Stimulation of resident dendritic cells to superactivate them for cancer immunotherapy

Abstract

The present application relates to cancer immunotherapy, for example, stimulating T cell-mediated antitumor therapy.Therefore, the present application describes a method for inducing or enhancing adaptive immune response against cancer in a subject, and a method for treating cancer in a subject.In some embodiments, the method superactivates dendritic cells (DCs), and the dendritic cells induce type I T helper (TH1) and cytotoxic T lymphocyte (CTL) responses in the absence of TH2 immunity.

Description

[Technical Field]

[0001] Priority claim This application claims the benefit of U.S. Provisional Application No. 62 / 937,073, filed November 18, 2019, the entire contents of which are incorporated herein by reference.

[0002] Statement of Federally Funded Research This invention was made with government support under Grant No. AI116550 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Technical Field This application relates to cancer immunotherapy, for example, stimulation of T cell-mediated anti-tumor therapy. [Background technology]

[0004] Background technology Central to understanding protective immunity against infection and cancer are dendritic cells (DCs), migratory phagocytes that patrol the body's tissues (D. Alvarez, EH et al., Immunity, Vol. 29, No. 3, pp. 325-42, September 2008). DCs survey the environment for threats to the host, most commonly for evidence of infection or tissue damage. This surveillance is achieved through the action of a superfamily of threat assessment receptors, known as pattern recognition receptors (PRRs), which typically recognize microbial products or host-encoded molecules indicative of tissue injury (S.W. Brubaker et al., Annu. Rev. Immunol., Vol. 33, pp. 257-90, 2015; C.A. Janeway and R. Medzhitov, Annu. Rev. Immunol., Vol. 20, pp. 197-216, January 2002). Microbial ligands for PRRs are classified as pathogen-associated molecular patterns (PAMPs), whereas host-derived PRR ligands are damage-associated molecular patterns (DAMPs) (P. Matzinger, Science, Vol. 296, No. 5566, pp. 301-305, April 2002).

[0005] Upon detecting PAMPs, PRRs unleash signaling pathways that fundamentally alter the physiology of DCs that express them (A. Iwasaki and R. Medzhitov, Nat. Immunol., Vol. 16, No. 4, pp. 343-53, April 2015; O. Joffre et al., Immunol. Rev., Vol. 227, No. 1, pp. 234-247, January 2009). For example, before PRR activation, DCs are typically considered non-inflammatory cells. Upon encountering extracellular PAMPs, PRRs stimulate the rapid and robust upregulation of numerous inflammatory mediators, including cytokines, chemokines, and interferons. Concurrent with these gene expressions is the upregulation of factors important for DC migration to draining lymph nodes (dLNs) and T cell activation, such as MHC and costimulatory molecules. Thus, PRR signaling processes shift DC activity from an unstimulated (naive) state to an "activated" state (K. Inaba et al., J. Exp. Med., Vol. 191, No. 6, pp. 927-36, March 2000; I. Mellman and R.M. Steinman, Cell, Vol. 106, No. 3, pp. 255-8, August 2001). Summary of the Invention [Problem to be solved by the invention]

[0006] overview There is a need to diversify current approaches to cancer immunotherapy. Accordingly, methods for inducing or enhancing an adaptive immune response against cancer in a subject and methods for treating cancer in a subject are described herein. In some embodiments, the methods superactivate dendritic cells (DCs), which induce type I T helper (TH1) and cytotoxic T lymphocyte (CTL) responses in the absence of TH2 immunity. The superactivating stimulus promotes a T cell response that protects against tumors that are sensitive or resistant to PD-1 blockade. This protective response depends on inflammasomes in DCs and can be elicited using tumor lysate as an immunogen. [Means for solving the problem]

[0007] Thus, provided herein is a method for inducing or enhancing an adaptive immune response against cancer in a subject, comprising administering to the subject effective amounts of (i) a Toll-like receptor (TLR) ligand, (ii) a non-canonical inflammasome-activating lipid, and (iii) a cancer immunogen.

[0008] Further provided herein is a method of treating cancer in a subject, comprising administering to the subject effective amounts of (i) a Toll-like receptor (TLR) ligand, (ii) a non-canonical inflammasome-activating lipid, and (iii) a cancer immunogen.

[0009] In some embodiments of the methods described herein, the cancer immunogen is an infectious agent immunogen, and infection by the infectious agent is associated with the development of cancer.

[0010] In some embodiments of the methods described herein, the cancer immunogen is derived from a cancer immunogenic cell.

[0011] In some embodiments of the methods described herein, the cancer immunogen is or comprises a whole tumor cell lysate.

[0012] In some embodiments of the methods described herein, the TLR ligand is selected from a TLR1 ligand, a TLR2 ligand, a TLR3 ligand, a TLR4 ligand, a TLR5 ligand, a TLR6 ligand, a TLR7 ligand, a TLR8 ligand, a TLR9 ligand, a TLR10 ligand, a TLR11 ligand, a TLR12 ligand, a TLR13 ligand, and combinations thereof.

[0013] In some embodiments of the methods described herein, the TLR ligand is a TLR4 ligand.

[0014] In some embodiments of the methods described herein, the TLR4 ligand is selected from monophosphoryl lipid A (MPLA), lipopolysaccharide (LPS), or a combination thereof.

[0015] In some embodiments of the methods described herein, the non-canonical inflammasome-activating lipid comprises a form of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC).

[0016] In some embodiments of the methods described herein, the non-canonical inflammasome-activating lipid is 2-[[(2R)-2-[(E)-7-carboxy-5-hydroxyhept-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium (HOdiA-PC), [(2R)-2-[(E)-7-carboxy-5-oxohept-6-enoyl]oxy-3-hexadecanoyloxypropyl]-2 -(Trimethylazaniumyl)ethyl phosphate (KOdiA-PC), 1-palmitoyl-2-(5-hydroxy-8-oxo-octenoyl)-sn-glycero-3-phosphorylcholine (HOOA-PC), 2-[[(2R)-2-[(E)-5,8-dioxooct-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium (KOOA-PC), [(2R)-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium Oxy-2-(5-oxopentanoyloxy)propyl]2-(trimethylazaniumyl)ethyl phosphate (POVPC), [(2R)-2-(4-carboxybutanoyloxy)-3-hexadecanoyloxypropyl]2-(trimethylazaniumyl)ethyl phosphate (PGPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[2-[(Z)-oct-2-enyl]-5-oxocyclopent-3-en-1-ylidene]methyl]methyl [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentylidene]methyl]oxiran-2-yl]butanoyloxy]propyl]2-(trimethylazaniumyl)ethyl phosphate (PECPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentylidene]methyl]oxiran-2-yl]butanoyloxy]propyl]2-(trimethylazaniumyl)ethyl phosphate (PEIPC), or a combination thereof.

[0017] In some embodiments of the methods described herein, the non-canonical inflammasome-activating lipid comprises [(2R)-2-(4-carboxybutanoyloxy)-3-hexadecanoyloxypropyl] 2-(trimethylazaniumyl)ethyl phosphate (PGPC).

[0018] In some embodiments of the methods described herein, the subject is a mammal. In some embodiments of the methods described herein, the subject is a human.

[0019] In some embodiments of the methods described herein, the TLR ligand, oxPAPC species, and cancer immunogen are administered as part of a pharmaceutical composition.

[0020] In some embodiments of the methods described herein, the immune response is a prophylactic immune response.

[0021] In some embodiments of the methods described herein, the immune response is a therapeutic immune response.

[0022] In some embodiments of the methods described herein, the adaptive immune response comprises T cell activation.

[0023] In some embodiments of the methods described herein, the methods further comprise treating the subject with one or more therapeutic interventions.

[0024] In some embodiments of the methods described herein, the TLR ligand, oxPAPC species, and cancer immunogen, and one or more therapeutic interventions are co-administered or sequentially administered.

[0025] In some embodiments of the methods described herein, the one or more therapeutic interventions comprise radiation, chemotherapy, surgery, a therapeutic antibody, an immunomodulatory agent, a proteasome inhibitor, a pan-deacetylase (DAC) inhibitor, a histone deacetylase (HDAC) inhibitor, a checkpoint inhibitor, adoptive cellular therapy, a vaccine, or a combination thereof.

[0026] In some embodiments of the methods described herein, adoptive cell therapy includes CAR-T cell therapy, CAR-NK cell therapy, T cells, dendritic cells, or a combination thereof. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention. Other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0027] Other features and advantages of the invention will become apparent from the following detailed description, drawings, and claims. [Brief explanation of the drawings]

[0028] [Figure 1A] Figure 1A-1F: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, oxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 1A: Release of cytokines, IL-1β and TNFα, was monitored by ELISA. Means and SDs from four mice are shown, and each panel is representative of two independent experiments. *P<0.05; **P<0.01; ***P<0.005. [Figure 1B]This is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. Figures 1A-1F: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, oxPAPC alone, or PGPC alone for 24 h; or BMDCs were primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 1B: Cell mortality was measured by LDH release into the cell supernatant. The mean and SD of four mice are shown; each panel is representative of two independent experiments. *P<0.05; **P<0.01; ***P<0.005. [Figure 1C] This is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. Figures 1A-1F: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, oxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 1C: BMDCs treated with the indicated stimuli as in Figure 1A were stained for CD11c and CD40 using a live-dead violet kit. The mean fluorescence intensity (MFI) of surface CD40 (among live CD11c+ cells) was measured by flow cytometry. The mean and SD of four mice are shown, and each panel is representative of two independent experiments. *P<0.05; **P<0.01; ***P<0.005. [Figure 1D]This is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. Figures 1A-1F: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, oxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 1D: BMDCs pretreated with the indicated stimuli were transferred onto CD40-coated plates and cultured for 24 h. IL-12p70 cytokine release was measured by ELISA. Mean and SD of four mice are shown; each panel is representative of two independent experiments. *P<0.05; **P<0.01; ***P<0.005. [Figure 1E] Figure 1A-1F: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, oxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 1E: BMDCs pretreated with the indicated stimuli as in Figure 1A were incubated with OVA protein for 2 h or with FITC-labeled OVA for 45 min. OVA-FITC uptake (left panel) was assessed by flow cytometry. Data are expressed as the percentage of OVA-associated CD11c BMDCs at 37°C and normalized to OVA-associated CD11c BMDCs at 4°C. OVA peptide presentation on MHC-I (right panel) was monitored using a PE-conjugated antibody against H-2Kb bound to the OVA peptide SIINFEKL (SEQ ID NO: 1). Data are expressed as the frequency of SIINFEKL (SEQ ID NO: 1)-associated DCs among live CD11c+ cells. Mean and SD from three replicates are shown, and data are representative of at least three independent experiments. Mean and SD from four mice are shown, and each panel is representative of two independent experiments. *P<0.05; **P<0.01; ***P<0.005. [Figure 1F]This is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. Figures 1A-1F: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, oxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 1F: BMDCs treated with the indicated stimuli as in Figure 1A were loaded (or not) with OVA protein or the OVA peptide SIINFEKL for 1 h and then incubated with splenic OT-II naive CD4+ T cells or OT-I naive CD8+ T cells for 4 days. Figure 1F: Supernatants were collected on day 4, and the release of cytokines IFNγ, IL-2, IL-10, TNFα, and IL-13 was measured by ELISA. The mean and SD of four mice are shown, and each panel is representative of two independent experiments. *P<0.05;**P<0.01;***P<0.005. [Figure 1G] This is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. Figure 1G: BMDCs were left untreated (none) or treated with LPC for 24 h, or BMDCs were primed with LPS for 3 h and then treated with PGPC or alum for 21 h. Treated BMDCs were co-cultured with splenic OT-I or OT-II T cells as in Figure 1F. Four days after co-culture, CD4+ and CD8+ T cells were stimulated with PMA and ionomycin for 5 h in the presence of brefeldin A and monensin. The frequency of TH1 cells (TNFα+IFNγ+) and the frequency of TH2 cells (Gata3+IL-4+IL-10+) among CD4+ T cells were measured by intracellular staining. Data are presented as the ratio of TH1 cells to TH2 cells (left panel). The frequency of IFNγ+ among CD8+ T cells is presented in the right panel. The mean and SD from three replicates are shown, and each panel is representative of at least two independent experiments. The mean and SD from four mice are shown, and each panel is representative of two independent experiments. *P<0.05; **P<0.01; ***P<0.005. [Figure 1H] (Figure 1H) is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. C57BL / 6 mice were subcutaneously injected with endofit-OVA protein alone or together with LPS, emulsified in either incomplete Freud's adjuvant (IFA) or alum, as indicated, into the right flank. Alternatively, mice were injected with endofit-OVA protein and LPS plus OxPAPC or PGPC, both emulsified in IFA. Forty days after immunization, CD4+ and CD8+ T cells were isolated from skin-draining lymph nodes (dLNs). T cells were then cultured with naive BMDCs loaded with or without OVA or SIINFEKL peptide for 5 days. IFNγ, IL-10, and IL-13 secretion were measured by ELISA. Means and SDs from four mice are shown, and each panel is representative of two independent experiments. *P<0.05;**P<0.01;***P<0.005. [Figure 2A] Figure 2A and Figure 2B are a series of graphs showing that superactive stimulation enhances the generation of memory T cells and augments antigen-specific IFNγ effector responses in an NLRP3-dependent manner. C57BL / 6 mice were injected subcutaneously (sc) into the right flank with endofit-OVA alone, with LPS, with PGPC, or with LPS plus OxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA). Seven or 40 days after immunization, T cells were isolated from skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD4 and anti-CD8 beads. Figure 2A: Percentages of CD44lowCD62Llow effector T cells (Teff), CD44highCD62Llow effector memory T cells (Tem), and CD44highCD62Lhigh central memory T cells (Tcm) among live CD3+CD4+ cells (left panel) or live CD3+CD8+ cells (right panel). Means and SDs from five mice are shown and are representative of three independent experiments. *P<0.05; **P<0.01; ***P<0.005, ****P<0.0005. [Figure 2B] This is a series of graphs showing that superactive stimulation enhances the generation of memory T cells and antigen-specific IFNγ effector responses in an NLRP3-dependent manner. Figures 2A and 2B: C57BL / 6 mice were injected subcutaneously (sc) into the right flank with endofit-OVA alone, with LPS, with PGPC, or with LPS plus OxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA). Seven or 40 days after immunization, T cells were isolated from the skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD4 and anti-CD8 beads. Figure 2B: CD8+ T cells were sorted from the dLNs 7 days after immunization and then treated with PMA plus ionomycin for 5 h or cocultured with B16OVA cells (target cells) at a 1:3 (effector:target) ratio. The percentage of CD107a+ among live CD8+ T cells was monitored by flow cytometry to assess degranulation. Means and SD of five mice are shown and are representative of three independent experiments. *P<0.05; **P<0.01; ***P<0.005, ****P<0.0005. [Figure 2C]Figure 2C-D: C57BL / 6 mice were subcutaneously injected with endofit-OVA alone, with LPS, or with LPS plus OxPAPC or PGPC, all emulsified in IFA, or with LPS plus alum, into the right flank. Alternatively, mice were injected with LPS plus PGPC alone (without OVA). NLRP3- / - or Casp1 / 11- / - mice were similarly injected with endofit-OVA emulsified in IFA with LPS plus PGPC. Figure 2C: Seven days after immunization, CD4+ and CD8+ T cells were sorted from the skin dLNs of immunized mice and cocultured with OVA-loaded (or unloaded) BMDCs at a ratio of 1:10 (DCs:T cells) for 7 days. The percentage of SIINFEKL+ (SEQ ID NO: 1) IFNγ+ among live CD8+ T cells (upper panel) and the percentage of AAHAEINEA+ (SEQ ID NO: 2) IFNγ+ among live CD4+ T cells (lower panel) were measured using OVA peptide tetramer staining and intracellular IFNγ staining. Mean and SD of 5 mice are shown. *P<0.05; **P<0.01; ***P<0.005, ****P<0.0005. [Figure 2D]Figure 2C-D: C57BL / 6 mice were subcutaneously injected with endofit-OVA alone, with LPS, or with LPS plus OxPAPC or PGPC, all emulsified in IFA, or with LPS plus alum into the right flank. Alternatively, mice were injected with LPS plus PGPC alone (no OVA). NLRP3- / - or Casp1 / 11- / - mice were similarly injected with endofit-OVA emulsified in IFA with LPS plus PGPC. Figure 2D: The percentage of CD44+ memory T cells in inguinal adipose tissue was assessed by flow cytometry. Mean and SD of five mice are shown. *P<0.05; **P<0.01; ***P<0.005, ****P<0.0005. [Figure 3A] A series of graphs and plots show that superactivated DCs induce inflammasome-dependent antitumor immunity. Figure 3A, Figure 3B: C57BL / 6 mice were injected subcutaneously (sc) into the right flank with PBS (unimmunized), B16OVA cell lysate alone (none), or with LPS, or B16OVA lysate plus LPS and OxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA). 15 days after immunization, mice were challenged subcutaneously with 3 × 10 live B16OVA cells in the upper left dorsal region. 150 days later, tumor-free mice were rechallenged subcutaneously with 5 × 10 live B16OVA cells in the dorsal region. Figure 3A: Tumor growth was monitored every 2 days (upper panel). In survival experiments (lower panel), tumors were allowed to reach a diameter of 20 mm (n = 8–15 mice per group). *P<0.05;**P<0.01;***P<0.005. Tx; T cell injection, WT; wild type. [Figure 3B]A series of graphs and plots show that superactivated DCs induce inflammasome-dependent antitumor immunity. Figure 3A, Figure 3B: C57BL / 6 mice were subcutaneously injected (sc) into the right flank with PBS (unimmunized), B16OVA cell lysate alone (none), or with LPS, or B16OVA lysate plus LPS and OxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA). 15 days after immunization, mice were challenged subcutaneously with 3 × 10 live B16OVA cells in the upper left back. 150 days later, tumor-free mice were rechallenged subcutaneously with 5 × 10 live B16OVA cells in the back. Figure 3B: At the end of tumor growth, tumors were harvested and dissociated to obtain single tumor cell suspensions. The percentage of tumor-infiltrating CD3+CD4+ and CD3+CD8+ T cells among enriched CD45+ live cells was assessed by flow cytometry (upper panel). Tumor-infiltrating CD3+ T cells were sorted and then stimulated for 24 h in the presence of anti-CD3 and anti-CD28 DYNABEADS™ (ThermoFisher Scientific). IFNγ release was measured by ELISA (lower panel) (n = 4 mice per group). *P < 0.05; **P < 0.01; ***P < 0.005. Tx: T cell-injected; WT: wild-type. [Figure 3C] (Figure 3C) is a series of graphs and plots showing that superactivated DCs induce inflammasome-dependent antitumor immunity. (Figure 3C) The absolute numbers of CD8+ T cells (upper panel) and CD69+CD103+ T-resident memory CD8+ T cells (lower panel) were assessed at the immunization or tumor injection site of survivor mice from Figure 3A and measured by flow cytometry (n=4 mice). *P<0.05; **P<0.01; ***P<0.005. Tx: T cell injection; WT: wild type. [Figure 3D](Figure 3D) shows that superactivated DCs induce inflammasome-dependent antitumor immunity. Figure 3D: The absolute numbers of total CD8+ T cells (upper panel), absolute numbers of SIINFEKL+ (SEQ ID NO: 1) among CD8+ T cells (middle panel), and absolute numbers of CD69+CD103+ (lower panel) in the spleen (left panel) or skin-inguinal adipose tissue (right panel) from survivor mice or age-matched, unimmunized tumor-bearing mice (n=5 mice per group). *P<0.05; **P<0.01; ***P<0.005. Tx: T cell injection; WT: wild-type. [Figure 3E] A series of graphs and plots show that superactivated DCs induce inflammasome-dependent antitumor immunity. Figures 3E-3F: Circulating memory CD8+ T cells (TCM) were isolated from the spleens of survivor mice or age-matched, unimmunized tumor-bearing mice, and resident memory CD8+ T cells (TRM) were isolated from the cutaneous inguinal adipose tissue. Figure 3E: TCM and TRM from survivor mice were cocultured with B16OVA, B16-F10, or CT26 tumor cells at a 1:5 (tumor cell:T cell) ratio for 5 h. Cell death mediated by cytolytic CD8+ T cells was measured by LDH release into the supernatant. *P<0.05; **P<0.01; ***P<0.005. Tx: T cell-injected; WT: wild-type. [Figure 3F]A series of graphs and plots show that superactivated DCs induce inflammasome-dependent antitumor immunity. Figures 3E-3F: Circulating memory CD8+ T cells (TCM) were isolated from the spleens of survivor mice or age-matched, unimmunized tumor-bearing mice, and resident memory CD8+ T cells (TRM) were isolated from the cutaneous inguinal adipose tissue. Figure 3F: C57BL / 6 recipient mice were left untreated (no Tx) or inoculated intravenously (iv) with 5 x 105 CD8+ TCM cells isolated from survivor mice or age-matched, unimmunized tumor-bearing mice and / or intradermally (id) with 5 x 105 CD8+ TRM cells. Seven days later, all mice were challenged with 3 x 105 live B16OVA cells. Survival was monitored every 2 days. Tumors were allowed to reach 20 mm in diameter (n = 5 mice per group). *P<0.05;**P<0.01;***P<0.005. Tx; T cell injection, WT; wild type. [Figure 3G] A series of graphs and plots show that superactivated DCs induce inflammasome-dependent antitumor immunity. Figure 3G: C57BL / 6 mice were left untreated (unimmunized) or immunized subcutaneously in the right flank with B16OVA tumor lysate alone, with MPLA, or with B16OVA lysate and MPLA + PGPC, with or without a neutralizing anti-mouse IL-1β antibody. 15 days after immunization, mice were challenged subcutaneously in the upper left back with 3 × 10 live B16OVA cells. 90 days later, tumor-free mice were rechallenged subcutaneously in the back with 5 × 10 live B16OVA cells. Survival was monitored every 2 days (n = 3-4 mice per group). *P < 0.05; **P < 0.01; ***P < 0.005. Tx: T cell-injected; WT: wild-type. [Figure 3H](Figure 3H) A series of graphs and plots showing that superactivated DCs induce inflammasome-dependent antitumor immunity. WT C57BL / 6, NLRP3- / -, Casp11- / -, or Casp1 / 11- / - mice were immunized subcutaneously in the right flank with B16OVA cell lysate, LPS, and PGPC, all emulsified in IFA. 15 days after immunization, mice were challenged subcutaneously in the upper left back with 3 x 105 live B16OVA cells. Survival rates are shown (n = 5 mice per group). *P < 0.05; **P < 0.01; ***P < 0.005. Tx: T cell injection; WT: wild-type. [Figure 3I] A series of graphs and plots show that superactivated DCs induce inflammasome-dependent antitumor immunity. Figures 3I-3J: C57BL / 6 mice were left untreated (naive) or immunized subcutaneously in the right flank with MC38OVA lysate + MPLA or MC38OVA lysate + MPLA + PGPC without intravenous injection of anti-mouse IL-1β antibody starting 2 days before immunization and continuing for 5 days. Alternatively, mice were immunized with OVA protein and MPLA + PGPC. 15 days later, mice were inoculated subcutaneously with 5 × 10 live MC38OVA cells in the upper left dorsal region. 50 days later, tumor-free mice were rechallenged subcutaneously with 1 × 10 MC38OVA cells in the dorsal region. (I) Survival rates are shown (n = 5 mice per group). *P < 0.05; **P < 0.01; ***P < 0.005. Tx: T cell injection; WT: wild-type. [Figure 3J]A series of graphs and plots show that superactivated DCs induce inflammasome-dependent antitumor immunity. Figures 3I-3J: C57BL / 6 mice were left untreated (naive) or immunized subcutaneously in the right flank with MC38OVA lysate + MPLA or MC38OVA lysate + MPLA + PGPC without intravenous injection of anti-mouse IL-1β antibody starting 2 days before immunization and continuing for 5 days. Alternatively, mice were immunized with OVA protein and MPLA + PGPC. 15 days later, mice were inoculated subcutaneously with 5 × 10 live MC38OVA cells in the upper left dorsal region. 50 days later, tumor-free mice were rechallenged subcutaneously with 1 × 10 MC38OVA cells in the dorsal region. (I) Survival rate is shown (n = 5 mice per group). Figure 3J: 170 days after the initial inoculation with MC38OVA tumor cells, survivor mice or age-matched naive mice were challenged with 3 x 10 live B16-F10 cells. Survival rates are shown. (n = 5 mice per group). *P < 0.05; **P < 0.01; ***P < 0.005. Tx: T cell-injected; WT: wild-type. [Figure 4A] Figure 4A is a series of diagrams and graphs showing that superactivation is a potent adjuvant for cancer immunotherapy. Figure 4A is a schematic diagram of the superactivation-based immunotherapy approach, with legends corresponding to the neutralizing antibodies used as follows: anti-IL-1β was injected intravenously (iv), and anti-CD4, anti-CD8a, and anti-PD1 antibodies were injected intraperitoneally. [Figure 4B] Figure 4B: A series of diagrams and graphs demonstrating that superactive stimulation is a potent adjuvant for cancer immunotherapy. C57BL / 6 WT mice were inoculated subcutaneously with 5 x 10 live MC38OVA cells in the upper left dorsum. 14 days later, mice were left untreated (unimmunized) or were injected subcutaneously in the right flank with syngeneic MC38OVA whole tumor lysate (WTL) plus LPS and PGPC, with or without injection of neutralizing antibodies as indicated in the diagram. Mice received two boost injections with WTL and LPS plus PGPC on days 37 and 55 after tumor inoculation. Tumors were allowed to reach 20 mm in diameter. Survival rates are shown (n = 10 mice per group). [Figure 4C]Figure 4C: C57BL / 6 WT mice were subcutaneously inoculated with 3 x 10 live B16OVA cells in the upper left dorsal region. Ten days later, mice were left untreated (unimmunized) or injected with anti-PD1 antibody as indicated in the diagram. Alternatively, mice were subcutaneously injected with syngeneic B16OVA WTL + LPS and PGPC in the right flank, with or without neutralizing antibody injection as indicated. Mice received two boost injections with B16OVA WTL + LPS and PGPC on days 17 and 24 after tumor inoculation. Survival rates are shown (n = 10 mice per group). [Figure 4D] Figure 4D: A series of diagrams and graphs demonstrating that superactive stimulation is a potent adjuvant for cancer immunotherapy. BALB / c WT mice were inoculated subcutaneously with 3 × 10 live CT26 cells in the left dorsal region. Seven days later, mice were left untreated (naive) or injected with anti-PD1 antibodies as indicated in the diagram. Alternatively, mice were injected subcutaneously in the right flank with syngeneic CT26 WT + LPS and PGPC, with or without the indicated neutralizing antibodies. Mice received two boost injections on days 14 and 21 after tumor inoculation. Survival rates are shown (n = 10 mice per group). [Figure 4E] Figure 4E: C57BL / 6 WT mice were inoculated subcutaneously with 3 x 10 live B16-F10 cells in the upper left dorsal region. Seven days later, mice were left untreated (unimmunized) or injected with anti-PD1 antibodies as indicated in the diagram. Alternatively, mice were immunized subcutaneously in the right flank with syngeneic B16-F10 WT + LPS and PGPC, with or without the indicated neutralizing antibodies. Mice received two boost injections on days 14 and 21 after tumor inoculation. Survival rates are shown (n = 10 mice per group). [Figure 4F]A series of diagrams and graphs demonstrating that superactive stimulation is a potent adjuvant for cancer immunotherapy. Figure 4F: C57BL / 6 WT mice were inoculated intravenously with 3 x 10 live B16-F10 cells. Five days later, mice were left untreated (unimmunized) or injected with B16F-10 syngeneic WT cells alone, with LPS, or with LPS + PGPC. 17 days after tumor cell inoculation, metastatic nodules in the lungs were counted (n = 5 mice per group). [Figure 5A] Figure 5A is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. Figure 5A, Figure 5B: BMDCs generated with GMCSF were left untreated (none) or treated with MPLA alone, alum alone, OxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with MPLA for 3 h and then treated with the indicated stimuli for 21 h. Figure 5A: Release of cytokines IL-1β and TNFα was monitored by ELISA. The mean and SD of three replicates are shown, and all panels are representative of at least three independent experiments. *P<0.05. [Figure 5B] Figure 5A and Figure 5B are a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. BMDCs generated with GMCSF were left untreated (none) or treated with MPLA alone, alum alone, OxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with MPLA for 3 h and then treated with the indicated stimuli for 21 h. Figure 5B: Cell mortality was measured by LDH release into the cell supernatant. The mean and SD of three replicates are shown, and all panels are representative of at least three independent experiments. *P<0.05. [Figure 5C]Figure 5C is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote a TH1-dominated immune response without evidence of TH2 immunity. Figure 5C: Release of cytokines IL-1β and TNFα was monitored by ELISA. The mean and SD of three replicates are shown, and all panels are representative of at least three independent experiments. *P<0.05. [Figure 5D] (Figure 5D) is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote a TH1-dominated immune response without evidence of TH2 immunity. (Figure 5D) Cell mortality was measured by LDH release into cell supernatants. The mean and SD of three replicates are shown, and all panels are representative of at least three independent experiments. *P<0.05. [Figure 5E] This is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. Figures 5E-5F: BMDCs generated with GMCSF and treated with the indicated stimuli as in A were stained with anti-CD11c, anti-CD80, anti-CD69, and anti-H2kb antibodies using a live-dead violet kit. Figure 5E: The mean fluorescence intensity (MFI) of surface CD80 (left panel), CD69 (middle panel), and H2kb (right panel) among live CD11c+ cells was measured by flow cytometry. The mean and SD of three replicates are shown, and all panels are representative of at least three independent experiments. *P<0.05. [Figure 6A]Figure 6A-6C: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, OxPAPC alone, or PGPC alone for 24 h. BMDCs were primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 6A: BMDCs were incubated with fixable FITC-labeled OVA at 37°C or 4°C for 45 min. BMDCs were then stained with a live-dead violet kit. Gating strategy for flow cytometry to identify the frequency of OVA-FITC-associated BMDCs at 37°C compared with OVA-associated BMDCs at 4°C. The mean and SD of four mice are shown, and each panel is representative of two independent experiments. ***P<0.005. [Figure 6B] Figure 6A-6C: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, OxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 6B: BMDCs were incubated with endofit-OVA protein for 2 h. Gating strategy for identifying the frequency of SIINFEKL (SEQ ID NO: 1) peptide bound to H2kb on the surface of viable BMDCs, as measured by flow cytometry using a PE-conjugated antibody against H-2kb bound to the OVA peptide SIINFEKL. Each panel represents three replicates from one of three experiments. The mean and SD of four mice are shown, and each panel is representative of two independent experiments. ***P<0.005. [Figure 6C]Figure 6A-6C: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, OxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 6C: C57BL / 6 mice were subcutaneously injected with endofit-OVA protein alone or with LPS, emulsified in either incomplete Freud's adjuvant (IFA) or alum as indicated, into the right flank. Alternatively, mice were injected with endofit-OVA protein and LPS plus OxPAPC or PGPC, both emulsified in IFA. Forty days after immunization, CD4+ T cells were isolated from skin-draining lymph nodes (dLNs). T cells were then cultured with naive BMDCs loaded with or without OVA for 5 days. IL-4 secretion was measured by ELISA. The mean and SD of four mice are shown, and each panel is representative of two independent experiments. ***P<0.005. [Figure 7] This is a series of plots showing that superactivated DCs are excellent antigen-presenting cells and promote a TH1-dominated immune response without evidence of TH2 immunity. BMDCs were left untreated (none), treated with LPC for 24 h, or primed with LPS for 3 h and then treated with PGPC or alum for 21 h. Treated BMDCs were then cocultured with splenic OT-II T cells at a 1:5 (BMDC:T cell) ratio. Four days after coculture, CD4+ T cells were stimulated with PMA+ionomycin in the presence of brefeldin A and monensin for 5 h. Gating strategy for identifying the frequency of IL-4+IL-10+ TH2 cells among live CD4+ T cells, as measured by intracellular staining. Each panel represents three replicates from one of three experiments. [Figure 8A]Figure 8A shows a series of plots and graphs demonstrating that superactive stimulation enhances the generation of memory T cells and augments antigen-specific IFNγ effector responses in an NLRP3-dependent manner. C57BL / 6 mice were injected subcutaneously (sc) with endofit-OVA alone, with LPS, with PGPC, or with LPS plus OxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA), into the right flank. Seven days after immunization, T cells were isolated from skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD4 and anti-CD8 beads. Figure 8A shows the gating strategy for identifying the percentage of CD44-low CD62L-low effector T cells (Teff), CD44-high CD62L-low effector memory T cells (TEM), and CD44-high CD62L-high central memory T cells (TCM) among live CD3+CD4+ cells. Each panel is representative of five replicates. [Figure 8B] Figure 8B: A series of plots and graphs showing that superactive stimulation enhances the generation of memory T cells and augments antigen-specific IFNγ effector responses in an NLRP3-dependent manner. C57BL / 6 mice were injected subcutaneously (sc) into the right flank with endofit-OVA alone, with LPS, with PGPC, or with LPS plus OxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA). Seven days after immunization, T cells were isolated from skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD4 and anti-CD8 beads. Figure 8B: The absolute numbers of Teff or TEM cells among total CD3+ viable cells in skin dLNs per mouse were assessed by flow cytometry. Each panel is representative of five replicates. [Figure 8C](Figure 8C) A series of plots and graphs showing that superactive stimulation enhances the generation of memory T cells and augments antigen-specific IFNγ effector responses in an NLRP3-dependent manner. C57BL / 6 mice were injected subcutaneously (sc) into the right flank with endofit-OVA alone, with LPS, with PGPC, or with LPS plus OxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA). Seven days after immunization, T cells were isolated from skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD4 and anti-CD8 beads. (Figure 8D) Sorting strategies and post-sort purity are shown for CD4+ and CD8+ T cell subsets. Each panel is representative of five replicates. [Figure 8D] (Figure 8D) A series of plots and graphs showing that superactive stimulation enhances the generation of memory T cells and antigen-specific IFNγ effector responses in an NLRP3-dependent manner. C57BL / 6 mice were injected subcutaneously (sc) with endofit-OVA alone, with LPS, with PGPC, or with LPS plus OxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA). Seven days after immunization, T cells were isolated from the skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD4 and anti-CD8 beads. (Figure 8D) CD4+ and CD8+ T cells were sorted from the dLNs 7 days after immunization and then cultured with BMDCs loaded with or without serial dilutions of OVA protein starting at 1000 μg / ml. IFNγ cytokine secretion was measured by ELISA. Means and SD of four replicates are shown. Each panel is representative of five replicates. [Figure 8E](Figure 8E) A series of plots and graphs showing that superactive stimulation enhances the generation of memory T cells and antigen-specific IFNγ effector responses in an NLRP3-dependent manner. C57BL / 6 mice were injected subcutaneously (sc) into the right flank with endofit-OVA alone, with LPS, with PGPC, or with LPS plus OxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA). Seven days after immunization, T cells were isolated from the skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD4 and anti-CD8 beads. (Figure 8E) CD8+ T cells were sorted from the dLNs 7 days after immunization and then treated with PMA plus ionomycin for 5 h or cocultured with B16OVA cells (target cells) at a 1:3 (effector:target) ratio. Gating strategy for identifying the percentage of CD107a+ cells among live CD8+ T cells by flow cytometry. Each panel is representative of five replicates. [Figure 9A] Figure 9A and Figure 9B are a series of graphs showing that superactivated DCs induce inflammasome-dependent antitumor immunity. C57BL / 6 mice were subcutaneously (sc) injected with PBS (unimmunized), B16OVA cell lysate alone (none), or with LPS, or B16OVA lysate plus LPS and OxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA), into the right flank. Fifteen days after immunization, mice were subcutaneously challenged with 3 x 10 live B16OVA cells in the upper left back. Tumor-free mice were rechallenged subcutaneously 150 days later with 5 x 10 live B16OVA cells in the back. Figure 9A: Percentage of tumor-free mice 300 days after tumor inoculation (n = 8–15 mice per group). [Figure 9B]Figure 9A and Figure 9B are a series of graphs showing that superactivated DCs induce inflammasome-dependent antitumor immunity. C57BL / 6 mice were subcutaneously (sc) injected into the right flank with PBS (unimmunized), B16OVA cell lysate alone (none), or with LPS, or B16OVA lysate plus LPS and OxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA). 15 days after immunization, mice were subcutaneously challenged with 3 x 10 live B16OVA cells in the upper left back. 150 days later, tumor-free mice were rechallenged subcutaneously with 5 x 10 live B16OVA cells in the back. (Figure 9B) C57BL / 6 mice were injected subcutaneously (sc) into the right flank with PBS (unimmunized), B16-F10 cell lysate alone (none), or with LPS, or B16-F10 lysate plus LPS and PGPC, all emulsified in incomplete Freud's adjuvant (IFA). 15 days after immunization, mice were challenged subcutaneously with 3 x 10 live B16-F10 cells in the upper left back. Survival rates are shown in the left panel, and the percentage of tumor-free mice 70 days after tumor inoculation is shown in the right panel (n = 5 mice per group). [Figure 9C] (C) A series of graphs showing that superactivated DCs induce inflammasome-dependent antitumor immunity. (D) C57BL / 6 mice were left untreated (unimmunized) or immunized subcutaneously in the right flank with B16OVA tumor lysate alone, with MPLA, or with B16OVA lysate and MPLA + PGPC, with or without a neutralizing anti-mouse IL-1β antibody. Fifteen days after immunization, mice were challenged subcutaneously in the left upper back with 3 x 10 live B16OVA cells. The percentage of tumor-free mice 150 days after tumor inoculation is shown in the right panel (n = 3-4 mice per group). [Figure 9D]Figure 9D: C57BL / 6 mice were left untreated (unimmunized) or subcutaneously immunized in the right flank with MC38OVA lysate and MPLA, or with MC38OVA lysate and MPLA+PGPC, without intravenous injection of anti-mouse IL-1β antibody 2 days prior to immunization and continuing for 5 days. Alternatively, mice were immunized with OVA protein and MPLA+PGPC. 14 days later, mice were subcutaneously inoculated with 5x10 live MC38OVA cells in the upper left back. 50 days later, tumor-free mice were re-challenged subcutaneously with 1x10 MC38OVA cells in the back. The percentage of tumor-free mice 150 days after tumor inoculation is shown (n=5 mice per group). [Figure 9E]

