Mammalian dendritic cell hyperactivator

Abstract

The present disclosure relates to lysophosphatidylcholine (LPC) compounds and their use in hyperactivating mammalian dendritic cells, such as human dendritic cells or canine dendritic cells. The present disclosure also relates to compositions comprising LPC and one or more of a pathogen recognition receptor agonist, an antigen, and a mammalian dendritic cell, as well as methods for making and using the compositions. In particular, the present disclosure provides a composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain and a TLR7 / 8 agonist, the acyl chain being a C13-C22 acyl chain or a C13-C24 acyl chain.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 246,740, filed September 21, 2021, and U.S. Provisional Patent Application No. 63 / 173,958, filed April 12, 2021, the disclosures of which are incorporated by reference in their entireties herein.

[0002] Submission of sequence listing as an ASCII text file none.

[0003] Field The present disclosure relates to lysophosphatidylcholine (LPC) compounds and their use in hyperactivating mammalian dendritic cells, such as human dendritic cells or canine dendritic cells. The present disclosure also relates to compositions comprising LPC and one or more of a pathogen recognition receptor agonist, an antigen, and human or canine dendritic cells, and methods for producing and using the compositions. [Background technology]

[0004] background Typically, maturation of dendritic cells (DCs) with vaccine adjuvants such as Toll-like receptor agonists does not lead to IL-1beta secretion. In some situations, such as inflammasome activation, IL-1beta secretion occurs, but at the expense of DC death by a lytic process of cell death called pyroptosis (Evavold et al., J Mol Biol, 430(2):217-237, 2018). However, maturation of DCs with the pathogen-associated molecular pattern (PAMP)-containing molecule lipopolysaccharide (LPS) and damage-associated molecular pattern (DAMP)-containing molecules, such as PGPC (1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine), results in the production and secretion of IL-1beta without pyroptosis, and these viable DCs are characterized as hyperactive (Zanoni et al., Science, 352(6290):1232-1236, 2016). Indeed, in mouse models, hyperactivated DCs have been shown to have an enhanced ability to induce immune responses compared to cells activated using LPS alone (Zhivaki et al., Cell Rep, 33(7):108381, 2020). However, little is known about effective stimuli for hyperactivating human DCs. Therefore, there is a need in the art for the identification of PAMPs and DAMPs suitable for the hyperactivation of human DCs. Furthermore, it is desirable to identify alternatives to the use of LPS and PGPC for the hyperactivation of mammalian DCs. In particular, LPS (endotoxin) is a potent PAMP, but its use in humans is contraindicated because it can cause septic shock. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Evavold et al., J Mol Biol,430(2):217-237,2018 [Non-Patent Document 2] Zanoni et al., Science, 352(6290):1232-1236, 2016 [Non-Patent Document 3] Zhivaki et al., Cell Rep,33(7):108381,2020 Summary of the Invention

[0006] Quick Overview The present disclosure relates to lysophosphatidylcholine (LPC) compounds and their use in hyperactivating mammalian dendritic cells, such as human dendritic cells or canine dendritic cells. The present disclosure also relates to compositions comprising LPC and one or more of a pathogen recognition receptor agonist, an antigen, and human or canine dendritic cells, and methods for producing and using the compositions.

[0007] In particular, the present disclosure provides compositions comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain and a TLR7 / 8 agonist, wherein the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl chain. In some embodiments, the acyl chain is a C18-C22 acyl chain, a C21-C24 acyl chain, or a C22 acyl chain. In some embodiments, the composition further comprises an antigen and / or a dendritic cell.

[0008] In some aspects, the present disclosure provides a composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain and an antigen, wherein the acyl chain is a C21-C24 acyl chain. In some embodiments, the composition further comprises a dendritic cell and / or a TLR7 / 8 agonist.

[0009] In some aspects, the present disclosure provides a composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain, the acyl chain being a C21-C24 acyl chain, and a dendritic cell. In some embodiments, the composition further comprises a TLR7 / 8 agonist and / or an antigen.

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

[0011] In some embodiments, the composition does not include liposomes. In some embodiments, the composition does not include LPS or MPLA. In some embodiments, the composition does not include oxPAPC or species of oxPAPC. In some embodiments, the composition does not include HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC, and / or PGPC.

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

[0013] In some embodiments, the present disclosure provides a pharmaceutical formulation comprising the composition of any of the preceding aspects and a pharma- ceutically acceptable excipient.

[0014] In other aspects, the disclosure provides a method for producing hyperactivated dendritic cells, the method comprising contacting dendritic cells with a composition comprising an effective amount of isolated lysophosphatidylcholine (LPC) having a single C13-C22 acyl chain or C13-C24 acyl chain and a TLR7 / 8 agonist for producing hyperactivated dendritic cells, the hyperactivated dendritic cells secreting IL-1 beta without undergoing pyroptosis. In some embodiments, the dendritic cells are contacted ex vivo with a composition or pharmaceutical formulation of any one of the preceding embodiments. In other embodiments, the dendritic cells are contacted in vivo with a pharmaceutical formulation comprising a composition of any one of the preceding embodiments. In some aspects, the disclosure provides a pharmaceutical formulation comprising a plurality of hyperactivated dendritic cells produced by the preceding embodiments and a pharma- ceutical acceptable excipient. In some embodiments, the plurality is at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 Pieces or 10 8 Contains overactivated DCs.

[0015] In another aspect, the disclosure provides a composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain and a pathogen recognition receptor (PRR) agonist, wherein the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl chain. In some embodiments, the PRR agonist is an agonist of a toll-like receptor (TLR), a NOD-like receptor (NLR), a RIG-I-like receptor (RLR), or a C-type lectin receptor (CLR). In some embodiments, the PRR agonist is an agonist of a cytoplasmic DNA sensor (CDS) or a stimulator of IFN genes (STING). In some embodiments, the PRR agonist comprises a TLR7 / 8 agonist. In some embodiments, the composition further comprises an antigen and / or a dendritic cell.

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

[0017] In some embodiments of the 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). In some embodiments, the LPC comprises LPC(22:0) and the TLR7 / 8 agonist comprises resiquimod (R848).

[0018] The present disclosure further provides a composition for the hyperactivation of human dendritic cells, comprising an isolated lysophosphatidylcholine (LPC) compound having a single acyl chain and a pathogen recognition receptor (PRR) agonist, the acyl chain being a C22 acyl chain, wherein the composition is effective in achieving a higher level of hyperactivation of dendritic cells than a control composition comprising PGPC instead of LPC. In some aspects, the hyperactivation occurs in vitro or ex vivo. In other embodiments, the hyperactivation occurs in vivo. In some embodiments, the higher level of hyperactivation of dendritic cells comprises inducing at least 2, 3, or 4-fold higher secretion of IL-1 beta from human dendritic cells in vitro when contacted with a composition comprising LPC and a PRR agonist than when contacted with a control composition comprising PGPC and a PRR agonist, wherein the PRR agonist is LPS. In some embodiments, the concentrations of LPC and PGPC are the same, optionally ranging from about 10 μM to about 80 μM, and LPS is present in both the composition and the control composition at a concentration of 1 μg / ml. In some embodiments, the higher level of dendritic cell hyperactivation comprises a lipid activity index for IL-1 beta secretion from human dendritic cells for a composition comprising LPC and a PRR agonist that is at least 4, 5, or 6 times higher, in activity units, than the lipid activity index for a control composition comprising LPC and a PRR agonist. [Brief description of the drawings]

[0019] [Figure 1] Figure 1 is a schematic diagram showing the effect of various stimuli on dendritic cell function. Depending on the stimulus, dendritic cells remain quiescent or are activated, undergo pyroptosis, or become hyperactivated.

[0020] [Diagram 2]Figure 2 shows IL-1β secretion by human monocyte-derived dendritic cells activated with 1 μg / mL LPS in the presence of various lysophosphatidylcholine compounds (LPC) at 82.5 μM. The results demonstrate that acyl chain length influences the ability of LPC to promote IL-1β secretion by DCs.

[0021] [Diagram 3] Figure 3 shows IL-1β secretion by human monocyte-derived dendritic cells activated with 1 μg / mL LPS in the presence of 82.5 μM of various lipids. The results demonstrate that lysophosphatidylcholine compounds (LPCs) with an acyl chain length of at least 12 carbons (12:0 Lyso PC, No. 29) can stimulate human DCs to secrete IL-1β at levels equal to or greater than DAMPs such as PGPCs identified by screening mouse DCs (22:0 Lyso PC, No. 42).

[0022] [Figure 4] Figure 4 shows the lipid activity index for various compounds, calculated by multiplying the maximum IL-1β signal observed at any concentration by the reciprocal of the lowest concentration at which IL-1β secretion by human monocyte-derived dendritic cells was 2-fold higher than the LPS-only control.

[0023] [Diagram 5] FIG. 5 shows IL-1β secretion by human monocyte-derived dendritic cells contacted with various PRR agonists in the presence or absence of 22:0 Lyso PC.

[0024] [Figure 6A-6C]Figures 6A-6B show IL-1β secretion by canine peripheral blood mononuclear cells (PBMCs) 2 days after activation with the indicated stimuli, shown as total concentration (Figure 6A) and fold change per donor (Figure 6B), respectively, compared to R848 alone. The results demonstrate that 22:0 LYSO PC in combination with R848 can stimulate canine PBMCs to secrete IL-1β at levels comparable to or higher than PGPC or DAMPs such as LPS and alum. Figure 6C shows the relative viability of canine PBMCs 2 days after activation with the indicated stimuli. The results demonstrate that canine PBMCs remain viable following treatment with 22:0 LYSO PC.

[0025] [Figure 7A-7C] Figures 7A-7B show IL-1β secretion by human PBMCs 2 days after activation with the indicated stimuli, shown as total concentration (Figure 7A) and fold change per donor compared to R848 alone (Figure 7B). The results demonstrate that 22:0 LYSO PC in combination with R848 can stimulate human PBMCs to secrete IL-1β at levels comparable to or higher than PGPC or DAMPs such as LPS and alum. Figure 7C shows the relative viability of human PBMCs 2 days after activation with the indicated stimuli. The results demonstrate that human PBMCs remain viable following treatment with 22:0 LYSO PC.

[0026] [Figure 8A-8B] Figures 8A-8B show IFNγ (Figure 8A) and TNFα (Figure 8B) secretion by human PBMCs 2 days after activation with the indicated stimuli, shown as fold change per donor compared to R848 alone. The results demonstrate that 22:0 LYSO PC in combination with R848 can stimulate human PBMCs to secrete other immunostimulatory cytokines at levels comparable to or higher than PGPC or DAMPs such as LPS and alum.

[0027] [Figure 9A-9B]Figures 9A-9B show the viability (Figure 9A) and IL-1β secretion (Figure 9B) by non-human primate monocyte-derived dendritic cells (moDCs) under various activation conditions (unstimulated or contacted with R848 in the presence or absence of 22:0 Lyso PC or PGPC).

[0028] [Figure 10A-10B] 10A-10B show IL-1β secretion by non-human primate PBMCs 2 days after activation with the indicated stimuli, shown as total concentration (FIG. 10A) and fold change compared to R848 alone (FIG. 10B).

[0029] [Figure 11A-11B] 11A-11B show IFN-γ secretion by non-human primate PBMCs 2 days after activation with the indicated stimuli, shown as total concentration (FIG. 11A) and fold change compared to R848 alone (FIG. 11B).

[0030] [Figure 12A-12B] 12A-12B show IL-17a secretion by non-human primate PBMCs 2 days after activation with the indicated stimuli, shown as total concentration (FIG. 12A) and fold change compared to R848 alone (FIG. 12B).

[0031] [Figure 13A-13B] Figures 13A-13B show IL-23 secretion by non-human primate PBMCs 2 days after activation with the indicated stimuli, shown as total concentration (Figure 13A) and fold change compared to R848 alone (Figure 13B).

[0032] [Figure 14A-14B] Figures 14A-14B show IFN-β secretion by non-human primate PBMCs 2 days after activation with the indicated stimuli, shown as total concentration (Figure 14A) and fold change compared to R848 alone (Figure 14B).

[0033] [Figure 15A-15B]Figures 15A-15B show IL-8 secretion by non-human primate PBMCs 2 days after activation with the indicated stimuli, shown as total concentration (Figure 15A) and fold change compared to R848 alone (Figure 15B).

[0034] [Figure 16A-16B] Figures 16A-16B show IL-6 secretion by non-human primate PBMCs 2 days after activation with the indicated stimuli, shown as total concentration (Figure 16A) and fold change compared to R848 alone (Figure 16B).

[0035] [Figure 17] FIG. 17 shows IFN-γ secretion by human memory CD4+ T cells.

[0036] [Figure 18A-18B] 18A-18B show secretion of IL-4 (FIG. 18A) and IL-13 (FIG. 18B) by human memory CD4+ T cells.

[0037] [Figures 19A-19C] Figures 19A-C show IL-17a (Figure 19A), IL-17f (Figure 19B) and IL-22 (Figure 19C) by human memory CD4+ T cells.

[0038] [Figure 20] FIG. 20 shows Th1 polarization of human naive CD4+ T cells as a result of co-culture with moDCs treated with R848 and 22:0 LYSO PC compared to co-culture with moDCs treated with R848 alone.

[0039] [Figure 21] FIG. 21 shows the viability of human moDCs cultured in the presence of PBS or various filtered or unfiltered lipid formulations.

[0040] [Figure 22] FIG. 22 shows IL-1β secretion by human moDCs cultured in the presence of PBS or various filtered or unfiltered lipid formulations.

[0041] [Figure 23A-23B] 23A-23B show IL-1β secretion by (FIG. 23A) and viability of (FIG. 23B) human moDCs cultured in the presence of PBS or various filtration formulations.

[0042] [Figure 24] FIG. 24 shows the characterization of 22:0 LYSOPC-containing particle size as determined by dynamic light scattering.

[0043] [Fig. 25A-25B] 25A-B show IL-1β secretion by murine FLT3L-differentiated DCs (FIG. 25A) and viability of the DCs (FIG. 25B) cultured under the indicated conditions.

[0044] [Figures 26A-26C] 26A-26C show TNF-alpha (FIG. 26A), IL-6 (FIG. 26B) and IL-12p40 (FIG. 26C) by murine FLT3L-differentiated DCs cultured under the indicated conditions.

[0045] [Figure 27] Figure 27 shows co-stimulatory molecule (CD40) expression by mouse FLT3L-differentiated cDC1 and cDC2 cells cultured under the indicated conditions. Mean values ​​and SD from at least two replicates are shown, and data represent at least two independent experiments. P values ​​of <0.05 (*), <0.01 (**) or <0.001 (***), %0.0001 (****) indicated significant differences between groups. Two-way ANOVA test was used.

[0046] [Fig. 28A-28B] 28A-28B show CCR7 (FIG. 28A) and CXCL16 (FIG. 28B) expression by mouse FLT3L-differentiated cDC1 and cDC2 cells cultured under the indicated conditions.

[0047] [Figure 29]Figure 29 shows MHC class I expression by mouse FLT3L-differentiated cDC1 and cDC2 cells cultured under the indicated conditions. Mean values ​​and SD from at least two replicates are shown, and data represent at least two independent experiments. P values ​​of <0.05 (*), <0.01 (**) or <0.001 (***), %0.0001 (****) indicated significant differences between groups. Two-way ANOVA test was used.

[0048] [Fig. 30A-30B] Figures 30A-30B show antigen uptake (Figure 30A) and antigen presentation (Figure 30B) by murine FLT3L-differentiated DCs cultured under the indicated conditions. Antigen uptake was assessed by measuring endocytosis of Red pHrodo dextran. Antigen presentation was assessed by measuring ovalbumin peptide bound to MHC class I, H-2Kb.

[0049] [Diagram 31] FIG. 31 shows DC infiltration in the skin-draining lymph nodes (dLN) of mice following subcutaneous injection of the indicated formulations containing R848, 22:0 LYSO PC and the surfactant, KP407.

[0050] [Diagram 32] FIG. 32 shows survival of tumor-bearing mice treated with PBS or a therapeutic cancer vaccine (eg, whole tumor lysate formulation).

[0051] [Diagram 33] FIG. 33 shows the kinetics of tumor growth in mice treated with PBS or a therapeutic cancer vaccine (eg, a whole tumor lysate formulation). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Detailed Description The present disclosure relates to lysophosphatidylcholine (LPC) compounds and their use in hyperactivating human dendritic cells.The present disclosure also relates to compositions comprising LPC and one or more of a pathogen recognition receptor agonist, an antigen, and human dendritic cells, and methods for producing and using the compositions.In further embodiments, the dendritic cells are non-human dendritic cells, with the proviso that the dendritic cells are not rodent dendritic cells.

