Stimulators of over-activated resident dendritic cells for cancer immunotherapy

Abstract

The present application relates to cancer immunotherapy, for example, T cell stimulation mediated anti-tumor therapy. Accordingly, disclosed herein are methods of inducing or enhancing an adaptive immune response to a cancer in a subject and methods of treating a cancer in a subject. In some embodiments, the methods hyperactivate dendritic cells (DCs) that induce Type I helper T cell (TH1) and cytotoxic T lymphocyte (CTL) responses in the absence of TH2 immunity.

Description

[0001] Priority Statement

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 937,073, filed November 18, 2019, the entire contents of which are incorporated herein by reference.

[0003] Federally sponsored research or development

[0004] This invention was developed with the support of the U.S. government under license number AI116550 granted by the National Institutes of Health. The U.S. government enjoys certain rights to this invention. Technical Field

[0005] This application relates to cancer immunotherapy, such as T-cell stimulation-mediated antitumor therapy. Background Technology

[0006] At the heart of our understanding of protective immunity against infection and cancer are dendritic cells (DCs), which are migratory phagocytes that patrol the body’s tissues (D. Alvarez, EH 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 signs of tissue damage. This surveillance 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 that indicate tissue damage) (SW. Brubaker, et al., Annu. Rev. Immunol., vol. 33, pp. 257–90, 2015; CA. Janeway and R. Medzhitov, Annu. Rev. Immunol., vol. 20, pp. 197–216, Jan. 2002). Microbial ligands of PRR are classified as pathogen-associated molecular patterns (PAMPs), while host-derived PRR ligands are classified as damage-associated molecular patterns (DAMPs) (P. Matzinger, Science, vol. 296, no. 5566, pp. 301–5, Apr. 2002).

[0007] Upon detection of PAMPs, PRR initiates signaling pathways that fundamentally alter the physiological function of dendritic cells (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 typically considered non-inflammatory cells. Upon encountering extracellular PAMPs, PRR stimulates a rapid and potent upregulation of many inflammatory mediators, including cytokines, chemokines, and interferons. Simultaneously with the expression of these genes, DCs migrate to draining lymph nodes (dLNs) and upregulate factors important for T cell activation, such as MHC and co-stimulatory molecules. Therefore, the PRR signaling process causes DC activity to change from a non-stimulated (naive) state to an "activated" state (K. Inaba, et al. J. Exp. Med., vol. 191, no. 6, pp. 927–36, Mar. 2000; I. Mellman and RMSteinman, Cell, vol. 106, no. 3, pp. 255–8, Aug. 2001). Summary of the Invention

[0008] There is a need to diversify current cancer immunotherapies. Therefore, this invention describes methods for inducing or enhancing adaptive immune responses to cancer in a subject, and methods for treating cancer in a subject. In some embodiments, the method involves overactivating dendritic cells (DCs) that induce type I T helper cell (TH1) and cytotoxic T lymphocyte (CTL) responses in the absence of TH2 immunity. The overactivated stimuli drive T cell responses that protect against tumors sensitive to or resistant to PD-1 inhibition. These protective responses depend on inflammasomes in the DCs and can be generated using tumor lysates as immunogens.

[0009] Therefore, this article provides a method for inducing or enhancing an adaptive immune response to cancer in a subject, the method comprising: administering an effective amount of (i) a Toll-like receptor (TLR) ligand; (ii) a non-classical inflammasome-activated lipid; and (iii) a cancer immunogen to the subject.

[0010] This article also provides a method for treating cancer in subjects, the method comprising: administering effective amounts of (i) Toll-like receptor (TLR) ligands; (ii) non-classical inflammasome-activated lipids and (iii) cancer immunogens to the subjects.

[0011] In some embodiments of the methods described herein, the cancer immunogen is an infectious agent immunogen, wherein infection in the presence of an infectious agent is associated with cancer development.

[0012] In some embodiments of the methods described herein, the cancer immunogen originates from cancer immunogenic cells.

[0013] In some embodiments of the methods described herein, the cancer immunogen is a whole tumor cell lysate or includes a whole tumor cell lysate.

[0014] In some embodiments of the methods described herein, 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.

[0015] In some embodiments of the methods described herein, the TLR ligand is a TLR4 ligand.

[0016] In some embodiments of the methods described herein, the TLR4 ligand is selected from monophospholipid A (MPLA), lipopolysaccharide (LPS), or a combination thereof.

[0017] In some embodiments of the methods described herein, the non-classical inflammasome-activated lipids include an oxidized 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphocholine (oxPAPC).

[0018] In some embodiments of the methods described herein, non-classical inflammasome-activated lipids 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- (trimethylammonium) ethyl phosphate (KOdiA-PC), 1-palmitoyl-2-(5-hydroxy-8-oxo-octenyl)-sn-glycerol-3-phosphocholine (HOOA-PC), 2-[[(2R)-2-[(E)-5,8-dioxooctyl-6-enyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylammonium (KOOA-PC), [(2R)-3-hexadecano ...]oxy-3-trimethylammonium (KOOA-PC), [(2R)-2-(5-oxopentanoyloxy)propyl]2-(trimethylammonium)ethyl phosphate (POVPC), [(2R)-2-(4-carboxybutyryloxy)-3-hexadecanoyloxypropyl]2-(trimethylammonium)ethyl phosphate (PGPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[2-[(Z)-oct-2-enyl]-5-oxocyclopent-3-en-1 [-methylene]methyl]ethylene oxide-2-yl]butyryloxy]propyl]2-(trimethylammonium)ethyl phosphate (PECPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentyl]methyl]ethylene oxide-2-yl]butyryloxy]propyl]2-(trimethylammonium)ethyl phosphate (PEIPC), or combinations thereof.

[0019] In some embodiments of the methods described herein, non-classical inflammasome-activated lipids include [(2R)-2-(4-carboxybutyryloxy)-3-hexadecanoyloxypropyl]2-(trimethylammonium)ethyl phosphate (PGPC).

[0020] In some embodiments of the methods described herein, the subjects are mammals.

[0021] In some implementations of the methods described herein, the subjects are humans.

[0022] In some embodiments of the methods described herein, TLR ligands, oxPAPCs, and cancer immunogens are administered as part of a pharmaceutical composition.

[0023] In some embodiments of the methods described herein, the immune response is a preventative immune response.

[0024] In some embodiments of the methods described herein, the immune response is a therapeutic immune response.

[0025] In some embodiments of the methods described herein, the adaptive immune response includes T cell activation.

[0026] In some embodiments of the method described herein, the method further includes treating the subject with one or more therapeutic interventions.

[0027] In some embodiments of the methods described herein, TLR ligands, oxPAPCs, and cancer immunogens, as well as one or more therapeutic interventions, are administered together or sequentially.

[0028] In some embodiments of the methods described herein, one or more therapeutic interventions include: radiation, chemotherapy, surgery, therapeutic antibodies, immunomodulators, proteasome inhibitors, pantothenic deacetylase (DAC) inhibitors, histone deacetylase (HDAC) inhibitors, checkpoint inhibitors, adoptive cell therapy, vaccines, or combinations thereof.

[0029] In some embodiments of the methods disclosed herein, adoptive cell therapy includes CAR-T cell therapy, CAR-NK cell therapy, T cells, dendritic cells, or combinations thereof. Unless otherwise stated, 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. The methods and materials used in this invention are described herein; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the specification including the definitions shall prevail.

[0030] Other features and advantages of the invention will be apparent from the following detailed description and drawings, as well as from the claims. Attached Figure Description

[0031] Figure 1A-1H This is a series of diagrams showing that, in the absence of signs of TH2 immunity, overactivated dendritic cells (DCs) are excellent antigen-presenting cells and drive a TH1-biased immune response. Figure 1A-1F WT BMDC can be left untreated (without treatment) or treated with LPS alone, or Alum alone, or oxPAPC or PGPC alone for 24 hours, or BMDC can be sensitized with LPS for 3 hours and then treated with the specified stimulant for 21 hours. Figure 1A : Monitoring the release of IL-1β and TNFα cytokines using ELISA. Figure 1BThe percentage of cell death was measured by the release of LDH from the cell supernatant. Figure 1C The live-dead violet kit, CD11c, and CD40 were used to... Figure 1A BMDC staining with specified stimuli. Surface CD40 (CD11c) was measured by flow cytometry. + The mean fluorescence intensity (MFI) in living cells. Figure 1D BMDCs pretreated with the specified stimulant were transferred to CD40-coated plates and cultured for 24 hours. IL-12p70 cytokine release was measured by ELISA. Figure 1E : will use Figure 1A BMDCs pretreated with the specified stimuli were incubated with OVA protein for 2 hours or with FITC-labeled OVA for 45 minutes. OVA-FITC uptake was assessed by flow cytometry (left figure). Data are represented as OVA-related CD11c at 37°C. + The percentage of BMDC and normalized to OVA-related CD11c at 4°C + BMDC. Monitoring of OVA peptide presentation on MHC-I using an antibody targeting the PE conjugate of H-2Kb bound to the OVA peptide SIINFEKL (SEQ ID NO:1) (right figure). Data are represented as CD11c. + Frequency of SIINFEKL (SEQ ID NO:1) associated DCs in live cells. Mean and SD from three replicates are shown, and the data represent at least three independent experiments. Figure 1F : will use Figure 1A BMDCs treated with the specified stimuli were loaded (or unloaded) with OVA protein or OVA peptide SIINFEKL for 1 hour, followed by treatment with spleen OT-II naive CD4. + T cells or OT-I immature CD8 + T cells were incubated for 4 days. Figure 1F On day 4, the supernatant was collected and the release of cytokines IFNγ, IL-2, IL-10, TNFα and IL-13 was measured by ELISA. Figure 1G BMDC can be left untreated (without treatment) or treated with LPS for 24 hours, or BMDC can be sensitized with LPS for 3 hours and then treated with PGPC or Alum for 21 hours. Then... Figure 1F BMDCs were cultured from spleen OT-I or OT-II T cells. Four days after co-culture, CD4 cells were stimulated with PMA and ionomycin in the presence of brevidin-A and monensin. + and CD8 + T cells were collected after 5 hours. CD4 levels were measured by intracellular staining. +T cells, TH1 cells, such as TNFα + IFNγ + and TH2 cells such as Gata3 + IL-4 + IL-10 + The frequency. Data is presented as the ratio of TH1 / TH2 cells (left figure). CD8 + IFNγ in T cells + The frequencies are shown in the right-hand plot. The plots show the mean and SD from three replicates, and each plot represents at least two independent experiments. Figure 1H As shown in the figure, C57BL / 6 mice were subcutaneously injected in the right flank into either endofit-OVA protein alone or endofit-OVA protein and LPS emulsified in incomplete Freud's adjuvant (IFA) or in Alum. Alternatively, mice were injected with endofit-OVA protein and LPS emulsified in IFA plus OxPAPC or PGPC. Forty days post-immunization, CD4+ was isolated from the skin drainage lymph nodes (dLN). + and CD8 + T cells. T cells were then cultured for 5 days in immature BMDCs loaded (or unloaded) with OVA or SIINFEKL peptide. IFNγ, IL-10, and IL-13 secretion were measured by ELISA. Mean values ​​and SDs for four mice are shown, and each figure represents two independent experiments. *P<0.05; **P<0.01; ***P<0.005.

[0032] Figures 2A-2D This is a series of diagrams illustrating how overactivating stimuli enhance memory T cell production and strengthen antigen-specific IFNγ effector responses in an NLRP3-dependent manner. Figure 2A , 2B C57BL / 6 mice were subcutaneously (sc) injected in the right flank with either endofit-OVA alone, endofit-OVA with LPS, endofit-OVA with PGPC, or endofit-OVA with LPS plus oxPAPC or PGPC. Seven or 40 days post-immunization, T cells were isolated from skin drainage lymph nodes (dLNs) by magnetic enrichment and the use of anti-CD4 and anti-CD8 beads. Figure 1A : in CD3 + CD4 + Live cells (left image), or CD3 + CD8 + The live cells (right image) show T effector cells (Teff) such as CD44. low CD62L lowT-effect memory cells (TEMs) such as CD44 hi CD62L low and T central memory cells (TCMs) such as CD44 hi CD62L hi The percentage. Figure 2B CD8 samples were sorted from dLN 7 days after immunization. + T cells were then treated with PMA and ionomycin, or co-cultured with B16OVA cells (target cells) at a ratio of 1:3 (effective cells:target cells) for 5 hours. Live CD8 cells were monitored using flow cytometry. + CD107a in T cells + The percentage of threshing was used to assess degranulation. The mean and SD of five mice are shown, and each represents three independent experiments. Figure 2C-2D C57BL / 6 mice were subcutaneously injected in the right flank with either endofit-OVA alone, endofit-OVA and LPS, endofit-OVA and LPS plus oxPAPC or PGPC, or endofit-OVA and LPS plus Alum, all emulsified in IFA. Optionally, mice were injected with LPS plus PGPC alone (without OVA). NLRP3 was then injected. - / - Or Casp1 / 11 - / - Mice were similarly injected with endo-fit-OVA emulsified in IFA and LPS plus PGPC. Figure 2C Seven days after immunization, CD4+ was sorted from skin dLN in immunized mice. + and CD8 + T cells were co-cultured with BMDCs loaded (or unloaded) with OVA at a ratio of 1:10 (DC:T cells) for 7 days. CD8 counts were measured using OVA peptide tetramer staining and intracellular IFNγ staining. + SIINFEKL in live T cells + (SEQ ID NO:1)IFNγ + Percentage (above), and CD4 + AHAEINEA in live T cells + (SEQ ID NO:2)IFNγ + The percentage (see chart below). Figure 2D Evaluation of CD44 in inguinal adipose tissue by flow cytometry + Percentage of memory T cells. Mean and SD are shown for 5 mice. *P<0.05; **P<0.01; ***P<0.005, ****P<0.0005.

[0033] Figure 3A-3JThis is a series of figures and graphs illustrating the inflammasome-dependent antitumor immunity induced by overactivated DCs. Figure 3A , 3B C57BL / 6 mice were subcutaneously (sc) injected in the right flank with PBS (unimmunized), B16OVA cell lysate alone (without), B16OVA lysate with LPS, or B16OVA lysate with LPS and oxPAPC or PGPC. Fifteen days post-immunization, the mice were treated with 3x10... 5 Live B16OVA cells were subcutaneously introduced into the left upper back of mice. After 150 days, 5 x 10⁶ cells were used to treat the mice. 5 Live B16OVA cells were subcutaneously injected into tumor-free mice on their backs. Figure 3A Tumor growth was monitored every 2 days (top image). For the survival experiment (bottom image), mice were induced to reach a diameter of 20 mm (n = 8-15 mice per group). Figure 3B Tumors were harvested at the endpoint of tumor growth and dissociated to obtain a single-cell tumor suspension. Enriched CD45 was assessed by flow cytometry. + CD3 infiltrating tumor cells + CD4 + and CD3 + CD8 + Percentage of T cells (top chart). Sorting of tumor-infiltrating CD3 cells. + T cells, then anti-CD3 and anti-CD28 DYNABEADS TM Stimulation was performed for 24 hours in the presence of ThermoFisher Scientific. IFNγ release was measured by ELISA (see figure below) (n = 4 mice per group). Figure 3C From Figure 3A Evaluation of surviving mice at the immunization or tumor injection site and measurement of CD8 by flow cytometry + T cells (top image) and CD69 + CD103 + T Residual Memory CD8 + The absolute number of T cells (see figure below) (n = 4 mice). Figure 3D Total CD8+ in the spleen (left image) or inguinal adipose tissue of the skin of surviving mice or age-matched non-immune tumor-bearing mice (right image) + T cells (top image), CD8 + SIINFEKL in T cells + (SEQ ID NO:1) (middle image) and CD69 + CD103 + (See the figure below) Absolute number (n = 5 mice per group). Figure 3E-3F: Isolate circulating memory CD8 from the spleen of surviving mice or age-matched non-immune tumor-bearing mice + T cells (TCM) and T-resident memory CD8 cells isolated from the inguinal adipose tissue of the skin. + Cells (TRM). Figure 3E TCM and TRM cells from surviving mice were co-cultured with B16OVA, B16-F10, or CT26 tumor cells at a ratio of 1:5 (tumor cells:T cells) for 5 hours. Cytolytic CD8+ was measured by LDH release from the supernatant. + T-cell-induced cell death. Figure 3F C57BL / 6 recipient mice were placed in untreated (Tx-free) or intravenously (iv) inoculated with 5 x 10 5 CD8 + TCM cells, and / or intradermal (id) seeding 5 x 10 5 CD8 isolates from surviving mice or age-matched non-immuno-bearing tumor mice + TRM cells. After 7 days, use 3x10 5 Live B16OVA cells were used to attack all mice. The survival rate was monitored every 2 days. Mice were induced to reach a diameter of 20 mm (n = 5 mice per group). Figure 3G C57BL / 6 mice were immunized subcutaneously in the right flank with untreated (unimmunized) or immunized with B16OVA tumor lysate alone, or B16OVA tumor lysate with MPLA, or B16OVA lysate and MPLA plus PGPC in the presence or absence of neutralizing anti-mouse IL-1β antibodies. Fifteen days post-immunization, mice were administered 3x10... 5 Live B16OVA cells were subcutaneously introduced into the left upper back of mice. After 90 days, 5 x 10⁶ cells were used to treat the mice. 5 Live B16 OVA cells were subcutaneously injected into the back of tumor-free mice. Survival was monitored every 2 days (n = 3-4 mice per group). Figure 3H : WT C57BL / 6, NLRP3 - / - Casp11 - / - Or Casp1 / 11 - / - Mice were subcutaneously immunized in the right flank with B16OVA cell lysates emulsified in IFA, LPS, and PGPC. Fifteen days post-immunization, mice were administered 3x10... 5 Live B16OVA cells were subcutaneously injected into mice in the upper left back. The percentage of survival is shown (n = 5 mice per group). Figure 3I-3JStarting two days before immunization, C57BL / 6 mice were subcutaneously immunized in the right flank for 5 days, either untreated (unimmunized), or immunized with MC38OVA lysate and MPLA, or MC38OVA lysate and MPLA plus PGPC, with or without intravenous injection of anti-mouse IL-1β antibody. Optionally, mice were immunized with OVA protein and MPLA plus PGPC. Fifteen days later, mice were subcutaneously inoculated with 5 x 10-1 slits in the left upper back. 5 One live MC38OVA cell. After 50 days, 1x10 6 MC38OVA cells were subcutaneously injected into tumor-free mice on their backs. (I) shows the percentage of survival (n = 5 mice per group). Figure 3J 170 days after initial seeding with MC38OVA tumor cells, 3x10 5 Live B16-F10 cells were used to attack surviving mice or age-matched juvenile mice. Percentage of survival is shown (n = 5 mice per group). *P<0.05; **P<0.01; ***P<0.005. Tx; T cell injection, WT; wild type.