[0033] Figure 9E: C57BL / 6 mice were left untreated (unimmunized) or subcutaneously (sc) immunized in the right flank with B16OVA lysate (WTL) + LPS and PGPC emulsified in IFA, or with B16OVA WTL + LPS and alum. 15 days after immunization, mice were challenged subcutaneously with 3 x 10 live B16OVA cells in the upper left back. Survival was monitored every 2 days (n = 5 mice per group). [Figure 9F] (Figure 9F) is a series of graphs showing that superactivated DCs induce inflammasome-dependent antitumor immunity. C57BL / 6 mice were subcutaneously immunized in the right flank with LPS and PGPC emulsified in IFA without any antigen. 15 days later, mice were challenged subcutaneously in the upper left back with either 3 x 10 live B16OVA cells, 3 x 10 live B16-F10 cells, or 5 x 10 live MC38OVA cells. Survival was monitored every 2 days (n = 5 mice per group). [Figure 10A]Figure 10A: Gating strategy for identifying the absolute numbers of CD8+ T cells and CD69+CD103+ resident memory CD8+ T cells measured by flow cytometry at the immunization or tumor injection site of survivor mice (from Figure 3A) previously immunized with B16OVA WTL and LPS+PGPC. Each panel is representative of four replicates. [Figure 10B] (B) Plots showing that superactivated DCs induce inflammasome-dependent antitumor immunity. (C) Gating strategy for determining the absolute number of total CD8+ T cells SIINFEKL+ (SEQ ID NO: 1) among live CD45+ cells in skin inguinal adipose tissue from survivor mice (from FIG. 3A) or age-matched, unimmunized tumor-bearing mice previously immunized with B16OVA WTL and LPS+PGPC. Each panel is representative of five replicates. [Figure 10C] Figure 10C: Gating strategy to identify the absolute numbers of CD8+ T cells and CD69+CD103+ T-resident memory CD8+ T cells in the skin and inguinal adipose tissue of survivor mice (from Figure 3A) previously immunized with B16OVA WTL and LPS+PGPC compared with age-matched, unimmunized tumor-bearing mice (each panel is representative of five replicates). [Figure 11A] Figure 11A shows a series of graphs and diagrams showing that superactivated DCs induce inflammasome-dependent antitumor immunity. C57BL / 6 mice were left untreated (unimmunized) or subcutaneously immunized in the right flank with MC38OVA lysate and MPLA, or with MC38OVA lysate and MPLA+PGPC, without intravenous injection of anti-mouse IL-1β antibody starting 2 days before immunization and continuing for 5 days. Alternatively, mice were immunized with OVA protein and MPLA+PGPC. After 15 days, each group was randomly divided into two sister cohorts. Figure 11A: Schematic representation of the experimental model. [Figure 11B]Figure 11B shows a series of graphs and diagrams demonstrating that superactivated DCs induce inflammasome-dependent antitumor immunity. C57BL / 6 mice were either left untreated (unimmunized) or subcutaneously immunized in the right flank with MC38OVA lysate and MPLA, or with MC38OVA lysate and MPLA+PGPC, without intravenous injection of anti-mouse IL-1β antibody starting 2 days before immunization and continuing for 5 days. Alternatively, mice were immunized with OVA protein and MPLA+PGPC. After 15 days, each group was randomly divided into two sister cohorts. Figures 11B-11E: In one cohort of mice, the skin-draining lymph nodes (dLNs) were dissected. Figure 11B: Absolute numbers of total CD8+ T cells (left panel) and SIINFEKL+ (SEQ ID NO: 1) CD8+ T cells (right panel) in the dLNs of immunized mice. The mean and SD of five mice are shown, and each panel is representative of two independent experiments. *P<0.05;**P<0.01;***P<0.005. [Figure 11C] Figure 11B-11E: Skin-draining lymph nodes (dLNs) were dissected from one cohort of mice. Figure 11C-11D: CD8+ T cells were enriched from the dLNs using anti-CD8 magnetic beads, followed by sorting of live CD3+CD8+ cells. Figure 11C: CD8+ T cells were cultured with MC38OVA cells (target cells) for 5 h. Cytotoxic CD8+ T cell responses were monitored by flow cytometry using a CD107a degranulation assay. The mean and SD of five mice are shown, and each panel is representative of two independent experiments. *P<0.05; **P<0.01; ***P<0.005. [Figure 11D]Figure 11B-11E: Superactivated DCs induce inflammasome-dependent antitumor immunity. C57BL / 6 mice were either left untreated (unimmunized) or subcutaneously immunized in the right flank with MC38OVA lysate and MPLA or with MC38OVA lysate and MPLA+PGPC without intravenous injection of anti-mouse IL-1β antibody for 5 days starting 2 days before immunization. Alternatively, mice were immunized with OVA protein and MPLA+PGPC. After 15 days, each group was randomly divided into two sister cohorts. Figures 11B-11E: Skin-draining lymph nodes (dLNs) were dissected from one cohort of mice. Figures 11C-11D: CD8+ T cells were enriched from the dLNs using anti-CD8 magnetic beads, followed by sorting of live CD3+CD8+ cells. (Figure 11D) CD8+ T cells were cocultured with naive BMDCs loaded (or not) with serial dilutions of OVA protein starting at 1000 μg / ml for 4 days. IFNγ release was monitored by ELISA. The mean and SD of five mice are shown, and each panel is representative of two independent experiments. *P<0.05; **P<0.01; ***P<0.005. [Figure 11E] Figure 11B-11E: A series of graphs and diagrams showing that superactivated DCs induce inflammasome-dependent antitumor immunity. C57BL / 6 mice were left untreated (unimmunized) or subcutaneously immunized in the right flank with MC38OVA lysate and MPLA, or with MC38OVA lysate and MPLA+PGPC, without intravenous injection of anti-mouse IL-1β antibody starting 2 days before immunization and continuing for 5 days. Alternatively, mice were immunized with OVA protein and MPLA+PGPC. After 15 days, each group was randomly divided into two sister cohorts. Figures 11B-11E: Skin-draining lymph nodes (dLNs) were dissected from one cohort of mice. The mean and SD of five mice are shown, and each panel is representative of two independent experiments. *P<0.05; **P<0.01; ***P<0.005. [Figure 12A]Figure 12A-B shows a series of graphs and diagrams showing that superactive cDC1s control tumor rejection induced by superactive-based immunotherapy. (Figures 12A-B) C57BL / 6 WT or Batf3- / - mice were inoculated subcutaneously (sc) with 3 x 10 live B16OVA cells in the left dorsal region. Ten days later, mice were left untreated (naive) or immunized subcutaneously in the right flank with syngeneic B16OVA tumor lysate plus LPS and PGPC. As shown in the diagram, mice received two boost injections on days 17 and 24 after tumor inoculation. Tumors were allowed to reach a diameter of 20 mm. (Figure 12A) Survival rates are shown (n = 5 mice per group). [Figure 12B] Figure 12A-B shows a series of graphs and diagrams showing that superactive cDC1s control tumor rejection induced by superactive-based immunotherapy. (Figures 12A-B) C57BL / 6 WT or Batf3- / - mice were subcutaneously (sc) inoculated with 3 x 10 live B16OVA cells in the left dorsal region. Ten days later, mice were left untreated (unimmunized) or immunized subcutaneously in the right flank with syngeneic B16OVA tumor lysate plus LPS and PGPC. As shown in the diagram, mice received two boost injections on days 17 and 24 after tumor inoculation. Tumors were allowed to reach a diameter of 20 mm. (Figure 12B) Fifteen days after tumor inoculation, skin-draining lymph nodes (dLNs), tumors, and spleen tissues were dissected from immunized mice. The percentages of antigen-specific CD8+ and CD4+ T cells were measured using SIINFEKL and AAHAEINEA tetramer staining, respectively (n=5 mice per group). [Figure 13A] Figure 13 shows results from mass spectrometry of synthetic lipids. Mass spectrometry of non-oxidized PAPC (Figure 13A). [Figure 13B] Figure 13 shows results from mass spectrometry of synthetic lipids. Mass spectrometry of oxPAPC (Figure 13B). [Figure 13C] Figure 13 shows results from mass spectrometry of synthetic lipids. Mass spectrometry of PEIPC-enriched oxPAPC (Figure 13C). [Figure 13D]Figure 13 shows results from mass spectrometry of synthetic lipids: Mass spectrometry of non-oxidized PAPC (Figure 13A), oxPAPC (Figure 13B), PEIPC-enriched oxPAPC (Figure 13C), and biotin-labeled oxPAPC (Figure 13D). [Figure 14A] Oxidized phospholipids induce hyperactive cDC1 and cDC2 cells that exhibit a hypermigratory phenotype. (A) Wild-type, NLRP3- / -, or Casp1 / 11- / - BMDCs generated using FLT3L were left untreated (none) or treated with LPS alone, alum alone, or PGPC alone for 24 h. BMDCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. IL-1β and TNFα release was monitored by ELISA. Cell mortality was measured by LDH release into the cell supernatant. Means and SD from three replicates are shown; data are representative of at least three independent experiments. [Figure 14B] Oxidized phospholipids induce hyperactive cDC1 and cDC2 cells that exhibit a hypermigratory phenotype. (B) Wild-type BMDCs generated using FLT3L were sorted as cDC1 or cDC2 cells and then treated with the indicated stimuli as in A. IL-1β and TNFα release was monitored by ELISA. Cell mortality was measured by LDH release into the cell supernatant. Mean and SD from three independent experiments performed in two different laboratories. [Figure 15A]Superactivated DCs induce significant CTL responses and durable antitumor immunity dependent on CCR7 expression and inflammasome activation. (A-B) Wild-type BMDCs generated using FLT3L were left untreated (DC naive) or treated with LPS alone (DC activated) for 18 h. Alternatively, BMDCs were primed with LPS for 3 h and then treated with PGPCs (DC superactivated) or alum (DC pyroptotic) for 15 h. Alternatively, BMDCs derived from NLRP3- / - or CCR7- / - mice were primed with LPS for 3 h and then treated with PGPCs for 15 h. 1.10e6 BMDCs were incubated with OVA protein for 1 h before subcutaneous injection into wild-type mice. Injection of BMDCs without OVA protein served as a control. Seven days after BMDC injection, skin-draining lymph nodes were dissected and stained with OVA peptide tetramer antibody, anti-CD45, anti-CD3, anti-CD8a, and anti-CD4 using a live-dead violet kit. (A) The percentage of SIINFEKL+CD8+ T cells (upper panel) and the percentage of live AAHAEINEA+CD4+ T cells were measured by flow cytometry. [Figure 15B]Superactivated DCs induce significant CTL responses and durable antitumor immunity dependent on CCR7 expression and inflammasome activation. (A-B) Wild-type BMDCs generated using FLT3L were left untreated (DC naive) or treated with LPS alone (DC activated) for 18 h. Alternatively, BMDCs were primed with LPS for 3 h and then treated with PGPCs (DC superactivated) or alum (DC pyroptotic) for 15 h. Alternatively, BMDCs derived from NLRP3- / - or CCR7- / - mice were primed with LPS for 3 h and then treated with PGPCs for 15 h. 1.10e6 BMDCs were incubated with OVA protein for 1 h before subcutaneous injection into wild-type mice. Injection of BMDCs without OVA protein served as a control. Seven days after BMDC injection, skin-draining lymph nodes were dissected and stained with OVA peptide tetramer antibody, anti-CD45, anti-CD3, anti-CD8a, and anti-CD4 using a live-dead violet kit. (B) The absolute numbers of SIINFEKL+CD8+ T cells (upper panel) and live AAHAEINEA+CD4+ T cells were measured by flow cytometry using CounterBright beads. [Figure 16A] Superactive stimulation induces significant CTL responses in an inflammasome-dependent manner. (A) C57BL / 6 mice were subcutaneously injected with OVA alone, together with LPS, with PGPC, or with LPS plus oxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA), into the right flank. Seven or 40 days after immunization, T cells were isolated from skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD8 beads. (A) Percentages of CD44-low CD62L-low effector T cells (Teff), CD44-high CD62L-low effector memory T cells (TEM), and CD44-high CD62L-high central memory T cells (TCM) are shown among live CD3+CD8+ cells. [Figure 16B](B) Superactive stimulation induces significant CTL responses in an inflammasome-dependent manner. CD8+ T cells were sorted from the dLN 7 days after immunization and then treated with PMA and ionomycin for 5 h or cocultured with B16OVA cells (target cells) at a 1:3 (effector:target) ratio. The percentage of CD107a+ cells among live CD8+ T cells was monitored using flow cytometry to assess CD8+ T cell degranulation. Mean and SD for 5-10 mice are shown. [Figure 16C] (C) Superactive stimulation induces significant CTL responses in an inflammasome-dependent manner. (D) Mice were subcutaneously injected with OVA alone, together with LPS, or together with LPS plus oxPAPC or PGPC, all emulsified in IFA, or with LPS plus alum. Alternatively, NLRP3- / - mice were injected with OVA emulsified in IFA together with LPS plus PGPC. Seven days after immunization, CD8+ T cells were sorted from the cutaneous dLNs of immunized mice and cocultured with OVA-loaded (or unloaded) BMDCs at a ratio of 1:10 (DC:T cells) for 7 days. The percentage of SIINFEKL+IFNγ+ among live CD8+ T cells was measured using OVA peptide tetramer staining followed by intracellular IFNγ staining. [Figure 16D]Superactive stimulation induces significant CTL responses in an inflammasome-dependent manner. (D-E) CD45.1 mice were irradiated and then bone marrow reconstituted with either WT, NLRP3- / -, Casp1 / 11- / -, or CCR7- / - mice (5:1 ratio), all on a CD45.2 C57BL / 6 background. Six weeks after reconstitution, chimeric mice were injected with tamoxifen every other day for 7 days. Chimeric mice were then immunized subcutaneously in the right flank with OVA and LPS plus PGPC emulsified in IFA. Seven days after immunization, CD8+ T cells were isolated from the skin-draining lymph nodes (dLN) or spleen by magnetic enrichment using anti-CD8 beads. (D) The percentages of Teff, TEM, TCM, and naive T cells in the skin dLN were measured by flow cytometry. [Figure 16E] Superactive stimulation induces significant CTL responses in an inflammasome-dependent manner. (D-E) CD45.1 mice were irradiated and then bone marrow reconstituted with either WT, NLRP3- / -, Casp1 / 11- / -, or CCR7- / - mice (5:1 ratio), all on a CD45.2 C57BL / 6 background. Six weeks after reconstitution, chimeric mice were injected with tamoxifen every other day for 7 days. Chimeric mice were then subcutaneously immunized in the right flank with OVA and LPS plus PGPC emulsified in IFA. Seven days after immunization, CD8+ T cells were isolated from the skin-draining lymph nodes (dLNs) or spleens by magnetic enrichment using anti-CD8 beads. (E) The percentage of SIINFEKL+ among live CD8+ T cells in the dLN (left panel) or spleen (right panel) was measured by flow cytometry using OVA peptide tetramer staining. Total CD8+ T cells were sorted from the dLN and cocultured with naive BMDCs (with or without OVA loading) at a ratio of 1:10 (DC:T cells) for 7 days. [Figure 17A]Immunization with superactive stimuli eradicates tumors with immunogenicity ranging from highly immunogenic (hot) to icy tumors. (A) C57BL / 6 mice were inoculated subcutaneously with 5 × 10 live MC38OVA cells in the upper left dorsum. 14 days later, mice were left untreated (naive) or subcutaneously injected in the right flank with syngeneic MC38OVA whole tumor lysate (WTL) plus LPS and PGPC, with or without intravenous (iv) injection of neutralizing anti-IL-1β antibody or intraperitoneal injection of anti-CD4 or anti-CD8a antibodies. Mice received two boost injections with WTL and LPS plus PGPC on days 37 and 55 after tumor inoculation. Tumors were allowed to reach 20 mm in diameter. Survival rates are shown (n = 10 mice per group). [Figure 17B] Immunization with superactive stimuli eradicates tumors with immunogenicity ranging from highly immunogenic (hot) to icy (icy) tumors. (B) C57BL / 6 mice were inoculated subcutaneously with 3 × 10 live B16OVA cells in the upper left dorsum. Ten days later, mice were left untreated (naive) or intraperitoneally injected with anti-PD1 antibody. Alternatively, mice were injected subcutaneously in the right flank with syngeneic B16OVA WTL + LPS and PGPC, with or without intravenous (i.v.) injections of neutralizing anti-IL-1β antibodies or intraperitoneal injections of anti-CD4 or anti-CD8a antibodies. Mice received two boost injections with B16OVA WTL + LPS and PGPC on days 17 and 24 after tumor inoculation. Survival rates are shown (n = 10 mice per group). [Figure 17C]Immunization with superactive stimuli eradicates tumors with immunogenicity ranging from highly immunogenic (hot) to icy (icy) tumors. (C) C57BL / 6 mice were inoculated subcutaneously with 3 × 10 live B16-F10 cells in the upper left dorsal region. Seven days later, mice were left untreated (naive) or injected intraperitoneally with anti-PD1 antibody. Alternatively, mice were immunized subcutaneously in the right flank with syngeneic B16-F10 wild-type T cells plus LPS and PGPC, with or without intravenous (i.v.) injections of neutralizing anti-IL-1β antibodies or intraperitoneal injections of anti-CD4 or anti-CD8a antibodies. Mice received two booster injections on days 14 and 21 after tumor inoculation. Survival rates are shown (n = 10 mice per group). [Figure 17D] Immunization with superactive stimuli eradicates tumors with immunogenicity ranging from hot to icy tumors. (D) BALB / c WT mice were inoculated subcutaneously with 3 × 10 live CT26 cells in the left dorsal region. Seven days later, mice were left untreated (naive) or intraperitoneally injected with anti-PD1 antibody. Alternatively, mice were injected subcutaneously in the right flank with syngeneic CT26 WT plus LPS and PGPC, with or without intravenous (i.v.) injections of neutralizing anti-IL-1β antibodies or intraperitoneal injections of anti-CD4 or anti-CD8a antibodies. Mice received two booster injections on days 14 and 21 after tumor inoculation. Survival rates are shown (n = 10 mice per group). [Figure 18A] Figure 1: Superactive cDC1s can use multiple antigen sources to stimulate T cell-mediated antitumor immunity. (A) Zbtb46DTR mice were subcutaneously injected with B16OVA cells. Mice were injected with diphtheria toxin (DTx) every other day for four consecutive injections, or with PBS. Seven days after tumor injection, all mice were immunized with B16OVA WTL + LPS and PGPC followed by two boost injections. Mouse survival is shown (n = 10 mice per group). [Figure 18B](B) Superactive cDC1s can utilize multiple antigen sources to stimulate T cell-mediated antitumor immunity. (C) CD45.1 mice were irradiated and then reconstituted with mixed BM derived from Zbtb46DTR mice plus either WT, Nlrp3- / -, Casp1 / 11- / -, or Ccr7- / - mice. Six weeks after reconstitution, chimeric mice were subcutaneously injected with B16OVA cells, and all mice received DTx injections three times a week for a total of 12 consecutive injections. Seven days after tumor inoculation, chimeric mice were immunized with B16OVA WTL and LPS+PGPC, followed by two boost injections. Mouse survival rates are shown (n = 5 mice per group). [Figure 18C] Superactive cDC1s can use multiple antigen sources to stimulate T cell-mediated antitumor immunity. (C-D) WT or Batf3- / - mice were subcutaneously injected with B16OVA cells. Seven days after tumor inoculation, mice were either left untreated or immunized with B16OVA WTL and LPS+PGPC followed by two boost injections. (C) Mouse survival rate (n = 10 mice per group). [Figure 18D] Superactive cDC1s can use multiple antigen sources to stimulate T cell-mediated antitumor immunity. (C-D) WT or Batf3- / - mice were subcutaneously injected with B16OVA cells. Seven days after tumor inoculation, mice were either left untreated or WT and Batf3- / - mice were immunized with B16OVA WTL and LPS+PGPC followed by two boost injections. (D) Twenty-one days after tumor inoculation, the percentage of OVA-specific CD8+ and CD4+ T cells was assessed using tetramer staining (n = 5 mice per group). [Figure 18E]Superactivated cDC1s can use multiple antigen sources to stimulate T cell-mediated antitumor immunity. (E-F) Batf3- / - mice were subcutaneously injected with B16OVA cells into the right flank. Seven days after tumor inoculation, mice were either left untreated (no cDC1 injection) or subcutaneously injected with FLT3-derived naive cDC1s, activated cDC1s treated with LPS, or superactivated cDC1s pretreated with LPS and PGPC into the left flank. All cDC1s were loaded with B16OVA WTL 1 hour before injection. (E) Mouse survival rate (n = 5 mice per group). [Figure 18F] Superactivated cDC1s can use multiple antigen sources to stimulate T cell-mediated antitumor immunity. (E-F) Batf3- / - mice were subcutaneously injected with B16OVA cells into the right flank. Seven days after tumor inoculation, mice were either left untreated (no cDC1 injection) or subcutaneously injected with FLT3-derived naive cDC1s, activated cDC1s treated with LPS, or superactivated cDC1s pretreated with LPS and PGPC into the left flank. All cDC1s were loaded with B16OVA WTL 1 hour before injection. (E) Mouse survival rates are shown (n = 5 mice per group). (F) Twenty-one days after tumor inoculation, OVA-specific CD8+ and CD4+ T cells were assessed using tetramer staining (n = 5 mice per group). [Figure 19A] Oxidized phospholipids induce inflammasome-dependent IL-1β secretion and promote a hypermigratory DC phenotype by cDC1 and cDC2 cells. (A) Wild-type BMDCs generated using FLT3L were left untreated (none) or treated with CpG1806 or PGPC alone for 24 h, or BMDCs were primed with CpG1806 for 3 h and then treated with the indicated stimuli for 21 h. IL-1β and TNFα release was monitored by ELISA. Cell mortality was measured by LDH release into the cell supernatant. Means and SD from three replicates are shown, and data are representative of at least three independent experiments. [Figure 19B](B) Gating strategy for isolating FLT3L-producing BMDC-derived or spleen-derived cDC1 or cDC2 from wild-type mice. The purity after sorting is shown for spleen cDCs or FLT3L DCs. (C) Oxidized phospholipids induce inflammasome-dependent IL-1β secretion by cDC1 and cDC2 cells and promote a hypermigratory DC phenotype. (D) Gating strategy for isolating FLT3L-producing or spleen-derived cDC1 or cDC2 from wild-type mice. The purity after sorting is shown for spleen cDCs or FLT3L DCs. [Figure 19C] Oxidized phospholipids induce inflammasome-dependent IL-1β secretion and promote a hypermigratory DC phenotype by cDC1 and cDC2 cells. (C) Splenic cDC1 or cDC2 cells were left untreated (none) or treated with LPS alone, alum alone, oxPAPC alone, or PGPC alone for 18 h, or BMDCs were primed with LPS for 3 h and then treated with the indicated stimuli for 15 h. IL-1β and TNFα release was monitored by ELISA. Cell mortality was measured by LDH release into the cell supernatant. Mean and SD from three independent experiments performed in two different laboratories. [Figure 20A]Superactivated DCs induce significant CTL responses and durable antitumor immunity dependent on CCR7 expression and inflammasome activation. Wild-type BMDCs generated using FLT3L were left untreated (DC naive) or treated with LPS alone (DC activated) for 18 h. Alternatively, BMDCs were primed with LPS for 3 h before PGPCs (DC superactivated) or alum (DC pyroptotic) were added to the culture medium for 15 h. Alternatively, BMDCs derived from NLRP3- / - or CCR7- / - mice were primed with LPS for 3 h before PGPCs were added to the culture medium for 15 h. BMDCs were washed and then incubated with FITC-labeled OVA for 45 min or non-fluorescent OVA protein for 2 h. (A) OVA peptide presentation on MHC-I was monitored using a PE-conjugated antibody against H-2Kb bound to the OVA peptide SIINFEKL. Data are expressed as the frequency of SIINFEKL-associated DCs among live CD11c+ cells. The mean and SD from three replicates are shown and data are representative of three independent experiments. [Figure 20B](B) Superactivated DCs induce significant CTL responses and durable antitumor immunity dependent on CCR7 expression and inflammasome activation. Wild-type BMDCs generated using FLT3L were left untreated (DC naive) or treated with LPS alone (DC activated) for 18 h. Alternatively, BMDCs were primed with LPS for 3 h before PGPCs (DC superactivated) or alum (DC pyroptotic) were added to the culture medium for 15 h. Alternatively, BMDCs derived from NLRP3- / - or CCR7- / - mice were primed with LPS for 3 h before PGPCs were added to the culture medium for 15 h. BMDCs were washed and then incubated with FITC-labeled OVA for 45 min or non-fluorescent OVA protein for 2 h. (C) Wild-type, NLRP3- / -, or CCR7- / - BMDCs generated using FLT3L were stimulated as described above. BMDCs were washed and then stained with a live-dead violet kit, CD11c, and CD40. The mean fluorescence intensity (MFI) of surface CD40 (among live CD11c+ cells) was measured by flow cytometry. [Figure 20C]Superactivated DCs induce significant CTL responses and durable antitumor immunity dependent on CCR7 expression and inflammasome activation. Wild-type BMDCs generated using FLT3L were left untreated (DC naive) or treated with LPS alone (DC activated) for 18 h. Alternatively, BMDCs were primed with LPS for 3 h and then cultured with PGPCs (DC superactivated) or alum (DC pyroptotic) for 15 h. Alternatively, BMDCs derived from NLRP3- / - or CCR7- / - mice were primed with LPS for 3 h and then cultured with PGPCs for 15 h. BMDCs were washed and then incubated with FITC-labeled OVA for 45 min or with non-fluorescent OVA protein for 2 h. (C) CCR7- / - BMDCs generated using FLT3L were left untreated (none) or treated with LPS alone, alum alone, or PGPC alone for 24 h. Alternatively, BMDCs were primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. IL-1β and TNFα release was monitored by ELISA. Cell mortality was measured by LDH release into the cell supernatant. Means and SD from three replicates are shown, and data are representative of at least three independent experiments. [Figure 21A] Superactivating stimuli enhance the generation of memory T cells and augment antigen-specific IFNγ effector responses in an inflammasome-dependent manner. C57BL / 6 mice were subcutaneously injected with OVA alone, together with LPS, with PGPC, or with LPS plus oxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA), into the right flank. Seven days after immunization, T cells were isolated from skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD8 beads. (A) Gating strategy for identifying the percentages of CD44-low CD62L-low effector T cells (Teff), CD44-high CD62L-low effector memory T cells (Tem), and CD44-high CD62L-high central memory T cells (Tcm). Each panel is representative of five mice. *P<0.05; **P<0.01. [Figure 21B] (B) Superactivating stimuli enhance the generation of memory T cells and augment antigen-specific IFNγ effector responses in an inflammasome-dependent manner. C57BL / 6 mice were subcutaneously injected with OVA alone, together with LPS, together with PGPC, or together with LPS plus oxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA), into the right flank. Seven days after immunization, T cells were isolated from skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD8 beads. (C) The absolute numbers of Teff or TEM cells among total CD3+ viable cells in skin dLNs per mouse were assessed by flow cytometry. Each panel is representative of five mice. *P<0.05; **P<0.01. [Figure 21C] (C) Superactivating stimulation enhances the generation of memory T cells and antigen-specific IFNγ effector responses in an inflammasome-dependent manner. OVA alone, together with LPS, with PGPC, or with LPS plus oxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA), was injected subcutaneously into the right flank of C57BL / 6 mice. Seven days after immunization, T cells were isolated from skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD8 beads. (C) Seven days after immunization, CD8+ T cells were sorted from dLNs and then cultured with naive BMDCs loaded with or without serial dilutions of OVA protein starting at 1000 μg / ml. IFNγ cytokine secretion was measured by ELISA. The mean and SD of five mice are shown. Each panel is representative of five mice. *P<0.05; **P<0.01. [Figure 21D](D) Superactivating stimulation enhances the generation of memory T cells and antigen-specific IFNγ effector responses in an inflammasome-dependent manner. C57BL / 6 mice were subcutaneously injected with OVA alone, together with LPS, with PGPC, or with LPS plus oxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA), into the right flank. Seven days after immunization, T cells were isolated from the skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD8 beads. (E) CD8+ T cells were sorted from the dLNs 7 days after immunization and then treated with PMA plus ionomycin for 5 h or cocultured with B16OVA cells (target cells) at a 1:3 (effector:target) ratio. Gating strategy for determining the percentage of CD107a+ cells among live CD8+ T cells by flow cytometry. Each panel is representative of five mice. *P<0.05; **P<0.01. [Figure 22A] Superactive stimulation enhances the generation of memory T cells and antigen-specific IFNγ effector responses in an inflammasome-dependent manner. (A-B) CD45.1 mice were irradiated and then bone marrow reconstituted with either WT, NLRP3- / -, Casp1 / 11- / -, or CCR7- / - mice (5:1 ratio), all on a CD45.2 C57BL / 6 background. Six weeks after reconstitution, chimeric mice were injected with tamoxifen every other day for 7 days. Chimeric mice were then subcutaneously immunized in the right flank with OVA and LPS plus PGPC emulsified in IFA. Seven days after immunization, CD8+ T cells were isolated from the skin-draining lymph nodes (dLN) or spleen by magnetic enrichment using anti-CD8 beads. (A) The percentages of Teff, TEM, TCM, and naive T cells in the skin dLN were measured by flow cytometry. Each panel is representative of 5 mice. [Figure 22B]Superactive stimulation enhances the generation of memory T cells and antigen-specific IFNγ effector responses in an inflammasome-dependent manner. (A-B) CD45.1 mice were irradiated and then bone marrow reconstituted with either WT or ZBTB46DTR mice (5:1 ratio) on a CD45.2 C57BL / 6 background plus either NLRP3- / -, Casp1 / 11- / -, or CCR7- / - mice. Six weeks after reconstitution, chimeric mice were injected with tamoxifen every other day for 7 days. Chimeric mice were then subcutaneously immunized in the right flank with OVA and LPS plus PGPC emulsified in IFA. Seven days after immunization, CD8+ T cells were isolated from the skin-draining lymph nodes (dLNs) or spleens by magnetic enrichment using anti-CD8 beads. (B) The percentage of SIINFEKL+ among live CD8+ T cells in the dLN (upper panel) or spleen (lower panel) was measured by flow cytometry using OVA peptide tetramer staining. [Figure 23A] (A-B) Mice were injected subcutaneously (sc) into the right flank with PBS (unimmunized), B16OVA cell lysate alone (none), or with LPS, or B16OVA lysate plus LPS and oxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA). 15 days after immunization, mice were challenged subcutaneously with 3 × 10 live B16OVA cells in the upper left back. 150 days later, tumor-free mice were rechallenged subcutaneously with 5 × 10 live B16OVA cells in the back. (A) Tumor growth was monitored every 2 days (upper panel). For survival experiments (lower panel), tumors were allowed to reach a diameter of 20 mm (n = 8–15 mice per group). (B-C) At the end of tumor growth, tumors were harvested and dissociated to obtain single tumor cell suspensions. [Figure 23B](A-B) Mice were injected subcutaneously (sc) into the right flank with PBS (unimmunized), B16OVA cell lysate alone (none), or with LPS, or B16OVA lysate plus LPS and oxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA). 15 days after immunization, mice were challenged subcutaneously with 3 × 10 live B16OVA cells in the upper left back. 150 days later, tumor-free mice were rechallenged subcutaneously with 5 × 10 live B16OVA cells in the back. (A) Tumor growth was monitored every 2 days (upper panel). For survival experiments (lower panel), tumors were allowed to reach a diameter of 20 mm (n = 8–15 mice per group). (B-C) At the end of tumor growth, tumors were harvested and dissociated to obtain single tumor cell suspensions. (B) The percentage of tumor-infiltrating CD3+CD4+ and CD3+CD8+ T cells among enriched CD45+ viable cells was assessed by flow cytometry. [Figure 23C] (B-C) Tumors were harvested at the end of tumor growth and dissociated to obtain a single tumor cell suspension. (C) Tumor-infiltrating CD3+ T cells were sorted and then stimulated for 24 h in the presence of anti-CD3 and anti-CD28 Dynabeads. IFNγ release was measured by ELISA (lower panel) (n = 4 mice per group). [Figure 24A] (A-B) The absolute numbers of CD8+ T cells and CD69+CD103+ T-resident memory CD8+ T cells were assessed at the immunization site or tumor injection site in survivor mice and measured by flow cytometry (n=4 mice). [Figure 24B] (A-B) The absolute numbers of CD8+ T cells and CD69+CD103+ T-resident memory CD8+ T cells were assessed at the immunization site or tumor injection site in survivor mice and measured by flow cytometry (n=4 mice). [Figure 24C](C-D) Circulating memory CD8+ T cells (TCM) were isolated from the spleens of survivor mice or age-matched, tumor-naive mice, and resident memory CD8+ T cells (TRM) were isolated from the inguinal adipose tissue. (C) TCM and TRM from survivor mice were co-cultured with B16OVA, B16-F10, or CT26 tumor cells at a 1:5 (tumor cell:T cell) ratio for 5 h. Cell death mediated by cytolytic CD8+ T cells was measured by LDH release into the supernatant. [Figure 24D] (C-D) Circulating memory CD8+ T cells (TCM) were isolated from the spleens of survivor mice or age-matched, unimmunized tumor-bearing mice, and resident memory CD8+ T cells (TRM) were isolated from the cutaneous inguinal adipose tissue. (D) Mice were left untreated (no Tx) or were inoculated intravenously (iv) with 5 x 105 CD8+ TCM cells isolated from survivor mice or age-matched, unimmunized tumor-bearing mice and / or intradermally with 5 x 105 CD8+ TRM cells. Seven days later, all mice were challenged with 3 x 105 live B16OVA cells. Survival was monitored every 2 days. Tumors were allowed to reach 20 mm in diameter (n = 5 mice per group). DETAILED DESCRIPTION OF THE INVENTION

