Over-activated dendritic cells enable long-lasting adoptive cell metastasis-based anti-tumor immunity

Abstract

The invention relates to an over-activated dendritic cell which can achieve durable anti-tumor immunity based on adoptive cell metastasis. The present application relates to cancer immunotherapies, such as T cell stimulation mediated anti-tumor therapies.

Description

[0001] This application is a divisional application of the patent application "Overactivated Dendritic Cells Enable Durable Adoptive-Cell-Transfer-Based Antitumor Immunity" with an application date of November 18, 2020, and an application number of 202080093397.7 (International Application No. PCT / US2020 / 061132).

[0002] Priority Claim

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 937,075, filed November 18, 2019. The foregoing is hereby incorporated by reference in its entirety.

[0004] Federally Sponsored Research or Development

[0005] This invention was made with government support under Grant No. AI116550 awarded by the National Institutes of Health. The government has certain rights in the invention. Technical Field

[0006] This application relates to cancer immunotherapy, e.g., T cell-stimulated antitumor therapy. Background Art

[0007] Central to the understanding of protective immunity to infection and cancer are dendritic cells (DCs), which are migratory phagocytes that patrol the body's tissues (D. Alvarez, E. H., et al., Immunity, vol. 29, no. 3, pp. 325-42, Sep. 2008). DCs monitor the environment for threats to the host, signs of most common infections or tissue damage. This monitoring is achieved through the activity of a superfamily of threat assessment receptors, classically known as pattern recognition receptors (PRRs), which recognize microbial products or host-encoded molecules indicative of tissue damage (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, Jan. 2002). Microbial ligands of PRRs are classified as pathogen-associated molecular patterns (PAMPs), while host-derived PRR ligands are damage-associated molecular patterns (DAMPs) (P. Matzinger, Science, vol. 296, no. 5566, pp. 301-5, Apr. 2002).

[0008] Upon detection of PAMPs, PRRs initiate signaling pathways that fundamentally alter the physiology of DCs expressing these receptors (A. Iwasaki and R. Medzhitov, Nat. Immunol., vol. 16, no. 4, pp. 343-53, Apr. 2015; O. Joffre, et al. Immunol. Rev., vol. 227, no. 1, pp. 234-247, Jan. 2009). For example, prior to PRR activation, DCs are generally considered non-inflammatory cells. Upon encountering extracellular PAMPs, PRRs stimulate rapid and robust upregulation of numerous inflammatory mediators including cytokines, chemokines, and interferons. Concurrent with the expression of these genes is the migration of DCs to the draining lymph node (dLN) and upregulation of factors important for T cell activation such as MHC and costimulatory molecules. Thus, the PRR signaling process results in a transition of DC activity from a non-stimulated (naïve) state to an "activated" state (K. Inaba, et al. J. Exp. Med., vol. 191, no. 6, pp. 927-36, Mar. 2000; I. Mellman and R. M. Steinman, Cell, vol. 106, no. 3, pp. 255-8, Aug. 2001). SUMMARY OF THE INVENTION

[0009] There is a need to diversify current cancer immunotherapies. Accordingly, the present invention relates to methods of generating populations of therapeutic dendritic cells, methods of inducing an immune response in a subject, methods of treating cancer, and methods of hyperactivating dendritic cells (DCs) that induce type I T helper cell (TH1) and cytotoxic T lymphocyte (CTL) responses in the absence of TH2 immunity. The hyperactivation stimuli drive T cell responses that protect against tumors sensitive or resistant to PD-1 inhibition. These protective responses are dependent on inflammasomes in DCs and can be generated using tumor lysates as immunogens.

[0010] In certain embodiments, a method of inducing a protective immune response to an immunogen in a subject includes obtaining dendritic cells, culturing the dendritic cells ex vivo with an effective amount of non-classical inflammasome-activated lipids, and administering an effective amount of the live dendritic cells to the subject to enhance the protective immune response, thereby inducing a protective immune response. In some embodiments, the therapeutically effective amount of non-classical inflammasome-activated lipids hyperactivates the dendritic cells.

[0011] In certain embodiments, the dendritic cells are optionally cultured ex vivo with an immunogen. In certain embodiments, the dendritic cells are optionally cultured ex vivo with cytokines.

[0012] In certain embodiments, lipids that activate non - classical inflammasomes include: 1 - palmitoyl - 2 - arachidonyl - sn - glycero - 3 - phosphocholine (PAPC), oxidized 1 - palmitoyl - 2 - arachidonyl - sn - glycero - 3 - phosphocholine (oxPAPC), oxPAPC species, components thereof, or combinations thereof.

[0013] In certain embodiments, the method further comprises administering a chemotherapeutic agent.

[0014] In certain embodiments, the therapeutic methods disclosed herein can also be combined with any of the following therapies: radiation, chemotherapy, surgery, therapeutic antibodies, immunomodulators, proteasome inhibitors, pan - deacetylase (DAC) inhibitors, histone deacetylase (HDAC) inhibitors, checkpoint inhibitors, adoptive cell therapies including CAR T and NK cell therapies, and vaccines.

[0015] Preferably, the methods described herein inhibit the growth or progression of cancers such as tumors, or viral infections, in a subject. For example, the methods described herein inhibit tumor growth by at least 1%, such as 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 a tumor by at least 1 mm in diameter, such as 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, most of the tumor has been resected from the subject.

[0016] Unless otherwise indicated, 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 pertains. Methods and materials for this invention are described herein; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0017] Other features and advantages of the present invention will be apparent from the following detailed description and drawings, as well as from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A-1H is a series of figures showing that in the absence of signs of TH2 immunity, over-activated DCs are excellent antigen-presenting cells and drive TH1-biased immune responses. Figure 1A-1F : WT BMDCs were left untreated (none) or treated with LPS alone, or Alum alone, or oxPAPC or PGPC alone for 24 hours, or BMDCs were primed with LPS for 3 hours and then treated with the indicated stimulants for 21 hours. Figure 1A : IL-1β and TNFα cytokine release was monitored by ELISA. Figure 1B : The percentage of cell death was measured by LDH release in the cell supernatant. Figure 1C : BMDCs treated with the indicated stimulants in Figure 1A were stained with a live-dead violet kit, CD11c, and CD40. The mean fluorescence intensity (MFI) of surface CD40 (in CD11c + live cells) was measured by flow cytometry. Figure 1D : BMDCs pretreated with the indicated stimulants were transferred to CD40-coated plates and cultured for 24 hours. IL-12p70 cytokine release was measured by ELISA. Figure 1E : BMDCs pretreated with the indicated stimulants in Figure 1A were incubated with OVA protein for 2 hours or with FITC-labeled OVA for 45 minutes. OVA-FITC uptake was evaluated by flow cytometry (left panel). Data are represented 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 was monitored using a PE-conjugated antibody against H-2Kb that binds to the OVA peptide SIINFEKL (SEQ ID NO:1) (right panel). Data are represented as the frequency of SIINFEKL (SEQ ID NO:1)-associated DCs in CD11c + live cells. Mean and SD from three replicates are shown, and the data represent at least three independent experiments. Figure 1F : BMDCs treated with the indicated stimulants in Figure 1A were loaded (or not loaded) with OVA protein or the OVA peptide SIINFEKL for 1 hour and then co-cultured with splenic OT-II naive cells or OT-I naive CD8+ The T cells were incubated for 4 days. Figure 1F : The supernatant was collected on day 4, and the cytokine release of IFNγ, IL-2, IL-10, TNFα and IL-13 was measured by ELISA. Figure 1G : The BMDCs were left untreated (none), or treated with LPS for 24 hours, or the BMDCs were sensitized with LPS for 3 hours and then treated with PGPC or Alum for 21 hours. Then the Figure 1F spleen OT-I or OT-II T cells in + were used to culture the treated BMDCs. Four days after co-culture, in the presence of brefeldin-A and monensin, CD4 + and CD8 + T cells were stimulated with PMA and ionomycin for 5 hours. The frequency of TH1 cells such as TNFα + IFNγ + and TH2 cells such as Gata3 + IL-4 + IL-10 + in CD4 + T cells was measured by intracellular staining. The data are presented as the ratio of TH1 / TH2 cells (left panel). The frequency of IFNγ + in CD8 Figure 1H T cells is shown in the right panel. The mean and SD from three replicates are shown, and each panel represents at least two independent experiments. + : As shown, C57BL / 6 mice were injected subcutaneously in the right flank with either the endofit-OVA protein alone emulsified in incomplete Freund's adjuvant (IFA) or Alum, or the endofit-OVA protein and LPS. Optionally, the mice were injected with the endofit-OVA protein and LPS plus OxPAPC or PGPC, both emulsified in IFA. Forty days after immunization, CD4 + and CD8

[0019] Figure 2 T cells were isolated from the skin-draining lymph nodes (dLN). Then, the T cells were cultured with naive BMDCs loaded (or not loaded) with OVA or SIINFEKL peptide for 5 days. The secretion of IFNγ, IL-10, and IL-13 was measured by ELISA. The mean and SD from four mice are shown, and each panel represents two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005. 5 live B16OVA cells. Seven, 14, and 21 days after tumor challenge, the mice were injected subcutaneously in the right flank with 5x10 6One untreated WT BMDC (DC 幼稚 ), or activated WT BMDC (DC LPS ) treated with LPS for 23 hours and then pulsed with B16OVAWTL for 1 hour, or WT, or NLRP3 - / - , or casp1 / 11 - / - BMDC (DC LPS+PGPC ) sensitized with LPS for 3 hours, treated with PGPC for 20 hours and then pulsed with B16OVA WTL for 1 hour. Survival was monitored daily (n = 5 mice per group).

[0020] Figure 3A-3E is a series of figures showing that, in the absence of signs of TH2 immunity, over-activated DCs are excellent antigen-presenting cells and drive TH1-biased immune responses. Figure 3A , 3B : BMDCs generated with GMCSF were left untreated (none), or treated with MPLA alone, Alum alone, or OxPAPC or PGPC alone, or BMDCs were sensitized with MPLA for 3 hours and then treated with the indicated stimuli for 21 hours. Figure 3A : IL-1β and TNFα cytokine release was monitored by ELISA. Figure 3B : The percentage of cell death was measured by LDH release in the cell supernatant. Figure 3C , 3D : Splenic CD11c + was sorted and left untreated (none), or treated with LPS alone, Alum alone, or PGPC alone, or DCs were sensitized with LPS for 3 hours and then treated with the indicated stimuli for 21 hours. Figure 3C : IL-1β and TNFα cytokine release was monitored by ELISA. Figure 3D : The percentage of cell death was measured by LDH release in the cell supernatant. Figure 3E : BMDCs generated with GMCSF treated with the indicated stimuli in A were stained with a live-dead violet kit, anti-CD11c, anti-CD80, anti-CD69, and anti-H2kb antibodies. Figure 3E : The mean fluorescence intensity (MFI) of surface CD80 (left panel), CD69 (middle panel), and H2Kb (right panel) in CD11c + live cells was measured by flow cytometry. Mean values and SD from three replicates are shown, and all figures represent at least three independent experiments. *P < 0.05.

[0021] Figure 4A-4CFigures and a series of planar graphs showing that, in the absence of signs of TH2 immunity, hyperactivated DCs are excellent antigen-presenting cells and drive TH1-biased immune responses. Figure 4A-4C : WT BMDCs were left untreated (none), or treated with LPS alone, Alum alone, or OxPAPC or PGPC alone for 24 hours, or BMDCs were sensitized with LPS for 3 hours and then treated with the indicated stimuli for 21 hours. Figure 4A : BMDCs were cultured with fixable FITC-labeled -OVA at 37°C or 4°C for 45 minutes. Then BMDCs were stained with a live-dead violet kit. The gating strategy was used to determine, by flow cytometry, the frequency of OVA-FITC-associated BMDCs at 37°C compared to OVA-associated BMDCs at 4°C. Figure 4B : BMDCs were cultured with endofit-OVA protein for 2 hours. The gating strategy was used to determine, by flow cytometry, using a PE-conjugated antibody against H-2Kb that binds to the OVA peptide SIINFEKL, the frequency of SIINFEKL (SEQ ID NO:1) peptide bound to H2bk on the surface of live BMDCs. Each graph represents triplicates of one of three experiments. Figure 4C : As shown, C57BL / 6 mice were injected subcutaneously in the right flank with either endofit-OVA protein alone or endofit-OVA protein and LPS emulsified in incomplete Freund's adjuvant (IFA) or in Alum. Optionally, 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). Then, the T cells were cultured with naive BMDCs loaded (or not loaded) with OVA for 5 days. IL-4 secretion was measured by ELISA. Mean and SD of four mice are shown, and each graph represents two independent experiments. ***P < 0.005.

[0022] Figure 5 A series of planar graphs showing that, in the absence of signs of TH2 immunity, hyperactivated DCs are excellent antigen-presenting cells and drive TH1-biased immune responses. BMDCs were left untreated (none), or treated with LPS for 24 hours, or BMDCs were sensitized with LPS for 3 hours and then treated with PGPC or Alum for 21 hours. Then the treated BMDCs were cultured with splenic OT-II T cells at a ratio of 1:5 (BMDC:T cell). Four days after co-culture, in the presence of brefeldin-A and monensin, CD4 +T cells were stimulated with PMA and ionomycin for 5 hours. Gating strategies were used to determine the frequency of TH2 cells such as IL-4 + IL-10 + in live CD4 + T cells by intracellular staining. Each plot represents three replicates of one of three experiments.

[0023] Figure 6A-6D are a series of plots and immunostainings showing that cDC1 and cDC2 cells achieve an in vitro overactivated state. ( Figure 6A-6B ) Splenic DCs (left panels) or FLT3-generated DCs (right panels) were sorted into cDC1 (CD11c + CD24 + ) or cDC2 (CD11c + Sirpa + ). DCs were left untreated (none), or treated with LPS alone, Alum alone, or OxPAPC or PGPC alone for 24 hours, or DCs were sensitized with LPS for 3 hours and then treated with the indicated stimulants for 21 hours. ( Figure 6A ) Cell death was measured by LDH release in the supernatant. ( Figure 6B ) IL-1β and TNFα cytokine release was monitored by ELISA. Mean and SD of 3 independent experiments are shown. ( Figure 6C ) FLT3-DCs treated with the indicated stimulants were stained with Phalloidin-FITC and DAPI. Images were obtained on a Zeiss confocal microscope using a 40X oil immersion lens. ( Figure 6D ) FLT3-DCs treated with the indicated stimulants were stained with a live-dead violet kit, CCR7 PE, and CD11c-APC. The mean fluorescence intensity (MFI) of CCR7 was measured by flow cytometry (gating on CD11c + live cells).

[0024] Figure 7 are a schematic diagram and a plan view showing that overactivated cDC1 controls tumor rejection induced by overactivation-based immunotherapy. WT mice were subcutaneously inoculated on the back with 3×10 5 live B16OVA cells. At 7, 14, and 21 days after tumor challenge, mice were left untreated, or mice were injected subcutaneously in the right flank with 1×10 6 untreated WT cDC1 or WT cDC1 treated with LPS for 23 hours (cDC1 活化 ), or WT cDC1 sensitized with LPS for 3 hours and then treated with PGPC for 20 hours (cDC1 过度活化)。Prior to injection, all DCs were pulsed with tumor lysate for 1 hour. Survival was monitored daily (n = 5 mice / group).

[0025] Figure 8A-8C are a series of graphs, planar views, and schematic diagrams showing that hyperactivated cDC1 controls tumor rejection and enhances the tumor infiltration of anti-tumor specific T cells. Figure 8A-8B : Batf3− / − mice were subcutaneously inoculated on the back with 3×10 5 viable B16OVA cells. At 7, 14, and 21 days after tumor challenge, the mice were subcutaneously injected in the right flank with 1×10 6 untreated WT cDC1, or WT cDC1 pulsed with B16OVA tumor lysate for 1 hour after being treated with LPS for 23 hours (cDC1 活化 ), or WT cDC1 sensitized with LPS for 3 hours then treated with PGPC for 20 hours then pulsed with tumor lysate for 1 hour (cDC1 过度活化 ). ( Figure 8C ) Survival was monitored daily (n = 5 mice / group). ( Figure 8B ) Fifteen days after tumor inoculation, skin-draining lymph nodes (dLNs), tumors, and spleen tissues were excised from the immunized mice. Antigen-specific CD8 + and CD4 + T cell percentages were measured using SIINFEKL and AAHAEINEA tetramer staining, respectively (n = 5 mice / group). ( Figure 8C ) Representative plots of SIINFEKL + CD8 + T cells in the tumors and dLNs of the treated mice.

[0026] Figure 9A and 9B are a series of schematic diagrams and planar views showing that hyperactivated cDC1 controls tumor rejection in an inflammasome-dependent manner. For ( Figure 9A ) Casp1 / 11 - / - mice, ( Figure 9B ) NLRP3 - / - mice were subcutaneously inoculated on the back with 3.105 viable B16OVA cells. At 7, 14, and 21 days after tumor challenge, the mice were subcutaneously injected in the right flank with 1.106 untreated WT cDC1 (cDC1 幼稚 ), or WT cDC1 pulsed with B16OVA tumor lysate for 1 hour after being treated with LPS for 23 hours (cDC1 活化 ), or WT or Casp1 / 11 - / - cDC1 sensitized with LPS for 3 hours then treated with PGPC for 20 hours (cDC1 过度活化)。Prior to injection, all DCs were pulsed with tumor lysate for 1 hour. At ( Figure 9A ) Casp1 / 11 - / - ) mice and ( Figure 9B ) NLRP3 mice were monitored daily for survival (n = 5 mice / group).

[0027] Figure 10A and 10B Show the mass spectra of synthetic lipids. Non-oxidized PAPC ( Figure 10A ), oxPAPC ( Figure 10B ), oxPAPC enriched in PEIPC ( Figure 10C ), and biotinylated oxPAPC ( Figure 10D ) were analyzed by mass spectrometry.

[0028] Figure 11A -B. Oxidized phospholipids induce hyperactivated cDC1 and cDC2 cells with a highly migratory phenotype. (A) Wild-type or NLRP3 - / - or Casp1 / 11 - / - BMDCs generated using FLT3L were left untreated (none), or treated with LPS alone, or Alum alone, or PGPC alone for 24 hours, or BMDCs were sensitized with LPS for 3 hours and then treated with the indicated stimuli for 21 hours. IL-1β and TNFα release were monitored by ELISA. The percentage of cell death was measured by LDH release in the cell supernatant. Mean and SD from three replicates are shown, and the data represent at least three independent experiments. (B) Wild-type BMDCs generated using FLT3L were sorted into cDC1 or cDC2 cells and then treated with the indicated stimuli in A. IL-1β and TNFα release were monitored by ELISA. The percentage of cell death was measured by LDH release in the cell supernatant. Mean and SD are from three independent experiments done in two different laboratories.

[0029] Figure 12A -B. Hyperactivated DCs induce strong CTL responses and long-term anti-tumor immunity that depend on CCR7 expression and inflammasome activation. (A-B) Wild-type BMDCs generated using FLT3L were left untreated (DC 幼稚 ), or treated with LPS alone (DC 活化 ) for 18 hours, or BMDCs were sensitized with LPS for 3 hours and then treated with PGPC (DC 过度活化 ) or Alum (DC 细胞焦亡 ) for 15 hours. Optionally, those from NLRP3 - / - or CCR7 - / -BMDCs from mice were sensitized with LPS for 3 hours and then treated with PGPC for 15 hours. 1.10e6 BMDCs were cultured with OVA protein for 1 hour and then injected subcutaneously into wild-type mice. BMDCs without loaded OVA protein were injected as a control group. Seven days after BMDC injection, the skin-draining lymph nodes were excised and stained with a live-dead violet kit, OVA peptide tetramer antibody, anti-CD45, anti-CD3, anti-CD8a, and anti-CD4. (A) The percentages of SIINFEKL+CD8+ T cells (upper panel) and AAHAEINEA+CD4+ live T cells were measured by flow cytometry. (B) The absolute numbers of SIINFEKL+CD8+ T cells (upper panel) and AAHAEINEA+CD4+ live T cells were measured by flow cytometry using CounterBright beads.