[0034] Figures 4A-4F These are a series of diagrams and charts illustrating that hyperactivating stimulants are powerful adjuvants for cancer immunotherapy. Figure 4A : A schematic diagram of an immunotherapy based on overactivation, and an illustration corresponding to the neutralizing antibodies used as follows: intravenous (iv) injection of anti-IL-1β, intraperitoneal injection of anti-CD4, anti-CD8a, and anti-PD1 antibodies. Figure 4B : C57BL / 6WT mice were subcutaneously inoculated with 5x10 cells in the upper left back. 5 Live MC38OVA cells were administered. Fourteen days later, mice were placed in the right flank and subcutaneously injected with whole MC38OVA tumor lysate (WTL), plus LPS and PGPC, with or without the neutralizing antibodies specified in the diagram. Mice received two booster injections of WTL and LPS plus PGPC on days 37 and 55 post-tumor inoculation. Tumors were induced to reach a diameter of 20 mm. Percentage of survival is shown (n = 10 mice per group). Figure 4B : C57BL / 6WT mice were subcutaneously inoculated with 3x10 cells in the upper left back. 5Live B16OVA cells were administered. Ten days later, mice were placed in untreated (unimmunized) or injected with anti-PD1 antibodies as specified in the diagram. Optionally, with or without the neutralizing antibodies shown, mice were subcutaneously injected with syngeneic B16OVA WTL, plus LPS and PGPC in the right flank. On days 17 and 24 post-tumor inoculation, mice received two booster injections of B16OVA WTL, plus LPS and PGPC. Percentage of survival is shown (n = 10 mice / group). Figure 4C BALB / c WT mice were subcutaneously inoculated with 3 x 10⁻⁶ cells in the left back. 5 Live CT26 cells were collected. Seven days later, mice were placed in untreated (unimmunized) or injected with the anti-PD1 antibody shown in the figure. Optionally, with or without the injection of the neutralizing antibody shown, mice were subcutaneously injected with syngeneic CT26 WTL, plus LPS and PGPC in the right flank. Mice received two booster injections on days 14 and 21 post-tumor inoculation. Percentage of survival is shown (n = 10 mice / group). Figure 4D : C57BL / 6WT mice were subcutaneously inoculated with 3x10 cells in the upper left back. 5 Live B16-F10 cells. Seven days later, mice were placed in untreated (unimmunized) or injected with the anti-PD1 antibody shown in the diagram. Optionally, with or without the neutralizing antibody shown, mice were immunized subcutaneously in the right flank with syngeneic B16-F10 WTL, plus LPS and PGPC. Mice received two booster injections on days 14 and 21 post-tumor inoculation. Percentage of survival is shown (n = 10 mice / group). Figure 4F C57BL / 6WT mice were intravenously injected with 3x10 5 Live B16-F10 cells were administered. Five days later, mice were placed in untreated (unimmunized) or injected with B16F-10 homologous WTL, B16F-10 homologous WTL with LPS, or B16F-10 homologous WTL with LPS plus PGPC. Metastatic nodules on the lungs were counted 17 days after tumor cell inoculation (n = 5 mice per group).

[0035] Figures 5A-5E This is a series of diagrams showing that, in the absence of signs of TH2 immunity, overactivated dendritic cells (DCs) are excellent antigen-presenting cells and drive a TH1-biased immune response. Figure 5A , 5B BMDCs generated with GMCSF can be left untreated (without treatment), or treated with MPLA alone, Alum alone, or OxPAPC or PGPC alone, or BMDCs can be sensitized with MPLA for 3 hours and then treated with the specified stimulant for 21 hours. Figure 5A: Monitoring the release of IL-1β and TNFα cytokines using ELISA. Figure 5B The percentage of cell death is measured by the release of LDH from the cell supernatant. Figure 5C , 5D : Spleen CD11c + Sorting and placing them untreated (without treatment), or treated with LPS alone, Alum alone, or PGPC alone, or sensitizing DCs with LPS for 3 hours and then treating them with the specified stimulant for 21 hours. Figure 5C : Monitoring the release of IL-1β and TNFα cytokines using ELISA. Figure 5D The percentage of cell death is measured by the release of LDH from the cell supernatant. Figure 5E -5F: BMDCs generated by GMCSF and treated with the stimuli specified in A will be stained with a live-dead purple kit, anti-CD11c, anti-CD80, anti-CD69, and anti-H2kb antibodies. Figure 5E CD11c was measured by flow cytometry. + Mean fluorescence intensity (MFI) of CD80 (left), CD69 (middle), and H2Kb (right) on the surface of live cells. The mean and SD are shown from three replicates, and all figures represent at least three independent experiments. *P<0.05.

[0036] Figures 6A-6C It is a diagram and a series of planar diagrams showing that, in the absence of signs of TH2 immunity, overactivated DCs are excellent antigen-presenting cells and drive a TH1-biased immune response. Figures 6A-6C WT BMDC can be left untreated (without treatment), or treated with LPS alone, Alum alone, or OxPAPC or PGPC alone for 24 hours, or BMDC can be sensitized with LPS for 3 hours and then treated with the specified stimulant for 21 hours. Figure 6A BMDCs were cultured with immobilizable FITC-labeled OVA at 37°C or 4°C for 45 minutes. The BMDCs were then stained with a live-dead purple staining kit. A gating strategy was employed to determine the frequency of OVA-FITC-associated BMDCs at 37°C compared to those at 4°C using flow cytometry. Figure 6B BMDCs were cultured with endofit-OVA protein for 2 hours. A gating strategy was used to determine the frequency of SIINFEKL (SEQ ID NO:1) peptides bound to H2kb on the surface of live BMDCs, as measured by flow cytometry, using an antibody conjugated to the PE of H-2kb that binds to the OVA peptide SIINFEKL. Each figure represents three replicates of one of three experiments. Figure 6CAs shown in the figure, C57BL / 6 mice were subcutaneously injected 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. Alternatively, mice were injected with endofit-OVA protein and LPS emulsified in IFA plus OxPAPC or PGPC. Forty days post-immunization, CD4+ was isolated from the skin drainage lymph nodes (dLN). + T cells. The T cells were then cultured for 5 days in naïve BMDCs loaded with (or unloaded) OVA. IL-4 secretion was measured by ELISA. Mean values ​​and SDs are shown for four mice, and each figure represents two independent experiments. ***P<0.005.

[0037] Figure 7 This is a series of diagrams illustrating how overactivated dendritic cells (DCs) are excellent antigen-presenting cells and drive a TH1-biased immune response in the absence of signs of TH2 immunity. BMDCs were placed untreated (without treatment), treated with LPS for 24 hours, or sensitized with LPS for 3 hours followed by treatment with PGPC or Alum for 21 hours. The treated BMDCs were then cultured with splenic OT-II T cells at a ratio of 1:5 (BMDC:T cells). After 4 days of co-culture, CD4+ cells were introduced into the cells in the presence of brevidin-A and monensin. + T cells were stimulated with PMA and iomycin for 5 hours. A gating strategy was used to determine the level of active CD4+ as measured by intracellular staining. + T cells, TH2 cells, such as IL-4 + IL-10 + The frequency of the experiment. Each figure represents three replicates of one of the three experiments.

[0038] Figures 8A-8E This is a series of figures and graphs illustrating how overactivating stimuli enhance memory T cell production and strengthen antigen-specific IFNγ effector responses in a NLRP3-dependent manner. C57BL / 6 mice were subcutaneously (sc) injected with either endofit-OVA alone, endofit-OVA with LPS, endofit-OVA with PGPC, or endofit-OVA with LPS plus oxPAPC or PGPC, all emulsified in incomplete Freund's adjuvant (IFA). Seven days post-immunization, T cells were isolated from skin drainage lymph nodes (dLNs) by magnetic enrichment and the use of anti-CD4 and anti-CD8 beads. Figure 8A : Set a gate strategy to determine CD3 + CD4 + T effector cells (Teff) in living cells, such as CD44 low CD62L low T-effect memory cells (TEMs) such as CD44hi CD62L low and T central memory cells (TCMs) such as CD44 hi CD62L hi The percentage. Figure 8B : In total CD3 + In live cells, the absolute number of Teff or TEM cells in the skin dLN of each mouse was assessed by flow cytometry. Figure 8C For CD4 + and CD8 + T cell subsets show sorting strategies and post-sorting purity. Figure 8D CD4 was sorted from dLN 7 days after immunization. + and CD8 + T cells were then cultured in BMDCs with serial dilutions (starting at 1000 μg / ml) loaded (or unloaded) with OVA protein. IFNγ cytokine secretion was measured by ELISA. The mean and SD of four replicates are shown. Figure 8E CD8 samples were sorted from dLN 7 days after immunization. + T cells were then treated with PMA and ionomycin, or co-cultured with B16OVA cells (target cells) at a ratio of 1:3 (effective cells:target cells) for 5 hours. A gating strategy was employed to identify live CD8+ cells by flow cytometry. + CD107a in T cells + The percentage. Each graph represents 4 repetitions.

[0039] Figures 9A-9F This is a series of diagrams illustrating the inflammasome-dependent antitumor immunity induced by overactivated DCs. Figure 9A , 9B C57BL / 6 mice were subcutaneously (sc) injected in the right flank with PBS (unimmunized), B16OVA cell lysate alone (without), B16OVA cell lysate with LPS, or B16OVA lysate with LPS and oxPAPC or PGPC. Fifteen days post-immunization, mice were administered 3x10 5 Live B16OVA cells were subcutaneously introduced into the left upper back of mice. After 150 days, 5 x 10⁶ cells were used to treat the mice. 5 Live B16OVA cells were subcutaneously injected into tumor-free mice on their backs. Figure 9A : Shows the percentage of tumor-free mice 300 days after tumor inoculation (n = 8-15 mice per group). Figure 9BC57BL / 6 mice were subcutaneously (sc) injected in the right flank with PBS (unimmunized), B16-F10 cell lysate alone (without immunization), B16-F10 cell lysate with LPS, or B16-F10 cell lysate with LPS and PGPC. Fifteen days post-immunization, mice were treated with 3x10... 5 Live B16-F10 cells were subcutaneously injected into mice on the upper left back. The percentage of survival is shown in the left figure, and the percentage of tumor-free mice at 70 days post-tumor inoculation is shown in the right figure (n = 5 mice per group). Figure 9C C57BL / 6 mice were immunized subcutaneously in the right flank with untreated (unimmunized) or immunized with B16OVA tumor lysate alone, or B16OVA tumor lysate with MPLA, or B16OVA lysate and MPLA plus PGPC in the presence or absence of neutralizing anti-mouse IL-1β antibodies. Fifteen days post-immunization, mice were administered 3x10... 5 Live B16OVA cells were subcutaneously injected into mice on the upper left back. The right figure shows the percentage of tumor-free mice 150 days after tumor inoculation (n = 3-4 mice per group). Figure 9D Starting two days before immunization, C57BL / 6 mice were subcutaneously immunized in the right flank for 5 days, either untreated (unimmunized), or with MC38OVA lysate and MPLA, or MC38OVA lysate and MPLA plus PGPC, with or without intravenous injection of anti-mouse IL-1β antibody. Optionally, mice were immunized with OVA protein and MPLA plus PGPC. Fourteen days later, mice were subcutaneously inoculated with 5 x 10-1 slits in the left upper back. 5 One live MC38OVA cell. After 50 days, 1x10 6 MC38OVA cells were subcutaneously injected into the back of tumor-free mice. The percentage of tumor-free mice is shown at 150 days post-tumor inoculation (n = 5 mice per group). Figure 9E C57BL / 6 mice were immunized subcutaneously in the right flank using either untreated (unimmunized) or B16OVA lysate (WTL) emulsified in IFA with LPS and PGPC, or B16OVA WTL with LPS and alum. Fifteen days post-immunization, mice were administered 3x10... 5 Live B16OVA cells were subcutaneously injected into mice in the upper left back. The survival rate was monitored every two days (n = 5 mice per group). Figure 9F In the absence of any antigen, C57BL / 6 mice were subcutaneously immunized in the right flank with LPS and PGPC emulsified in IFA. Fifteen days later, they were administered B16OVA WTL plus LPS and alum. Fifteen days post-immunization, they were given 3x10 5 One live B16OVA cell, or 3 x 10 5One live B16-F10 cell, or 5 x 10 5 Live MC38OVA cells were subcutaneously injected into mice in the upper left back. The survival rate was monitored every two days (n = 5 mice per group).

[0040] Figures 10A-10C This is a diagram illustrating inflammasome-dependent antitumor immunity induced by overactivated DCs. Figure 10A Gating strategies were used to identify surviving mice (from which cells were previously immunized with B16OVA WTL and LPS plus PGPC, as measured by flow cytometry) Figure 3A CD8 at the site of immune or tumor injection + T cells and CD69 + CD103 + T Residual Memory CD8 + The absolute number of T cells. The figure represents 4 replicates. Figure 10B : Gating strategy to identify surviving mice previously immunized with B16OVA WTL and LPS plus PGPC (from Figure 3A CD45 in the skin and inguinal adipose tissue of age-matched, non-immunized tumor-bearing mice + Total CD8 in living cells + T cells SIINFEKL + The absolute number of (SEQ ID NO:1). The figure represents 5 repetitions. Figure 10C A gating strategy was used to identify surviving mice (from) previously immunized B16OVA WTL and LPS plus PGPC, compared with age-matched unimmunized tumor-bearing mice. Figure 3A CD8 in the skin and groin adipose tissue + T cells and CD69 + CD103 + T Residual Memory CD8 + The absolute number of T cells (the figure represents 5 replicates).

[0041] Figure 11A-11E This is a series of figures and diagrams illustrating overactivated DCs-induced inflammasome-dependent antitumor immunity. Starting two days prior to immunization, C57BL / 6 mice were subcutaneously immunized in the right flank for 5 days, either untreated (unimmunized), or with MC38OVA lysate and MPLA, or MC38OVA lysate and MPLA plus PGPC, with or without intravenous injection of anti-mouse IL-1β antibodies. Optionally, mice were immunized with OVA protein and MPLA plus PGPC. After 15 days, each group was randomly assigned to two sister populations. Figure 11A : A schematic representation of the experimental model. Figure 11B-11E :) In a group of mice, skin draining lymph nodes (dLN) were excised. Figure 11BTotal CD8+ in dLN of immunized mice + T cells (left image) and SIINFEKL + (SEQ ID NO:1)CD8 + The absolute number of T cells (right image). Figure 11C-11D Enriching CD8 from dLN using anti-CD8 beads + T cells, then sorted CD3 + CD8 + Living cells. Figure 11C : Culture CD8 cells using MC38OVA cells (target cells) + T cells were collected 5 hours later. Cytotoxicity of CD8 was monitored by flow cytometry using the CD107a degranulation assay. + T cell response. Figure 11D CD8 + T cells were co-cultured with naive BMDCs loaded (or unloaded) with serially diluted OVA protein (starting at 1000 μg / ml) for 4 days. IFNγ release was monitored by ELISA. Mean values ​​and SDs are shown for 5 mice; each figure represents two independent experiments. *P<0.05; **P<0.01; ***P<0.005.

[0042] Figure 12A and 12B This is a series of diagrams and schematics illustrating the control of tumor rejection induced by overactivated cDC1 by immunotherapy based on overactivated cDC1. Figure 12A-12B C57BL / 6WT mice or Batf3 mice - / - Mice were subcutaneously (sc) injected with 3 × 10⁻⁶ mice on their left back. 5 Live B16OVA cells were administered. Ten days later, mice were immunized subcutaneously in the right flank with either untreated (unimmunized) or syngeneic B16OVA tumor lysate, supplemented with LPS and PGPC. As shown in the diagram, mice received two booster injections on days 17 and 24 post-tumor inoculation to achieve a tumor diameter of 20 mm. Figure 12A The percentage of survivors is shown (n = 5 mice / group). Figure 12B Fifteen days after tumor inoculation, skin draining lymph nodes (dLNs), tumors, and spleen tissue were excised from immunized mice. Antigen-specific CD8+ was measured using SIINFEKL and AAHAEINEA tetramer staining, respectively. + and CD4 + Percentage of T cells (n = 5 mice / group).

[0043] Figures 13A-13D The results of the mass spectrum from the synthetic lipids are shown. Non-oxidized PAPC ( Figure 13A ), oxPAPC ( Figure 13B), oxPAPC enriched by PEEPC ( Figure 13C ) and biotin-labeled oxPAPC ( Figure 13D Mass spectrometry analysis.

[0044] Figure 14A -B. Oxidized phospholipids induce overactivated cDC1 and cDC2 cells exhibiting a high migration phenotype. (A) Wild-type or NLRP3 cells generated using FLT3L. - / - Or Casp1 / 11 - / - BMDCs were placed untreated (without treatment), 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 specified stimulus 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. Means and SDs 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 specified stimulus 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. Means and SDs are from three independent experiments performed in two different laboratories.

[0045] Figure 15A -B. Overactivated DCs induce a strong CTL response and long-term antitumor immunity dependent on CCR7 expression and inflammasome activation. (AB) Wild-type BMDCs generated using FLT3L were placed in untreated (DCs) 幼稚 ), or use separate LPS processing (DC) 活化 )18 hours, or sensitize BMDC with LPS for 3 hours and then with PGPC (DC) 过度活化 ) or Alum (DC) 细胞焦亡 Processing time is 15 hours. Optionally, the material from NLRP3... - / - Or CCR7 - / -Mice's BMDCs 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 subcutaneously injected into wild-type mice. BMDCs without OVA protein loading were used as a control group. Seven days after BMDC injection, the skin drainage lymph nodes were excised and stained with a live-dead purple kit, OVA peptide tetramer antibody, anti-CD45, anti-CD3, anti-CD8a, and anti-CD4. (A) The percentage of SIINFEKL+CD8+ T cells (top) and AHAEINEA+CD4+ live T cells was measured by flow cytometry. (B) The absolute number of SIINFEKL+CD8+ T cells (top) and AHAEINEA+CD4+ live T cells was measured by flow cytometry using CounterBright beads.

[0046] Figure 16A -E. Overactivated stimuli induce strong CTL responses in an inflammasome-dependent manner. (A) C57BL / 6 mice were subcutaneously injected in the right flank with either single OVA, OVA with LPS, OVA with PGPC, or OVA with LPS plus oxPAPC or PGPC, all emulsified in incomplete Freund's adjuvant (IFA). T cells were isolated from skin drainage lymph nodes (dLN) 7 or 40 days post-immunization by magnetic enrichment using anti-CD8 beads. (A) T effector cells (Teff) such as CD44 are shown in CD3+CD8+ live cells. low CD62L low T-effect memory cells (TEMs) such as CD44 hi CD62L low (A) Percentage of T central memory cells (TCMs) such as CD44hi and CD62Lhi. (B) Seven days post-immunization, CD8+ T cells were sorted from dLN and then treated with PMA plus iomycin, or co-cultured with B16 OVA cells (target cells) at a ratio of 1:3 (effective cells:target cells) for 5 hours. CD8+ T cell degranulation was assessed by monitoring the percentage of CD107a+ in live CD8+ T cells using flow cytometry. The mean and SD of five to ten mice are shown. (C) Mice were subcutaneously injected in the right flank into the mice with OVA alone, or OVA with LPS, or OVA with LPS plus oxPAPC or PGPC, or OVA with LPS plus Alum, all emulsified in IFA. Optionally, NLRP3 - / -Mice were injected with OVA emulsified in IFA and LPS plus PGPC. Seven days post-immunization, CD8+ T cells were sorted from skin dLNs of immunized mice and co-cultured with BMDCs loaded (or unloaded) with OVA at a ratio of 1:10 (DC:T cells) for 7 days. The percentage of SIINFEKL+IFNγ+ in viable CD8+ T cells was measured using OVA peptide tetramer staining followed by intracellular IFNγ staining. (DE) CD45.1 mice were irradiated and then bone marrow ZBTB46DTR mice were irradiated with WT or NLRP3. - / - Or Casp1 / 11 - / - Or CCR7 - / - (5:1 ratio) Reconstruction, all against a CD45.2 C57BL / 6 background. For 6 weeks post-reconstruction, chimeric mice were injected with tamoxifen every other day for 7 days. Then, the chimeric mice were subcutaneously immunized in the right flank with OVA emulsified in IFA with LPS and PGPC. Seven days post-immunization, CD8+ T cells were isolated from skin drained lymph nodes (dLN) or spleen using anti-CD8 beads via magnetic enrichment. (D) Percentages of Teff, TEM, TCM, and immature T cells in skin dLN were measured by flow cytometry. (E) Percentage of SIINFEKL+ CD8+ viable T cells in dLN (left panel) or spleen (right panel) was measured by flow cytometry using OVA peptide tetramer staining. Total CD8+ T cells were sorted from dLN and co-cultured for 7 days with untreated BMDCs loaded (or unloaded) with OVA at a ratio of 1:10 (DC:T cells).