[0029] Detailed Description This disclosure is based, in part, on the discovery that stimuli that activate dendritic cells (DCs) or promote DC pyroptosis induce mixed T cell responses consisting of type I and type II T helper (Th) cells. In contrast, stimuli that superactivate DCs selectively stimulate TH1 and cytotoxic T lymphocyte (CTL) immune responses without evidence of TH2-induced immunity. The TH1-biased immunity elicited by superactivated DCs confers the unique ability of TH1 cells to mediate long-term protective antitumor immunity, even when complex antigen sources (e.g., tumor cell lysates) are used. Conventional DC activation states or stimuli that promote pyroptosis lack the ability to support tumor cell lysates and confer minimal protection against tumors. This novel property is essential for superactivating DCs and depends on IL-1β and inflammasome components. The resulting tumor-specific T cells can be transferred into recipient mice and confer complete protection from subsequent challenge. Superactivating stimuli induce protective immunity against tumors that are sensitive or resistant to PD-1 checkpoint inhibitors. Together, these findings establish the physiological importance of the hyperactive state of DCs and pave the way for novel strategies for cancer immunotherapy that are independent of the nature of tumor antigens.

[0030] Thus, provided herein are methods for generating or enhancing an adaptive immune response in a subject and methods for treating cancer in a subject. The methods are useful, for example, for therapeutic and / or prophylactic cancer vaccination.

[0031] Provided herein are methods for inducing or enhancing an adaptive immune response against cancer in a subject, comprising administering to the subject effective amounts of (i) a Toll-like receptor (TLR) ligand, (ii) a non-canonical inflammasome-activating lipid, and (iii) a cancer immunogen.

[0032] Further provided herein is a method of treating cancer in a subject, comprising administering to the subject effective amounts of (i) a Toll-like receptor (TLR) ligand, (ii) a non-canonical inflammasome-activating lipid, and (iii) a cancer immunogen.

[0033] Preferably, the method described herein inhibits the growth or progression of cancer, such as tumor, or viral infection in a subject.For example, the method described herein inhibits tumor growth by at least 1%, for example, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%. In other cases, the methods described herein reduce the size of the tumor by at least 1 mm in diameter, e.g., at least 2 mm in diameter, at least 3 mm in diameter, at least 4 mm in diameter, at least 5 mm in diameter, at least 6 mm in diameter, at least 7 mm in diameter, at least 8 mm in diameter, at least 9 mm in diameter, at least 10 mm in diameter, at least 11 mm in diameter, at least 12 mm in diameter, at least 13 mm in diameter, at least 14 mm in diameter, at least 15 mm in diameter, at least 20 mm in diameter, at least 25 mm in diameter, at least 30 mm in diameter, at least 40 mm in diameter, at least 50 mm in diameter, or more. In some cases, the subject has had a majority of the tumor resected.

[0034] Other aspects are described below. definition The articles "a" and "an" are used herein in reference to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. Thus, for example, reference to "a cell" includes a plurality of such cells. Furthermore, the terms "including," "includes," "having," "has," "with," or variations thereof, when used in either the detailed description and / or claims, are intended to be inclusive in a manner similar to the term "comprising."

[0035] As used herein, "about," when referring to a measurable value, e.g., an amount, length of time, etc., is meant to include variations of + / - 20%, + / - 10%, + / - 5%, + / - 1%, or + / - 0.1% from the specified value, provided such variations are appropriate for carrying out the disclosed method. Alternatively, the term can mean within a five-fold and two-fold order of magnitude of the value, particularly with respect to biological systems or processes. When particular values ​​are described in the application and claims, unless otherwise specified, the term "about" is intended to mean within an acceptable range of error for the particular value.

[0036] "Cancer," as used herein, as known in the art, means a disease, condition, trait, genotype, or phenotype characterized by uncontrolled cell growth or replication, and includes, for example, colorectal cancer, as well as leukemias such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia, AIDS-related cancers such as Kaposi's sarcoma; breast cancer; bone cancers such as osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, giant cell tumor, adamantinoma, and chordoma; brain cancers such as meningioma, glioblastoma, low-grade astrocytoma, oligodendrocytoma, pituitary tumor, schwannoma, and metastatic brain cancer; various lymphomas such as mantle cell lymphoma, leukemia, and lymphomas of the thyroid gland. "Metastatic" means cancers of the head and neck, including lymphoma, non-Hodgkin's lymphoma, adenoma, squamous cell carcinoma, laryngeal cancer, gallbladder and bile duct cancer, retinal cancers such as retinoblastoma, esophageal cancer, gastric cancer, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, lung cancer, bladder cancer, prostate cancer, lung cancer (including non-small cell lung cancer), pancreatic cancer, sarcoma, Wilms' tumor, cervical cancer, head and neck cancer, skin cancer, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adenocarcinoma, parotid gland cancer, endometrial sarcoma, multidrug resistant cancer; and proliferative diseases and conditions, such as tumor angiogenesis-associated angiogenesis, macular degeneration (e.g., wet / dry AMD), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration, and other proliferative diseases and conditions.

[0037] As used herein, the term "pattern recognition receptor ligand" refers to a molecular compound that activates one or more members of the Toll-like receptor (TLR) family, the RIG-I-like receptor (RLR) family, the nucleotide-binding leucine-rich repeat-containing (NLR) family, cGAS, STING, or AIM2-like receptor (ALR). Specific examples of pattern recognition receptor ligands include natural or synthetic bacterial lipopolysaccharides (LPS), natural or synthetic bacterial lipoproteins, natural or synthetic DNA or RNA sequences, natural or synthetic cyclic dinucleotides, and natural or synthetic carbohydrates. Cyclic dinucleotides include cyclic GMP-AMP (cGAMP), cyclic diAMP, and cyclic diGMP.

[0038] As used herein, the term "cancer therapy" refers to a therapy useful for treating cancer. Examples of anti-cancer therapeutic agents include, for example, surgical procedures, chemotherapeutic agents, immunotherapy, growth inhibitors, cytotoxic agents, agents used in radiotherapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer, such as anti-HER-2 antibodies (e.g., HERCEPTIN™), anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (TARCEVA™)), These include, but are not limited to, platelet-derived growth factor inhibitors (e.g., GLEEVEC™ (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following receptors: ErbB2, ErbB3, ErbB4, PDGFRβ, BlyS, APRIL, BCMA, or VEGF, TRAIL / Apo2, and other bioactive and organic chemical agents. Combinations thereof are also contemplated for use in the methods described herein.

[0039] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents are erlotinib (TARCEVA™, Genentech / OSI Pharm.), bortezomib (VELCADE™, Millennium), and cefotaxime (Ciprofen). Pharm.), fulvestrant (FASLODEX™, Astrazeneca), Sutent (SU11248, Pfizer), letrozole (FEMARA™, Novartis), imatinib mesylate (GLEEVEC™, Novartis), PTK787 / ZK222584 (Novartis), oxaliplatin (Eloxatin™, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE™, Wyeth), lapatinib (GSK572016, GlaxoSmithKline), lonafarnib (SCH66336), sorafenib (BAY43-9006, Bayer Labs.) and gefitinib (IRESSA™, Astrazeneca), AG1478, AG1571 (SU5271; Sugen), alkylating agents such as thiotepa and CYTOXAN™ cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylameramines (me), including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine thylamelamine); acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CBI-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatin;Nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin γ1 and calicheamicin ω1 (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186; dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; as well as neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ (doxorubicin), including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, and deoxydoxorubicin ), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; florinic acid Folic acid replenishers such as aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziconazole; elfornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidammol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK™ polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triazicone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL™ paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ Cremophor-free, an albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE™ doxetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR™ gemcitabine;6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE™ vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0040] Also included in this definition of "chemotherapeutic agent" are: (i) antihormonal agents that act to regulate or inhibit hormone action on tumors (e.g., antiestrogens and selective estrogen receptor modulators (SERMs), including tamoxifen (including NOLVADEX™ (tamoxifen)), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and FARESTON™ (toremifene)); (ii) aromatase inhibitors, which inhibit the aromatase enzyme that controls estrogen production in the adrenal glands, such as 4(5)-imidazoles, aminoglutethimide, MEGASE™ (megestrol acetate), AROMASIN™ (exemestane), formestany, fadrozole, RIVISOR™ (vorozole), FEMARA™ (letrozole), and ARIMIDEX™ (anastrozole); (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, loxazosinamide, thiazolinone, thiazolinone, thiazolinone, thiazolinone, thiazolinone, thiazolinone; (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in cell proliferation disorders, such as PKC-α, Ralf, and H-Ras; (viii) ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME™ (ribozyme)) and HE R2 expression inhibitors; (ix) vaccines, e.g., gene therapy vaccines such as ALLOVECTIN™ vaccine, LEUVECTIN™ vaccine, and VAXID™ vaccine; PROLEUKIN™ rIL-2; LURTOTECAN™ topoisomerase 1 inhibitors; ABARELIX™ rmRH; (x) anti-angiogenic agents, e.g., bevacizumab (AVASTIN™, Genentech); and (xi) pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0041] The term "checkpoint inhibitor" refers to a class of drugs that inhibit CD4 + T cells and / or CD8 +"Immune checkpoint inhibitor therapy" refers to a group of molecules on the cell surface of T cells. Immune checkpoint proteins are well known in the art and include, without limitation, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, and A2aR (see, for example, WO 2012 / 177624). "Anti-immune checkpoint inhibitor therapy" refers to the use of agents that inhibit immune checkpoint inhibitors. In order to more effectively treat cancer, the inhibition of one or more immune checkpoint inhibitors can prevent or neutralize inhibitory signal transduction, thereby upregulating the immune response. Exemplary agents useful for inhibiting immune checkpoint inhibitors include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands that can bind to and / or inactivate or inhibit immune checkpoint proteins or fragments thereof; and RNA interference, antisense, nucleic acid aptamers, etc., that can downregulate the expression and / or activity of nucleic acids or fragments of immune checkpoint inhibitors. Exemplary agents that upregulate an immune response include antibodies against one or more immune checkpoint inhibitor proteins that interfere with the interaction between the proteins and their natural receptors; inactive forms of one or more immune checkpoint inhibitor proteins (e.g., dominant negative polypeptides); small molecules or peptides that interfere with the interaction between one or more immune checkpoint inhibitor proteins and their natural receptors; fusion proteins that bind to their natural receptors (e.g., the extracellular portion of an immune checkpoint inhibitor protein fused to the Fe moiety of an antibody or immunoglobulin); nucleic acid molecules that interfere with the transcription or translation of immune checkpoint inhibitor nucleic acids, and the like.Such agents may directly interfere with the interaction between one or more immune checkpoint inhibitors and their natural receptors (e.g., antibodies) to disrupt inhibitory signaling and upregulate the immune response. Alternatively, agents may indirectly interfere with the interaction between one or more immune checkpoint proteins and their natural receptors to disrupt inhibitory signaling and upregulate the immune response. For example, a soluble form of an immune checkpoint protein ligand, e.g., a stabilized extracellular domain, may bind to the receptor and indirectly reduce the effective concentration of the receptor for binding to the appropriate ligand. In one embodiment, an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-CTLA-4 antibody are used, either alone or in combination.

[0042] As used herein, the terms "comprising," "comprise," or "comprised," and variations thereof, in connection with a defined or described element, such as an item, composition, apparatus, method, process, system, etc., mean inclusive or open-ended, allowing for additional elements, and thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements, or their equivalents, as appropriate, and that other elements may be included and still fall within the scope / definition of the defined item, composition, apparatus, method, process, system, etc.

[0043] As used herein, "administered in conjunction" means administering two compounds sufficiently close in time to achieve a combined immunological effect. Thus, co-administration can be by sequential administration or simultaneous administration (e.g., co-administration in a common or identical carrier).

[0044] As used herein, "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0045] "Immunogen" and "antigen" are used interchangeably and refer to any compound that is the subject of a cellular or humoral immune response. Non-viable immunogens include, for example, tumor cell lysates, tumor proteins, tumor lipids, tumor carbohydrates, killed immunogens, subunit vaccines, recombinant proteins, or peptides, etc. The adjuvants described herein can be used with any suitable immunogen. Exemplary immunogens of interest include those composed of or derived from viruses, mycoplasmas, bacteria, parasites, protozoans, prions, etc. Thus, immunogens of interest may be derived, without limitation, from human papillomavirus, herpesviruses, such as herpes simplex virus or varicella zoster virus, retroviruses, such as human immunodeficiency virus 1 or 2, hepatitis virus, influenza virus, rhinovirus, respiratory syncytial virus, cytomegalovirus, adenovirus, Mycoplasma pneumoniae, bacteria of the genera Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium, amoeba, malaria parasites, and / or Trypanosoma cruzi.

[0046] As used herein, the term "in combination" refers to the administration of a therapy to a subject, and refers to the use of two or more therapies for therapeutic benefit. The administration term "in combination" can also refer to the prophylactic use of a therapy to a subject when used with at least one additional therapy. The use of the term "in combination" does not limit the order in which the therapies (e.g., a first and a second therapy) are administered to a subject. Therapy can be administered to the subject with cancer, with cancer, or susceptible to cancer before (for example, 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), at the same time, or after (for example, 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after).The therapy is administered to the subject in order and within a period that allows the therapy to act together. In certain embodiments, the therapies are administered to a subject in a sequence and within a time period that results in increased efficacy than if the therapies were administered otherwise. Any additional therapy may be administered in any order and with other additional therapies.

[0047] For example, "modulation" of a symptom, molecular level, biological activity, etc., refers to, for example, a detectably increased or decreased symptom or activity, etc. Such an increase or decrease can be observed in a subject treated relative to a subject not treated with an adjuvant lipid (non-canonical inflammasome-activating lipid) described herein, provided that the untreated subject (e.g., a subject to which an immunogen is administered in the absence of the adjuvant lipid) has the same or a similar disease or infection as the treated subject, or is susceptible to developing the same or a similar disease or infection as the treated subject. Such an increase or decrease can be at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 1000%, or more, or within any range between any two of these values. Modulation can be ascertained subjectively or objectively, e.g., by subject self-assessment, by clinician evaluation, or by performance of a suitable assay or measurement, including, e.g., assessing the degree and / or quality of immune stimulation in a subject achieved by administration of an immunogen in the presence of an adjuvant lipid (non-canonical inflammasome-activating lipid) described herein. Modulation may be transient, long-term, or permanent, or may vary in time relative to during or after administration of the adjuvant lipids described herein to a subject, or during or after use of the adjuvant lipids described herein in assays or other methods described herein or in the cited references, for example, within the times described below, or about 12 to 24 or 48 hours after administration or use of the adjuvant lipids described herein, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28 days, or 1, 3, 6, 9 or more months after the subject has received such immune stimulatory composition / treatment.

[0048] As used herein, the term "non-canonical inflammasome-activating lipid" refers to a lipid that can induce hyperactivation of dendritic cells or macrophages. Exemplary "non-canonical inflammasome-activating lipids" include PAPC, oxPAPC, and oxPAPC species (e.g., HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC, POVPC, PGPC).