[0030] Figure 13A -E. Hyperactivated stimuli induce strong CTL responses in an inflammasome-dependent manner. (A) C57BL / 6 mice were injected subcutaneously in the right flank with either OVA alone or OVA emulsified in incomplete Freund's adjuvant (IFA) with LPS or OVA with PGPC, or OVA with LPS plus oxPAPC or PGPC. Seven or 40 days after immunization, T cells were isolated from skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD8 beads. (A) Among CD3+CD8+ live cells, the percentages of T effector cells (Teff) such as CD44 low CD62L low 、T effector memory cells (TEM) such as CD44 hi CD62L low 、and T central memory cells (TCM) such as CD44hiCD62Lhi are shown. (B) Seven days after immunization, CD8+ T cells were sorted from dLNs and then treated with PMA and ionomycin or co-cultured with B16OVA cells (target cells) at a ratio of 1:3 (effector cell:target cell) for 5 hours. Degranulation of CD8+ T cells was evaluated by monitoring the percentage of CD107a+ among live CD8+ T cells using flow cytometry. The mean and SD of five to ten mice are shown. (C) Mice were injected subcutaneously in the right flank with either OVA alone or OVA with LPS, or OVA with LPS plus oxPAPC or PGPC, or OVA with LPS plus Alum. Optionally, for NLRP3 - / -Mice were injected with OVA emulsified in IFA with LPS plus PGPC. Seven days after immunization, CD8+ T cells were sorted from the skin dLNs of immunized mice and co-cultured with BMDCs loaded (or not loaded) with OVA at a ratio of 1:10 (DC:T cells) for 7 days. The percentage of SIINFEKL+IFNγ+ in CD8+ live T cells was measured using OVA peptide tetramer staining followed by intracellular IFNγ staining. (D-E) CD45.1 mice were irradiated and then reconstituted with bone marrow ZBTB46DTR mice plus WT or NLRP3 - / - or Casp1 / 11 - / - or CCR7 - / - (ratio 5:1), all on a CD45.2 C57BL / 6 background. Six weeks after reconstitution, tamoxifen was injected into the chimeric mice every other day for 7 days. Then, the chimeric mice were immunized subcutaneously in the right flank with OVA emulsified in IFA with LPS plus PGPC. Seven days after immunization, CD8+ T cells were isolated from the skin draining lymph nodes (dLNs) or from the spleen by magnetic enrichment using anti-CD8 beads. (D) The percentages of Teff, TEM, TCM, and T naive cells in the skin dLNs were measured by flow cytometry. (E) The percentage of SIINFEKL+ in CD8+ live T cells in the dLNs (left panel) or spleen (right panel) was measured by flow cytometry using OVA peptide tetramer staining. Total CD8+ T cells were sorted from the dLNs and co-cultured with untreated BMDCs loaded (or not loaded) with OVA at a ratio of 1:10 (DC:T cells) for 7 days.

[0031] Figure 14A -D. Immunization with an over-activating stimulant abrogates immunogenic tumors from hot to cold tumors. (A) C57BL / 6 mice were subcutaneously inoculated with 5x10 5 live MC38OVA cells in the upper left back. Fourteen days later, the mice were left untreated (non-immunized), or injected subcutaneously in the right flank with syngeneic MC38OVA whole tumor lysate (WTL), plus LPS and PGPC, with or without intravenous (i.v.) injection of neutralizing anti-IL-1β, or with or without intraperitoneal injection of anti-CD4, or anti-CD8a. The mice received 2 booster injections of WTL and LPS plus PGPC on days 37 and 55 after tumor inoculation. The tumors were allowed to reach 20 mm in diameter. The percentage of survival is shown (n = 10 mice per group). (B) C57BL / 6 mice were subcutaneously inoculated with 3X10 5live B16OVA cells. Ten days later, the mice were left untreated (non-immunized) or injected intraperitoneally with anti-PD1 antibody. Optionally, the mice were subcutaneously injected in the right flank with syngeneic B16OVA WTL, plus LPS and PGPC, with or without intravenous (i.v.) injection of a neutralizing antibody against IL-1β, or with or without intraperitoneal injection of anti-CD4 or anti-CD8a. On days 17 and 24 after tumor inoculation, the mice received two booster injections of B16OVA WTL, plus LPS and PGPC. The percentage of survival is shown (n = 10 mice / group). (C) C57BL / 6 mice were subcutaneously inoculated in the upper left back with 3X10 5 live B16-F10 cells. Seven days later, the mice were left untreated (non-immunized) or injected intraperitoneally with anti-PD1 antibody. Optionally, the mice were immunized subcutaneously in the right flank with syngeneic B16-F10 WTL, plus LPS and PGPC, with or without intravenous (i.v.) injection of a neutralizing antibody against IL-1β, or with or without intraperitoneal injection of anti-CD4 or anti-CD8a. On days 14 and 21 after tumor inoculation, the mice received two booster injections. The percentage of survival is shown (n = 10 mice per group). (D) BALB / c WT mice were subcutaneously inoculated in the left back with 3X10 5 live CT26 cells. Seven days later, the mice were left untreated (non-immunized) or injected intraperitoneally with anti-PD1 antibody. Optionally, the mice were subcutaneously injected in the right flank with syngeneic CT26 WTL, plus LPS and PGPC, with or without intravenous (i.v.) injection of a neutralizing antibody against IL-1β, or with or without intraperitoneal injection of anti-CD4 or anti-CD8a. On days 14 and 21 after tumor inoculation, the mice received two booster injections. The percentage of survival is shown (n = 10 mice per group).

[0032] Figure 15A -F. Overactivated cDC1 can use a complex antigen source to stimulate T cell-mediated anti-tumor immunity. (A) B16OVA cells were subcutaneously injected into Zbtb46DTR mice. The mice were injected with diphtheria toxin (DTx) every other day for 4 consecutive injections, or injected with PBS. Seven days after tumor injection, all mice were immunized with B16OVA WTL plus LPS and PGPC, followed by two booster injections. The percentage of mouse survival is shown (n = 10 mice per group). (B) CD45.1 mice were irradiated and then received cells from Zbtb46DTR mice plus WT or Nlrp3 - / - or Casp1 / 11 - / - or Ccr7 - / -Mixed BM reconstitution of mice. Six weeks after reconstitution, B61OVA cells were subcutaneously injected into the mouse chimeras, and then all mice received DTx three times a week for a total of 12 consecutive injections. Seven days after tumor inoculation, the chimeric mice were immunized with B16OVA WTL and LPS plus PGPC and received 2 booster injections. The percentage of surviving mice is shown (n = 5 mice per group). (C-D) B16OVA cells were subcutaneously injected into WT or Batf3 - / - mice. Seven days after tumor inoculation, the mice were left untreated, or WT and Batf3 - / - mice were immunized with B16OVA WTL and LPS plus PGPC followed by 2 booster injections. (C) The percentage of surviving mice is shown (n = 10 mice per group). (D) Twenty-one days after tumor inoculation, the percentages of OVA-specific CD8+ T cells and CD4+ T cells were evaluated using tetramer staining (n = 5 mice per group). (E-F) Batf3 - / - mice were subcutaneously injected with B16OVA cells in the right flank. Seven days after tumor inoculation, the mice were left untreated (no cDC1 injection), or the mice were subcutaneously injected with FLT3-derived naive cDC1 or LPS-treated activated cDC1 or LPS plus PGPC-pretreated hyperactivated cDC1 in the left flank. Before injection, all cDC1 were loaded with B16OVA WTL for 1 hour. (E) The percentage of surviving mice is shown (n = 5 mice per group). (F) Twenty-one days after tumor inoculation, OVA-specific CD8+ T cells and CD4+ T cells were evaluated using tetramer staining (n = 5 mice per group).

[0033] Figure 16A-C. Oxidized phospholipids induce inflammasome-dependent IL-1β secretion in cDC1 and cDC2 cells and promote a highly migratory DC phenotype. (A) Wild-type BMDCs generated using FLT3L were left untreated (none), or treated with CpG 1806 alone, or PGPC alone for 24 h, or BMDCs were sensitized with CpG1806 for 3 h and then treated with the indicated stimuli for 21 h. IL-1β and TNFα release were monitored by ELISA. The percentage of cell death was measured by LDH release in the cell supernatant. Mean and SD from triplicates are shown, and data represent at least three independent experiments. (B) Gating strategy to isolate cDC1 or cDC2 from FLT3L-generated BMDCs or from the spleen of wild-type mice. Purity post-sorting is shown for splenic cDCs or FLT3L DCs. (C) Splenic cDC1 or cDC2 were left untreated (none), or treated with LPS alone, or Alum alone, or oxPAPC or PGPC alone for 18 h, or BMDCs were sensitized with LPS for 3 h and then treated with the indicated stimuli for 15 h. IL-1β and TNFα release were monitored by ELISA. The percentage of cell death was measured by LDH release in the cell supernatant. Mean and SD are from three independent experiments done in two different laboratories.

[0034] Figure 17A -C. Over-activated DCs induce strong CTL responses and long-term anti-tumor immunity that are dependent on CCR7 expression and inflammasome activation. Wild-type BMDCs generated using FLT3L were left untreated (DC 幼稚 ), or treated with LPS alone (DC 活化 ) for 18 h, or BMDCs were sensitized with LPS for 3 h and then PGPC (DC 过度活化 ) or Alum (DC 细胞焦亡 ) was added to the medium for 15 h. Optionally, BMDCs from NLRP3 - / - or CCR7 - / - mice were sensitized with LPS for 3 h and then PGPC was added to the medium for 15 h. BMDCs were washed and then cultured with FITC-labeled-OVA for 45 min or with non-fluorescent OVA protein for 2 h. (A) OVA peptide presentation on MHC-I was monitored using a PE-conjugated antibody against H-2Kb that binds the OVA peptide SIINFEKL. Data are represented as the frequency of SIINFEKL-associated DCs in CD11c+ live cells. Mean and SD from triplicates are shown, and data represent three independent experiments. (B) Wild-type or NLRP3 - / - or CCR7 - / -BMDCs. BMDCs were washed and then stained with a live-dead violet kit, CD11c, and CD40. The mean fluorescence intensity (MFI) of surface CD40 (in CD11c+ live cells) was measured by flow cytometry. (C) CCR7 generated using FLT3L - / - BMDCs were left untreated (none), or treated with LPS alone, or Alum alone, or PGPC alone for 24 h. Optionally, BMDCs were sensitized with LPS for 3 h and then treated with the indicated stimuli for 21 h. IL-1β and TNFα release was monitored by ELISA. The percentage of cell death was measured by LDH release in the cell supernatant. Mean and SD from triplicates are shown and the data represent at least three independent experiments.

[0035] Figure 18A -D. Hyperactivated stimuli enhance memory T cell generation and augment antigen-specific IFNγ effector responses in an inflammasome-dependent manner. C57BL / 6 mice were injected subcutaneously in the right flank with either OVA alone emulsified in incomplete Freund's adjuvant (IFA), or OVA with LPS, or OVA with PGPC, or OVA with LPS plus oxPAPC, or PGPC. Seven days after immunization, T cells were isolated from skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD8 beads. (A) Gating strategy to determine the percentage of T effector cells (Teff) such as CD44 low CD62L low , T effector memory cells (TEM) such as CD44 high CD62L low , and T central memory cells (TCM) such as CD44 high CD62L high . (B) The absolute number of Teff or TEM cells in the skin dLNs of each mouse was evaluated by flow cytometry in total CD3+ live cells. (C) Seven days after immunization, CD8+ T cells were sorted from the dLNs and then cultured with untreated BMDCs loaded (or not loaded) with serial dilutions (starting at 1000 μg / ml) of OVA protein. IFNγ cytokine secretion was measured by ELISA. Mean and SD of five mice are shown. (D) Seven days after immunization, CD8+ T cells were sorted from the dLNs and then treated with PMA and ionomycin, or co-cultured with B16OVA cells (target cells) at a ratio of 1:3 (effector cell:target cell) for 5 h. Gating strategy to determine the percentage of CD107a+ in live CD8+ T cells by flow cytometry. Each panel represents five mice. *P < 0.05; **P < 0.01.

[0036] Figure 19A-B. Hyperactivated stimuli increase memory T cell generation and enhance antigen-specific IFNγ effector responses in an inflammasome-dependent manner. (A–B) Irradiated CD45.1 mice were then reconstituted with bone marrow ZBTB46DTR mice plus WT or NLRP3 - / - or Casp1 / 11 - / - or CCR7 - / - (at a ratio of 5:1), all on a CD45.2 C57BL / 6 background. Six weeks after reconstitution, tamoxifen was injected into the chimeric mice every other day for 7 days. Then, the chimeric mice were immunized subcutaneously in the right flank with OVA emulsified in IFA plus LPS and PGPC. Seven days after immunization, CD8+ T cells were isolated from skin-draining lymph nodes (dLNs) or from the spleen by magnetic enrichment using anti-CD8 beads. (A) Percentages of Teff, TEM, TCM, and T naive cells in skin dLNs were measured by flow cytometry. Each plot represents five mice. (B) Percentages of SIINFEKL+ among CD8+ live T cells in dLNs (upper panel) or spleen (lower panel) were measured by flow cytometry using OVA peptide tetramer staining.

[0037] Figure 20A -C. Mice were injected subcutaneously (s.c.) in the right flank with PBS (non-immunized), B16OVA cell lysate alone emulsified in incomplete Freund's adjuvant (IFA) (none), or B16OVA cell lysate and LPS, or B16OVA lysate plus LPS and oxPAPC or PGPC. Fifteen days after immunization, mice were challenged subcutaneously in the upper left back with 3 x 10 5 viable B16OVA cells. One hundred and fifty days later, tumor-free mice were rechallenged subcutaneously on the back with 5 x 10 5 viable B16OVA cells. (A) Tumor growth was monitored every 2 days (upper panel). For the survival experiment (lower panel), mice were allowed to reach a 20 mm diameter (n = 8–15 mice per group). (B–C) Tumors were harvested at the end point of tumor growth and dissociated to obtain single-cell tumor suspensions. (B) Percentages of tumor-infiltrating CD3+CD4+ and CD3+CD8+ T cells among enriched CD45+ live cells were evaluated by flow cytometry. (C) Tumor-infiltrating CD3+ T cells were sorted and then stimulated in the presence of anti-CD3 and anti-CD28 dynabeads for 24 h. IFNγ was measured by ELISA (lower panel) (n = 4 mice per group).

[0038] Figure 21A-D. (A - B) was evaluated at the immune or tumor injection sites in surviving mice and the absolute numbers of CD8+ T cells and CD69+CD103+ tissue-resident memory CD8+ T cells were measured by flow cytometry (n = 4 mice). (C - D) Circulating memory CD8+ T cells (TCM) were isolated from the spleens of surviving mice or age-matched non-immunized tumor-bearing mice, and tissue-resident memory CD8+ cells (TRM) were isolated from skin inguinal adipose tissue. (C) TCM and TRM from surviving mice were co-cultured with B16OVA or B16-F10 or CT26 tumor cells at a ratio of 1:5 (tumor cells: T cells) for 5 hours. Cell death induced by cytotoxic CD8+ T cells was measured by LDH release in the supernatant. (D) Mice were left untreated (no Tx), or were inoculated intravenously (i.v.) with 5x10 5 CD8+ TCM cells and / or intradermally (i.d.) with 5x10 5 CD8+ TRM cells isolated from surviving mice or age-matched non-immunized tumor-bearing mice. Seven days later, all mice were challenged with 3x10 5 viable B16OVA cells. The percentage of survival was monitored every 2 days. Mice were allowed to reach 20 mm in diameter (n = 5 mice per group). Detailed Description

[0039] The innate immune system is generally thought to function in an all-or-none manner, with DCs either initiating an inflammatory response that promotes adaptive immunity or not. Thus, Toll-like receptors (TLRs) expressed by DCs are thought to play an important role in determining the immunogenic potential of these cells. The mammalian immune system is responsible for detecting microbes and activating protective responses that limit infection. The key to this task lies with dendritic cells, which sense microbes and subsequently promote T cell activation. Dendritic cells have been shown to gauge the threat of any infection and direct a proportional response (Blander, J.M. (2014). Nat Rev Immunol 14, 601 - 618; Vance, R.E. et al., (2009) Cell host & microbe 6, 10 - 21), but the mechanisms by which these immunomodulatory activities can occur remain unclear.

[0040] PRRs serve to directly or indirectly detect molecules common to a wide range of microbial classes. These molecules are commonly referred to as pathogen-associated molecular patterns (PAMPs) and include factors such as bacterial lipopolysaccharide (LPS), bacterial flagellin, or viral double-stranded RNA.

[0041] An important property of PRRs as immune regulators is their ability to recognize specific microbial products. As such, PRR-mediated signaling events can provide a clear indication of infection. It is assumed that activation of PRRs expressed on DCs promotes a "GO" signal for inflammation and T cell-mediated immunity. Interestingly, several groups have recently proposed that DCs do not function in this all-or-none manner (Blander, J.M., and Sander, L.E. (2012). Nat Rev Immunol 12, 215-225; Vance, R.E. et al., (2009) Cell hostµbe 6, 10-21). Rather, DCs may have the ability to gauge the threat (or virulence) posed by any potential infection and mount a proportional response. The most commonly discussed way to gauge virulence is based on the ability of virulent pathogens to activate more distinct PRRs than non-pathogens. However, not all microbes have a common set of PRR activators, and not all PRR activators have equal potency. Thus, the number of PRRs activated during infection is not an ideal measure of virulence. In addition, increasing the number of PRRs activated during infection will generally result in a larger inflammatory response, which can indirectly promote a larger T cell response. Conditions previously shown to enhance the DC activation state (e.g., by using virulent pathogens as stimuli) are also expected to enhance the MΦ activation state (Vance, R.E. et al., (2009) Cellhostµbe 6, 10-21). Thus, it remains unclear whether the immune system (i.e., DCs) truly has a mechanism for specifically gauging the threat of infection.

[0042] One possible way to assess the threat of infection is through a known coincidence detection process, where independent inputs result in a response different from that elicited by any single input. In the case of PRRs, regardless of the threat of virulence, one such input must be microbial products that serve as indicators of infection. To measure the virulence threat, a second input must be present. Without wishing to be bound by theory, it is currently thought that the putative second input is a molecule produced at the site of tissue damage, as cellular damage is a characteristic typically associated with highly pathogenic microorganisms. Candidate molecules that can provide a second stimulus to DCs are molecules of different families called damage-associated molecular patterns (DAMPs), which are also known as alarmins (Kono, H., and Rock, K. L. (2008) Nat Rev Immunol 8, 279-289; Pradeu, T., and Cooper, E. L. (2012) Front Immunol 3, 287). DAMPs are found at sites of infectious or non-infectious tissue damage and are thought to regulate the immune response, although their mechanism of action remains unclear. A representative of one such class of DAMPs is oxidized phospholipids derived from 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), which are collectively called oxPAPC. These lipids are produced at sites of both infectious and non-infectious tissue damage (Berliner, J. A., and Watson, A. D. (2005). N Engl J Med 353, 9-11; Imai, Y. et al. (2008) Cell 133, 235-249; Shirey, K. A. et al. (2013) Nature 497, 498-502) and are found at very high levels in the membranes of dying cells (Chang, M. K. 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 tissues (Leitinger, N. (2003) Curr Opin Lipidol 14, 421-430), where local concentrations can be as high as 10-100 μM (Oskolkova, O. V. et al. (2010) J Immunol 185, 7706-7712). The association between oxPAPC and dying cells increases the likelihood that these lipids can be used as a general indicator of tissue health. Thus, in the presence of microbial product(s), oxPAPC can indicate an increased threat of infection.