[0047] Figure 17A -D. Immunogenic tumors from hot to cold tumors eliminated by overactivating stimuli. (A) C57BL / 6 mice were subcutaneously inoculated with 5 x 10 saturates in the left upper back. 5 Live MC38OVA cells. After 14 days, mice were placed either untreated (unimmunized) or subcutaneously injected with syngeneic MC38OVA whole tumor lysate (WTL), plus LPS and PGPC, with intravenous (iv) or no injection of neutralizing anti-IL-1β, or intraperitoneal or no injection of anti-CD4 or anti-CD8a. Mice received two booster injections of WTL and LPS plus PGPC on days 37 and 55 post-tumor inoculation. Tumors were induced to reach a diameter of 20 mm. Percentage of survival is shown (n = 10 mice per group). (B) C57BL / 6 mice were subcutaneously inoculated with 3 x 10 cells in the left upper back. 5Live B16OVA cells. Ten days later, mice were placed in untreated (unimmunized) or intraperitoneally injected with anti-PD1 antibody. Optionally, mice were subcutaneously injected with syngeneic B16OVA WTL, plus LPS and PGPC in the right flank, with intravenous (iv) or no injection of neutralizing antibody anti-IL-1β, or intraperitoneally or no injection of anti-CD4, or anti-CD8a. On days 17 and 24 post-tumor inoculation, mice received two booster injections of B16OVA WTL, plus LPS and PGPC. Percentage of survival is shown (n = 10 mice / group). (C) C57BL / 6 mice were subcutaneously inoculated with 3 x 10 cells in the left upper back. 5 Live B16-F10 cells. Seven days later, mice were placed either untreated (unimmunized) or intraperitoneally injected with anti-PD1 antibody. Optionally, mice were immunized subcutaneously in the right flank with syngeneic B16-F10 WTL, plus LPS and PGPC, while simultaneously receiving intravenous (iv) or no injection of neutralizing antibody anti-IL-1β, or intraperitoneally or no injection of anti-CD4, or anti-CD8a. Mice received two booster injections on days 14 and 21 post-tumor inoculation. Percentage of survival is shown (n = 10 mice per group). (D) BALB / c WT mice were subcutaneously inoculated with 3 x 10 cells in the left back. 5 Live CT26 cells were administered. Seven days later, mice were placed either untreated (unimmunized) or intraperitoneally injected with anti-PD1 antibody. Optionally, mice were subcutaneously injected with syngeneic CT26 WTL, plus LPS and PGPC in the right flank, with intravenous (iv) or no injection of neutralizing antibody anti-IL-1β, or intraperitoneally or no injection of anti-CD4, or anti-CD8a. Mice received two booster injections on days 14 and 21 post-tumor inoculation. Percentage of survival is shown (n = 10 mice per group).

[0048] Figure 18A -F. Overactivated cDC1 can be used to stimulate T cell-mediated antitumor immunity using complex antigen sources. (A) Subcutaneous injection of B16OVA cells into Zbtb46DTR mice. Mice were injected with diphtheria toxin (DTx) every other day for four consecutive times, 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 mice surviving is shown (n = 10 mice per group). (B) Irradiation of CD45.1 mice, followed by irradiation with WT or Nlrp3 from Zbtb46DTR mice. - / - Or Casp1 / 11 - / - or Ccr7 - / -Hybrid BM remodeling in mice. Six weeks after remodeling, chimeric mice were subcutaneously injected with B61OVA cells, followed by 12 consecutive injections of DTx three times a week. Seven days after tumor inoculation, chimeric mice were immunized with B16OVA WTL and LPS plus PGPC, and received two booster injections. The percentage of mouse survival is shown (n = 5 mice per group). (CD) against WT or Batf3 - / - Mice were subcutaneously injected with B16OVA cells. Seven days after tumor inoculation, the mice were placed in untreated or WT and Batf3-treated environments. - / - Mice were immunized with B16OVA WTL and LPS plus PGPC, followed by two booster injections. (C) shows the percentage of mice that survived (n = 10 mice per group). (D) 21 days after tumor inoculation, the percentage of OVA-specific CD8+ T cells and CD4+ T cells was assessed using tetramer staining (n = 5 mice per group). (EF) Batf3... - / - Mice were subcutaneously injected with B16OVA cells in the right flank. Seven days after tumor inoculation, mice were placed either untreated (without cDC1 injection) or subcutaneously injected with FLT3-derived naive cDC1, LPS-treated activated cDC1, or LPS-pretreated PGPC-pre-activated cDC1 in the left flank. All cDC1 cells were loaded with B16OVA WTL for 1 hour prior to injection. (E) shows the percentage of mice surviving (n = 5 mice per group). (F) OVA-specific CD8+ T cells and CD4+ T cells were assessed using tetramer staining 21 days after tumor inoculation (n = 5 mice per group).

[0049] Figure 19A-C. Oxidized phospholipids induce inflammasome-dependent IL-1β secretion in cDC1 and cDC2 cells and promote a high-migratory DC phenotype. (A) Wild-type BMDCs generated using FLT3L were placed untreated (without treatment), or treated with CpG 1806 alone, or PGPC alone for 24 h, or BMDCs were sensitized with CpG 1806 for 3 h and then treated with the specified stimulus 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. Means and SD from three replicates are shown, and data represent at least three independent experiments. (B) Gating strategies were used 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 cells were placed untreated (without treatment), or treated with LPS alone, or Alum alone, or oxPAPC or PGPC alone for 18 hours, or BMDCs were sensitized with LPS for 3 hours and then treated with the specified stimulus for 15 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 are from three independent experiments performed in two different laboratories.

[0050] Figure 20A -C. Overactivated DCs induce a strong CTL response and long-term antitumor immunity dependent on CCR7 expression and inflammasome activation. Wild-type BMDCs generated using FLT3L were placed in untreated (DCs) 幼稚 ), or use separate LPS processing (DC) 活化 )18 hours, or sensitize BMDC with LPS for 3 hours, then PGPC (DC) 过度活化 ) or Alum (DC) 细胞焦亡 Add to the culture medium for 15 hours. Optionally, add the NLRP3... - / - Or CCR7 - / - Mouse BMDCs were sensitized with LPS for 3 hours, then PGPC was added to the culture medium for 15 hours. BMDCs were washed and then cultured with FITC-labeled -OVA for 45 minutes or with non-fluorescent OVA protein for 2 hours. (A) OVA peptide presentation on MHC-I was monitored using a PE-conjugated antibody against H-2Kb, which binds to the OVA peptide SIINFEKL. Data are expressed as the frequency of SIINFEKL-associated DCs in CD11c+ live cells. Mean and SD from three replicates are shown, and data represent three independent experiments. (B) Stimulation was performed as described above using wild-type or NLRP3 generated by FLT3L. - / - Or CCR7- / - BMDCs. BMDCs were washed and then stained with a live-dead purple kit, CD11c, and CD40. The mean fluorescence intensity (MFI) of surface CD40 was measured by flow cytometry (CD11c+ live cells). (C) CCR7 generated using FLT3L - / - BMDCs were placed untreated (without treatment), or treated with LPS alone, or Alum alone, or PGPC alone for 24 hours. Optionally, BMDCs were sensitized with LPS for 3 hours and then treated with the specified stimulus 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. Means and SDs from three replicates are shown, and the data represent at least three independent experiments.

[0051] Figure 21A -D. Overactivating stimuli enhance memory T cell production and boost antigen-specific IFNγ effector responses in an inflammasome-dependent manner. C57BL / 6 mice were subcutaneously injected in the right flank with either a single OVA, or OVA with LPS, or OVA with PGPC, or OVA with LPS plus oxPAPC or PGPC, emulsified entirely in incomplete Freund's adjuvant (IFA). Seven days post-immunization, T cells were isolated from skin drainage lymph nodes (dLNs) using magnetic enrichment and anti-CD8 beads. (A) Gating strategies were used to identify T effector cells (Teff) such as CD44. low CD62L low T-effect memory cells (TEMs) such as CD44 high CD62L low and T central memory cells (TCMs) such as CD44 high CD62L high (B) The absolute number of Teff or TEM cells in the skin dLN of each mouse was assessed by flow cytometry in total CD3+ viable cells. (C) Seven days post-immunization, CD8+ T cells were sorted from the dLN and then cultured in untreated BMDCs loaded (or unloaded) with serial dilutions of OVA protein (starting at 1000 μg / ml). IFNγ cytokine secretion was measured by ELISA. Mean and SD of five mice are shown. (D) Seven days post-immunization, CD8+ T cells were sorted from the dLN and then treated with PMA plus iomycin, or co-cultured with B16OVA cells (target cells) at a ratio of 1:3 (effect cells:target cells) for 5 hours. Gating strategies were used to determine the percentage of CD107a+ in viable CD8+ T cells by flow cytometry. Each figure represents five mice. *P<0.05; **P<0.01.

[0052] Figure 22A-B. Overactivating stimuli enhance memory T cell production and boost antigen-specific IFNγ effector responses in an inflammasome-dependent manner. (AB) Irradiate CD45.1 mice, followed by bone marrow ZBTB46DTR mice with WT or NLRP3. - / - Or Casp1 / 11 - / - Or CCR7 - / - (5:1 scale) Reconstruction, all against a CD45.2 C57BL / 6 background. Six weeks post-reconstruction, chimeric mice were injected with tamoxifen every other day for seven days. The chimeric mice were then subcutaneously immunized in the right flank with OVA emulsified in IFA with LPS and PGPC. Seven days post-immunization, CD8+ T cells were isolated from skin drained lymph nodes (dLNs) or spleen using magnetic enrichment and anti-CD8 beads. (A) Percentages of Teff, TEM, TCM, and immature T cells in skin dLNs were measured by flow cytometry. Each figure represents five mice. (B) Percentages of SIINFEKL+ CD8+ viable T cells in dLNs (top) or spleen (bottom) were measured by flow cytometry using OVA peptide tetramer staining.

[0053] Figure 23A -C. Mice were subcutaneously (sc) injected in the right flank with PBS (unimmunized), B16OVA cell lysate alone (without), B16OVA cell lysate and LPS, or B16OVA lysate plus LPS and oxPAPC or PGPC. Fifteen days post-immunization, mice were treated with 3x10... 5 Live B16OVA cells were subcutaneously introduced into the left upper back of mice. After 150 days, 5 x 10⁶ cells were used to treat the mice. 5 Live B16OVA cells were subcutaneously injected into tumor-free mice on their backs. (A) Tumor growth was monitored every 2 days (top panel). For the survival assay (bottom panel), mice were induced to reach a diameter of 20 mm (n = 8–15 mice per group). (BC) Tumors were harvested at the endpoint of tumor growth and isolated to obtain single-cell tumor suspensions. (B) The percentage of tumor-infiltrating CD3+CD4+ and CD3+CD8+ T cells in enriched CD45+ live cells was assessed by flow cytometry. (C) Tumor-infiltrating CD3+ T cells were sorted and then stimulated for 24 hours in the presence of anti-CD3 and anti-CD28 dynabeads. IFNγ release was measured by ELISA (bottom panel) (n = 4 mice per group).

[0054] Figure 24A-D. (AB) Assess and measure the absolute number of CD8+ T cells and CD69+CD103+ T-resident memory CD8+ T cells by flow cytometry at the immunization or tumor injection site in surviving mice (n = 4 mice). (CD) Isolate circulating memory CD8+ T cells (TCM) from the spleen of surviving mice or age-matched unimmunized tumor-bearing mice, and isolate T-resident memory CD8+ T cells (TRM) from skin inguinal adipose tissue. (C) Co-culture TCM and TRM from surviving mice with B16OVA or B16-F10 or CT26 tumor cells at a ratio of 1:5 (tumor cells:T cells) for 5 hours. Measure cell death induced by cytolytic CD8+ T cells by LDH release in the supernatant. (D) Place mice in untreated (without Tx) or intravenously (iv) inoculated with 5 x 10⁵ cells. 5 5 x 10 CD8+ TCM cells and / or intradermal (id) seeding 5 CD8+ TRM cells were isolated from surviving mice or age-matched non-immunized tumor-bearing mice. After 7 days, 3x10⁻⁶ cells were used... 5 Live B16OVA cells were used to attack all mice. The survival rate was monitored every 2 days. Mice were induced to reach a diameter of 20 mm (n = 5 mice per group). Detailed Implementation

[0055] This disclosure is based in part on the finding that stimuli that activate dendritic cells (DCs) or promote DC pyroptosis induce a mixed T cell response consisting of type I and type II helper T (Th) cells. Conversely, stimuli that overactivate DCs selectively stimulate TH1 and cytotoxic T lymphocyte (CTL) immune responses, without any indication of TH2-induced immunity. Even when using a complex antigen source (e.g., tumor cell lysate), the TH1-biased immunity generated by overactivated DCs endows these cells with the unique ability to mediate long-term protective antitumor immunity. Stimuli that promote conventional DC activation or pyroptosis do not have the ability to help tumor cell lysates and provide minimal protection against tumors. These novel properties are inherent to overactivated DCs and depend on IL-1β and inflammasome components. The resulting tumor-specific T cells can be transferred to recipient mice and confer complete protection against subsequent attacks. The overactivation stimuli induce protective immunity against tumors that are sensitive to or resistant to PD-1 checkpoint blockade. These joint findings establish the physiological importance of the overactivated state of dendritic cells (DCs) and pave the way for new strategies for cancer immunotherapy in cases where the identification of tumor antigens is unknown.

[0056] This article provides methods for generating or enhancing adaptive immune responses in subjects and methods for treating cancer in subjects. These methods are useful, for example, for therapeutic and / or preventative cancer vaccination.

[0057] This article provides a method for inducing or enhancing an adaptive immune response to cancer in subjects, the method comprising: administering an effective amount of (i) a Toll-like receptor (TLR) ligand; (ii) a non-classical inflammasome-activated lipid; and (iii) a cancer immunogen to the subject.

[0058] This article also provides a method for treating cancer in subjects, the method comprising: administering an effective amount of (i) a Toll-like receptor (TLR) TLR ligand; (ii) a non-classical inflammasome-activated lipid; and (iii) a cancer immunogen to the subject.

[0059] Preferably, the method described herein is a method for inhibiting the growth or progression of cancer, such as tumors, or viral infections in a subject. For example, the method described herein inhibits 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 the tumor by at least 1 mm in diameter, for example, at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm, at least 7 mm, at least 8 mm, at least 9 mm, at least 10 mm, at least 11 mm, at least 12 mm, at least 13 mm, at least 14 mm, at least 15 mm, at least 20 mm, at least 25 mm, at least 30 mm, at least 40 mm, or at least 50 mm or more. In some cases, a large portion of the tumor in the subject has been removed.

[0060] Other aspects are described below.

[0061] definition

[0062] As used herein, the articles “a” and “an” refer to one or more (i.e., at least one) grammatical objects of the article. For example, “an element” refers to one or more elements. Thus, for example, a reference to “a cell” includes multiple cells of the same type. Furthermore, with regard to the terms “including,” “includes,” “having,” “has,” “with,” or variations thereof used in the specification and / or claims, such terms are intended to be included in a form similar to the term “comprising.”

[0063] When referring to measurable values ​​such as quantity and time interval, as used herein, "about" means including a variation of + / -20%, + / -10%, + / -5%, + / -1%, or + / -0.1% from the specified value, as such variation is suitable for carrying out the disclosed methods. Optionally, particularly relating to biological systems or processes, the term may mean on the order of five times the value, and may also mean on the order of two times the value. Where specific values ​​are described in this application and claims, unless otherwise indicated, the term "about" should be assumed to mean within an acceptable range of error for the specific value.

[0064] As used herein, “cancer” 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 leukemias 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 cancers such as osteosarcoma, chondrosarcoma, and Ewing's sarcoma. Sarcoma, fibrosarcoma, giant cell tumor, amelioma (Adamantinomas), and chordomas; brain cancers such as meningiomas, glioblastomas, low-grade astrocytomas, oligodendrogliomas, pituitary adenomas, Schwannomas, and metastatic brain cancers; head and neck cancers including various lymphomas such as mantle cell lymphoma, non-Hodgkin's lymphoma, adenomas, squamous cell carcinoma, laryngeal cancer, gallbladder and bile duct cancer, retinal cancers such as retinoblastoma, esophageal cancer, gastric cancer, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, and testicular cancer. Cancer, endometrial cancer, melanoma, lung cancer, bladder cancer, prostate cancer, lung cancer (including non-small cell lung cancer), pancreatic cancer, sarcoma, Wilms' tumor, cervical cancer, head and neck cancer, skin cancer, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adenocarcinoma, parotid gland tumor, endometrial sarcoma, multidrug-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.

[0065] As used herein, the term "pattern recognition receptor ligand" refers to a molecular compound that activates one or more members of the Toll-like receptor (TLR) family, the RIG-I-like receptor (RLR) family, the nucleotide-binding leucine-rich repeat (NLR) family, cGAS, STING, or AIM2-like receptor (ALR). Specific examples of pattern recognition receptor ligands include natural or synthetic bacterial lipopolysaccharides (LPS), natural or synthetic bacterial lipoproteins, natural or synthetic DNA or RNA sequences, natural or synthetic cyclic dinucleotides, and natural or synthetic sugars. Cyclic dinucleotides include cyclic GMP-AMP (cGAMP), cyclic bisAMP, and cyclic bisGMP.

[0066] As used herein, the term "cancer therapy" refers to a therapy used to treat cancer. Examples of anticancer therapeutic agents include, but are not limited to, surgical agents, chemotherapy agents, immunotherapy, growth inhibitors, cytotoxic agents, agents used in radiotherapy, anti-angiogenic agents, apoptosis agents, anti-microtubule agents, and other agents used to treat 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), and 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 receptor, TRAIL / Apo2, and other biologically active and organic chemical agents, etc. Combinations thereof are also intended for use with the methods disclosed herein.