[0049] OxyPAPC species are known and described in the art, see, e.g., Ni et al., "Evaluation of Air Oxidized PAPC: A Multi Laboratory Study by LC-MS / MS," Free Radical Biology and Medicine 144:156-66 (2019); Table 1.

[0050] In some embodiments, the non-canonical inflammasome-activating lipid is 2-[[(2R)-2-[(E)-7-carboxy-5-hydroxyhept-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium (HOdiA-PC), [(2R)-2-[(E)-7-carboxy-5-oxohept-6-enoyl]oxy-3-hexadecanoyloxypropyl]2-(trimethylazanium) umyl)ethyl phosphate (KOdiA-PC), 1-palmitoyl-2-(5-hydroxy-8-oxo-octenoyl)-sn-glycero-3-phosphorylcholine (HOOA-PC), 2-[[(2R)-2-[(E)-5,8-dioxooct-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium (KOOA-PC), [(2R)-3-hexadecanoyloxy-2-(5 -oxopentanoyloxy)propyl]2-(trimethylazaniumyl)ethyl phosphate (POVPC), [(2R)-2-(4-carboxybutanoyloxy)-3-hexadecanoyloxypropyl]2-(trimethylazaniumyl)ethyl phosphate (PGPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[2-[(Z)-oct-2-enyl]-5-oxocyclopent-3-en-1-ylidene]methyl] [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentylidene]methyl]oxiran-2-yl]butanoyloxy]propyl]2-(trimethylazaniumyl)ethyl phosphate (PECPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentylidene]methyl]oxiran-2-yl]butanoyloxy]propyl]2-(trimethylazaniumyl)ethyl phosphate (PEIPC), or a combination thereof.

[0051] In some embodiments, the oxPAPC species is an oxPAPC species listed in Table 1 or a combination thereof.

[0052] [Table 1-1]

[0053] Table 1-2

[0054] Table 1-3

[0055] Table 1-4

[0056] Table 1-5

[0057] Table 1-6

[0058] Table 1-7

[0059] Table 1-8

[0060] Table 1-9

[0061] Table 1-10

[0062] Table 1-11

[0063] [Table 1-12]

[0064] [Table 1-13]

[0065] [Table 1-14]

[0066] As used herein, the term "hyperactive dendritic cells" or "hyperactive macrophages" refers to cells that retain viability and have the ability to secrete interleukin-1, a process typically associated with the assembly of inflammasomes within the hyperactive cells.

[0067] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0068] As used in this specification and the appended claims, the term "or" is generally used inclusive of "and / or" unless the content clearly dictates otherwise.

[0069] As used herein, the term "oxPAPC" or "oxidized PAPC" refers to lipids produced by oxidation of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC), resulting in a mixture of oxidized phospholipids containing either fragmented or full-length oxygenated sn-2 residues. Well-characterized oxidative fragmentation species contain 5-carbon sn-2 residues bearing omega-aldehyde or omega-carboxyl groups. Oxidation of arachidonic acid residues also produces phospholipids containing esterified isoprostanes. oxPAPC includes species such as HOdiA-PC, KOdiA-PC, HOOA-PC, PGPC, POVPC, and KOOA-PC, among other oxidation products present in oxPAPC.

[0070] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection or infusion techniques.

[0071] The terms "patient" or "individual" or "subject" are used interchangeably herein and refer to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods described herein are used in laboratory animals, veterinary applications, and in the development of animal models for disease, including, but not limited to, rodents, including mice, rats, hamsters, and primates.

[0072] As used herein, a "pharmaceutically acceptable" ingredient / carrier, etc. is one that is suitable for use in humans and / or animals without undue adverse side effects (e.g., toxicity, irritation, or allergic response) and for a reasonable benefit / risk ratio.

[0073] An "appropriate dose level" refers to a dose level that provides a reasonable therapeutic balance between beneficial effects and adverse effects (e.g., an immunogen administered in the presence of an adjuvant lipid described herein provides sufficient immunostimulatory activity and sufficiently low levels of macrophage stimulation). For example, the dose level can refer to, for example, the peak or mean serum level in a subject of anti-immunogen antibodies produced after administration of an immunogenic composition (including an adjuvant lipid described herein) at a particular dose level.

[0074] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of a disease or disorder, e.g., cancer, experienced by a subject.

[0075] "Treatment" is an intervention made to prevent the occurrence of a disorder or to alter the pathology or symptoms of a disorder. Thus, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. "Treatment" may also be designated as palliative care. Those in need of treatment include those already with the disorder as well as those in whom the disorder is to be prevented. Thus, "treating" or "treatment" of a condition, disorder, or condition includes (1) preventing or delaying the onset of clinical symptoms of the condition, disorder, or condition occurring in a human or other mammal that may be afflicted with or prone to the condition, disorder, or condition but that has not yet experienced or exhibited clinical or subclinical symptoms of the condition, disorder, or condition; (2) inhibiting the condition, disorder, or condition, i.e., arresting, alleviating, or delaying the onset of the disease or its recurrence (if treatment is maintained) or at least one clinical or subclinical symptom thereof; or (3) palliating the disease, i.e., causing regression of the condition, disorder, or condition, or at least one clinical or subclinical symptom thereof. The benefit to an individual to be treated is statistically significant, or at least perceptible to the patient or to the physician.

[0076] As defined herein, a "therapeutically effective" amount (i.e., an effective dose) of a compound or agent means an amount sufficient to produce a therapeutically (e.g., clinically) desired result. The composition may be administered one or more times per day, including once every other day, up to one or more times per week. One of ordinary skill in the art will understand that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the subject's general health and / or age, and other current illnesses, may affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a compound described herein may include a single treatment or a series of treatments.

[0077] Gene: All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species to which the compositions and methods disclosed herein are applicable. When a gene or gene product from a particular species is disclosed, the disclosure is intended to be illustrative only and not limiting unless the context in which it appears dictates otherwise. Thus, for example, with respect to the genes or gene products disclosed herein, it is intended to include homologous and / or orthologous genes and gene products from other species.

[0078] Ranges: Throughout this disclosure, various embodiments may be presented in a range format. The description in range format is understood to be merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claims. Accordingly, the description of a range is considered to have all possible subranges explicitly disclosed, as well as individual numerical values ​​within that range. For example, a description of a range, such as 1 to 6, is considered to have explicitly stated subranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This is true regardless of the breadth of the range.

[0079] Any composition or method provided herein can be combined with any one or more of the other compositions and methods provided herein.

[0080] Dendritic cells (DCs) and the regulation of pattern recognition receptors (PRRs) The innate immune system has traditionally been viewed as operating in an all-or-none fashion, with DCs either acting to initiate inflammatory responses that promote adaptive immunity or not. Thus, the Toll-like receptors (TLRs) expressed by DCs appear to be central in determining their immunogenic potential. The mammalian immune system is responsible for detecting microorganisms and activating defensive responses that limit infection. Central to this task are dendritic cells, which promote T cell activation following microbial sensing. It has been proposed that dendritic cells can assess any infectious threat and direct a proportionate response (Blander, JM (2014) Nat Rev Immunol 14, pp. 601–618; Vance, RE et al. (2009) Cell Hostµbe 6, pp. 10–21), but the mechanisms that may trigger their immunomodulatory activity remain unclear.

[0081] PRRs act directly or indirectly to detect molecules common to a wide range of microorganisms, traditionally referred to as pathogen-associated molecular patterns (PAMPs), including factors such as bacterial lipopolysaccharide (LPS), bacterial flagellin, or viral double-stranded RNA, among others.

[0082] A key characteristic of PRRs as immune regulators is their ability to recognize specific microbial products. Therefore, PRR-mediated signaling events should provide a definitive indication of infection. It has been proposed that "GO" signals are activated by PRRs expressed on DCs that promote inflammation and T cell-mediated immunity. Interestingly, several groups have recently proposed that DCs do not simply act in an all-or-none manner (Blander, JM and Sander, LE (2012). Nat Rev Immunol 12, 215-225; Vance, RE et al. (2009) Cell Hostµbe 6, 10-21). Rather, DCs may have the ability to assess the threat (or pathogenicity) of any possible infection and mount a proportionate response. The most commonly discussed means by which pathogenicity can be assessed is based on the ability of virulent pathogens to activate a wider variety of PRRs than nonpathogens. However, not all microorganisms share a common set of PRR activators, and not all PRR activators are equally potent. Therefore, the number of PRRs activated during infection is not an ideal assessment of virulence. Furthermore, an increase in the number of PRRs activated during infection generally results in a significant inflammatory response, which may indirectly promote a significant T cell response. Situations previously proposed to enhance DC activation (e.g., by using virulent pathogens as stimuli) are also expected to enhance MΦ activation (Vance, RE et al., (2009) Cell Host Microbiology 6, 10–21). Therefore, it remains unclear whether mechanisms exist for the immune system (i.e., DCs) to specifically assess the threat of infection.

[0083] One possible means by which infection threat can be assessed is by coincidence detection, a well-known process in which independent inputs result in a response that differs from that elicited by any single input. For PRRs, one such input must be a microbial product as an indicator of infection, regardless of pathogenic threat. To assess virulence threat, a second input must be present. Without wishing to be bound by theory, this putative second input is thought to be a molecule produced at the site of tissue injury, since cellular damage is a feature frequently associated with highly pathogenic microorganisms. Candidate molecules that could provide a second stimulus for DCs are a diverse family of molecules called damage-associated molecular patterns (DAMPs), also known as alarmins (Kono, H. and Rock, KL (2008) Nat Rev Immunol 8, 279-289; Pradue, T. and Cooper, EL (2012) Front Immunol 3, 287). DAMPs are found at sites of infectious and non-infectious tissue injury and have been proposed to regulate inflammatory responses, although their mechanisms of action remain unclear. One such class of DAMPs is represented by oxidized phospholipids derived from 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC), collectively known as oxPAPC. These lipids are produced at sites of both infectious and non-infectious tissue injury (Berliner, JA and Watson, AD (2005) N Engl J Med 353, 9-11; Imai, Y. et al. (2008) Cell 133, 235-249; Shirey, KA et al. (2013) Nature 497, 498-502) and are found at high levels in the membranes of dying cells (Chang, MK et al. (2004) J Exp Med 200, 1359-1370).oxPAPC is also an active component of oxidized low-density lipoprotein (oxLDL) aggregates that promote inflammation in atherosclerotic tissue (Leitinger, N. (2003) Curr Opin Lipidol 14, pp. 421-430), and local concentrations can be 10-100 μM (Oskolkova, OV et al. (2010) J Immunol 185, pp. 7706-7712). The association between oxPAPC and dying cells raises the possibility that this lipid could be used as a standard indicator of tissue health. Therefore, in the presence of microbial products, oxPAPC may indicate an increased infectious threat.

[0084] Because of these characteristics, activated DCs have a superior ability to stimulate antigen-specific T cell responses, and numerous strategies have been attempted to promote DC activation in order to stimulate protective immunity. These strategies generally involve the use of synthetic or natural microbial products that stimulate the Toll-like receptor (TLR) family of PRRs. A notable example is the monophosphoryl lipid A (MPLA) molecule, an FDA-approved TLR4 ligand that is being used to adjuvant an increasing number of vaccines (J. Paavonen, Lancet, Vol. 374, No. 9686, pp. 301-314, July 2009; M. Kundi, Expert Rev. Vaccines, Vol. 6, No. 2, pp. 133-140, April 2007; A.M. Diierlaurent et al., J. Immunol., Vol. 183, No. 10, pp. 6186-6197, November 2009). Notably, TLRs alone do not upregulate all the molecular signals necessary to promote T cell-mediated immunity. Members of the interleukin-1 (IL-1) family of cytokines are critical regulators of many aspects of T cell differentiation, persistent memory T cell generation, and effector function (SZ Ben-Sasson et al., Proc. Natl. Acad. Sci. USA, Vol. 106, No. 17, pp. 7119-24, April 2009; SZ Ben-Sasson et al., J. Exp. Med., Vol. 210, No. 3, pp. 491-502, March 2013; A. Jain et al., Nat. Commun., Vol. 9, No. 1, pp. 1-13, 2018). Expression of IL-1β, a well-characterized family member, is highly induced by TLR signaling, but this cytokine lacks an N-terminal secretion signal and is therefore not released from cells via the conventional biosynthetic pathway. Rather, IL-1β accumulates in an inactive state in the cytosol of DCs activated by TLR ligands (C. Garlanda et al., Immunity, Vol. 39, No. 6, pp. 1003-1018, December 2013). The lack of IL-1β release from activated DCs raises the possibility that TLR signaling alone is not sufficient to maximally stimulate T cell responses and protective immunity.

[0085] The DC activation state is not the only cell fate that DCs can achieve upon PRR signaling. Indeed, different PRRs stimulate different DC fates. One such fate is the inflammatory form of cell death known as pyroptosis. Pyroptosis is a regulated process resulting from the action of inflammasomes, supramolecular assembly centers (SMOCs) that assemble in the cytosol of DCs and other cells (A. Lu et al., Cell, Vol. 156, No. 6, pp. 1193-1206, March 2014; J.C. Kagan et al., Nat. Rev. Immunol., Vol. 14, No. 12, pp. 821-826, December 2014). Inflammasome assembly is typically stimulated in response to the detection of PAMPs or DAMPs in the host cell cytosol; therefore, cytosolic PRRs are responsible for linking cytosolic threat assessment to inflammasome-dependent pyroptosis (KJ Kieser and JC Kagan, Nat. Rev. Immunol., Vol. 17, No. 6, pp. 376–390, May 2017; M. Lamkanfi and V.M. Dixit, Cell, Vol. 157, No. 5, pp. 1013–22, May 2014). The pyroptotic process results in the release of IL-1β and other IL-1 family members from the cell, providing a signal to T cells that TLRs cannot provide. Despite this increased activity in promoting IL-1β release, pyroptotic cells die and therefore lose their ability to participate in the multi-day process required to stimulate and differentiate naive T cells in the dLN (TR Mempel et al., Nature, Vol. 427, No. 6970, pp. 154-159, January 2004).Indeed, stimuli that promote pyroptosis, such as the commonly used vaccine adjuvant alum (SC Isenbarth et al., Nature, Vol. 453, No. 7198, pp. 1122-1126, June 2008; M. Kool et al., J. Immunol., Vol. 181, No. 6, pp. 3755-3759, September 2008), are well understood for their ability to stimulate type 2 immune responses (P. Marrack et al., Nat. Rev. Immunol., Vol. 9, No. 4, pp. 287-293, April 2009), which are inappropriate for the elimination of many microbial infections or cancers.

[0086] In certain embodiments, a method of treating cancer comprises administering to a subject in need thereof a therapeutically effective amount of a composition comprising a dendritic cell hyperactivation stimulus together with a tumor cell lysate that serves as an immunogen, thereby treating the cancer. In certain embodiments, the method further comprises administering a chemotherapeutic agent, an immunogen, or a combination thereof.

[0087] In certain embodiments, the superactivating stimulus, chemotherapeutic agent, immunogen, or combination thereof, are co-administered or sequentially administered.

[0088] In certain embodiments, the superactivating stimulus comprises a combination of a pattern recognition receptor ligand and 1-palmitoyl-2-(5-glutaryl)-sn-glycero-3-phosphocholine (PGPC).

[0089] In certain embodiments, the dendritic cell superactivating stimulus comprises a pattern recognition receptor ligand and 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC), oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC), oxPAPC species, components thereof, or combinations thereof.

[0090] TLR4 ligand In some embodiments of the methods described herein, the TLR ligand is selected from a TLR1 ligand, a TLR2 ligand, a TLR3 ligand, a TLR4 ligand, a TLR5 ligand, a TLR6 ligand, a TLR7 ligand, a TLR8 ligand, a TLR9 ligand, a TLR10 ligand, a TLR11 ligand, a TLR12 ligand, a TLR13 ligand, and combinations thereof.

[0091] In some embodiments of the methods described herein, the TLR ligand is a TLR4 ligand.

[0092] In some embodiments of the methods described herein, the TLR4 ligand is selected from monophosphoryl lipid A (MPLA), lipopolysaccharide (LPS), or a combination thereof.

[0093] immunogen Immunogens, e.g., cancer immunogens, and their uses, e.g., in cancer vaccines, are described in the art. See, for example, Michael J.P. Lawman and Patricia D. Lawman (eds.), "Cancer Vaccines, Methods and Protocols," Methods in Molecular Biol. 1136 (2014); Chiang et al., "Whole Tumor Antigen Vaccines: Where Are We?" Vaccines (Basel) 3(2):344-72 (2015); Thumann et al., "Antigen Loading of Dendritic Cells with Whole Tumor Cell Preparations," J. Immunol. Methods 277:1-16 (2003); Kamigaki et al., "Immunotherapy of Autologous Tumor Lysate-Loaded Dendritic Cell Vaccines by a Closed-Flow Electroporation System for Solid Tumors," Anticancer Res. 33:2971-6 (2013); U.S. Patent Nos. 3,823,126; 3,960,827; and 4,160,018.

[0094] In some embodiments, the immunogen is a cancer antigen. In some embodiments, the cancer antigen is selected from tumor lysate, apoptotic bodies, peptides, tumor RNA, tumor-derived exosomes, tumor DC fusion, or a combination thereof.

[0095] In some embodiments, the immunogen is a whole tumor lysate. In some embodiments, whole tumor lysates are prepared by irradiation, boiling and / or freeze-thaw lysis.

[0096] In some embodiments, the immunogen is autologous. In some embodiments, the immunogen is allogeneic.

[0097] In some embodiments of the methods of inducing an immune response in a subject, the immunogen is a tumor lysate from a cell donor.

[0098] In some embodiments of the methods described herein, the cancer immunogen is an infectious agent immunogen, and infection by the infectious agent is associated with the development of cancer.

[0099] In some embodiments of the methods described herein, the cancer immunogen is derived from a cancer immunogenic cell.

[0100] In some embodiments of the methods described herein, the cancer immunogen is or comprises a whole tumor cell lysate.

[0101] The immunogenic compositions containing the adjuvants described herein can be administered to a subject using any known form of vaccine, e.g., tumor antigens, tumor cell lysates, attenuated viruses, proteins, nucleic acids, etc., to produce a vaccine in the subject, and an amount of the selected immunogen effective in inducing a therapeutic or prophylactic immune response to the target antigen in the subject. The subject can be a human or non-human subject. Animal subjects include, without limitation, non-human primates, dogs, cats, equines (horses), ruminants (e.g., sheep, goats, cattle, camels, alpacas, llamas, deer), pigs, poultry (e.g., chickens, turkeys, quail), rodents, and chirodoptera. The subject can be treated for any purpose, including, without limitation, to elicit a protective immune response or to generate antibodies (or B cells) for collection and use for other purposes.

[0102] The immunogen of interest is expressed in disease target cells (e.g., neoplastic cells, infected cells) and is expressed at lower levels or not at all in other tissues. Examples of target cells include cells derived from neoplastic diseases, including, but not limited to, sarcoma, lymphoma, leukemia, carcinoma, melanoma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, colon cancer, lung cancer, glioblastoma, and astrocytoma. Alternatively, the target cells may be infected with, for example, a virus, mycoplasma, parasite, protozoan, prion, etc. Thus, immunogens of interest may be derived, without limitation, from human papillomavirus (see below), herpesviruses, e.g., herpes simplex virus or varicella zoster virus, retroviruses, e.g., human immunodeficiency virus 1 or 2, hepatitis virus, influenza virus, rhinovirus, respiratory syncytial virus, cytomegalovirus, adenovirus, Mycoplasma pneumoniae, bacteria of the genera Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium, amoeba, malaria parasites, and Trypanosoma cruzi.

[0103] In addition to tumor antigens, tumor cell lysates, and antigens of infectious agents, mutant forms of tumor suppressor gene products, including but not limited to p53, BRCA1, BRCA2, retinoblastoma, and TSG101, or oncogene products such as, for example, without limitation, RAS, WT, MYC, ERK, and TRK, may also provide target antigens for use with the present disclosure. Target antigens may also be autoantigens, such as those associated with cancer or neoplastic disease. In some embodiments, the immunogen is a peptide derived from a heat shock protein (hsp)-peptide complex of a diseased cell, or the hsp-peptide complex itself.

[0104] The immunogenic compositions described herein may comprise an immunogen and an adjuvant lipid and can be administered for therapeutic and / or prophylactic purposes. For therapeutic applications, the immunogenic compositions described herein are administered in an amount sufficient to induce an effective immune response and / or hyperactivated dendritic cells to treat or prevent the progression and / or symptoms of a disease. The dose of the adjuvant described herein may vary depending on the nature of the immunogen and the condition of the subject. However, the dose is sufficient to enhance the effectiveness of the immunogen in eliciting an immunogenic response. For therapeutic or prophylactic treatments, the amount of adjuvant administered may range from 0.05 mg, 0.1 mg, 0.5 mg, or 1 mg per kg of body weight to about 10 mg, 50 mg, or 100 mg per kg of body weight or more. The adjuvants described herein are generally non-toxic and can generally be administered in relatively large amounts without causing life-threatening side effects.

[0105] The term "therapeutic immune response" as used herein refers to an increase in humoral and / or cellular immunity against a target antigen, as measured by standard techniques. Preferably, the level of induced immunity against the target antigen is at least four times, preferably at least five times, the level before administration of the immunogen. Furthermore, immune responses can also be measured qualitatively. In this case, the inhibition of progression or remission of neoplastic disease or infectious disease in a subject using an appropriate in vitro or in vivo assay is considered to indicate the induction of a therapeutic immune response.

[0106] In some embodiments of the methods described herein, a composition comprising an immunogen and an adjuvant described herein combined in a therapeutically effective amount is administered to a mammal in need thereof. The term "administering," as used herein, means delivering an immunogen and an adjuvant described herein to a mammal by any method capable of achieving the desired result. The immunogens and adjuvants described herein may be administered, for example, intravenously or intramuscularly. The term "mammal," as used herein, is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and livestock. A "therapeutically effective amount" refers to an amount of immunogen and adjuvant effective to produce a desired therapeutic effect when administered to a mammal.

[0107] The compositions comprising the immunogen and adjuvant described herein can be administered cutaneously, subcutaneously, intravenously, intramuscularly, parenterally, pulmonary, vaginally, rectally, nasally, or topically. The compositions may also be delivered by injection, orally, by spray, or by particle bombardment.

[0108] The composition for administration may further contain various additional substances, such as pharmaceutically acceptable carriers. Suitable carriers include any standard pharmaceutically acceptable carrier, such as phosphate buffered saline solution, water, emulsions, such as oil / water emulsions or triglyceride emulsions, various types of wetting agents, tablets, coated tablets, and capsules. Typically, such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid, talc, vegetable oils, gums, glycols, or other known excipients. Such carriers may further contain flavorings, colorings, or other ingredients. The compositions described herein may further contain suitable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions may be in liquid form or may be lyophilized or otherwise dry formulations, and may contain diluents of varying buffer (e.g., Tris-HCl, acetate, phosphate) content, pH, and ionic strength, additives to prevent absorption to surfaces, such as albumin or gelatin, surfactants (e.g., Tween 20, Tween 80, Pluronic® F68, bile salts), solubilizers (e.g., glycerol, polyethyleneglycerol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), antiseptics, and the like. These may include preservatives (e.g., thimerosal, benzyl alcohol, parabens), bulking substances or tonicity adjusting agents (e.g., lactose, mannitol), covalent attachment of polymers, such as polyethylene glycol, to the protein, complexation with metal ions, or incorporation of the substance into or onto particulate preparations such as polymeric compounds, e.g., polylactic acid, polyglycolic acid, hydrogels, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will affect the physical state, solubility, stability, in vivo release rate, and in vivo clearance rate.

[0109] Combination therapy In certain embodiments, it may be preferable to administer one or more other therapeutically beneficial agents to the subject. These agents include, without limitation, chemotherapeutic agents, chemical compounds, cytokine antagonists, cytokine receptor antagonists, cytokines, adoptive cellular therapies, antiviral agents, checkpoint inhibitors, adjuvants, or combinations thereof. In certain embodiments, the compound comprises at least one amidoamine compound. Amidoamines are a class of compounds formed from lipids and diamines. One example of an amidoamine compound is myristamidopropyldimethylamine (Aldox).

[0110] Immune checkpoint modulation: In certain embodiments, immune checkpoint modulators are co-administered with superactivated dendritic cells. Immune checkpoints relate to inhibitory pathways of the immune system responsible for maintaining self-tolerance and regulating the duration and breadth of physiological immune responses.

[0111] Certain cancer cells thrive by exploiting immune checkpoint pathways, which are a major mechanism of immune tolerance, particularly for T cells specific to tumor antigens. For example, certain cancer cells overexpress one or more immune checkpoint proteins that are responsible for inhibiting cytotoxic T cell responses. Therefore, immune checkpoint regulators can be administered to overcome the inhibitory signals and allow and / or enhance immune attacks against cancer cells. Immune checkpoint regulators can promote immune cell responses against cancer cells by reducing, inhibiting, or suppressing signaling by negative immune cell response regulators (e.g., CTLA4), or can stimulate or enhance signaling by positive regulators of the immune response (e.g., CD28).

[0112] Immunotherapeutic agents targeting immune checkpoint regulators can be administered to promote immune attack targeted to cancer cells. The immunotherapeutic agent can be or include an antibody agent that targets (e.g., is specific for) an immune checkpoint regulator. Examples of immunotherapeutic agents include antibody agents that target one or more of CTLA-4, PD-1, PD-L1, GITR, OX40, LAG-3, KIR, TIM-3, CD28, CD40, and CD137. Specific examples of antibody agents can include monoclonal antibodies. Specific monoclonal antibodies that target immune checkpoint regulators are available. For example, ipilimumab targets CTLA-4, tremelimumab targets CTLA-4, and pembrolizumab targets PD-1.

[0113] The programmed death 1 (PD-1) protein is an inhibitory member of the extended CD28 / CTLA-4 family of T cell regulators (Okazaki et al. (2002) Curr Opin Immunol 14:391-779-82; Bennett et al. (2003) J. Immunol. 170:711-718). Other members of the CD28 family include CD28, CTLA-4, ICOS, and BTLA. Two cell surface glycoprotein ligands for PD-1 have been identified: programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2). PD-L1 and PD-L2 have been shown to bind to PD-1 and downregulate T cell activation and cytokine secretion (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43; Ohigashi et al. (2005) Clin Cancer Res 11:2947-53).

[0114] PD-L1 (also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1)) is a 40-kDa type 1 transmembrane protein. PD-L1 binds to its receptor PD-1, found on activated T cells, B cells, and myeloid cells, to regulate activation or inhibition. Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not to CD28 or CTLA-4 (Blank et al. (2005) Cancer Immunol Immunother. 54:307-14). PD-L1 binding to its receptor PD-1 on T cells delivers a signal that inhibits TCR-mediated activation of IL-2 production and T cell proliferation. The mechanism involves inhibition of ZAP70 phosphorylation and its association with CD3 zeta (Sheppard et al. (2004) FEBS Lett. 574:37-41). PD-1 signaling attenuates TCR signaling-induced PKC-θ activation loop phosphorylation, which is required for activation of the transcription factors NF-κB and AP-1 and production of IL-2. PD-L1 also binds to the costimulatory molecule CD80 (B7-1), but not CD86 (B7-2) (Butte et al. (2008) Mol Immunol. 45:3567-72).

[0115] Expression of PD-L1 on the cell surface has been shown to be upregulated via IFN-γ stimulation. PD-L1 expression is found in many human cancers, including lung, ovarian, and colon cancers and various myelomas, and is frequently associated with poor prognosis (Iwai et al. (2002) PNAS 99:12293-7; Ohigashi et al. (2005) Clin Cancer Res 11:2947-53; Okazaki et al. (2007) Intern. Immun. 19:813-24; Thompson et al. (2006) Cancer Res. 66:3381-5). PD-L1 has been proposed to be important in tumor immunity by increasing apoptosis of antigen-specific T cell clones (Dong et al. (2002) Nat Med 8:793-800). PD-L1 may be involved in intestinal mucosal inflammation, and it has been proposed that inhibition of PD-L1 suppresses colitis-associated wasting (Kanai et al. (2003) J Immunol 171:4156-63).

[0116] Exemplary anti-PD1 antibodies include pembrolizumab (MK-3475, Merck), nivolumab (BMS-936558, Bristol-Myers Squibb), and pidilizumab (CT-011, Curetech LTD.). Anti-PD1 antibodies are commercially available from, for example, ABCAM™ (AB137132), BIOLEGEND™ ​​(EH12.2H7, RMP1-14), and Affymetrix Ebioscience (J105, J116, MIH4).

[0117] Anti-cancer agents: In certain embodiments, the method further comprises administering an anti-cancer agent. In some embodiments, the anti-cancer agent is a chemotherapeutic agent, a growth inhibitory agent, a targeted therapeutic agent, a chimeric antigen receptor-expressing T cell, an antibody or antigen-binding fragment thereof, an antibody-drug conjugate, an angiogenesis inhibitor, an anti-tumor agent, a cancer vaccine, an adjuvant, and combinations thereof.

