 (m, 2H), 1.28 (brs, 40H), 0.87 (t, J = 6.4 Hz, 3H).

[1071] Synthesis of (S)-1-(Docosyloxy)-3-hydroxypropan-2-yl methylcarbamate (7-8)

[1072] At 0 °C, camphorsulfonic acid (2.98 g, 12.865 mmol) was added to a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-yl methylcarbamate (9.0 g, 12.865 mmol) in 100 mL of MeOH and DCM (1:1). 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 (300.0 mL) and washed with water (200 mL x 2), then the organic layer was dried over anhydrous sodium sulfate and concentrated to give the crude compound. The crude material was washed with pentane to give the pure title compound (2.8 g, pure) as an off-white solid and characterized by 1H 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 = 4.8 Hz, 3H), 1.57 - 1.52 (m, 2H), 1.28 (s, 40H), 0.88 (t, J = 6.4 Hz, 3H).

[1073] Synthesis of (R)-1-(docosyloxy)-3-((2-oxo-1,3,2-dioxaphospholan-2-yl)oxy)propan-2-yl methylcarbamate (7 - 9)

[1074] At 0 °C, 2-chloro-1,3,2-dioxaphospholane 2-oxide (0.18 mL, 1.967 mmol) was added to a stirred solution of (S)-1-(docosyloxy)-3-hydroxypropan-2-yl methylcarbamate (0.3 g, 0.655 mmol) and Et3N (0.27 mL, 1.967 mmol) in THF (5 mL), and the reaction mixture was stirred for 1 h. Completion of the reaction was monitored by TLC. The reaction mixture was filtered, the filtrate was concentrated in vacuo, and the crude compound obtained (0.3 g) was characterized by 1H 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).

[1075] ((R)-3-(Docosyloxy)-2-((methylcarbamoyl)oxy)propyl (2-(trimethylammonio)ethyl)phosphate: Synthesis of Compound 4

[1076] A solution of 2 M trimethylamine (3 mL) was added to a stirred solution of (R)-1-(docosyloxy)-3-((2-oxo-1,3,2-dioxaphospholan-2-yl)oxy)propan-2-yl methylcarbamate (0.3 g, 0.532 mmol) in ACN (15 mL) at 0 °C, and the reaction mixture was stirred at 65 °C for 16 h. Completion of the reaction was monitored by TLC. The reaction mixture was cooled to 0 °C, and a solid precipitated, which was filtered and dried in vacuo to give the crude material. The crude compound was washed with water (15 mL) and acetonitrile (30 mL) to give the title compound as an off-white solid (0.23 g, pure) and characterized by 1 1H NMR: 1 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 = 5.6 Hz, 3H). 31P NMR (CD3OD, 162 MHz): 0.17 ppm. HRMS = 622.6053.

[1077] Example S-8: Synthesis of Compound 14, Compound 15 and Compound 16

[1078]

[1079] Synthesis of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (8-3):

[1080] 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 to a stirred solution of 1-bromodocosane (16.96 g, 128.3664 mmol) in toluene. The reaction mixture became a viscous substance and stirring ceased. The reaction mixture was warmed to room temperature and 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. After completion of the reaction, diethyl ether was added to the reaction mixture and stirred for 10 minutes. An aqueous brine solution was added to the reaction mixture and extracted with diethyl 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. Confirmation by 1H NMR: 1 1H 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). The crude 1H NMR showed the desired product along with impurities; 1H NMR values were assigned based on the product peaks in the subsequent steps.

[1081] Synthesis of Compound 9:

[1082] Concentrated HCl (125 mL) was added to a stirred solution of (R)-4-((docosyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (60 g, 136.1315 mmol) in MeOH (500 mL) and heated to 70 °C for 16 hours. Completion of the reaction was monitored by TLC. The reaction mixture was diluted with water and filtered, and the filtered solid was stirred with water again and filtered to give a solid. The solid was stirred with hexane and filtered to give the product. The product contained water, so acetonitrile was added to the product and evaporated three times to remove the water, giving Compound 9 (30 g, 55%, over two steps) as an off-white solid. Confirmation by 1 1H NMR (CDCl3) 400 MHz): 1 1H 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).