[0053] General Techniques and Definitions The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0054] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless otherwise indicated. For example, "a" excipient includes one or more excipients.

[0055] As used herein, the phrase "comprising" is open ended and indicates that such embodiments may include additional elements. In contrast, the phrase "consisting of" is closed and indicates that such embodiments do not include additional elements (except for trace impurities). The phrase "consisting essentially of" is partially closed and indicates that such embodiments may further include elements that do not materially alter the basic characteristics of such embodiments.

[0056] The term "about" as used herein in reference to a value includes 90% to 110% of that value (eg, a molecular weight of about 900 daltons refers to a molecular weight of 910 daltons to 990 daltons).

[0057] An "effective amount" or "sufficient amount" of a substance is an amount sufficient to produce a beneficial or desired result, including a clinical result, and thus 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 includes sufficient antigen and one or both of a lysophosphatidylcholine (LPC) compound and a PRR agonist to stimulate an immune response (e.g., antigen-reactive antibody and / or cellular immune response) against the antigen.

[0058] The terms "individual" and "subject" refer to a mammal. "Mammals" include, but are not limited to, humans, non-human primates (e.g., monkeys), farm animals, sport animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats). In some embodiments, the subject is a human patient, such as a human patient suffering from cancer and / or an infectious disease.

[0059] The term "dose" as used herein with respect to an immunogenic composition refers to the measured portion of the immunogenic composition taken (administered or received) by a subject at any one time.

[0060] The terms "isolated" and "purified" as used herein refer to a material that is removed from at least one component with which it is naturally associated (e.g., removed from its original environment). As an example, when used in reference to LPC, an isolated LPC is at least 90%, 95%, 96%, 97%, 98% or 99% pure as measured by thin layer chromatography or gas chromatography. As a further example, when used in reference to a recombinant protein, an isolated protein refers to a protein that has been removed from the culture medium of a host cell that produced the protein.

[0061] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the individual to whom the formulation or composition is administered. Such formulations or compositions are intended to be sterile.

[0062] As used herein, "excipient" includes a pharma- ceutically acceptable excipient, carrier, vehicle, or stabilizer that is non-toxic to cells or mammals exposed thereto at the dosages and concentrations used. Often, the physiologically acceptable excipient is an aqueous pH buffered solution.

[0063] The term "antigen" refers to a substance that is specifically recognized and bound by an antibody or a T cell antigen receptor. Antigens may include peptides, polypeptides, proteins, glycoproteins, polysaccharides, glycoconjugates, sugars, gangliosides, lipids and phospholipids; portions thereof and combinations thereof. Antigens, when present in the compositions of the present disclosure, may 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 "antigens." A "hapten" is a low molecular weight compound that is not immunogenic in itself, but generally becomes immunogenic when combined with a larger immunogenic molecule (carrier).

[0064] "Polypeptide antigens" may include purified natural peptides, synthetic peptides, recombinant peptides, crude peptide extracts, or partially purified or unpurified active peptides (e.g., peptides that are part of attenuated or inactivated viruses, microorganisms, or cells), or fragments of such peptides. Polypeptide antigens are preferably at least 8 amino acid residues in length.

[0065] The term "agonist" is used in the broadest sense and includes any molecule that activates signal transduction through a receptor. In some embodiments, the agonist binds to the receptor. For example, the TLR8 agonist binds to the TLR8 receptor and activates the TLR8 signal transduction pathway.

[0066] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group. Cx alkyl refers to an alkyl group having x carbon atoms. Cx-Cy alkyl or Cx-y alkyl refers to an alkyl group having between x and y carbon atoms, inclusive.

[0067] "Alkylene" refers to a divalent saturated aliphatic hydrocarbyl group.

[0068] "Alkenyl" refers to a monovalent hydrocarbyl group having at least one double bond (>C=C<). Cx alkenyl refers to an alkenyl group having x carbon atoms. Cx-Cy alkenyl or Cx-y alkenyl refers to an alkenyl group having between x and y carbon atoms, inclusive.

[0069] "Stimulation" of a response or parameter includes eliciting and / or enhancing that response or parameter when compared to otherwise identical conditions except for the parameter of interest, or when compared to another condition (e.g., increased TLR signaling in the presence of a TLR agonist compared to the absence of a TLR agonist). For example, "stimulation" of an immune response means an increase in the response. Depending on the parameter measured, this increase can be 2-2,000-fold, or 5-500-fold or greater, or from 2, 5, 10, 50, or 100-fold to 500, 1,000, 2,000, 5,000, or 10,000-fold.

[0070] Conversely, "inhibition" of a response or parameter includes reducing and / or suppressing that response or parameter when compared to otherwise identical conditions except for the parameter of interest, or when compared to another condition (e.g., a reduction in abnormal cell proliferation following administration of a composition comprising an LPC compound and one or more of a pathogen recognition receptor agonist, an antigen, and human dendritic cells, when compared to administration of a placebo composition or no treatment). For example, "inhibition" of an immune response refers to a reduction in the response. Depending on the parameter measured, this reduction can be 2-2,000-fold, or 5-500-fold or greater, or 2, 5, 10, 50, or 100-fold to 500, 1,000, 2,000, 5,000, or 10,000-fold.

[0071] The relative terms "higher" and "lower" refer to a measurable increase or decrease in a response or parameter, respectively, when compared to otherwise identical conditions, except for the parameter of interest, or when compared to another condition. For example, "higher levels of DC hyperactivation" refers to a level of DC hyperactivation resulting from a treatment condition (including the LPC compounds of the present disclosure) that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than the level of DC hyperactivation resulting from a control condition (e.g., without LPC, PGPC, oxPAPC, etc.). Similarly, "lower levels of DC hyperactivation" refers to a level of DC hyperactivation resulting from a treatment condition (including the LPC compounds of the present disclosure) that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times lower than the level of DC hyperactivation resulting from a control condition (e.g., without LPC, PGPC, oxPAPC, etc.).

[0072] As used herein, the term "immunization" refers to the process of increasing the response of a mammalian subject to an antigen, thus improving its ability to resist or overcome infection and / or resist disease.

[0073] As used herein, the term "vaccination" refers to the introduction of a vaccine into the body of a mammalian subject.

[0074] "Adjuvant" refers to a substance that, when added to a composition containing an antigen, enhances or boosts the immune response to the antigen in a mammalian recipient upon exposure.

[0075] The term "treating" or "treatment" of a disease refers to carrying out a protocol that may include administering one or more therapeutic agents to an individual (human or otherwise) 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 amelioration of one or more signs or symptoms of the disease, reduction in the extent of the disease, stabilization of the disease state (i.e., not worsening), prevention of the spread of the disease, delay or slowing of the progression of the disease, improvement or alleviation of the disease state, and remission (whether partial or total). "Treatment" may also mean prolonging survival compared to the expected survival of an individual not receiving treatment. Furthermore, "treating" and "treatment" may be performed by administration of a single dose of one or more therapeutic agents, or by administration of a series of doses of one or more therapeutic agents. In addition, "treating" or "treatment" does not require complete relief of signs or symptoms, does not require a cure, and specifically includes protocols that provide only a palliative effect to the patient. "Ameliorating" a disease or disorder means reducing the extent and / or undesirable clinical symptoms of the disease or disorder and / or slowing the time course of progression of the disease or disorder compared to the expected untreated outcome.

[0076] I. Lysophosphatidylcholine Compounds "Lysophosphatidylcholine" (LPC) or "lysophosphatidylcholine molecule" refers to a glycerol molecule having one phosphocholine group on a hydroxyl group of the glycerol and one acyl group on one of the other two hydroxyl groups of the glycerol. The remaining hydroxyl group is unsubstituted.

[0077] In some embodiments, the isolated lysophosphatidylcholine (LPC) having a single acyl chain has the following form: [ka] It is.

[0078] In some embodiments, the isolated lysophosphatidylcholine (LPC) having a single acyl chain has the following form: [ka] It is.

[0079] The alkyl or alkenyl chain together with the carbonyl carbon forms an acyl chain that is one carbon atom longer than the alkyl or alkenyl chain. For example, a (C23 alkyl)-C(=O)- group forms a C24 acyl chain. Thus, if the group "(alkyl or alkylene)" is a C12-C23 alkyl group (e.g., a C12-C19 alkyl group or a C20-C23 alkyl group), the (C12-C23 alkyl-C(=O)- group forms a C13-C24 acyl chain (e.g., a C13-C20 acyl chain or a C21-C24 acyl chain). If the group "(alkyl or alkylene)" is a C12-C23 alkenyl group (e.g., a C12-C19 alkenyl group or a C20-C23 alkenyl group), the (C12-C23 alkyl-C(=O)- group forms a C13-C24 acyl chain (e.g., a C13-C20 acyl chain or a C21-C24 acyl chain). In the case of a C12-C23 alkenyl-C(=O)- group, the C12-C23 alkenyl-C(=O)- group forms a C13-C24 acyl chain (e.g., a C13-C20 acyl chain or a C21-C24 acyl chain). The acyl chain can be referred to as saturated or unsaturated acyl to distinguish between alkyl- and alkenyl-containing acyl groups. Standard delta or omega notation can be used to indicate the position of one or more double bonds in an unsaturated acyl chain.

[0080] The lysophosphatidylcholine (LPC) compounds of the present disclosure have a single acyl chain, where the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl chain. In some embodiments, the acyl chain is a C18-C22 acyl chain or a C21-C24 acyl chain. In some embodiments, the acyl chain is a C21 acyl chain or a C22 acyl chain. In some preferred embodiments, the acyl chain is a C22 acyl chain. The names and structures of exemplary LPC compounds of the present disclosure, as well as their Chemical Abstract Service (CAS) registry numbers, are shown in Table I as #30-43, and optionally #30-42. [Table I-1] [Table I-2] [Table I-3] [Table I-4]

[0081] II. Pathogen Recognition Receptor Agonists The compositions and methods of the present disclosure may further comprise a pathogen recognition receptor (PRR) agonist. In some embodiments, the PRR agonist comprises a toll-like receptor (TLR), a NOD-like receptor (NLR), a RIG-I-like receptor (RLR) or a C-type lectin receptor (CLR) agonist. In other embodiments, the PRR agonist comprises a cytoplasmic DNA sensor (CDS) agonist or a stimulator of IFN genes (STING) agonist. In some embodiments, the PRR agonist comprises a TLR7 / 8 agonist.

[0082] A. TLR7 / 8 agonists The term "TLR7 / 8 agonist" as used herein refers to an agonist of TLR7 and / or TLR8. In one embodiment, the TLR7 / 8 agonist is a TLR7 agonist. In another embodiment, the TLR7 / 8 agonist is a TLR8 agonist. In a further embodiment, the TLR7 / 8 agonist is an agonist of both TLR7 and TLR8. The TLR7 / 8 agonist of the present disclosure is suitable for hyperactivating human dendritic cells in the presence of LPC.

[0083] 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 large molecule, such as a recombinant protein or a synthetic oligonucleotide, regulatable by the Center for Biologics Evaluation and Research of the U.S. FDA. Rather, the small molecule TLR7 / 8 agonist is regulatable by the Center for Drug Evaluation and Research of the FDA. In some embodiments, the molecular weight of the small molecule is 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).

[0084] B. Other PRR agonists In some aspects, the pathogen recognition receptor (PRR) agonist comprises a Toll-like receptor (TLR) agonist, provided that the TLR agonist does not comprise a TLR7 / 8 agonist. In some embodiments, the TLR agonist comprises one or more agonists of TLR2, TLR3, TLR4, TLR5, TLR9 and TLR13. In some embodiments, the PRR agonist is a TLR2 / 6 agonist, such as Pam2CSK4. In other embodiments, the TLR agonist is a TLR4 agonist, such as monophosphoryl lipid A (MPLA). However, in preferred embodiments, the TLR agonist is not an agonist of TLR2, TLR4 and / or TLR9. For example, in preferred embodiments, the TLR9 agonist is not a TLR4 ligand, such as LPS (endotoxin).

[0085] In other embodiments, the PRR agonist comprises a NOD-like receptor (NLR) agonist. In further embodiments, the PRR agonist comprises a RIG-I-like receptor (RLR) agonist. In further embodiments, the PRR agonist comprises a C-type lectin receptor (CLR) agonist. In yet further embodiments, the PRR agonist comprises a CDS agonist or a STING agonist.

[0086] III. Antigen The compositions and methods of the present disclosure may further include an antigen. In some embodiments, the antigen comprises a proteinaceous antigen. The terms "polypeptide" and "protein" are used interchangeably herein to refer to a proteinaceous antigen comprising a peptide chain of at least 8 amino acids in length. In some embodiments, the proteinaceous antigen is 8-1800 amino acids, 9-1000 amino acids, or 10-100 amino acids in length. In some aspects, the antigen comprises a synthetic or recombinant protein. In other embodiments, the antigen comprises a protein purified from a biological sample. The polypeptide may be post-translationally modified, such as by phosphorylation, hydroxylation, sulfonation, palmitoylation, and / or glycosylation.

[0087] In some embodiments, the antigen is a tumor antigen comprising the amino acid sequence of at least one full-length protein or a fragment thereof. In some embodiments, the tumor antigen comprises an amino acid sequence from a cancer protein or a fragment thereof. In some embodiments, the mammalian antigen is a neo-antigen or is encoded by a gene comprising a mutation compared to a gene present in a normal cell from a mammalian subject. Neo-antigens are believed to be particularly useful in enabling T cells to distinguish between cancer cells and non-cancer cells (see, e.g., Schumacher and Schreiber, Science, 348:69-74, 2015). In other embodiments, the tumor antigen comprises a viral antigen, such as an antigen of a virus that causes cancer.

[0088] In some embodiments, the tumor antigen is a fusion protein comprising two or more polypeptides, where each polypeptide comprises an amino acid sequence from a different tumor antigen or a non-contiguous amino acid sequence from the same tumor antigen. In some of these embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, where each polypeptide comprises a non-contiguous amino acid sequence from the same tumor antigen.

[0089] In some embodiments, the antigen is a microbial antigen. In some embodiments, the microbial antigen comprises a viral antigen, a bacterial antigen, a protozoan antigen, a fungal antigen, or a combination thereof. In some embodiments, the microbial antigen comprises a surface protein or other antigenic subunit of a microorganism. In other embodiments, the microbial antigen comprises an inactivated or attenuated microorganism. For example, the microbial antigen may comprise an inactivated virus, such as a chemically or genetically inactivated virus. Alternatively, the microbial antigen may comprise a virus-like particle.

[0090] In some embodiments, the antigen may be present in a biological sample obtained from an individual, such as a human patient. For example, the antigen may include a cancer cell. In another aspect, the antigen may include a microbially infected cell, such as a virally infected cell.

[0091] IV. Dendritic cells The compositions and methods of the present disclosure may further include dendritic cells (DCs), which are antigen-presenting cells believed to bridge the innate and adaptive immune systems of mammals. In a preferred embodiment, the DCs are subset 1 conventional DCs (previously called myeloid DCs, cDC1s), as opposed to plasmacytoid DCs (pDCs).

[0092] In some embodiments, the DCs are hyperactivated DCs that express high levels of CD40 and IL-12p70. As used herein, the term "hyperactivated dendritic cells" refers to a cellular state in which DCs can secrete IL-1β while maintaining cell viability (e.g., without undergoing pyroptosis). In this way, hyperactivated dendritic cells can stimulate robust T cell immunity (Figure 1), apparently combining the advantages of activated and pyroptotic dendritic cells (Zhivaki et al., Cell Reports, 33(7), 2020, 108381).

[0093] V. Pharmaceutical Preparations Some compositions of the present disclosure are pharmaceutical formulations comprising a pharma- ceutically acceptable excipient and an LPC compound, 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 suspension. Alternatively, the pharmaceutical formulations may be dehydrated solids (e.g., freeze-dried or spray-dried solids). The pharmaceutical formulations of the present disclosure are preferably sterile and preferably essentially endotoxin-free. The term "pharmaceutical formulation" is used interchangeably herein with the terms "medicine" and "medicine". In some embodiments, the pharmaceutical formation comprises a specific ratio of various components based on the intended purpose of the formulation. In some embodiments, the pharmaceutical formation comprises an LPC compound and a non-ionic surfactant. In some embodiments, the non-ionic surfactant comprises an ethylene oxide-propylene oxide copolymer, such as poloxamer-407 (CAS Registry Number 977057-91-2).