[0118] In some embodiments, the anticancer agent is a chemotherapeutic agent or growth inhibitor.For example, the chemotherapeutic agent or growth inhibitor can include alkylating agents, anthracyclines, antihormones, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic agents, antitumor antibiotics, proteasome inhibitors, microtubule inhibitors, EGFR antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors, and combinations thereof.

[0119] Examples of chemotherapeutic agents include erlotinib (TARCEVA™, Genentech / OSI Pharm.), bortezomib (VELCADE™, Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX™, AstraZeneca), sunitinib (SUTENT™, Pfizer / Sugen), letrozole (FEMARA™, Novartis), imatinib mesylate (GLEEVEC™, Novartis), and rituximab (RITC). rtis), finasunate (VATALANIB™, Novartis), oxaliplatin (ELOXATIN™, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE™, Wyeth), lapatinib (TYKERB™, GSK572016, GlaxoSmithKline), lonafamib (SCH66336), sorafenib (NEXAVAR™, Bayer Labs.), gefitinib (IRESSA™, AstraZeneca), AG1478, alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylameramines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); can Ptothecins (including topotecan and irinotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and baizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); corticosteroids (including prednisone and prednisolone); cyproterone acetate; 5α-deductase inhibitors including finasteride and dutasteride; vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat, dolastatins;Antibiotics such as aldesleukin, talc, duocarmycins (including synthetic analogs, KW-2189 and CBI-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatin; enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin γ1I and calicheamicin ω1I (Angew Chem. Intl. Ed. Engl. 1994, 33:183-186); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; as well as neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabimycin, caminomycin, cardi nophylline, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ (doxorubicin) (morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivine pomycin, peplomycin, porfiromycin, puromycin, chelamycin, rhodrubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine , thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as furoic acid; aceglatone;Aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziconazole; elfomithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK™ polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ (Cremophor-free), an albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE™ (docetaxel; Sanofi-Aventis); chlorambucil; GEMZAR™ (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE™ (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA™); ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO);Retinoids, such as retinoic acid, may be included, as well as pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0120] In some embodiments, chemotherapeutic agents may include alkylating agents (including monofunctional and bifunctional alkylating agents), such as thiotepa, CYTOXAN™, cyclophosphamide, nitrogen mustards, such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; temozolomide; and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0121] In some embodiments, chemotherapeutic agents may include anthracyclines such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0122] In some embodiments, chemotherapeutic agents may include antihormonal agents (e.g., antiestrogens and selective estrogen receptor modulators (SERMs), including tamoxifen (NOLVADEX™; including tamoxifen citrate), raloxifene, droloxifene, iodoxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and FARESTON™ (toremifene citrate)); and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0123] In some embodiments, chemotherapeutic agents may include aromatase inhibitors, which inhibit the aromatase enzyme that controls estrogen production in the adrenal glands, such as 4(5)-imidazoles, aminoglutethimide, MEGASE™ (megestrol acetate), AROMASIN™ (exemestane; Pfizer), formestany, fadrozole, RIVISOR™ (vorozole), FEMARA™ (letrozole; Novartis), and ARIMIDEX™ (anastrozole; AstraZeneca), as well as pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0124] In some embodiments, chemotherapeutic agents may include antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all-trans-retinoic acid, fenretinide; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0125] In some embodiments, the chemotherapeutic agent may include a protein kinase inhibitor, a lipid kinase inhibitor, or an antisense oligonucleotide, particularly one that inhibits the expression of genes in signal transduction pathways involved in cell proliferation disorders, such as PKC-α, Ralf, and H-Ras.

[0126] In some embodiments, chemotherapeutic agents may include ribozymes, such as VEGF expression inhibitors (eg, ANGIOZYME™) and HER2 expression inhibitors.

[0127] In some embodiments, chemotherapeutic agents may include cytotoxic agents or antitumor antibiotics, such as dactinomycin, actinomycin, bleomycin, plicamycin, mitomycin, e.g., mitomycin C, and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0128] In some embodiments, chemotherapeutic agents may include proteasome inhibitors such as bortezomib (VELCADE™, Millennium Pharm.), epoxomicins such as carfilzomib (KYPROLIS™, Onyx Pharm.), marizomib (NPI-0052), MLN2238, CEP-18770, oprozomib, and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0129] In some embodiments, chemotherapeutic agents may include microtubule inhibitors, such as vinca alkaloids, including vincristine, vinblastine, vindesine, and vinorelbine; taxanes, including paclitaxel and docetaxel; podophyllotoxins; and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0130] In some embodiments, chemotherapeutic agents may include "EGFR antagonists," which refer to compounds that bind to or otherwise directly interact with EGFR and prevent or reduce its signaling activity, alternatively referred to as "EGFRi." Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb579 (ATCC CRL HB 8506), MAb455 (ATCC CRL HB8507), MAb225 (ATCC CRL 8508), MAb528 (ATCC CRL 8509) (see U.S. Pat. No. 4,943,533, Mendelsohn et al.) and their variants, such as chimerized 225 (C225 or cetuximab; ERBUTIX) and reshaped human 225 (H225) (see WO 96 / 40210, Imclone Systems, Inc.). Inc.); IMC-11F8, an antibody targeted to fully human EGFR (Imclone); antibodies that bind to type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind to EGFR, as described in U.S. Pat. No. 5,891,996; and human antibodies that bind to EGFR, such as ABX-EGF or panitumumab (see WO 98 / 50433, Abgenix / Amgen); EMD55900 (Stragliotto et al., Eur. J. Cancer 32A:636-640 (1996); EMD7200 (matuzumab), a humanized EGFR antibody against EGFR that competes with both EGF and TGF-α for binding to EGFR (EMD / Merck); human EGFR antibodies, HuMax-EGFR (GenMab); fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E7.6.3, and described in U.S. Pat. No. 6,235,883; MDX-447 (Medarex Inc); and mAb806 or humanized mAb806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)).Anti-EGFR antibodies may be conjugated to a cytotoxic agent, thus forming an immunoconjugate (see, e.g., European Patent Application Publication No. 659439, Merck Patent GmbH). EGFR antagonists may be small molecules, e.g., those described in U.S. Patent Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, and 6,140,332. Nos. 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, and the following PCT publications: WO 98 / 14451, WO 98 / 50038, WO 99 / 09016, and WO 99 / 24037.Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA™ Genentech / OSI Pharmaceuticals); PD183805 (CI1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA™) 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca; ZM105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim; PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol); (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine; CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3- cyano-7-ethoxy-6-quinolinyl]-4-(-dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU5271; Pfizer); dual EGFR / HER2 tyrosine kinase inhibitors, such as lapatinib (TYKERB™, GSK572016 or N-[3-chloro-4-[(3fluorophenyl)methoxy]phenyl]-6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).

[0131] In some embodiments, the chemotherapeutic agent may comprise a tyrosine kinase inhibitor, such as an EGFR-targeted drug as noted in the preceding paragraph; a small molecule HER2 tyrosine kinase inhibitor, e.g., TAK165 available from Takeda; CP-724,714, a selective oral inhibitor of ErbB2 receptor tyrosine kinase (Pfizer and OSI); a dual HER inhibitor, e.g., EKB-569 (available from Wyeth), which preferably binds to EGFR but inhibits both HER2-overexpressing and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); a pan-HER inhibitor, e.g., canertinib (CI-1033; Pharmacia); a Raf-1 inhibitor, e.g., ISIS, which inhibits Raf-1 signaling. antisense agent ISIS-5132 available from GlaxoSmithKline Pharmaceuticals; non-HER-targeted TK inhibitors such as imatinib mesylate (GLEEVEC™, available from GlaxoSmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib (SUTENT™, available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787 / ZK222584, Novartis / Schering available from AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, e.g., PD153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, e.g., CGP59326, CGP60261, and CGP62706; pyrazolopyrimidine, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostins containing a nitrothiophene moiety; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to HER-encoding nucleic acids); quinoxalines (U.S. Pat. No. 5,804,396); tyrphostins (U.S. Pat. No. 5,804,396); ZD6474 (Astra Zeneca);PTK-787 (Novartis / Schering AG); pan-HER inhibitors, such as CI-1033 (Pfizer); Affinitac (ISIS3521; Isis / Lilly); imatinib mesylate (GLEEVEC™); PKI166 (Novartis); GW2016 (GlaxoSmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone), including rapamycin (sirolimus, RAPAMUNE™); or the following patent publications: U.S. Pat. No. 5,804,396; WO 1999 / 09016 (American Cyanamid); WO 1998 / 43960 (American Cyanamid; 1997 / 38983 (Warner Lambert); 1999 / 06378 (Warner Lambert); 1999 / 06396 (Warner Lambert); 1996 / 30347 (Pfizer, Inc); 1996 / 33978 (Zeneca); 1996 / 3397 (Zeneca) and 1996 / 33980 (Zeneca);

[0132] In some embodiments, the chemotherapeutic agent may include a retinoid, such as retinoic acid, as well as pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0133] In some embodiments, chemotherapeutic agents can include antimetabolites.Examples of antimetabolites can include folic acid analogs and antifolates, such as denopterin, methotrexate, pteropterin, trimethotrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as 5-fluorouracil (5-FU), ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; nucleoside analogs; and nucleotide analogs.

[0134] In some embodiments, the chemotherapeutic agent can include a topoisomerase inhibitor.Examples of topoisomerase inhibitors can include topoisomerase 1 inhibitors, such as LURTOTECAN™ and ABARELIX™ rmRH; topoisomerase II inhibitors, such as doxorubicin, epirubicin, etoposide, and bleomycin; and topoisomerase inhibitor RFS 2000.

[0135] In some embodiments, the chemotherapeutic agent may include a histone deacetylase (HDAC) inhibitor, such as vorinostat, romidepsin, belinostat, mocetinostat, valproic acid, panobinostat, and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0136] Chemotherapeutic agents further include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone 17-butyrate, 17- hydrocortisone valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone 17-butyrate, clobetasol 17-propionate, fluocortolone caproate, fluocortolone pivalate, and fluprednidene acetate; immunoselective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and its D-isomer form (feG) (IMULAN) BioTherapeutics, LLC; antirheumatic drugs such as azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide minocycline, sulfasalazine, tumor necrosis factor alpha (TNFα) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), cetolithumab pegol (Cimzia), golimumab (Simponi), interleukin 1 (IL-1) blockers such as anakinra (K ineret), T cell costimulation blockers, e.g., abatacept (Orencia), interleukin 6 (IL-6) blockers, e.g., tocilizumab (ACTEMERA™); interleukin 13 (IL-13) blockers, e.g., lebrikizumab; interferon alpha (IFN) blockers, e.g., rontalizumab; beta 7 integrin blockers, e.g., rhuMAb beta 7; IgE pathway blockers, e.g., anti-M1 prime; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa1 / β2 blockers, e.g., anti-lymphotoxin alpha (LTa); radioisotopes (e.g., 211 At, 131 I, 125 I, 90Y、 186 Re 188 Re 212 Hello 32 Q 212Pb, and Lu radioisotopes; various investigational agents, such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols, such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavin, flavanols, procyanidins, betulinic acid and its derivatives; autophagy inhibitors, such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL (trade name)) beta-lapachone; lapachol; colchicine; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin; podophyllotoxin; tegafur (UFTORAL™); bexarotene (TARGRETIN™); bisphosphonates, such as clodronate (e.g., BONEFOS™ or OSTAC™), etidronate (DIDROCAL™), NE-58095, zoledronic acid / zoledronate (ZOMETA™), alendronate (FOSAMAX™), (trademark), pamidronate (AREDIA™), tiludronate (SKELID™), or risedronate (ACTONEL™); and epidermal growth factor receptor (EGF-R); vaccines, such as the THERATOPE™ vaccine; perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors, such as oblimersen sodium (GENASENS E™); pixantrone; farnesyltransferase inhibitors, such as lonafarnib (SCH6636, SARASAR™); and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing; and combinations of two or more of the foregoing, such as CHOP (short for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone); and FOLFOX (short for the treatment regimen using oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin).

[0137] Chemotherapeutic agents can also include non-steroidal anti-inflammatory drugs with analgesic, antipyretic, and anti-inflammatory effects.NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase.Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, and valdecoxib. NSAIDs may be indicated for the symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthropathy, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headache and migraine, postoperative pain, mild to moderate pain due to inflammation and tissue injury, fever, intestinal obstruction, and renal colic.

[0138] Pharmaceutical Therapeutics In one embodiment, the pharmaceutical composition is administered systemically, for example, by formulation in a pharmaceutically acceptable buffer, such as physiological saline. Preferred routes of administration include, for example, intravesical instillation, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intratumoral injection, or intradermal injection, which provide continuous, sustained, or effective levels of the composition in the patient. Human patients or other animals are treated using a therapeutically effective amount of the therapeutic agent identified herein in a physiologically acceptable carrier. Suitable carriers and their formulations are described, for example, in Remington's Pharmaceutical Sciences by E.W. Martin. The amount of therapeutic agent to be administered will vary depending on the method of administration, the age and weight of the patient, and the clinical symptoms of the cancer. Generally, the amount will be in the range of amounts used for other agents used in the treatment of other diseases associated with cancer, although in certain cases, smaller amounts may be required due to the increased specificity of the compound. The compound is administered at a dose that enhances the subject's immune response or reduces the proliferation, survival, or invasiveness of neoplastic or infected cells, as determined by methods known to those skilled in the art.

[0139] The compositions embodied herein can be administered by any suitable means that results in a concentration of the therapeutic agent, in combination with other components, that is effective in ameliorating, alleviating, or stabilizing cancer. The compositions can be prepared in a dosage form suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, intravesicular, intratumoral, or intraperitoneal) administration. For example, pharmaceutical compositions can be prepared according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), A.R. Gennaro, Lippincott Williams & Wilkins, eds., 2000, and Encyclopedia of Pharmaceutical Technology, J.Swarbrick and J.C.B. Boylan, eds., 1988-1999, Marcel Dekker, New York).

[0140] The dosage for humans is initially determined by extrapolating the amount of compound used in mice or non-human primates. Because those skilled in the art recognize that modifying dosages for humans compared with animal models is routine in the art, dosages can vary from about 1 μg compound / kg body weight to about 5,000 mg compound / kg body weight; or from about 5 mg / kg body weight to about 4,000 mg / kg body weight, or from about 10 mg / kg body weight to about 3,000 mg / kg body weight; or from about 50 mg / kg body weight to about 2,000 mg / kg body weight; or from about 100 mg / kg body weight to about 1,000 mg / kg body weight; or from about 150 mg / kg body weight to about 500 mg / kg body weight. For example, dosages may range from about 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1,000 mg, 1,050 mg, 1,100 mg, 1,200 mg, 1,300 mg, 1,400 mg, 1,500 mg, 2,600 mg, 2,700 mg, 2,800 mg, 3,900 mg, 3,950 mg, 4,100 mg, 4,100 mg, 5,200 mg, 5,300 mg, 6,100 mg, 7,100 mg, 8,100 mg, 9,100 mg, 1,100 mg, 1,200 mg, 1,300 mg, 1,400 mg, 1,500 mg, 1,600 mg, 1,700 mg, 1,800 mg, 1,900 mg, 2,100 mg, 2,200 mg, 2,300 mg, 2,400 mg, 2,500 mg, 3,500 mg, 3,600 mg, 3,700 mg, 4,100 mg, 4,100 mg, 5,200 mg, 5,200 mg, 5,300 mg, 5,400 mg, 5,500 mg, 6,100 mg, 6,100 The dosage ranges from about 5 mg compound / kg body weight to about 20 mg compound / kg body weight. In other examples, the dosage ranges from about 8 mg, 10 mg, 12 mg, 14 mg, 16 mg, or 18 mg / kg body weight. Naturally, this dosage can be adjusted upward or downward, depending on the results of initial clinical trials and the needs of a particular patient, as is routine in such treatment protocols.

[0141] The pharmaceutical composition is formulated with appropriate excipients into a pharmaceutical composition that releases the therapeutic agent in a controlled manner after administration, examples of which include single- or multiple-unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

[0142] The pharmaceutical compositions embodied herein may be administered parenterally by injection, infusion, or implantation (subcutaneous, intravenous, intramuscular, intratumoral, intravesicular, intraperitoneal) of the formulation, or via a suitable delivery device or implant containing conventional non-toxic, pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations may be found in Remington: The Science and Practice of Pharmacy, supra.

[0143] In certain embodiments, the composition is presented in the form of a solution, suspension, emulsion, injection device, or implantable delivery device, or as a dry powder that can be reconstituted with water or other suitable vehicle before use. In addition to the active agent that reduces or improves cancer, the composition contains a suitable parenterally acceptable carrier and / or excipient. The active therapeutic agent may be incorporated into microspheres, microcapsules, nanoparticles, or liposomes for controlled release. In addition, the composition may contain suspending agents, solubilizing agents, stabilizing agents, pH adjusting agents, tonicity adjusting agents, and / or dispersing agents.

[0144] As described above, pharmaceutical compositions may be in a form suitable for sterile injection. To prepare such compositions, the appropriate active therapeutic agent is dissolved or suspended in a parenterally acceptable liquid vehicle. Acceptable vehicles and solvents that can be used include water, water adjusted to an appropriate pH by adding an appropriate amount of hydrochloric acid or sodium hydroxide, or appropriate buffers, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution, and dextrose solution. Aqueous formulations may further contain one or more preservatives (e.g., methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, or n-propyl p-hydroxybenzoate). If one of the compounds is sparingly or poorly soluble in water, a solubility enhancer or solubilizer may be added, or the solvent may contain 10-60% (w / w) propylene glycol.

[0145] Provided herein is a method for treating cancer or a symptom thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition. Thus, provided herein is a method for treating a subject suffering from or susceptible to cancer. The method may include administering a therapeutic amount of a composition described herein to a mammal under conditions that treat the disease or disorder, in a dose sufficient to treat the disease or disorder or a symptom thereof.

[0146] The methods herein can include administering to a subject (including a subject identified as needing such treatment) an effective amount of a compound described herein, or a composition described herein that produces such an effect. Identification of a subject in need of such treatment can be the judgment of the subject or a medical professional, and can be subjective (e.g., opinion) or objective (e.g., measurable by testing or diagnostic methods).

[0147] Therapeutic methods (including prophylactic treatments) described herein generally involve administration of a therapeutically effective amount of a compound described herein, e.g., a compound formulated herein, to a subject (e.g., animal, human), including a mammal, particularly a human, in need thereof. Such treatments would be suitably administered to a subject, particularly a human suffering from, having, susceptible to, or at risk for cancer or a condition thereof. Determination of such an "at risk" subject can be made by any objective or subjective determination, such as by diagnostic testing or the subject's or health care provider's opinion (e.g., genetic testing, enzyme or protein markers, markers (described herein), family history, etc.). The fusion protein complexes described herein can be used in the treatment of any other disorder in which an increased immune response is desired.

[0148] Further provided herein are methods for monitoring the course of treatment. The methods may include determining the level of a diagnostic marker (marker) (e.g., any target, protein, or indicator thereof described herein that is modulated by a compound herein), or performing a diagnostic measurement (e.g., a screen, an assay) in a subject suffering from or susceptible to a cancer-related disorder or a symptom thereof, wherein the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disorder or its symptom. The level of the marker determined by this method can be compared with the known level of the marker in a healthy control or other affected patient to establish the subject's condition. In some cases, a second level of the marker in the subject is determined at a later time point than the first level, and the two levels are compared to monitor the course of the disease or the effectiveness of the therapy. In certain embodiments, a pre-treatment level of the marker in the subject is determined before the initiation of a treatment described herein. This pre-treatment level of the marker may then be compared with the level of the marker in the subject after the initiation of treatment to determine the effectiveness of the treatment.

[0149] The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration.

[0150] The practice of the present methods employs, unless otherwise indicated, known techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture, and transgenic biology, which are within the skill of the art. See, for example, Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Sambrook et al., 1989, Molecular Cloning, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Sambrook and Russell, 2001, Molecular Cloning, 3rd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Ausubel et al., 1992, Current Protocols in Molecular Biology (John Wiley & Sons, with periodic updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988, Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Jakoby and Pastan, 1979; Nucleic Acids Hybridization (eds. B.D. Hames & S.J. Higgins, 1984); Transcription And Translation (eds. B.D. Hames & S.J. Higgins, 1984); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the monograph, Methods In Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors For Mammalian Cells (eds. J.H. Miller and M.P. Calos, 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, vols. 154 and 155 (eds. Wu et al.), Immunochemical Methods In Cell And Molecular Biology (eds. Mayer and Walker, Academic Press, London, 1987); Handbook Of Experimental Immunology, vols. I-IV (eds. D.M. Weir and C.C. Blackwell, 1986); Riott, Essential Immunology, 6th ed., Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986; Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), (4th ed., University of Oregon Press, Eugene, 2000).

[0151] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.Methods and materials similar or equivalent to those described herein can be used in the practice or testing of this invention, and suitable methods and materials are described below.It is understood and expected that those skilled in the art can make modifications to the principles of the invention disclosed herein, and such modifications are intended to be included within the scope of this invention.

[0152] Exemplary Embodiments 1. A method for preparing an immunogenic composition, comprising: a) removing leukocytes from a suspension of cells prepared from the tumor to obtain a tumor cell enriched suspension; b) lysing cells from the enriched tumor cell suspension to obtain a tumor cell lysate; c) contacting the tumor cell lysate with an oxidized lipid and a toll-like receptor 4 (TLR4) agonist to obtain the immunogenic composition A method comprising:

[0153] 2. The method of embodiment 1, wherein the leukocytes are removed in step a) by negative selection using an anti-CD45 antibody.

[0154] 3. The method of embodiment 1, wherein the cells are lysed in step b) by one or more freeze-thaw cycles.

[0155] 4. The method of embodiment 1, wherein the oxidized lipid comprises at least one oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC).

[0156] 5. The method of embodiment 4, wherein the at least one phospholipid comprises at least one of the group consisting of POVPC, PGPC, PECPC, and PEIPC, and optionally the at least one phospholipid comprises PGPC.

[0157] 6. The method of embodiment 1, wherein the TLR4 agonist comprises monophosphoryl lipid A (MPLA).

[0158] 7. The method of embodiment 6, wherein the TLR4 agonist is present in an adjuvant, the adjuvant further comprising one or more of an aluminum salt, a saponin, and a liposome, and optionally the adjuvant is AS01B or AS04.

[0159] 8. The method of embodiment 1, further comprising, prior to step a), obtaining a sample from the tumor and preparing a suspension of the cells.

[0160] 9. An immunogenic composition prepared by the method according to any one of embodiments 1 to 8. 10. A method for inducing an anti-cancer immune response, comprising: Administering an effective amount of the immunogenic composition of embodiment 9 to a mammalian subject having cancer. A method comprising:

[0161] 11. The method of embodiment 10, wherein the anti-cancer immune response comprises a cellular immune response. 12. The method of embodiment 10, wherein the anti-cancer immune response comprises cancer antigen-induced IL-1β secretion and activation of CD8+ T lymphocytes.

[0162] 13. The method of any one of embodiments 10 to 12, wherein the cancer is a non-hematopoietic cancer.

[0163] 14. The method of embodiment 13, wherein the non-hematopoietic cancer is carcinoma, sarcoma, or melanoma. 15. The method of any one of embodiments 10-12, wherein the cancer is lymphoma.

[0164] 16. A method for treating cancer, comprising: a) preparing an immunogenic composition comprising a tumor cell lysate, an oxidized lipid, and a toll-like receptor 4 (TLR4) agonist, the immunogenic composition being prepared or having been prepared from a tumor sample obtained from a mammalian subject with cancer; b) administering an effective amount of the immunogenic composition to a subject. A method comprising:

[0165] 17. A method of treating a mammalian subject having cancer, comprising: a) preparing an immunogenic composition comprising a tumor cell lysate, an oxidized lipid, and a toll-like receptor 4 (TLR4) agonist, the immunogenic composition being prepared or having been prepared from a tumor sample obtained from said mammalian subject with cancer; b) administering an effective amount of the immunogenic composition to a subject. A method comprising:

[0166] 18. The method of any one of embodiments 10-17, wherein the oxidized lipid comprises at least one phospholipid of oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC).

[0167] 19. The method of embodiment 18, wherein the at least one phospholipid comprises at least one of the group consisting of POVPC, PGPC, PECPC and PEIPC, and optionally the at least one phospholipid comprises PGPC.

[0168] 20. The method of any one of embodiments 10-17, wherein the TLR4 agonist comprises monophosphoryl lipid A (MPLA).

[0169] 21. The method of embodiment 20, wherein the TLR4 agonist is present in an adjuvant, the adjuvant further comprising one or more of an aluminum salt, a saponin, and a liposome, and optionally the adjuvant is AS01B or AS04.

[0170] 22. The method of any one of embodiments 10-17, wherein the at least one phospholipid comprises PGPC and the TLR4 agonist comprises monophosphoryl lipid A (MPLA).

[0171] 23. The method of any one of claims 10 to 22, further comprising administering to the subject an effective amount of an additional therapeutic agent.

[0172] 24. The method of embodiment 23, wherein the additional therapeutic agent comprises one or more of the group consisting of an immune checkpoint inhibitor, an anti-tumor agent, and radiation therapy.

[0173] 25. The method of any one of embodiments 10 to 23, wherein the cancer was resistant to immune checkpoint inhibitors prior to administration of the immunogenic composition. [Example]

[0174] Example 1: Superactive dendritic cells stimulate long-lasting antitumor immunity against complex antigen mixtures An ideal strategy for stimulating protective immunity would combine the benefits of activated and pyroptotic DCs, allowing activated cells to maintain viability while retaining the ability to release IL-1β. We recently identified a novel DC activation state that exhibits these characteristics. DCs achieve a persistent state of "hyperactivation" upon exposure to PAMPs (e.g., TLR ligands) and a group of oxidized phospholipids (DAMPs) released from dying cells (I. Zanoni et al., Science, Vol. 352, No. 6290, pp. 1232-1236, 2016; I. Zanoni et al., Immunity, Vol. 47, No. 4, pp. 697-709 e3, 2017). This group of oxidized lipids is known as oxPAPC (oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine). Superactivated DCs exhibit the activity of activated DCs with respect to cytokine release (e.g., TNFα) but have also acquired the ability to release IL-1β over several days. Consistent with their designation as "superactivated" DCs, superactivated DCs are superior to their activated counterparts in their ability to stimulate T cell responses against model antigens.

[0175] The DAMP in question (oxPAPC) can bind to and stimulate the cytosolic PRR caspase-11, defining the mechanism underlying the hyperactive state of DCs (I. Zanoni et al., 2016). Stimulation of caspase-11 leads to the activation of NLRP3 and the assembly of inflammasomes, which does not result in pyroptosis but rather in the release of IL-1β from live cells. IL-1β release from hyperactive cells is mediated by the pore-forming protein gasdermin D, which serves as a conduit for secreting these cytokines (C.E. Vavold et al., Immunity, 2018, January 16;48(1):35-44e6; X. Liu et al., Nature, Vol. 535, No. 7610, pp. 153-158, 2016; R.A. Aglietti et al., Proc. Natl. Acad. Sci. USA, Vol. 113, No. 28, pp. 7858-63, July 2016; N. Kayagaki et al., Nature, Vol. 526, No. 7575, pp. 666-671, September 2015). To remove the gasdermin D pore, the cell membrane is thought to be repaired in a manner that ensures cell viability (S. Ruehl et al., Science, Vol. 362, No. 6417, pp. 956-960, November 2018). However, in other instances (e.g., alum stimulation), membrane repair pathways are overwhelmed and pyroptosis ensues. Despite this insight into how IL-1β can be released from living cells, the physiological benefits of a hyperactive cellular state for the command of adaptive immunity remain poorly defined.

[0176] material and method Mouse strains and tumor cell lines: C57BL / 6J (Jax000664), caspase-1 / -11 dKO mice (Jax016621), NLRP3KO (Jax021302), Casp11KO (Jax024698), OT-I (Jax003831) and OT-II (Jax004194), and BALB / c (Jax000651) mice were purchased from Jackson Labs. For the syngeneic tumor model in C57BL / 6J, two melanoma cell lines were used: the parental cell line, B16.F10, and the OVA-expressing cell line, B16.F10OVA. For the syngeneic colorectal model, the OVA-expressing MC-38 cell line, derived from C57BL6 mouse colon adenocarcinoma cells, was used. These cell lines were a gift from the Arlene Sharpe Laboratory. For the syngeneic colon cancer model in BALB / c mice, the CT26 cell line was used (a gift from the Jeff Karp laboratory).