[0043] Due to the aforementioned characteristics of activated DCs, these cells have the ability to stimulate antigen-specific T cell responses and have adopted many strategies to promote DC activation to drive protective immunity. These strategies typically involve the use of synthetic or natural microbial products that stimulate pattern recognition receptors (PRRs) of the Toll-like receptor (TLR) family, notable examples being the molecule monophosphoryl lipid A (MPLA, an FDA-approved TLR4 ligand used in an increasing number of vaccines) (J. Paavonen, Lancet, vol. 374, no. 9686, pp. 301-314, Jul. 2009; M. Kundi, Expert Rev. Vaccines, vol. 6, no. 2, pp. 133-140, Apr. 2007; A. M. Didierlaurent, et al. J. Immunol., vol. 183, no. 10, pp. 6186-6197, Nov. 2009). Note that individual TLRs are unable to upregulate all of the molecular signals required to promote T cell-mediated immunity. Members of the interleukin-1 (IL-1) family of cytokines are key regulators of many aspects of T cell differentiation, the generation of long-lived memory T cells, and effector functions (S. Z. Ben-Sasson, et al. Proc. Natl. Acad. Sci. U.S.A., vol. 106, no. 17, pp. 7119-24, Apr. 2009; S. Z. Ben-Sasson, et al. J. Exp. Med., vol. 210, no. 3, pp. 491-502, Mar. 2013; A. Jain, et al. Nat. Commun., vol. 9, no. 1, pp. 1-13, 2018). The 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 thus is not released from the cell via the conventional biosynthetic pathway. However, IL-1β accumulates in an inactive state in the cytoplasm of DCs that have been activated by TLR ligands (C. Garlanda, et al. Immunity, vol. 39, no. 6, pp. 1003-1018, Dec. 2013). The lack of release of IL-1β from activated DCs increases the likelihood that TLR signaling alone is insufficient to maximally stimulate T cell responses and protective immunity.

[0044] The DC activation state is not the only cell fate achievable by DCs upon PRR signaling. Indeed, different PRRs stimulate different fates in these cells. One such fate is commitment to an inflammatory form of cell death called pyroptosis. Pyroptosis is a regulated process caused by the activity of inflammasomes, which are supramolecular organizing centers (SMOCs) assembled in the cytoplasm of DCs and other cells (A. Lu, et al Cell, vol. 156, no. 6, pp. 1193-1206, Mar. 2014; J. C. Kagan, et al Nat. Rev. Immunol., vol. 14, no. 12, pp. 821-826, Dec. 2014). Inflammasome assembly is typically stimulated upon detection of PAMPs or DAMPs in the cytoplasm of host cells, such that cytoplasmic PRRs are responsible for linking the threat assessment in the cytoplasm to inflammasome-dependent pyroptosis (K. J. Kieser and J. C. 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 process of pyroptosis results in the release of IL-1β and other IL-1 family members from the cell, thus providing signals to T cells that TLRs cannot. In addition to this increased activity, in terms of promoting IL-1β release, pyroptotic cells are dead and thus lose the ability to participate in the multi-day processes required to stimulate and differentiate naive T cells in the dLN (T. R. Mempel, et al. Nature, vol. 427, no. 6970, pp. 154-159, Jan. 2004). Indeed, stimulants that promote pyroptosis, such as the commonly used vaccine adjuvant alum (S. C. Eisenbarth, et al Nature, vol. 453, no. 7198, pp. 1122-1126, Jun. 2008; M. Kool, et al J. Immunol., vol. 181, no. 6, pp. 3755-3759, Sep. 2008), are widely praised for their ability to stimulate type 2 immune responses (P. Marrack, et al Nat. Rev. Immunol., vol. 9, no. 4, pp. 287-293, Apr. 2009), which are not applicable to the elimination of many microbial infections or cancers).

[0045] Adoptive cell therapies (ACTs) (including allogeneic and autologous hematopoietic stem cell transplantation (HSCT) and recombinant cell (i.e., CAR T) therapies) are treatment options for many malignancies (for reviews of HSCT and adoptive cell therapy approaches, see, Rager & Porter, Ther Adv Hematol (2011) 2(6) 409 - 428; Roddie & Peggs, Expert Opin. Biol. Ther. (2011) 11(4):473 - 487; Wang et al Int. J. Cancer. (2015) 136, 1751 - 1768; and Chang, Y.J. and X.J. Huang, Blood Rev, 2013. 27(1):55 - 62). Such adoptive cell therapies include, but are not limited to, allogeneic and autologous hematopoietic stem cell transplantation, donor leukocyte (or lymphocyte) infusion (DLI), adoptive transfer of tumor - infiltrating lymphocytes, or adoptive transfer of T cells or NK cells (including recombinant cells, i.e., CAR T, CAR NK, gene - edited T cells or NK cells, see Hu et al. Acta Pharmacologica Sinica (2018) 39:167 - 176, Irving et al Front Immunol. (2017) 8:267). In addition to the necessity of donor - derived cells to reconstitute hematopoiesis after radiotherapy and chemotherapy, immune reconstitution from the transferred cells is important for eliminating residual tumor cells. The efficacy of ACT as a treatment option for malignancies is influenced by many factors, including the source, composition, and phenotype of donor cells (lymphocyte subsets, activation status), underlying diseases, pre - transplant conditioning regimens, and post - transplant immune support (i.e., IL - 2 therapy) and the graft - versus - tumor (GVT) effect mediated by donor cells within the graft. In addition, these factors must be balanced against transplantation - related mortality, which is typically caused by the conditioning regimen and / or excessive immune activity of donor cells within the host (i.e., graft - versus - host disease, cytokine release syndrome, etc.).

[0046] This application is in part based on the discovery that stimuli that activate dendritic cells (DCs), or that promote DC pyroptosis, induce a mixed T - cell response consisting of type 1 and type 2 T helper (Th) cells. In contrast, stimuli that over - activate DCs selectively stimulate TH1 and cytotoxic T lymphocyte (CTL) immune responses, without signs of TH2 - induced immunity. Even when using a complex antigen source (e.g., tumor cell lysates), the TH1 - biased immunity generated by over - activated DCs confers on these cells the unique ability to mediate long - term protective anti - tumor immunity. As described herein, over - activated DCs can be generated ex vivo and used, for example, in adoptive cell therapy.

[0047] Accordingly, the present disclosure provides methods for generating a population of therapeutic dendritic cells, the methods comprising obtaining live dendritic cells from a cell donor, sensitizing the dendritic cells ex vivo with a TLR ligand, culturing the sensitized dendritic cells ex vivo with a lipid activated by a non-canonical inflammasome, and loading the dendritic cells with an immunogen, thereby generating a population of therapeutic dendritic cells.

[0048] The present disclosure also provides methods for inducing an immune response in a subject, the methods comprising obtaining live dendritic cells from a cell donor, sensitizing the dendritic cells ex vivo with a TLR ligand, culturing the sensitized dendritic cells ex vivo with a lipid activated by a non-canonical inflammasome, loading the dendritic cells with an immunogen, thereby generating a population of therapeutic dendritic cells, and administering the population of therapeutic dendritic cells to the subject, thereby inducing an immune response in the subject.

[0049] The present disclosure also provides methods for treating cancer, the methods comprising obtaining live dendritic cells from a cell donor, sensitizing the dendritic cells ex vivo with a TLR ligand, culturing the sensitized dendritic cells ex vivo with a lipid activated by a non-canonical inflammasome, loading the dendritic cells with an immunogen, thereby generating a population of therapeutic dendritic cells, and administering the population of therapeutic dendritic cells to the subject, thereby treating cancer in the subject.

[0050] Dendritic cells

[0051] The methods disclosed herein relate to obtaining live dendritic cells from a cell donor. The dendritic cells obtained from the cell donor can be immature or mature. Dendritic cells can be differentiated in vivo or in vitro.

[0052] In some embodiments, obtaining live dendritic cells from a cell donor comprises harvesting progenitor cells from the cell donor and culturing the progenitor cells ex vivo under conditions effective to induce differentiation, thereby obtaining dendritic cells from the cell donor. Methods for the in vitro differentiation of progenitor cells into dendritic cells are known in the art. See, e.g., Ardavin et al., “Origin and Differentiation of Dendritic Cells,” TRENDS in Immunol. 22(12):691-700 (2001).

[0053] In some embodiments, the progenitor cells are lymphoid progenitor cells. In some embodiments, the progenitor cells are myeloid progenitor cells. In some embodiments, the progenitor cells are blood monocytes.

[0054] In some embodiments, the progenitor cells are derived from bone marrow. In some embodiments, the progenitor cells are derived from blood. In some embodiments, the progenitor cells are derived from peripheral blood monocytes. In some embodiments, the progenitor cells are derived from umbilical cord blood.

[0055] In some embodiments, culturing progenitor cells ex vivo under conditions effective to induce differentiation comprises culturing the progenitor cells in the presence of one or more cytokines.

[0056] In some embodiments, culturing progenitor cells ex vivo under conditions effective to induce differentiation comprises culturing the progenitor cells in the presence of granulocyte - macrophage colony - stimulating factor (GM - CSF), interleukin - 4 (IL - 4), tumor necrosis factor α (TNF - α), transforming growth factor β (TGF - β), interleukin 7 (IL - 7), stem cell factor (SCF), fms - like tyrosine kinase 3 ligand (FLT3 - L), interleukin 1 (IL - 1), or a combination thereof.

[0057] In some embodiments, the progenitor cells are cultured ex vivo for between about 1 and about 48 hours. In some embodiments, the progenitor cells are cultured for about 6 to about 48, about 12 to about 48, about 18 to about 48, about 24 to about 48, about 30 to about 48, about 36 to about 48, about 42 to about 48, about 1 to about 42, about 6 to about 42, about 12 to about 42, about 18 to about 42, about 24 to about 42, about 30 to about 42, about 36 to about 42, about 1 to about 36, about 6 to about 36, about 12 to about 36, about 18 to about 36, about 24 to about 36, about 30 to about 36, about 1 to about 30, about 6 to about 30, about 12 to about 30, about 18 to about 30, about 24 to about 30, about 1 to about 24, about 6 to about 24, about 12 to about 24, about 18 to about 24, about 1 to about 18, about 6 to about 18, about 12 to about 18, about 1 to about 12, about 6 to about 12, or about 1 to about 6 hours.

[0058] In some embodiments, the progenitor cells are blood monocytes. In some embodiments, the blood monocytes are cultured in the presence of GM - CSF and / or IL - 4.

[0059] In some embodiments, obtaining dendritic cells from a cell donor comprises harvesting in vivo - differentiated dendritic cells from the cell donor. In some embodiments, the in vivo - differentiated dendritic cells are immature dendritic cells. In some embodiments, the in vivo - differentiated dendritic cells are mature dendritic cells.

[0060] In some embodiments, the in vivo - differentiated dendritic cells are harvested from the spleen of the cell donor. In some embodiments, the in vivo - differentiated dendritic cells are harvested from the lymph nodes of the cell donor. In some embodiments, the in vivo - differentiated dendritic cells are harvested from the thymus of the cell donor. In some embodiments, the in vivo - differentiated dendritic cells are harvested from the blood of the cell donor. In some embodiments, the in vivo - differentiated dendritic cells are harvested from the skin of the cell donor.

[0061] In some embodiments, obtaining dendritic cells from a subject includes cryopreserved progenitors and / or in vivo differentiated dendritic cells.

[0062] The methods disclosed herein include ex vivo sensitization of dendritic cells with TLR ligands. Suitable TLR ligands are described herein. Sensitizing dendritic cells can include culturing progenitors, ex vivo differentiated dendritic cells, and / or in vivo differentiated dendritic cells in the presence of a TLR ligand.

[0063] In some embodiments, dendritic cells are ex vivo sensitized with a TLR ligand for about 1 to about 24 hours. In some embodiments, dendritic cells are ex vivo sensitized with a TLR ligand for about 3 to about 24, about 6 to about 24, about 9 to about 24, about 12 to about 24, about 15 to about 24, about 18 to about 24, about 21 to about 24, about 1 to about 21, about 3 to about 21, about 6 to about 21, about 9 to about 21, about 12 to about 21, about 15 to about 21, about 18 to about 21, about 1 to about 18, about 3 to about 18, about 6 to about 18, about 9 to about 18, about 12 to about 18, about 15 to about 18, about 1 to about 15, about 3 to about 15, about 6 to about 15, about 9 to about 15, about 12 to about 15, about 1 to about 12, about 3 to about 12, about 6 to about 12, about 9 to about 12, about 1 to about 9, about 3 to about 9, about 6 to about 9, about 1 to about 6, about 3 to about 6, or about 1 to about 3 hours.

[0064] In embodiments where progenitors are ex vivo differentiated, dendritic cells can be sensitized before ex vivo culturing progenitors under conditions effective to induce differentiation. In some embodiments, dendritic cells can be sensitized after ex vivo culturing progenitors under conditions effective to induce differentiation. Sensitizing dendritic cells can occur simultaneously with ex vivo culturing progenitors under conditions effective to induce differentiation.

[0065] The methods disclosed herein include culturing sensitized dendritic cells ex vivo with non-canonical inflammasome-activating lipids. Suitable non-canonical inflammasome-activating lipids are described herein. Culturing sensitized dendritic cells can include culturing progenitors, ex vivo differentiated dendritic cells, and / or in vivo differentiated dendritic cells in the presence of a non-canonical inflammasome-activating lipid.

[0066] In some embodiments, the lipid-activated non-canonical inflammasome is used to culture the sensitized dendritic cells ex vivo for between about 1 and about 48 hours. In some embodiments, the progenitor cells are cultured for about 6 to about 48, about 12 to about 48, about 18 to about 48, about 24 to about 48, about 30 to about 48, about 36 to about 48, about 42 to about 48, about 1 to about 42, about 6 to about 42, about 12 to about 42, about 18 to about 42, about 24 to about 42, about 30 to about 42, about 36 to about 42, about 1 to about 36, about 6 to about 36, about 12 to about 36, about 18 to about 36, about 24 to about 36, about 30 to about 36, about 1 to about 30, about 6 to about 30, about 12 to about 30, about 18 to about 30, about 24 to about 30, about 1 to about 24, about 6 to about 24, about 12 to about 24, about 18 to about 24, about 1 to about 18, about 6 to about 18, about 12 to about 18, about 1 to about 12, about 6 to about 12, or about 1 to about 6 hours.

[0067] In some embodiments, culturing the lipid-activated non-canonical inflammasome-sensitized dendritic cells ex vivo is performed simultaneously with culturing the TLR ligand-sensitized dendritic cells ex vivo. In some embodiments, culturing the lipid-activated non-canonical inflammasome-sensitized dendritic cells ex vivo is performed after culturing the TLR ligand-sensitized dendritic cells ex vivo.

[0068] The methods disclosed herein include loading dendritic cells with an immunogen. Suitable immunogens are described herein. Loading dendritic cells with an immunogen can include culturing the dendritic cells with the immunogen.

[0069] In some embodiments, loading dendritic cells with an immunogen can be performed for about 1 to about 24 hours. In some embodiments, loading dendritic cells with an immunogen can be performed for about 3 to about 24, about 6 to about 24, about 9 to about 24, about 12 to about 24, about 15 to about 24, about 18 to about 24, about 21 to about 24, about 1 to about 21, about 3 to about 21, about 6 to about 21, about 9 to about 21, about 12 to about 21, about 15 to about 21, about 18 to about 21, about 1 to about 18, about 3 to about 18, about 6 to about 18, about 9 to about 18, about 12 to about 18, about 15 to about 18, about 1 to about 15, about 3 to about 15, about 6 to about 15, about 9 to about 15, about 12 to about 15, about 1 to about 12, about 3 to about 12, about 6 to about 12, about 9 to about 12, about 1 to about 9, about 3 to about 9, about 6 to about 9, about 1 to about 6, about 3 to about 6, or about 1 to about 3 hours.

[0070] In some embodiments, loading dendritic cells with an immunogen is performed simultaneously with culturing dendritic cells ex vivo with a lipid that activates a non-canonical inflammasome. In some embodiments, loading dendritic cells with an immunogen is performed after culturing dendritic cells ex vivo with a lipid that activates a non-canonical inflammasome.

[0071] In some embodiments of methods of generating a population of therapeutic dendritic cells and / or methods of inducing an adaptive immune response, the dendritic cells and / or progenitors are cryopreserved. In some embodiments, the dendritic cells and / or progenitors are cryopreserved before being loaded with an immunogen. In some embodiments, the dendritic cells are cryopreserved after being loaded with an immunogen.

[0072] Methods of obtaining dendritic cells and loading dendritic cells with, for example, cancer immunogens are described in the art, such as in US20060134067A1, US9694059B2, US9962433B2, US20080254537A1, US9701942B2, US20060057129A1, US20160263206A1, US20150352200A1, US20070292448A1, US6251665B1, WO2003010292A3, US2017036325A1, US20040197903A1, and US10731130B2.

[0073] TLR ligand

[0074] 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 oligomerization domain (NOD)-like receptor (NLR) family, cGAS, STING, or the AIM2-like receptor (ALR). Specific examples of pattern recognition receptor ligands include natural or synthetic bacterial lipopolysaccharide (LPS), natural or synthetic bacterial lipoproteins, natural or synthetic DNA or RNA sequences, natural or synthetic cyclic dinucleotides, and natural or synthetic saccharides. Cyclic dinucleotides include cyclic GMP-AMP (cGAMP), cyclic di-AMP, and cyclic di-GMP.

[0075] In some embodiments, the TLR ligand is selected from TLR1 ligand, TLR2 ligand, TLR3 ligand, TLR4 ligand, TLR5 ligand, TLR6 ligand, TLR7 ligand, TLR8 ligand, TLR9 ligand, TLR10 ligand, TLR11 ligand, TLR12 ligand, TLR13 ligand, and combinations thereof.

[0076] In some embodiments, the TLR ligand is a TLR4 ligand. In some embodiments, the TLR4 ligand is LPS. In some embodiments, the TLR4 ligand is MPLA.

[0077] Oxidized phospholipids

[0078] As used herein, the term “non-classical inflammasome-activating lipid” refers to a lipid capable of eliciting an inflammatory response in the caspase-11-dependent inflammasome of a cell. Exemplary “non-classical inflammasome-activating lipids” include PAPC, oxPAPC, and oxPAPC classes (e.g., HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC, POVPC, PGPG), as well as Rhodo LPS (LPS-RS or LPS from Rhodobacter sphaeroides).

[0079] As used herein, the term “oxPAPC” or “oxidized PAPC” refers to a lipid produced by the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), which results in a mixture of oxidized phospholipids containing fragmented or full-length, oxidized sn-2 residues. Well-characterized oxidized fragmentation species contain penta-carbon sn-2 residues carrying ω-aldehydes or ω-carboxylic acids. Oxidation of the arachidonic acid residue also produces phospholipids containing esterified isoprostanes. oxPAPC includes the classes of HOdiA-PC, KOdiA-PC, HOOA-PC, and KOOA-PC, as well as other oxidation products present in oxPAPC.

[0080] In some embodiments, the non-classical inflammasome-activating lipid includes a class of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC).

[0081] The oxPAPC classes 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.

[0082] In some embodiments, lipids that activate non-canonical inflammasomes include 2-[[(2R)-2-[(E)-7-carboxy-5-hydroxyhept-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylammonium (HOdiA-PC), [(2R)-2-[(E)-7-carboxy-5-oxohept-6-enoyl]oxy-3-hexadecanoyloxypropyl] 2-(trimethylammonio)ethyl phosphate (KOdiA-PC), 1-palmitoyl-2-(5-hydroxy-8-oxooct-1-enoyl)-sn-glycero-3-phosphocholine (HOOA-PC), 2-[[(2R)-2-[(E)-5,8-dioxooct-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylammonium (KOOA-PC), [(2R)-3-hexadecanoyloxy-2-(5-oxovaleryloxy)propyl] 2-(trimethylammonio)ethyl phosphate (POVPC), [(2R)-2-(4-carboxybutyryloxy)-3-hexadecanoyloxypropyl] 2-(trimethylammonio)ethyl phosphate (PGPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[2-[(Z)-oct-2-enyl]-5-oxocyclopent-3-ene-1-ylidene]methyl]oxiranyl]butyryloxy]propyl] 2-(trimethylammonio)ethyl phosphate (PECPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentylidene]methyl]oxiranyl]butyryloxy]propyl] 2-(trimethylammonio)ethyl phosphate (PEIPC), or a combination thereof.