[0094] A. Excipients Pharmaceutically acceptable excipients of the present disclosure include, for example, solvents, buffers, tonicity adjusters, bulking agents, and preservatives (see, for example, Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, pharmaceutical formulations may include one or more of excipients that function as solvents, buffers, tonicity adjusters, and bulking agents (e.g., sodium chloride in saline can function as both an aqueous vehicle and a tonicity adjuster). Pharmaceutically acceptable excipients of the present disclosure also include detergents, wetting agents, emulsifiers, foaming agents, and dispersing agents, as well as surfactants.

[0095] Many of the lipids disclosed herein are poorly soluble in water.Surfactants can be used to solubilize lipids in aqueous formulations.A wide variety of surfactants are available, and they can be classified as anionic surfactants, nonionic surfactants, cationic surfactants, and zwitterionic surfactants.

[0096] Some examples of non-ionic surfactants include poloxamers, which have the general formula: HO-[CH2CH2-O-] a -[CH2CH(CH3)-O-] b -[CH2-CH2-O-] a -H It is a triblock copolymer of ethylene oxide and propylene oxide. Some poloxamers are sold under the trade name Pluronic® (PLURONIC is a registered trademark of BASF SE, Ludwigshafen, Germany). Examples of poloxamers are Poloxamer 407 (KP407; a=101, b=56); Poloxamer 188 (KP188; a=80, b=27); Pluronic® P84 (P-84; a=19, b=39); and Pluronic® P123 (P-123; a=20, b=70) (the values ​​of a and b above may vary slightly).

[0097] Other nonionic surfactants include the Cremophor® series (CREMAPHOR is a registered trademark of BASF SE, Ludwigshafen, Germany). Cremophor® surfactants include Cremophor® EL (K EL), a mixture of polyoxyethylated triglycerides produced by reacting castor oil with ethylene oxide in a molar ratio of about 1:35, and Cremophor® RH40 (also known as Kolliphor® RH40; KOLLIPHOR is a registered trademark of BASF), which is obtained by reacting 40 moles of ethylene oxide with 1 mole of hydrogenated castor oil.

[0098] In some embodiments, the pharmaceutical formulation comprises an aqueous vehicle as a solvent. Suitable vehicles include, for example, sterile water, saline, phosphate-buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic.

[0099] The pharmaceutical formulation may include a buffering agent. The buffering agent controls the pH to inhibit degradation of the active agent during processing, storage, and, if necessary, reconstitution. Suitable buffers include salts, including, for example, acetate, citrate, phosphate, or sulfate. Other suitable buffers include amino acids, such as, for example, arginine, glycine, histidine, and lysine. The buffering agent may further include hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the composition within the range of 6-9. In some embodiments, the pH is greater than (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 6-9, with the lower limit being less than the upper limit.

[0100] The pharmaceutical composition may include a tonicity adjuster. Suitable tonicity adjusters include, for example, dextrose, glycerol, sodium chloride, glycerin and mannitol.

[0101] Pharmaceutical preparations may contain bulking agents. Bulking agents are particularly useful when the pharmaceutical composition is freeze-dried before administration. In some embodiments, bulking agents are protective agents that help stabilize and prevent the degradation of active agents during freeze-drying or spray-drying and / or storage. Suitable bulking agents are sugars (monosaccharides, disaccharides and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbital, glucose and raffinose.

[0102] The pharmaceutical preparation may contain a preservative. Suitable preservatives include, for example, antioxidants and antimicrobial agents. However, in a preferred embodiment, the pharmaceutical preparation is prepared under sterile conditions and is in a single-use container, so there is no need to include a preservative.

[0103] The pharmaceutical formulations of the present disclosure are suitable for parenteral administration, i.e., they are not intended for enteral administration (e.g., not for oral, gastric, or rectal administration).

[0104] B. Adjuvants The pharma- ceutically acceptable adjuvants of the present disclosure include, for example, aluminum salt adjuvants, squalene-in-water emulsions, saponins, or combinations thereof.In some embodiments, the adjuvant is an aluminum salt adjuvant selected from the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, and combinations thereof.In other embodiments, the adjuvant is a squalene-in-water emulsion, such as MF59 or AS03.In other embodiments, the adjuvant is a saponin, such as Quil A or QS-21, as well as AS01 or AS02.

[0105] VI. Manufacturing method The present disclosure relates in some aspects to methods for preparing hyperactivated dendritic cells and methods for preparing immunogenic compositions suitable for in vitro, ex vivo, or in vivo hyperactivation of dendritic cells.

[0106] In one aspect, the present disclosure provides a method for producing hyperactivated dendritic cells (DCs), comprising contacting dendritic cells with an effective amount of isolated lysophosphatidylcholine (LPC) having a single acyl chain and a PRR agonist to produce hyperactivated dendritic cells, which do not undergo pyroptosis and secrete IL-1 beta. In some aspects, the DCs are isolated, and in other aspects, the DCs are present in a biological sample obtained from a mammalian subject, such as a human patient. In some aspects, the DCs are monocyte-derived DCs, preferably cDC1s.

[0107] In another aspect, the present disclosure provides a method for producing an immunogenic composition, the method comprising combining an antigen with an effective amount of isolated lysophosphatidylcholine (LPC) having a single acyl chain and a PRR agonist to produce an immunogenic composition. In some aspects, the antigen comprises a proteinaceous antigen present in or purified from a biological sample obtained from a mammalian subject. In some aspects, the proteinaceous antigen is a synthetic or recombinant protein. In a preferred embodiment, the antigen is a tumor antigen. In a preferred embodiment, the antigen is a microbial antigen.

[0108] In certain embodiments, the present disclosure provides a method for producing an immunogenic composition, the method comprising: a) depleting leukocytes from a suspension of cells prepared from a tumor to obtain a tumor cell-enriched suspension; b) lysing cells from the tumor cell enriched suspension to obtain a tumor cell lysate; and c) contacting the tumor cell lysate with an isolated lysophosphatidylcholine (LPC) having a single acyl chain and a PRR agonist to obtain said immunogenic composition. In some embodiments, the tumor cell enriched cell suspension is depleted of leukocytes by contacting the tumor cell enriched suspension with an antibody specific for leukocytes. In some embodiments, the tumor cell enriched suspension is depleted of leukocytes by contacting the tumor cell enriched suspension with an anti-CD45 antibody. In some aspects, the cells are lysed by a cell lysis method based on physical disruption, such as, but not limited to, mechanical lysis, liquid homogenization, sonication, freeze-thawing, or manual grinding. In some preferred embodiments, the cells are lysed by one or more freeze-thaw cycles.

[0109] In some embodiments of the aforementioned method, the acyl chain of the LPC is a C13-C22 acyl chain or a C13-C24 acyl chain. In some embodiments, the acyl chain of the LPC is a C18-C22 acyl chain or a C18-C24 acyl chain. In some preferred embodiments, the acyl chain is fully saturated. In some preferred embodiments, the acyl chain of the LPC is a C22 acyl chain. In some preferred embodiments, the LPC is 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)]. In some embodiments, the PRR agonist is a TLR7 / 8 agonist. In some preferred embodiments, the TLR7 / 8 agonist is an imidazoquinoline compound, which in a particularly preferred embodiment is resiquimod (R848).

[0110] VII.How to use In some aspects, the disclosure relates to a method of using any one of the compositions or formulations described herein, including LPC compounds, PRR agonists, dendritic cells, antigens, adjuvants, or any combination thereof. The method of use is suitable for multiple uses involving the stimulation of an immune response. In some embodiments, the method of use includes a method of treating cancer. In some embodiments, the method of use includes a method of inhibiting abnormal cell proliferation. In some embodiments, the method of use includes a method of treating infectious disease. The method includes 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 method of use includes clinical use, while in other embodiments, the method of use includes preclinical and / or veterinary use. For preclinical use, the mammalian subject may be a non-human primate (e.g., monkey or ape) or rodent (e.g., mouse or rat). For veterinary uses, the mammalian subject can be a farm animal (eg, a cow), a sports animal (eg, a horse), or a pet (eg, a companion animal such as a dog or cat).

[0111] A. Stimulation of the immune response Briefly, the present disclosure provides a method of stimulating an immune response in an individual, comprising administering to the individual a sufficient amount of a composition or formulation described herein to stimulate an immune response in the individual. "Stimulating" an immune response (used interchangeably with "eliciting" an immune response) means increasing an immune response that may result from eliciting a de novo immune response (e.g., as a result of an initial vaccination regimen) or enhancing an existing immune response (e.g., as a result of a booster vaccination regimen). In some embodiments, stimulating an immune response includes one or more of the following group: stimulating cytokine production; stimulating B lymphocyte proliferation; stimulating interferon pathway-related gene expression; stimulating chemoattractant-related gene expression; stimulating dendritic cell DC maturation. Methods for measuring the stimulation of an immune response are known in the art.

[0112] For example, the present disclosure provides a method of inducing an antigen-specific immune response in an individual by administering to the individual a composition or formulation described herein in an amount sufficient to induce an antigen-specific immune response in the individual. In a preferred embodiment, the composition or formulation comprises an antigen. In some embodiments, the composition or formulation is administered to a tissue of the individual that contains the antigen. The immune response may comprise one or both of an antigen-specific antibody response and an antigen-specific cytotoxic T lymphocyte (CTL) response. "Inducing" an antigen-specific antibody response means increasing the titer of antigen-specific antibodies above a threshold level, such as a baseline titer or serum protection level, before administration. "Inducing" an antigen-specific CTL response means increasing the frequency of antigen-specific CTLs found in peripheral blood above the baseline frequency before administration.

[0113] Analysis of the immune response (both qualitative and quantitative) may be by any method known in the art, including, but not limited to, measuring antigen-specific antibody production (including measuring specific antibody subclasses), activation of specific populations of lymphocytes such as B cells and helper T cells, production of cytokines such as IFN-alpha, IFN-gamma, IL-6, IL-12, and / or release of histamine. Methods for measuring antigen-specific antibody responses include enzyme-linked immunosorbent assays (ELISAs). Activation of specific populations of lymphocytes can be measured by proliferation assays and fluorescence-activated cell sorting (FACS). Cytokine production can also be measured by ELISAs. In some embodiments, methods of stimulating an immune response include stimulating interleukin-1 beta (IL-1β), interferon gamma (IFN-γ), and / or tumor necrosis factor alpha (TNF-α) secretion by monocyte-derived dendritic cells or peripheral blood mononuclear cells. In some embodiments, the method of stimulating an immune response comprises stimulating secretion of one or more of IFN-γ, IL-17a, IL-17f, and IL-22 by memory CD4+ T cells. In some embodiments, the method of stimulating an immune response comprises increasing Th1 differentiation of naive CD4+ T cells. In some preferred embodiments, at least 50%, 55%, 60%, 65%, 70% or 75% of cells contacted with a composition of the present disclosure remain viable 40-56 hours (or about 48 hours) after contact.

[0114] In some embodiments, the method is suitable for stimulating an anti-tumor immune response. In other embodiments, the method is suitable for stimulating an anti-microbial immune response. In some embodiments, the anti-microbial response is an anti-bacterial immune response. In some embodiments, the anti-microbial response is an anti-fungal immune response. In some embodiments, the anti-microbial response is an anti-viral immune response. In some embodiments, the anti-microbial response is an anti-protozoan immune response.

[0115] B. Treatment or Prevention of Disease The present disclosure further provides a method of treating or preventing a disease in an individual, comprising administering to the individual a composition or formulation described herein in an amount sufficient to treat or prevent the disease in the individual. In some embodiments, the disease is cancer. In some embodiments, the disease is abnormal cell proliferation. In other embodiments, the disease is an infectious disease.

[0116] In one embodiment, the method may comprise administering to a subject in need thereof a composition comprising an LPC compound, a PRR agonist, an antigen, an adjuvant, or any combination thereof. In another embodiment, the method comprises adoptive cell therapy, comprising administering to a subject in need thereof a composition comprising a dendritic cell, such as a hyperactivated dendritic cell, and an LPC compound, a PRR agonist, an antigen, an adjuvant, or any combination thereof.

[0117] In some embodiments, the method includes treating cancer in an individual or otherwise treating a mammalian subject having cancer. In some embodiments, the method includes a) preparing an immunogenic composition comprising a tumor cell lysate, an isolated lysophosphatidylcholine (LPC) having a single acyl chain, and a toll-like receptor 7 / 8 (TLR7 / 8) agonist, where the tumor cell lysate is or has been prepared from a tumor sample obtained from a subject having cancer, and the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl 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 a sarcoma, carcinoma, or melanoma. In some embodiments, the cancer is malignant.

[0118] In some embodiments, the method includes inhibiting abnormal cell growth in an individual. "Abnormal cell growth" refers to the growth of a benign or malignant tumor. A malignant tumor can be a metastatic tumor.