[1083] (R)-1-(Docosyloxy)-3-(trityloxy)propan-2-ol (8-4) synthesis:

[1084] At 0 °C, trityl chloride (13.91 g, 49.913 mmol) was added 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 and heated to 120 °C in a sealed tube for 16 h. (In this step, the starting material, pyridine, and trityl chloride should be anhydrous; if the reaction mixture contains any moisture, no reaction will occur). The completion of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was evaporated under reduced pressure. The crude product was purified by combiflash chromatography using 5% EtOAc / 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 a white solid as the desired compound contaminated with trityl chloride. The desired product 8-4 (19.4 g, 60%) as a white solid was confirmed by 1H NMR. 1 1H NMR (400 MHz, CDCl3): δ = 7.25 - 7.43 (m, 15H), 3.94 (m, 1H), 3.42 - 3.52 (m, 4H), 3.18 (m, 2H), 1.22 - 1.48 (m, 38H), 0.87 (m, 3H).

[1085] (R)-1-(Docosyloxy)-3-(trityloxy)propyl pyridin-2-yl carbonate (8-6) synthesis:

[1086] At room temperature, Et3N (4.3 mL, 31.1036 mmol) was added to a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-ol (5 g, 7.7759 mmol) in THF, followed by bis(pyridin-2-yl) carbonate (3.36 g, 15.5518 mmol), and stirred at 80 °C in a sealed tube for 16 h. The completion of the reaction was monitored by TLC. Since the product reaction mixture was unstable, this crude mixture was used in the next step without further workup to give (R)-1-(docosyloxy)-3-(trityloxy)propyl pyridin-2-yl carbonate 8-6 (5 g, crude) as a light brown reaction mixture. 11H 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). 1H NMR of the crude compound showed the desired product along with impurities; 1H NMR values were assigned based on the product peaks in the subsequent steps.

[1087] Synthesis of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-yl dimethylcarbamate (8 - 7):

[1088] To 50 mL of THF containing the crude product (R)-1-(docosyloxy)-3-(trityloxy)propyl pyridin-2-yl carbonate (8 - 6) was added THF (40 mL) containing 7% dimethylamine, and the mixture was heated to 80 °C for 16 h. The completion of the reaction was monitored by TLC. The reaction mixture was evaporated under reduced pressure. The crude product was dissolved in EtOAc (200 mL) and washed with water (2 x 50 mL), and the organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to give the product. The crude compound was purified by combiflash chromatography. The product was eluted in 1% triethylamine / hexane containing 5% EtOAc. The fractions containing the product were concentrated and dried completely to give the title product 7 (6 g, pure) as an off-white solid and characterized by 1H NMR. 1 1H 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).

[1089] Synthesis of (S)-1-(docosyloxy)-3-hydroxypropan-2-yl dimethylcarbamate (Compound 14):

[1090] At 0 °C, camphorsulfonic acid (1.46 g, 6.3017 mmol) was added to a stirred solution of (R)-1-(docosyloxy)-3-(trityloxy)propan-2-yl dimethylcarbamate (3.0 g, 4.2011 mmol) in 30 mL of MeOH and DCM (1:1). 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 x 2), dried over sodium sulfate and concentrated to dryness. The crude compound from the previous batch was mixed with the batch of the present invention and purified by combiflash chromatography. The product was eluted in 40% EtOAc / hexanes. The fractions containing the product were concentrated and dried completely to give the title compound 14 (3.2 g, pure) as an off-white foam solid and was characterized by 1H NMR. 1 1H 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.

[1091] Synthesis of (R)-1-((diethoxyphosphoryl)oxy)-3-(docosyloxy)propan-2-yl dimethylcarbamate (8-8)

[1092] At 0 °C, DIPEA (1.84 mL, 10.5983 mmol) and DMAP (0.51 g, 4.2392 mmol) were added to a stirred solution of (S)-1-(docosyloxy)-3-hydroxypropyl dimethylcarbamate compound 14 (1 g, 2.1196 mmol) in THF (10 mL). Subsequently, diethyl chlorophosphate (1.52 mL, 10.5983 mmol) was added dropwise, and the mixture was stirred at room temperature for 24 h. TLC indicated the formation of the starting material along with the product. Additional DIPEA (0.92 mL, 5.299 mmol), DMAP (0.26 g, 2.1196 mmol), and diethyl chlorophosphate (0.76 mL, 5.299 mmol) were added, and the mixture was stirred at room temperature for an additional 24 h. The completion of the reaction was monitored by TLC. The reaction mixture was diluted with ethyl acetate (100 mL) and washed with water (2 x 50 mL). 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 in 30% EtOAc / hexane. The fractions containing the product were concentrated and dried completely to give the title compound (1 g, pure) as a pale pink waxy solid and was characterized by 1H NMR and 31P NMR: 1 1H 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 singlet at -0.437 was observed in CDCl3.