[0083] In some embodiments, the lipid that activates non-canonical inflammasomes includes [(2R)-2-(4-carboxybutyryloxy)-3-hexadecanoyloxypropyl] 2-(trimethylammonio)ethyl phosphate (PGPC).

[0084] In some embodiments, the oxPAPC classes are the oxPAPC classes listed in Table 1, or a combination thereof.

[0085] Table 1. Oxidized PAPC molecular classes identified in Ni et al., and corresponding elemental composition (neutral), exact mass, adducts, m / z, ID, and proposed structures. Nomenclature: “The lipid nomenclature is based on that recommended by the LIPID MAPS Consortium

[31] . For example, the shorthand notation PC 36:4 denotes a phosphatidylcholine lipid containing 36 carbons and 4 double bonds. When the fatty acid characteristics and sn positions are known, as in our case, a slash delimiter is used (e.g., PC 16:0 / 20:4). Since there is no unified nomenclature available for oxidized lipids, the shorthand notation provided by the LPPtiger tool is used

[28] . Short-chain oxidized lipids are represented by the corresponding termini enclosed within angle brackets (e.g., “<” and “>”), and the truncation site represented by the number of carbon atoms (e.g., <COOH@C9> and <CHO@C12). For long-chain products, our recommendation is to indicate the number of oxygen additions after the fully identified parent lipid when the type of addition is unknown (e.g., PC 16:0 / 20:4+1O), or to indicate the known functional groups within parentheses (e.g., PC16:0 / 20:4[1xOH@C11]). 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) at 2.7.

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095] Immunogen

[0096] "Immunogen" and "antigen" are used interchangeably and refer to any compound to which a cellular or humoral immune response is directed. Non-living immunogens include, for example, inactivated immunogens, subunit vaccines, recombinant proteins or peptides, etc. The adjuvants disclosed herein can be used with any suitable immunogen. Exemplary immunogens of interest include those that consist of or are derived from viruses, mycoplasmas, parasites, protozoa, or prions, etc. Thus, immunogens of interest can be from, but are not limited to: human papillomavirus, herpes viruses such as herpes simplex or varicella zoster, retroviruses such as human immunodeficiency virus 1 or 2, hepatitis viruses, influenza viruses, rhinoviruses, respiratory syncytial virus, cytomegalovirus, adenovirus, Mycoplasma pneumoniae, Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, bacteria of the genus Mycobacterium, amoeba, Plasmodium, and / or Trypanosoma cruzi.

[0097] The immunogens of interest are expressed by diseased target cells (e.g., neoplastic cells, infected cells) and are expressed at lower levels or not at all in other tissues. Examples of target cells include cells from neoplastic diseases, including but not limited to, sarcomas, lymphomas, leukemias, carcinomas, melanomas, breast cancer, prostate cancer, ovarian cancer, cervical cancer, colon cancer, lung cancer, glioblastoma, and astrocytoma. Optionally, the target cells may be infected by, for example, viruses, mycoplasmas, bacteria, parasites, protozoa, and prions. Thus, the immunogens of interest can be derived from, without limitation: human papillomavirus (see below), herpes viruses such as herpes simplex or varicella zoster, retroviruses such as human immunodeficiency virus 1 or 2, hepatitis viruses, influenza viruses, rhinoviruses, respiratory syncytial virus, cytomegalovirus, adenovirus, Mycoplasma pneumoniae, Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, bacteria of the genus Mycobacterium, amoeba, Plasmodium, and Trypanosoma cruzi.

[0098] In some embodiments, infection by an infectious agent is associated with the development of cancer. See, e.g., Kuper et al., “Infections as a Major Preventable Cause of Human Cancer,” Journal of International Medicine 249(S741):61-74(2001).

[0099] In addition to tumor antigens and antigens of infectious agents, mutant tumor suppressor gene products including but not limited to p53, BRCA1, BRCA2, retinoblastoma, and TSG101, or oncogene products such as but not limited to RAS, WT, MYC, ERK, and TRK can also provide target antigens for use according to the present disclosure. The target antigen can be an autoantigen, such as an antigen associated with cancer or a neoplastic disease. In one embodiment, the immunogen is a peptide from a heat shock protein (hsp)-peptide complex from diseased cells, or the hsp-peptide complex itself.

[0100] "Cancer" as used herein refers to a disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as known in the art; including colorectal cancer, and for example leukemia, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia, AIDS-related cancers such as Kaposi's sarcoma; breast cancer; bone cancer such as osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, giant cell tumor, Adamantinomas, and chordomas; brain cancer such as meningioma, glioblastoma, low-grade astrocytoma, oligodendroglioma, pituitary tumor, Schwannomas, and metastatic brain cancer; head and neck cancer including various lymphomas such as mantle cell lymphoma, non-Hodgkin's lymphoma, adenoma, squamous cell carcinoma, laryngeal cancer, gallbladder and bile duct cancer, retinoblastoma 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 cancer, liposarcoma, epithelial cancer, renal cell carcinoma, gallbladder adenocarcinoma, parotid tumor, endometrial sarcoma, multi-drug resistant cancer; and proliferative diseases and conditions, such as neovascularization associated with tumor angiogenesis, macular degeneration (e.g., dry / wet AMD), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration and other proliferative diseases and conditions.

[0101] Immunogens such as cancer immunogens and their uses, for example, in loaded dendritic cells, are known and 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); US3823126A; US3960827A; and US4160018A.

[0102] In some embodiments, the immunogen is a cancer antigen. In some embodiments, the cancer antigen is selected from tumor lysates, apoptotic bodies, peptides, tumor RNA, tumor-derived exosomes, tumor-DC fusions, or combinations thereof.

[0103] In some embodiments, the immunogen is a whole tumor lysate.

[0104] In some embodiments, the whole tumor lysate is prepared by irradiation, boiling, and / or freeze-thaw lysis.

[0105] In some embodiments, the immunogen is autologous. In some embodiments, the immunogen is allogeneic.

[0106] In some embodiments of a method of inducing an immune response in a subject, the immunogen is a tumor lysate derived from a cell donor.

[0107] Cell donor and subject

[0108] The terms "patient" and "individual" or "subject" are used interchangeably herein and refer to a mammalian subject to be treated, preferably a human patient. In some instances, the methods disclosed herein can be used for experimental animals, veterinary applications, and the development of animal models of disease, including but not limited to rodents such as mice, rats, hamsters, and primates.

[0109] In some embodiments, the cell donor and / or the subject is a mammalian subject. As used herein, the term "mammal" is intended to include, but not be limited to, humans, laboratory animals, domestic pets, and farm animals.

[0110] In some embodiments, the cell donor and / or the subject is a human subject.

[0111] In some embodiments of the methods of inducing an immune response in a subject, the cell donor is the subject. In some embodiments, the cell donor is not the subject. In some embodiments, the progenitor cells and / or in vivo differentiated dendritic cells are autologous. In some embodiments, the progenitor cells and / or in vivo differentiated dendritic cells are allogeneic.

[0112] Administration

[0113] In the methods disclosed herein, a population of therapeutic dendritic cells is administered to a subject. In some embodiments, a therapeutically effective amount of live dendritic cells is administered to the subject.

[0114] The dose of the population of therapeutic dendritic cells disclosed herein will vary depending on the nature of the immunogen and the condition of the dendritic cells, but should be sufficient to enhance the efficacy of the live dendritic cells in eliciting an immunogenic response. For therapeutic and prophylactic treatments, the amount of live dendritic cells administered can range from 1×10 3 、1×10 4 、1×10 5 、1×10 6 、1×10 7 、1×10 8 、1×10 9 、1x10 10 or 1×10 11 cells or more per dose. The dendritic cells of the present disclosure are generally non-toxic and are typically administered as live cells in relatively large amounts without causing life-threatening side effects.

[0115] The methods include off-the-shelf methods. In some embodiments, the methods include separating cells from a subject, preparing, processing, culturing, and reintroducing them into the same patient before and after cryopreservation, as described herein.

[0116] Administration of the populations of therapeutic dendritic cells disclosed herein is by any suitable means that results in a cellular concentration that effectively ameliorates, reduces, or stabilizes cancer. The populations of therapeutic dendritic cells can be provided in a dosage form suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-vascular, intratumoral, or intraperitoneal) routes of administration.

[0117] Human doses are initially determined by extrapolation from the amounts of the populations of therapeutic dendritic cells disclosed herein used for mice or non-human primates, and as recognized by those of ordinary skill in the art, it is routine in the art to modify the dose for humans as compared to animal models. For example, the dose can vary between about 1×10 3 、1×10 4 、1×10 5 、1×10 5 、1×10 6 、1×10 7 、1×10 8 、1×10 9 and about 1×10 11 cells or more per dose.

[0118] A "suitable dose level" refers to a dose level that provides a therapeutically reasonable balance between pharmacological efficacy and deleterious effects (e.g., sufficient immunostimulatory activity conferred by administration of the dendritic cells disclosed herein, and a sufficiently low level of macrophage stimulation). For example, the dose level can be related to the peak or mean serum level of anti-immunogen antibodies produced in a subject, e.g., following administration of an immunogenic composition (including the dendritic cells disclosed herein) at a particular dose level.

[0119] As defined herein, a "therapeutically effective" amount (i.e., an effective dose) of a compound or agent is an amount sufficient to produce a therapeutically (e.g., clinically) desired result. The compositions can be administered once or more times per day, or once or more times per week; including once every other day. Those of ordinary skill in the art will understand that certain factors can affect the dose and time required to effectively treat a subject, including but not limited to the severity of the disease or condition, previous treatment, the overall health and / or age of the subject, and the presence of other diseases. In addition, treating a subject with a therapeutically effective amount of the live dendritic cells disclosed herein can include a single treatment or a series of treatments.

[0120] The population of therapeutic dendritic cells disclosed herein is administered parenterally by injection, infusion, or transplantation (subcutaneous, intravenous, intramuscular, intratumoral, intra-arterial, intraperitoneal), in a dosage form, formulation, or by a suitable delivery device or implant containing a conventional non-toxic pharmaceutically acceptable carrier. The preparation and formulation of such carriers are well known to those skilled in the art of pharmaceutical formulations. Formulations can be found in Remington: The Science and Practice of Pharmacy, as described above.

[0121] As used herein, a "pharmaceutically acceptable" component / carrier, etc. is a component / carrier that is suitable for use in humans and / or animals without undue adverse side effects (e.g., toxicity, irritation, and allergic reactions) and is commensurate with a reasonable benefit / risk ratio.

[0122] The present invention provides a method for treating cancer or its symptoms, the method comprising administering a population of therapeutic dendritic cells. Thus, one embodiment is a method for treating a subject suffering from or susceptible to cancer. The method comprises the steps of: under conditions for treating a disease or disorder, administering to the subject a therapeutically effective amount of a population of therapeutic dendritic cells disclosed herein in a dose sufficient to treat the disease or disorder or its symptoms.

[0123] An "effective amount" as used herein refers to an amount that provides a therapeutic or prophylactic benefit.

[0124] As used herein, the term "treating" a disease means reducing the frequency or severity of at least one sign or symptom of a disease or disorder such as cancer in a subject.

[0125] "Treatment" is an intervention performed with the intention of preventing the development of a disease or altering the pathology or symptoms of a disease. Thus, "treatment" refers to both therapeutic treatment and prophylactic or preventive measures. "Treatment" can also refer to palliative therapy. Patients in need of treatment include those who already have a disorder as well as those who want to prevent a disorder. Thus, "treating" or "treatment" of a condition, disorder, or disease state can include: (1) preventing or delaying the appearance of clinical symptoms of a condition, disorder, or disease state that develops in a human or other mammal that may be subject to or susceptible to the condition, disorder, or disease state but has not yet experienced or manifested the clinical or subclinical symptoms of the condition, disorder, or disease state; (2) inhibiting a condition, disorder, or disease state, i.e., preventing, reducing, or delaying the development of a disease or its recurrence (in the case of maintenance therapy) or at least one of its clinical or subclinical symptoms; or (3) alleviating a disease, i.e., causing the regression of a condition, disorder, or disease state or at least one of its clinical or subclinical symptoms. The benefit to the individual to be treated is statistically significant or at least perceptible to the patient or the physician.

[0126] Thus, in the case of cancer, "treatment" can include: (1) reducing the size and / or number of tumors; (2) reducing the number of circulating tumor cells; (3) reducing the risk of metastasis; (4) reducing the risk of cancer occurrence and / or recurrence.

[0127] "Modulation", such as of a symptom, level, or molecular biological activity, for example, refers to a symptom or activity, such as, that can be detectably increased or decreased. Such increases and decreases are observed in treated subjects compared to untreated subjects, where the untreated subjects (e.g., subjects administered an immunogen in the absence of an adjuvant lipid) have or have developed the same or a similar disease or infection as the treated subjects. Such increases and decreases 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%, more than 1000%, or any range between any two of these values. Modulation can be determined subjectively or objectively, e.g., by self-assessment of the subject, by assessment of a clinician, or by performing appropriate tests or measurements, including, for example, assessment of the degree and / or quality of immune stimulation in a subject achieved by administering a dendritic cell disclosed herein. Modulation can be transient, long-term, or permanent, or its duration can be variable during or after the administration of a dendritic cell disclosed herein to a subject or during or after a test or other method described herein or in the cited references, e.g., within the times described below, or from about 12 hours to about 24 or 48 hours after administration of an adjuvant lipid disclosed herein to a subject having received such an immunostimulatory composition / treatment for about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28 days, or 1, 3, 6, 9 months or more.

[0128] The present disclosure includes methods of inducing an immune response. In some embodiments, the immune response is an adaptive immune response.

[0129] In some embodiments, the immune response is a therapeutic immune response. As used herein, the term "therapeutic immune response" refers to an increase in humoral and / or cellular immunity that is directed directly against a target antigen and measured by standard techniques. Preferably, the level of immunity induced that is directed directly against the target antigen is at least 4-fold, and preferably at least 5-fold, the level before administration of the immunogen. The immune response can also be measured qualitatively, where the arrest or remission of the progression of a neoplastic or infectious disease in a subject is considered to indicate the induction of a therapeutic immune response by means of a suitable in vitro or in vivo test.

[0130] The methods of the present disclosure include administering to a subject, including a subject identified as in need of such treatment, an effective amount of a population of the therapeutic dendritic cells disclosed herein to produce such an effect. Identifying a subject in need of such treatment can be a determination by the subject or a healthcare professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method).

[0131] The treatment methods disclosed herein, which include prophylactic treatment, generally include administering a therapeutically effective amount of a population of the therapeutic dendritic cells disclosed herein to a subject in need thereof (e.g., an animal, a human), including a mammal, particularly a human. Such treatment will be appropriately administered to a subject, particularly a human, suffering from, having, predisposed to, or at risk of having cancer or its symptoms. Determining that these subjects are "at risk" is made by any objective or subjective determination by diagnostic tests or the opinion of the subject or a healthcare provider (e.g., genetic testing, enzyme or protein markers, markers (as defined herein), and family history, etc.).

[0132] The present disclosure also provides methods of monitoring a course of treatment. The method includes the step of determining the level of a diagnostic marker (e.g., any target, protein, or its indicator described herein that is modulated by the compounds described herein) or a diagnostic measurement (e.g., screening, assay) in a subject suffering from or predisposed to a cancer-related disorder or its symptoms, wherein a therapeutically effective amount of the compounds described herein has been administered to the subject. The level of the marker determined in the method can be compared to a known marker level in a healthy normal control or other diseased patients to determine the disease state of the subject. In some cases, a second level of the marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of the disease or the efficacy of the therapy. In certain methods, a pre-treatment level of the marker in the subject is determined prior to the initiation of treatment according to the methods disclosed herein; then, the pre-treatment level of the marker is compared to the level of the marker in the subject after the start of treatment to determine the efficacy of the treatment.

[0133] In some embodiments, the population of the therapeutic dendritic cells disclosed herein is administered as part of a pharmaceutical composition.

[0134] In some embodiments, the pharmaceutical composition is for systemic administration, e.g., formulated in a pharmaceutically acceptable buffer such as physiological saline. Preferred routes of administration include, for example, bladder instillation, subcutaneous, intravenous, intraperitoneal, intramuscular, intratumoral, or intradermal injection to provide a continuous, sustained, or effective level of the composition in a patient. Treatment of human patients or other animals is carried out with 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 varies according to the method of administration, the age and weight of the patient, and the clinical manifestation of the cancer. Generally, the amount will be within the range of amounts used for other agents in the treatment of other diseases associated with cancer, although in some cases, due to the increased specificity of the compound, lower amounts will be required. Live dendritic cells are administered in a dose determined by methods known to those skilled in the art to enhance the immune response of a subject, or to reduce the proliferation, survival, or invasion of neoplastic or infected cells.

[0135] Compositions comprising a population of the therapeutic dendritic cells disclosed herein can be administered transdermally, subcutaneously, intravenously, intramuscularly, parenterally, intrapulmonarily, intravaginally, rectally, nasally, or topically. The composition can be delivered by injection, orally, by aerosol, or particle bombardment.

[0136] A pharmaceutical composition of a population of the therapeutic dendritic cells disclosed herein can be included in a kit, container, package, or dispenser together with instructions for administration.

[0137] Combination therapy

[0138] As used herein, the term "combination" in the context of administering a therapy to a subject refers to the use of more than one therapy for a therapeutic benefit. The term "combination" in the context of administration also refers to the prophylactic use of a therapy when used in combination with at least one additional therapy. The use of the term "combination" does not limit the order in which the therapies (e.g., a first and a second therapy) are administered to a subject. The therapies can be administered before (e.g., 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), simultaneously with, or after (e.g., 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 administration of a second therapy to a subject who has had, has, or is susceptible to cancer. The therapies are administered sequentially and at intervals such that the therapies can act together. In certain embodiments, the therapies are administered sequentially and at intervals such that they provide an increased benefit compared to administration in other ways. Any additional therapy can be administered in any order with any other additional therapy.

[0139] As used herein, the term "cancer therapy" refers to a therapy for treating cancer. Examples of anti-cancer therapeutic agents include, but are not limited to, for example, surgery, chemotherapeutic agents, immunotherapy, growth inhibitors, cytotoxic agents, agents for radiotherapy, anti-angiogenic agents, apoptosis agents, anti-tubulin agents, and other agents for treating cancer, such as anti-HER-2 antibodies (e.g., HERCEPTIN TM ), anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (TARCEVA TM ), platelet-derived growth factor inhibitors (e.g., GLEEVEC TM (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA, or VEGF receptors, TRAIL / Apo2, and other bioactive and organic chemical agents, etc. Combinations thereof are also contemplated for use with the methods disclosed herein.

[0140] Some embodiments of methods of inducing an immune response in a subject include administering an anti-cancer agent to the subject. In some embodiments, the anti-cancer agent is a chemotherapeutic agent. In some embodiments, the anti-cancer agent is an immune checkpoint modulator.

[0141] Anti-cancer agent: In certain embodiments, the method further includes administering an anti-cancer agent. In some embodiments, the anti-cancer agent is a chemotherapeutic agent or a growth inhibitor, a T cell expressing a chimeric antigen receptor, an antibody or an antigen-binding fragment thereof, an antibody-drug conjugate, an angiogenesis inhibitor, and combinations thereof.

[0142] In some embodiments, the anti-cancer agent is a chemotherapeutic agent or a growth inhibitor. For example, the chemotherapeutic agent or growth inhibitor may include an alkylating agent, an anthracycline, an anti-hormonal agent, an aromatase inhibitor, an anti-androgen, a protein kinase inhibitor, a lipid kinase inhibitor, an antisense oligonucleotide, a ribozyme, an antimetabolite, a topoisomerase inhibitor, a cytotoxic agent or an antitumor antibiotic, a proteasome inhibitor, an anti-microtubule agent, an EGFR antagonist, a retinoid, a tyrosine kinase inhibitor, a histone deacetylase inhibitor, and combinations thereof.