[0119] In some embodiments, the method comprises treating or preventing an infectious disease in an individual. In some embodiments, the infectious disease is caused by a viral infection. In other embodiments, the infectious disease is caused by a bacterial infection. In further embodiments, the infectious disease is caused by a fungal infection. In yet further embodiments, the infectious disease is caused by a protozoan infection. Of particular interest 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 pathogens are transmitted to humans via an intermediate species (vector). Enumerated Embodiments Embodiment 1. A composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain and a TLR7 / 8 agonist, wherein the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl chain. Embodiment 2. The composition of embodiment 1, wherein the acyl chain is a C18-C22 acyl chain or a C21-C24 acyl chain. Embodiment 3. The composition of embodiment 1 or embodiment 2, further comprising an antigen. Embodiment 4. The composition of any one of embodiments 1 to 3, further comprising dendritic cells. Embodiment 5. A composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain and an antigen, wherein the acyl chain is a C21-C24 acyl chain. Embodiment 6 The composition of embodiment 5, further comprising dendritic cells. Embodiment 7. The composition of embodiment 5 or embodiment 6, further comprising a TLR7 / 8 agonist. Embodiment 8. A composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain and a dendritic cell, wherein the acyl chain is a C21-C24 acyl chain. Embodiment 9. The composition of embodiment 8, further comprising a TLR7 / 8 agonist. Embodiment 10 The composition of embodiment 8 or embodiment 9, further comprising an antigen. Embodiment 11. The composition of any one of embodiments 1 to 10, wherein the acyl chain is a C22 acyl chain. Embodiment 12. The composition of any one of embodiments 1 to 11, wherein the acyl chains are fully saturated. Embodiment 13. The composition of any one of embodiments 1 to 12, wherein the LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)]. Embodiment 14. The composition of any one of embodiments 1 to 13, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 Daltons or less. Embodiment 15. The composition of embodiment 14, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound. Embodiment 16 The composition of embodiment 15, wherein the TLR7 / 8 agonist comprises resiquimod (R848). Embodiment 17. The composition of embodiment 14 or embodiment 15, wherein the TLR7 / 8 agonist does not inhibit NLR family pyrin domain containing 3 (NLRP3). Embodiment 18 The composition of embodiment 13, wherein the LPC comprises LPC(22:0) and the TLR7 / 8 agonist comprises resiquimod (R848). Embodiment 19. The composition of any one of embodiments 1 to 18, wherein the antigen is present in a biological sample obtained from an individual. Embodiment 20 The composition of embodiment 19, wherein the biological sample comprises biopsy tissue. Embodiment 21 The composition of embodiment 19, wherein the biological sample comprises cells. Embodiment 22 The composition of embodiment 19, wherein the biological sample does not contain cells. Embodiment 23 The composition of embodiment 19, wherein the biological sample comprises pus from an abscess. Embodiment 24 The composition of any one of embodiments 1 to 23, wherein the antigen comprises a proteinaceous antigen. Embodiment 25 The composition of embodiment 24, wherein the antigen comprises a tumor antigen. Embodiment 26 The compound of embodiment 25, wherein the tumor antigen comprises a synthetic or recombinant neoantigen. Embodiment 27 The composition of embodiment 26, wherein the tumor antigen comprises a tumor cell lysate. Embodiment 28. The composition of embodiment 24, wherein the tumor antigen comprises a microbial antigen, the microbial antigen comprising one or more of a viral antigen, a bacterial antigen, a protozoal antigen, and a fungal antigen. Embodiment 29 The composition of embodiment 28, wherein the microbial antigen comprises a purified or recombinant surface protein. Embodiment 30 The composition of embodiment 28, wherein the microbial antigen comprises an inactivated whole virus. Embodiment 31 The composition of any one of embodiments 1 to 30, wherein the composition does not contain liposomes. Embodiment 32 The composition of any one of embodiments 1 to 31, wherein the composition does not include LPS or MPLA. Embodiment 33. A composition described in any one of embodiments 1 to 32, wherein the composition does not contain oxPAPC or a species of oxPAPC. Embodiment 34 The composition of embodiment 33, wherein the composition does not include HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC, and / or PGPC. Embodiment 35. The composition of any one of embodiments 1 to 34, further comprising an adjuvant, wherein the adjuvant comprises an aluminum salt adjuvant, a squalene-in-water emulsion, a saponin, or a combination thereof. Embodiment 36 A pharmaceutical formulation comprising the composition of any one of embodiments 1 to 35 and a pharma- ceutical acceptable excipient. Embodiment 37. A method for producing hyperactivated dendritic cells, the method comprising contacting the dendritic cells with a composition comprising an effective amount of isolated lysophosphatidylcholine (LPC) having a single C13-C22 acyl chain or a single C13-C24 acyl chain and a TLR7 / 8 agonist for producing hyperactivated dendritic cells, wherein the hyperactivated dendritic cells secrete IL-1 beta without undergoing pyroptosis. Embodiment 38 The method of embodiment 37, wherein the dendritic cells are contacted ex vivo with the composition of any one of embodiments 1 to 35 or the formulation of embodiment 36. Embodiment 39 The method of embodiment 37, wherein the dendritic cells are contacted with the formulation of embodiment 36 in vivo. Embodiment 40. At least 10 produced by the method of embodiment 38. 3 , 10 4 , 10 5 Pieces or 10 6 A pharmaceutical formulation comprising said hyperactivated dendritic cells and a pharma- ceutical acceptable excipient. Embodiment 41. A method of stimulating an immune response to an antigen, comprising administering to an individual in need thereof an effective amount of the formulation of embodiment 36 to stimulate the immune response to the antigen. Embodiment 42. A method of treating cancer, comprising administering to an individual in need thereof an effective amount of the formulation of embodiment 36 to treat said cancer. Embodiment 43. A method of inhibiting abnormal cell growth comprising administering to an individual in need thereof an effective amount of the formulation of embodiment 36 to inhibit abnormal cell growth. Embodiment 44. A method of treating an infectious disease comprising administering to an individual in need thereof an effective amount of the formulation of embodiment 36 to treat the infectious disease. Embodiment 45 Use of the formulation of embodiment 36 to induce an immune response against the antigen in an individual in need thereof. Embodiment 46. Use of the formulation of embodiment 36 for inducing an anti-tumor immune response in an individual in need thereof, wherein the individual is or has been tumor-bearing. Embodiment 47. Use of the formulation of embodiment 36 to induce an anti-microbial immune response in an individual in need thereof, wherein the individual is infected with or has not been exposed to the microorganism. Embodiment 48. A composition, formulation, method or use according to any one of embodiments 19 to 47, wherein the individual is a mammalian subject. Embodiment 49. The composition, formulation, method or use of any one of embodiments 19 to 47, wherein the individual is a human subject. Embodiment 50. A method for preparing an immunogenic composition, the method comprising: a) depleting leukocytes from a suspension of cells prepared from a tumor to obtain a tumor cell enriched suspension; b) lysing cells from said tumor cell enriched suspension to obtain a tumor cell lysate; c) contacting the tumor cell lysate with an isolated lysophosphatidylcholine (LPC) having a single acyl chain and a toll-like receptor 7 / 8 (TLR7 / 8) agonist to obtain the immunogenic composition, wherein the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl chain; The method includes: Embodiment 51. The method of embodiment 50, wherein in step a) the leukocytes are depleted by negative selection using an anti-CD45 antibody. Embodiment 52. The method of embodiment 50 or embodiment 51, wherein the cells are lysed in step b) by one or more freeze-thaw cycles. Embodiment 53. The method of any one of embodiments 50 to 52, wherein the acyl chain is a fully saturated C18 to C22 acyl chain or a fully saturated C18 to C24 acyl chain. Embodiment 54. The method of embodiment 53, wherein the LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)]. Embodiment 55. The method of any one of embodiments 50 to 54, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 daltons or less. Embodiment 56 The method of embodiment 55, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound. Embodiment 57 The method of embodiment 56, wherein the TLR7 / 8 agonist comprises resiquimod (R848). Embodiment 58. The method of embodiment 55 or embodiment 56, wherein the TLR7 / 8 agonist does not inhibit NLR family pyrin domain containing 3 (NLRP3). Embodiment 59. The method of embodiment 54, wherein the LPC comprises LPC(22:0) and the TLR7 / 8 agonist comprises resiquimod (R848). Embodiment 60. The method of any one of embodiments 50 to 59, further comprising, prior to step a), obtaining a sample from said tumor from a mammalian subject having cancer and preparing said suspension of cells from said sample. Embodiment 61. An immunogenic composition prepared by the method of any one of embodiments 50 to 60. Embodiment 62. A method of eliciting an anti-cancer immune response, comprising: Administering to a mammalian subject having cancer an effective amount of the immunogenic composition of embodiment 61. A method comprising: Embodiment 63 The method of embodiment 62, wherein the anti-cancer immune response comprises a cellular immune response. Embodiment 64 The method of embodiment 63, wherein the anti-cancer immune response comprises cancer antigen-induced IL-1 beta secretion and / or activation of CD8+ T lymphocytes. Embodiment 65 The method of any one of embodiments 62 to 64, wherein the cancer is a non-hematological cancer. Embodiment 66 The method of embodiment 65, wherein the non-hematological cancer is carcinoma, sarcoma or melanoma. Embodiment 67 The method of any one of embodiments 62 to 64, wherein the cancer is lymphoma. Embodiment 68. A method of treating cancer, comprising: a) preparing an immunogenic composition comprising a tumor cell lysate, an isolated lysophosphatidylcholine (LPC) having a single acyl chain, and a toll-like receptor 7 / 8 (TLR7 / 8) agonist, wherein the tumor cell lysate is or has been prepared from a tumor sample obtained from a mammalian subject having cancer, and the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl chain; b) administering to the subject an effective amount of the immunogenic composition; A method comprising: Embodiment 69. The method of any one of embodiments 62 to 68, wherein the acyl chain is a fully saturated C18 to C22 acyl chain or a fully saturated C18 to C24 acyl chain. Embodiment 70. The method of embodiment 68, wherein the LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)]. Embodiment 71. The method of any one of embodiments 62 to 70, wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 daltons or less. Embodiment 72 The method of embodiment 71, wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound. Embodiment 73 The method of embodiment 72, wherein the TLR7 / 8 agonist comprises resiquimod (R848). Embodiment 74 The method of embodiment 70, wherein the LPC comprises 22:0 LPC and the TLR7 / 8 agonist comprises resiquimod (R848). Embodiment 75 The method of any one of embodiments 68 to 74, further comprising administering to the subject an effective amount of an additional therapeutic agent. Embodiment 76. The method of embodiment 75, wherein the additional therapeutic agent comprises one or more of the group consisting of an immune checkpoint inhibitor, an anti-neoplastic agent, and radiation therapy. Embodiment 77. A composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain and a pathogen recognition receptor (PRR) agonist, wherein the acyl chain is a C13-C22 acyl chain or a C13-C24 acyl chain. Embodiment 78. The composition of embodiment 77, wherein the PRR agonist is an agonist of a toll-like receptor (TLR), a NOD-like receptor (NLR), a RIG-I-like receptor (RLR), or a C-type lectin receptor (CLR). Embodiment 79. The composition of embodiment 77, wherein the PRR agonist is an agonist of a cytoplasmic DNA sensor (CDS) or a stimulator of IFN genes (STING). Embodiment 80. The composition of embodiment 77, wherein the PRR agonist comprises one or more of R848, TL8-506, LPS, Pam2CSK4, and ODN2336. Embodiment 81. The composition of any one of embodiments 77 to 80, further comprising an antigen. Embodiment 82 The composition of any one of embodiments 77 to 81, further comprising dendritic cells. Embodiment 83. A pharmaceutical formulation comprising the composition of any one of embodiments 77 to 82 and a pharma- ceutically acceptable excipient. Embodiment 84. A pharmaceutical formulation comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain and a pharma- ceutically acceptable excipient, wherein the acyl chain is a C21-C24 acyl chain. Embodiment 85. A pharmaceutical formulation of embodiment 83 or embodiment 84, wherein the acyl chain is a fully saturated C22 acyl chain. Embodiment 86 The pharmaceutical formulation of embodiment 85, wherein the LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)]. Embodiment 87. A composition for hyperactivating human dendritic cells comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain and a pathogen recognition receptor (PRR) agonist, wherein the acyl chain is a C22 acyl chain, and the composition is effective in achieving a higher level of dendritic cell hyperactivation than a comparative control composition comprising PGPC instead of the LPC. Embodiment 88. The composition of embodiment 87, wherein the high level of dendritic cell hyperactivation comprises induction of at least 2, 3 or 4 times higher levels of IL-1 beta secretion from human dendritic cells in vitro when contacted with the composition comprising the LPC and the PRR agonist than when contacted with the control composition comprising the PGPC and the PRR agonist, and the PRR agonist is LPS. Embodiment 89. The composition of embodiment 88, wherein the concentration of LPC and the concentration of PGPC are the same within the range of about 10 μM to about 80 μM, and the LPS is present at a concentration of 1 μg / ml in both the composition and the comparative control composition. Embodiment 90. The composition of embodiment 88, wherein the high level of dendritic cell hyperactivation comprises a lipid activity index for IL-1 beta secretion from human dendritic cells for the composition comprising the LPC and the PRR agonist, which is at least 4, 5 or 6 times higher in activity units than the lipid activity index of the comparative control composition comprising the LPC and the PRR agonist. Embodiment 91 The composition, formulation, method or use of any one of embodiments 19 to 47, wherein the individual is a canine subject. Embodiment 92. The composition, formulation, method or use of any one of embodiments 60 to 90, wherein the mammalian subject is a human patient. Embodiment 93 The composition, formulation, method or use of any one of embodiments 60 to 90, wherein the mammalian subject is a non-human patient. Embodiment 94. The composition, formulation, method or use of any one of embodiments 60 to 90, wherein the mammalian subject is a canine patient. Embodiment 95. A composition, formulation, method or use according to any one of embodiments 1 to 90 or 92, wherein the dendritic cells are human dendritic cells. Embodiment 96 The composition, formulation, method or use of any one of embodiments 1 to 91 or 94, wherein the dendritic cells are canine dendritic cells. Embodiment 97. The composition, method or use of embodiment 95 or embodiment 96, wherein the dendritic cells are present in a composition comprising peripheral blood mononuclear cells (PBMCs). Embodiment 98. A composition, method or use according to any one of embodiments 37 to 49 or embodiment 91, wherein the hyperactivated dendritic cells secrete one or both of IFNγ and TNFα. Embodiment 99. A composition, formulation, method or use according to any one of embodiments 1 to 98, comprising a surfactant. Embodiment 100. The composition, formulation, method or use of embodiment 99, wherein the surfactant comprises a non-ionic surfactant. Embodiment 101. A composition, formulation, method or use according to embodiment 100, wherein the nonionic surfactant comprises an ethylene oxide-propylene oxide copolymer. Embodiment 102. A composition, formulation, method or use according to embodiment 100, wherein the non-ionic surfactant comprises one or more of poloxamer 407, poloxamer 188 and P123. Embodiment 103. The composition, formulation, method or use of embodiment 100, wherein the non-ionic surfactant comprises poloxamer 407. Embodiment 104. A composition, formulation, method or use described in any one of embodiments 100 to 103, wherein i) the LPC is dissolved in alcohol to form an alcoholic solution of LPC; ii) the alcoholic solution of LPC 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 LPC and the non-ionic surfactant. Embodiment 105. A composition, formulation, method or use according to any one of embodiments 100 to 104, 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). Embodiment 106. A composition, formulation, method or use according to any one of embodiments 100 to 105, wherein the LPC and non-ionic surfactant are present in particles having a diameter of about 1000 to 2000 nanometers, optionally having a diameter of about 1500 nanometers. EXAMPLES

[0120] Abbreviations: BM (bone marrow); BMDC (bone marrow-derived dendritic cells); CDS (cytoplasmic DNA sensor); CLR (C-type lectin receptor); DAMP (damage-associated molecular pattern); DC (dendritic cell); dLN (draining lymph node); HOdiA-PC (1-palmitoyl-2-(5-hydroxy-8-oxo-6-octenedioyl)-sn-glycero-3-phosphatidylcholine); HOOA-PC (1-palmitoyl-2-(5-hydroxy-8-oxo-6-octenedioyl)-sn-glycero-3-phosphatidylcholine); 1-(palmitoyl)-sn-glycero-3-phosphocholine;IFNγ(Interferon gamma);IL-1b / IL1-beta / IL-1β(Interleukin-1 beta);KOdiA-PC(1-(palmitoyl)-2-(5-keto-6-octenedioyl)phosphatidylcholine);KOOA-PC(1-palmitoyl-(5-keto-8-oxo-6-octenoyl)-sn-glycero-3-phosphocholine);KP407(Poloxamer 407);LPC / Lyso PC(Lysophosphatidylcholine);Lyso PC(22:0)(1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine);LPS(lipopolysaccharide);MFI(mean fluorescence intensity);moDC(monocyte-derived dendritic cells);MPLA(monophosphoryl lipid A);NLR(NOD-like receptor);oxPAPC(oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine);PAMP(pathogen-associated molecular patterns);PBMC(peripheral blood mononuclear cells);PGP C (1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine); POVPC (1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine); PRR (pathogen recognition receptor); RLR (RIG-I-like receptor); R848 (resiquimod); STING (stimulator of IFN genes); TNFα (tumor necrosis factor-alpha); TLR (toll-like receptor); and WTL (whole tumor lysate).

[0121] Although the present disclosure has been described in some detail by way of illustration and example for clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be implemented. Therefore, the following examples should not be construed as limiting the scope of the present disclosure, which is defined by the appended claims.

[0122] Example 1: Identification of DAMPs and PAMPs for dendritic cell hyperactivation This example describes the identification of lipid DAMPs and small molecule PAMPs that can in combination hyperactivate human dendritic cells.

[0123] material and method Differentiation of human monocyte-derived dendritic cells (moDCs). Human monocytes were isolated from Leukopaks (purchased from Miltenyi) using the StraightFrom Leukopak CD14 MicroBead Kit (Miltenyi) according to the manufacturer's instructions. Monocytes were then aliquoted and frozen in fetal bovine serum containing 10% dimethylsulfoxide. For studies of hyperactivating lipids using moDC cultures, monocytes were thawed and cultured in RPMI medium containing 10% FBS, 50 units / mL penicillin, 50 mg / mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 mM beta-mercaptoethanol (R10 medium). Human monocytes were differentiated using recombinant human GM-CSF (50 ng / mL) and IL-4 (25 ng / mL), which were added to the R10 medium. The cells were cultured with GM-CSF and IL-4 for 6 days, and on day 3 the cells were further fed with R10 containing GM-CSF and IL-4.

[0124] Danger-associated molecular patterns (DAMPs) screening. After 6 days of differentiation into moDCs, cells were harvested and counted. Cells were plated at 100,000 cells / well in R10 medium in 96-well flat-bottom tissue culture treated plates. LPS, serotype O55:B5 (Enzo Life Sciences) was added to a final concentration of 1 μg / mL in each well. After addition of LPS, lipids were prepared and added at a final concentration of 82.5 μM or 41.3 μM. Cells were incubated at 37° C., 5% CO2 for 2 days. Cell cultures were then used for endpoint analysis.

[0125] Lipid titration. After 6 days of differentiation into moDCs, cells were harvested and counted. Cells were replated at 100,000 cells / well in R10 medium in 96-well flat-bottom tissue culture treated plates. LPS, serotype O55:B5 (Enzo Life Sciences) was added to a final concentration of 1 μg / mL in each well. After addition of LPS, lipids were prepared. A working stock of the highest lipid concentration tested (82.5 μM) was made in R10, followed by serial 2-fold dilutions down to a final concentration tested of 1.3 μM. After 2 days of culture, cell cultures were used for endpoint analysis.

[0126] Pathogen-associated molecular pattern (PAMP) screening. Innate immune signaling pathway agonists were purchased from Invivogen. Lyophilized stocks were reconstituted and stored at the recommended concentrations according to the manufacturer's instructions. After 6 days of differentiation into moDCs, cells were harvested and counted. Cells were replated at 100,000 cells / well in R10 medium in 96-well flat-bottom tissue culture-treated plates. Innate immune signaling agonists were diluted to the manufacturer's recommended working concentrations in R10 medium and then added to the cells. 22:0 Lyso PC was then reconstituted from lyophilized stocks in R10 and then further diluted. Lipids were added to the cells at a final concentration of 20 μM. After incubating the cells for 2 days, cultures were harvested for endpoint analysis.