[1093] (R)-1-(Docosyloxy)-3-(phosphonyloxy)propyl dimethylcarbamate: Synthesis of compound 15:

[1094] At 0 °C, TMSBr (1.3 mL, 9.8708 mmol) was added to a stirred solution of (R)-1-((diethoxyphosphoryl)oxy)-3-(docosyloxy)propan-2-yl dimethylcarbamate (1 g, 1.6451 mmol) in DCM (10 mL), 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 and stirred at 0 °C for another 10 min, saturated NaHCO3 solution (50 ml) was added to the reaction mixture, and washed three times with EtOAc (3x50 mL). The aqueous layer was slowly acidified with 6N HCl solution at 0 °C and extracted with diethyl ether. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a crude gummy white solid. The gummy white solid was stirred with acetonitrile and methanol and filtered to give the title compound as an off-white solid (0.35 g, pure), and characterized by 1H NMR. 1 1H 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). A 31P NMR singlet was observed at 1.312 in CD3OD. HRMS: (M+1) = 551.044.

[1095] (2-(Trimethylammonio)ethyl) phosphate (R)-2-((dimethylcarbamoyl)oxy)-3-ethoxypropyl ester - docosane (1 / 1), Synthesis of Compound 16:

[1096]

[1097] Synthesis of (R)-1-(docosyloxy)-3-((2-oxo-1,3,2-dioxaphospholan-2-yl)oxy)propan-2-yl dimethylcarbamate (8 - 9):

[1098] At 0 °C, triethylamine (0.44 mL, 3.1795 mmol) was added to a stirred solution of (S)-1-(docosyloxy)-3-hydroxypropan-2-yl dimethylcarbamate compound 14 (0.5 g, 1.0598 mmol) in THF. Then, 2-chloro-1,3,2-dioxaphospholane 2-oxide (0.3 mL, 3.1795 mmol) was added, and the mixture was stirred at 0 °C for 4 h. The completion of the reaction mixture was monitored 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 material. Due to the unstable nature of the product, based on TLC and crude NMR, the crude product was used in the next step without any post-treatment and purification.

[1099] (2-(Trimethylammonio)ethyl) phosphate (R)-2-((dimethylcarbamoyl)oxy)-3-ethoxypropyl ester - eicosane (1 / 1), synthesis of compound 16:

[1100] Trimethylamine (5 mL) was added to a stirred solution of (R)-1-(docosyloxy)-3-((2-oxo-1,3,2-dioxaphospholan-2-yl)oxy)propan-2-yl dimethylcarbamate (0.62 g, 1.073 mmol) in acetonitrile, and the mixture was heated to 65 °C in a sealed tube for 16 h. The progress of the reaction was monitored by TLC. The reaction mixture was filtered and the solid was dried to give the product. The crude product was purified by combiflash chromatography. The product was eluted with 40% MeOH / DCM as the eluent. The fractions containing the product were concentrated and dried completely to give an impure compound. The compound was stirred with acetonitrile containing 10% THF and dried to give the title compound (0.1 g, pure) as an off-white solid and characterized by 1H NMR and HRMS. 1 1H NMR (CD3OD, 400 MHz) δ ppm 4.982 - 4.905 (m, 1H), 4.259 (bs, 2H), 4.02 - 3.98 (m, 2H), 3.61 - 3.60 (m, 4H), 3.49 - 3.43 (m, 2H), 3.215 (s, 9H), 2.93 - 2.89 (m, 6H), 1.54 - 1.53 (m, 2H), 1.26 (BS, 40H), 0.89 (t, J = 6.4 Hz, 3H). A 31P NMR singlet was observed at 0.893 in CD3OD. HRMS: (M + 1) = 636.6127.

[1101] Example P-1: Preparation of lipid nanopartic...