[0067] Chemotherapy agents are compounds used to treat cancer. Examples of chemotherapy agents include erlotinib (TARCEVA). TM Genentech / OSI Pharm.), bortezomib (VELCADE) TM Millennium Pharm.), fulvestrant (FASLODEX) TM AstraZeneca, Sutent (SU11248, Pfizer), Letrozole (FEMARA) TMNovartis), imatinib mesylate (GLEEVEC) TM Novartis, PTK787 / ZK 222584 (Novartis), oxaliplatin (ELOXATIN) TM Sanofi, 5-FU (5-fluorouracil), leucovorin, rapamycin (Sirolimus, RAPAMUNE) TM Wyeth), Lapatinib (GSK572016, GlaxoSmithKline), Lonafamb (SCH66336), Sorafenib (BAY43-9006, Bayer Labs.), and Gefitinib (IRESSA) TM Astrazeneca), AG1478, AG1571 (SU5271; Sugen), alkylating agents such as thiotepa and CYTOXAN TMCyclophosphamides; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; azacyclopropanes such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylene melamine, triethylenephosphamide, triethylenethiophosphamide, and tris(hydroxymethyl)melamine; anechoic acid lactones (ac etogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozcicsin, carzcicsin, and bizcicsin); nostocins (especially...) Nostocin 1 and Nostocin 8; dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, oxynitrogen mustard hydrochloride, melphalan, novobichin, phenesterine, prednimustine, trofosfamide, uracil Mustard); nitrosoureas such as carmustine, chlorozotocin, formosine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calichimycin, especially calichimycin γ1I and calichimycin ω1 (Angew Chem. Intl. Ed. Engl.).(1994)33:183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromogenin ethynylene antibiotic chromophore), aclacinomysin, actinomycin, autramycin, azaserine, bleomycins, actinomycin C, carabicin, caminomycin, carzinophilin, chromomycinis, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine, ADRIAMYCIN. TMDoxorubicin includes (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, and potfibromycin. ycin), puromycin, quelamycin, rodorubicin, streptoigrin, streptozocin, tubcrcidin, ubenimcx, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil ( 5-FU; folic acid analogs, such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluxuridine; androgens, such as calusterone and dromostanolone. Propionate, epitiostanol, mepitiostane, testolactone; antiadrenergic drugs, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid;Eniluracil; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elfornithine; 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; PSK; TM Polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A) A) and anguidine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; pipebroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, such as TAXOL TMPaclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE TM (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE TM Docetaxel (Rhone-Poulenc Rorer, Antony, France); Chloranmbucil; GEMZAR TM Gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Navelbine TM Vinorelbine; novantrone hydrochloride; teniposide; edatrexate; daunomycin; aminopterin; capecitabine; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives thereof.

[0068] The definition of "chemotherapy agents" also includes (1) anti-hormonal drugs that regulate or inhibit the effects of hormones on tumors, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX). TM Tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, raloxifene hydrochloride, LY117018, onapristone, and FARESTON TM (ii) Inhibitors of aromatase enzymes that regulate estrogen production in the adrenal glands, such as 4(5)-imidazoles, aminoglutethimide, and megase. TM(megestrol acetate), AROMASIN TM (exemestane), formestanie, fadrozole, Rivisor TM (vorozole), FEMARA TM (letrozole) and ARIMIDEX TM (iii) Anastrozole; (iv) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxane cytosine analog); (v) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC-α, Ralf, and H-Ras; (viii) ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME). TM (ribozymes) and HER2 expression inhibitors; (ix) vaccines, such as gene therapy vaccines, such as ALLOVECTIN TM Vaccines, LEUVECTIN TM Vaccines and VAXID TM Vaccine; PROLEUKIN TM rIL-2; LURTOTECAN TM Topoisomerase 1 inhibitor; ABARELIX TM rmRH; (x) anti-angiogenic agents, such as bevacizumab (AVASTIN) TM Genentech); and (xi) any of the pharmaceutically acceptable salts, acids, or derivatives of the above.

[0069] The term "checkpoint inhibitor" refers to CD4+ inhibitors that fine-tune the immune response by downregulating or inhibiting the anti-tumor immune response. + and / or CD8 +A group of molecules on the cell surface of T cells. Immune checkpoint proteins are well known in the art and include, but are not limited to, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, and A2aR (see, for example, WO 2012 / 177624). “Anti-immune checkpoint inhibitor therapy” refers to the use of agents that inhibit immune checkpoint inhibitors. Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling, thereby upregulating the immune response to treat cancer more effectively. Exemplary agents for inhibiting immune checkpoint inhibitors include antibodies, small molecules, peptides, peptide mimics, natural ligands, and derivatives of natural ligands, or fragments thereof, that can bind to and / or inactivate or inhibit immune checkpoint proteins; and RNA interference, antisense, nucleic acid aptamers, etc., or fragments thereof, that can downregulate the expression and / or activity of immune checkpoint inhibitor nucleic acids. Exemplary agents for upregulating immune responses include antibodies targeting one or more immune checkpoint inhibitor proteins that block the interaction between proteins and their natural receptors; inactive forms of one or more immune checkpoint inhibitor proteins (e.g., dominant-negative peptides); small molecules or peptides that block the interaction between one or more immune checkpoint inhibitor proteins and their natural receptors; fusion proteins that bind to their natural receptors (e.g., extracellular portions of immune checkpoint inhibitor proteins fused to the Fe moiety of antibodies or immunoglobulins); and nucleic acid molecules that block the transcription or translation of immune checkpoint inhibitor nucleic acids. Such agents can directly block the interaction between one or more immune checkpoint inhibitors and their natural receptors (e.g., antibodies) to prevent inhibitory signal transduction and upregulate immune responses. Optionally, the agent can indirectly block the interaction between more than one immune checkpoint protein and its natural receptor to prevent inhibition of signal transduction and upregulation of the immune response. For example, soluble forms of immune checkpoint protein ligands, such as stabilized extracellular domains, can bind to their receptors, thereby indirectly reducing the effective concentration of receptors binding to appropriate ligands. In one embodiment, anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody are used alone or in combination.

[0070] As used herein, with respect to elements of the definition or description of an item, composition, apparatus, method, process, system, etc., the terms “comprising,” “comprise,” or “comprised,” and variations thereof, mean included or open-ended, allowing for additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes these specified elements—or, where appropriate, their equivalents—and may contain other elements, and still falls within the scope / definition of the defined item, composition, apparatus, method, process, system, etc.

[0071] As used herein, “co-administration” means that two compounds are administered at sufficiently close temporal proximity to achieve a combined immunizing effect. Therefore, co-administration can be performed by sequential or simultaneous administration (e.g., simultaneous administration in a common or identical carrier).

[0072] As used in this article, “effective amount” refers to the amount that provides therapeutic or preventative benefits.

[0073] "Immunogen" and "antigen" are used interchangeably and refer to any compound that is directly targeted by a cellular or humoral immune response. Non-in vivo immunogens include, for example, tumor cell lysates, tumor proteins, tumor lipids, tumor glycosides, killer 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 constitute or are derived from viruses, mycoplasma, bacteria, parasites, protozoa, or prions, etc. Therefore, immunogens of interest may be derived from, but are not limited to: human papillomavirus, herpesviruses such as herpes simplex or herpes 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, Mycobacterium, amoebae, Plasmodium, and / or Trypanosoma cruzi.

[0074] As used herein, the term “combination” in the context of administering a therapy to a subject means the use of more than one therapy for therapeutic benefit. The term “combination” in the context of administration also refers to the preventative use of a therapy on a subject when used in conjunction with at least one additional therapy. The use of the term “combination” does not limit the order in which therapies (e.g., first and second therapies) are administered to the subject. 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) a subject who has had, has had, or is susceptible to cancer. Administering therapies sequentially and at time intervals to the subject allows the therapies to work together. In certain embodiments, administering therapies sequentially and at time intervals to the subject provides increased benefit compared to other methods of administration. Any additional treatment may be administered in any order with other additional treatments.

[0075] For example, “modulation” of symptoms, levels, or molecular biological activity refers to, for example, a detectable increase or decrease in symptoms or activity. Such increases or decreases were observed in treated subjects compared to those not treated with the adjuvant lipids (non-classical inflammasome-activated lipids) described herein, where untreated subjects (e.g., subjects given the immunogen in the absence of adjuvant lipids) had or were predisposed to developing the same or similar diseases or infections as 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%, 1000% or more, or any range between any two of these values. The modulation can be determined subjectively or objectively, for example, through subject self-assessment, clinician assessment, or by performing appropriate tests or measurements, including, for example, assessment of the degree and / or quality of immunostimulation in the subject achieved by administering the immunogen in the presence of the adjuvant lipids (non-classical inflammasome-activated lipids) described herein. The modulation can be transient, long-term, or permanent, and its duration during or after the administration of the adjuvant lipids disclosed herein to the subject or for use in the tests or other methods described herein or cited references is variable, for example, within the timeframes described below, or from approximately 12 hours to approximately 24 or 48 hours after administration or use of the adjuvant lipids disclosed herein to approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28 days, or more than 1, 3, 6, 9 months after the subject receives such an immunostimulatory composition / treatment.

[0076] As used herein, the term “non-classical inflammasome-activated lipids” refers to lipids that can induce excessive activation of dendritic cells or macrophages. Exemplary “non-classical inflammasome-activated lipids” include PAPCs, oxPAPCs, and oxPAPC classes (e.g., HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC, POVPC, PGPC).

[0077] The oxPAPC class is known and described in this art, see, for example, Ni et al., “Evaluation of Air OxidizedPAPC: A Multi Laboratory Study by LC-MS / MS,” Free Radical Biology and Medicine 144:156–66 (2019); Table 1.

[0078] In some embodiments, non-classical inflammasome-activated lipids 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-(trimethylammonium) ) Ethyl phosphate (KOdiA-PC), 1-palmitoyl-2-(5-hydroxy-8-oxo-octenyl)-sn-glycerol-3-phosphocholine (HOOA-PC), 2-[[(2R)-2-[(E)-5,8-dioxooctyl-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylammonium (KOOA-PC), [(2R)-3-hexadecanoyloxy [(2R)-2-(5-oxopentanoyloxy)propyl]2-(trimethylammonium)ethyl phosphate (POVPC), [(2R)-2-(4-carboxybutyryloxy)-3-hexadecanoyloxypropyl]2-(trimethylammonium)ethyl phosphate (PGPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[2-[(Z)-oct-2-enyl]-5-oxocyclopent-3-en-1-ene] [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentyl]methyl]ethylene oxide-2-yl]butyryloxy]propyl]2-(trimethylammonium)ethyl phosphate (PECPC), or combinations thereof.

[0079] In some implementations, the oxPAPC class is the oxPAPC class listed in Table 1, or a combination thereof.

[0080] Table 1. Oxidized PAPC molecular species 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 notations provided by the LPPtiger tool are 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 denote the number of oxygen additions after the fully identified parent lipid when the type of addition is unknown (e.g., PC16:0 / 20:4+1O), or the known functional groups within parentheses (e.g., PC16:0 / 20:4[1xOH@C11]). Ni et al., “Evaluation of Air Oxidized PAPC: A MultiLaboratory Study by LC-MS / MS,” Free Radical Biology and Medicine 144:156–66 (2019) at 2.7.

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] As used herein, the term “hyperactivated dendritic cell” or “hyperactivated macrophage” refers to a cell that has the ability to secrete interleukin-1 while maintaining viability. This process is typically associated with the assembly of inflammasomes within the hyperactivated cells.

[0090] "Optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes instances where the event or situation occurs and instances where the event or situation does not occur.

[0091] As used in this specification and the appended claims, unless otherwise expressly indicated, the term "or" is generally used in its meaning to include "and / or".

[0092] As used herein, the term "oxPAPC" or "oxidized PAPC" refers to lipids produced by the oxidation of 1-palmitoyl-2-arachidonico-sn-glycerol-3-phosphocholine (PAPC), yielding a mixture of oxidized phospholipids containing fragmented or fully oxidized sn-2 residues. Well-characterized oxidative fragmentation types include pent-carbon sn-2 residues carrying ω-aldehydes or ω-carboxyl groups. Oxidation of arachidonic acid residues also yields phospholipids containing esterified isoprostanes. In addition to other oxidation products present in oxPAPC, oxPAPC also includes HOdiA-PC, KOdiA-PC, HOOA-PC, PGPC, POVPC, and KOOA-PC classes.

[0093] "Parenteral" administration of the immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, or infusion techniques.

[0094] The terms “patient” or “individual” or “subject” are used interchangeably herein and refer to a mammalian subject to be treated, preferably a human patient. In some cases, the methods described herein can be used for laboratory animals, veterinary applications, and for the development of animal models of disease, including but not limited to rodents such as mice, rats, and hamsters, and primates.

[0095] As used herein, a “pharmaceuticalally acceptable” component / carrier is a component / carrier suitable for human and / or animal use without excessively adverse side effects (e.g., toxicity, irritation, and allergic reactions) and in proportion to a reasonable benefit / risk ratio.

[0096] "Appropriate dose level" refers to a dose level that provides a therapeutically reasonable balance between pharmacological efficacy and adverse effects (e.g., adequate immunostimulatory activity conferred by administration of the immunogen in the presence of adjuvant lipids as described herein, and sufficiently low levels of macrophage stimulation). For example, this dose level may be related to, for example, the peak or mean serum level of antiimmunogen antibodies generated after administration of a specific dose level of an immunogenic composition (including adjuvant lipids as described herein) in a subject.

[0097] As used in this article, “treatment” refers to reducing the frequency or severity of at least one sign or symptom of a disease or condition, such as cancer, experienced by the subject.

[0098] "Treatment" is an intervention aimed at preventing the development of a disease or altering its pathology or symptoms. Therefore, "treatment" refers to both therapeutic treatment and preventative or preventative measures. "Treatment" can also refer to palliative care. Patients requiring treatment include those already suffering from the disease and those seeking to prevent it. Therefore, "treating" or "treatment" of a state, condition, or illness includes: (1) preventing or delaying the onset of clinical symptoms of a state, condition, or illness that develops in humans or other mammals who are susceptible to or prone to the state, condition, or illness but have not yet experienced or exhibited clinical or subclinical symptoms; (2) suppressing a state, condition, or illness, i.e., preventing, reducing, or delaying the development of the disease or its recurrence (in the case of maintenance treatment) or at least one of its clinical or subclinical symptoms; or (3) alleviating the disease, i.e., causing the resolution of the state, condition, or illness 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 physician.

[0099] As defined herein, a “therapeuticly effective” amount (i.e., effective dose) of a compound or pharmaceutical agent is an amount sufficient to produce the desired therapeutic (e.g., clinically) outcome. Compositions may be administered from once or more daily to once or more weekly; including every other day. Those skilled in the art will understand that certain factors can influence the dose and duration required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, prior treatment, the subject’s overall health and / or age, and any other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of a compound as described herein may comprise monotherapy or a series of treatments.

[0100] Genes: All genes, gene names, and gene products disclosed herein are intended to correspond to any kind of homologue to which the compositions and methods disclosed herein are applicable. It should be understood that when genes or gene products from a particular kind are disclosed, this disclosure is intended to be illustrative only and not to be construed as limiting, unless the context in which it appears clearly indicates otherwise. Thus, for example, the use of genes or gene products disclosed herein is intended to cover homologous and / or orthologous genes and gene products from other kinds.

[0101] Scope: In this disclosure, various aspects may be presented in the form of scope. It should be understood that the description in the form of scope is for convenience and brevity only and should not be construed as an immutable limitation on the scope of the invention. Therefore, the description of scope should be considered to specifically disclose all possible sub-scopes and the various values ​​within the scope. For example, the description of scope such as 1 to 6 should be considered to specifically disclose, for example, sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the various values ​​within the scope such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the scope.

[0102] Any composition or method provided herein may be combined with one or more of any other compositions and methods provided herein.

[0103] Regulation of dendritic cells (DCs) and pattern recognition receptors (PRRs)

[0104] The innate immune system is generally thought to function in an all-or-nothing manner, with dendritic cells (DCs) acting to elicit an inflammatory response that promotes adaptive immunity, or not. Therefore, Toll-like receptors (TLRs) expressed by DCs are considered to play a crucial 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. Dendritic cells are key to this task, sensing microbes and subsequently promoting T cell activation. It has been shown that dendritic cells can measure the threat of any infection and instruct a proportional response (Blander, JM (2014). Nat Rev Immunol 14, 601-618; Vance, RE et al., (2009) Cell hostµbe 6, 10-21), but the mechanisms by which these immunomodulatory activities occur remain unclear.

[0105] PRR serves to detect molecules common to a wide range of microbial classes, either directly or indirectly. These molecules are often referred to as pathogen-associated molecular patterns (PAMPs) and include factors such as bacterial lipopolysaccharide (LPS), bacterial flagellin, or viral double-stranded RNA.

[0106] A key characteristic of PRRs as immune regulators is their ability to recognize specific microbial products. Thus, PRR-mediated signaling events can provide a clear indication of infection. It is assumed that the “GO” signal is activated by PRRs expressed on dendritic cells (DCs) that promote inflammation and T-cell-mediated immunity. Interestingly, several teams have recently proposed that DCs do not merely function in this all-or-nothing manner (Blander, JM, and Sander, LE (2012). Nat Rev Immunol 12, 215-225; Vance, RE et al., (2009) Cell hostµbe 6, 10-21). More precisely, DCs may have the ability to measure the threat (or virulence) of any possible infection and elicit a proportional response. The most frequently discussed method for measuring virulence is based on the ability of virulent pathogens to activate more diverse PRRs than non-pathogens. However, not all microorganisms share a common set of PRR activators, and not all PRR activators are equally potent. Therefore, the number of PRRs activated during infection is not an ideal measure of virulence. Furthermore, an increased number of PRRs activated during infection typically leads to a larger inflammatory response, which can indirectly promote a larger T-cell response. Conditions that previously indicated enhanced DC activation (e.g., using virulent pathogens as stimulants) were also expected to enhance MΦ activation (Vance, RE et al., (2009) Cellhostµbe 6, 10-21). Therefore, it remains unclear whether the immune system (i.e., DCs) truly possesses a mechanism for specifically measuring the threat of infection.

[0107] One possible method for assessing the threat of infection is through the well-known coincidence detection process, where an independent input leads to a response distinct from that elicited by any single input. In the case of PRR, regardless of the virulence threat, one such input must be a microbial product that serves as an indicator of infection. To measure the virulence threat, a second input must be present. Not wishing to be limited by theory, this hypothetical second input is currently considered to be a molecule generated at the site of tissue injury, as cellular damage is typically a hallmark of highly pathogenic microorganisms. Candidate molecules that could provide a second stimulus to DCs are molecules from different families called damage-associated molecular patterns (DAMPs), also known as alarmins (Kono, H., and Rock, KL (2008) Nat Rev Immunol 8, 279-289; Pradeu, T., and Cooper, EL (2012) Front Immunol 3, 287). DAMPs are found at sites of both infectious and non-infectious tissue injury and are thought to modulate inflammatory responses, although their mechanisms of action remain unclear. One representative class of such DAMPs are oxidized phospholipids derived from 1-palmitoyl-2-arachidonico-sn-glycerol-3-phosphocholine (PAPC), collectively referred to as oxPAPC. These lipids are produced at sites of both infectious and non-infectious tissue damage (Berliner, JA, and Watson, AD (2005). N Engl J Med 353, 9-11; Imai, Y. et al. (2008) Cell 133, 235-249; Shirey, KA et al. (2013) Nature 497, 498-502) and are found at very high levels in the membranes of dying cells (Chang, MK et al. (2004) J Exp Med 200, 1359-1370). oxPAPC is also an active component of oxidized low-density lipoprotein (oxLDL) aggregates that promote inflammation in atherosclerotic tissues (Leitinger, N. (2003) Curr OpinLipidol 14, 421-430), with local concentrations reaching up to 10-100 μM (Oskolkova, OV 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 general indicators of tissue health. Therefore, in the presence of microbial products, oxPAPC may indicate an increased threat of infection.