[0127] Endpoint Analysis. After culturing moDCs with PAMPs and DAMPs for 2 days, supernatants and cell samples were collected for analysis. Cells in culture were pelleted by centrifugation at 400×g for 5 min. Half of the medium volume in the wells was collected for cytokine quantification by enzyme-linked immunosorbent assay (ELISA), and the remaining medium and cells were used to quantify cell viability by assessing metabolic activity.

[0128] Quantification of IL-1 beta secretion. IL-1 beta secretion was assessed using the ELISA MAX Deluxe Set Human IL-1 beta kit (Biolegend). ELISA was performed according to the manufacturer's instructions with the following modifications: i) the total sample volume for incubation was reduced from 100 mL to 50 mL (25 mL / well of sample or 25 mL / well of assay buffer D with standard); ii) the upper standard was prepared at 500 pg / mL and diluted 2-fold to 7.8 pg / mL; iii) sample incubation was completed overnight at 4 °C on an orbital shaker. Absorbance was measured at 450 nm with 570 nm correction using a Spectramax iD3 plate reader (Molecular Devices). To determine IL-1 beta concentration in the supernatant, the IL-1 beta concentration of the samples was interpolated using a standard curve from 4PL analysis in GraphPad Prism 9 (GraphPad Software). The interpolated results for the samples were then adjusted for any dilutions made to the supernatant.

[0129] Quantification of cell viability. Cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) by quantifying the presence of ATP as an indicator of metabolically active cells. Metabolic activity was assessed according to the manufacturer's instructions. CellTiter-Glo reagent was mixed with the cell pellet and remaining supernatant and transferred to a white opaque 96-well plate. Luminescence was measured at all wavelengths using an integration time of 500 ms on a Spectramax iD3 plate reader (Molecular Devices). Viability was calculated relative to the control condition of moDCs treated with LPS.

[0130] Statistical analysis. For each data set, multiple donors were tested, and the numbers per test are shown in the table corresponding to each test. For each test condition, cells from each donor were plated and tested in triplicate. Triplicate measurements for each donor were averaged, and the average was included as one donor measurement when IL-1beta was quantified or viability was measured. On the graphs, each data point represents the mean value for the donor. To test for differences between test conditions, test results were compared to the control condition shown in each table. P values ​​were calculated using repeated measures one-way ANOVA with correction for multiple comparisons using Dunnett's test.

[0131] Results – Lipids with defined structure have hyperstimulatory capabilities Typically, maturation of dendritic cells (DCs) with vaccine adjuvants such as Toll-like receptor agonists (TLRs) does not result in IL-1β secretion. During inflammasome activation, IL-1β secretion occurs, but may be accompanied by a lytic programmed cell death process called pyroptosis (Evavold et al., J Mol Biol., 430(2):217-237, 2019). However, when DCs are matured using the PAMP LPS and the DAMP PGPC, they are able to produce and secrete IL-1β without pyroptosis (see Figure 1), and these viable DCs can be characterized as hyperactive DCs (Zhivaki et al., Cell Rep., 33(7):108-381, 2020). In mouse models, it has been observed that hyperactivated DCs have an improved ability to induce immune responses compared to cells activated using LPS alone (Zhivaki et al., Cell Rep., 33(7):108381, 2020).

[0132] To identify additional lipids capable of hyperactivating DCs (e.g., hyperactivating lipids), a library of phosphatidylcholine (PC) lipids was selected for screening. The library of PC lipids included mixed acyl PC lipids, Lyso PC lipids, and oxidized PC lipids. All three types of PC lipids are structurally similar to PGPC due to the presence of a phosphocholine head group, but each type differs in the number and length of acyl chains, fatty acid saturation, and oxidation state. When designing a lipid screening strategy, the importance of these properties was unclear, since the use of these lipids to hyperactivate DCs, let alone human DCs, had not been reported.

[0133] To identify lipid species most relevant for clinical use, we screened PC lipids using human DCs. moDCs are activated by LPS and produce precursors of IL-1β (e.g., immature IL-1β), but do not secrete IL-1β in response to LPS alone. When exposed to PGPC in combination with LPS, moDCs secrete a cleaved, active form of IL-1β, but moDCs remain viable. Secreted IL-1β can be detected in cell culture supernatants 1 day after DC overactivation, but cell viability was assessed 2 days after overactivation to ensure viability remains unresolved long after IL-1β secretion. moDC viability is essential, since cells that die due to pyroptosis or lipid toxicity cannot interact with other immune cells and therefore cannot stimulate adaptive T cell responses.

[0134] For each data set, human monocytes from multiple donors were tested. Briefly, human monocyte samples were differentiated into moDCs using GM-CSF and IL-4. Cells were plated and then activated with LPS. Lipids in the library were then added to the cell cultures at 82.5 μM and cells were hyperactivated for 2 days. Many lipid species induced the production of IL-1 beta, primarily in the Lyso PC lipid group (Table 1-1). Lipids also had various effects on cell viability, with lipids that induced IL-1 beta secretion sometimes decreasing cell viability (Table 1-1).

[0135] To identify the relationship between lipid structure and hyperactivation potential, the largest group of lipids stimulating IL-1 beta secretion (Lyso PC) was further analyzed. All Lyso PC lipids tested have a single acyl chain ranging from 6 to 26 carbons in length. For all donors tested, IL-1 beta secretion was detectable above LPS-treated control conditions when lipids had a single acyl chain longer than 12 carbons in length (Figure 2). This indicates that PC lipids with a single acyl chain between 12 and 22 carbons in length can stimulate IL-1 beta secretion. Surprisingly, unsaturated acyl chains between 12 and 22 carbons in length did not induce IL-1 beta secretion, as was seen in studies involving lipids 36 and 37 (Table 1-1 and Figure 2). In contrast, lipids with similarly long saturated acyl chains (lipid studies 35 and 38) were able to induce IL-1 beta secretion (Table 1-1 and Figure 2). The double bonds found in lipid species 36 and 37 render these lipids inactive, presumably due to alterations in their structure.

[0136] Furthermore, as shown by lipid studies 38 and 39 (Table 1-1 and Figure 2), the position of the single acyl chain does not appear to be important. Instead, the number of acyl chains appears to play a larger role. Lipids within the mixed acyl-PC subset have two acyl chains of various lengths. Despite having acyl chains within the 12-22 carbon length range, these lipids were largely inactive. [Table 1-1-1] [Table 1-1-2] [Table 1-1-3]

[0137] Results – 22:0 Lyso PC has powerful hyperstimulatory capabilities An initial screen of hyperactivating lipids was performed using a lipid concentration of 82.5 μM with moDCs obtained from five human donors. However, different lipid species may have hyperactivating effects across a range of concentrations. Specifically, several of the lipids tested at 82.5 μM caused a decrease in cell viability, indicating that lower lipid concentrations may be optimal for maintaining cell viability while promoting IL-1β secretion. The lipid library was rescreened at 41.3 μM using moDCs obtained from three human donors. The results are shown in Tables 1-2. [Table 1-2-1] [Table 1-2-2] [Table 1-2-3]

[0138] At 41.3 μM, lipids that induced IL-1β secretion largely confirmed the results observed at 82.5 μM (Tables 1-2, compare with Table 1). As hypothesized, viability of moDCs treated with lipids that induced IL-1β secretion was improved at 41.3 μM compared to 82.5 μM (Tables 1-2).

[0139] Next, several lipids were selected for use in additional studies (Tables 1-3 and Figure 3). From the LYSO PC subset, 22:0 LYSO PC and 19:0 LYSO PC were selected for additional studies based on their robust induction of IL-1β secretion. Additionally, 12:0 LYSO PC was selected because it was the minimum acyl chain lipid for stimulating IL-1β secretion. 10:0 LYSO PC and 24:0 Lyso PC were selected as negative control lipids because the acyl chain length was outside the range of IL-1β secretion activity. From the oxidized lipid group, PGPC, POVPC and oxPAPC were kept for further studies because the screening was based on studies involving these lipids. PAzePC was selected for further studies because it promoted IL-1β secretion, was structurally similar to other oxidized lipids, and had the longest second acyl chain tested while retaining activity. To more clearly demonstrate the differences observed in these active and inactive lipid species, the results of the selected lipids are shown in Tables 1-3 and Figure 3. [Table 1-3]

[0140] To further understand the potency of the hyperactivating lipids, we studied IL-1β secretion and cell viability over a wider range of lipid concentrations. moDCs from four human donors were activated with LPS. The lipid of interest was added to the cells at 82.5 μM and the concentration was diluted 2-fold to a final test concentration of 1.3 μM. The lipid activity index was calculated by multiplying the maximum IL-1β signal observed at any concentration by the reciprocal of the lowest concentration that induced IL-1β secretion at a level two-fold higher than cells treated with LPS, to compare the potency of each lipid. The results for selected lipids are shown in Tables 1-4 and Figure 4. In summary, 22:0 Lyso PC had the most potent activity by far, followed by 19:0 Lyso PC. PGPC and PAzePC had similar activity indexes. [Table 1-4]

[0141] The titration of lipids also expanded our knowledge of the activity of selected lipids. At 10.3 μM, 22:0 Lyso PC retained the ability to induce IL-1 beta secretion, whereas all other lipids tested failed to do so (Tables 1-5). At 10.3 μM, moDCs treated with 22:0 Lyso PC retained high viability (Tables 1-5), thus displaying both properties of hyperactivated dendritic cells. At 20.6 μM, 22:0 Lyso PC increased IL-1 beta secretion, whereas the other lipids tested did not (Tables 1-6), although this slightly reduced cell viability. [Table 1-5] [Table 1-6]

[0142] At 10.3 μM, 22:0 Lyso PC stimulates IL-1 beta secretion at a concentration comparable to PGPC at 82.5 μM (Tables 1-5 and 1-1). Thus, 22:0 Lyso PC can be used at nearly 8-fold lower concentrations than PGPC, making 22:0 Lyso PC the most potent hyperactivating lipid identified to date.

[0143] Results – Innate immune signaling pathway agonists can hyperactivate dendritic cells Previous experiments have shown that two signals are required to hyperactivate dendritic cells, namely, an innate immune agonist (e.g., PAMPs such as LPS) that produces an immature form of IL-1β, and a lipid (e.g., DAMPs such as PGPC) that enables IL-1β secretion. Although the above DAMP screen used LPS as the innate immune agonist, LPS has not been used as a clinical agonist due to toxicity concerns. Therefore, a selection of clinically relevant innate immune signaling pathway agonists were tested in combination with 22:0 Lyso PC for their ability to hyperactivate human moDCs.

[0144] The PRR agonists tested were primarily focused on TLR signaling pathway agonists, but several cGAS / STING signaling pathway agonists and C-type lectin family receptor agonists were also tested. Agonists were initially tested using moDCs from 10 different human donors (Tables 1-7). moDCs from 5 of the 10 human donors were also used to test agonists at lower concentrations (Tables 1-8). PAMPs were added to post-differentiation moDC cultures along with the DAMP, 22:0 Lyso PC. Cells were stimulated for 2 days, after which cell viability and IL-1β secretion were measured. [Table 1-7-1] [Table 1-7-2]

[0145] Table 7 and Figure 5 show that four agonists: R848, TL8-506, Pam2CSK4 and ODN2336 enabled IL-1β secretion. These agonists had little or no effect on cell viability (Table 7). Table 8 shows the results of low-dose PAMP treatment. [Table 1-8-1] [Table 1-8-2] [Table 1-8-3]

[0146] As shown in Table 1-7 and Figure 5, four agonists (R848, TL8-506, Pam2CSK4, and ODN2336) induced IL-1β secretion by human DCs in the presence of 22:0 Lyso PC. These PRR agonists had little to no effect on cell viability (Table 1-7). At the low agonist concentrations tested, IL-1β secretion was only observed when R848 or Pam2CSK4 was used in combination with 22:0 Lyso PC (Table 1-8). Neither R848 nor Pam2CSK4 affected cell viability at the lower concentrations tested (Table 1-8). From this small screen of clinically relevant innate immune signaling pathway agonists, a handful were found to be able to hyperactivate human moDCs when combined with 22:0 Lyso PC. Based on IL-1β secretion, R848 is the most potent clinically relevant candidate as an agonist to promote hyperactivation of human DCs (eg, to secrete IL-1β and maintain cell viability).

[0147] Example 2: Combination of Lysophosphatidylcholine (LPC) with a Single Acyl Chain and a TLR7 / 8 Agonist for Hyperactivation of Mammalian Peripheral Blood Mononuclear Cells This example describes the hyperactivation of canine and human peripheral blood mononuclear cells (PBMCs) by lipid DAMPs in combination with small molecule PAMPs.

[0148] material and method Isolation of PBMCs from whole blood. PBMCs were isolated from whole blood using density gradient centrifugation with Ficoll-Paque PLUS (Cytivia). Whole blood was diluted 1:1 with PBS, layered on top of Ficoll-Paque PLUS, and centrifuged at 1000 x g for 30 minutes at room temperature. PBMCs were collected, washed twice with PBS, and incubated with Ack lysis buffer (Lonza) to remove remaining red blood cells.

[0149] Cell culture and stimulation. Immediately after isolation, PBMCs were plated in RPMI medium containing 10% FBS, 50 units / mL penicillin, 50 mg / mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate and 50 mM beta-mercaptoethanol (R10 medium). Cells were plated at 1 × 10 per well in 96-well flat-bottom tissue culture plates. 5 (dog cells) or 1 × 10 6 (human cells). Lyophilized Vaccigrade R848 (Invivogen) was reconstituted, diluted according to the manufacturer's recommendations, and added to the cells at a final concentration of 1 μg / mL. Immediately after, 22:0 LYSO PC was added to the cells at a final concentration of 82.5 μM. Additional natural agonists were diluted in R10 medium according to the manufacturer's recommendations and added to the cells as follows: human GM-CSF (Peprotech) was added at a final concentration of 10 ng / mL; 2'3'cGAMP (Invivogen) was added at a final concentration of 15 μg / mL; LPS, serotype O55:B5 (Enzo Life Sciences) was added at a final concentration of 1 μg / mL; alum hydroxide (Invivogen) was added at a final concentration of 30 μg / mL. The cells were incubated at 37 °C, 5% CO2 for 2 days. The cell cultures were then used for endpoint analysis.

[0150] Endpoint Analysis. After 2 days of culturing PBMCs with PAMPs and DAMPs, supernatants and cell samples were collected for analysis. Cells in culture were pelleted by centrifugation at 400xg for 5 minutes. Half of the medium volume in the wells was collected for cytokine quantification by enzyme-linked immunosorbent assay (ELISA) or Lumit™ bioluminescence assay, and the remaining medium and cells were used to quantify cell viability by assessing metabolic activity.

[0151] Quantification of cytokine secretion. IL-1β secretion from human PBMC was assessed using one of the following kits: ELISA MAX Deluxe Set Human IL-1β kit (Biolegend), Invitrogen Human IL-1β kit, or Lumit™ Human IL-1β Immunoassay (Promega). IFNγ secretion from human PBMC was assessed using ELISA MAX Deluxe Set Human IFNγ (Biolegend) and TNFα secretion from human PBMC was assessed using Human TNFα Uncoated ELISA kit (Invitrogen). ELISAs were performed according to the manufacturer's instructions with the following modifications: i) total sample + buffer volume for incubation was reduced from 100 μL to 50 μL; ii) top standard was prepared at 500 pg / mL and diluted 2-fold to 7.8 pg / mL; iii) sample incubation was completed overnight at 4° C. on an orbital shaker. Lumit™ assays were performed according to the manufacturer's instructions. IL-1β secretion from canine PBMCs was assessed using the Canine IL-1β / IL-1F2 DuoSet ELISA (R&D) according to the manufacturer's instructions with the following modifications: i) the total sample + buffer volume for incubation was reduced from 100 μL to 50 μL; ii) sample incubation was completed overnight at 4°C on an orbital shaker. For all ELISAs, absorbance was measured at 450 nm with 570 nm correction using a Spectramax M5e plate reader (Molecular Devices). For Lumit™ assays, luminescence was measured at all wavelengths using a Spectramax M5e plate reader (Molecular Devices) with an integration time of 500 ms. To determine cytokine concentrations in the supernatants, sample concentrations were interpolated using a standard curve from 4PL analysis in GraphPad Prism 9 (GraphPad Software). The interpolated results of the samples were then adjusted for any dilutions made to the supernatants.

[0152] Quantification of cell viability. Cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) by quantifying the presence of ATP as an indicator of metabolically active cells. Metabolic activity was assessed according to the manufacturer's instructions. CellTiter-Glo reagent was mixed with cell pellets and fresh medium and then transferred to a white opaque 96-well plate. Luminescence was measured at all wavelengths using an integration time of 500 ms on a Spectramax M5e plate reader (Molecular Devices). Viability was calculated relative to the control condition of R848-treated PBMCs.