[0177] Reagents: E. coli LPS (serotype O55:B5-TLRGRADE™) was purchased from Enzo and used at 1 μg / ml for cell culture or 10 μg / mouse for in vivo use. Monophosphoryl lipid A (MPLA) from S. minnesota R595 was purchased from Invivogen and used at 1 μg / ml for cell culture or 20 μg / mouse for in vivo use. OxPAPC was purchased from Invivogen, resuspended in prewarmed serum-free medium, and used at 100 μg / ml for cell stimulation or 65 μg / mouse for in vivo use. POVPC and PGPC were purchased from Cayman Chemical. Reconstitution of commercially available POVPC and PGPC was performed as previously described (CL Evavold et al., Immunity, 2018, January 16;48(1):35-44). Briefly, the ethanol solvent was evaporated using a gentle stream of nitrogen gas. Prewarmed serum-free medium was then immediately added to the dried lipid to a final concentration of 1 mg / ml. The reconstituted lipid was incubated at 37°C for 5–10 minutes and sonicated for 20 seconds before adding to the cells. POVPC or PGPC was used at 100 μg / ml for cell stimulation or 65 μg / mouse for in vivo use. EndoFit chicken egg ovalbumin protein, with endotoxin levels <1 EU / mg, and OVA257-264 peptide were purchased from Invivogen at a concentration of 200 μg / mouse for in vivo use or 500 μg or 100 μg / ml for in vitro use. Incomplete Freud's adjuvant (F5506) was purchased from Sigma and used at a working concentration of 1:4 (IFA:antigen emulsion) for in vivo immunization. Alum, an alhydrogel, was purchased from Accurate Chemical and used at a working concentration of 2 mg / mouse for in vivo immunization. In some experiments, Addavax, a squalene-oil-in-water adjuvant, was used in place of IFA at a working concentration of 1:2 (AddVax:antigen).

[0178] Cell culture: BMDCs were generated by bone marrow differentiation in IMDM (Gibco), 10% B16-GM-CSF-derived supernatant, 2 μM 2-mercaptoethanol, 100 U / ml penicillin, 100 μg / ml streptomycin (Sigma-Aldrich), and 10% FBS. After 6 days of culture, BMDCs were washed with PBS and resuspended in IMDM containing 10% FBS at 1 × 10 6 The cells were replated in a final volume of 100 μl at a concentration of 100 cells / ml. CD11c was detected by flow cytometry using a BD Fortessa. + DC purity was assessed and was typically >80%. Splenic DCs from mice injected with B16-FLT3 for 15 days were cultured using CD11c + MHC + Purified as live cells and then cultured at 1 x 10 in complete IMDM. 6 DCs were plated at a concentration of 100 cells / ml in a final volume of 100 μl. To induce hyperactivated or pyroptotic BMDCs, DCs were primed with LPS (1 μg / ml) for 3 hours and then stimulated with OxPAPC or PGPC (100 μg / ml) or alum (100 μg / ml) in complete IMDM for 21 hours. In some cases, activated BMDCs were restimulated for an additional 24 hours on plate-bound agonist anti-CD40 using Ultra-LEAF anti-mouse CD40 (clone 1C10; BioLegend). T cells were cultured in RPMI-1640 (Gibco) supplemented with 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin (Sigma-Aldrich), and 50 μM β-mercaptoethanol (Sigma-Aldrich). All tumor cell lines were cultured in DMEM supplemented with 10% FBS. For OVA-expressing cell lines, puromycin (2 μg / ml) was added to the medium.

[0179] LDH assay and ELISA: After BMDC stimulation, fresh supernatants were clarified by centrifugation and then subjected to LDH release assays using the Pierce LDH Cytotoxicity Colorimetric Assay Kit (Life Technologies) according to the manufacturer's protocol. Absorbance measurements were performed at wavelengths of 490 nm and 680 nm using a Tecan plate reader. To measure secreted cytokines, supernatants were collected, clarified by centrifugation, and stored at -20°C. ELISAs for IL-1β, TNFα, IL-10, IL-12p70, IFNγ, IL-2, IL-13, IL-4, and IL-17 were performed using eBioscience Ready-SET-Go! (ThermoFisher) ELISA kits according to the manufacturer's protocol.

[0180] Flow cytometry: After FcR blockade, day 7 BMDCs were resuspended in MACS buffer (PBS containing 1% FCS and 2 mM EDTA) and stained with the following fluorescently labeled antibodies (BioLegend): anti-CD11c (clone N418), anti-IA / IE (clone M5 / 114.15.2), anti-CD40 (clone 3 / 23), anti-CD80 (16-10A1), anti-CD69 clone (H1.2F3), and anti-H-2Kb (clone AF6-88.5). Single-cell suspensions from tumors, draining inguinal lymph nodes, or skin-inguinal adipose tissue were resuspended in MACS buffer (PBS containing 1% FCS and 2 mM EDTA) and stained with the following fluorescently labeled antibodies (BioLegend): anti-CD8α (clone 53-6.7), anti-CD4 (clone RM4-5), anti-CD44 (clone IM7), anti-CD62L (MEL-14), anti-CD3 (17A2), anti-CD103 (2E7), anti-CD69 clone (H1.2F3), and anti-CD45 (A20 or 30F11). To determine cell viability, cells were stained in PBS at 4°C for 20 minutes using the LIVE / DEAD™ Fixable Violet Dead Cell Stain Kit (Molecular Probes). T cells from draining inguinal lymph nodes were stained with OVA peptide tetramer for 1 hour at room temperature. PE-conjugated H2K(b)SIINFEKL (OVA257-264; SEQ ID NO: 1) and APC-conjugated IA(b)AAHAEINEA (OVA329-337; SEQ ID NO: 2) were used. IA(b) and H2K(b) associated with the CLIP peptide were used as isotype controls. Tetramers were purchased from the NIH Tetramer Core Facility. In some experiments, FITC anti-CD8.1 (clone Lyt-2.1 CD8-E1) purchased from Accurate Chemical was used with the tetramers. To determine absolute cell numbers, COUNTBRIGHT counting beads (Molecular probes) were used according to the manufacturer's protocol. Appropriate isotype controls were used as staining controls. Data were acquired using a BD FACS ARIA or BD Fortessa. Data were analyzed using FlowJo software.

[0181] Antigen uptake assay: FITC-labeled chicken OVA (FITC-OVA) (Invitrogen-Molecular Probes) was used to examine the antigen uptake and endocytosis capacity of BMDCs in different activation states (activated, superactivated, or pyroptotic). Briefly, pretreated BMDCs were incubated with either FITC-OVA or AF488-dextran (0.5 mg / ml) for 45 min at 37°C or 4°C (as a control for antigen surface binding). BMDCs were then washed and stained with the Live / Dead Fixable Violet Dead Cell Stain Kit (Molecular Probes) to distinguish live from dead cells. Cells were then fixed with BD fixative and resuspended in MACS buffer (PBS containing 1% FCS and 2 mM EDTA). FITC fluorescence of live cells was measured every 15 min using a Fortessa flow cytometer (Becton-Dickenson). Fluorescence values ​​of BMDCs incubated at 37°C were reported as the percentage of OVA-FITC- or Dextran-AF488-associated cells, and data were normalized to the percentage of OVA-FITC-associated cells incubated at 4°C.

[0182] OVA antigen presentation assay: To measure the efficiency of OVA antigen presentation on MHC-I, 0.5 × 10 cells were treated with activation (LPS), superactivation (LPS + PGPC or LPS + OxPAPC), or pyroptotic stimulation (LPS + alum). 6 BMDCs were incubated with Endofit-OVA protein (0.5 mg / ml) at 37°C for 2 hours. The cells were then washed with MACS buffer and incubated on ice for 20-30 minutes with APC anti-mouse H-2K. b H-2K bound to antibody (clone AF6-88.5, BioLegend) and OVA peptide SIINFEKL b The cells were stained with a PE-conjugated antibody (SEQ ID NO: 1; clone 25-D1.16, BioLegend) that binds to total surface H-2K. Appropriate isotype controls were used as staining controls. bThe percentage of cells expressing OVA peptide on MHC-I was calculated. Data were acquired using a Fortessa flow cytometer (Becton-Dickenson) and analyzed with FlowJo software (Tree Star).

[0183] In vitro T cell stimulation of OT-I and OT-II: splenic CD8 + T cells and CD4 + T cells were sorted from OT-I and OT-II mice by magnetic cell sorting using anti-CD8 or anti-CD4 beads (Miltenyi Biotech), respectively. Sorted T cells were then seeded onto 96-well plates at a concentration of 100,000 cells per well in the presence of 20,000 or 10,000 DCs (5:1 or 10:1 ratios) pretreated with either LPS (activating stimulus), LPS + PGPC (superactivating stimulus), or LPS + alum (pyroptotic stimulus) and pulsed (or not) with OVA protein or SIINFEKL (SEQ ID NO: 1) peptide at 100 μg / ml for 2 hours. After 5 days of culture, supernatants were collected and clarified by centrifugation for short-term storage at -20°C and cytokine measurement by ELISA.

[0184] Intracellular Staining: For intracellular cytokine staining, cells were stimulated with 50 ng / ml phorbol 12-myristate 13-acetate (PMA) and 500 ng / ml ionomycin (Sigma-Aldrich) for 4–5 h in the presence of GolgiStop (BD) and Brefeldin A. Cells were then washed twice with PBS and stained with the LIVE / DEAD™ Fixable Violet or Green Dead Cell Stain Kit (Molecular probes) in PBS for 20 min at 4°C. Cells were washed with MACS buffer and stained for the appropriate surface markers for 20 min at 4°C. After two washes, cells were fixed and permeabilized using the BD Cytofix / Cytoperm kit according to the manufacturer's protocol for 20 min at 4°C, then washed with 1X permeabilization wash buffer (BD). Intracellular cytokine staining was performed in 1X permeabilization buffer at 4°C for 20–30 min using the following conjugated antibodies, all purchased from BioLegend: anti-Ki67 (clone 16A8), anti-IFN-γ (clone XMG1.2), anti-TNFα (clone MP6-XT22), anti-Gata3 (16E10A23), anti-IL4 (11B11), and anti-IL10 (clone JES5-16E3). Data were acquired using a BD FACS ARIA or BD Fortessa. Data were analyzed using FlowJo software.

[0185] In vivo immunization and T cell costimulation: Eight-week-old female C57BL / 6J mice were immunized subcutaneously (sc) in the left lower back with 150 μg / mouse endotoxin-free OVA emulsified in incomplete Freud's adjuvant plus 10 μg / mouse LPS, or 150 μg / mouse endotoxin-free OVA emulsified in incomplete Freud's adjuvant plus 65 μg / mouse oxPAPC or PGPC plus 10 μg / mouse LPS. In some experiments, mice were injected sc with OVA alone or with LPS emulsified in alum. Seven or 40 days after immunization, CD4 T cells were isolated from the draining lymph nodes of immunized mice by magnetic cell sorting using anti-CD4 or anti-CD8 beads and columns (Miltenyi Biotech).+ T cells and CD8 + T cells were isolated and the enriched cells were then analyzed using a FACS ARIA for CD45 + CD3 + CD4 + Live cells or CD45 + CD3 + CD8 + Viable cells were sorted. The purity after sorting was >98%. Sorted cells were then plated onto 96-well plates at a concentration of 100,000 cells per well, 10–20 × 10 cells pulsed with serial dilutions of OVA starting at 1 mg / ml. 3 After 5 days, secretion of IFNγ, IL-10 and IL-2 was measured by ELISA.

[0186] CD107a degranulation assay: CD8 + To assess the effector antitumor activity of T cells, we assessed the surface exposure of the lysosome-associated protein CD107a by flow cytometry. Briefly, CD8 T cells were isolated from the skin-draining lymph nodes of immunized mice by magnetic cell enrichment using anti-CD8 beads and columns (Miltenyi Biotech). + T cells were isolated and then analyzed using a FACS ARIA (BD) for CD3 + CD8 + Sorted as live cells. Freshly sorted CD8 + T cells were cultured at 1 x 10 in complete RPMI. 6 The T cells were resuspended at a concentration of 100,000 cells / ml. PerCP / Cy5.5 anti-mouse CD107a (LAMP-1) antibody (clone 1D4B, BioLegend) was added to the medium at a concentration of 1 μg / ml in the presence of GolgiStop (BD). The T cells were then immediately seeded at 100,000 cells onto 10,000 MC38OVA or B16OVA tumor cells / well in a 96-well plate. Alternatively, CD8 +T cells were plated alone and stimulated with 50 ng / ml phorbol 12-myristate 13-acetate (PMA) and 500 ng / ml ionomycin (Sigma-Aldrich). After 5 hours of culture, cells were washed with MACS buffer and stained with the LIVE / DEAD™ Fixable Violet Dead Cell Stain Kit (Molecular probes) and APC anti-CD8 (clone 53-6.7, BioLegend). Cells were then fixed with BD fixative for 20 minutes at 4°C and resuspended in MACS buffer. CD107a and CD107b were detected by flow cytometry using a Fortessa flow cytometer (BD). + The percentage of cells was determined.

[0187] In vitro cytotoxicity assay: CD8 derived from spleen or skin-inguinal adipose tissue of survivor mice + T cells were isolated using anti-CD8 MACS beads and columns (Miltenyi Biotec). Enriched T cells were then analyzed using a FACS ARIA for CD45 + CD3 + CD8 + Viable cells were sorted. The purity after sorting was >97%. Tumor cell lines, e.g., B16OVA, B16F-10, or CT26 cells, were plated in 96-well plates (2 × 10 cells) at least 5 hours before co-culture with T cells. 4 Cells were seeded in complete DMEM on 10 wells (10 cells / well). 5 CD8 + Twelve hours after seeding the T cells onto the tumor cells, cytotoxicity was assessed by LDH release assay using the Pierce LDH Cytotoxicity Colorimetric Assay Kit (Life Technologies) according to the manufacturer's protocol.

[0188] Preparation of whole tumor cell lysates: To prepare whole tumor cell lysates (WTL) for immunization, tumor cell lines were cultured in complete DMEM for 4–5 days. When cells reached confluence, the supernatant was collected, and the cells were washed and dissociated using trypsin-EDTA (Gibco). Then, tumor cell lines were cultured in the collected culture supernatant at 5 × 106 Cells were resuspended at 1000 cells / ml and lysed by three freeze-thaw cycles.

[0189] Syngeneic whole tumor lysates of melanoma or colon adenocarcinoma tumors were prepared from tumor explants in naive tumor-bearing mice. Briefly, tumors were mechanically disaggregated using a gentleMACS dissociator (Miltenyi Biotec) and then heated in a 42°C water bath for 15 minutes. They were then digested using a tumor dissociation kit (Miltenyi Biotec) according to the manufacturer's protocol. After digestion, tumors were washed with PBS, passed through a 70-μm filter and a 30-μm filter, and then purified using CD45 microbeads (Miltenyi Biotec). + The cells were removed. Tumor cells were added at 5 × 10 6 The WTL preparations were centrifuged at 12,000 rpm for 15 minutes, passed through 70 μm and 30 μm filters, and stored in aliquots at -20°C until use. 2.5 × 10 cells / ml per mouse were used as described in the next section. 5 WTLs were used for immunization, immunotherapy or DCs at a concentration equivalent to that of tumor cells.

[0190] In vivo immunization and tumor challenge: For pre-tumor immunization, C57BL / 6 mice were injected subcutaneously (sc) into the right flank with PBS (naive), WTL alone or with LPS, or WTL plus LPS and OxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA). In some experiments, LPS was replaced with MPLA. 15 days after immunization, mice received 3 × 10 immunizations as indicated in the left flank. 5 B16OVA live cells, or 3 x 10 5 viable B16-F10 cells, or 5 x 10 5 Tumor-free mice were challenged sc with live MC38-OVA cells. Tumor-free mice were challenged sc with a lethal dose of 5 × 10 cells in the upper back as indicated. 51 x 10 live B16OVA cells or B16F-10 live cells, or 1 x 10 6 Mice were rechallenged sc with live MC38-OVA cells. In some experiments, mice received 100 μg of LEAF anti-mouse / rat IL-1β antibody (BioLegend) by intravenous injection 2 and 1 days before immunization. Antibody treatment continued 1, 2, and 3 days after immunization to ensure chronic clearance of circulating IL-1β.

[0191] For immunization of immunotherapeutic approaches, C57BL / 6J mice were immunized with 3 × 10 5 B16OVA live cells, or 3 x 10 5 viable B16-F10 cells, or 5 x 10 5 Instead, BALB / c mice were injected with 5 × 10 live MC38-OVA cells in the left flank. 5 Each mouse was injected with live CT26 cells. Following tumor inoculation, mice were left untreated (unimmunized) or immunized with WTL plus LPS and PGPC emulsified in incomplete Freund's adjuvant (IFA), as indicated. Immunization was followed by two boost injections, as indicated by the immunization schedule at the top of each survival graph. Where indicated, mice received 100 μg of antibody intraperitoneally (ip) on the same day of immunization or boost injection, followed by four injections every three days using the following antibodies: anti-PD-1 (clone 29F.1A12), Ultra-LEAF anti-CD4 (clone GK1.5), and anti-CD8a (clone 53-6.7). Alternatively, mice received 100 μg of LEAF anti-mouse / rat IL-1β antibody by iv injection two and one days before receiving the immunization / boost. To ensure chronic clearance of circulating IL-1, anti-IL-1β treatment was continued on days 1, 2, and 3 after immunization as described above. Control mice received isotype-matched rat IgG. All antibodies were purchased from BioLegend.

[0192] Tumor size was assessed every 2 days in a blinded, coded fashion and measured as tumor area (length x width) using a caliper. Mice were randomly assigned to receive a tumor with a size greater than 2 cm.3 The animals were sacrificed when they reached the maximum temperature or when ulcers formed.

[0193] In vivo immunization and B16-F10 lung colonization: To induce experimental lung colonization, 3 × 10 5 B16-F10 tumor cells were injected intravenously via the tail vein in a volume of 100 μl. Two days before tumor inoculation, mice were left untreated (naive) or immunized sc in the right flank with WTL alone, with LPS, or with WTL plus LPS and PGPC, all emulsified in Addavax. Mice received a boost injection 5 days after tumor inoculation. Mice were then sacrificed 18 days after tumor injection, and lung tissue was isolated and fixed in Fekete's solution. The number of metastatic lung nodules present on the lung surface for each mouse was counted.

[0194] Tumor infiltration: To assess the frequency of tumor-infiltrating lymphocytes (TILs) in immunized mice, tumors were harvested when they reached 1.8–2 cm in size. Tumors were dissociated using a tumor dissociation kit (Milteny Biotec) and a gentleMACS dissociator according to the manufacturer's protocol. After digestion, tumors were washed with PBS and passed through a 70 μm filter and a 30 μm filter. CD45 microbeads (Milteny Biotec) were used to dissociate tumors. + Cells were positively selected, and T cell infiltration was assessed by flow cytometry. Tumor-infiltrating T cells were cultured with Dynabeads Mouse T Activator CD3 / CD28 (Gibco) to activate and expand T cells.

[0195] Adoptive cell transfer: For T cell transfer, CD8 T cells derived from the spleen or skin and inguinal adipose tissue of survivor mice were used. + T cells were isolated using anti-CD8 MACS beads and columns (Milteny Biotec). The enriched cells were then analyzed using a FACS ARIA for CD45 + CD3 + CD8 +Viable cells were sorted. The purity after sorting was >97%. The sorted T cells were then plated onto 24-well plates (~2 × 10 cells) coated with anti-CD3 (4 μg / ml) and anti-CD28 (4 μg / ml). 6 5 × 10 cells / well) and stimulated for 24 h in the presence of IL-2 (50 ng / ml). 5 Activated circulating splenic CD8 + T cell or cutaneous inguinal fat resident CD8 + T cells were transferred into naive recipient mice by iv or intradermal (id) injection, respectively, with some mice receiving both T cell subsets.

[0196] Statistical analysis: Statistical significance for experiments with three or more groups was tested by two-way analysis of variance with Tukey's multiple comparison test correction. Adjusted p values ​​calculated using Prism (Graphpad) are indicated by an asterisk: <0.05 ( * );<0.0005( *** );≦0.0001( **** )

[0197] result Superactivating stimuli upregulate several activities important for DCs to stimulate T cell immunity Virtually all studies of DC superactivation have focused on the ability of DCs to release IL-1β while maintaining viability. The extent to which DC function is affected by superactivating stimuli has not been defined. To investigate this scope, bone marrow-derived DCs (BMDCs) were primed with LPS and subsequently treated with oxPAPC or a specific, pure lipid component of oxPAPC called PGPC (I. Zanoni et al., Science, Vol. 352, No. 6290, pp. 1232–1236, 2016). The resulting superactivated cells were compared with conventionally activated BMDCs (treated with LPS) or pyroptotic BMDCs (primed with LPS and then treated with alum). In contrast to the activating stimuli that did not induce IL-1β release as expected, pyroptotic or superactivating stimuli promoted IL-1β release into the extracellular medium (Figure 1A). All stimuli examined promoted secretion of the cytokine TNFα (Figure 1A). This finding is consistent with previous studies (I. Zanoni et al., 2016) that established that LPS-activated BMDCs release TNFα but not IL-1β. IL-1β secretion, assessed by the release of the cytosolic enzyme lactate dehydrogenase (LDH) (Figure 1B), coincided with cell death in pyroptotic DCs. In contrast, IL-1β secretion occurred in the absence of LDH release in superactivated cells (Figure 1B). Similar behavior of BMDCs was observed when LPS was replaced with MPLA, an FDA-approved TLR4 ligand used in vaccines against human papillomavirus (HPV) and hepatitis B virus (HBV) (Figures 5A and 5B). To determine whether the behavior of superactivated BMDCs extended to differentiated DCs in vivo, we investigated the activity of CD11c ... + Similar to the behavior of GMCSF-derived BMDCs, treatment with LPS and PGPC increased splenic CD11c activity in the absence of cell death as assessed by LDH release. + This resulted in the release of TNFα and IL-1β from DCs (Figures 5C and 5D). These results demonstrate that the superactivating stimulator PGPC can be used to induce IL-1β release from viable DCs differentiated in vitro or in vivo.

[0198] We examined several signals important for T cell differentiation, such as the expression of the costimulatory molecules CD80, CD69, and CD40, as well as the secretion of the p70 subunit of IL-12. Surface expression of CD80 was similar in DCs responding to all activating stimuli (Figure 5E). In contrast, CD40 expression was significantly affected by the activating stimuli. Compared with the activating stimulus LPS, superactivating stimuli significantly induced CD40 expression (Figure 1C). Pyroptotic stimuli were very weak inducers of CD40 and CD69, even within the range of 20–30% of viable cells after LPS-alum treatment (Figure 1C and Figure 5E). When cultured on agonist anti-CD40-coated plates, differential expression of CD40 correlated with superactivating DCs with the greatest ability to secrete IL-12p70 (Figure 1D).

[0199] Although superactivated BMDCs were not superior to their activated counterparts in antigen capture, as assessed by comparable internalization of fluorescent ovalbumin (OVA-FITC) (Figures 6A and 6B), the former cell populations displayed significantly higher amounts of the OVA-derived SIINFEKL peptide on cell surface MHC-I molecules (Figures 1E and 6C). The total amount of surface MHC-I did not differ between activated and superactivated cells (Figure 5E). Collectively, compared with other stimuli of DCs, superactivated BMDCs exhibit enhanced several activities important for T cell differentiation.

[0200] Superactive DCs stimulate a TH1-focused immune response without evidence of TH2 immunity To assess the effect of DC activation state on T cell command, BMDCs were treated as described above and then loaded with OVA. These cells were then exposed to naive OT-II or OT-I T cells. OT-II cells express T cell receptors (TCRs) specific for the MHC-II-restricted OVA peptide (OVA323-339), whereas OT-I cells express TCRs specific for the MHC-I-restricted OVA peptide (OVA257-264) (KA Hogquist et al., Cell, Vol. 76, No. 1, pp. 17-27, January 1994; MJ Burnden et al., Immunol. Cell Biol., Vol. 76, No. 1, pp. 34-40, February 1998). To identify T cell polarization toward a TH1 response (IFNγ production) or a TH2 response (IL-10, IL-4, or IL-13 production), the activity of responding T cells was assessed by ELISA. Regardless of DC activation status, OVA-treated BMDCs stimulated IFNγ production from OT-II T cells. The magnitude of IFNγ production varied slightly among the activation stimuli tested (Fig. 1F). Similarly, TNFα production by OT-II cell responses was comparable across all DC activation statuses (Fig. 1F). These results indicate that in vitro TH1 responses are generally induced regardless of the activation status of antigen-presenting cells (APCs). In contrast, TH2 responses differed significantly across DC activation statuses. Stimuli inducing BMDC activation (LPS) or pyroptosis (LPS + alum) promoted the release of large amounts of IL-10 and IL-13, whereas superactivating stimuli resulted in minimal production of TH2-associated cytokines (Figure 1F). Intracellular staining of single cells for TH1 cytokines (IFNγ and TNFα) and TH2 cytokines (IL-4 and IL-10) and the TH2 lineage-defining transcription factor GATA3 allowed calculation of the ratio of TH1 to TH2 cells generated by different DC activation stimuli. This analysis revealed that superactivating BMDCs induced a significant bias of individual T cells toward the IFNγ-producing TH1 lineage (Figure 1G and Figure 7). The ratio of TH1 to TH2 cells under superactivating conditions was greater than 100:1 (Figure 1G).In contrast, all other activating stimuli induced mixed T cell responses, with pyroptotic stimulation resulting in a nearly 1:1 ratio of TH1 to TH2 cells (Fig. 1G).

[0201] CD8 + Similar studies using OT-I T cells revealed a slight enhancement of IFNγ production by superactivating BMDCs compared with activating or pyroptotic stimulation (Figure 1F). IL-2 production in response to OT-I cells was comparable across all DC activation states (Figure 1F). Together, these results indicate that the superactivating BMDC state in vitro results in a markedly TH1-biased T cell response and a slightly enhanced CD8 T cell response. In contrast, activating or pyroptotic stimulation resulted in a mixed TH1 and TH2 response.

[0202] To confirm whether the in vitro observations using superactivated BMDCs and OT-I and OT-II T cells correspond to an endogenous scenario in vivo, we investigated the ability of superactivating stimuli to promote antigen-specific T cell responses in vivo. Mice were immunized with either OVA alone, together with an activating stimulus (LPS), together with a superactivating stimulus (LPS + oxPAPC or PGPC), or together with a pyroptotic stimulus (LPS + alum or alum alone). Forty days after immunization, CD4 + T cells or CD8 + T cells were restimulated ex vivo with naive BMDCs loaded with or without OVA. Immunization with the superactivating stimulus resulted in greater IFNγ production by T cell responses than immunization with LPS or pyroptotic stimuli (LPS + alum) (Figure 1H). Notably, IL-10, IL-4, or IL-13 were not produced by T cells from mice immunized with the superactivating stimulus (Figure 1H and Figure 7). Thus, similar to the in vitro observations using transgenic T cells, the superactivating stimulus induced a TH1-biased T cell response and CD8 T cell proliferation in vivo from endogenous T cells.+ It can induce T cell responses.

[0203] In contrast to the findings with the superactivating stimulus, the activating stimulus (LPS) resulted in a mixed TH1 and TH2 phenotype, with T cells producing IFNγ, IL-10, IL-4, and IL-13 (Figure 1H and Figure 7). Immunization with OVA and alum alone resulted in a significantly biased IL-13 response, and no IFNγ was detected in the T cell response (Figure 1H). These results are consistent with previous studies showing that alum is particularly weak at promoting type I immunity (E. Oleszycka et al., Eur. J. Immunol., Vol. 48, No. 4, pp. 705-715, April 2018; M. J. Newman et al., J. Immunol., Vol. 148, No. 8, pp. 2357-2357, April 1992) and that alum induces a T2-biased immune response (M. Kool et al., J. Exp. Med., Vol. 205, No. 4, pp. 869-882, April 2008; T. Marichal et al., Nat. Med., Vol. 17, No. 8, pp. 996-1002, August 2011).

[0204] One difference between LPS and LPS + oxPAPC is the latter's ability to induce IL-1β secretion, as previously described. Because alum and oxPAPC treatment result in NLRP3-dependent IL-1β release (L. Franchi and G. Nunez, Eur. J. Immunol., Vol. 38, No. 8, pp. 2085-2089, August 2008; H. Li et al., J. Immunol., Vol. 178, No. 8, pp. 5271-5276, April 2007), we asked whether LPS + alum immunization could phenocopy the T cell responses induced by LPS + oxPAPC or PGPC immunization. The answer to this question was no. LPS + alum immunization resulted in a balanced TH1:TH2 response, with ex vivo stimulated T cells producing large amounts of IFNγ, IL-10, IL-4, and IL-13 (Figure 1H and Figure 7). These results indicate that not all NLRP3 agonists stimulate a TH2-focused immune response.