[0108] Due to the aforementioned characteristics of activated dendritic cells (DCs), these cells are capable of stimulating antigen-specific T cell responses, and numerous strategies have been employed to promote DC activation to drive protective immunity. These strategies typically involve the use of synthetic or natural microbial products that stimulate the Toll-like receptor (TLR) family. Notable examples include the molecule monophospholipid A (MPLA, an FDA-approved TLR4 ligand used to assist 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 Didierlaurent, et al., J. Immunol., vol. 183, no. 10, pp. 6186–6197, Nov. 2009). Note that TLRs alone cannot upregulate all 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, long-lived memory T cell production, and effector function (SZBen-Sasson, et al., Proc. Natl. Acad. Sci. USA, vol. 106, no. 17, pp. 7119–24, Apr. 2009; SZBen-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). IL-1β (a well-characterized family member) expression is highly induced by TLR signaling, but this cytokine lacks an N-terminal secretion signal and is therefore not released from the cell via conventional biosynthetic pathways. However, IL-1β accumulates in an inactive state in the cytosol of dendritic cells (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 IL-1β release from activated DCs increases the likelihood that TLR signaling alone is insufficient to maximally stimulate T cell responses and protective immunity.

[0109] DC activation is not the only cell fate that DCs can achieve under PRR signaling. In fact, different PRR stimuli result in different cell fates. One such fate is a commitment to an inflammatory form of cell death called pyroptosis. Pyroptosis is a regulated process resulting from the activity of inflammasomes, supramolecular organizing centers (SMOCs) assembled in the cytosol of DCs and other cells (A. Lu, et al. Cell, vol. 156, no. 6, pp. 1193–1206, Mar. 2014; J. C. Gan, et al. Nat. Rev. Immunol., vol. 14, no. 12, pp. 821–826, Dec. 2014). Inflammatory body assembly is typically stimulated when PAMP or DAMP is detected in the host cell cytosol. Thus, the cytosol PRR is responsible for associating threat assessment in the cytosol with inflammatory body-dependent pyroptosis (KJ Kieser and JCKagan, 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). Pyroptosis results in the release of IL-1β and other members of the IL-1 family from the cell, providing T cells with signals that TLRs cannot provide. In addition to the increased activity, pyroptosis is a process that promotes IL-1β release, and thus loses the ability to participate in the multi-day process required to stimulate and differentiate naive T cells in the dLN (TRMempel, et al. Nature, vol. 427, no. 6970, pp. 154–159, Jan. 2004). In fact, stimuli that promote pyroptosis, such as the commonly used vaccine adjuvant alum (SCEisenbarth 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), are not suitable for the elimination of many microbial infections or cancers.

[0110] In a particular embodiment, a method of treating cancer includes administering to a subject in need a therapeutically effective amount of a composition comprising a dendritic cell hyperactivation stimulant and tumor cell lysate as an immunogen, thereby treating the cancer. In a particular embodiment, the method further includes administering a chemotherapeutic agent, an immunogen, or a combination thereof.

[0111] In certain implementation schemes, the overactivating stimulant, chemotherapeutic agent, immunogen, or combination thereof are administered together or sequentially.

[0112] In a particular embodiment, the overactivation stimulant comprises a combination of a pattern recognition receptor ligand and 1-palmityl-2-(5-glutaryl)-sn-glycerol-3-phosphate choline (PGPC).

[0113] In a particular embodiment, the dendritic cell overactivation stimulant includes a pattern recognition receptor ligand and 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphate choline (PAPC), oxidized 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphate choline (oxPAPC), oxPAPC class, components thereof, or combinations thereof.

[0114] TLR4 ligand

[0115] In some embodiments of the methods described herein, 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.

[0116] In some embodiments of the methods described herein, the TLR ligand is a TLR4 ligand.

[0117] In some embodiments of the methods described herein, the TLR4 ligand is selected from monophospholipid A (MPLA), lipopolysaccharide (LPS), or a combination thereof.

[0118] Immunogen

[0119] This art describes immunogens such as cancer immunogens and their uses, for example, in cancer vaccines. See, for example, Michael J. 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.

[0120] 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 exogenous bodies, tumor-DC fusions, or combinations thereof.

[0121] In some implementations, the immunogen is a whole tumor lysate.

[0122] In some implementations, whole tumor lysates are prepared by irradiation, boiling, and / or freeze-thaw lysate preparation.

[0123] In some implementations, the immunogen is autologous. In other implementations, the immunogen is allogeneic.

[0124] In some implementations of methods for inducing an immune response in subjects, the immunogen is a tumor lysate derived from a cell donor.

[0125] In some embodiments of the methods described herein, the cancer immunogen is an infectious agent immunogen, wherein infection in the presence of an infectious agent is associated with cancer development.

[0126] In some embodiments of the methods described herein, the cancer immunogen originates from cancer immunogenic cells.

[0127] In some embodiments of the methods described herein, the cancer immunogen is a whole tumor cell lysate or includes a whole tumor cell lysate.

[0128] Immunogenic compositions comprising adjuvants as described herein may be administered to subjects using any known form of vaccine, such as tumor antigens, tumor cell lysates, attenuated viruses, proteins, nucleic acids, etc., to generate in subjects an amount of the selected immunogen that effectively induces a therapeutic or prophylactic immune response against a target antigen in the subject. Subjects may be human or non-human. Animal subjects include, but are not limited to, non-human primates, dogs, cats, equines (horses), ruminants (e.g., sheep, goats, cattle, camels, alpacas, llamas, deer), pigs, birds (e.g., chickens, turkeys, quails), rodents, and chiropterans. Subjects may be treated for any purpose, including but not limited to, inducing a protective immune response or producing antibodies (or B cells) for collection or other purposes.

[0129] The immunogen of interest is expressed by diseased target cells (e.g., neoplastic cells, infected cells) and expressed at low 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 cancers, prostate cancers, ovarian cancers, cervical cancers, colon cancers, lung cancers, glioblastomas, and astrocytomas. Optionally, the target cells may be infected by, for example, viruses, mycoplasma, parasites, protozoa, and prions. Therefore, immunogens of interest may come from, but are not limited to: human papillomavirus (see below), herpesviruses such as herpes simplex or herpes zoster, retroviruses such as human immunodeficiency virus 1 or 2, hepatitis viruses, influenza viruses, rhinoviruses, respiratory syncytial virus, cytomegalovirus, adenoviruses, Mycoplasma pneumoniae, Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium, amoebae, Plasmodium, and Trypanosoma cruzi.

[0130] In addition to tumor antigens, tumor cell lysates, and antigens of infectious agents, target antigens for use according to this disclosure may also be provided, including but not limited to, tumor suppressor gene products of p53, BRCA1, BRCA2, retinoblastoma, and TSG101, or mutants of oncogene products such as, but not limited to, RAS, WT, MYC, ERK, and TRK. Target antigens may be autoantigens, such as antigens associated with cancer or neoplastic diseases. In some embodiments, the immunogen is a peptide from a heat shock protein (hsp)-peptide complex derived from diseased cells, or the hsp-peptide complex itself.

[0131] The immunogenic compositions described herein may include an immunogen and an adjuvant lipid and may be administered for therapeutic and / or prophylactic purposes. In therapeutic applications, the immunogenic compositions described herein may be administered in amounts sufficient to elicit an effective immune response and / or overactivate dendritic cells to treat disease or inhibit progression and / or symptoms. The dosage of the adjuvant described herein may vary depending on the nature of the immunogen and the condition of the subject, but should be sufficient to enhance the efficacy of the immunogen in evoking an immunogenic response. For therapeutic or prophylactic treatment, the amount of adjuvant administered may range from 0.05, 0.1, 0.5, or 1 mg per kg body weight, up to approximately 10, 50, or more than 100 mg per kg body weight. The adjuvants described herein are generally non-toxic and can generally be administered in relatively large amounts without causing life-threatening side effects.

[0132] As used herein, the term "therapeutic immune response" refers to an increase in humoral and / or cellular immunity directly against a target antigen, as measured by standard techniques. Preferably, the level of induced immunity directly against the target antigen is at least four times, and preferably at least five times, the level prior to administration of the immunogen. The immune response can also be measured qualitatively, wherein the inhibition or relief of the progression of a neoplastic or infectious disease in the subject, as determined by appropriate in vitro or in vivo assays, indicates the induction of a therapeutic immune response.

[0133] In some embodiments of the methods described herein, a composition containing a therapeutically effective amount of the immunogen and adjuvant as described herein is administered to a mammal in need. As used herein, the term "administration" means the delivery of the immunogen and adjuvant as described herein to a mammal by any method that achieves the desired result. These may be administered, for example, intravenously or intramuscularly. As used herein, the term "mammal" is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and farm animals. "Therapeutically effective amount" refers to the amount of immunogen and adjuvant that, when administered to a mammal, effectively produces the desired therapeutic effect.

[0134] Compositions containing the immunogen and adjuvant as described herein may be administered dermally, subcutaneously, intravenously, intramuscularly, parenterally, intrapulmonaryly, intravaginally, rectally, nasally, or topically. The compositions may be delivered by injection, orally, via aerosol, or by particle bombardment.

[0135] Compositions for application may further include various additional materials, such as pharmaceutically acceptable carriers. Suitable carriers include any standard pharmaceutically acceptable carrier, such as phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions or triglyceride emulsions, various types of wetting agents, tablets, coated tablets, and capsules. Typically, such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid, talc, vegetable fats or oils, gums, ethylene glycol, or other known excipients. Such carriers may also include flavor and color additives or other ingredients. The compositions described herein may also include suitable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions may be in liquid form or lyophilized or otherwise dried formulations, and may include various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength diluents, additives to prevent surface absorption such as albumin or gelatin, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile salts), solubilizers (e.g., glycerol, polyethylene glycol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), fillers or tension modifiers (e.g., lactose, mannitol), covalent linkages of polymers such as polyethylene glycol to proteins that coordinate with metal ions, or the introduction of materials into or onto granular formulations of polymers such as polylactic acid, polyglycolic acid, hydrogels, or onto liposomes, microemulsions, micelles, monolayers or multilayers of vesicles, erythrocyte shadows, or protoplasts. Such compositions will affect physical state, solubility, stability, rate of release in vivo, and rate of clearance in vivo.

[0136] combination therapy

[0137] In certain embodiments, it is preferred to administer one or more other agents with therapeutic benefits to the subject. These include, but are not limited to, chemotherapeutic agents, compounds, cytokine antagonists, cytokine receptor antagonists, cytokines, adoptive cell therapy, antiviral drugs, checkpoint inhibitors, adjuvants, or combinations thereof. In certain embodiments, the compound includes at least one amide-amine compound. Amide-amines are a class of compounds formed from fatty acids and diamines. An example of an amide-amine compound is myristamidopropyl dimethylamine (Aldox).

[0138] Immune checkpoint modulation: In certain implementation schemes, immune checkpoint modulators are co-administered with overactivated dendritic cells. Immune checkpoints are inhibitory pathways of the immune system responsible for maintaining self-tolerance and regulating the duration and extent of physiological immune responses.

[0139] Some cancer cells thrive by utilizing immune checkpoint pathways as a major mechanism of immune resistance, particularly those involving T cells specific to tumor antigens. For example, some cancer cells overexpress more than one immune checkpoint protein responsible for suppressing cytotoxic T cell responses. Therefore, immune checkpoint modulators can be administered to overcome inhibitory signals and allow and / or enhance immune attack against cancer cells. Immune checkpoint modulators can promote immune cellular responses against cancer cells by reducing, inhibiting, or eliminating signaling through negative immune response modulators (e.g., CTLA4), or by stimulating or enhancing the signaling of positive immune response modulators (e.g., CD28).

[0140] Immunotherapy agents targeting immune checkpoint modulators can be administered to promote immune attack against cancer cells. Immunotherapy agents can be or include antibody agents targeting immune checkpoint modulators (e.g., specific to immune checkpoint modulators). Examples of immunotherapy agents include antibody agents targeting 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 may include monoclonal antibodies. Some monoclonal antibodies targeting immune checkpoint modulators are available. For example, ipilimumab targets CTLA-4; tremelimumab targets CTLA-4; pembrolizumab targets PD-1, etc.

[0141] 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 for PD-1 have been identified: programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2). It has been shown that PD-L1 and PD-L2 downregulate T cell activation and cytokine secretion when they bind to PD-1 (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43; Ohigashi et al. (2005) Clin Cancer Res 11:2947-53).

[0142] PD-L1 (also known as differentiation cluster 274 (CD274) or B7 homologue 1 (B7-H1)) is a 40 kDa type I transmembrane protein. PD-L1 binds to its receptor PD-1, found on activated T cells, B cells, and myeloid cells, to regulate activation or inhibition. Both PD-L1 and PD-L2 are B7 homologues that bind to PD-1 but not to CD28 or CTLA-4 (Blank et al. (2005) Cancer Immunol Immunother. 54:307-14). The binding of PD-L1 to its receptor PD-1 on T cells delivers a signal that inhibits TCR-mediated activation, suppressing IL-2 production and T cell proliferation. This mechanism involves the 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 PKC-θ activation cyclic phosphorylation, which is essential for the activation of transcription factors NF-κB and AP-1, and for IL-2 production. PD-L1 also binds to the co-stimulatory molecule CD80 (B7-1) but not to CD86 (B7-2) (Butte et al. (2008) Mol Immunol. 45:3567-72).

[0143] PD-L1 expression on the cell surface has been shown to be upregulated by IFN-γ stimulation. PD-L1 expression has been found in many cancers, including human lung cancer, ovarian cancer, colon cancer, and various myelomas, and it is often 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) NatMed 8:793-800). It has also been shown that PD-L1 may be involved in intestinal mucosal inflammation, and that inhibition of PD-L1 suppresses wasting disease associated with colitis (Kanai et al. (2003) J Immunol 171:4156-63).

[0144] Exemplary anti-PD1 antibodies include pembrolizumab (MK-3475, Merck), nivolumab (BMS-936558, Bristol-Myers Squibb), and pidilizumab (CT-011, Curetech LTD.). These anti-PD1 antibodies are commercially available, for example, from ABCAM. TM (AB137132), BIOLEGEND TM (EH12.2H7,RMP1-14) and Affymetrix Ebioscience (J105,J116,MIH4).

[0145] Anticancer agent: In certain embodiments, the method further includes administering an anticancer agent. In some embodiments, the anticancer agent is a chemotherapeutic agent or growth inhibitor, a targeted therapeutic agent, a T cell expressing a chimeric antigen receptor, an antibody or an antigen-binding fragment thereof, an antibody-drug conjugate, an angiogenesis inhibitor, an antitumor drug, a cancer vaccine, an adjuvant, or a combination thereof.

[0146] In some implementations, the anticancer agent is a chemotherapeutic agent or a growth inhibitor. For example, chemotherapeutic agents or growth inhibitors may include alkylating agents, anthracyclines, antihormones, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic agents or antitumor antibiotics, proteasome inhibitors, antimicrotubule agents, EGFR antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors, and combinations thereof.

[0147] Examples of chemotherapy agents may include erlotinib (TARCEVA). TM Genentech / OSIPharm.), Bortezomib (VELCADE) TM Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), rhizobacterin, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX) TM AstraZeneca), sunitinib (SUTENT) TM Pfizer / Sugen), letrozole (FEMARA) TM Novartis), imatinib mesylate (GLEEVEC) TM Novartis, finasunate (VATALANIB) TM Novartis, oxaliplatin (ELOXATIN) TM Sanofi, 5-FU (5-fluorouracil), leucovorin, rapamycin (Sirolimus, RAPAMUNE) TM Wyeth), Lapatinib (TYKERB) TM GSK572016, Glaxo Smith Kline), Lonafamib (SCH 66336), Sorafenib (NEXAVAR) TM Bayer Labs), gefitinib (IRESSA) TMAstraZeneca, AG1478; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; azacyclopropanes such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylene melamine, triethylene ethylphosphonamide, triethylene thiophosphonamide, and tris(hydroxymethyl)melamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); nostocins (especially nostocin 1 and nostocin 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., calichimycin, especially calichimycin γ1I and calichimycin ω1I (Angew Chem. Intl. Ed. Engl.)).1994 33:183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromogenin ethynylene antibiotic chromophore), aclacinomysin, actinomycin, autramycin, azaserine, bleomycins, actinomycin C, carabicin, caminomycin, carzinophilin, chromomycinis, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine, ADRIAMYCIN. TM(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, fluxuridine; and androgens, such as calusterone, dromostanolone. Propionate, epitiostanol, mepitiostane, testolactone; antiadrenergic drugs, 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; PSK; TMPolysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A) A) and anguidine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; pipebroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, such as TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE TM (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE TM (docetaxel, doxetaxel; Sanofi-Aventis; chloranmbucil; GEMZAR) TM (Gemcitabine); 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs, such as cisplatin and carboplatin; Vinblastine; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Navelbine TM (vinorelbine); novantrone hydrochloride; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA) TMIbandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and any of the above pharmaceutically acceptable salts, acids and derivatives.

[0148] In some implementations, the chemotherapeutic agent may include an alkylating agent (including monofunctional and bifunctional alkylating agents), such as thiotepa or cytoxan. TM Cyclophosphamides, nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, oxynitrogen mustard, melphalan, novombhichin, 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.

[0149] In some implementations, the chemotherapeutic agent may include anthracyclines such as daunorubicin, doxorubicin, epirubicin, edabacin, mitoxantrone, valrubicin, and any of the pharmaceutically acceptable salts, acids, and derivatives thereof.

[0150] In some implementations, the chemotherapy agent may include anti-hormonal drugs, such as anti-estrogens, and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX). TM Tamoxifen citrate, raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, raloxifene hydrochloride, LY117018, onapristone, and FARESTON TM(toremifine citrate); and any of the pharmaceutically acceptable salts, acids and derivatives thereof.

[0151] In some implementations, the chemotherapeutic agent may include aromatase inhibitors that inhibit aromatase (which regulates estrogen production in the adrenal glands), such as 4(5)-imidazoles, aminoglutethimide, and megase. TM (medroxyprogesterone acetate), AROMASIN TM (Exemestane; Pfizer), Formestanie, Fadrozole, Rivisor TM (vorozole), FEMARA TM (letrozole; Novartis), and ARIMIDEX TM (Anastrozole; AstraZeneca); and any of the pharmaceutically acceptable salts, acids and derivatives thereof.