[0143] "Chemotherapeutic agents" are compounds used for the treatment of cancer. Examples of chemotherapeutic agents can 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), finasunate (VATALANIB™, Novartis), oxaliplatin (ELOXATIN™, Sanofi), 5-FU (5-fluorouracil), leucovorin, sirolimus (RAPAMUNE™, Wyeth), lapatinib (TYKERB™, GSK572016, Glaxo Smith Kline), lonafamib (SCH 66336), 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 methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan); bryostatin; callystatin;CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); adrenocorticosteroids (including prednisone and prednisolone); cyproterone acetate; 5α-reductases, including finasteride and dutasteride; vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat, dolastatin; aldesleukin, talc duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ω1I (Angew Chem Intl Ed Engl 1994 33:183-186)); dynemicin, including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysin, actinomycin, authramycin, azaserine, bleomycins, actinomycin C, carabicin, caminomycin, carzinophilin, chromomycinis, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCINTM (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, 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 analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKTM polysaccharide complex (JHS Natural Products, Eugene, Oreg.);Razoxane; Rhizoxin; Sizofuran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2',2”-Trichloroethylamine; Trichothecenes (especially T-2 toxin, Verracurin A, Roridin A, and Anguidine); Urethan; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Cytarabine (“Ara-C”); Cyclophosphamide; Thiotepa; Taxoids, e.g., TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ (Cremophor-free), 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; and any pharmaceutically acceptable salts, acids, and derivatives of the foregoing.

[0144] In some embodiments, chemotherapeutic agents can include alkylating agents (including monofunctional and bifunctional alkylating agents), such as thiotepa, CYTOXAN™ cyclophosphamide, nitrogen mustards, such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mannomustine, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; temozolomide; and any pharmaceutically acceptable salts, acids, and derivatives thereof.

[0145] In some embodiments, chemotherapeutic agents can include anthracyclines, such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin; and any pharmaceutically acceptable salts, acids, and derivatives thereof.

[0146] In some embodiments, chemotherapeutic agents can include antihormonal agents, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX™; tamoxifen citrate), raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON™ (toremifine citrate); and any pharmaceutically acceptable salts, acids, and derivatives thereof.

[0147] In some embodiments, chemotherapeutic agents can include aromatase inhibitors that inhibit aromatase (which regulates estrogen production in the adrenal glands), such as 4(5)-imidazoles, aminoglutethimide, MEGASETM (megestrol acetate), AROMASINTM (exemestane; Pfizer), formestanie, fadrozole, RIVISORTM (vorozole), FEMARATM (letrozole; Novartis), and ARIMIDEXTM (anastrozole; AstraZeneca); and any pharmaceutically acceptable salts, acids, and derivatives thereof.

[0148] In some embodiments, chemotherapeutic agents can include antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; buserelin, triptorelin, medroxyprogesterone acetate, diethylstilbestrol, Premarin, fluoxymesterone, all-trans retinoic acid, fenretinide, and troxacitabine (a 1,3-dioxolane nucleoside cytosine analogue); and any pharmaceutically acceptable salts, acids, and derivatives thereof.

[0149] In some embodiments, chemotherapeutic agents can include protein kinase inhibitors, lipid kinase inhibitors, or antisense oligonucleotides, particularly those that inhibit gene expression in signal pathways involved in abnormal cell proliferation, such as, for example, PKC-α, Ralf, and H-Ras.

[0150] In some embodiments, chemotherapeutic agents can include ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYMETM) and HER2 expression inhibitors.

[0151] In some embodiments, chemotherapeutic agents can include cytotoxic agents or antitumor antibiotics, such as actinomycin D, actinomycin, bleomycin, plicamycin, mitomycins such as mitomycin C, and any pharmaceutically acceptable salts, acids, and derivatives thereof.

[0152] In some embodiments, the chemotherapeutic agent can include proteasome inhibitors such as bortezomib (VELCADETM, Millennium Pharm.), epoxomicins such as carfilzomib (KYPROLISTM, Onyx Pharm.), marizomib (NPI-0052), MLN2238, CEP-18770, oprozomib, and any pharmaceutically acceptable salts, acids, and derivatives thereof.

[0153] In some embodiments, the chemotherapeutic agent can include anti-microtubule agents, such as vinca alkaloids, including vincristine, vinblastine, vindesine, and vinorelbine; taxanes, including paclitaxel and docetaxel; podophyllotoxin; and any pharmaceutically acceptable salts, acids, and derivatives thereof.

[0154] In some embodiments, the chemotherapeutic agent may include an “EGFR antagonist,” which refers to a compound that binds to EGFR or otherwise directly interacts with EGFR and prevents or reduces its signaling activity, or is referred to as “EGFR i.” Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Patent No. 4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBUTIX) and reshaped human 225 (H225) (see, WO 96 / 40210, Imclone Systems Inc.); IMC-11F8, a fully human EGFR-targeted antibody (Imclone); an antibody that binds to a type II mutant EGFR (U.S. Patent No. 5,212,290); humanized and chimerized antibodies that bind to EGFR as described in U.S. Patent No. 5,891,996; human antibodies that bind to EGFR, such as ABX-EGF or Panitumumab (see, WO98 / 50433, Abgenix / Amgen); EMD 55900 (Stragliotto et al Eur.J.Cancer 32A:636-640 (1996)); EMD7200 (matuzumab), a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF-α for EGFR binding (EMD / Merck); a human EGFR antibody, 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. Patent No. 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanized mAb 806 (Johns et al, J.Biol.Chem. 279(29):30375-30384 (2004)). Anti-EGFR antibodies may be conjugated with cytotoxic agents to produce immunoconjugates (see, for example, EP659439A2, Merck Patent GmbH).EGFR antagonists include small molecules, such as the compounds described in the following: 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, 6,140,332, 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, as well as the following PCT publications: WO98 / 14451, WO98 / 50038, WO99 / 09016, and WO99 / 24037.Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA™ Genentech / OSI Pharmaceuticals); PD 183805 (CI 1033, 2-acrylamide, 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); ZM 105180 ((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 (SU 5271; Pfizer); dual EGFR / HER2 tyrosine kinase inhibitors such as lapatinib (TYKERB™, GSK572016 or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[2-methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).

[0155] In some embodiments, the chemotherapeutic agent may include tyrosine kinase inhibitors, including the EGFR-targeted drugs mentioned in the foregoing paragraphs; small molecule HER2 tyrosine kinase inhibitors such as TAK165 available from Takeda; CP-724,714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual HER inhibitors such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits both HER2 and EGFR overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as the antisense drug ISIS-5132 available from ISIS Pharmaceuticals, which inhibits Raf-1 signaling; non-HER targeted TK inhibitors such as imatinib mesylate (GLEEVECTM, available from Glaxo SmithKline); multi-target tyrosine kinase inhibitors such as sunitinib (SUTENTTM, available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787 / ZK222584, available from Novartis / Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferuloyl methane, 4,5-bis(4-fluoroanilino)phthalimide); tyrosine phosphorylation inhibitors (tyrphostines) containing a nitrothiophene moiety; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to nucleic acids encoding HER); quinoxalines (U.S. Patent No. 5,804,396); tryphostins (U.S. Patent No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis / Schering AG); pan-HER inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis / Lilly);Imatinib mesylate (GLEEVECTM); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone), sirolimus (RAPAMUNETM); or as described in any of the following patent disclosures: U.S. Patent No. 5,804,396; WO 1999 / 09016 (American Cyanamid); WO 1998 / 43960 (American Cyanamid); WO 1997 / 38983 (Warner Lambert); WO 1999 / 06378 (Warner Lambert); WO 1999 / 06396 (Warner Lambert); WO 1996 / 30347 (Pfizer, Inc); WO 1996 / 33978 (Zeneca); WO 1996 / 3397 (Zeneca) and WO 1996 / 33980 (Zeneca).;

[0156] In some embodiments, the chemotherapeutic agent can include retinoids such as retinoic acid and any pharmaceutically acceptable salts, acids, and derivatives thereof as described above.

[0157] In some embodiments, the chemotherapeutic agent can include antimetabolites. Examples of antimetabolites include folic acid analogs and antifolates, such as methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thioguanine; pyrimidine analogs, such as 5-fluorouracil (5-FU), cytarabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, floxuridine, enocitabine, fluorodeoxyuridine; nucleoside analogs; and nucleotide analogs.

[0158] In some embodiments, the chemotherapeutic agent can include topoisomerase inhibitors. Examples of topoisomerase inhibitors can include topoisomerase 1 inhibitors such as LURTOTECANTM and ABARELIXTM rmRH; topoisomerase II inhibitors such as doxorubicin, epirubicin, etoposide, and bleomycin; and the topoisomerase inhibitor RFS2000.

[0159] In some embodiments, the chemotherapeutic agent can include histone deacetylase (HDAC) inhibitors such as vorinostat, romidepsin, belinostat, mocetinostat, valproic acid, panobinostat, and any pharmaceutically acceptable salts, acids, and derivatives thereof.

[0160] Chemotherapeutic agents may also 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, hydrocortisone-17-valerate, alclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasol-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-isomeric form (feG) (IMULAN BioTherapeutics, LLC); antirheumatic drugs, such as azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide, minocycline, sulfasalazine, tumor necrosis factor α (TNFα) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab (Cimzia), golimumab (Simponi), interleukin 1 (IL-1) blockers such as anakinra (Kineret), T cell costimulation blockers such as abatacept (Orencia), interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA™); interleukin 13 (IL-13) blockers such as lebrikizumab; interferon α (IFN) blockers such as rontalizumab; β7 integrin blockers such as rhuMAb Beta7; IgE pathway blockers such as anti-M1 prime; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa1 / β2 blockers such as anti-lymphotoxin α (LTa); radioisotopes (e.g., radioisotopes of 211At, 131I, 125I, 90Y, 186Re, 188Re, 212Bi, 32P, 212Pb, and Lu);Other investigational drugs such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavins, flavanols, procyanidins, betulinic acid and its derivatives; autophagy inhibitors such as chloroquine; Δ9-tetrahydrocannabinol (dronabinol, MARINOLTM); β-lapachone; toosendanin; colchicines; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin); podophyllotoxin; tegafur (UFTORALTM); bexarotene (TARGRETINTM); bisphosphonates such as clodronate (e.g., BONEFOSTM or OSTACTM), etidronate (DIDROCALTM), NE-58095, zoledronic acid / zoledronate (ZOMETATM), alendronate (FOSAMAXTM), pamidronate (AREDIATM), tiludronate (SKELIDTM), or risedronate (ACTONELTM); and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPETM vaccine; perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteasome inhibitors (e.g., PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors such as oblimersen sodium (GENASENSETM); pixantrone; farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASAR; TM )); any of the above pharmaceutically acceptable salts, acids, and derivatives; and combinations of two or more of the above, such as CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisone; and FOLFOX, an abbreviation for a treatment regimen combining oxaliplatin (ELOXATIN TM ) with 5-FU and leucovorin.

[0161] Chemotherapeutic agents may also include non-steroidal anti-inflammatory drugs having analgesic, antipyretic and anti-inflammatory effects. NSAIDs include non-selective inhibitors of 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, valdecoxib. NSAIDs can be used for symptomatic relief of, for example, the following conditions: 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 damage, fever, intestinal obstruction, and renal colic.

[0162] Immune checkpoint modulation: In certain embodiments, an immune checkpoint modulator is co-administered with over-activated dendritic cells. An immune checkpoint refers to an inhibitory pathway of the immune system that is responsible for maintaining self-tolerance and regulating the duration and extent of a physiological immune response.

[0163] Certain cancer cells thrive by exploiting immune checkpoint pathways as a major mechanism of immune resistance, particularly with respect to T cells specific for tumor antigens. For example, certain cancer cells can overexpress more than one immune checkpoint protein responsible for inhibiting cytotoxic T cell responses. Accordingly, immune checkpoint modulators can be administered to overcome inhibitory signals and permit and / or enhance the immune attack against cancer cells. Immune checkpoint modulators can promote immune cell responses against cancer cells by reducing, inhibiting, or eliminating signaling by negative immune response modulators (e.g., CTLA4), or can stimulate or enhance signaling by positive modulators of the immune response (e.g., CD28).

[0164] Immunotherapeutic agents that target immune checkpoint modulators can be administered to promote the immune attack against cancer cells. The immunotherapeutic agent can be or include an antibody agent that targets an immune checkpoint modulator (e.g., is specific for the immune checkpoint modulator). 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. Certain monoclonal antibodies that target immune checkpoint modulators are available. For example, ipilumimab targets CTLA-4; tremelimumab targets CTLA-4; pembrolizumab targets PD-1, etc.

[0165] 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 14:391779-82; Bennett et al. (2003) J. Immunol. 170:711-8). Other members of the CD28 family include CD28, CTLA-4, ICOS, and BTLA. Two cell surface glycoprotein ligands have been identified for PD-1, programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2). PD-L1 and PD-L2 have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et al. (2000) J Exp Med192:1027-34; 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).

[0166] PD-L1 (also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1)) is a 40 kDa type I transmembrane protein. PD-L1 binds to its receptor PD-1, which is 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). Binding of PD-L1 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 binding to CD3.zeta (Sheppard et al. (2004) FEBS Lett. 574:37-41). PD-1 signaling attenuates TCR signaling-induced cyclic phosphorylation of the PKC-θ activation loop, which is required for activation of the transcription factors NF-κB and AP-1 and for IL-2 production. PD-L1 also binds to the costimulatory molecule CD80 (B7-1) but not to CD86 (B7-2) (Butte et al. (2008) Mol Immunol. 45:3567-72).

[0167] Upregulation of PD-L1 expression on the cell surface has been shown to be stimulated by IFN-γ. PD-L1 expression has been found in many cancers, including human lung cancer, ovarian cancer, and colon cancer and various myelomas, and it is generally 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 shown to play a role in tumor immunity by increasing apoptosis of antigen-specific T cell clones (Dong et al. (2002) Nat Med 8:793-800). It has also been shown that PD-L1 may be involved in intestinal mucosal inflammation, and inhibition of PD-L1 suppresses wasting diseases associated with colitis (Kanai et al. (2003) J Immunol 171:4156-63).

[0168] 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, for example from ABCAMTM (AB137132), BIOLEGEND TM (EH12.2H7, RMP1-14) and Affymetrix Ebioscience (J105, J116, MIH4).

[0169] Unless otherwise indicated, the practice of the present disclosure employs conventional techniques in chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture, and transgenic biology within the skill of the art. See, for example, Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989, Molecular Cloning, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Ausubel et al., 1992), Current Protocols in Molecular Biology (John Wiley & Sons, including 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, N.Y.); Jakoby and Pastan, 1979; Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); Transcription And Translation (B.D. Hames & S.J. Higgins eds. 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); treatises, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J.(edited by H. Miller and M. P. Calos, 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Volumes 154 and 155 (edited by Wu et al.); Immunochemical Methods In Cell And Molecular Biology (edited by Mayer and Walker, Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I - IV (edited by D. M. Weir and C. C. Blackwell, 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), (4th Edition, Univ. of Oregon Press, Eugene, 2000).

[0170] Genes: All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species applicable to the compositions and methods disclosed herein. It should be understood that when a gene or gene product from a particular species is disclosed, the disclosure is intended to be illustrative only and not limiting, unless specifically indicated in the context in which it appears. Thus, for example, for a gene or gene product disclosed herein, homologs and / or orthologs from other species are intended to be encompassed.

[0171] Ranges: In this disclosure, various aspects of the disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an immutable limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible sub - ranges as well as individual numerical values within the range. For example, a range description such as 1 to 6 should be considered to specifically disclose sub - ranges 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 numerical values within the range such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0172] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0173] Unless otherwise indicated, 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. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. It is to be understood and expected that those skilled in the art can make variations to the principles of the invention disclosed herein, and such modifications are expected to be included within the scope of the present invention.

[0174] Examples

[0175] The present invention is further described in the following examples, which do not limit the scope of the present invention described in the claims.

[0176] Example 1: Persistent anti-tumor immunity to a complex antigen mixture by stimulation with over-activated dendritic cells

[0177] The ideal strategy to stimulate protective immunity is to combine the benefits of activated and pyroptotic DCs, such that the activated cells will have the ability to release IL-1β while maintaining viability. The present inventors recently identified a new activation state of DCs that exhibits these properties. When DCs are exposed to PAMPs (e.g., TLR ligands) and some oxidized phospholipids (DAMPs) released from dying cells, the cells achieve a long-term "over-activated" state (I. Zanoni, et al. Science, vol. 352, no. 6290, pp. 1232-1236, 2016; I. Zanoni, et al. Immunity, vol. 47, no. 4, p. 697-709.e3, 2017). These oxidized lipids are referred to as oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine). Over-activated DCs exhibit the activity of activated DCs in terms of cytokine (e.g., TNFα) release, but they acquire the ability to also release IL-1β over the course of several days. Consistent with their task as "over-activated" DCs, these cells are superior to their activated counterparts in their ability to stimulate T cell responses to model antigens.

[0178] The underlying mechanisms defining the hyperactivated state of DCs, such as the DAMP (oxPAPC) discussed, can bind and stimulate the cytosolic PRR caspase-11 (I. Zanoni, et al 2016). Caspase-11 stimulation leads to the activation of NLRP3 and the assembly of the inflammasome, which does not result in pyroptosis but leads to the release of IL-1β from live cells. The release of IL-1β from hyperactivated cells is mediated by the pore-forming protein gasdermin D, which serves as a conduit for the secretion of these cytokines (C.L. Evavold, et al Immunity, 2018 Jan 16;48(1):35-44.e6.; X. Liu, et al Nature, vol. 535, no. 7610, pp. 153-158, 2016; R.A. Aglietti, et al Proc. Natl. Acad. Sci. U.S.A., vol. 113, no. 28, pp. 7858-63, Jul. 2016; N. Kayagaki, et al Nature, vol. 526, no. 7575, pp. 666-671, Sep. 2015). It is thought that the plasma membrane is repaired to remove gasdermin D pores in a manner that ensures cell viability (S. Rühl, et al Science, vol. 362, no. 6417, pp. 956-960, Nov. 2018), however in other situations (alum stimulation), the membrane repair pathway may be overwhelmed and pyroptosis ensues. Although the mechanism by which IL-1β is released from live cells is understood, the physiological benefits of the hyperactivated cell state in guiding adaptive immunity remain poorly defined.

[0179] Materials and Methods

[0180] Mouse strains and tumor cell lines: C57BL / 6J (Jax 000664), caspase-1 / -11 dKO mice (Jax016621), NLRP3KO (Jax 021302), Casp11KO (Jax 024698), OT-I (Jax 003831) and OT-II (Jax004194) and BALB / c (Jax 000651) mice were purchased from Jackson Labs. For the syngeneic tumor models in C57BL / 6J, two melanoma cell lines were used. Parental cell line: B16.F10 and OVA-expressing cell line: B16.F10OVA. For the syngeneic colorectal model, the OVA-expressing MC-38 cell line derived from C57BL6 murine 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 (a gift from the Jeff Karp laboratory) was used.

[0181] Reagents: Escherichia coli (E.coli) LPS (serotype O55:B5 - TLRGRADE TM) 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 Salmonella 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 pre-warmed 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 (C. L. Evavold et al., Immunity, 2018 Jan 16;48(1):35-44). Briefly, the ethanol solvent was evaporated using a gentle stream of nitrogen gas. Then pre-warmed serum-free medium was immediately added to the dried lipids to a final concentration of 1 mg / ml. The reconstituted lipids were incubated at 37 °C for 5 - 10 minutes and sonicated for 20 seconds before addition to cells. POVPC or PGPC was used at 100 μg / ml for cell stimulation and 65 μg / mouse for in vivo use. EndoFit chicken ovalbumin and OVA 257 - 264 peptide with endotoxin levels <1 EU / mg were purchased from Invivogen and used at a concentration of 200 μg / mouse for in vivo use or 500 or 100 μg / ml for in vitro use. Incomplete Freund's adjuvant (F5506) was purchased from Sigma and used at a working concentration of 1:4 (IFA:antigen emulsion) for in vivo immunization. Aluminum hydroxide gel (Alhydrogel), referred to as alum, 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-in-water-in-oil adjuvant, was used instead of IFA at a working concentration of 1:2 (AddaVax:antigen).