[0205] Superactive stimulation enhances the generation of memory T cells and enhances antigen-specific IFNγ effector responses in an inflammasome-dependent manner We hypothesized that the enhanced T cell responses induced by superactivating stimuli could be explained by the generation of memory T cells and enhanced effector memory T cell responses. To investigate this possibility, mice were immunized with OVA alone, OVA plus an activating stimulus (LPS), or OVA plus a superactivating stimulus (LPS plus oxPAPC or PGPC). Seven and 40 days after immunization, CD44 低 CD62L 低 effector T cells (Teff) and CD44 高 CD62L 低 Effector memory T cells (TEMs) and CD44 高 CD62L 高 Memory and effector T cell generation in the dLN was assessed by flow cytometry using the CD62L and CD44 markers, which distinguish T cells from central memory T cells (TCM) (SZ Ben-Sasson, Cold Spring Harb. Symp. Quant. Biol., Vol. 78, No. 0, pp. 117-124, January 2013). Seven days after immunization, the superactivating stimulus was administered to the dLNs, resulting in the generation of CD4 + Teff cells and CD8 + The superactivating stimulus was superior to the activating stimulus in inducing Teff cells (Fig. 2A, upper panel and Fig. 8A, Fig. 8B). At this early time point, both stimuli induced comparable, albeit smaller, amounts of TEM cells (Fig. 2A, middle panel and Fig. 8A, Fig. 8B). Forty days after immunization, mice exposed to the superactivating stimulus had ample TCM cells, whereas mice immunized with OVA alone or LPS coimmunized with OVA had fewer TCM cells (Fig. 2A, lower panel). These data indicate that the superactivating stimuli oxPAPC and PGPC enhance the magnitude of effector and memory T cell generation. The increased frequency of Teff cells 7 days after immunization was observed in CD4 T cells isolated from the dLN of immunized mice and restimulated ex vivo in the presence of OVA-loaded naive BMDCs. + T cells and CD8 +This correlated with an enhanced T cell IFNγ response (Fig. 8C, Fig. 8D). + T cells exhibit increased degranulation capacity, an activity associated with functional CTLs (Figure 2B). + T cells isolated from mice immunized with a superactivating stimulus and co-cultured with the OVA-expressing B16 tumor cell line (B16OVA) expressed CD8 + T cells were isolated from mice immunized with OVA alone or OVA + LPS. + They exhibited enhanced degranulation activity compared to T cells (Fig. 2B, Fig. 8E).

[0206] To compare the antigen specificity of T cells resulting from immunization with activating, pyroptotic, or superactivating stimuli, mice were injected with OVA alone or together with activating stimuli (LPS), pyroptotic stimuli (LPS + alum), or superactivating stimuli (LPS + oxPAPC or PGPC). Alternatively, mice were immunized with LPS + PGPC without the OVA antigen. Seven days after immunization, CD4 T cells were isolated from the skin dLN of immunized mice. + T cells and CD8 + T cells were isolated and restimulated ex vivo for 7 days with OVA-loaded (or unloaded) naive BMDCs to enrich for OVA-specific T cell subsets. T cell effector function of OVA-specific T cells was assessed by intracellular staining for IFNγ. TCR specificity was assessed by staining with MHC-restricted OVA peptide tetramers. H2 kb The restricted SIINFEKL (OVA257-264) peptide and the IA(d)OVA peptide (OVA329-337) tetramer were used. + IFNγ + The frequency of double-positive cells was determined by CD4 + T cell subsets and CD8 + As expected, T cells isolated from mice immunized with LPS+OVA resulted in an in vitro enrichment of OVA-specific T cells, with a higher CD8 T cell count compared to mice immunized with OVA alone. +IFNγ effector function of T cells was induced (Fig. 2C, upper panel). + T cells showed higher OVA-specific IFN+ T cells in the presence of a high OVA antigen load compared with T cells from mice immunized with OVA alone (Fig. 2C, lower panel).

[0207] Notably, OVA with superactivating stimuli (LPS + OxPAPC or PGPC) was superior in inducing antigen-specific T cells, and OVA antigen was associated with CD4 + T cells or CD8 + Restimulation of T cells results in a higher frequency of tetramers. + IFNγ + The Pyroptotic stimulation (LPS + alum) resulted in an antigen-specific IFNγ response (Figure 2C). Pyroptotic stimulation (LPS + alum) was the weakest inducer of the antigen-specific IFNγ response (Figure 2C). These results are consistent with those described herein and elsewhere, which show that alum is an effective adjuvant for promoting humoral immunity and TH2 responses but not TH1 or CTL responses (M.J. Newman et al., J. Immunol., Vol. 148, No. 8, pp. 2357-2357, April 1992; J.M. Brewer et al., J. Immunol., Vol. 163, No. 12, pp. 6448-644, December 1999; A. Mori et al., Eur. J. Immunol., Vol. 42, No. 10, pp. 2709-2719, October 2012).

[0208] Previous studies have shown that recombinant IL-1β enhances antigen-specific T cell responses during immunization and increases the potency of weak vaccines (SZBen-Sasson, K. et al., Cold Spring Harb. Symp. Quant. Biol., Vol. 78, No. 0, pp. 117-124, January 2013; SZBen-Sasson et al., J. Exp. Med., Vol. 210, No. 3, pp. 491-502, March 2013). To determine whether the antigen-specific T cell responses induced by superactive stimulation depend on the inflammasome-IL-1β axis, we performed a randomized controlled trial of WT mice versus NLRP3 mice. - / -A controlled comparison of T cell activity following immunization with superactive stimuli was performed in mice. The superactive conditions were characterized by the expression of NLRP3 - / - In mice, tetramers were reduced compared to WT mice. + IFNγ + These results demonstrate that NLRP3 activation is critical for antigen-specific T cell generation and effector function under hyperactive conditions.

[0209] A recent study on bacterial infection showed that white adipose tissue constitutes a storage compartment for antigen-specific memory T cells that persist for several months after antigen contraction (S.-J. Han et al., Immunity, Vol. 47, No. 6, pp. 1154-1168 e6, 2017). To test the ability of different DC activation stimuli to induce memory T cells in subcutaneous adipose tissue, mice were subcutaneously immunized with OVA and different activation stimuli as described above. Eight days after immunization, CD44 expression in the cutaneous inguinal adipose tissue was significantly elevated. + The presence of memory T cells was assessed by flow cytometry. Interestingly, the superactivating condition resulted in higher CD44 expression compared to the activating stimulus. + The frequency of memory T cells was induced (Figure 2D). Notably, when mice were immunized with OVA and pyroptotic stimulation, no T cells were detected in the fat compartment (Figure 2D). Furthermore, the accumulation of memory T cells depends on the activation of the NLRP3 inflammasome, because NLRP3 - / - Mice are CD44 + These results suggest that memory T cells generated by superactive stimulation are not only restricted to the cutaneous dLN of immunized mice, but also induce memory T cells in the subcutaneous adipose tissue compartment.

[0210] Overall, these data suggest that superactivating stimuli significantly biased functional memory CD4 + T cells and CD8 + Provides evidence for generating large populations of T cells.

[0211] Superactive DCs can use complex antigen sources to stimulate T cell-mediated antitumor immunity Based on these findings, it was reasoned that superactive stimulation may be particularly useful in strategies where immunization has had difficulty achieving clinical (or preclinical) benefit. One area of ​​interest relates to cancer immunotherapy. Current efforts to stimulate antitumor immunity include strategies that stimulate resident T cell populations (e.g., PD-1 blockade) or personalized cancer vaccine strategies that stimulate the induction of de novo T cell responses against tumor-specific antigens (TSAs) (REF). The latter approach has been hampered by the unavailability of tumor cell lysates, a source of TSAs, also known as neoantigens. Therefore, efforts are underway to advance the identification of neoantigens that can be used in pure form to induce T cell-mediated antitumor immunity. Although these efforts have yielded success (DB Keskin et al., Nature, Vol. 565, No. 7738, pp. 234-239, January 2019; Z. Hu, P.A. Ott, and C.J. Wu, Nat. Rev. Immunol., Vol. 18, No. 3, pp. 168-182, December 2017; P.A. Ott et al., Nature, Vol. 547, No. 7662, pp. 217-221, 2017), the path to neoantigen identification requires routes to discover mutant and aberrantly expressed TSAs (C.M. Laumont et al., Sci. Transl. Med., Vol. 10, No. 470, p. 5516, December 2018), which is challenging and does not represent the natural history of events. Naturally, DCs never encounter pure antigens but rather stimulate T cell responses in complex environments.

[0212] Because superactivated DCs are excellent stimulators of T cell responses, it was reasoned that superactivated stimulation might circumvent the need for neoantigen identification and allow the use of whole tumor cell lysates (WTLs) as an antigen source. WTLs serve as an attractive alternative source of antigens because they offer a range of mutant and aberrantly expressed TSAs and might allow the generation of a broad repertoire of T cells specific for tumor-associated antigens.

[0213] To address the possibility that superactivating stimuli might support WTL, mice were immunized in the right flank with WTL alone, WTL mixed with the activating stimuli LPS, or WTL mixed with the superactivating stimuli LPS + oxPAPC or LPS + PGPC. The source of WTL was OVA-expressing B16 melanoma cells (B16OVA). Fifteen days after immunization, mice were subcutaneously (sc) challenged with B16OVA parental cells in the upper left back. Naive mice or mice immunized with WTL alone did not exhibit any protection, and all mice contained large tumors and died by 24 days after tumor inoculation (Figure 3A). Similarly, WTL + LPS immunization offered minimal protection. Although two of eight mice immunized with WTL + LPS were tumor-free, they relapsed quickly after rechallenge with B16OVA (Figure 3A), indicating that a simple DC-activating stimulus did not confer protective immunity. In contrast, WTL immunization in the presence of LPS and oxPAPC induced a significant delay in tumor growth and provided significant protection against subsequent lethal rechallenge with B16OVA parental tumor cells, with 50% of immunized mice being completely protected (Figures 3A and 9A).

[0214] To determine whether the protective responses induced by the superactivating stimuli correlated with T cell responses, tumors were harvested from mice that received each activating stimulus. Tumors from mice immunized with the superactivating stimulus contained substantially higher amounts of CD4 + T cells and CD8 + The tumors contained T cells (Figure 3B). In response to anti-CD3 and anti-CD28 stimulation, enriched T cells from these tumors secreted large amounts of IFNγ (Figure 3B). Thus, the predominant restriction of tumor growth induced by the superactivating stimulus (LPS + oxPAPC) was consistent with the infiltration of inflammatory T cells into the tumor.

[0215] Notably, the protective phenotype induced by PGPC, a pure oxPAPC component, was superior to that of oxPAPC. WTL immunization in the presence of LPS + PGPC rendered 100% of mice tumor-free for 150 days after tumor challenge. These mice completely rejected a lethal rechallenge with B16OVA cells and remained tumor-free for 300 days after the initial tumor challenge (Figures 3A and 9A).

[0216] Among memory T cell subsets, resident memory T cells (TRM) are defined by the expression of the CD103 integrin in addition to the C-type lectin CD69, which contributes to their resident properties in peripheral tissues (REF). + TRM cells have recently attracted much attention because they accumulate at tumor sites in various human cancer tissues and correlate with more favorable clinical outcomes (J.R. Webb et al., Clin. Cancer Res., Vol. 20, No. 2, pp. 434-444, January 2014; F. Djenidi et al., J. Immunol., Vol. 194, No. 7, pp. 3475-3476, April 2015; S.L. Park et al., Nature, Vol. 565, No. 7739, pp. 366-371, January 2019). In experimental cutaneous melanoma models, cutaneous CD8 + TRM cells promoted sustained protection against melanoma progression.

[0217] We examined the presence of TRM cells at the tumor injection site as well as in immunized skin biopsies of survivor mice previously immunized with the superactivating stimulus LPS+PGPC. Interestingly, 200 days after tumor inoculation, CD8 + CD69 + CD103 +TRM cells were highly enriched at the tumor injection site but were scarce at the immunization site in all survivor mice (Figure 3C and Figure S10A). These data are consistent with clinical and experimental reports linking the presence of high levels of TRM to long-term tumor control, potentially resulting from the long-term maintenance of TRM at the tumor injection site (SL Park et al., Nature, Vol. 565, No. 7739, pp. 366-371, January 2019; CM Koebel et al., Nature, Vol. 450, No. 7171, pp. 903-907, December 2007).

[0218] CD8 + TRM cells accumulate in white adipose tissue after antigen contraction and are recruited to the site of infection upon secondary challenge (S.-J. Han et al., Immunity, Vol. 47, No. 6, pp. 1154-1168 e6, 2017). We analyzed the T cell compartment in the subcutaneous adipose tissue surrounding the inguinal LN draining from the immunization site. We found high frequencies of CD8 + T cells were observed, and antigen-specific CD8 T cells were also observed in the adipose tissue of mice immunized with WTL and the superactivating stimulus LPS + PGPC. + TRM cells were observed in the inguinal adipose tissue of unimmunized mice (Fig. 3D, left panel and Fig. 10B, Fig. 10C). In contrast, only a small number of TRM cells were observed in the inguinal adipose tissue of unimmunized mice (Fig. 3D, left panel and Fig. 10B, Fig. 10C). Furthermore, circulating memory T cells derived from the spleens of WTL and survivor mice immunized with the superactivating stimulus LPS + PGPC contained a large population of OVA-specific T cells compared with their unimmunized counterparts.

[0219] To investigate the functional specificity of these T cells, we monitored their ex vivo cytotoxic lymphocyte (CTL) activity. + T cells and TRM cells were isolated from the spleen or skin adipose tissue of survivor mice that had received a previous superactivation stimulus. These cells were cultured with B16OVA cells, non-OVA-expressing B16 cells, or the unrelated cancer cell line CT26. CTL activity, assessed by LDH release, was observed in CD8 +This was observed only when T cells were mixed with B16OVA or B16 cells (Figure 3E). No killing of CT26 cells was observed (Figure 3E), thus demonstrating the functional and antigen-specific nature of the hyperactivation-induced T cell response.

[0220] Based on the antigen-specific T cell responses induced by superactivating stimulation, we determined whether T cells were sufficient to protect against tumor progression. + T cells were transferred from survivor mice to naive mice, which were subsequently challenged with the parental tumor cell line used as the primary immunogen. + TRM cells or circulating CD8 + T cell transfer conferred significant protection from subsequent tumor challenge, with the TRM subset playing a dominant role in protection (Figure 3F). Transfer of both T cell subsets from survivor mice to naive mice 1 week before tumor inoculation protected recipient mice 100% from subsequent tumor challenge (Figure 3F). Together, these data suggest that PGPCs mediate the uptake of circulating and resident antitumor CD8 T cells. + Induction of T cell responses is shown to be the primary bioactive superactivating stimulus that confers optimal protection in the B16 melanoma model.

[0221] To confirm whether the benefits of PGPC-based superactivation stimulation extend to other mouse cancer models, similar experiments were performed using B16-F10 parental melanoma cells. Unlike B16OVA melanoma, which contains a large amount of neoantigens in the form of OVA, with a few exceptions (JC Castle et al., Cancer Res., Vol. 72, No. 5, pp. 1081-91, March 2012; MO Mohsen et al., Front. Immunol., Vol. 10, pp. 1015, 2019), the tumor-specific antigens present in B16-F10 cells are poorly defined. Therefore, B16-F10 cells represent a common clinical scenario in which minimal neoantigen identification has occurred. Notably, the PGPC-based superactivation strategy induced protection against B16-F10 tumor growth (Figure 9B).

[0222] Similar findings were observed when LPS+PGPC was substituted with MPLA+PGPC; 100% of mice immunized in this manner remained tumor-free 90 days after tumor challenge, and 75% of mice rejected a lethal rechallenge with B16OVA cells (Figure 3G). These data indicate that superactivating stimuli, unlike activating stimuli, can complement complex antigen mixtures (e.g., WTL) to promote long-lasting, protective antitumor immunity.

[0223] Superactivating stimuli induce inflammasome-dependent antitumor immunity. The defining characteristic of superactivating stimuli is their ability to induce IL-1β secretion from viable cells. To confirm whether IL-1β and its upstream inflammasome regulators are important for antitumor responses, several procedures were performed. First, immunization was performed as described above, except that a neutralizing anti-IL-1β antibody was injected intravenously (iv) two days before immunization. This was followed by three consecutive injections on days 0, 1, and 2 after immunization to ensure chronic IL-1β clearance. 15 days after immunization, mice were challenged with parental tumor cells.

[0224] Neutralization of IL-1β completely abolished protection of mice from tumor growth in the B16OVA melanoma model (Figures 3G and 9C). Similar to the phenotype observed upon neutralization of IL-1β, superactive stimulation of NLRP3 - / - , Casp1 - / - / 11 - / - or Casp11 - / -Immunization of each mouse with IL-1β did not confer protection from tumor challenge (Figure 3H). Similar results were obtained using a different cancer model—the MC38 colon adenocarcinoma model (Figure 3I and Figure S10D), in which IL-1β neutralization eliminated the ability of the superactivating stimulus to confer protection from challenge with the OVA-expressing MC38 strain (MC38OVA). These functional data establish a link between the in vitro activity of the superactivating stimulus and its in vivo T cell-promoting activity. Notably, not all NLRP3 agonists conferred antitumor immunity, and LPS plus alum failed to support wild-type T cells (WT T cells) to confer protection against tumor growth (Figure S10E). These findings highlight the importance of superactivated DCs (but not pyroptotic DCs) in inducing antitumor immunity.

[0225] Superactive stimuli can supplement either WTL or neoantigens to induce antitumor immunity. The ability of superactive stimuli to induce protective immunity against complex antigen mixtures raises the question of how superior this protective response is compared to immunization with pure neoantigens. To address this question, control immunizations were performed with pure OVA or OVA present in tumor lysates. Mice were injected with MC38OVA cell-derived WTL in the presence or absence of the activating stimuli MPLA or the superactivating stimuli MPLA+PGPC. These injections were compared with injections in which pure OVA replaced WTL as the antigen. 15 days after immunization, mice from each group were blindly divided into two sister cohorts. One cohort was challenged with MC38OVA cells, and the other was challenged with CD8 + The mice were sacrificed and dissected to assess the T cell response (Fig. 11A). Compared with mice immunized with WTL alone or with MPLA alone, the superactive stimulation resulted in an increased number of CD8 + Induced a large absolute number of T cells (Fig. 11B, left panel), SIINFEKL-specific CD8 + It was found that CD8 T cells isolated from the dLN of mice immunized with the superactive stimulus were induced in large absolute numbers (Fig. 11B, right panel). +T cells exhibited the highest degranulation capacity when co-cultured with MC38OVA cells (Fig. 11C). These cells also produced higher levels of IFNγ upon ex vivo restimulation with OVA-loaded BMDCs (Fig. 11D). Furthermore, CD8 T cells stimulated with PMA and ionomycin produced higher levels of IFNγ. + Single cell analysis using intracellular cytokine staining of T cells revealed that CD8 + Within the T cell compartment, we demonstrated highly polyfunctional T cells. These CD8 + T cells were able to simultaneously produce multiple cytokines, including IL-2, TNFα, and IFNγ (Fig. 11E).

[0226] The enhanced T cell activity present in mice immunized with the superactivating stimulus correlated with survival over 150 days after challenge with MC38OVA cells (Figure 3I). It is noteworthy that compared with immunization with pure OVA, WTL-based immunization conferred superior protection after tumor challenge (Figure 3I). The inability of a single antigen to confer robust protection from cancer is consistent with recent studies demonstrating the value of using multiple neoantigens (up to 20 peptides) in personalized cancer vaccines (PAOtt et al., Nature, Vol. 547, No. 7662, pp. 217-221, 2017; JC Castle et al., Cancer Res., Vol. 72, No. 5, pp. 1081-91, March 2012).

[0227] To confirm whether the antitumor responses generated during superactivation-based immunization in the MC38 cancer model were antigen-specific, survivor mice were rechallenged with an unrelated tumor cell line. Survivor mice that rejected MC38OVA tumors died shortly after challenge with B16-F10 cells (Figure 3J). Furthermore, in all cases of superactivation-based antitumor immunization, protection against B16-F10, B16OVA, or MC38OVA cells was conferred only when the mice were immunized with the corresponding WTL (Figure 9F). These findings demonstrate the ability of superactivation stimuli to promote antigen-specific antitumor responses, even when the experimenter is blinded to (and unaware of) the nature of the neoantigen.

[0228] The superactivating stimulus uses WTL to confer protection from metastasis to the lung. To determine whether the superactivating stimulus can be used as a cancer immunotherapy, antitumor responses were examined in mice bearing tumors that were growing before any additional treatment. For this study, rather than using cultured tumor cells as the antigen source, syngeneic tumors from unimmunized mice were used, and harvested 10 mm tumors were dissociated and then transfected with CD45. + We generated cell-free ex vivo WTLs. Mice were inoculated subcutaneously (sc) with tumor cells in the upper left dorsal region. When tumors reached a size of 3–4 mm, tumor-bearing mice were either left untreated (naive) or received a therapeutic injection consisting of ex vivo WTLs and LPS+PGPC in the right flank. Two subsequent sc boosts of the therapeutic injections were administered (Figure 4A). Interestingly, these superactivation-based therapeutic injections induced tumor eradication in B16OVA and B16F10 melanoma models, as well as MC38OVA and CT26 colon cancer tumor models (Figures 4B–4E). In all of these models, a high percentage of mice receiving the immunotherapy regimen remained tumor-free long after tumor inoculation (Figures 4B–4E). The efficacy of immunotherapy was dependent on IL-1β in all tumor models tested, as neutralization of IL-1β abolished the protection conferred by the superactivation stimulus plus ex vivo WTLs (Figures 4B–4E). In addition, CD8 +T cells were important for protection against immunogenic tumor models, e.g., B16OVA or MC38OVA tumors, while CD4 + T cells and CD8 + Both T cells were required for protection against less immunogenic tumors, such as CT26 and B16F-10 tumors (Figures 4B-E).

[0229] To assess the efficacy of superactivation-based immunotherapy compared with PD-1 blockade, we performed a comparative study. Superactivation-based immunotherapy was as effective as anti-PD-1 therapy in the immunogenic B16OVA model, but was even more effective in tumor models insensitive to anti-PD-1 treatment, such as CT26 and B16F-10 tumors (Figures 4C-E).

[0230] Superactivation-based immunotherapy not only protected mice against subcutaneously implanted tumors. In fact, superactivation-based immunotherapy protected mice against B16 lung metastases compared with immunization with WTL injection alone or WTL + LPS (Figure 4F). These observations indicate that superactivation induced antitumor immunity that was not limited to a local response but also contributed to protective systemic immunity.

[0231] Hyperactivation of DC inflammasome components is sufficient to confer protective antitumor immunity. Because DCs are the primary cells responsible for promoting de novo T cell-mediated immunity, we sought to determine whether conditions that specifically activate DCs are sufficient to confer antitumor immunity. We addressed this possibility by adoptively transferring BMDCs stimulated ex vivo with different activation stimuli and with wild-type T cells into mice. BMDCs were chosen because 1) their hyperactivation state has been well characterized and 2) they are considered a model for monocyte-derived DCs, the most common APCs used in DC-based immunotherapy in humans (RL Sabado et al., Cell Res., Vol. 27, No. 1, pp. 74–95, January 2017).

[0232] BMDCs were treated with various activation stimuli along with WTL and then injected sc into B16OVA tumor-bearing mice every 7 days for 3 weeks. LPS-activated BMDCs pulsed with B16OVA WTL conferred modest protection from B16OVA-induced lethality compared with mice injected with naive BMDCs, and 25-30% of mice receiving DC transplants rejected tumors and remained tumor-free long after the final / third DC transplant. Notably, hyperactivated BMDCs induced complete rejection of B16OVA tumors in 100% of tumor-bearing mice. The antitumor activity of hyperactivated DCs depended on the inflammasome in these cells because of the NLRP3 - / - and Casp1 - / - 11 - / - Transplantation of BMDCs induced only minor rejection, comparable to that of activated DCs. Thus, these data indicate that superactivated DCs are sufficient to induce long-lasting, protective antitumor immunity and that inflammasomes within DCs are essential for this process.

[0233] Consideration This study expanded the range of immunological activities upregulated by treatment of DCs with superactivating stimuli. Superactivating stimuli not only elicit IL-1β release from live cells, but also surpass other activating stimuli in their ability to induce CD40 expression and IL-12p70 secretion. Furthermore, cells exposed to superactivating stimuli exhibit elevated surface expression of MHC-peptide complexes. Together, these findings underscore the superactivating nature of DCs exposed to oxPAPC or its pure component, PGPC, and provide evidence of their enhanced ability to promote adaptive immunity. While superactivated DCs are indeed superior stimulators of T cell responses to activated or pyroptotic cells, the most notable aspect of their activity may be their ability to stimulate both TH1-focused and CTL-focused responses. Indeed, superactivating DC stimuli resulted in a 100:1 ratio of TH1:TH2 cells, and no other DC activation strategy has induced such a polarized T cell response.

[0234] It is noteworthy that alum, a clear inflammasome stimulus, does not exhibit the same activity as oxPAPC or PGPC. In fact, alum is well known to induce TH2 immunity. This finding was verified in the present study, as alum treatment or alum + LPS treatment induced robust TH2 immunity. A possible reason for the lack of TH1-focused immunity in alum-treated cells is based on our findings that alum is a weak inducer of several signals essential for TH1 differentiation, such as CD40 expression and IL-12p70 secretion. Notably, CD40 expression was significantly lower even when DCs that did not undergo pyroptosis in response to alum + LPS were examined. Perhaps due to the lack of high-level expression of this factor, pyroptotic stimulation is a weak inducer of TH1 responses and therefore antitumor immunity. Without wishing to be bound by theory, we propose that the TH1-focused immunity induced by hyperactivated DCs is due to the action of inflammasomes as well as several other characteristics of hyperactivated DCs. These additional characteristics include increased antigen-presenting capacity, CD40 expression, IL-12p70 expression, and increased survival rate, each of which is likely important for DC function as APCs and contributes to the marked TH1-focused immune response observed under conditions of DC hyperactivation.

[0235] These results may explain why certain chemotherapeutic agents (e.g., oxaliplatin) induce cell death and inflammasome-dependent antitumor T cell immunity (F. Ghiringhelli et al., Nat. Med., Vol. 15, No. 10, pp. 1170-1178, 2009). Oxaliplatin is a robust stimulator of reactive oxygen species (ROS) production, which can oxidize cell membranes and generate a complex mixture of different oxidized phospholipid species, including PGPC. Therefore, the protective immunity induced by oxaliplatin may be due to the action of hyperactivated DCs, which prime antitumor T cell responses.

[0236] We have discovered that superactivating stimuli can be utilized as immunotherapy using complex mixtures of antigens. WTLs are an intriguing source of antigens for several reasons, particularly important from a practical standpoint. A key benefit of a WTL-based approach is that it alleviates the need for neoantigen identification. Despite the potential utility offered by WTL-based immunotherapy, previous studies in this field have yielded mixed results. Our discovery that superactivating stimuli can uniquely support WTLs to elicit potent antitumor immunity may explain the lack of success in previous studies, as the DC activation strategy discovered here has not been previously considered. Regarding the latter point, it is noteworthy that the DC superactivation strategy can protect mice from the lethality associated with tumors sensitive to and resistant to PD-1 inhibition. While the full range of tumors susceptible to treatment with superactivating stimuli has not yet been defined, this study urges further investigation into the importance of DC-centered strategies for cancer immunotherapy.

[0237] Example 2: cDC1 regulates tumor rejection induced by hyperactivation-based immunotherapy Based on our previous findings that superactivated DCs are excellent stimulators of antigen-specific Th1 and CTL responses, we reasoned that superactivated DCs could be utilized as an immunotherapeutic strategy for cancer. To examine the effect of superactivated DC injection on antitumor immunity, tumor-bearing mice (containing 4 mm tumors in the right flank) were injected sc with ex vivo whole tumor lysate (WTL) and the superactivated stimuli LPS + PGPC into the left flank. Two subsequent sc boost injections with whole tumor lysate (WTL) and LPS + PGPC were administered. Superactivated DC injections induced tumor rejection in B16OVA, and a high percentage of WT mice receiving the immunotherapy regimen remained tumor-free (>40 days) after tumor inoculation. Superactivated DC-induced protection was dependent on cDC1 cells because Batf3 cells received the immunization regimen. - / - Mice lacking cDC1 cells were unable to induce tumor control. Furthermore, tumor-specific CD4 +T cells and CD8 + In contrast to immunized WT mice, which showed high frequencies of T cells, Batf3 - / - Mice express OVA-specific CD8 + lacking T cells and lower OVA-specific CD4 + Overall, these data demonstrate that 1) a superactivating stimulus (LPS + PGPC) can uniquely support WTLs to induce potent antitumor immunity, and 2) cDC1 cells are the primary antigen-presenting cells that initiate superactivation-induced antitumor immunity in vivo.