Claims

1. A compound of formula (IV-F): wherein R 2 is H, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5; R 3 is C 21- C 24 a normal alkyl group; and Each R 5 is independently a C1-C4 alkyl group; or its protonated or deprotonated form; or its salt.

2. The compound according to claim 1, wherein R 2 is H.

3. The compound according to claim 1, wherein R 2 is -(C=O)-NH2.

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

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

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

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

8. A compound according to any one of claims 1 to 2 or 4 to 7, wherein R 5 is -CH3.

9. A compound of the following formula: or its protonated or deprotonated form, or its salt.

10. A compound of the following formula: or its protonated or deprotonated form, or its salt.

11. A compound of the following formula: or its protonated or deprotonated form, or its salt.

12. A compound of the following formula: or its protonated or deprotonated form, or its salt.

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

14. A composition comprising the compound according to any one of claims 1 to 13 and a TLR agonist.

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

16. The composition according to any one of claims 1 to 15, further comprising an antigen.

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

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

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

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

21. A composition comprising an isolated ether lipid (ETL) of formula (I): wherein: R 1 is H or 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; wherein R 4 is H or (CH3)3N + -(CH2)2-; and Each R 5 independently is a C1-C4 alkyl group; or its protonated or deprotonated form; or its pharmaceutically acceptable salt; and a TLR agonist.

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

23. The composition according to claim 21 or claim 22, wherein R 3 is C 18 -C 22 -n-alkyl or C 21 -C 24 -n-alkyl.

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

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

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

27. A composition comprising an isolated ether lipid (ETL) of formula (I): wherein: R 1 is H or 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 a normal alkyl group; wherein R 4 is H or (CH3)3N + -(CH2)2-; and Each R 5 independently is a C1-C4 alkyl group; or its protonated or deprotonated form; or its pharmaceutically acceptable salt; and an antigen.

28. The composition according to claim 27, further comprising dendritic cells.

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

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

31. A composition comprising an isolated ether lipid (ETL) of formula (I): wherein: R 1 is H or 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 a normal alkyl group; wherein R 4 is H or (CH3)3N + -(CH2)2-; and Each R 5 independently is a C1-C4 alkyl group; or its protonated or deprotonated form; or its pharmaceutically acceptable salt; and dendritic cells.

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

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

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

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

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

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

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

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

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

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

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

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

46. The composition according to claim 44, wherein the biological sample comprises cells.

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

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

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

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

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

52. The composition according to claim 50, wherein the tumor antigen comprises a tumor cell lysate.

53. The composition according to claim 49, wherein the antigen comprises a microbial antigen, and the microbial antigen comprises one or more of a viral antigen, a bacterial antigen, a protozoal antigen, and a fungal antigen.

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

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

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

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

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

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

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

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

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

63. A method for generating hyperactivated dendritic cells, the method comprising contacting the dendritic cells with a composition comprising an effective amount of a separated ether lipid (ETL) as follows: i) Formula (I): Wherein: R 1 is H or 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 a normal alkyl group; wherein R 4 is H or (CH3)3N + -(CH2)2-; and Each R 5 independently is a C1-C4 alkyl group; Or its protonated or deprotonated form; or its pharmaceutically acceptable salt; Or ii) Formula (IV-F): wherein R 2 is H, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5; R 3 is C 21- C 24 a normal alkyl group; and Each R 5 independently is a C1-C4 alkyl group; Or its protonated or deprotonated form; or its salt; And A TLR7 / 8 agonist to generate hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1β without undergoing pyroptosis.

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

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

66. A pharmaceutical preparation comprising at least 10 3 , 10 4 , 10 5 or 10 6 over-activated dendritic cells produced by the method according to claim 64 and a pharmaceutically acceptable excipient.

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

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

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

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

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

72. Use of the formulation according to claim 62 for inducing an anti-tumor immune response in an individual in need thereof, wherein the individual has or has had a tumor. Use of the preparation according to claim 62 for inducing an anti-microbial immune response in an individual in need thereof, wherein the individual is infected with the microbe or has not been exposed to the microbe.