[0152] In some embodiments, the chemotherapeutic agent may include antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all-trans retinoic acid, fenretinide, and troxacitabine (a 1,3-dioxolane-cytosine analog); and any of the pharmaceutically acceptable salts, acids, and derivatives thereof.

[0153] In some implementations, the chemotherapeutic agent may include protein kinase inhibitors, lipid kinase inhibitors, or antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC-α, Ralf, and H-Ras.

[0154] In some implementations, the chemotherapeutic agent may include ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME). TM ) and HER2 expression inhibitors.

[0155] In some implementations, the chemotherapeutic agent may include cytotoxic agents or antitumor antibiotics, such as actinomycin D, actinomycin, bleomycin, plicamycin, mitomycin such as mitomycin C, and any of the pharmaceutically acceptable salts, acids and derivatives thereof.

[0156] In some implementations, the chemotherapeutic agent may include a proteasome inhibitor such as bortezomib (VELCADE). TM Millennium Pharm.), epoxomicins such as carfilzomib (KYPROLIS) TM Onyx Pharm.), marizomib (NPI-0052), MLN2238, CEP-18770, opprozomib, and any of the pharmaceutically acceptable salts, acids and derivatives thereof.

[0157] In some implementations, the chemotherapeutic agent may include antimicrotubule agents, such as vinca alkaloids, including vincristine, vinblastine, vindesine, and vinorelbine; taxanes, including paclitaxel and docetaxel; podophyllotoxin; and any of the pharmaceutically acceptable salts, acids, and derivatives thereof.

[0158] In some implementations, the chemotherapeutic agent may include an “EGFR antagonist,” which is a compound that binds to 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 chimeric 225 (C225 or cetuximab; ERBUTIX) and remodeled human 225 (H225) (see WO 96 / 40210, Imclone Systems). Inc.); IMC-11F8, a fully human EGFR-targeting antibody (Imclone); an antibody binding to type II mutant EGFR (US Patent No. 5,212,290); humanized and chimeric antibodies binding EGFR as described in US Patent No. 5,891,996; human antibodies binding 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 that directly targets EGFR and competes with both EGF and TGF-α for EGFR binding (EMD / Merck); 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 can be conjugated with cytotoxic agents to produce immunoconjugates (see, for example, EP659439A2, Merck Patent GmbH).EGFR antagonists include small molecules, such as 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, and the following PCT disclosures: WO98 / 14451, WO98 / 50038, WO99 / 09016, and WO99 / 24037. Certain small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA). TM Genentech / OSI Pharmaceuticals); PD 183805 (CI 1033, 2-Acrylates, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-dihydrochloride, Pfizer Inc.); ZD1839, Gefitinib (IRESSA) TM ), 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); ZM105180((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382(N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimidino[5,4--d]pyrimidin-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]pyrimidin); 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) TMGSK572016 or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[2-methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).

[0159] In some implementations, the chemotherapeutic agent may include tyrosine kinase inhibitors, including EGFR-targeting drugs mentioned in the preceding paragraphs; small molecule HER2 tyrosine kinase inhibitors such as TAK165 available from Takeda; CP-724,714, oral selective inhibitors of ErbB2 receptor tyrosine kinases (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; and non-HER-targeting TK inhibitors such as imatinib mesylate (GLEEVEC). TM Available from Glaxo SmithKline; multi-target tyrosine kinase inhibitors such as sunitinib (SUTENT) TMAvailable 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-chloroaniline)quinazoline; pyridopyrimidines; pyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferoylmethane). methane, 4,5-bis(4-fluoroaniline)phthalimide; tyrphostines containing a nitrothiophene moiety; PD-0183805 (Warner-Lamber); antisense molecules (e.g., those that bind to HER-encoded nucleic acids); quinoxalines (US Patent No. 5,804,396); tryphostins (US 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 (GLEEVEC) TM PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone); Rapamycin (sirolimus, RAPAMUNE) TM); or as described in any of the following patent publications: 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).

[0160] In some implementations, the chemotherapeutic agent may include retinoids such as retinoic acid, as well as any of the pharmaceutically acceptable salts, acids, and derivatives described above.

[0161] In some implementations, the chemotherapeutic agent may include antimetabolites. Examples of antimetabolites include folic acid analogs and antifolates, such as folate, methotrexate, pteroxetine, and trimethoprim; purine analogs, such as fludarabine, 6-mercaptopurine, thioimidazoline, and thioguanine; pyrimidine analogs, such as 5-fluorouracil (5-FU), cyclocytidine, azacytidine, 6-azauridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, and fludeoxyuridine; nucleoside analogs; and nucleotide analogs.

[0162] In some embodiments, the chemotherapeutic agent may include a topoisomerase inhibitor. Examples of topoisomerase inhibitors may include topoisomerase 1 inhibitors such as LURTOTECAN. TM and ABARELIX TM rmRH; topoisomerase II inhibitors such as doxorubicin, epirubicin, etoposide, and bleomycin; and topoisomerase inhibitor RFS 2000.

[0163] In some implementations, the chemotherapeutic agent may include histone deacetylase (HDAC) inhibitors such as vorinostat, romidesin, belistat, moxistat, valproic acid, parbistat, and any of the pharmaceutically acceptable salts, acids, and derivatives thereof.

[0164] Chemotherapy agents may also include hydrocortisone, hydrocortisone acetate, tecortisone valerate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, and fluocinolone acetate. Acetone, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, acmetasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasol-17-butyrate, clobetasol-17-propionate, fluocortolone hexanoate, fluocortolone neopentylate, and fluprednidazole acetate; immunoselective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and its D-isomer (feG) (IMULANBioTh... Erapeutics, 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), cetuzumab (Cimzia), golimumab (Simponi); interleukin-1 (IL-1) blockers such as anaerobiculin (Kineret); T-cell co-stimulation blockers such as abatacept (Orencia); interleukin-6 (IL-6) blockers such as tocilizumab (ACTEMERA). TM Interleukin-13 (IL-13) blockers such as levozumab; interferon-alpha (IFN) blockers such as rontalizumab; β7 integrin blockers such as rhuMAb Beta7; IgE pathway blockers such as anti-M1 prime; secretory homotrimer LTa3 and membrane-bound heterotrimer LTa1 / β2 blockers such as anti-lymphotoxin alpha (LTa); radioisotopes (e.g., 211 At、 131 I, 125 I, 90 Y、 186 Re、 188 Re、212 Bi、 32 P, 212 Radioactive isotopes of Pb and Lu; other research drugs such as thioplatin, PS-341, phenyl butyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, leucine, epigallocatechin gallate, theaflavins, flavanols, procyanidins, betulinic acid and its derivatives; autophagy inhibitors such as chloroquine; Δ9-tetrahydrocannabinol (drocannabinol, MARINOL) TM ); β-Lapaquinone; succinyl quinone; colchicine; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin; podophyllotoxin; tegafur TM ); Targretin TM ); bisphosphonates such as chlorophosphonates (e.g., Bonefos) TM or OSTAC TM ), etidronate (DIDROCAL) TM ), NE-58095, zoledronic acid / zoledronic acid salt (ZOMETA) TM alendronate (FOSAMAX) TM ), pamidronate (AREDIA) TM ), tiluphosphonate (SKELID) TM ), or risedronate (ACTONEL) TM ); and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE TM Vaccines; perifoxine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteasome inhibitors (e.g., PS341); CCI-779; tilpifanib (R11577); orafenib, ABT510; Bcl-2 inhibitors such as oblimersen sodium (GENASENSE) TM Pixantrone; Farnesyltransferase inhibitors such as lonafazil (SCH 6636, SARASAR) TM ); any pharmaceutically acceptable salts, acids, and derivatives thereof; and combinations of two or more of the above, such as CHOP (an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone); and FOLFOX (an abbreviation for oxaliplatin). TM () is an abbreviation for a treatment regimen combining 5-FU and folinic acid.

[0165] Chemotherapy agents may also include nonsteroidal anti-inflammatory drugs (NSAIDs) with 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, and diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, and isoxicam, and fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, and tolfenamic acid. NSAIDs include celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, and valdecoxib. NSAIDs can be used to relieve symptoms in conditions such as rheumatoid arthritis, osteoarthritis, inflammatory joint disease, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, migraine, headache and migraine, postoperative pain, mild to moderate pain due to inflammation and tissue damage, fever, intestinal obstruction, and renal colic.

[0166] Drug treatment

[0167] On the one hand, the pharmaceutical composition is administered systemically, for example, formulated in a pharmaceutically acceptable buffer such as physiological saline. Preferred routes of administration include, for example, bladder infusion, 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 performed using a therapeutically effective amount of the therapeutic agent identified herein in a physiologically acceptable carrier. Suitable carriers and their formulations are described, for example, in Remington's Pharmaceutical Sciences, EW Martin. The amount of the therapeutic agent to be administered varies depending on the method of administration, the patient's age and weight, and the clinical presentation of the cancer. Generally, the amount will be within the range used for other agents in the treatment of other cancer-related diseases, although in some cases, a lower amount will be required due to the increased specificity of the compound. The compound is administered at a dose known to those skilled in the art to enhance the immune response of the subject or to reduce the proliferation, survival, or invasion of proliferative or infected cells.

[0168] The administration of the compositions illustrated herein is by any suitable means of producing a therapeutic agent, in combination with other components, that effectively improves, reduces, or stabilizes the concentration of cancer. The compositions may be provided in dosage forms suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, intravascular, intratumoral, or intraperitoneal) administration. For example, the pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, for example, Remington: The Science and Practice of Pharmacy (20th edition), ed. ARGennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JCBoylan, 1988–1999, Marcel Dekker, New York).

[0169] Human doses are initially determined by extrapolation from the amount of the compound used in mice or non-human primates. As those skilled in the art will recognize, it is routine practice in the art to vary the dose for humans compared to animal models. For example, doses may vary between about 1 μg compound / kg body weight and about 5000 mg compound / kg body weight; or about 5 mg / kg body weight and about 4000 mg / kg body weight; or about 10 mg / kg body weight and about 3000 mg / kg body weight; or about 50 mg / kg body weight and about 2000 mg / kg body weight; or about 100 mg / kg body weight and about 1000 mg / kg body weight; or about 150 mg / kg body weight and about 500 mg / kg body weight. For example, the dosage is approximately 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150, 1,200, 1,250, 1,300, 1,350, 1,400, 1,450, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 mg / kg body weight. Optionally, the dose is in the range of about 5 mg compound / kg body weight to about 20 mg compound / kg body weight. In another example, the dose is about 8, 10, 12, 14, 16, or 18 mg / kg body weight. Of course, as is routinely done in such treatment regimens, the dose may be increased or decreased depending on the results of the initial clinical trial and the specific needs of the patient.

[0170] Pharmaceutical compositions can be formulated with suitable excipients to produce a controlled release of the therapeutic agent upon administration. Examples include single or multi-unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

[0171] The pharmaceutical compositions described herein can be administered parenterally by injection, infusion, or transplantation (subcutaneous, intravenous, intramuscular, intratumoral, intravascular, intraperitoneal) in dosage forms, formulations, or via suitable delivery devices or grafts containing conventional, non-toxic, pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. For formulations, see Remington: The Science and Practice of Pharmacy, as cited above.

[0172] In certain embodiments, the composition may be in the form of a solution, suspension, emulsion, infusion device, delivery device for transplantation, or presented as a dry powder reconstituted with water or another suitable solvent prior to use. In addition to an active agent that reduces or improves cancer, the composition includes a suitable parenteral acceptable carrier and / or excipient. The active therapeutic agent may be incorporated into microspheres, microcapsules, nanoparticles, or liposomes for controlled release. Furthermore, the composition may contain suspending agents, thickeners, stabilizers, pH adjusters, tension modifiers, and / or dispersants.

[0173] As described above, the pharmaceutical composition may be in a form suitable for sterile injection. To prepare such a composition, a suitable active therapeutic agent is dissolved or suspended in a parenteral acceptable liquid solvent. Acceptable solvents and media that may be used are water, water adjusted to a suitable pH by adding appropriate amounts of hydrochloric acid, sodium hydroxide, or a suitable buffer solution, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride and glucose solutions. Aqueous formulations may also contain more than one preservative (e.g., methylparaben, ethylparaben, or n-propylparaben). When a compound is only slightly or negligibly soluble in water, a solubilizer or dissolving agent may be added, or the solvent may contain 10-60% w / w propylene glycol.

[0174] This document provides a method for treating cancer or its symptoms, comprising administering a therapeutically effective amount of a pharmaceutical composition. Therefore, this document describes a method for treating a subject who has or is susceptible to cancer. The method may include the step of administering a therapeutic amount of the composition described herein to a mammal at a dose sufficient to treat the disease or condition or its symptoms, under conditions for treating the disease or condition.

[0175] The methods described herein include administering an effective amount of the compound or composition described herein to a subject (including a subject identified as needing such treatment) to produce such an effect. Identifying a subject needing such treatment can be a judgment of the subject or a healthcare professional and can be subjective (e.g., opinion) or objective (e.g., measurable by test or diagnostic methods).

[0176] The treatments described herein (including prophylactic treatments) generally involve administering a therapeutically effective amount of the compounds described herein, such as those in the protocols described herein, to subjects in need (e.g., animals, humans), including mammals, particularly humans. Such treatments will be appropriately administered to subjects, particularly humans, who suffer from, have, are susceptible to, or are at risk of having cancer or its symptoms. The determination that these subjects are “at risk” is made by any objective or subjective determination through diagnostic testing or the opinion of the subject or healthcare provider (e.g., genetic testing, enzyme or protein markers, markers (as defined herein), and family history, etc.). Fusion protein complexes as described herein can be used to treat any other condition in which an increased immune response is desired.

[0177] This article also provides a method for monitoring the course of treatment. The method includes the steps of determining the level of a diagnostic marker (e.g., any target, protein, or indicator thereof described herein regulated by the compound described herein) or a diagnostic measurement (e.g., screening, testing) in a subject suffering from or susceptible to a cancer-related condition or its symptoms, wherein a therapeutic amount of the compound described herein has been administered to the subject sufficient to treat the disease or its symptoms. The marker level determined in this method may be compared with known marker levels in healthy controls or other patients with the disease to determine the subject's disease status. 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 progression of the disease or the efficacy of the treatment. In some aspects, a pre-treatment level of the marker in the subject is determined before the initiation of treatment as described herein; then, this pre-treatment level of the marker is compared with the level of the marker in the subject after the initiation of treatment to determine the efficacy of the treatment.

[0178] The pharmaceutical composition may be included in a kit, container, package, or dispenser along with instructions for use.

[0179] Unless otherwise indicated, the practice of this method employs known techniques within the scope of the art, including those in chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture, and transgenic biology. See, for example, Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Sambrook et al., 1989, Molecular Cloning, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Sambrook and Russell, 2001, Molecular Cloning, 3rd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Ausubel et al., 1992, Current Protocols in Molecular Biology (John Wiley & Sons, including regular updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988, Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Jakoby and Pastan, 1979; Nucleic AcidHybridization (edited by BD Hames & S.J. Higgins. 1984); Transcription And Translation (edited by BD Hames & S.J. Higgins. 1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, APractical Guide To Molecular Cloning (1984); Thesis, Methods In Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors For Mammalian Cells (edited by J.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 ed., Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986); Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), (4th ed., Univ. of Oregon) Press, Eugene, 2000.

[0180] Unless otherwise stated, 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. While similar or equivalent methods and materials may be used to practice or test this invention, suitable methods and materials are described below. It should be understood and anticipated that those skilled in the art can make changes to the principles of the invention disclosed herein, and such modifications are intended to be included within the scope of this invention.

[0181] Exemplary Implementation

[0182] 1. A method for preparing an immunogenic composition, the method comprising:

[0183] a) Deplete leukocytes from a cell suspension prepared from tumor cells to obtain a suspension enriched with tumor cells;

[0184] b) Lyse cells from a suspension enriched with tumor cells to obtain tumor cell lysates;

[0185] c) Contact tumor cell lysates with oxidized lipids and Toll-like receptor 4 (TLR4) agonists to obtain an immunogenic composition.

[0186] 2. The method according to embodiment 1, wherein the leukocytes are depleted in step a) by using negative selection with an anti-CD45 antibody.

[0187] 3. The method according to embodiment 1, wherein the cells are lysed in step b) by one or more freeze-thaw cycles.

[0188] 4. The method according to embodiment 1, wherein the oxidized lipid comprises at least one phospholipid of oxidized 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphocholine (oxPAPC).

[0189] 5. The method according to embodiment 4, wherein the at least one phospholipid comprises at least one of the group consisting of POVPC, PGPC, PECPC, and PEEPC, optionally wherein the at least one phospholipid comprises PGPC.

[0190] 6. The method according to embodiment 1, wherein the TLR4 agonist comprises monophospholipid A (MPLA).

[0191] 7. The method according to embodiment 6, wherein the TLR4 agonist is present in an adjuvant, the adjuvant further comprising one or more of an aluminum salt, a saponin, and a liposome, optionally wherein the adjuvant is AS01B or AS04.

[0192] 8. The method according to embodiment 1, further comprising obtaining a sample from the tumor and preparing a suspension of cells prior to step a).

[0193] 9. An immunogenic composition prepared by any one of embodiments 1-8.

[0194] 10. A method for inducing an anticancer immune response, the method comprising:

[0195] An effective amount of the immunogenic composition of embodiment 9 was administered to a mammalian subject with cancer.

[0196] 11. The method according to embodiment 10, wherein the anticancer immune response includes a cellular immune response.

[0197] 12. The method according to embodiment 10, wherein the anticancer immune response includes cancer antigen-induced IL-1β secretion and CD8+ T lymphocyte activation.

[0198] 13. The method according to any one of embodiments 10-12, wherein the cancer is a non-hematologic cancer.

[0199] 14. The method according to embodiment 13, wherein the non-hematologic cancer is a tumor, sarcoma, or melanoma.

[0200] 15. The method according to any one of embodiments 10-12, wherein the cancer is lymphoma.

[0201] 16. A method for treating cancer, the method comprising:

[0202] a) Prepare an immunogenic composition containing tumor cell lysate, oxidized lipids, and a Toll-like receptor 4 (TLR4) agonist, wherein the tumor cell lysate is prepared from or has been prepared from a sample of a tumor obtained from a mammalian subject with cancer.

[0203] b) Administer an effective amount of the immunogenic composition to the subject.

[0204] 17. A method for treating a mammalian subject with cancer, the method comprising:

[0205] a) Prepare an immunogenic composition containing tumor cell lysate, oxidized lipids, and a Toll-like receptor 4 (TLR4) agonist, wherein the tumor cell lysate is prepared from or has been prepared from a sample of a tumor obtained from a mammalian subject with cancer.

[0206] b) Administer an effective amount of the immunogenic composition to the subject.

[0207] 18. The method according to any one of embodiments 10-17, wherein the oxidized lipid comprises at least one phospholipid of oxidized 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphocholine (oxPAPC).

[0208] 19. The method according to embodiment 18, wherein the at least one phospholipid comprises at least one of the group consisting of POVPC, PGPC, PECPC, and PEEPC, optionally wherein the at least one phospholipid comprises PGPC.

[0209] 20. The method according to any one of embodiments 10-17, wherein the TLR4 agonist comprises monophospholipid A (MPLA).