[0238] Example 3: Oxidized phospholipids induce hyperactive cDC1 and cDC2 cells Virtually all studies evaluating the state of cell superactivation have focused on the ability of bone marrow-derived DCs (BMDCs) generated with the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) to release IL-1β while maintaining viability [24, 31, 32, 33, 34]. Recent studies have demonstrated that monocyte-derived macrophages, rather than DCs, are responsible for inflammasome activation and IL-1β secretion

[35] . To examine whether conventional DCs can achieve a superactivation state, we used BMDCs generated using the DC hematopoietin Fms-like tyrosine kinase 3 ligand (Flt3L). To assess superactivation, FLT3-DCs were primed with LPS and subsequently treated with the oxidized phospholipid oxPAPC, or the pure lipid component of oxPAPC, known as PGPC

[36] . Alternatively, FLT3-DCs were stimulated with conventional activating stimuli, such as LPS alone, or primed with LPS followed by treatment with a pyroptotic stimulus, such as alum. In contrast to activation stimuli that did not induce IL-1β release from DCs, pyroptotic DCs promoted the release of IL-1β into the extracellular medium (Figure 14A). IL-1β secretion, assessed by the release of the cytosolic enzyme lactate dehydrogenase (LDH) (Figure 14B), coincided with cell death in pyroptotic DCs. Interestingly, stimulation with the superactivating stimuli LPS + PGPC, or to a lesser extent LPS + oxPAPC, induced IL-1β secretion from DCs in the absence of LDH release (Figure 14A). All DCs primed or stimulated with LPS promoted the secretion of the cytokine TNFα (Figure 14A). IL-1β secretion in both pyroptotic and superactivating DCs depended on the inflammasome components NLRP3 and caspase 1 / 11 (Figure 14A). These findings are consistent with previous studies that defined the mechanism underlying the hyperactive state of DCs, whereby oxPAPC binds to and stimulates the cytosolic PRR caspase-11, resulting in NLRP3 activation and assembly of nonpyroptic inflammasomes, leading to the release of IL-1β from live cells [ 24 ].Similar behavior of DCs was observed when DCs were primed with other TLR agonists, such as the TLR9 agonist CpG (Figure 19A). Therefore, these data indicate that FLT3 DCs can achieve a hyperactivated state. DCs are divided into two major subsets, cDC1 and cDC2. cDC1 are typical DCs that can cross-present tumor-associated antigens and prime CD8+ T cells

[37] ,

[38] . On the other hand, cDC2 regulate type 2 immune responses against parasites and activate Th2 immunity. To confirm whether the hyperactivated behavior of DCs extends to cDC1 or cDC2, we isolated cDC1 or cDC2 from wild-type naive FLT3 DCs or from the spleen (Figure 19B). Similar to the behavior of FLT3-derived DCs, treatment with LPS and PGPC, and to a lesser extent LPS and oxPAPC, resulted in the release of TNFα and IL-1β from FLT3-derived cDC1s and cDC2s in the absence of cell death as assessed by LDH release (Figure 14A). These data indicate that PGPC is the bioactive component of oxPAPC that induces the superactivation of cDC1s and cDC2s. Furthermore, similar behavior was observed for splenic cDC2s, which produced IL-1β in response to pyroptotic stimuli LPS and alum, concomitant with pyroptotic cell death, but produced IL-1β in response to superactivating stimuli LPS and PGPC, in the absence of cell death (Figure 19C). In contrast, splenic cDC1s produced minimal amounts of IL-1β in response to pyroptotic or superactivating stimuli. Because splenic cDC1 were highly susceptible to cell death after sorting and could not be primed with LPS (Figure 19C). Overall, these results demonstrate that superactivating stimuli can be used to induce IL-1β release from viable DCs differentiated in vitro or in vivo. For practical reasons, we continued to use FLT3-derived DCs as the DC source in this paper.

[0239] Example 4: Superactivated DCs enhance CTL responses in an inflammasome-dependent manner IL-1β is a critical regulator of T cell differentiation, persistent memory T cell generation, and effector function [12–14]. We were interested to determine whether superactivated DCs, which produce IL-1β for several days in the dLN, could enhance CD8+ T cell stimulation. To test this, we sought to measure OVA-specific CD8+ T cells in the dLN after subcutaneous (sc) adoptive transfer of DCs containing OVA protein. First, we examined the ability of different DC conditions to internalize OVA protein and cross-present the OVA peptide SIINFEKL on H2kb molecules. We found that all DCs in different conditions internalized OVA to a similar extent, as indicated by comparable internalization of fluorescent ovalbumin (OVA-FITC). However, we found that both activated and superactivated DCs primed with LPS or CpG exhibited enhanced SIINFEKL cross-presentation when loaded with OVA protein compared to naive counterparts. This is consistent with previous studies showing that DC maturation enhances their antigen-presenting capacity. Surprisingly, pyroptotic stimulation significantly reduced the cross-presentation capacity of DCs, suggesting that pyroptotic DCs are not suitable for optimal T cell stimulation. Accordingly, when 1.10e6 OVA-loaded naive, activated, pyroptotic, or superactivated DCs were injected into WT mice, we observed that superactivated DCs induced the highest frequency and absolute number of SIINFEKL+CD8+ T cells in the dLN of recipient mice compared with naive, activated, or pyroptotic DCs (Figures 15A and 20B). The increased CD8+ T cell response mediated by superactivated DCs depended on inflammasome activation, as injection of NLRP3- / - DCs treated with LPS and PGPC induced weak OVA-specific T cell responses.

[0240] Example 5: Superactivating stimuli enhance the generation of memory T cells and augment antigen-specific IFNγ effector responses in an inflammasome-dependent manner We hypothesized that superactivating stimuli might be a potent adjuvant that could enhance the effects of superactivated DC injections. To investigate this possibility, mice were immunized sc with OVA alone, OVA plus an activating stimuli (LPS), or OVA plus a superactivating stimuli (LPS plus oxPAPC or PGPC). Seven and 40 days after immunization, memory and effector T cell generation in the dLN was assessed by flow cytometry using CD44 and CD62L markers, which distinguish between CD44-low CD62L-low effector T cells (Teff), CD44-high CD62L-low effector memory T cells (TEM), and CD44-high CD62L-high central memory T cells (TCM)

[47] . Seven days after immunization, superactivating stimuli were superior to activating stimuli in inducing CD8+ Teff cells (Figure 16A, upper panel and Figures 21A-B). Furthermore, at this early time point, the superactivating stimulus induced the highest amount of CD8+ TEM cells (Figure 16A, middle panel and Figures 21A-B). At 40 days after immunization, mice exposed to the superactivating stimulus had abundant TCM cells, whereas mice immunized with OVA alone or LPS were less abundant (Figure 16A, bottom panel). Conversely, Teff and TEM cells were more abundant at 40 days after immunization in mice immunized with OVA alone or LPS compared with mice immunized with OVA+. Thus, these data indicate that the superactivating stimuli oxPAPC and PGPC enhance the magnitude of effector and memory T cell generation. Furthermore, the increased frequency of Teff cells at 7 days after immunization correlated with enhanced IFNγ responses of CD8+ T cells upon ex vivo restimulation in the presence of OVA-loaded naive BMDCs isolated from the dLNs of mice immunized with the OVA+ superactivating stimulus (Figure 21C). Furthermore, when total CD8+ T cells were isolated from mice immunized with the superactivating stimulus and cocultured with the OVA-expressing B16 tumor cell line (B16OVA), the CD8+ T cells exhibited enhanced degranulation activity compared with CD8+ T cells isolated from mice immunized with OVA alone or OVA plus LPS (Figures 16B and 21D), indicating that the superactivating stimulus enhances CTL function.

[0241] To evaluate the antigen specificity of T cells resulting from sc immunization with different activation stimuli, mice were injected with OVA alone, together with an activation stimulus (LPS), together with a pyroptotic stimulus (LPS + alum), or together with a superactivating stimulus (LPS + oxPAPC or PGPC). Alternatively, mice were immunized sc with LPS + PGPC without the OVA antigen. Seven days after immunization, CD8+ T cells were isolated from the cutaneous dLN of immunized mice and restimulated ex vivo for 7 days with naive BMDCs loaded with or without OVA to enrich for OVA-specific T cell subsets. T cell effector function of OVA-specific T cells was assessed by intracellular staining for IFNγ. TCR specificity was assessed by staining with an MHC-restricted OVA peptide tetramer. An H2kb-restricted SIINFEKL (OVA257-264) peptide tetramer was used. The frequencies of tetramer+IFNγ+ double-positive cells were measured for CD4+ and CD8+ T cell subsets. Notably, OVA with superactivating stimuli was superior in inducing antigen-specific T cells, because oxPAPC- or PGPC-based immunization induced the highest frequency of tetramer+IFNγ+ responses upon CD8+ T cell restimulation with OVA antigen (Figure 16C). In contrast, pyroptotic stimulation (LPS + alum) was the weakest inducer of antigen-specific IFNγ responses (Figure 16C). These results are consistent with previous studies showing that alum is an effective adjuvant for promoting humoral immunity and Th2 responses, but not Th1 or CTL responses [39, 42, 43].

[0242] Previous studies have shown that co-immunization with recombinant IL-1β, a cytokine whose physiological activity is naturally regulated by the inflammasome, can enhance antigen-specific T cell responses [47, 13]. However, despite both superactivating and pyroptotic stimulation inducing IL-1β secretion, how is it that superactivating stimulation induces high antigen-specific T cell activity, while pyroptotic stimulation does not? To determine whether inflammasome-mediated events regulate T cell responses triggered by superactivating stimulation, we performed a controlled comparison of T cell activity in WT and NLRP3- / - mice. Notably, we found that NLRP3 is required for the superactivation-induced increase in antigen-specific responses by CD8+ T cells (Figure 16C). Thus, these data indicate that pyroptotic versus nonpyroptic inflammasome activation following immunization with superactivating or pyroptotic stimulation, respectively, induces distinctly different adaptive immune T cell regulation.

[0243] Our previous results using a DC injection strategy showed that DCs stimulated with pyroptotic stimuli lost their ability to migrate to the adjacent dLN and stimulate T cell activation, whereas DCs exposed to superactive stimuli supermigrated to the dLN and enhanced CTL responses (Figures 15A-B). However, it is unknown whether endogenous DCs achieve in vivo superactivation following immunization with superactive stimuli. To assess this, chimeric mice were generated using Zbtb46DTR and WT mice, Zbtb46DTR and NLRP3- / - mice, or Zbtb46DTR and Casp1 / 11- / - mice. To this end, 4-week-old CD45.1-irradiated mice were reconstituted on a CD45.2 background using mixed bone marrow from 80% Zbtb46DTR mice and 20% WT, 20% NLRP3- / -, or 20% Casp1 / 11- / - mice, as previously described

[51] . Six weeks after reconstitution, the efficacy of BM reconstitution was assessed in all mice by flow cytometry using CD45.1 versus CD45.2 markers. To eliminate Zbtb46+ conventional DCs, chimeric mice were treated with diphtheria toxin (DT) every other day, resulting in either WT DCs capable of hyperactivation or inflammasome-deficient (NLRP3- / - or Casp1 / 11- / -) DCs that could not hyperactivate, respectively. To test the effect of endogenous DC hyperactivation on CD8+ T cell responses, all chimeric mice received three consecutive DT injections followed by sc immunization with OVA, LPS, and PGPC. Seven days after immunization, we evaluated the CD8+ T cell response from dLNs. Interestingly, we found that the amount of Teff CD8+ T cells was significantly reduced in chimeric mice containing DCs that could not become hyperactive, such as NLRP3- / - chimeric mice and Casp1 / 11- / - chimeric mice, compared with chimeric mice containing WT DCs that could become hyperactive (Figure 16D, Figure 22A).Furthermore, we found that the frequency of SIINFEKL+CD8+ T cells in the dLN or spleen was reduced in NLRP3- / - and Casp1 / 11- / - chimeric mice containing DCs that could not become hyperactivated, whereas abundant SIINFEKL+CD8+ cells were observed in chimeric mice containing WT DCs (Figures 16E and 22B). Thus, these data clearly demonstrate that 1) endogenous DCs can reach a state of hyperactivation in vivo and enhance CTL responses following immunization with hyperactivating stimuli, and 2) inflammasome activation in endogenous DCs is important for hyperactivation-mediated protective CTL responses.

[0244] Example 6: Infiltration of superactivated DCs into lymphoid tissues is essential for superactivation-mediated CTL responses We previously demonstrated that DCs stimulated with superactive stimuli hypermigrated to the dLN and enhanced CTL responses (Figures 15A-15B). To assess whether superactivation-mediated CTL responses required the entry of endogenous hyperactive DCs into the dLN, chimeric mice were generated as described above using Zbtb46DTR and WT or Zbtb46DTR and CCR7- / - BM (Figure 15D). Overall, endogenous transport of hyperactive DCs into the dLN is essential for superactivation-mediated CTL function.

[0245] Example 7: Superactive cDC1 can use complex antigen sources to stimulate preventative T cell-mediated antitumor immunity Current efforts to stimulate antitumor immunity include strategies to stimulate resident T cell populations (e.g., PD-1 blockade) or personalized cancer vaccine strategies to stimulate de novo T cell responses against tumor-specific antigens (TSAs)

[46] . The latter approach has been hampered by the unavailability of tumor cell lysates, a source of TSAs, also known as neoantigens. Therefore, efforts are underway to advance the identification of neoantigens that can be used in pure form to elicit T cell-mediated antitumor immunity. While these efforts have yielded success, the path to neoantigen identification requires the discovery of mutated TSAs as well as aberrantly expressed TSAs

[54] , which are challenging and do not represent the natural history of events. As discussed above, WTLs are an intriguing alternative source of antigens because this lysate provides the numerous antigens required for personalized antitumor immune responses. However, fundamental questions remain unanswered, such as what the most effective types of adjuvants (including those associated with different types of antigens) are that can be used in cancer vaccines.

[0246] To address the possibility that the superactivating stimulus could be an adjuvant against WTL, mice were immunized in the right flank with WTL alone, WTL mixed with the activating stimulus LPS, or WTL mixed with the superactivating stimuli LPS + oxPAPC or LPS + PGPC. The source of WTL was B16OVA cells. Fifteen days after immunization, mice were subcutaneously challenged in the upper left back with B16OVA parental cells. Naive mice or mice immunized with WTL alone did not show any protection, and all mice contained large tumors and died by 24 days after tumor inoculation (Figure 23A). Similarly, WTL + LPS immunization showed minimal protection. Although two of eight mice immunized with WTL + LPS were tumor-free, they relapsed quickly after rechallenge with B16OVA (Figure 23A), indicating that a stimulus that simply activates DCs does not confer protective immunity. In contrast, WTL immunization in the presence of LPS and oxPAPC induced a significant delay in tumor growth and provided significant protection against subsequent lethal rechallenge with B16OVA parental tumor cells, with 50% of immunized mice being completely protected (Figure 23A). To determine whether the protective response induced by oxPAPC correlated with T cell responses, tumors were harvested from mice that received each activating stimulus. Tumors from mice immunized with LPS + oxPAPC contained substantially more CD4+ and CD8+ T cells than those immunized with LPS (Figure S7B). Furthermore, when comparing the same number of T cells derived from their tumors, oxPAPC-based immunization resulted in intratumoral T cells that secreted the greatest amounts of IFNγ upon anti-CD3 and anti-CD28 stimulation (Figure 23C). Thus, the predominant restriction of tumor growth induced by the superactivating stimulus (LPS + oxPAPC) was consistent with the infiltration of inflammatory T cells into the tumor.

[0247] Notably, the protective phenotype induced by PGPC, a pure oxPAPC component, was superior to that of oxPAPC. WTL immunization in the presence of LPS + PGPC rendered 100% of mice tumor-free for 150 days after tumor challenge. These mice completely rejected a lethal rechallenge with B16OVA cells and remained tumor-free for 300 days after the initial tumor challenge (Figure 20A). Because these mice never relapsed, we were intrigued by how tumor cell growth remained controlled at the tumor injection site in mice immunized with WTL + LPS + PGPC.

[0248] Among memory T cell subsets, resident memory T cells (TRM) are defined by the expression of the CD103 integrin in addition to the C-type lectin CD69, which contributes to their resident properties in peripheral tissues

[55] . CD8+ TRM cells have recently attracted considerable attention because they accumulate at tumor sites in various human cancer tissues and correlate with more favorable clinical outcomes [54, 55, 56]. In an experimental cutaneous melanoma model, cutaneous CD8+ TRM cells promoted sustained protection against melanoma progression

[58] .

[0249] We examined the presence of TRM cells at tumor injection sites and in immunized skin biopsies of survivor mice previously immunized with the superactivating stimulus LPS+PGPC. Interestingly, 200 days after tumor inoculation, CD8+CD69+CD103+ TRM cells were highly enriched at the tumor injection site but were scarce at the immunization sites of all survivor mice (Figures 24A-B). These data are consistent with clinical and experimental reports linking high levels of TRM to long-term tumor control, potentially resulting in long-term maintenance of TRM cells at the tumor injection site [56, 57]. Thus, immunization with both WTL and superactivating stimuli appears to generate TRM cells that can stave off tumor cells.

[0250] To examine the functional specificity of these T cells, we monitored their cytotoxic lymphocyte (CTL) activity ex vivo. Circulating memory CD8+ T cells and TRM cells were isolated from the spleens or skin adipose tissue of survivor mice that had previously received a superactivating stimulus. These cells were cultured with B16OVA cells, non-OVA-expressing B16 cells, or the unrelated cancer cell line CT26. CTL activity, assessed by LDH release, was observed only when CD8+ T cells were mixed with B16OVA cells or B16 cells (Figure 24C). Killing of CT26 cells was not observed (Figure 24C), thus demonstrating the functional and antigen-specific nature of the superactivation-induced T cell response.

[0251] Based on the antigen-specific T cell responses induced by the superactivating stimulation, we confirmed whether T cells were sufficient to protect against tumor progression. CD8+ T cells were transferred from survivor mice to naive mice, which were subsequently challenged with the parental tumor cell line used as the primary immunogen. Transfer of CD8+ TRM cells or circulating CD8+ T cells from survivor mice to naive recipient mice conferred significant protection from subsequent tumor challenge, with the TRM subset playing a dominant role in protection (Figure 24D). Transfer of both T cell subsets from survivor mice to naive mice 1 week before tumor inoculation 100% protected recipient mice from subsequent tumor challenge (Figure 24D). Together, these data indicate that PGPC-based superactivating stimulation confers optimal protection in the B16 melanoma model by significantly inducing circulating and resident antitumor CD8+ T cell responses.

[0252] Example 8: Superactive stimulation provides protection against anti-PD1 resistant established tumors To determine whether superactivation stimuli could be used as a cancer immunotherapy, we examined antitumor responses in mice bearing tumors that were still growing before any additional treatment. For this study, rather than using cultured tumor cells as the antigen source, we used syngeneic tumors from unimmunized mice and generated ex vivo WTLs by dissociating harvested 10 mm tumors and then depleting CD45+ cells. Mice were inoculated subcutaneously (sc) with tumor cells in the upper left dorsum. When tumors reached a size of 3–4 mm, tumor-bearing mice were either left untreated (unimmunized) or received a therapeutic injection consisting of ex vivo WTLs and LPS+PGPC in the right flank. Two subsequent sc boosts of the therapeutic injection were administered (Figure 17A). Interestingly, superactivation-based therapeutic injections induced tumor eradication in a wide range of tumors, including B16OVA and B16F10 melanoma models and MC38OVA and CT26 colon cancer tumor models (Figures 17B–D). In all of these models, a high percentage of mice receiving the immunotherapy regimen remained tumor-free long after tumor inoculation (Figures 17B-17D). The efficacy of immunotherapy depended on IL-1β in all tumor models tested, as neutralization of IL-1β abolished the protection conferred by the superactivating stimulus plus ex vivo WTL (Figures 17B-17D). Furthermore, CD8+ T cells were important for protection against immunogenic tumor models, such as B16OVA or MC38OVA tumors, while both CD4+ and CD8+ T cells were required for protection against less immunogenic tumors, such as CT26 and B16F-10 tumors (Figures 17B-17D)

[63] . To determine the efficacy of superactivation-based immunotherapy compared with PD-1 blockade-based therapy, a control evaluation was performed. Superactivation-based immunotherapy was as effective as anti-PD-1 therapy in the immunogenic B16OVA model, but was even more effective in tumor models insensitive to anti-PD-1 treatment, such as CT26 and B16F-10 (Figures 17B-D).

[0253] Example 9: Endogenous hyperactive DCs stimulate long-lasting T cell-mediated antitumor immunity Adoptive transfer of superactivated DCs into tumor-bearing mice induces significant antitumor immunity (Figures 15A-B). To test whether endogenous DCs can initiate hyperactivation-mediated antitumor responses, we used Zbtb46DTR mice, in which conventional DCs had been eliminated by DT injection. Zbtb46DTR or WT mice were injected sc with B16OVA cells. DT was then injected every other day to completely eliminate resident DCs in Zbtb46DTR mice before immunization. When tumors reached a size of 4 mm, Zbtb46DTR or WT mice were immunized with B16OVA WT + superactivating stimuli LPS + PGPC. In contrast to WT mice, which rejected tumors in 90% of mice, Zbtb46DTR mice (lacking DCs) failed to reject tumors. These data confirm that DCs are the initiators of hyperactivation-mediated defense (Figure 18A).

[0254] Example 10: Superactive cDC1 can use complex antigen sources to stimulate T cell-mediated antitumor immunity We demonstrated in vitro that both cDC1 and cDC2 cells can reach a hyperactivated state in response to LPS+PGPC, producing IL-1β while maintaining viability. These data necessitate further definition of the specific DC subsets that initiate hyperactivation-mediated antitumor responses in vivo. Given the importance of the cDC1 subset in tumor rejection, we hypothesized that cDC1 cells play a central role in inducing hyperactivation-mediated antitumor defense. To test this idea, we used Batf3- / - mice (which lack cDC1 but contain cDC2 cells)

[65] . To this end, we immunized Batf3- / - or WT tumor-bearing mice (containing 3–4 mm B16 OVA) with LPS+PGPC and WT. These mice received two sc boost injections 7 days apart. Unimmunized Batf3 − / − mice exhibited more severe tumor growth than unimmunized WT mice, and all mice died of tumors shortly after 18 days post-tumor inoculation. This data supports previous studies showing that rejection of highly immunogenic tumors is significantly impaired in Batf3 − / − mice lacking cDC1 cells

[65] . Interestingly, although immunization of Batf3 − / − mice improved their survival by several days compared with unimmunized Batf3 − / − mice, all Batf3 − / − mice died of tumor growth by 25 days post-tumor inoculation. In contrast, WT mice rejected tumors in 100% of tumor-bearing mice (Figure 18C). Thus, cDC1s play a crucial role in hyperactivation-mediated antitumor immunity. Furthermore, immunized WT mice induced high frequencies of antigen-specific CD8+ and CD4+ T cells in the TME and skin dLNs, whereas immunized Batf3- / - mice induced slightly reduced antigen-specific CD4+ T cells in the TME, but notably, no antigen-specific CD8+ T cells (Fig. 18D).

[0255] To further confirm the role of superactive cDC1s in inducing durable antitumor defenses, we sought to assess the ability of superactive cDC1s to restore antitumor defenses in Batf3- / - mice. Naive, activated, or superactive cDC1 cells were adoptively transferred into Batf3- / - mice. To this end, FLT3-derived cDC1s were sorted from C57BL / 6J mice as B220-MHC-II+CD11c+CD24+ cells as previously described. cDC1s were treated in vitro and loaded with B16OVA WTL as described above, and 1.10e6 cells were injected sc into tumor-bearing Batf3- / - mice. In contrast to naive or activated cDC1s, which provided only a slight improvement in mouse survival compared with uninjected mice, superactivated cDC1s induced tumor rejection in 100% of tumor-bearing mice, which remained tumor-free for more than 60 days after tumor inoculation (Figure 18E). It is noteworthy that superactivated cDC1 injection restored CD8+ T responses in Batf3- / - mice, as measured by SIINFEKL tetramer staining in tumors and skin dLNs (Figure 18F). In contrast, injection of naive or activated cDC1s failed to restore antigen-specific CD8+ T cells. cDC1-mediated tumor rejection depended on inflammasome activation, because injection of NLRP3- / - cDC1s treated with LPS and PGPC did not confer any antitumor protection and abolished the ability of superactivated cDC1s to restore CD8+ T cell responses (Figure 18F).

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[68] F. Ghiringhelli et al., “Activation of the NLRP3 inflammasome in dendritic cells induces IL- 1β-dependent adaptive immunity against tumors,” Nat. Med., vol. 15, no. 10, pp. 1170- 1178, 2009. Another embodiment While the present invention has been described in connection with its detailed description, the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0257] The patent and scientific literature referred to herein constitutes knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and foreign patent applications cited herein are incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated by reference.

[0258] Although the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail may be made in the preferred embodiments without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. 1. An immunogenic composition comprising a Toll-like receptor (TLR) ligand, a non-canonical inflammasome-activating lipid, and a cancer immunogen for use in a method of inducing or enhancing an adaptive immune response to cancer in a subject, the immunogenic composition comprising: The method comprises administering to the subject an effective amount of the immunogenic composition, wherein the non-canonical inflammasome-activating lipid comprises [(2R)-2-(4-carboxybutanoyloxy)-3-hexadecanoyloxypropyl]2-(trimethylazaniumyl)ethyl phosphate (PGPC).

2. 1. An immunogenic composition comprising a Toll-like receptor (TLR) ligand, a non-canonical inflammasome-activating lipid, and a cancer immunogen for use in a method of treating cancer in a subject, the immunogenic composition comprising: The method comprises administering to the subject an effective amount of the immunogenic composition, wherein the non-canonical inflammasome-activating lipid comprises [(2R)-2-(4-carboxybutanoyloxy)-3-hexadecanoyloxypropyl]2-(trimethylazaniumyl)ethyl phosphate (PGPC).

3. 3. The immunogenic composition for use according to claim 1 or claim 2, wherein the cancer immunogen is an infectious agent immunogen, and infection by the infectious agent is associated with the development of cancer.

4. The immunogenic composition for use according to claim 1 or 2, wherein the cancer immunogen is derived from a cancer cell.

5. 5. The immunogenic composition for use according to claim 4, wherein the cancer immunogen is or comprises a whole cancer cell lysate.

6. 6. The immunogenic composition for use according to any one of claims 1 to 5, wherein the TLR ligand is selected from a TLR1 ligand, a TLR2 ligand, a TLR3 ligand, a TLR4 ligand, a TLR5 ligand, a TLR6 ligand, a TLR7 ligand, a TLR8 ligand, a TLR9 ligand, a TLR10 ligand, a TLR11 ligand, a TLR12 ligand, a TLR13 ligand, and combinations thereof.

7. The immunogenic composition for use according to any one of claims 1 to 5, wherein said TLR ligand is a TLR4 ligand.

8. 8. The immunogenic composition for use according to claim 7, wherein the TLR4 ligand is selected from monophosphoryl lipid A (MPLA), lipopolysaccharide (LPS), or a combination thereof.

9. The non-canonical inflammasome-activating lipids include 2-[[(2R)-2-[(E)-7-carboxy-5-hydroxyhept-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium (HOdiA-PC), [(2R)-2-[(E)-7-carboxy-5-oxohept-6-enoyl]oxy-3-hexadecanoyloxypropyl]2-(trimethyl azaniumyl)ethyl phosphate (KOdiA-PC), 1-palmitoyl-2-(5-hydroxy-8-oxo-octenoyl)-sn-glycero-3-phosphorylcholine (HOOA-PC), 2-[[(2R)-2-[(E)-5,8-dioxooct-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium (KOOA-PC), [(2R)-3- Hexadecanoyloxy-2-(5-oxopentanoyloxy)propyl]2-(trimethylazaniumyl)ethyl phosphate (POVPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[2-[(Z)-oct-2-enyl]-5-oxocyclopent-3-en-1-ylidene]methyl]oxiran-2-yl]butanoyloxy]propyl]2-(trimethylazaniumyl)ethyl phosphate 9. The immunogenic composition for use according to any one of claims 1 to 8, further comprising [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentylidene]methyl]oxiran-2-yl]butanoyloxy]propyl]2-(trimethylazaniumyl)ethyl phosphate (PEIPC), ... or a combination thereof.

10. The immunogenic composition for use according to any one of claims 1 to 9, wherein the subject is a mammal.

11. The immunogenic composition for use according to claim 10, wherein the subject is a human.

12. The immunogenic composition for use according to any one of claims 1 to 11, wherein the TLR ligand, non-canonical inflammasome-activating lipid, and cancer immunogen are administered as part of a pharmaceutical composition.

13. 13. The immunogenic composition for use according to any one of claims 1 or 3 to 12, wherein the adaptive immune response is a prophylactic immune response, or wherein the adaptive immune response is a therapeutic immune response and / or wherein the adaptive immune response comprises T cell activation.

14. An immunogenic composition for use according to any one of claims 1 to 13, wherein the method further comprises treating the subject with one or more therapeutic interventions.

15. 15. The immunogenic composition for use according to any one of claims 1 to 14, wherein the TLR ligand, non-canonical inflammasome-activating lipid, and cancer immunogen, and one or more therapeutic interventions, are co-administered or sequentially administered.

16. 16. The immunogenic composition for use according to claim 14 or claim 15, wherein the one or more therapeutic interventions comprise radiation, chemotherapy, surgery, a therapeutic antibody, an immunomodulator, a proteasome inhibitor, a pan-deacetylase (DAC) inhibitor, a histone deacetylase (HDAC) inhibitor, a checkpoint inhibitor, adoptive cellular therapy, a vaccine, or a combination thereof.

17. 17. The immunogenic composition for use according to claim 16, wherein the adoptive cell therapy comprises CAR-T cell therapy, CAR-NK cell therapy, T cells, dendritic cells, or a combination thereof.

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