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

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

76. A method for preparing an immunogenic composition, the method comprising: a) removing white blood cells from a cell suspension prepared from a tumor to obtain a tumor cell-rich suspension; b) lysing the cells from the tumor cell-rich suspension to obtain a tumor cell lysate; and c) contacting the tumor cell lysate with the following separated ether lipids (ETL): i) formula (I): Where: R 1 is H or 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 a normal alkyl group; wherein R 4 is H or (CH3)3N + -(CH2)2-; and Each R 5 is independently a C1-C4 alkyl group; Or its protonated or deprotonated form; or its pharmaceutically acceptable salt; or ii) formula (IV-F): wherein R 2 is H, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5; R 3 is C 21- C 24 a normal alkyl group; and Each R 5 independently is a C1-C4 alkyl group; Or its protonated or deprotonated form; or its salt; And contacting with a toll-like receptor (TLR) agonist to obtain the immunogenic composition.

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

78. The method according to claim 76 or claim 77, wherein in step a), the white blood cells are removed by negative selection using an anti-CD45 antibody.

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

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

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

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

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

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

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

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

88. The method according to 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. The method according to any one of claims 76 to 88, the method further comprising obtaining a sample from the tumor of a mammalian subject suffering from cancer and preparing a cell suspension from the sample before step a).

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

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

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

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

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

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

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

97. A method of treating cancer, the method comprising: a) preparing an immunogenic composition comprising a tumor cell lysate, an isolated ether lipid (ETL) as follows: i) Formula (I): Where: R 1 is H or 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; wherein R 4 is H or (CH3)3N + -(CH2)2-; and Each R 5 independently is a C1-C4 alkyl group; Or its protonated or deprotonated form; or its pharmaceutically acceptable salt; Or ii) Formula (IV-F): Where R 2 is H, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5; R 3 is C 21- C 24 is a normal alkyl group; and Each R 5 independently is a C1-C4 alkyl group; Or its protonated or deprotonated form; or its salt; And a toll-like receptor (TLR) agonist, wherein the tumor cell lysate is prepared from or has been prepared from a tumor sample obtained from a mammalian subject having cancer; and b) administering to the subject an effective amount of the immunogenic composition.

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

99. The method according to claim 97 or claim 98, wherein R in formula (I) 3 is C 18 -C 22 alkyl chain or C 18 -C 24 alkyl chain.

100. The method according to claim 97 or claim 98, wherein R in formula (I) 3 is C 16 -C 20 alkyl.

101. The method according to claim 97 or claim 98, wherein R 3 is C 21 -C 24 alkyl 102. The method according to 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 according to any one of claims 97 to 102, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 daltons or less.

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

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

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

109. The method of claim 108, wherein the additional therapeutic agent comprises one or more of the group consisting of immune checkpoint inhibitors, anti-neoplastic agents, and radiotherapy.

110. A composition comprising an isolated ether lipid (ETL) as follows: i) Formula (I): Where: R 1 is H or 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; wherein R 4 is H or (CH3)3N + -(CH2)2-; and Each R 5 independently is a C1-C4 alkyl group; or its protonated or deprotonated form; or a pharmaceutically acceptable salt thereof; or ii) Formula (IV-F): where R 2 is H, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5; R 3 is C 21- C 24 a normal alkyl group; and Each R 5 independently is a C1-C4 alkyl group; or its protonated or deprotonated form; or a salt thereof; and a pathogen recognition receptor (PRR) agonist.

111. The composition according to claim 110, wherein the PRR agonist is an agonist of a toll-like receptor (TLR), NOD-like receptor (NLR), RIG-I-like receptor (RLR), or C-type lectin receptor (CLR).

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

113. The composition according to claim 110, wherein the PRR agonist comprises one or more of R848, TL8-506, LPS, Pam2CSK4, and ODN 2336.

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

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

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

117. A pharmaceutical preparation comprising the following isolated ether lipid (ETL): i) Formula (I): Wherein: R 1 is H or 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; wherein R 4 is H or (CH3)3N + -(CH2)2-; and Each R 5 independently is a C1-C4 alkyl group; or its protonated or deprotonated form; or a pharmaceutically acceptable salt thereof; or ii) Formula (IV-F): where R 2 is H, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5; R 3 is C 21- C 24 is a normal alkyl group; and Each R 5 independently is a C1-C4 alkyl group; or its protonated or deprotonated form; or a salt thereof; and a pharmaceutically acceptable excipient.