[0153] Statistical analysis. For each condition, cells from each donor were plated in triplicate for testing. For cytokine quantification, triplicate values ​​were used for interpolation and data were plotted as total concentration (pg / mL) or fold change per donor compared to the R848 alone control condition. For viability quantification, triplicate values ​​for each donor were averaged and the average value was used as a single donor measurement. Multiple donors were tested and each data point in the bar graph represents a donor's value. To test for differences between test conditions, test results were compared to the R848 alone control condition. P values ​​were calculated using a mixed-effects one-way ANOVA with correction for multiple comparisons using Dunnett's test.

[0154] Results - Treatment with 22:0 LYSO PC and R848-hyperactivated canine PBMC As described in Example 1, the combination of 22:0 LYSO PC (DAMP) and TLR7 / 8 agonist R848 (PAMP) was found to have potent hyperstimulatory activity in human moDCs. To assess whether this hyperstimulatory activity also applies to other clinically relevant species, the ability of 22:0 LYSO PC+R848 to hyperactivate PBMCs isolated from canine whole blood was evaluated. For each data set, PBMCs from multiple donors were used in place of moDCs, as there are no canine-specific reagents available to induce bone fide canine moDCs. Briefly, PBMCs were isolated from whole blood using density gradient centrifugation and then cultured with the hyperactivating stimulus of interest for 2 days.

[0155] After 2 days of culture, hyperactivation was assessed by quantification of IL-1β in cell culture supernatants and by measurement of cell viability. When treated with 22:0 LYSO PC and R848 together, canine PBMCs secreted similar or higher levels of IL-1β, both in terms of concentration per mL compared to all other stimuli tested, as well as in terms of fold change per donor compared to R848 alone (Figures 6A-6B). Consistent with previous studies showing that monocytes, which make up 5-10% of PBMCs, can release IL-1β in response to activation with R848, canine PBMCs exhibited elevated levels of IL-1β secretion with R848 alone compared to untreated cells. The pyroptotic combination of LPS+alum elicited higher levels of IL-1β, as expected. Notably, PGPC+R848 elicited similar levels of IL-1β compared to R848 alone, but neither GM-CSF nor 2'3'cGAMP induced significant IL-1β secretion from canine PBMC compared to untreated cells.

[0156] Although IL-1β can be detected one day after overactivation of canine PBMC in cell culture supernatant, cell viability was evaluated two days after overactivation to ensure that viability remains unresolved long after IL-1β secretion. 22:0 LYSO+R848 did not significantly reduce relative cell viability (Figure 6C). Interestingly, PGPC in combination with R848 proved to be somewhat toxic to canine PBMC, but was not observed to be toxic to human moDC (Example 1) or human PBMC (Figure 7C). Taken together, these data demonstrate that 22:0 LYSO+R848 elicits high levels of IL-1β secretion from canine PBMC, indicating overactivation.

[0157] Results – Treatment with 22:0 LYSO PC and R848 hyperactivated human PBMC The initial screen in which 22:0 LYSO PC and R848 were identified as having strong hyperactivation potential was performed with human moDCs as described in Example 1. To confirm that the observations made with canine PBMCs and human moDCs could be reproduced in human PBMCs, hyperactivation experiments were also performed with PBMCs isolated from whole blood obtained from human donors. Briefly, PBMCs were isolated from whole blood by density gradient centrifugation from multiple human donors and cultured with the hyperactivating stimulus of interest for two days.

[0158] Human PBMCs, like human moDCs and canine PBMCs, secreted IL-1β at levels higher or comparable to all other stimuli tested (Figures 7A-7B). Like canine PBMCs, human PBMCs secreted IL-1β in response to R848 alone, due to monocyte activation, which was elevated by the addition of 22:0 LYSO PC. The pyroptotic combination of LPS+alum elicited high levels of IL-1β as expected. Consistent with the observations in canine PBMCs, PGPC+R848 did not induce substantially higher levels of IL-1β than R848 alone. GM-CSF did not induce IL-1β secretion levels from human PBMCs significantly above background levels produced by untreated cells.

[0159] To ensure that the long-lasting viability of human PBMCs after IL-1β secretion remains intact, we also assessed the viability of human PBMCs 2 days after hyperactivation. No significant decrease in human PBMC viability was observed after treatment with either stimulus (Figure 7C). Taken together, these data demonstrate that both human and canine PBMCs are hyperactivated by 22:0 LYSO PC+R848. Interestingly, canine PBMCs are more highly hyperactivated by 22:0 LYSO PC+R848 than by PGPC+R848.

[0160] Since activated human PBMCs can secrete other cytokines in addition to IL-1β, secretion of the pro-inflammatory cytokines IFNγ and TNFα in cell culture supernatants was measured 2 days after hyperactivation. The combination of 22:0 LYSO PC+R848 induced the greatest fold change per donor in both IFNγ and TNFα secretion compared to R848 alone compared to all other stimuli tested (Figures 8A-8B). Notably, LPS+Alum induced high levels of IL-1β secretion from human PBMCs (Figure 7B), but this combination of stimuli did not induce a fold increase in IFNγ or TNFα secretion. Furthermore, neither GM-CSF nor 2'3'cGAMP elicited a substantial fold change in IFNγ secretion over R848 alone. These data indicate that the combination of 22:0 LYSO PC+R848 is superior in inducing secretion of the pro-inflammatory cytokines IFNγ and TNFα from human PBMCs.

[0161] Example 3: 22:0 LYSO PC hyperactivates non-human primate dendritic cells and peripheral blood mononuclear cells This example describes the hyperactivation of non-human primate moDCs and peripheral blood mononuclear cells (PBMCs) by lipid DAMPs in combination with a small molecule PAMP (R848).

[0162] material and method Differentiation and hyperactivation of non-human primate moDCs. Whole blood from rhesus monkeys was diluted with an equal volume of PBS and layered on Ficoll-Paque PLUS solution. Cells were rotated uninterrupted at 1000rcf for 30 minutes at room temperature. The buffy coat layer was collected, washed twice with PBS and counted. To isolate monocytes from PBMCs, Miltenyi human CD14 microbeads were used according to the manufacturer's recommendations. The isolated monocytes were then differentiated into moDCs using recombinant human GM-CSF (50ng / mL) and recombinant human IL-4 (25ng / mL) for 6 days. Cells were grown in R10 medium consisting of RPMI, 10% FBS, penicillin, streptomycin, L-glutamine, sodium pyruvate and beta-mercaptoethanol.

[0163] Differentiated moDCs were collected, counted, and plated in 96-well plates at 1 x 10 5 Cells were plated at 1000 cells / well. Cells were treated with or without a hyperactivating stimulus. Hyperactivating cultures were incubated for 2 days before assessing cell viability and IL-1beta secretion. ATP was quantified as a measure of cell viability using Promega Cell Titer-Glo according to the manufacturer's instructions. An ELISA kit specific for non-human primate IL-1beta was purchased from Invitrogen and the assay was completed according to the manufacturer's instructions.

[0164] Hyperactivation of non-human primate PBMCs. Whole blood from cynomolgus monkeys was diluted with an equal volume of PBS. A solution of 90% Ficoll-Paque PLUS and 10% RPMI was made and the diluted whole blood was layered on top of the diluted Ficoll. Cells were centrifuged at 1000rcf for 30 minutes at room temperature without interruption. The buffy coat layer was collected and washed twice with PBS. If red blood cell contamination was present, cells were incubated in ACK lysis buffer for 2 minutes and then washed with PBS to remove lysed red blood cells.

[0165] 1 x 10 isolated PBMCs 6 Cells were plated at 1000 x g / well and cultured with or without hyperactivating stimuli. After 2 days of incubation, cells were assessed for viability and cell culture supernatants were used to measure cytokine production. Cytokines of interest were quantified using commercially available kits such as the Biolegend LegendPlex kit as directed by the manufacturer's protocol.

[0166] Statistical analysis. Rhesus moDCs were generated from four animals. Each activation condition was tested in triplicate, and reported data points are the average of triplicate animal donor samples. Note that due to limited cell numbers, overactivation with R848+41.3uM PGPC was only tested on two samples. Data were tested for statistical significance using ordinary one-way ANOVA.

[0167] PBMCs were obtained from 13 cynomolgus monkeys. Each activation condition was tested in triplicate, and the averages of the triplicate animal samples are graphed. Statistical significance was tested using a repeated measures one-way ANOVA, followed by Dunnett's multiple comparison test.

[0168] result If hyperactivation is an important biological function of the innate immune system, we would expect to find it evolutionarily conserved across multiple species. Furthermore, further development of 22:0 LPC as a therapeutic agent may require testing in relevant disease animal models. We hypothesize that closely related species, such as non-human primates, would also respond to 22:0 LPC-mediated hyperactivation.

[0169] To test our hypothesis, we isolated monocytes from rhesus monkey whole blood. Using the same differentiation protocol as for human cells, monocytes were differentiated into moDCs using recombinant human GM-CSF and IL-4. Cells were plated and stimulated with or without R848 and with or without hyperactivating lipids. After 2 days of incubation, IL-1 beta was detected in the cell culture supernatant. Interestingly, 22:0 LPC induced detectable IL-1 beta at 82.5uM and 41.3uM, whereas IL-1 beta was only detectable at 82.5uM using PGPC (Figure 9B). These results are consistent with the data generated using human moDCs, demonstrating that 22:0 Lyso PC is more potent than PGPC. Furthermore, cells remained viable across the conditions tested (Figure 9A).

[0170] Studies using human and canine PBMCs have demonstrated that cells within the population respond to hyperactivation. Because non-human primate moDCs can be hyperactivated, we sought to test whether NHP-derived PBMCs could also be hyperactivated. Whole blood from cynomolgus monkeys was processed to isolate PBMCs. Cells were cultured at 1 × 10 6PBMCs were plated at 1000 cells / well and hyperactivated with 22:0 Lyso PC or PGPC at concentrations of 82.5 and 41.3 μM. As a negative control for hyperactivation, PBMCs were left untreated or stimulated only with 1 μg / mL of R848. Cell viability was maintained in all conditions tested. Similar to canine and human PBMCs, R848 stimulation induced IL-1 beta secretion. Addition of either 82.5 uM 22:0 Lyso PC or PGPC to induce hyperactivation increased IL-1 beta production (Figure 10A). This effect was particularly evident when quantifying the fold change in IL-1 beta output in the hyperactivated state compared to R848 stimulation alone (Figure 10B).

[0171] Similar to human PBMCs, cynomolgus PBMCs also showed a significant increase in IFN-gamma production when overactivated with 82.5uM 22:0 Lyso PC or PGPC for 2 days. To extend these observations and identify other cytokines that may be increased by overactivation, we quantified inflammatory cytokines using a multiplex bead-based assay. Biolegend's LegendPlex kit works similarly to an ELISA, but the primary antibody is bound to beads rather than the plate surface. The different bead populations allow for simultaneous quantification of multiple cytokines within one reaction. Using the bead assay, we identified multiple cytokines that increased with overactivation. IFN-gamma, IL-17a, IL-23, IFN-beta and IL-8 were increased when PBMCs were overactivated with 82.5uM 22:0 Lyso PC or PGPC compared to treatment with R848 alone (Figures 11A-15A). These increases were especially evident when fold changes were calculated for each animal sample to compare overactivation with R848 activation (Figures 11B-15B). The increase in cytokine levels was dependent on the amount of lipid used. Fold changes increased less markedly when 22:0 Lyso PC or PGPC was used at 41.3 uM, and at this concentration, increases in cytokine secretion were not always statistically significant. Interestingly, not all cytokines were affected by overactivation. For example, IL-6 secretion remained relatively unchanged with the addition of overactivation lipid (Figure 16A). The data graphed as fold changes appear to be very different from the trends observed for overactivation-dependent cytokines (Figure 16B).

[0172] Taken together, these data indicate that 22:0 Lyso PC is a hyperactivating lipid in both rhesus and cynomolgus monkeys. Furthermore, non-human primate cells can be hyperactivated, similar to what has been observed in other species. These data are particularly interesting because cytokines such as IFN-gamma and IL-17a are unlikely to be produced by phagocytes, which we believe are the primary responding cells to the hyperactivating stimuli we use. If this is true, hyperactivated cells may be inducing secondary effects on neighboring cells, such as T cells, that can express IFN-gamma and IL-17a.

[0173] Example 4: Hyperactivated dendritic cells stimulate potent CD4+ Th1 cell responses This example describes the effect of overactivated human moDCs on human memory and naive CD4+ T cells.

[0174] material and method Co-culture of human moDCs and memory CD4 T cells. Human monocytes were isolated from the leukapheresis product using Miltenyi StraightFrom LeukoPak CD14 microbeads according to the manufacturer's instructions. Red blood cells and PBMCs were separated from granulocytes from the same blood product using a custom PBMC isolation microbead kit from Miltenyi. After PBMC purification, the CD45RO+ fraction was negatively selected using the Miltenyi human memory CD4+ T cell isolation kit. Cells were stored frozen until ready for use in experiments.

[0175] Human monocytes were differentiated into dendritic cells using recombinant human GM-CSF and IL-4 for 6 days as described above. For hyperactivation, moDCs were plated in R10 medium at 5E4 cells / well in 96-well U-bottom microplates. R848 and lipids were added to the cell cultures and hyperactivation was incubated overnight. T cell stimulating anti-CD3 antibodies were added to the cultures before adding T cells (anti-IL-1beta antibodies may or may not be blocking). Autologous frozen memory CD4 T cells were thawed and resuspended in R10 medium and 2.5 × 10 5 DCs were added at 1:5 cells / well (ratio 1:5, DC:T cells). After 2 days of incubation, cell culture supernatants were collected and used to measure cytokine secretion. Cytokines of interest were quantified using commercially available kits such as Biolegend LegendPlex kits as instructed by the manufacturer's protocol.

[0176] Four donors were tested, and each experimental condition was tested in triplicate. Each data point represents the mean of triplicate donors. Data were tested for statistical significance using two-way ANOVA followed by Tukey's multiple comparison test.

[0177] Mixed lymphocyte reaction. Human monocytes were isolated from leukapheresis products using Miltenyi StraightFrom LeukoPak CD14 microbeads according to the manufacturer's instructions. To isolate naive CD4 T cells, whole blood was diluted with an equal volume of PBS. The diluted blood was layered on top of Ficoll-Paque PLUS solution and spun uninterrupted at 1000rcf for 20 minutes at room temperature. The buffy coat layer was collected, washed, and counted. The Miltenyi Human Naive CD4 T Cell Microbead Isolation Kit was used to isolate cells of interest from PBMCs according to the manufacturer's instructions. Recovered cells were cryopreserved in 90% FBS and 10% DMSO.

[0178] Monocytes were differentiated into moDCs. moDCs were counted and plated in U-bottom plates at 2 × 10 4After 1 day of culture, 1 × 10 cells / well were plated and treated with hyperactivating stimuli or control conditions. 5 Naïve CD4 T cells at 100 ng / mL per well were thawed and added to the cultures containing 100 ng / mL IL-2. Cells were co-cultured for 5 days and IL-2 was given on day 3 of co-culture. After 5 days, cells were reactivated for 4 hours using a leukocyte activation cocktail containing BD GolgiPlug (BD Biosciences). Cells were stained to determine the polarization of the activated T cells obtained by flow cytometry. Intracellular staining targets required the use of the Transcription Factor Buffer set from BD Biosciences to fix and permeabilize the cells prior to the intracellular staining step.

[0179] Ten moDC donor samples were tested in the MLR assay. Staining results were analyzed using FlowJo software, and a consistent gating strategy was applied to all samples. The frequency of activated cells was graphed as a percentage of CD4 T cells in each sample. Statistical significance was determined using a paired t-test.

[0180] Results: Hyperactivated dendritic cells potently reactivate Th1 memory CD4 T cells One important feature of hyperactivated dendritic cells is that they secrete mature IL-1beta while remaining viable. In contrast, non-hyperactivated cells undergo pyroptosis upon secreting IL-1b. These two features are particularly interesting because continued cell survival allows dendritic cells the opportunity to interact with other cells, such as T cells. These cell-cell interactions provide opportunities for DCs to present antigens and provide costimulatory and inflammatory signals to T cells. As mentioned above in other examples, hyperactivation of PBMCs can potentially activate other cell types. Interestingly, it has been reported that DCs more potently reactivate memory CD4 T cells compared to non-cell-based methods, such as using plate-bound anti-CD3 and anti-CD28 agonistic antibodies (Jain et al., Nat Commun,9(1):3185,2018). The mechanism of enhanced T cell reactivation in the published study relied on IL-1beta signaling from DCs to T cells. However, the impact of different DC activation states on T cell responses was not investigated. Given that hyperactivated DCs potently produce IL-1beta and their viability allows for continued cell-cell interactions, we hypothesized that hyperactivation is the most potent cellular state of dendritic cells to reactivate memory CD4 T cells.