[0210] 21. The method according to embodiment 20, wherein the TLR4 agonist is present in an adjuvant, the adjuvant further comprising one or more of an aluminum salt, a saponin, and a liposome, optionally wherein the adjuvant is AS01B or AS04.

[0211] 22. The method according to any one of embodiments 10-17, wherein the at least one phospholipid comprises PGPC and the TLR4 agonist comprises monophospholipid A (MPLA).

[0212] 23. The method according to any one of embodiments 10-22, further comprising administering an effective amount of an additional therapeutic agent to the subject.

[0213] 24. The method according to embodiment 23, wherein the additional therapeutic agent comprises one or more of the group consisting of immune checkpoint inhibitors, antitumor drugs, and radiotherapy.

[0214] 25. The method according to any one of embodiments 10-23, wherein the cancer is resistant to immune checkpoint inhibitors prior to the application of the immunogenic composition.

[0215] Example

[0216] Example 1: Durable antitumor immunity to complex antigen mixtures stimulated by overactivated dendritic cells

[0217] An ideal strategy for stimulating protective immunity is to combine the benefits of activated and pyroptotic dendritic cells (DCs), thereby enabling activated cells to release IL-1β while maintaining viability. The inventors have recently identified a novel activated state of DCs exhibiting these properties. When DCs are exposed to PAMPs (e.g., TLR ligands) and some oxidized phospholipids (DAMPs) released from dying cells, cells achieve a prolonged state of “hyperactivation” (I. Zaoni, et al., Science, vol. 352, no. 6290, pp. 1232–1236, 2016; I. Zaoni, et al., Immunity, vol. 47, no. 4, p. 697–709, e3, 2017). These oxidized lipids are referred to as oxPAPC (oxidized 1-palmitoyl-2-arachidonico-sn-glycerol-3-phosphocholine). Hyperactivated DCs exhibit the activity of activated DCs in terms of cytokine (e.g., TNFα) release, but they also acquire the ability to release IL-1β over a period of several days. Consistent with their role as “overactivated” DCs, these cells outperform their activated counterparts in their ability to stimulate T-cell responses to model antigens.

[0218] The underlying mechanisms of the hyperactivated state of dendritic cells (DCs) have been defined, such as the DAMP (oxPAPC) discussed here, which can bind to and stimulate cytoplasmic PRR caspase-11 (I. Zanoni, et al., 2016). Caspase-11 stimulation leads to the activation of NLRP3 and the assembly of inflammasomes, which does not result in pyroptosis but does lead to the release of IL-1β from living cells. Release of IL-1β from overactivated cells is mediated by the pore-forming protein gasdermin D, which acts as a conduit for the secretion of these cytokines (CLEvavold, 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. Aglietti, et al. Proc. Natl. Acad. Sci. USA, vol.113, no.28, pp.7858–63, Jul. 2016; N. Kayagaki, et al. Nature, vol.526, no.7575, pp.666–671, Sep. 2015). The removal of dermal D pores is thought to be a way to repair the cell membrane and thus ensure 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, leading to pyroptosis. Although the mechanisms by which IL-1β is released from living cells are understood, the physiological benefits of an overactivated cellular state in guiding adaptive immunity remain poorly defined.

[0219] Materials and methods

[0220] Mouse strains and tumor cell lines: C57BL / 6J (Jax 000664), caspase-1 / -11dKO mice (Jax016621), NLRP3KO (Jax 021302), Casp11KO (Jax 024698), OT-I (Jax 003831), OT-II (Jax004194), and BALB / c (Jax 000651) mice were purchased from Jackson Labs. For the syngeneic tumor model 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 MC-38 cell line expressing OVA, derived from C57BL6 mouse colon adenocarcinoma cells, was used. These cell lines were a gift from Arlene Sharpe Laboratory. For the syngeneic colon cancer model in BALB / c mice, the CT26 cell line (a gift from Jeff Karp laboratory) was used.

[0221] Reagent: Escherichia coli (E. coli) LPS (serotype O55:B5-TLRGRADE) TM) Purchased from Enzo and used at 1 μg / ml for cell culture or at 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 at 20 μg / mouse for in vivo use. OxPAPC was purchased from Invivogen, resuspended in preheated serum-free medium and used at 100 μg / ml for cell stimulation or at 65 μg / mouse for in vivo use. POVPC and PGPC were purchased from Cayman Chemical. Commercially available POVPC and PGPC were reconstituted as previously described (CLEvavold et al., Immunity, 2018 Jan 16; 48(1):35-44). Briefly, the ethanol solvent was evaporated using a gentle nitrogen stream. The preheated serum-free medium was then 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 being added to cells. POVPC or PGPC were used for cell stimulation at 100 μg / ml and for in vivo use at 65 μg / mouse. EndoFit chicken ovalbumin and OVA257-264 peptide with endotoxin levels <1 EU / mg were purchased from Invivogen and used in vivo at a concentration of 200 μg / mouse or in vitro at concentrations of 500 or 100 μg / ml. Incomplete Freund's adjuvant (F5506) was purchased from Sigma and used for in vivo immunization at a working concentration of 1:4 (IFA:antigen emulsion). Alhydrogel called alum was purchased from Accurate Chemical and used for in vivo immunization at a working concentration of 2 mg / mouse. In some experiments, Addavax as a squalene-oil-in-water adjuvant was used instead of IFA at a working concentration of 1:2 (AddaVax:antigen).

[0222] Cell culture: Bone marrow-derived cells (BMDCs) were generated by differentiating them from IMDM (Gibco), 10% B16-GM-CSF-derived supernatant, 2 μM 2-mercaptoethanol, 100 U / ml penicillin, 100 μg / ml streptomycin (Sigma-Aldrich), and 10% FBS. After 6 days of culture, BMDCs were washed with PBS and cultured at 1 x 10⁻⁶ cells / mL. 6 CD11c was plated at a concentration of 100 μl cells / ml in IMDM containing 10% FBS. + DC purity was assessed by flow cytometry using BD Fortessa and was routinely above 80%. Splenic DCs from mice injected with B16-FLT3 for 15 days were purified to CD11c. + MHC +Live cells, then at 1x10 6 Cells were plated in complete IMDM at a concentration of 100 μl cells / ml. To induce overactivated or pyroptotic BMDCs, DCs were sensitized in complete IMDM with LPS (1 μg / ml) for 3 h, followed by stimulation with OxPAPC or PGPC (100 μg / ml) or alum (100 μg / ml) for 21 h. In some cases, activated BMDCs were restimulated on plate-bound agonist anti-CD40 for an additional 24 h using Ultra-LEAF anti-mouse CD40 (clone 1C10; BioLegend). T cells were cultured in RPMI-1640 (Gibco) supplemented with 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin (Sigma-Aldrich), and 50 μM β-mercaptoethanol (Sigma-Aldrich). All tumor cell lines were cultured in DMEM supplemented with 10% FBS. For OVA-expressing cell lines, puromycin (2 μg / ml) was added to the culture medium.

[0223] LDH Detection and ELISA: Following BMDC stimulation, the fresh supernatant was clarified by centrifugation, and LDH release was detected using the Pierce LDH Cytotoxicity Colorimetric Assay Kit (Life Technologies) according to the manufacturer's protocol. Absorbance readings were measured at 490 nm and 680 nm using a Tecan plate reader. To measure secreted cytokines, the supernatant was collected, clarified by centrifugation, and stored at -20°C. ELISA for IL-1β, TNFα, IL-10, IL-12p70, IFNγ, IL-2, IL-13, IL-4, and IL-17 was performed using the eBioscience Ready-SET-Go! (ThermoFisher) ELISA kit according to the manufacturer's protocol.

[0224] Flow cytometry: After FcR blockade, 7-day-old 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-IA / IE (clone M5 / 114.15.2), anti-CD40 (clone 3 / 23), anti-CD80 (16-10A1), anti-CD69 clone (H1.2F3), and anti-H-2Kb (clone AF6-88.5). Single-cell suspensions from tumors or draining inguinal lymph nodes, or skin and 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 (clones 53–6.7), anti-CD4 (clones RM4–5), anti-CD44 (clones IM7), anti-CD62L (MEL-14), anti-CD3 (17A2), anti-CD103 (2E7), anti-CD69 clone (H1.2F3), and anti-CD45 (A20 or 30F11). LIVE / DEAD TM A Fixable Violet Dead Cell Stain Kit (Molecular probes) 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 (OVA257-264; SEQ ID NO:1) and APC-conjugated IA(b)AAHAEINEA (OVA329-337; SEQ ID NO:2) were used. IA(b) and H2K(b) associated with CLIP peptides were used as isotype controls. Tetramers were purchased from the NIH Tetramer Core Facility. In some experiments, FITC anti-CD8.1 (clone Lyt-2.1CD8-E1) purchased from Accurate Chemical was used in conjunction with the tetramers. To determine the absolute number of cells, COUNTBRIGHT counting beads (Molecular probes) were used according to the manufacturer's protocol. Appropriate isotype controls were used as staining controls. Data were obtained from BD FACS ARIA or BD Fortessa. Data were analyzed using FlowJo software.

[0225] Antigen Uptake Assay: To examine the antigen uptake and endocytosis capabilities of BMDCs during different activation states (activated, hyperactivated, or pyroptotic), FITC-labeled chicken OVA (FITC-OVA) (Invitrogen-Molecular Probes) was used. Briefly, BMDCs pretreated with FITC-OVA or AF488-dextran (0.5 mg / ml) were cultured at 37°C or 4°C (as controls for antigen surface binding) for 45 minutes. The BMDCs were then washed and stained with a live / dead fixable purple dead cell staining kit (Molecular probes) to distinguish live cells from dead cells. Cells were then fixed with BD fixation solution and resuspended in MACS buffer (PBS with 1% FCS and 2 mM EDTA). 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-related cells, and the data were normalized to the percentage of OVA-FITC-related cells cultured at 4°C.

[0226] OVA antigen presentation assay: To measure the efficiency of OVA antigen presentation on MHC-I, Endofit-OVA protein (0.5 mg / ml) was used at 37°C to treat cells treated with activating stimulant (LPS), hyperactivating stimulant (LPS+PGPC or LPS+OxPAPC), or pyroptosis stimulant (LPS+Alum) (0.5 × 10⁻⁶ cells / ml). 6 BMDCs were cultured for 2 hours. Cells were then washed with MACS buffer and treated on ice with APC anti-mouse H-2K. b Antibody (clone AF6-88.5, BioLegend), and H-2K bound to OVA peptide SIINFEKL (SEQ ID NO:1; Clone 25-D1.16, BioLegend). b Stain with the conjugated PE antibody for 20 to 30 minutes. Use a suitable isotype control as a staining control. Calculate the total surface H-2K. b The percentage of cells associated with the OVA peptide on MHC-I, and the percentage of cells associated with it. Data were acquired on a Fortessa flow cytometer (Becton-Dickenson) and analyzed using FlowJo software (Tree Star).

[0227] OT-I and OT-II in vitro T cell stimulation: Splenic CD8 cells were isolated from OT-I and OT-II mice using magnetic cells sorted with anti-CD8 beads or anti-CD4 beads (Miltenyi Biotech), respectively.+ and CD4 + T cells. Then, sorted T cells were pretreated with LPS (activating stimulant), LPS+PGPC (overactivating stimulant), or LPS+Alum (pyroptosis stimulant) and seeded at a concentration of 100,000 cells per well in 96-well plates in the presence of 20,000 or 10,000 DC (5:1 or 10:1 ratio) pulsed (or not pulsed) with 100 μg / ml OVA protein or SIINFEKL (SEQ ID NO:1) peptide for 2 hours. After 5 days of culture, the supernatant was collected, clarified by centrifugation, and stored at -20°C for short-term storage. Cytokine levels were measured by ELISA.

[0228] Intracellular staining: For intracellular cytokine staining, cells were stimulated for 4–5 hours with 50 ng / ml phorbol 12-tetradecanoate 13-acetate (PMA) and 500 ng / ml iomycin (Sigma-Aldrich) in the presence of GolgiStop (BD) and brevidin A. Cells were then washed twice with PBS and incubated at 4°C in PBS with LIVE / DEAD. TM Cells were stained for 20 minutes using a purple or green molecular probes staining kit. Cells were washed with MACS buffer and stained with appropriate surface markers at 4°C for 20 minutes. After two washes, cells were fixed and permeabilized for 20 minutes at 4°C using the BD Cytofix / Cytoperm kit according to the manufacturer's protocol, followed by washing with 1X perm wash buffer (BD). Intracellular cytokine staining was performed at 4°C in 1X perm buffer for 20–30 minutes using the following conjugated antibodies, all purchased from BioLegend: anti-Ki67 (clone 16A8), anti-IFN-γ (clone XMG1.2), anti-TNFα (clone MP6-XT22), anti-Gata3 (16E10A23), anti-IL4 (11B11), and anti-IL10 (clone JES5-16E3). Data were obtained on BD FACSARIA or BD Fortessa. Data were analyzed using FlowJo software.

[0229] In vivo immunization and T-cell restimulation: Eight-week-old female C57BL / 6J mice were immunized subcutaneously (sc) in the left lower back with 150 μg / mouse endotoxin-free OVA emulsified in incomplete Freund's adjuvant plus 10 μg / mouse LPS, or with 150 μg / mouse endotoxin-free OVA emulsified in incomplete Freund's adjuvant plus 65 μg / mouse oxPAPC or PGPC plus 10 μg / mouse LPS. In some experiments, mice were subcutaneously injected with OVA alone or OVA and LPS emulsified in Alum. Seven or 40 days post-immunization, CD4+ was isolated from the draining lymph nodes of immunized mice using magnetic cells sorted by anti-CD4 beads or anti-CD8 beads and columns (Miltenyi Biotech). + T cells and CD8 + T cells. The enriched cells were then sorted into live CD45 cells using FACS ARIA. + CD3 + CD4 + Cells or live CD45 + CD3 + CD8 + Cells. Purity after sorting was >98%. Then, they were pulsed 10–20 x 10⁻⁶ times with a series of dilutions starting at 1 mg / ml of OVA. 3 In the presence of individual dendritic cells (DCs), sorted cells were seeded in 96-well plates at a concentration of 100,000 cells per well. After 5 days, the secretion of IFNγ, IL-10, and IL-2 was measured by ELISA.

[0230] CD107a threshing test: In order to evaluate CD8 + The antitumor activity of T cell effectors was assessed by evaluating surface exposure of the lysosomal-associated protein CD107a using flow cytometry. In short, CD8+ was isolated from the skin drainage lymph nodes of immunized mice via magnetic cell enrichment using anti-CD8 beads and columns (Miltenyi Biotech). + T cells were then sorted into CD3 cells on FACS ARIA (BD). + CD8 + Live cells. Freshly sorted CD8 cells + T cells at a rate of 1x10 6 Resuspended in complete RPMI at a concentration of 1 cell / ml. PerCP / Cy5.5 anti-mouse CD107a (LAMP-1) antibody (clone 1D4B, BioLegend) was added to the medium at a concentration of 1 μg / ml in the presence of GolgiStop (BD). T cells were then immediately seeded at a concentration of 100,000 cells per well into 10,000 MC38OVA or B16OVA tumor cells per well in a 96-well plate. Optionally, CD8+ cells were added...+ T cells were seeded individually and stimulated with 50 ng / ml phorbol 12-tetradecanoate 13-acetate (PMA) and 500 ng / ml iomycin (Sigma-Aldrich). After 5 hours of culture, the cells were washed with MACS buffer and subjected to LIVE / DEAD. TM Cells were fixed using a purple dead cell staining kit (Molecular probes) and APC anti-CD8 (clone 53-6.7, BioLegend) staining. Cells were then fixed with BD fixative for 20 minutes at 4°C and resuspended in MACS buffer. CD107a was identified by flow cytometry on a Fortessa flow cytometer (BD). + Percentage of cells.

[0231] In vitro cytotoxicity assay: CD8 inhibitors were isolated from the spleen or inguinal adipose tissue of surviving mice using anti-CD8 MACS beads and columns (Miltenyi Biotech). + T cells. Then, FACS ARIA was used to sort the enriched T cells into live CD45 cells. + CD3 + CD8 + Cells. Purity after sorting >97%. Tumor cell lines such as B16OVA, B16F-10, or CT26 cells were seeded into 96-well plates (2x10⁻¹²) in complete DMEM at least 5 hours before co-culturing with T cells. 4 (cells / well). 10 5 CD8 + T cells were inoculated onto tumor cells 12 hours later, and then cytotoxicity was assessed using the Pierce LDH cytotoxicity assay kit (Life Technologies) according to the manufacturer's protocol.

[0232] Preparation of whole tumor cell lysates (WTL): To prepare whole tumor cell lysates (WTL) for immunization, tumor cell lines were cultured in complete DMEM for 4–5 days. When the cells reached confluence, the supernatant was collected, the cells were washed, and the cells were dissociated using trypsin-EDTA (Gibco). The tumor cell lines were then cultured at 5 x 10⁻⁶ cells / day. 6 Cells / ml were resuspended in their collected culture supernatant and then lysed by three cycles of freeze-thaw.

[0233] Synthetic whole tumor lysates of melanoma or colonic adenocarcinoma tumors were prepared from transplanted tumors in tumor-bearing mice that had never been immunized. In short, the tumors were mechanically dissociated using a gentleMACS dissociator (Miltenyi Biotec), heated in a water bath at 42°C for 15 minutes, and then digested using a tumor dissociation kit (Miltenyi Biotec) according to the manufacturer's instructions. After digestion, the tumors were washed with PBS and passed through 70-μm and 30-μm filters, and then CD45 was depleted using CD45 microbeads (Miltenyi Biotec). + Cells. Tumor cells were packaged at 5x10... 6 Cells / ml were resuspended and then lysed by three cycles of freeze-thaw. WTL preparations were centrifuged at 12,000 rpm for 15 min, passed through 70 μm and 30 μm filters, and then stored in aliquots at -20°C until use. WTL was prepared at a concentration equivalent to 2.5 x 10⁻⁶ cells per mouse. 5 The concentration of tumor cells is used for the immunization, immunotherapy, or DC as described in the following sections.

[0234] In vivo immunization and tumor challenge: For pre-tumor immunization, C57BL / 6 mice were subcutaneously (sc) injected in the right flank with PBS (unimmunized), WTL alone, WTL with LPS, or WTL plus LPS and OxPAPC or PGPC. In some experiments, MPLA was used instead of LPS. Fifteen days post-immunization, as shown, 3x10 5 One live B16OVA cell, or 3 x 10 5 One B16-F10 cell, or 5 x 10 5 Live MC38-OVA cells were subcutaneously injected into mice in the left flank. As shown, 5 x 10⁸ live cells were injected. 5 One live B16OVA or B16F-10 cell, or 1 x 10 6 Live MC38-OVA cells were subcutaneously injected into tumor-free mice on the upper back. In some experiments, mice were administered 100 μg of LEAF anti-mouse / rat IL-1β antibody (BioLegend) via intravenous injection 2 days and 1 day prior to immunization. Post-immunization, antibody treatment continued for 1, 2, and 3 days to ensure chronic depletion of circulating IL-1β.