118. The pharmaceutical preparation according to claim 117, wherein R 3 is C 22 n-alkyl.

119. The pharmaceutical preparation according to claim 118, wherein the ETL comprises one or both of DGPC and DGP or a pharmaceutically acceptable salt thereof.

120. A composition for the overactivation of human dendritic cells, the composition comprising the following isolated ether lipid (ETL): i) Formula (I): Wherein: R 1 is H or 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; wherein R 4 is H or (CH3)3N + -(CH2)2-; and Each R 5 is independently a C1-C4 alkyl group; or its protonated or deprotonated form; or a pharmaceutically acceptable salt thereof; or ii) Formula (IV-F): wherein R 2 is H, -(C=O)-NH2, -(C=O)-NH(R 5 ), -(C=O)-N(R 5 )2 or -CH2-C6H5; R 3 is C 21- C 24 a normal alkyl group; and Each R 5 independently is a C1-C4 alkyl group; or its protonated or deprotonated form; or a salt thereof; and a pathogen recognition receptor (PRR) agonist, wherein the alkyl chain is a C22 normal alkyl chain, and wherein the composition effectively achieves a higher level of dendritic cell overactivation compared to a comparative composition comprising PGPC instead of ETL.

121. The composition according to claim 120, wherein R 3 is C 22 n-alkyl.

122. The composition according to claim 120 or claim 121, wherein the higher level of dendritic cell overactivation comprises at least 2, 3, or 4 times higher levels of in vitro induction of IL-1β secretion from the human dendritic cells upon contact with the composition comprising the ETL and the PRR agonist compared to upon contact with the comparative composition comprising the PGPC and the PRR agonist, wherein the PRR agonist is LPS.

123. The composition according to claim 122, wherein the concentration of the ETL and the concentration of the PGPC are the same concentration in the range of about 10 μM to about 80 μM, and the LPS is present in both the composition and the comparative composition at a concentration of 1 μg / ml.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

139. The composition, formulation, method or use according to any one of claims 135 to 138, wherein i) the ETL is dissolved in an 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.

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

142. An isolated ether lipid (ETL) of formula (I): wherein R 1 is H or 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; wherein R 4 is H or (CH3)3N + -(CH2)2-; and Each R 5 independently is a C1-C4 alkyl group; or its protonated or deprotonated form; or its salt.

143. The isolated ether lipid according to claim 142, wherein the isolated ether lipid is a compound of formula (II): wherein R 1 is H or 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; wherein R 4 is H or (CH3)3N + -(CH2)2-; and Each R 5 independently is a C1-C4 alkyl group; or its protonated or deprotonated form; or its salt.

144. The isolated ether lipid according to claim 142, wherein the isolated ether lipid is a compound of formula (III): wherein 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 is a normal alkyl group; and Each R 5 independently is a C1-C4 alkyl group; or its salt.

145. The isolated ether lipid according to claim 142, wherein the isolated ether lipid is an isolated ether phospholipid (ETPL) compound of formula (IV): wherein 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-; and Each R 5 independently is a C1-C4 alkyl group; or its protonated or deprotonated form; or its salt.

146. The isolated ether lipid according to claim 142, wherein the isolated ether lipid is an isolated ether phospholipid (ETPL) compound of formula (IV-A): wherein 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 a normal alkyl group; and Each R 5 independently is a C1-C4 alkyl group; or its protonated or deprotonated form; or its salt.

147. The isolated ether lipid according to claim 142, wherein the isolated ether lipid is an isolated ether phospholipid (ETPL) compound of formula (IV-B): wherein 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; and Each R 5 independently is a C1-C4 alkyl group; or its protonated form; or its salt.

148. The isolated ether lipid according to claim 142, wherein the isolated ether lipid is an isolated ether phospholipid (ETPL) compound of formula (IV-C): wherein R 3 is C 13- C 24 n-alkyl; and R 4 is H or (CH3)3N + -(CH2)2-; or its protonated or deprotonated form; or its salt.

149. A compound of formula 2: or its protonated form; or its pharmaceutically acceptable salt.

150. The compound according to claim 149, wherein the compound is isolated.

151. An isolated compound 1 of formula 1: or its protonated form; or its pharmaceutically acceptable salt.

152. A compound of formula (III-A-1), wherein: R 2 is -(C=O)-NH2, -(C=O)-NH(R 5 ) or -(C=O)-N(R 5 )2; R 3 is C 21- C 24 is a normal alkyl group; and Each R 5 independently is a C1-C4 alkyl group; or its pharmaceutically acceptable salt.