[0181] To test our hypothesis, human monocytes were differentiated into moDCs. moDCs were left unstimulated, activated with R848, overactivated with R848 and 22:0 LPC, or mock-treated with non-overactivated lipids (R848 and 10:0 LPC). After 1 day of incubation, autologous T cells were added to the cultures. To simulate peptide-MHC recognition, TCR-stimulating anti-CD3 antibodies were added at very specific concentrations (already noted to be very dilute, 300-fold lower than those used in Jain et al., supra, 2018). To test whether the T cell response was mediated by IL-1b, further experimental conditions were set up in which anti-IL-1beta blocking antibodies were added to the cultures to inhibit IL-1beta signaling. As an additional control, no T cells were added to some moDC stimuli to ensure that the measured cytokine response was not directly from the moDCs. Stimuli were also plated in empty wells, followed by addition of T cells, to ensure that the stimuli did not directly affect T cells without requiring moDCs.

[0182] T cell response cytokines were measured from culture supernatants 2 days after the addition of T cells. IFN-gamma was minimally detected when moDCs or T cells were cultured alone. Upon co-culture, IFN-gamma was produced, indicating a Th1 response from T cells. Given the limited TCR stimulation by anti-CD3 antibodies, R848-treated moDCs did not generate a significantly stronger Th1 response compared to unstimulated moDCs. However, combining R848 with the overactivating lipid 22:0 LPC produced a significantly stronger Th1 response (Figure 17). These elevated IFN-gamma levels were not detected when non-overactivating lipids such as 10:0 LPC were used. Furthermore, when anti-IL-1beta was added to the co-cultures, the overactivating co-cultures showed significantly reduced IFN-gamma production, suggesting that the enhanced Th1 response is dependent on IL-1beta signaling.

[0183] In contrast, the Th2 cytokines IL-4 and IL-13 were not enhanced by moDC overactivation. moDCs treated with various stimuli resulted in similar IL-4 and IL-13 responses from cocultured T cells, but not statistically significant (Figures 18A-18B). Th17 cytokines IL-17a, IL-17f, and IL-22 were also measured. Th17 cytokines followed a similar trend to that observed with IFN-gamma (Figures 19A-19C). Overactivated moDCs induced a statistically significant increase in Th17 cytokine production, whereas non-overactivated moDCs did not. Anti-IL-1beta antibodies significantly reduced cytokine levels, so the enhancement of Th17 cytokine production was dependent on IL-1beta signaling. These results suggested that overactivated moDCs preferentially enhanced Th1 and Th17 responses, while Th2 responses remained unaffected. In the context of antitumor responses, hyperactivated DCs may be more suitable to reactivate tumor-responsive memory CD4 T cells because they can provide IL-1beta in the right context during antigen presentation. Given the low anti-CD3 stimulation used in our experimental system, hyperactivated DCs may have a better ability to stimulate reactivation, despite weaker TCR interactions with peptide-MHC as seen in cancer.

[0184] Results – Hyperactive moDCs bias naive CD4 T cells towards Th1 responses Hyperactivation improves the ability of moDCs to reactivate Th1 and Th17 memory CD4 T cell responses. In some disease situations, CD4 T cells may not have been previously activated and therefore may be naive. To examine the effect of hyperactivated moDCs on naive CD4 T cells, a widely used immunological assay, the mixed lymphocyte reaction (MLR), was used. moDCs were activated with R848 or hyperactivated with R848 and 22:0 LPC. After 1 day of incubation, naive CD4 T cells from allogeneic donors were added to the cultures. Mismatch between TCR and peptide-MHC provides TCR stimulation to some T cells. IL-2 was added to provide a basal T cell growth signal. Cells were co-cultured for 5 days before staining and flow cytometry analysis was performed. Flow cytometry was chosen because this technique collects data with specificity at the single cell level. To determine CD4 T cell polarization, activated cells were stained for effector cytokines and associated polarizing transcription factors.

[0185] To analyze Th1 polarization, cells were stained for the transcription factor Tbet and the cytokines IFN-gamma and TNF-alpha. Compared to R848 activation, overactivation with 22:0 LPC increased the frequency of Th1 cells (Figure 20). Furthermore, polyfunctional Th1 polarized cells (as indicated by their dual expression of IFN-gamma and TNF-alpha) increased in frequency when cultured with overactivated moDCs (Figure 20). Th2 polarization was studied by staining for Gata3 and IL-4. Compared to Th1 responses, minimal Th2 polarization occurred (Figure 20). Interestingly, cells were also stained for RORg and IL-17, but these cells were not detected. The lack of Th17 cell differentiation from naive CD4 T cells contrasts with the results observed when memory CD4 T cells were cocultured with moDCs. Taken together, the results of these studies indicate that overactivation with 22:0 LPC enhances the polarization of naive CD4 T cells towards a Th1 response upon activation.

[0186] Example 5: Incorporation of 22:0 LYSO PC into micelles This example describes the preparation and testing of surfactant-containing formulations of 22:0 Lyso PC.

[0187] material and method Formulation of 22:0 Lyso PC with Poloxamers. The following surfactants were dissolved in water at 1%, 2%, or 5.5%: Poloxamer 407 (KP407), Poloxamer 188 (KP188), Cremophor EL (K EL), Cremophor RH40 (K RH), Pluronic® P84 (P-84), and Pluronic® P123 (P-123). 22:0 Lyso PC was resuspended in the surfactant solution and stirred at 4° C. for 1 hour. The solution was then brought to room temperature to form micelles. Salt was added to the solution to stabilize molecular interactions and obtain physiologically relevant osmolality using 10x concentrated PBS. The lipid solution was left unfiltered or filtered through 0.45um pore size to remove insoluble lipid flakes. The lipid was further diluted and added to human moDC cell cultures along with 1ug / mL of R848. The target lipid concentration is based on a scenario in which lipids are fully bioavailable before filtration. However, filtration of insoluble lipids reduces the supply of bioavailable lipids. Therefore, IL-1 beta as a measure of hyperactivity can be used as an indicator of 22:0 Lyso PC incorporation into micelles and thus into solution. After one day of hyperactivation, cell culture supernatants are collected to measure cytokine secretion and cells are used to measure viability by Cell Titer-Glo.

[0188] The solvent evaporation method utilizes methanol or ethanol to completely dissolve 22:0 Lyso PC before mixing with KP407. The dissolved lipid solution is mixed with 5.5% KP407 with stirring at room temperature for 90 minutes to evaporate the alcohol solvent and induce particle formation with poloxamer and lipid. Salt is added to the solution to stabilize molecular interactions and bring the solution to a physiologically appropriate osmolarity. The solution is then left unfiltered or filtered through a 0.45um pore size to remove insoluble lipid flakes or particle aggregates. The lipid is then added to human moDC cultures along with 1ug / mL of R848. One day after hyperactivation, cell culture supernatants were collected to measure cytokine secretion and cells were used to measure cell viability.

[0189] Particle size was quantified by dynamic light scattering (DLS). Particle size was assessed for particles in solution after 0.45 um filtration of micelles made using resuspension in KP407 or solvent evaporation particle synthesis.

[0190] Data from these studies in poloxamer formulations are shown from one human moDC donor sample, with each data point representing the average of triplicates.

[0191] Results – Incorporation of 22:0 LYSO PC into micelles 22:0 Lyso PC is a lipid that is nearly insoluble in aqueous solutions. Our initial method of adding 22:0 Lyso PC to cell cultures was to simply resuspend the lipid in the culture medium. However, the lipid material was clearly visible as flakes in the solution. To administer a consistent dose, it would be ideal to solubilize the lipid. This would be paramount in animal studies and also as a developed treatment for human use. Given that the overactivating molecule is a lipid, we hypothesized that 22:0 Lyso PC could be incorporated into micelles.

[0192] To test our hypothesis, we screened a set of surfactants that could aid in the micellization of 22:0 Lyso PC. Lyophilized lipids were mixed with an aqueous solution containing 1 or 2% surfactant. The solution was refrigerated during mixing to allow the surfactant to monomerize and interact with the lipid. The solution was then warmed to room temperature to allow micellization. Salt was added to the solution to stabilize the molecular interactions and bring the solution to a physiologically relevant osmolarity using 10x concentrated PBS. The 22:0 Lyso PC micelles were then left unfiltered or passed through a 0.45um filter to remove insoluble lipids. These lipid stocks were then further diluted and added to human moDC cultures to hyperactivate the cells. Some surfactants, such as KEL, KRH, and P-84, caused a decrease in cell viability and were not ideal for use (Figure 21). In comparison, KP407, KP188, and P-123 did not impair cell viability. We then quantified IL-1 beta secretion from these various stimulation conditions where cell viability was maintained. When lipids were left unfiltered, high IL-1 beta secretion was observed, as was our standard method of resuspending 22:0 Lyso PC in PBS and adding it to the cells (Figure 22). Filtration of lipids significantly reduced IL-1 beta secreted by moDCs, suggesting that insoluble lipids also contribute to hyperactivation (Figure 22). Formulation of 22:0 Lyso PC with KP407 or P-123 and filtering improved IL-1 beta secretion compared to filtered 22:0 Lyso PC in PBS (Figure 22). These data demonstrate that 22:0 Lyso PC solubilized as micelles maintains the ability to induce DC hyperactivation, and improved solubility increases DC hyperactivation. This initial study demonstrated that a consistent solution of 22:0 Lyso PC could be achieved by incorporating lipids into micelles using poloxamer and Pluronic®.

[0193] KP407 was selected as the poloxamer for further study and optimization. Since 22:0 Lyso PC is mostly insoluble in aqueous solutions, we hypothesized that fully solubilized lipids would more easily associate with KP407. 22:0 Lyso PC is soluble in ethanol or methanol, both of which are miscible with water. To optimize the incorporation of 22:0 Lyso PC into particles, lipids were first dissolved in methanol or ethanol. The dissolved lipid solution was mixed with 5.5% KP407 under stirring and stirred at 150 rpm for 90 minutes at room temperature to evaporate the alcohol solvent and induce particle formation with the poloxamer and lipid. Water was added after evaporation to bring the KP407 concentration back to 5.5%. 10x concentrated PBS was used to stabilize particle formation and salt was added to reach a physiologically relevant osmolality, resulting in a final concentration of KP407 in PBS of 5%. After filtering through a 0.45um pore size, the lipid solution was diluted and used to hyperactivate cells at a theoretical target concentration of 82.5uM. 22:0 Lyso PC not incorporated into KP407 micelles induced the least amount of IL-1beta because most of the material was filtered out (Figure 23A). However, mixing KP407 increased IL-1beta secretion (Figure 23A). IL-1beta secretion increased further when methanol or ethanol was used to first solubilize the lipids, suggesting that more lipid was incorporated into the particles using this solvent evaporation strategy. Although the majority of cells remained viable in each condition, the solvent evaporation method reduced cell viability by approximately 25% (Figure 23B). Interestingly, the viability of samples treated using the vehicle solvent evaporation method did not decrease as much. This suggests that by increasing the bioavailability of 22:0 Lyso PC, lower doses of 22:0 Lyso PC can be used.

[0194] Dynamic light scattering was used to characterize particle size. Readings performed with 22:0 Lyso PC in PBS and 22:0 Lyso PC in 5% KP407 resulted in large variations in sample readings and poor quality sizing data due to inconsistent particle size. In contrast, particles generated using solvent evaporation were consistently approximately 1500 nm in diameter (Figure 24), while preparations of micelles lacking 22:0 Lyso PC were devoid of particles of this size. Thus, the interaction between 22:0 Lyso PC and KP407 allowed the formation of stable particles approximately 1500 nm in diameter. These particles are not solid and, considering the measured particle size, may deform, allowing them to pass through smaller filter pores.

[0195] Example 6: Hyperactivation of mouse dendritic cells in vitro and in vivo This example describes the testing of KP407-containing formulations of 22:0 Lyso PC and / or R848 on mouse cells in in vitro and in vivo mouse tumor models.

[0196] material and method Generation of mouse FLT3L-differentiated bone marrow-derived DCs (BMDCs). Leg femurs and tibias were removed from mice, cut with scissors, and drained into sterile tubes. Bone marrow suspensions were treated with ACK for 1 min and then passed through a 40um cell strainer. Cells were counted and resuspended in medium consisting of complete IMDM containing 10% FBS, penicillin and streptomycin, and supplements of L-glutamine and sodium pyruvate (I10). Cells were then plated in P12 plates at 5E6 bone marrow cells per well. Recombinant mouse FLT3L (Miltenyi) was added to the cultures at 200ng / mL. Differentiated cells were used for subsequent assays on day 9. Efficiency of differentiation was monitored by flow cytometry using a BD Symphony A3, and CD11c+MHC-II+ cells were always greater than 80% of live cells. For each experiment, 5 mice were used to collect BM and generate DCs.

[0197] Mouse FLT3L-differentiated BMDC (FLT3L-BMDC) hyperactivation. BMDC were washed with PBS and diluted to 1.5 × 10 5 The cells were replated in I10 containing FLT3L at a concentration of 1000 cells / well. Stimuli were added to the cultures at the indicated concentrations to a final volume of 200 μL / well. After 24 h of stimulation, cell culture supernatants were collected after centrifugation and stored at -20°C for measuring cytokine secretion. IL-1b, IL-6, IL-12p40 and TNF-alpha ELISAs were performed using eBioscience Ready-SET-Go! (now ThermoFisher) ELISA kits according to the manufacturer's protocol. Cell viability was measured using the Cell Titer-Glo reagent kit from Promega.

[0198] Flow cytometry. After FcR blockade, treated FLT3L-BMDCs were washed and stained with Live Dead Fixable dye (ThermoFisher) in PBS for 20 min at 4°C. Cells were then washed again and stained for 20 min at 4°C in MACS buffer (PBS with 1% FCS and 2 mM EDTA) containing the following fluorescently conjugated antibodies purchased from BioLegend: anti-CD11c, anti-IA / IE, anti-H-2Kb, anti-SIRPa, anti-CD24, anti-CD40, anti-CD45R, anti-CXCL16 and anti-CCR7. Data were acquired on a BD FACS Symphony (Becton-Dickenson). Data were analyzed using FlowJo software (Tree Star). Experimental conditions were tested in triplicate and conditions were tested in two or three independent experiments.

[0199] DC infiltration in skin dLN. To evaluate the infiltration of DC in skin-draining lymph nodes (dLN), mice were subcutaneously injected with R848 combined with 22:0 Lyso PC resuspended in KP407 at 10%, 15%, or 20%. 24 hours after injection, skin-draining lymph nodes (dLN) were dissected out. Single cell suspensions were prepared and cells were stained with Live Dead Fixable dye (ThermoFisher) in PBS for 20 min at 4°C. Cells were then washed again and stained in MACS buffer (PBS with 1% FCS and 2 mM EDTA) containing the following fluorescently conjugated antibodies: anti-CD11c and anti-IA / IE (MHC-II) for 20 min at 4°C. To determine the absolute number of CD11c+MHC-IIhigh in live cells, countBright counting beads (ThermoFisher) were used according to the manufacturer's protocol. Data were acquired on a BD FACS Symphony (Becton-Dickenson). Data were analyzed using FlowJo software (Tree Star). Five mice were used in each experimental group.

[0200] Antigen uptake and presentation assay. To examine the antigen uptake and endocytosis ability of BMDCs, pHrodo™ Red dextran 10,000 MW was used (ThermoFisher). Briefly, FLT3L-derived BMDCs previously cultured in medium alone or treated with R848 alone or in combination with 22:0 Lyso PC for 24 hours were incubated with pHrodo™ Red dextran (40ug / ml) for 45 minutes at 37°C or (as a control for antigen surface binding) at 4°C. BMDCs were then washed and stained with Live / Dead Fixable Violet dye (ThermoFisher) to distinguish between live and dead cells. Cells were then fixed with BD fixative and resuspended in MACS buffer. Red (APC) fluorescence of live cells was measured by flow cytometry. Fluorescence values ​​of BMDCs incubated at 37 °C were reported as the mean fluorescence intensity (MFI) of pHrodo™ Red dextran-associated cells normalized to the MFI of pHrodo™ Red dextran-associated cells incubated at 4 °C. To measure the efficiency of OVA peptide presentation on MHC-I, FLT3L-BMDCs were treated as above and incubated with Endofit-OVA protein (0.5 mg / ml) for 1 h at 37 °C. Cells were then washed with MACS buffer and stained with APC-conjugated anti-mouse H-2Kb antibody (BioLegend) and PE-conjugated antibody binding to H-2Kb bound to the OVA peptide "SIINFEKL" (BioLegend) for 20-30 min at 4 °C. Non-OVA-treated DCs served as negative controls, and isotype controls were used as staining controls. The percentage of cells associated with the OVA peptide on MHC-I was calculated by flow cytometry. Data were acquired on a Symphony A3 flow cytometer (Becton-Dickenson) and analyzed with FlowJo software (Becton-Dickenson). Experimental conditions were tested in triplicate.