[0182] Cell culture: BMDC were generated by differentiating bone marrow 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, BMDC were washed with PBS and re - plated at a concentration of 1 x 10 6 cells / ml and a final volume of 100 μl in IMDM with 10% FBS. CD11c + DC purity was evaluated by flow cytometry using a BD Fortessa and routinely exceeded 80%. Splenic DC from mice injected with B16 - FLT3 for 15 days were purified to CD11c + MHC+ Live cells were then plated at a concentration of 1x10 6 cells / ml in a final volume of 100 μl in complete IMDM. To induce over-activated or pyroptotic BMDCs, DCs were sensitized with LPS (1 μg / ml) for 3 hours in complete IMDM and then stimulated with OxPAPC or PGPC (100 μg / ml) or alum (100 μg / ml) for 21 hours. In some cases, activated BMDCs were restimulated on plate-bound agonistic anti-CD40 with Ultra-LEAF anti-mouse CD40 (clone 1C10; BioLegend) for an additional 24 hours. 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.

[0183] LDH assay and ELISA: After BMDC stimulation, fresh supernatants were clarified by centrifugation and then subjected to LDH release assay using the Pierce LDH Cytotoxicity Colorimetric Assay Kit (Life Technologies) according to the manufacturer's protocol. Absorbance readings were measured on a Tecan plate reader at wavelengths of 490 nm and 680 nm. 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 the eBioscience Ready-SET-Go! (now ThermoFisher) ELISA kit according to the manufacturer's protocol.

[0184] Flow cytometry: After FcR blockade, 7-day BMDCs were resuspended in MACS buffer (PBS with 1% FCS and 2 mM EDTA) and stained with the following fluorescently conjugated antibodies (BioLegend): anti-CD11c (clone N418), anti-I-A / I-E (clone M5 / 114.15.2), anti-CD40 (clone 3 / 23), anti-CD80 (16-10A1), anti-CD69 clone (H1.2F3), anti-H-2Kb (clone AF6-88.5). Single cell suspensions from tumors or draining inguinal lymph nodes, or skin inguinal adipose tissue were resuspended in MACS buffer (PBS with 1% FCS and 2 mM EDTA) and stained with the following fluorescently conjugated 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), anti-CD45 (A20 or 30F11). LIVE / DEAD TM The Fixable Violet Dead Cell Stain Kit (Molecularprobes) was used to determine cell viability and cells were stained in PBS at 4 °C for 20 minutes. Draining inguinal lymph node T cells were stained with OVA-peptide tetramers for 1 hour at room temperature. PE-conjugated H2K(b)SIINFEKL (OVA 257-264; SEQ ID NO:1) and APC-conjugated I-A(b)AAHAEINEA (OVA 329-337; SEQ ID NO:2) were used. I-A(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) from Accurate Chemical was used together with the tetramers. To determine the absolute number of cells, CountBright counting beads (Molecularprobes) were used according to the manufacturer's protocol. Appropriate isotype controls were used as staining controls. Data were acquired on a BD FACS ARIA or BD Fortessa. Data were analyzed using FlowJo software.

[0185] Antigen uptake assay: To examine the antigen uptake and endocytosis ability of BMDCs during different activation states (activated, over-activated or pyroptotic states), FITC-labeled chicken OVA (FITC-OVA) (Invitrogen - Molecular Probes) was used. Briefly, pre-treated BMDCs were cultured with FITC-OVA or AF488-dextran (0.5 mg / ml) for 45 minutes at 37 °C or 4 °C (as a control for surface binding of the antigen). Then, the BMDCs were washed and stained with the Live / Dead Fixable Violet Dead Cell Staining Kit (Molecular probes) to distinguish live cells from dead cells. Subsequently, the cells were fixed with BD Fixation Solution and resuspended in MACS buffer (PBS with 1% FCS and 2 mM EDTA). The FITC fluorescence of live cells was measured every 15 minutes using a Fortessa flow cytometer (Becton - Dickenson). The fluorescence values of BMDCs cultured at 37 °C were reported as the percentage of OVA-FITC or dextran-AF488 associated cells, and the data were normalized to the percentage of OVA-FITC associated cells cultured at 4 °C.

[0186] OVA antigen presentation assay: To measure the efficiency of OVA antigen presentation on MHC-I, BMDCs treated with an activating stimulant (LPS), an over-activating stimulant (LPS + PGPC or LPS + OxPAPC), or a pyroptotic stimulant (LPS + Alum) (0.5 × 10 6 ) were cultured with Endofit-OVA protein (0.5 mg / ml) for 2 hours at 37 °C. Then the cells were washed with MACS buffer and stained on ice for 20 to 30 minutes with an APC anti-mouse H-2K b antibody (clone AF6-88.5, BioLegend), and a PE-conjugated antibody that binds to H-2K b bound to the OVA peptide SIINFEKL (SEQ ID NO:1; Clone 25-D1.16, BioLegend). Appropriate isotype controls were used as staining controls. The percentage of total surface H-2K b and the percentage of cells associated with the OVA peptide on MHC-I were calculated. Data were acquired on a Fortessa flow cytometer (Becton - Dickenson) and analyzed using FlowJo software (Tree Star).

[0187] In vitro T cell stimulation of OT-I and OT-II: Splenic CD8+ and CD4 + T cells. Then, in the presence of 20,000 or 10,000 DCs (at a 5:1 or 10:1 ratio) pretreated with LPS (activation stimulant) or LPS + PGPC (over-activation stimulant) or LPS + Alum (pyroptosis stimulant) and pulsed (or not pulsed) with 100 μg / ml of OVA protein or SIINFEKL (SEQ ID NO:1) peptide for 2 hours, the sorted T cells were seeded in a 96-well plate at a concentration of 100,000 cells per well. Five days after culture, the supernatant was collected, clarified by centrifugation for short-term storage at -20°C, and cytokine measurements were performed by ELISA.

[0188] 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 hours in the presence of GolgiStop (BD) and brefeldin A. Then, the cells were washed twice with PBS and stained with the LIVE / DEAD TM Fixable Violet or Green Dead Cell Staining Kit (Molecular probes) for 20 minutes at 4°C. The cells were washed with MACS buffer and stained for 20 minutes at 4°C with appropriate surface markers. After two washes, the cells were fixed and permeabilized for 20 minutes at 4°C using the BD Cytofix / Cytoperm kit according to the manufacturer's protocol and then washed with 1X perm wash buffer (BD). Intracellular cytokine staining was performed for 20 - 30 minutes at 4°C in 1X perm buffer 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), anti-IL10 (clone JES5-16E3). Data were acquired on a BD FACSARIA or BD Fortessa. Data were analyzed using FlowJo software.

[0189] CD107a degranulation assay: To evaluate the effector anti-tumor activity of CD8 + T cells, the surface exposure of the lysosome-associated protein CD107a was evaluated by flow cytometry. Briefly, CD8 + T cells from the skin-draining lymph nodes of immunized mice were isolated by magnetic cell enrichment using anti-CD8 beads and columns (Miltenyi Biotech), and then sorted into CD3 +CD8 + Live cells. Freshly sorted CD8 + T cells were resuspended in complete RPMI at a concentration of 1 x 10 6 cells / ml. In the presence of GolgiStop (BD), PerCP / Cy5.5 anti-mouse CD107a (LAMP-1) antibody (clone 1D4B, BioLegend) was added to the medium at a concentration of 1 μg / ml. Then, immediately, the T cells were seeded at 100,000 cells onto 10,000 MC38OVA or B16OVA tumor cells / well in a 96-well plate. Optionally, CD8 + T cells were seeded alone and stimulated with 50 ng / ml phorbol 12-myristate 13-acetate (PMA) and 500 ng / ml ionomycin (Sigma-Aldrich). Five hours after incubation, the cells were washed with MACS buffer and stained with LIVE / DEAD TM Fixable Violet Dead Cell Staining Kit (Molecular probes), and APC anti-CD8 (clone 53-6.7, BioLegend). Then, the cells were fixed with BD Fixation Buffer for 20 minutes at 4°C and resuspended in MACS buffer. The percentage of CD107a + cells was determined by flow cytometry on a Fortessa flow cytometer (BD).

[0190] In vitro cytotoxicity assay: CD8 + T cells were isolated from the spleens or inguinal fat tissues of the skin of surviving mice using anti-CD8 MACS beads and columns (Miltenyi Biotech). Then, the enriched T cells were sorted into live CD45 + CD3 + CD8 + cells using FACS ARIA. The purity after sorting was >97%. At least 5 hours before co-culture with T cells, tumor cell lines such as B16OVA, B16F-10 or CT26 cells were seeded onto a 96-well plate in complete DMEM (2 x 10 4 cells / well). 10 5 CD8 + T cells were seeded onto the tumor cells for 12 hours, and then cytotoxicity was evaluated by LDH release assay using Pierce LDH Cytotoxicity Colorimetric Detection Kit (Life Technologies) according to the manufacturer's protocol.

[0191] 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 the cells were confluent, the supernatant was collected, the cells were washed and dissociated with trypsin - EDTA (Gibco). Then, the tumor cell lines were resuspended at 5x10 6 cells / ml in their collected culture supernatants and then lysed by 3 cycles of freeze - thaw.

[0192] Tumor infiltration: To evaluate the frequency of tumor - infiltrating lymphocytes (TIL) in immunized mice, tumors were harvested when the tumor size reached 1.8 - 2 cm. According to the manufacturer's protocol, the tumors were dissociated using a tumor dissociation kit (Milteny Biotec) and a gentleMACS dissociator. After digestion, the tumors were washed with PBS and passed through 70 - μm and 30 - μm filters. CD45 + cells were positively selected using CD45 microbeads (Milteny Biotec), and T - cell infiltration was evaluated by flow cytometry. Tumor - infiltrating T cells were cultured with dynabeads mouse T - Activator CD3 / CD28 (Gibco) for T - cell activation and expansion.

[0193] Adoptive cell transfer: For T - cell transfer, CD8 + T cells were isolated from the spleens or inguinal fat tissue of the skin of surviving mice using anti - CD8 MACS beads and columns (Miltenyi Biotech). Then, the enriched T cells were sorted into live CD45 + CD3 + CD8 + cells using FACS ARIA. The purity after sorting was >97%. Then, in the presence of IL - 2 (50 ng / ml), the sorted T cells were stimulated for 24 hours in 24 - well plates coated with anti - CD3 (4 μg / ml) and anti - CD28 (4 μg / ml) (~2×10 6 cells / well). 5x10 5 activated cycling splenic or inguinal fat - resident CD8 + T cells were transferred to naive recipient mice by intravenous or intradermal (i.d.) injection, respectively. Some mice received both T - cell subsets.

[0194] For DC transfer, BMDCs were harvested on day 6, and 5x10 6Cells were seeded into 6-well plates. DC activation was induced by culturing with over-activating stimuli (LPS + PGPC) or activating stimuli (LPS). Tumor lysates were added to the DC culture plates at a ratio of 1 DC to 2 tumor cell equivalents (i.e., 1:2) for 1 hour. Unloaded naive DCs were used as negative controls.

[0195] Statistical analysis: Two-way ANOVA with Tukey multiple comparison test correction was used to test the statistical significance of experiments with more than two groups. Adjusted p-values calculated using Prism (Graphpad) were marked with asterisks: < 0.05 (*); < 0.0005 (***); ≤ 0.0001 (****).

[0196] Results

[0197] Over-activating stimuli upregulate several activities important for DCs to stimulate T cell immunity.

[0198] Almost all studies on DC over-activation have focused on the ability of these cells to release IL-1β while maintaining viability. The spectrum of DC functions affected by over-activating stimuli has not been defined. To examine this spectrum, bone marrow-derived DCs (BMDCs) were sensitized with LPS and then treated with oxPAPC or a specific and pure lipid fraction of oxPAPC called PGPC (I. Zanoni, et al Science, vol. 352, no. 6290, pp. 1232 - 1236, 2016). The resulting over-activated cells were compared with conventionally activated BMDCs (treated with LPS) or pyroptotic BMDCs (sensitized with LPS and then treated with alum). In contrast to activating stimuli that do not induce the release of IL-1β as expected, pyroptotic or over-activating stimuli promoted the release of IL-1β into the extracellular medium ( Figure 1A ). All stimuli tested promoted the secretion of the cytokine TNFα ( Figure 1A ). These findings are consistent with previous work that determined that LPS-activated BMDCs release TNFα but not IL-1β (I. Zanoni et al, 2016. IL-1β secretion was consistent with cell death in pyroptotic DCs as evaluated by the release of cytoplasmic lactate dehydrogenase (LDH) ( Figure 1B ). In contrast, IL-1β occurred in over-activated cells in the absence of LDH release ( Figure 1B ). Similar behavior of BMDCs was observed when LPS was replaced with MPLA ( Figure 3A 、 3B), the MPLA is an FDA-approved TLR4 ligand in vaccines against human papillomavirus (HPV) and hepatitis B virus (HBV). To determine whether the behavior of over-activated BMDCs extends to DCs differentiated in vivo, the activity of CD11c + DCs isolated from the spleens of mice injected with B16-FLT3 was examined. Similar to the behavior of GMCSF-derived BMDCs, as evaluated by LDH release, treatment with LPS and PGPC resulted in the release of TNFα and IL-1β from splenic CD11c + DCs in the absence of cell death ( Figure 3C , 3D ). These results indicate that the over-activating stimulant PGPC can be used to induce the release of IL-1β from live DCs differentiated in vitro or in vivo.

[0199] Several signals important for T cell differentiation were examined, e.g., the expression of co-stimulatory molecules CD80, CD69, and CD40, and the secretion of the p70 subunit of IL-12. CD80 surface expression was similar in DCs responding to all activating stimulants ( Figure 3E ). In contrast, CD40 expression was highly affected by the activating stimulants. The over-activating stimulant induced more CD40 expression compared to the activating stimulant LPS ( Figure 1C ). The pyroptosis stimulant was a very weak inducer of CD40 and CD69, even in the 20 - 30% of live cells remaining after LPS-alum treatment ( Figure 1C and 3E ). When cultured on agonist anti-CD40-coated plates, the differential expression of CD40 was associated with over-activated DCs having the greatest ability to secrete IL-12p70 ( Figure 1D ).

[0200] As evaluated by the equivalent internalization of fluorescent ovalbumin (OVA-FITC), over-activated BMDCs were no better than their activated counterparts in antigen capture ( Figure 4A , 6B ), but the former cell population showed a greater abundance of OVA-derived SIINFEKL peptides on MHC-I molecules on the cell surface ( Figure 1E and 4C ). The total surface MHC-I abundance was not different between activated and over-activated cells ( Figure 3E ). Taken together, compared to other stimulants of DCs, the over-activating stimulant exhibits enhanced several activities important for T cell differentiation.

[0201] Over-activated DCs stimulate a TH1-focused immune response without signs of TH2 immunity.

[0202] To assess the impact of DC activation status on T cell guidance, BMDCs were treated as described above and then loaded with OVA. These cells were exposed to naive OT-II or OT-I T cells. OT-II cells express a T cell receptor (TCR) specific for the MHC-II-restricted OVA peptide (OVA323-339), while OT-I cells express a TCR specific for the MHC-I-restricted OVA peptide (OVA257-264) (K.A. Hogquist, et al Cell, vol.76, no.1, pp.17-27, Jan. 1994; M.J. Barnden, et al Immunol. Cell Biol., vol.76, no.1, pp.34-40, Feb. 1998). The activity of responding T cells was evaluated by ELISA to determine whether T cell polarization tended towards a TH1 response (IFNγ production) or a TH2 response (IL-10, IL-4 or IL-13 production). Regardless of the DC activation status, OVA-treated BMDCs stimulated the production of IFNγ from OT-II T cells. The extent of IFNγ production varied moderately between the activation stimuli tested ( Figure 1F ). Similarly, TNFα production by responding OT-II cells was comparable when all DC activation states were compared ( Figure 1F ). These results indicate that, regardless of the activation status of the antigen-presenting cell (APC), a TH1 response is generally induced in vitro. In contrast, when comparing DC activation states, TH2 responses were significantly different. Stimuli that induced BMDC activation (LPS) or pyroptosis (LPS + alum) promoted the substantial release of IL-10 and IL-13, whereas over-activation stimuli led to minimal production of these TH2-related cytokines ( Figure 1F ). Intracellular staining of single cells for TH1 (IFNγ and TNFα) and TH2 (IL-4 and IL-10) cytokines, as well as the TH2 lineage-restricted transcription factor GATA3, made it possible to calculate the proportions of TH1 and TH2 cells generated by different DC activation stimuli. This analysis showed that over-activated BMDCs induced a strong bias of individual T cells towards the IFNγ-producing TH1 lineage ( Figure 1G and 5 ). The ratio of TH1 to TH2 cells under over-activation conditions was greater than 100:1 ( Figure 1G ). In contrast, all other activation stimuli induced a mixed T cell response, with the pyroptosis stimulus resulting in a TH1 to TH2 cell ratio close to 1:1 ( Figure 1G ).

[0203] Using CD8 + OT-I T cells to perform similar studies, which showed that compared with activators of activated or pyroptotic cells, activators that overactivate BMDCs led to a slightly enhanced production of IFNγ ( Figure 1F ). When comparing all DC activation states, the production of IL-2 induced by responding OT-I cells was comparable ( Figure 1F ). These combined results indicate that in vitro, the overactivated BMDC state leads to a highly TH1-biased T cell response and a slightly enhanced CD8 T cell response. In contrast, activators of activation or pyroptosis produce mixed TH1 and TH2 responses.

[0204] Overactivation of inflammasome-competent DCs is sufficient to confer protective anti-tumor immunity.

[0205] Since DCs are the main cells responsible for stimulating de novo T cell-mediated immunity, an attempt was made to determine whether the conditions for specifically overactivating DCs are sufficient to confer anti-tumor immunity. This possibility was addressed by adoptively transferring BMDCs ex vivo stimulated with different activation stimuli and WTL to mice. BMDCs were selected because these cells 1) are well characterized as overactivated and 2) are considered a model of monocyte-derived DCs, which are the most commonly used APCs for DC-based immunotherapy in humans (R.L. Sabado et al., Cell Res., vol. 27, no. 1, pp. 74-95, Jan. 2017).

[0206] BMDCs were treated with various activation stimuli, together with WTL, and then injected subcutaneously into B16OVA tumor-bearing mice every 7 days for 3 consecutive weeks. Compared with mice injected with naive BMDCs, BMDCs activated with LPS and pulsed with B16OVA WTL provided a slight protection against B16OVA-induced lethality; 25-30% of the mice receiving DC transfer rejected the tumor and remained tumor-free for a long time after the last / third DC transfer procedure ( Figure 2 ). Note that in 100% of the tumor-bearing mice, overactivated BMDCs induced complete rejection of B16OVA tumors ( Figure 2 ). In these cells, the anti-tumor activity of overactivated DCs depends on the inflammasome, because NLRP3 - / - and Casp1 - / - 11 - / - BMDC transfer only induced a smaller rejection compared with activated DCs ( Figure 2)。Therefore, these data indicate that hyperactivated DCs are sufficient to induce durable protective anti-tumor immunity and that inflammasomes within DCs are essential for this process.

[0207] Discussion

[0208] In this study, the immunological activities upregulated in the context of DC treatment with hyperactivating stimuli were extended. Not only were these stimuli able to trigger the release of IL-1β from live cells, but hyperactivating stimuli exceeded other activating stimuli in their ability to induce CD40 expression and IL-12p70 secretion. Additionally, cells exposed to hyperactivating stimuli exhibited enhanced surface expression of MHC-peptide complexes. These collective findings underscore the hyperactivated nature of DCs exposed to oxPAPC or its pure component PGPC and provide evidence of the ability to enhance the stimulation of adaptive immunity. It was also found that hyperactivated DCs are actually better stimulators of T cell responses than activated or pyroptotic cells, and the most important aspect of their activity is their ability to stimulate TH1- and CTL-focused responses. Indeed, stimuli of hyperactivated DCs result in a 100:1 ratio of TH1:TH2 cells; other strategies of DC activation do not induce such a biased T cell response.