153. The compound according to claim 152, wherein R 2 is -(C=O)-NH2.

154. The compound according to claim 152, wherein R 2 is -(C=O)-NH-CH3.

155. The compound according to claim 152, wherein R 2 is -(C=O)-N(CH3)2.

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

157. A compound 7 of formula 7: or its pharmaceutically acceptable salt.

158. The compound according to claim 157, wherein the compound is isolated.

159. A compound 8 of formula 8: or its pharmaceutically acceptable salt.

160. The compound according to claim 159, wherein the compound is isolated.

161. A compound of the following formula: or its protonated form; or its salt.

162. The compound according to claim 161, wherein the compound is isolated.

163. A composition comprising the compound according to any one of claims 142 to 162 and a pharmaceutically acceptable excipient.

164. The composition according to claim 163, which further comprises a surfactant.

165. The composition according to claim 164, wherein the surfactant is selected from the group consisting of: nonionic surfactants, wetting agents, P407, P188, polysorbate 80, thickeners, and carboxymethyl cellulose.

166. The composition according to any one of claims 163 to 165, which comprises particles having a diameter of less than about 5 microns to about 20 microns (D 50 <5 microns to 20 microns), wherein the particles comprise an ether lipid and a nonionic surfactant.

167. The composition according to any one of claims 163 to 166, wherein the pharmaceutically acceptable excipient comprises phosphate buffered saline.

168. The composition according to any one of claims 163 to 167, wherein the pharmaceutically acceptable excipient comprises an aqueous solution of ethylene oxide-propylene oxide copolymer (poloxamer), or further comprises ethylene oxide-propylene oxide copolymer.

169. The composition according to any one of claims 163 to 165, wherein the pharmaceutically acceptable excipient comprises phosphate buffered saline, and at least one of poloxamer 407, poloxamer 188, and P123.

170. The composition according to any one of claims 163 to 169, wherein the composition is sterile.

171. An article comprising a container encapsulating a liquid preparation of the compound according to any one of claims 142 to 170 and a pharmaceutically acceptable excipient.

172. The article according to claim 171, wherein the container is a syringe.

173. The article according to claim 172, wherein the syringe is further contained within an injection device.

174. The article according to claim 173, wherein the injection device is an autoinjector.

175. A composition comprising an ether lipid (ETL) or ether phospholipid (ETPL) 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) as disclosed herein, 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, where possible, its protonated or deprotonated form, or its pharmaceutically acceptable salt; and at least one other lipid, wherein the at least one other lipid is selected from the group consisting of: ionizable lipids, cationic lipids, another phospholipid, polyethylene glycolated lipids, structured lipids, and mixtures thereof.

176. The composition according to claim 175, wherein the ETL or ETPL and the at least one other lipid are part of a lipid nanoparticle (LNP).

177. The composition according to claim 175 or claim 176, which further comprises an antigen.

178. The composition according to any one of claims 175 to 177, which further comprises dendritic cells.

179. The composition according to any one of claims 175 to 178, which further comprises a TLR agonist.

180. The composition according to any one of claims 175 to 178, which further comprises a TLR7 / 8 agonist.

181. A composition, method or use according to any one of claims 16, 25, 27, 34, 67, 71, 114 or 177, wherein the antigen comprises one or more viral antigens.

182. A composition, method or use according to claim 181, wherein the one or more viral antigens comprise one or both of an influenza A antigen and an influenza B antigen.

183. A composition, method or use according to claim 182, wherein one or both of the influenza A antigen and the influenza B antigen comprise one or both of a hemagglutinin and a nucleoprotein.

184. A composition, method or use according to any one of claims 181 to 183, wherein the viral antigen comprises inactivated virus particles, optionally wherein the inactivated virus particles comprise inactivated split virus particles.

185. A composition, method or use according to any one of claims 182 to 184, which comprises both an influenza A antigen and an influenza B antigen of H1N1 influenza A virus, H3N2 influenza A virus, Victoria lineage influenza B virus and Yamagata lineage influenza B virus.

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