[0201] Preparation of ex vivo whole tumor lysates. Syngeneic whole tumor lysates (WTL) were prepared from tumor explants of unimmunized tumor-bearing mice. Briefly, tumors from unimmunized mice with tumors of 10-12 mm size were mechanically disaggregated using a gentle MACS dissociator (Miltenyi Biotec) and enzymatically digested using a tumor dissociation kit (Miltenyi Biotec) according to the manufacturer's protocol. After digestion, the tumor cell suspension was washed with PBS and passed through a 70um and then a 30um filter. CD45+ cells were removed from the single cell suspension using anti-CD45 TIL microbeads (Miltenyi Biotec). Tumor cells were then counted and eluted at 1 × 10 7 After resuspension at 100 cells / ml, cells were lysed by 3-4 cycles of freeze-thaw. Lysed cells were further disrupted by repeatedly passing the material through an 18G, then a 21G, and finally a 25G needle. The lysate was filtered again through 70um and 30um cell strainers and stored in aliquots at -80°C until use. WTL was used for immunotherapy at a concentration equivalent to 5.75E5 tumor cells per mouse.

[0202] Immunotherapy immunization and tumor challenge. For immunization in the context of immunotherapy approaches, C57BL / 6J were cultured at 5 × 10 4 B16F10 cells were injected into the left flank. If tumors were palpable, mice were either left unimmunized, immunized with WTL alone, R848 in combination with WTL, or R848 and 22:0 LPC in combination with WTL prepared in PBS or KP407. Mice received two boost injections at 7-day intervals.

[0203] Quantitative and statistical analysis. For in vivo studies, n refers to the number of animals per condition from one or two independent experiments. Statistical differences were calculated using unpaired two-tailed Student's t test, or one-way ANOVA with Tukey post-hoc test. Dependent samples were analyzed with paired t test. Statistical significance of experiments with more than two groups was tested with two-way ANOVA with Tukey multiple comparison test correction. All experiments were analyzed using Prism 7 (GraphPad Software). Graph data are shown as mean values, and error bars indicate SD or SEM. <0.05 ( * ), <0.01( ** ) or <0.001( *** ), %0.0001( **** ) P values ​​indicated significant differences between groups.

[0204] result Mice are an important experimental model, especially for testing cancer treatments. To evaluate the therapeutic effect of 22:0 Lyso PC in a mouse tumor-bearing model, we first sought to determine whether 22:0 LPC would overactivate mouse dendritic cells. Dendritic cells were differentiated from mouse bone marrow using mouse FLT3L recombinant protein. To overactivate mouse DCs, 22:0 Lyso PC was prepared using two methods. 22:0 Lyso PC was either resuspended in PBS and added to the cells, or lipids were resuspended in 5% Kolliphor P407 (KP407). When combined with R848 (1ug / mL), various concentrations of 22:0 Lyso PC resuspended in PBS were unable to induce IL-1 beta secretion. In contrast, 22:0 Lyso PC formulated in KP407 induced IL-1 beta secretion in a dose-dependent manner, demonstrating that mouse cells could be overactivated by 22:0 Lyso PC (Figure 25A). As expected, untreated DCs or DCs treated with R848 alone or 22:0 Lyso PC without R848 were unable to induce IL-1beta secretion. Of the lipid concentrations tested, 41uM 22:0 Lyso PC formulated in KP407 was superior in supporting IL-1beta secretion while maintaining cell viability (Figure 25B). These data suggest that incorporation of 22:0 Lyso PC into micelles makes lipids bioavailable to mouse dendritic cells. Highly activated DCs will gain the ability to release IL-1beta while remaining viable, but should share similar properties to activated DCs treated with PAMPs alone. To test whether hyperactivated DCs treated with R848 and 22:0 Lyso PC retained their classical DC functions (proinflammatory cytokine secretion, antigen uptake, antigen presentation, costimulatory molecule expression and chemokine receptors), DCs were treated as above with medium or R848 alone, or DCs were treated with a combination of R848 and 41 uM 22:0 Lyso PC for 24 hours.As expected, R848 stimulation induced higher levels of TNF-alpha secretion or secretion of pro-inflammatory cytokines such as IL-6 compared to naive DCs treated with medium alone. Similarly, overactivated DCs treated with a combination of 22:0 LPC and R848 resuspended in PBS or KP407 induced high TNF-alpha and IL-6 secretion, with a slight increase in TNF-alpha secretion following DC stimulation with R848 and 22:0 Lyso PC in KP407 (Figures 26A-26B). Notably, IL-12p40 cytokine, a key driver of Th1 responses, was induced by R848 stimulation in the presence or absence of 22:0 LPC (Figure 26C). Overall, these data highlight that 22:0 Lyso PC does not impede NF-kB-mediated responses in DCs. As a result, overactivated DCs share similar properties to activated DCs treated with the PAMP R848 alone, but add IL-1 beta to their cytokine secretion repertoire.

[0205] FLT3L-DCs are divided into two major subsets: cDC1 and cDC2. Of these subsets, cDC1 is capable of unique antigen cross-presentation and can stimulate not only naive CD8+ T cells but also CD4+ T cells. In contrast, cDC2 activates Th2 and Th17 immunity. To determine the behavior of 22:0 Lyso PCs in cDC1 and cDC2 subsets, we analyzed FLT3L-differentiated DCs, cDC1 and cDC2, by flow cytometry. To identify cDC1 and cDC2, FLT3L DCs were stained with CD11c, SIRPa, CD24, MHC-II and CD45R. cDC1 is defined as CD11c+MHC-II+CD45R-CD24+SIRPa-, and cDC2 is defined as CD11c+MHC-II+CD45R-CD24lowSIRPa+. We analyzed the expression of costimulatory molecules such as CD40, which is important for DC and T cell interaction. As expected, 24 hours after stimulation, R848 induced upregulation of CD40 expression compared to naive cDC1 and cDC2 cells. Interestingly, we found that CD40 expression was strongly enhanced in hyperactivated cDC1 and cDC2 treated with R848 in combination with 41 uM 22:0 LPC resuspended in 5% KP407 (Figure 27).

[0206] DC migration is a key step in activating adaptive immune responses. CCR7 is a chemokine receptor required for DC migration to lymph nodes. When cells were overactivated using 22:0 Lyso PC in KP407 formulation, cDC1 cells significantly increased CCR7 expression compared to R848 alone (Figure 28A). A similar trend was observed for CXCL16, a T cell chemoattractant that plays a key role in antitumor T cell-DC interactions in the tumor microenvironment (Figure 28B). Furthermore, overactivation enhanced MHC class I expression in both cDC1 and cDC2 subsets compared to R848 stimulation alone (Figure 29).

[0207] MHC class I (MHC-I) expression on DCs is important for antigen cross-presentation to CD8+ T cells. Therefore, we analyzed the ability of DCs to take up and cross-present antigens on MHC-I, a function of DCs required for stimulating antigen-specific T cells. To first test the endocytic ability of DCs, FLT3L-DCs were stimulated as above for 24 hours and then incubated with Red pHrodo dextran. When dextran is endocytosed, red fluorescence can be detected by flow cytometry. DCs that did not receive dextran served as negative controls. We found that when DCs were treated with R848 in the presence or absence of 22:0 Lyso PC, DCs took up antigens similarly, indicating that 22:0 Lyso PC does not impair the antigen uptake ability of DCs (Figure 30A). To test antigen processing and antigen cross-presentation, DCs were stimulated as above for 24 hours and then treated with ovalbumin whole protein for 45 minutes at 4°C or 37°C. Next, we measured the expression of ovalbumin peptides on MHC-I molecules by using an antibody specific for H-2Kb bound to SIINFEKL. We found that DCs treated with R848 strongly enhanced their ability to process and cross-present OVA antigen. Notably, 22:0 Lyso PC did not prevent these functions, as overactive DCs were still able to process antigen and cross-present OVA peptides on MHC-I molecules (Figure 30B). Taken together, these data indicate that overactive DCs are potent in inducing adaptive T cell immune responses.

[0208] We hypothesize that by combining 22:0 Lyso PC with R848, we can harness endogenous DCs to initiate antitumor immune responses. As observed in vitro, micellization of 22:0 Lyso PC with KP407 is necessary to hyperactivate DCs. To optimize the amount of KP407 required for in vivo vaccination of mice, various ratios were used for subcutaneous injection. Mice were subcutaneously immunized with R848 combined with 22:0 Lyso PC resuspended in KP407 at 10%, 15% or 20%. With 15% KP407, we observed the highest influx of DCs defined as CD11c+MHChigh in dLN, indicating that 15% KP407 is the optimal concentration for inducing DC trafficking to lymph nodes (Figure 31). This initial proof-of-concept study helped optimize the vaccination protocol for subsequent studies.

[0209] 22:0 Lyso PC was then tested as a therapeutic tumor vaccine. Mice were inoculated subcutaneously in the left flank with B16-F10, a murine melanoma tumor cell line. As a source of vaccine antigen, whole tumor lysate (WTL) from B16-F10 cells was used after 3-4 freeze / thaw cycles to release antigens from the tumor cells. To treat tumor-bearing mice, 7 days after tumor injection, mice were immunized in the right flank with 80ug of WTL in combination with 100ug of R848 per mouse and 65ug of 22:0 Lyso PC in KP407 (15%). Mice then received two boosts every 7 days. Unimmunized mice died from tumor growth within 3 weeks of tumor inoculation. No significant benefit was observed when mice were vaccinated with tumor lysate combined with R848 in PBS or with tumor lysate combined with R848 and 22:0 Lyso PC. In contrast, mice immunized with WTL and R848 in combination with 22:0 Lyso PC formulated with 15% KP407 survived longer (Figure 32) and tumor growth kinetics was delayed (Figure 33). This initial study shows that 22:0 Lyso PC has a hyperactivating effect on mouse DCs and can mount a protective immune response against tumor challenge.

Claims

1. A composition comprising an isolated lysophosphatidylcholine (LPC) having a single acyl chain and a TLR7 / 8 agonist, wherein the acyl chain is a C13-C24 acyl chain.

2. The composition of claim 1, wherein the acyl chain is a C18-C22 acyl chain or a C21-C24 acyl chain.

3. The composition of claim 2 , further comprising an antigen and / or a dendritic cell.

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

5. The composition of claim 4, wherein the LPC comprises 1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine [LPC(22:0)].

6. The composition of claim 1 , wherein the TLR7 / 8 agonist is a small molecule having a molecular weight of 900 Daltons or less.

7. The composition of claim 6 , wherein the TLR7 / 8 agonist comprises an imidazoquinoline compound.

8. The composition of claim 7 , wherein the TLR7 / 8 agonist comprises resiquimod (R848).

9. 9. The composition of claim 8, wherein the LPC comprises LPC(22:0) and the TLR7 / 8 agonist comprises resiquimod (R848).

10. The composition described in claim 3, further comprising an antigen, wherein the antigen comprises a proteinaceous antigen.

11. The composition of claim 10 , wherein the antigen comprises a tumor antigen.

12. The composition of claim 11 , wherein the tumor antigen comprises a synthetic or recombinant neo-antigen.

13. The composition of claim 11 , wherein the tumor antigen comprises a tumor cell lysate.

14. 11. The composition of claim 10, wherein the antigen comprises a microbial antigen, the microbial antigen comprising one or more of a viral antigen, a bacterial antigen, a protozoal antigen, and a fungal antigen.

15. The composition of claim 14 , wherein the microbial antigen comprises a purified or recombinant surface protein.

16. 15. The composition of claim 14, wherein the microbial antigen comprises an inactivated whole virus.

17. The composition i) liposomes; ii) LPS or MPLA; and / or iii) oxPAPC or species of oxPAPC The composition of claim 1 , wherein the composition is free of

18. 10. The composition of claim 1, further comprising an adjuvant, the adjuvant comprising an aluminum salt adjuvant, a squalene in water emulsion, a saponin, or a combination thereof.

19. A pharmaceutical formulation comprising the composition of claim 1 and a pharma- ceutically acceptable excipient.

20. 20. An ex vivo method for producing hyperactivated dendritic cells, the method comprising contacting dendritic cells ex vivo with an effective amount of a composition according to any one of claims 1 to 18 or a formulation according to claim 19 for producing hyperactivated dendritic cells, the hyperactivated dendritic cells secreting IL-1 beta without undergoing pyroptosis.

21. A composition according to any one of claims 1 to 18 or a formulation according to claim 19 for use in a method for producing hyperactivated dendritic cells, the method comprising contacting dendritic cells in vivo with an effective amount of the composition or formulation for producing hyperactivated dendritic cells, the hyperactivated dendritic cells secreting IL-1 beta without undergoing pyroptosis.

22. At least 10 produced by the method of claim 20 ３ pieces, 10 ４ pieces, 10 ５ Pieces or 10 ６ A pharmaceutical formulation comprising said hyperactivated dendritic cells and a pharma- ceutical acceptable excipient.

23. 20. The formulation of claim 19 for use in a method of stimulating an immune response to an antigen, said method comprising administering to an individual in need thereof an effective amount of said formulation to stimulate said immune response to said antigen.

24. The formulation of claim 19 for use in a method of treating cancer, the method comprising administering to an individual in need thereof an effective amount of the formulation to treat the cancer.

25. 20. The formulation of claim 19 for use in a method of inhibiting abnormal cell growth, said method comprising administering to an individual in need thereof an effective amount of said formulation to inhibit abnormal cell growth.

26. 20. The formulation of claim 19 for use in a method of treating an infectious disease, said method comprising administering to an individual in need thereof an effective amount of said formulation to treat said infectious disease.

27. 1. A method for preparing an immunogenic composition, the method comprising: a) preparing a tumor cell-enriched suspension from a sample obtained from a tumor of a mammalian subject having cancer; b) lysing cells from said tumor cell enriched suspension to obtain a tumor cell lysate; c) contacting said tumor cell lysate with the formulation of claim 19 to obtain said immunogenic composition; The method includes:

28. 28. The method of claim 27, wherein in step a) the leukocytes are depleted by negative selection using an anti-CD45 antibody and / or the cells are lysed in step b) by one or more freeze-thaw cycles.

29. 28. An immunogenic composition prepared by the method of claim 27.

30. 30. The immunogenic composition of claim 29 for use in a method of eliciting an anti-cancer immune response, the method comprising: administering to a mammalian subject having cancer an effective amount of said immunogenic composition. An immunogenic composition comprising:

31. The immunogenic composition for use according to claim 30, wherein the anti-cancer immune response comprises a cellular immune response.

32. The immunogenic composition for use according to claim 31, wherein the anti-cancer immune response comprises cancer antigen-induced IL-1 beta secretion and / or activation of CD8+ T lymphocytes.

33. 32. The immunogenic composition for use according to claim 31 , wherein the cancer is a non-hematological cancer.

34. 34. The immunogenic composition for use according to claim 33, wherein the non-hematological cancer is carcinoma, sarcoma or melanoma.

35. The immunogenic composition for use according to claim 31 , wherein the cancer is lymphoma.

36. The composition of any one of claims 1 to 18, further comprising a surfactant.

37. 37. The composition of claim 36, wherein the surfactant comprises a non-ionic surfactant.

38. The composition of claim 37, wherein the nonionic surfactant comprises an ethylene oxide-propylene oxide copolymer.

39. 38. The composition of claim 37, wherein the non-ionic surfactant comprises one or more of poloxamer 407, poloxamer 188, and P123.

40. 38. The composition of claim 37, wherein i) the LPC is dissolved in an alcohol to form an alcoholic solution of LPC; ii) the alcoholic solution of LPC 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 LPC and the non-ionic surfactant.

41. 38. The composition of claim 37, wherein the non-ionic surfactant is present in an amount of about 2.5% to 25% (w / w).

42. 42. The composition of claim 41, wherein the LPC and non-ionic surfactant are present in particles having a diameter of about 1000 to 2000 nanometers.

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