[0209] Notably, the well-defined inflammasome stimulant alum does not exhibit the same activity as oxPAPC or PGPC. Indeed, alum is known to induce TH2 immunity. These findings were confirmed in this study, as alum or alum + LPS treatment induced strong TH2 immunity. A possible reason for the lack of TH1-focused immunity in alum-treated cells is based on the findings in this article: alum is a poor inducer of several signals required for TH1 differentiation such as CD40 expression and IL-12p70 secretion. Note that even when examining DCs that did not undergo pyroptosis in response to alum + LPS, CD40 expression was significantly low. The lack of high-level expression of these factors may render pyroptosis stimulants weak inducers of TH1 responses, and subsequently anti-tumor immunity. Without wishing to be bound by theory, it is proposed that TH1-focused immunity induced by hyperactivated DCs is caused by the activity of inflammasomes, as well as several other characteristics of these cells. These additional characteristics include enhanced antigen presentation ability, CD40 expression, IL-12p70 expression, and increased viability. Perhaps each of these enhanced activities is important for the function of DCs as APCs and may contribute to the strong TH1-focused immune responses observed under conditions of DC hyperactivation.

[0210] These results may help to explain why certain chemotherapeutic agents, such as oxaliplatin, induce tumor cell death and inflammasome-dependent anti-tumor T cell immunity (F. Ghiringhelli et al., Nat. Med., vol. 15, no. 10, pp. 1170-1178, 2009). Oxaliplatin is a strong stimulator of reactive oxygen species (ROS) production, which can oxidize biological membranes and generate a complex mixture of diverse oxidized phospholipids including PGPC. Thus, the protective immunity that may be induced by oxaliplatin is caused by the activity of hyperactivated DCs that initiate anti-tumor T cell responses.

[0211] It has been found that hyperactivated stimulants can be utilized as immunotherapies using complex mixtures of antigens. WTL is an attractive antigen source for several reasons, the most important of which is from a practical perspective. A significant benefit of the WTL-based approach is that it alleviates the need for neoantigen recognition. Although WTL-based immunotherapy offers potential benefits, previous work in the field has yielded disparate results. It is found herein that hyperactivated stimulants uniquely can assist WTL to initiate strong anti-tumor immunity, which may explain the lack of success in previous work because the DC activation strategy discovered herein was not considered before. In the latter, it is notable that the DC hyperactivation strategy protects mice from lethality associated with tumors that are sensitive and resistant to PD-1 blockade. The full spectrum of tumors that can be treated by hyperactivated stimulants has not been determined, but these studies provide an indication of the value of further exploring DC-centered strategies for cancer immunotherapy.

[0212] Example 2: Hyperactivated cDC1 Controls Tumor Rejection in an Inflammasome-Dependent Manner

[0213] Conventional dendritic cells (cDCs) are proficient at presenting exogenous and endogenous antigens to T cells and regulating T cell proliferation, survival, and effector functions. cDCs are divided into two major subsets, called cDC1 and cDC2. Resident cDC1 in the spleen and lymph nodes (LN) express CD8α, CD24, and XCR1, while cDC2 express CD4 and Sirpα. cDC1 is the classical DC that cross-presents tumor-associated antigens and initiates Th1 immunity and anti-tumor CD8 + T cells to effectively reject tumors. On the other hand, cDC2 controls type 2 immune responses against parasites in which Th2 immunity is activated.

[0214] To investigate whether resident cDCs can achieve a state of hyperactivation, cDC1 or cDC2 were sorted from the spleens of WT mice and left untreated, or treated with LPS for 20 h, or sensitized with LPS for 3 h and then treated with the hyperactivation stimuli oxPAPC or PGPC, or the pyroptosis stimulus Alum for 21 h. As measured by their LDH release, splenic cDC1 died rapidly after isolation compared with splenic cDC2 ( Figure 6A Left panel). Thus, cDC1 cells could not be sensitized with LPS and failed to produce the TNFa cytokine in response to the activating stimulus (LPS), hyperactivation stimuli (LPS + OxPAPC / PGPC), or pyroptosis stimulus (LPS + Alum) ( Figure 6B Left panel). In response to the hyperactivation stimulus (LPS + PGPC), a small amount of IL-1β release was observed ( Figure 6B Left panel). In contrast, splenic cDC2 cells were effectively sensitized with LPS and achieved a state of hyperactivation, as confirmed by their ability to produce IL-1β without undergoing cell death ( Figure 6A-6B Left panel). Since resident cDC1 are highly sensitive to ex vivo isolation and in vitro stimulation, we alternatively used the cytokine FLT3 ligand (FLT3L) to generate cDCs from bone marrow (BM) progenitors. Nine days after culture, FLT3L-generated cDC1 and cDC2 were sorted and then treated with the activating, hyperactivation, or pyroptosis stimuli as described above. As measured by their release of large amounts of IL-1β and TNFa while maintaining their viability, FLT3L-generated cDC1 and cDC2 were effectively sensitized with LPS and achieved a state of hyperactivation in response to their stimulation with LPS + PGPC, but not LPS + oxPAPC, compared with resident cDC1 cells isolated from the spleen ( Figure 6A-6B Right panel). These data indicate that the pure form of the oxidized phospholipid PGPC can hyperactivate both cDC1 and cDC2 subsets.

[0215] Hyperactivated FLT3L-generated cDC1 exhibited more stellate dendrites compared with their naive, activated, or pyroptotic counterparts, suggesting a higher migratory potential. Indeed, hyperactivated cDC1 and cDC2 upregulated the chemokine receptor CCR7, which guides the migration of DCs to lymph nodes for T cell stimulation ( Figure 6C-6D ). Collectively, these results demonstrate that cDC1 and cDC2 subsets can achieve a state of hyperactivation in vitro and exhibit unique properties compared with their classically activated counterparts.

[0216] The unique functions of cDC1 cells are crucial in the context of cancer, where cDC1s uptake tumor antigens and cross-present them to T cells either within the tumor microenvironment (TME) or after migrating to the draining lymph nodes. The fact that cDC1s become hyperactivated in vitro provides an indication for further exploring the value of the cDC hyperactivated state for cancer immunotherapy. Thus, to investigate the role of the hyperactivated state of cDC1 in controlling tumor rejection, mice were inoculated subcutaneously (s.c.) on the left dorsal flank with B16OVA cells. At 7, 14, and 21 days after tumor challenge, the mice were left untreated, or the mice were injected subcutaneously in the right flank with 1×10 6 untreated WT cDC1s (cDC1 naive), or WT cDC1s treated with LPS for 23 h (cDC1 activated), or WT cDC1s sensitized with LPS for 3 h and then treated with PGPC for 20 h (cDC1 hyperactivated). Before injection, all cDCs were pulsed with B16OVA tumor lysate for 1 h. Surprisingly, adoptive transfer of hyperactivated cDC1s conferred strong and long-lasting protection against tumor growth to the mice, while naive or activated cDC1 transfer only induced minor tumor rejection( Figure 7 ). These data demonstrate the first evidence for the superior role of hyperactivated cDC1s in durable tumor rejection.

[0217] To further define the crucial role of hyperactivated cDC1s in tumor control, we used Batf3 + mice that lack CD8 - / - cDC1s and are defective in cross-presentation, and thus Batf3 - / - mice lack anti-tumor antigen-specific CD8 + T cell responses. Thus, compared with WT mice, Batf3 - / - mice inoculated with B16OVA cells were unable to control tumor growth( Figure 8A ). However, when tumor-bearing Baft3 - / - mice were supplemented with WT cDC1s by subcutaneous injection at days 7, 14, and 21 after tumor challenge, we found that, in contrast to naive or activated cDC1s, only hyperactivated cDC1s could completely eliminate the tumors. This protection correlated with a higher frequency of tumor-infiltrating OVA-specific CD8 - / - and CD4 + T cells restored in Batf3 + mice when injecting WT hyperactivated cDC1s instead of WT activated or naive cDC1 cells( Figure 8B-8C ). Collectively, these data provide strong evidence that hyperactivated cDC1s control tumor rejection by enhancing the tumor infiltration of anti-tumor specific T cells.

[0218] The underlying mechanisms that have sufficiently defined the over-activated state of DCs, as discussed, oxidized phospholipids (oxPAPC / PGPC) can bind and stimulate the cytoplasmic pathogen recognition receptor (PRR) caspase-11. Stimulation of caspase-1 / 11 leads to the activation and assembly of the NLRP3 inflammasome, which results in the release of IL-1β from live cells through the gasdermin D pore. To evaluate the role of IL-1β in the anti-tumor activity of over-activated cDC1s, Casp1 / 11 - / - mice with defective IL-1β secretion or NLRP3 - / - mice were used. Casp1 / 11 - / - or NLRP3 - / - mice were inoculated with B16OVA cells on the left back. On days 7, 14, and 21 after tumor challenge, the mice were left untreated (no DC injection), or the mice were inoculated subcutaneously in the right flank with 1.10 6 untreated WT cDC1s (cDC1 幼稚 ), or WT cDC1s treated with LPS for 23 hours (cDC1 活化 ), or WT or Casp1 / 11 - / - cDC1s sensitized with LPS for 3 hours and then treated with PGPC for 20 hours (cDC1 过度活化 ). Before injection, all DCs were pulsed with B16OVA tumor lysate for 1 hour. Interestingly, we found that adoptive transfer of WT over-activated cDC1s to Casp1 / 11 - / - and NLRP3 - / - recipient mice completely eliminated tumor growth in 100% of the tumor-bearing animals, compared to WT naive or activated cDC1s that induced only minimal protection. This protection was dependent on the inflammasome mechanism, as cDC1s from casp1 / 11 - / - or NLRP3 - / - that were unable to respond to over-activation stimuli (LPS + PGPC) to induce IL-1β secretion were unable to induce tumor rejection. In summary, adoptive transfer of over-activated DCs is sufficient to induce a durable anti-tumor response and recapitulates the protection observed with over-activation-based vaccines.

[0219] In summary, with respect to the activation state employed in adoptive cell transfer-based immunotherapy, the data herein have the potential to transform the mindset in DC-based immunotherapy and thus could re-inspire attempts to "regulate" DCs in vitro to generate effective cancer immunotherapies.

[0220] Example 3: Oxidized phospholipids induce over-activated cDC1 and cDC2 cells

[0221] Nearly all studies evaluating the state of hyperactivated cells 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]. Current reports suggest that monocyte-derived macrophages, rather than DCs, are responsible for inflammasome activation and IL-1β secretion

[35] . To test whether conventional DCs can achieve a hyperactivated state, we used BMDCs generated with DC erythropoietin Fms-like tyrosine kinase 3 ligand (Flt3L). To assess hyperactivation, FLT3-DCs were sensitized with LPS and subsequently treated with oxidized phospholipid oxPAPC or a pure lipid component of oxPAPC called PGPC

[36] . Optionally, FLT3-DCs were stimulated with conventional activation stimuli such as LPS alone, or FLT3-DCs were sensitized with LPS and then treated with pyroptosis stimuli such as alum. In contrast to conventional activation stimuli that do not induce IL-1β release from DCs, pyroptotic DCs promoted IL-1β release into the extracellular medium ( Figure 11A ). IL-1β secretion was consistent with cell death in pyroptotic DCs as assessed by the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) ( Figure 11B ). Interestingly, stimulation with the hyperactivation stimuli LPS + PGPC or to a lesser extent with LPS + oxPAPC induced IL-1β secretion from DCs, which occurred in the absence of LDH release ( Figure 11A ). All DCs sensitized or stimulated with LPS promoted the secretion of the cytokine TNFα ( Figure 11A ). IL-1β secretion in pyroptotic or hyperactivated DCs was dependent on the inflammasome components NLRP3 and caspase-1 / 11 in both cases ( Figure 11A ). These findings are consistent with previous work defining the underlying mechanisms of the hyperactivated state of DCs, where oxPAPC binds and stimulates the cytoplasmic PRR caspase-11, leading to NLRP3 activation and the assembly of non-pyroptotic inflammasomes, which results in the release of IL-1β from live cells

[24] . Similar behavior of DCs was observed when DCs were sensitized with other TLR agonists such as the TLR9 agonist CpG ( Figure 16A)。Therefore, these data indicate that FLT3 DCs can achieve a state of overactivation. DCs are divided into two major subsets, called cDC1 and cDC2. cDC1 are classical DCs that can cross-present tumor-associated antigens and prime CD8+ T cells [

[37] ], [

[38] ]. On the other hand, cDC2 control type 2 immune responses against parasites that activate Th2 immunity. To determine whether the behavior of overactivated DCs extends to cDC1 or cDC2, we isolated cDC1 or cDC2 from FLT3-DCs or from the spleens of wild-type naive mice ( Figure 16B ). Similar to the behavior of FLT3-derived DCs, as evaluated by LDH release, treatment with LPS and PGPC and to a lesser extent with LPS and oxPAPG led to the release of TNFα and IL-1β from FLT3-derived cDC1 and cDC2 in the absence of cell death ( Figure 11A ). These data indicate that PGPC is the bioactive component of oxPAPC that induces overactivation of cDC1 and cDC2. We also observed similar behavior in splenic cDC2, which produced IL-1β in response to the pyroptosis stimuli LPS and alum accompanied by pyroptotic cell death and also produced IL-1β in response to the overactivation stimuli LPS and PGPC in the absence of cell death ( Figure 16C ). In contrast, splenic cDC1 produced minimal amounts of IL-1β in response to pyroptosis or overactivation stimuli because these cells are very sensitive to cell death after sorting and cannot be primed by LPS ( Figure 16C ). Collectively, these results indicate that overactivation stimuli can be used to induce the release of IL-1β from live DCs differentiated in vitro or in vivo. For practical reasons, we continue to use FLT3-derived DCs as the source of DCs in this article.

[0222] Example 4: Overactivated DCs enhance CTL responses in an inflammasome-dependent manner

[0223] IL-1β is a key regulator of T cell differentiation, generation of long-lived memory T cells, and effector functions

[12] -

[14] . We wondered whether hyperactivated DCs that produce IL-1β over the course of several days in the dLN could enhance CD8+ T cell stimulation. To test this idea, we attempted to adoptively transfer subcutaneously (s.c.) DCs loaded with OVA protein and then measure OVA-specific CD8+ T cells in the dLN. First, we tested the ability of different DC states to uptake OVA protein and cross-present the OVA peptide SIINFEKL on the H2kb molecule. We found that all DCs in different states took up OVA to a similar extent as confirmed by internalization of an equivalent of fluorescent ovalbumin (OVA-FITC). However, we found that both activated DCs and hyperactivated DCs sensitized with LPS or with CpG showed enhanced SIINFEKL cross-presentation upon OVA protein loading compared to their naive counterparts. This is consistent with previous work showing that DC maturation enhances their antigen-presenting ability. Surprisingly, the pyroptosis stimulant alum strongly reduced the cross-presentation ability of DCs, indicating that pyroptotic DCs are not optimal for T cell stimulation. Thus, when injecting 1.106 DCs of naive, activated, pyroptotic, or hyperactivated DCs loaded with OVA into WT mice, we observed that hyperactivated DCs induced the highest frequency and absolute number of SIINFEKL+ CD8+ T cells in the dLN of recipient mice ( Figure 12A and 17B ). Since injection of NLRP3- / - DCs treated with LPS+PGPC induced a weak OVA-specific T cell response, the enhanced CD8+ T cell response mediated by hyperactivated DCs depends on inflammasome activation.

[0224] Example 5: Hyperactivation stimulants enhance memory T cell generation and antigen-specific IFNγ effector responses in an inflammasome-dependent manner

[0225] We hypothesized that hyperactivated stimuli could represent potent adjuvants that recapitulate the effects of injecting hyperactivated DCs. To test this possibility, mice were immunized subcutaneously with OVA alone, or OVA plus an activating stimulus (LPS), or OVA plus hyperactivated stimuli (LPS+oxPAPC or PGPC). At 7 and 40 days after immunization, memory and effector T cell generation in the dLNs was evaluated by flow cytometry using CD44 and CD62L markers that discriminate T effector cells (Teff) as CD44lowCD62Llow, T effector memory cells (TEM) as CD44hiCD62Llow, and T central memory cells (TCM) as CD44hiCD62Lhi

[47] . At 7 days after immunization, hyperactivated stimuli were superior to activating stimuli in inducing CD8+ Teff cells ( Figure 13A upper panel and Figure 18A-18B ). Additionally, at this early time point, hyperactivated stimuli induced the highest abundance of CD8+ TEM ( Figure 13A middle panel and Figure 18A-18B ). At 40 days after immunization, substantial TCM cells were observed in mice exposed to hyperactivated stimuli; however, these cells were less abundant in mice immunized with OVA alone or with OVA and LPS ( Figure 13A lower panel). Conversely, at 40 days after immunization, Teff and TEM cells were more abundant in mice immunized with OVA alone or with OVA and LPS compared to mice immunized with OVA plus. Thus, these data demonstrate the extent to which hyperactivated stimuli oxPAPC and PGPC enhance effector and memory T cell generation. Additionally, when restimulated ex vivo in the presence of OVA-loaded naive BMDCs, the increased frequency of Teff cells at 7 days after immunization was associated with enhanced IFNγ responses of CD8+ T cells isolated from the dLNs of mice immunized with OVA plus hyperactivated stimuli ( Figure 18C ). Furthermore, when all CD8+ T cells were isolated from mice immunized with hyperactivated stimuli and co-cultured with the OVA-expressing B16 tumor cell line (B16OVA), the CD8+ T cells exhibited enhanced degranulation activity compared to CD8+ T cells isolated from mice immunized with OVA alone or OVA plus LPS ( Figure 13B and 18D ), indicating that hyperactivated stimuli enhance CTL function.

[0226] To evaluate the antigen specificity of T cells resulting from subcutaneous immunization with different activating stimuli, mice were injected with OVA alone, or OVA and an activating stimulus (LPS), or OVA and a pyroptosis stimulus (LPS+alum) or OVA and a hyperactivation stimulus (LPS+oxPAPC or PGPC). Optionally, mice were subcutaneously immunized with LPS+PGPC without the OVA antigen. Seven days after immunization, CD8+ T cells were isolated from the skin dLNs of immunized mice and restimulated ex vivo with naive BMDCs loaded (or not loaded) with OVA for 7 days to enrich the OVA-specific T cell subset. The T cell effector function of OVA-specific T cells was evaluated by intracellular staining for IFNγ. TCR specificity was evaluated by staining with MHC-restricted OVA peptide tetramers. The H2kb-restricted SIINFEKL (OVA 257-264) peptide tetramer was used. The frequency of tetramer+IFNγ+ double-positive cells was measured for CD4+ and CD8+ T cell subsets. Significantly, OVA with hyperactivation stimuli was superior in inducing antigen-specific T cells, as immunization based on oxPAPC or PGPC resulted in the generation of the highest frequency of tetramer+IFNγ+ responses in the case of restimulating CD8+ T cells with the OVA antigen ( Figure 13C ). In contrast, the pyroptosis stimulus (LPS+alum) was the weakest inducer of antigen-specific IFNγ responses ( Figure 13C ). These results are consistent with previous studies showing that alum is an adjuvant that effectively promotes humoral immunity and Th2 responses, but not Th1 or CTL responses [39,42,43].

[0227] Previous studies have shown that antigen-specific T cell responses can be enhanced by co-immunization with recombinant IL-1β [47,13], a cytokine whose bioactivity is naturally controlled by the inflammasome. However, although both hyperactivation and pyroptosis stimuli induce IL-1β secretion, how do hyperactivation stimuli, but not pyroptosis stimuli, induce higher antigen-specific T cells? To determine whether inflammasome-mediated events control T cell responses generated with hyperactivation stimuli, a parallel comparison of T cell activity was performed in WT and NLRP3- / - mice. Note that we found that the enhancement of hyperactivation-induced antigen-specific responses by CD8+ T cells requires NLRP3 ( Figure 13C ). Thus, these data indicate that non-pyroptotic versus pyroptotic inflammasome activation after immunization with hyperactivation stimuli or pyroptosis stimuli, respectively, induces significantly different adaptive immune T cell control.

[0228] Our previous results using the DC injection strategy showed that DCs stimulated with pyroptosis stimuli lose their ability to migrate to neighboring dLNs and stimulate T cell activation, while DCs exposed to hyperactivation stimuli hypermigrate to dLNs and enhance the CTL response ( Figure 12A-12B ). However, it was unknown whether endogenous DCs could achieve hyperactivation in vivo after immunization with hyperactivation stimuli. To evaluate this, we generated mouse chimeras using Zbtb46DTR and WT mice or Zbtb46DTR and NLRP3− / − mice or Zbtb46DTR and Casp1 / 11− / − mice. For this, 4-week-old CD45.1-irradiated mice on a CD45.2 background were reconstituted with a mixture of bone marrow from 80% Zbtb46DTR and 20% WT mice or 20% NLRP3− / − or 20% Casp1 / 11− / − mice as previously described

[51] . Six weeks after reconstitution, the efficacy of BM reconstitution in all mice was evaluated by flow cytometry using the cD45.1 and CD45.2 markers. Chimeric mice were treated with diphtheria toxin (DT) every other day to deplete Zbtb46+ conventional DCs, generating mice carrying WT or inflammasome-deficient (NLRP3− / − or Casp1 / 11− / −) DCs that could or could not be hyperactivated, respectively. To test the effect of endogenous DC hyperactivation on CD8+ T cell responses, all chimeric mice were immunized subcutaneously with OVA plus LPS+PGPC after three consecutive DT injections. Seven days after immunization, CD8+ T cell responses from dLNs were evaluated. Interestingly, we found that the abundance of Teff CD8+ T cells was strongly reduced in chimeric mice carrying non-hyperactivatable DCs such as NLRP3− / − and Casp1 / 11− / − chimeric mice compared with chimeric mice carrying hyperactivatable WT DCs ( Figure 13D , Figure 19A ). In addition, we found that the frequency of SIINFEKL+ CD8+ T cells in dLNs or spleen was reduced in NLRP3− / − and Casp1 / 11− / − chimeric mice carrying non-hyperactivatable DCs, while high SIINFEKL+ CD8+ cells were observed in chimeras carrying WT DCs ( Figure 13E , Figure 19B ). Thus, these data clearly show that: 1) endogenous DCs can achieve a state of hyperactivation in vivo and enhance the CTL response after immunization with hyperactivation stimuli, and 2) inflammasome activation within endogenous DCs is crucial for hyperactivation-mediated protective CTL responses.

[0229] Example 6: Hyperactivated DC entry into lymphoid tissue is essential for hyperactivation-mediated CTL responses

[0230] We previously showed that DCs stimulated with hyperactivating stimuli hypermigrate to the dLN and enhance the CTL response ( Figure 12A-12B ). To evaluate whether endogenous hyperactivated DCs entering the dLN are required for hyperactivation-mediated CTL responses, we generated mouse chimeras using Zbtb46DTR and WT or Zbtb46DTR and CCR7− / − BM, as described above ( Figure 13D ). In summary, endogenous delivery of hyperactivated DCs to the dLN is required for hyperactivation-mediated CTL function.

[0231] Example 7: Hyperactivating stimuli can use a complex antigen source to stimulate T cell-mediated anti-tumor immunity of pyroptosis

[0232] Current efforts to stimulate anti-tumor immunity include strategies to activate resident T cell populations (e.g., PD-1 blockade) or personalized cancer vaccine strategies that stimulate the generation of de novo T cell responses to tumor-specific antigens (TSAs)

[46] . The latter effort is hampered by the inability to use tumor cell lysates as a source of TSAs, also known as neoantigens. Thus, efforts have been made to improve the identification of neoantigens, which can be used in pure form to initiate T cell-mediated anti-tumor immunity. Although these efforts have been successful [50,49,51], the pathway to neoantigen identification requires avenues for the discovery of mutated, as well as aberrantly expressed, TSAs

[54] , which is laborious and does not represent the natural course of events. As previously discussed, WTL represents an attractive alternative source of antigens because these lysates provide a large number of antigens required to initiate personalized anti-tumor immune responses. However, fundamental questions remain unanswered, such as what is the most effective type of adjuvant for cancer vaccines (including the type of adjuvant associated with different types of antigens).

[0233] To address the possibility that hyperactivating stimuli could be an adjuvant to WTL, mice were immunized in the right flank with either WTL alone, or WTL mixed with the activating stimulus LPS or the hyperactivating stimuli LPS + oxPAPC or LPS + PGPC. The source of WTL was B16OVA cells. Fifteen days after immunization, mice were challenged subcutaneously in the upper left back with parental B16OVA cells. Non-immunized mice or mice immunized with WTL alone did not exhibit any protection, and all mice carried large tumors and died by day 24 after tumor inoculation ( Figure 20A ). Similarly, WTL + LPS immunization provided minimal protection. Two out of eight mice immunized with WTL + LPS were tumor-free, but rapidly relapsed after B16OVA rechallenge ( Figure 20A), indicating that the stimulant that only activates DCs does not provide protective immunity. In contrast, WTL immunization in the presence of LPS and oxPAPC induces a significant delay in tumor growth and confers strong protection against subsequent lethal rechallenge with parental B16OVA tumor cells; 50% of the immunized mice were fully protected( Figure 20A ). To determine whether the protective response induced by oxPAPC is related to the T cell response, tumors were harvested from mice receiving each activating stimulant. Compared to LPS immunization, tumors from mice immunized with LPS + oxPAPC contained a large abundance of CD4+ and CD8+ T cells (Figure S7B). Additionally, when equal numbers of T cells from these tumors were compared, oxPAPC-based immunization led to intratumoral T cells that secreted the highest amount of IFNγ in the case of anti-CD3 and anti-CD28 stimulation( Figure 20C ). Thus, the better restriction of tumor growth induced by the over-activating stimulant (LPS + oxPAPC) is consistent with the infiltration of inflammatory T cells into the tumor.

[0234] Note that the protective phenotype of oxPAPC was replaced by those elicited by the pure oxPAPC component PGPC. WTL immunization in the presence of LPS + PGPC led to 100% of the mice being tumor-free 150 days after tumor challenge. These mice completely rejected the lethal rechallenge with B16OVA cells and remained tumor-free 300 days after the initial tumor challenge( Figure 20A ). Since these mice never relapsed, we wondered how tumor cell growth was kept under control at the tumor injection site in mice immunized with WTL plus LPS + PGPC?

[0235] Among memory T cell subsets, tissue-resident memory T cells (TRM) are defined by the expression of the CD103 integrin together with the C-type lectin CD69, which contributes to their tissue-resident properties in peripheral tissues

[55] . CD8+ TRM cells have recently received much attention because these cells accumulate at the tumor site in various human cancer tissues and are associated with more favorable clinical outcomes [54, 55, 56]. In an experimental cutaneous melanoma model, CD8+ TRM cells in the skin promote durable protection against melanoma development

[58] .

[0236] We examined the presence of TRM cells at the tumor injection site and immunofluorescence skin biopsies in surviving mice previously immunized with the over-activating stimulant LPS + PGPC. Interestingly, 200 days after tumor inoculation, CD8+CD69+CD103+ TRM cells were highly enriched at the tumor injection site in all surviving mice, but were less abundant at the immunization site( Figure 21A-21B)。These data are consistent with clinical and experimental reports associating high levels of TRM with long-term tumor control, where TRM may be maintained long-term to study tumor injection sites [56,57]. Thus, immunization with WTL and hyperactivating stimuli may generate TRM that keep tumor cells in check.

[0237] To test the functional specificity of these T cells, we monitored cytotoxic lymphocyte (CTL) activity ex vivo. Circulating memory CD8+ T cells and TRM cells were isolated from the spleens or skin adipose tissues of surviving mice previously exposed to hyperactivating stimuli. These cells were cultured with B16OVA cells, or B16 cells that do not express OVA, or the unrelated cancer cell line CT26. CTL activity was observed only when CD8+ T cells were mixed with B16OVA or B16 cells, as assessed by LDH release ( Figure 21C ). No killing of CT26 cells was observed ( Figure 21C ), thus demonstrating the functional and antigen-specific nature of the hyperactivation-induced T cell response.

[0238] Based on the antigen-specific T cell response induced by hyperactivating stimuli, we determined whether T cells were sufficient to protect against tumor progression. CD8+ T cells were transferred from surviving mice to naive mice, which were then challenged with the parental tumor cell line used as the primary immunogen. Transfer of CD8+ TRM or circulating CD8+ T cells from surviving mice to naive recipients conferred strong protection against subsequent tumor challenge, with the TRM subset playing a major protective role ( Figure 21D ). Transfer of both T cell subsets from surviving mice to naive mice one week prior to tumor inoculation provided 100% protection of recipient mice from subsequent tumor challenge ( Figure 21D ). These combined data indicate that PGPC-based hyperactivating stimuli confer optimal protection in the B16 melanoma model by inducing strong circulating and resident anti-tumor CD8+ T cell responses.

[0239] Example 8: Hyperactivating stimuli protect against established anti-PD1-resistant tumors

[0240] To determine whether hyperactivated stimulants can be used as cancer immunotherapies, we examined the anti-tumor responses in mice bearing growing tumors prior to any additional treatment. For these studies, instead of using cultured tumor cells as the antigen source, syngeneic tumors from naive mice were used to generate ex vivo WTL, in which harvested tumors of 10 mm were dissociated and CD45+ cells were depleted. Mice were subcutaneously (s.c.) inoculated with tumor cells in the upper left back. When the tumors reached a size of 3 - 4 mm, tumor-bearing mice were left untreated (naive) or received therapeutic injections consisting of ex vivo WTL and LPS + PGPC in the right flank. Subsequently, 2 subcutaneous booster therapeutic injections ( Figure 14A ) were given. Interestingly, hyperactivation-based therapeutic injections induced tumor elimination in many tumor models such as the B16OVA and B16F10 melanoma models, and in the MC38OVA and CT26 colon cancer tumor models ( Figure 14B-14D ). In all of these models, a high percentage of mice receiving the immunotherapy regimen remained tumor-free for a long time after tumor inoculation ( Figure 14B-14D ). In all tested tumor models, the efficacy of immunotherapy was dependent on IL-1β, as neutralization of IL-1β abrogated the protection conferred by hyperactivated stimulants plus ex vivo WTL ( Figure 14B-14D ). Additionally, CD8+ T cells were crucial 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 ( Figure 14B-14D )

[63] . To determine how hyperactivation-based immunotherapy compares in efficacy to PD-1 blockade-based therapies, parallel evaluations were conducted. Hyperactivation-based immunotherapy was as effective as anti-PD-1 therapy in the immunogenic B16OVA model, but more effective in tumor models such as CT26 and B16F-10 that are insensitive to anti-PD-1 treatment ( Figure 14B-14D ).

[0241] Example 9: Endogenous hyperactivated DCs stimulate T cell-mediated durable anti-tumor immunity

[0242] Adoptive transfer of hyperactivated DCs into tumor-bearing mice induced strong anti-tumor immunity ( Figure 12A-12B)。To test whether endogenous DCs can initiate overactivation-mediated anti-tumor responses, we used Zbtb46DTR mice in which conventional DCs were depleted by DT injection. Zbtb46DTR or WT mice were subcutaneously injected with B16OVA cells. Then, prior to immunization of Zbtb46DTR mice, DT was injected every other day to completely deplete resident DCs in Zbtb46DTR mice. When the tumors reached 4 mm in size, Zbtb46DTR or WT mice were immunized with B16OVA WTL plus the overactivation stimulator LPS + PGPC. We found that, contrary to WT mice that rejected tumors in 90% of the mice, Zbtb46DTR mice (lacking DCs) could not reject tumors. These results confirmed that DCs are initiators of overactivation-mediated protection( Figure 15A )。

[0243] Example 10: Overactivated cDC1 can use a complex antigen source to stimulate T cell-mediated anti-tumor immunity

[0244] We demonstrated in vitro that both cDC1 and cDC2 can achieve a state of overactivation, as these cells produce IL-1β in response to LPS + PGPC while maintaining their viability. These data provide an indication for further defining the specific DC subsets that initiate overactivation-mediated anti-tumor responses in vivo. Given the importance of the cDC1 subset in tumor rejection, we hypothesized that cDC1 plays an important role in inducing overactivation-mediated anti-tumor protection. To test this idea, we used Batf3- / - mice that lack cDC1 but carry cDC2 cells

[65] . For this purpose, Batf3- / - or WT tumor-bearing mice (carrying 3-4 mm B16OVA tumors) were immunized with LPS + PGPC and WTL. These mice received 2 subcutaneous booster injections every 7 days. We observed that unimmunized Batf3- / - mice showed more severe tumor growth than unimmunized WT mice, and all mice died of tumors immediately 18 days after tumor inoculation. This data corroborates previous studies showing that the rejection of highly immunogenic tumors is strongly impaired in Batf3- / - mice lacking cDC1 cells

[65] . Interestingly, although the immunization of Batf3- / - mice increased their survival by a few days compared to unimmunized Batf3- / - mice, all Batf3- / - mice died of tumor growth 25 days after tumor inoculation. In contrast, WT mice rejected tumors in 100% of the tumor-bearing mice( Figure 15C)。Therefore, cDC1 plays a crucial role in anti-tumor immunity mediated by over-activation. In addition, although immunized WT mice induced high frequencies of antigen-specific CD8+ and CD4+ cells in TEM and in skin dLN, immunized Batf3− / − induced slightly reduced antigen CD4+ T cells, but no significant antigen-specific CD8+ T cells in TEM ( Figure 15D )。

[0245] To further confirm the role of over-activated cDC1 in inducing long-term anti-tumor protection, we sought to evaluate the ability of over-activated cDC1 to restore anti-tumor protection in Batf3− / −. We adoptively transferred naive, activated, or over-activated cDC1 cells into Batf3− / − mice. For this purpose, FLT3-derived cDC1 were sorted from C57BL / 6J mice as B220-MHC-II+CD11c+CD24+ cells as previously described. cDC1 were processed in vitro and loaded with B16OVA WTL as described above, and then 1.10e6 cells were injected subcutaneously into tumor-bearing Batf3− / − mice. We observed that, in contrast to naive or activated cDC1, which provided only a slightly improved mouse survival compared to non-injected mice, over-activated cDC1 induced tumor rejection in 100% of tumor-bearing mice, which remained tumor-free for more than 60 days after tumor inoculation ( Figure 15E )。Note that over-activated cDC1 injection restored CD8+ T cell responses in Batf3− / − as measured by SIINFEKL tetramer staining in tumors and skin dLN ( Figure 15F )。In contrast, naive or activated cDC1 injection failed to restore antigen-specific CD8+ T cells. cDC1-mediated tumor rejection depends on inflammasome activation, as injection of NLRP3− / − cDC1 treated with LPS+PGPC did not provide any anti-tumor protection and abolished the ability of over-activated cDC1 to restore CD8+ T cell responses ( Figure 15F )。

[0246] In addition to their ability to produce IL-1 from live cells, over-activated DCs highly migrate to neighboring dLN to enhance CD8+ T cell responses ( Figure 12A-12B )。

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[0316] Other embodiments

[0317] It should be understood that although the present invention has been described in connection with the detailed description thereof, the foregoing description is intended to be illustrative and not limiting of the scope of the present invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A method for generating a population of therapeutic dendritic cells, the method comprising: obtaining live dendritic cells from a cell donor; sensitizing the dendritic cells ex vivo with a TLR ligand; culturing the sensitized dendritic cells ex vivo with a lipid that activates a non-canonical inflammasome; and loading the dendritic cells with an immunogen, thereby generating a population of therapeutic dendritic cells.

2. A method for inducing an immune response in a subject, the method comprising: obtaining live dendritic cells from a cell donor; sensitizing the dendritic cells ex vivo with a TLR ligand; culturing the sensitized dendritic cells ex vivo with a lipid that activates a non-canonical inflammasome; loading the dendritic cells with an immunogen, thereby generating a population of therapeutic dendritic cells; and administering the live dendritic cells to the subject, thereby inducing an immune response in the subject.

3. A method for treating cancer in a subject, the method comprising: obtaining live dendritic cells from a cell donor; sensitizing the dendritic cells ex vivo with a TLR ligand; culturing the sensitized dendritic cells ex vivo with a lipid that activates a non-canonical inflammasome; loading the dendritic cells with an immunogen, thereby generating a population of therapeutic dendritic cells; and administering the live dendritic cells to the subject, thereby treating cancer in the subject.

4. The method according to any one of claims 1-3, wherein the cell donor and / or the subject is a mammalian subject.

5. The method according to claim 4, wherein the cell donor and / or the subject is a human subject.

6. The method according to any one of claims 1-5, wherein obtaining dendritic cells from a cell donor comprises: harvesting progenitor cells from the cell donor; and culturing the progenitor cells ex vivo under conditions effective to induce differentiation, thereby obtaining dendritic cells from the cell donor.

7. The method according to any one of claims 1-5, wherein obtaining dendritic cells from a subject comprises harvesting in vivo differentiated dendritic cells from the cell donor.

8. The method according to any one of claims 1-7, wherein the immunogen is an immunogen from an infectious agent associated with the development of cancer.

9. The method according to any one of claims 1-7, wherein the immunogen is a cancer antigen.

10. The method according to any one of claims 1-7, wherein the immunogen is a whole tumor lysate.

11. The method according to claim 9 or 10, wherein the immunogen is autologous.

12. The method according to any one of claims 1-11, wherein the sensitization and the culturing occur simultaneously.

13. The method according to any one of claims 1-11, wherein the sensitization occurs before the culturing.

14. The method according to any one of claims 1-13, wherein the TLR ligand is selected from TLR1 ligand, TLR2 ligand, TLR3 ligand, TLR4 ligand, TLR5 ligand, TLR6 ligand, TLR7 ligand, TLR8 ligand, TLR9 ligand, TLR10 ligand, TLR11 ligand, TLR12 ligand, TLR13 ligand, and combinations thereof.

15. The method according to any one of claims 1-13, wherein the TLR ligand is a TLR4 ligand.

16. The method according to claim 15, wherein the TLR4 ligand is selected from monophosphoryl lipid A (MPLA), lipopolysaccharide (LPS), or a combination thereof.

17. The method according to any one of claims 1-16, wherein the lipid that activates the non-canonical inflammasome comprises a class of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC).

18. The method according to any one of claims 1-16, wherein the lipid that activates the non-canonical inflammasome comprises 2-[[(2R)-2-[(E)-7-carboxy-5-hydroxyhept-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylammonium (HOdiA-PC), [(2R)-2-[(E)-7-carboxy-5-oxohept-6-enoyl]oxy-3-hexadecanoyloxypropyl]2-(trimethylammonio)ethyl phosphate (KOdiA-PC), 1-palmitoyl-2-(5-hydroxy-8-oxooctenoyl)-sn-glycero-3-phosphocholine (HOOA-PC), 2-[[(2R)-2-[(E)-5,8-dioxooct-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylammonium (KOOA-PC), [(2R)-3-hexadecanoyloxy-2-(5-oxopentanoyloxy)propyl]2-(trimethylammonio)ethyl phosphate (POVPC), [(2R)-2-(4-carboxybutanoyloxy)-3-hexadecanoyloxypropyl]2-(trimethylammonio)ethyl phosphate (PGPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[2-[(Z)-oct-2-enyl]-5-oxocyclopent-3-ene-1-ylidene]methyl]oxirane-2-yl]butanoyloxy]propyl]2-(trimethylammonio)ethyl phosphate (PECPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentylidene]methyl]oxirane-2-yl]butanoyloxy]propyl]2-(trimethylammonio)ethyl phosphate (PEIPC), or a combination thereof.

19. The method according to any one of claims 1-16, wherein the lipid that activates the non-canonical inflammasome comprises [(2R)-2-(4-carboxybutanoyloxy)-3-hexadecanoyloxypropyl]2-(trimethylammonio)ethyl phosphate (PGPC).

20. The method according to any one of claims 2-19, further comprising administering an anti-cancer agent to the subject.

21. The method according to claim 20, wherein the anti-cancer agent is a chemotherapeutic agent.

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