[0235] For immunization in the context of immunotherapy, inject 3x10 C57BL / 6J into the left flank. 5 One live B16OVA cell, or 3 x 10 5 One B16-F10 cell, or 5 x 10 55 live MC38-OVA cells. Optionally, BALB / c mice were injected with 5 x 10 cells into the left flank. 5 Live CT26 cells. In the specified post-tumor inoculation scenario, mice were placed in untreated (unimmunized) or immunized with WTL emulsified in incomplete Freund's adjuvant (IFA) plus LPS and PGPC. Two booster injections followed immunization, as shown in the immunization schedule at the top of each survival chart. As indicated, the following antibodies—anti-PD-1 (clone 29F.1A12), Ultra-LEAF anti-CD4 (clone GK1.5), and anti-CD8a (clone 53-6.7)—were administered intraperitoneally (ip) to mice on the same day as immunization or booster injections, followed by a total of four injections every three days. Optionally, mice were administered 100 μg of LEAF anti-mouse / rat IL-1β antibody intravenously 2 days and 1 day prior to immunization / booster injection. Anti-IL-1β treatment was continued on days 1, 2, and 3 post-immunization, as previously mentioned, to ensure chronic depletion of circulating IL-1. Control mice received isotype-matched rat IgG. All antibodies were purchased from BioLegend.

[0236] Every two days, the size of the tumor is assessed using a blinded, coded method and recorded as the tumor area (length × width) with calipers. When the tumor reaches 2cm... 3 Alternatively, euthanize the mouse when it develops an ulcer.

[0237] In vivo immunity and B16-F10 lung colonization: For induction of experimental lung colonization, 3 x 10 μL were injected intravenously via the tail vein at a volume of 100 μL. 5 Two days prior to tumor inoculation, mice were subcutaneously immunized in the right flank with untreated (unimmunized) or WTL alone, or WTL with LPS, or WTL plus LPS and PGPC, all emulsified in Addavax. Five days post-inoculation, mice received a booster injection. Then, on day 18 post-inoculation, mice were sacrificed, lung tissue was dissected and fixed in Fekete solution. Metastatic nodules appearing on the lung surface of each mouse were counted.

[0238] Tumor Infiltration: To assess the frequency of tumor-infiltrating lymphocytes (TILs) in immunized mice, tumors were harvested when they reached a size of 1.8–2 cm. Tumors were dissected using a tumor dissociation kit (Milteny Biotec) and a gentleMACS dissociator, according to the manufacturer's protocol. After digestion, the tumors were washed with PBS and passed through 70-μm and 30-μm filters. CD45 positive selection CD45 microbeads (Milteny Biotec) were used. +Tumor-infiltrating T cells were cultured using dynabeads mouse T-Activator CD3 / CD28 (Gibco) for T-cell activation and expansion. T-cell infiltration was assessed by flow cytometry.

[0239] Adoptive cell transfer: For T cell transfer, anti-CD8 MACS beads and columns (Miltenyi Biotech) were used to isolate CD8 cells from the spleen of surviving mice or from the inguinal adipose tissue of the skin. + T cells. The enriched cells were then sorted into live CD45 cells using FACS ARIA. + CD3 + CD8 + Cells. The purity after sorting was >97%. Then, in the presence of IL-2 (50 ng / ml), cells were cultured in 24-well plates (~2 × 10⁻⁶) coated with anti-CD3 (4 μg / ml) and anti-CD28 (4 μg / ml). 6 Sorted T cells will be stimulated for 24 hours in wells (5 x 10 cells / well). 5 An activated circulating spleen or skin groin fat deposit CD8 + T cells were transferred to naive recipient mice via intravenous or intradermal (id) injection. Some mice received two T cell subsets.

[0240] Statistical analysis: Experiments with more than two groups were statistically significant using a two-way ANOVA with Tukey multiple comparison test correction. Adjusted p-values ​​calculated using Prism (Graphpad) are marked with an asterisk: <0.05 (*); <0.0005 (***); ≤0.0001 (****).

[0241] result

[0242] Overactivation stimuli upregulate several important activities of dendritic cells (DCs) to stimulate T-cell immunity.

[0243] Almost all studies on DC overactivation have focused on the ability of these cells to release IL-1β while maintaining viability. The spectrum of DC function affected by overactivation stimuli is undefined. To examine this spectrum, bone marrow-derived DCs (BMDCs) were sensitized with LPS and subsequently treated with a specific and purified lipid fraction of oxPAPC or oxPAPC called PGPC (I. Zanoni, et al., Science, vol. 352, no. 6290, pp. 1232–1236, 2016). The resulting overactivated cells were compared with conventionally activated BMDCs (treated with LPS) or pyroptotic BMDCs (sensitized with LPS and subsequently treated with alum). Contrary to activating stimuli that were not expected to induce IL-1β release, pyroptotic or overactivation stimuli promoted the release of IL-1β into extracellular mediators (…). Figure 1A All stimuli tested promoted the secretion of the cytokine TNFα. Figure 1A These findings are consistent with previous work that determined LPS-activated BMDCs release TNFα but not IL-1β (I. Zanoni et al., 2016. As assessed by the release of cytoplasmic lactate dehydrogenase (LDH), IL-1β secretion is consistent with cell death in pyroptotic DCs). Figure 1B Conversely, IL-1β occurs in overactivated cells in the absence of LDH release. Figure 1B Similar BMDC behavior was observed when LPS was replaced with MPLA. Figure 5A , 5B The MPLA mentioned is an FDA-approved TLR4 ligand used in vaccines against human papillomavirus (HPV) and hepatitis B virus (HBV). To determine whether the behavior of overactivated BMDCs extends to differentiated DCs in vivo, CD11c isolated from the spleens of mice injected with B16-FLT3 was examined. + DC activity. Similar to the behavior of BMDCs derived from GMCSF, as assessed by LDH release, treatment with LPS and PGPC resulted in the release of TNFα and IL-1β from spleen CD11c cells in the absence of cell death. + DC release ( Figure 5C , 5D These results indicate that the overactivated stimulant PGPC can be used to induce the release of IL-1β from live dendritic cells differentiated in vitro or in vivo.

[0244] Several signals important for T cell differentiation were examined, such as 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 stimuli. Figure 5EConversely, CD40 expression is highly influenced by activating stimuli. Compared to the activating stimulus LPS, overactivating stimuli induce greater CD40 expression. Figure 1C Pyroptosis stimulants are very weak inducers of CD40 and CD69, even in the remaining 20-30% of viable cells after LPS-alum treatment. Figure 1C and 5E When cultured on plates coated with an agonist anti-CD40 agent, differential expression of CD40 was associated with overactivated DCs possessing the greatest capacity to secrete IL-12p70. Figure 1D ).

[0245] As assessed by equivalent internalization of fluorescent ovalbumin (OVA-FITC), overactivated BMDCs are no better than their activated counterparts at capturing antigens. Figure 6A , 6B However, the former cell population showed a greater abundance of OVA-derived SIINFEKL peptides on the MHC-I molecules on the cell surface. Figure 1E and 6C Total surface MHC-I abundance did not differ between activated and hyperactivated cells. Figure 5E In summary, compared with other DC stimuli, the overactivating stimuli exhibited enhanced activity in several activities important for T cell differentiation.

[0246] Overactivated dendritic cells stimulate a TH1-focused immune response, but show no signs of TH2 immunity.

[0247] To assess the impact of DC activation status on T cell-directed responses, BMDCs were treated as described above and then loaded with OVA. These cells were then exposed to naive OT-II or OT-I T cells. OT-II cells expressed T cell receptors (TCRs) specific to MHC-II-restricted OVA peptides (OVA 323-339), while OT-I cells expressed TCRs specific to MHC-I-restricted OVA peptides (OVA 257-264) (KA Hogquist, et al., Cell, vol. 76, no. 1, pp. 17–27, Jan. 1994; MJ Barnden, et al., Immunol. Cell Biol., vol. 76, no. 1, pp. 34–40, Feb. 1998). The activity of responding T cells was assessed by ELISA to determine whether T cell polarization tended toward a TH1 response (IFNγ production) or a TH2 response (IL-10, IL-4, or IL-13 production). Regardless of DC activation state, OVA-treated BMDCs stimulated IFNγ production from OT-II T cells. The level of IFNγ production varied moderately among the activating stimuli tested. Figure 1F Similarly, TNFα production induced by responding OT-II cells was comparable when comparing all DC activation states. These results indicate that TH1 responses are generally induced in vitro regardless of the activation state of antigen-presenting cells (APCs). Conversely, TH2 responses differed significantly when comparing DC activation states. Stimuli inducing BMDC activation (LPS) or pyroptosis (LPS+ alum) promoted the massive release of IL-10 and IL-13; however, over-activation stimuli resulted in 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-restricting transcription factor GATA3, made it possible to calculate the proportions of TH1 and TH2 cells generated by different DC-activating stimuli. This analysis showed that overactivated BMDCs induced a strong shift of individual T cells toward the IFNγ-producing TH1 lineage. Figure 1G and 7 Under conditions of overactivation, the ratio of TH1 to TH2 cells is greater than 100:1. Figure 1G Conversely, all other activating stimuli induce mixed T cell responses, with pyroptosis stimuli resulting in a near 1:1 ratio of TH1 to TH2 cells. Figure 1G ).

[0248] Use CD8 +Similar studies were performed on OT-1 T cells, showing that stimuli that over-activate BMDCs resulted in a slight increase in IFNγ production compared to stimuli that activate or cause pyroptosis. Figure 1F When comparing all DC activation states, IL-2 production induced by OT-I responding cells was comparable. Figure 1F These combined results indicate that, in vitro, an overactivated BMDC state leads to a highly TH1-biased T cell response and a slightly enhanced CD8 T cell response. Conversely, activation or pyroptosis stimuli produce a mixed TH1 and TH2 response.

[0249] To determine whether in vitro observations using overactivated BMDCs and OT-I and OT-II T cells were applicable to in vivo endogenous settings, the ability of the overactivation stimulant to promote antigen-specific T cell responses in vivo was examined. Mice were immunized with OVA alone, or OVA and an activating stimulant (LPS), or OVA and an overactivation stimulant (LPS + oxPAPC or PGPC), or a pyroptosis stimulant (LPS + alum, or alum alone). Forty days post-immunization, CD4 cells were restimulated in vitro with naive BMDCs loaded (or unloaded) with OVA. + or CD8 + T cells. Immunization with overactive stimuli results in a greater amount of IFNγ produced by responding T cells than immunity with LPS or pyroptosis stimuli (LPS+alum). Figure 1H Note that T cells from mice immunized with overactive stimulants do not produce IL-10, IL-4, or IL-13. Figure 1H and 7 Therefore, similar to in vitro observations using transgenic T cells, overactivation stimulants can induce TH1-biased responses and CD8+ responses from endogenous T cells in vivo. + T cell response.

[0250] Contrary to the findings with overactivating stimulants, activating stimulants (LPS) resulted in a mixed TH1 and TH2 phenotype, in which T cells produced IFNγ, IL-10, IL-4, and IL-13 ( Figure 1H and 7 Immunization with OVA and alum alone resulted in a highly skewed IL-13 response, with IFNγ not detectable by responding T cells. Figure 1HThese results are consistent with previous studies that showed alum was particularly poor at promoting type I immunity (E. Oleszycka, et al., Eur. J. Immunol., vol. 48, no. 4, pp. 705–715, Apr. 2018; MJ Newman, et al., J. Immunol., vol. 148, no. 8, pp. 2357–62, Apr. 1992) and that alum caused a TH2-biased immune response (M. Kool, et al., J. Exp. Med., vol. 205, no. 4, pp. 869–882, Apr. 2008; T. Marichal, et al., Nat. Med., vol. 17, no. 8, pp. 996–1002, Aug. 2011).

[0251] As mentioned above, one difference between LPS and LPS+oxPAPC is the latter's ability to induce IL-1β secretion. Treatment with alum and oxPAPC leads to NLRP3-dependent release of IL-1β (L. Franchi and G.). Eur. J. Immunol., vol. 38, no. 8, pp. 2085–9, Aug. 2008; H. Li, et al. J. Immunol., vol. 178, no. 8, pp. 5271–6, Apr. 2007), thus questioning whether LPS+alum immunity can phenotype a T-cell response induced by LPS+oxPAPC or PGPC immunity. The answer to this question is no; LPS+alum immunity leads to a balanced TH1:TH2 response, in which ex vivo stimulated T cells produce large amounts of IFNγ, IL-10, IL-4, and IL-13 ( Figure 1H and 7 These results indicate that not all NLRP3 agonists stimulate the TH2-focused immune response.

[0252] Overactivating stimulants enhance memory T cell production and strengthen antigen-specific IFNγ effector responses in an NLRP3-dependent manner.

[0253] It is hypothesized that the enhanced T cell response induced by hyperactivating stimuli can be explained by enhanced memory T cell production and effector memory T cell responses. To test this possibility, mice were immunized with OVA alone, OVA plus activating stimuli (LPS), or OVA plus hyperactivating stimuli (LPS + oxPAPC or PGPC). At 7 and 40 days post-immunization, mice were analyzed by flow cytometry using differential T effector cells (Teff) such as CD44. low CD62L low T-effect memory cells (TEMs) such as CD44hi CD62L low and T central memory cells (TCMs) such as CD44 hi CD62L hi CD62L and CD44 markers were used to assess memory and effector T cell production in dLN (SZBen-Sasson, Cold Spring Harb. Symp. Quant. Biol., vol. 78, no. 0, pp. 117–124, Jan. 2013). Seven days post-immunization, overactivating stimulants induced CD44 + and CD8 + Teff cells are superior to activating stimulants ( Figure 2A The above figure and figures 8A and 8B). At this early time point, both stimuli induced a low but considerable number of TEM cells ( Figure 2A (Intermediate figure and 8A, 8B). Forty days post-immunization, abundant TCM cells were observed in mice exposed to the overactivating stimulant, while these cells were less abundant in mice immunized with OVA alone or with OVA and LPS. Figure 2A (See figure below). These data indicate the extent to which the overactivating stimulants oxPAPC and PGPC enhance the effects and production of memory T cells. The increased frequency of Teff cells 7 days post-immunization is correlated with CD4+. + and CD8 + The enhanced IFNγ response of T cells is associated with CD4. + and CD8 + T cells were isolated from dLNs of immunized mice and restimulated in vitro in the presence of naive BMDCs loaded with OVA. Figure 8C , 8D Similarly, CD8 + T cells exhibit enhanced degranulation ability, an activity associated with functional CTLs. Figure 2B When the total CD8 + When T cells were isolated from mice immunized with an overactive activator and co-cultured with the B16 tumor cell line expressing OVA (B16OVA), the CD8+ cells... + T cells and CD8 cells isolated from mice immunized with OVA alone or OVA plus LPS + T cells showed enhanced degranulation activity compared to T cells. Figure 2B , 8E ).

[0254] To compare the antigen specificity of T cells induced by immunization with activating stimulants, pyroptosis stimulants, or hyperactivating stimulants, mice were injected with OVA alone or with activating stimulants (LPS), pyroptosis stimulants (LPS + alum), or hyperactivating stimulants (LPS + oxPAPC or PGPC). Optionally, mice were immunized with LPS + PGPC without the OVA antigen. Seven days post-immunization, CD4 counts... + and CD8 + T cells were isolated from dLN skin cells of immunized mice and restimulated in vitro for 7 days with naive BMDCs loaded (or unloaded) with OVA to enrich OVA-specific T cell subsets. T cell effector function of OVA-specific T cells was assessed by intracellular staining for IFNγ. TCR specificity was assessed by MHC-restricted OVA peptide tetramer staining. H2 was used. kb Restricted SIINFEKL (OVA 257-264) peptides and IA(d)OVA peptides (OVA 329-337) tetramers. For CD4 + and CD8 + T cell subsets were measured for tetramers. + IFNγ + Frequency of double-positive cells. As expected, T cells isolated from mice immunized with LPS+OVA resulted in enrichment of OVA-specific T cells in vitro and induced CD8 compared to mice immunized with OVA alone. + Higher IFNγ effector function in T cells Figure 2C (See above). Similarly, compared to T cells from mice immunized with OVA alone, CD4 cells isolated from mice immunized with LPS+OVA... + T cells exhibit higher OVA-specific IFN+ T cell activity in the presence of high OVA antigen load. Figure 2C (See image below).

[0255] Significantly, OVA and overactivating stimulants (LPS + OxPAPC or PGPC) were superior in inducing antigen-specific T cells, and in restimulating CD4 cells with OVA antigen. + or CD8 + In the case of T cells, this leads to a higher frequency of tetramers. + IFNγ + The generation of response ( Figure 2C Pyroptosis stimulant (LPS+ alum) is the weakest inducer of antigen-specific IFNγ response. Figure 2CThese results are consistent with those described in this paper and elsewhere (MJ Newman, et al., J. Immunol., vol. 148, no. 8, pp. 2357–62, Apr. 1992; JMBrewer, et al., J. Immunol., vol. 163, no. 12, pp. 6448–54, Dec. 1999; A. Mori, et al., Eur. J. Immunol., vol. 42, no. 10, pp. 2709–2719, Oct. 2012), indicating that alum is an effective adjuvant that promotes humoral immunity and TH2 responses but not TH1 or CTL responses.

[0256] Previous studies have shown that recombinant IL-1β enhances antigen-specific T cell responses during immunization and increases the capacity of attenuated vaccines (SZBen-Sasson, K. et al. Cold Spring Harb. Symp. Quant. Biol., vol. 78, no. 0, pp. 117–124, Jan. 2013; SZBen-Sasson, et al. J. Exp. Med., vol. 210, no. 3, pp. 491–502, Mar. 2013). To determine whether antigen-specific T cell responses induced by overactive stimulants depend on the inflammasome-IL-1β axis, WT and NLRP3 were performed. - / - Parallel comparison of T cell activity in mice immunized with an overactivating stimulant. Compared with WT mice, the overactivation condition at NLRP3... - / - Tetramer reduced by induction + IFNγ + answer( Figure 2C These results reveal that under conditions of overactivation, NLRP3 activation is crucial for antigen-specific T cell production and effector function.

[0257] In recent studies of viral infection, white adipose tissue has been shown to constitute a reservoir of antigen-specific memory T cells that persist for months after antigen contraction (S.-J. Han, et al., Immunity, vol. 47, no. 6, p. 1154–1168.e6, 2017). To test the ability of different DC-activating stimuli to induce memory T cells in subcutaneous adipose tissue, mice were subcutaneously immunized with OVA and different activating stimuli as previously described. Eight days post-immunization, CD44 in the inguinal adipose tissue of the skin was assessed by flow cytometry. + The presence of memory T cells. Interestingly, overactivation conditions induced a higher frequency of CD44 cells compared to activating stimuli. + Memory T cells ( Figure 2DNote that T cells were not detected in the fat compartment when mice were immunized with OVA or pyroptosis stimulants. Figure 2D Furthermore, memory T cell accumulation depends on the activation of the NLRP3 inflammasome, because NLRP3... - / - Decreased CD44 in mice + Frequency of memory T cells ( Figure 2D In summary, these results indicate that memory T cells generated by overactivating stimuli are not limited to the skin dLNs of immunized mice, but also induce memory T cells in the subcutaneous adipose tissue compartment.

[0258] In summary, these data provide evidence that overactivating stimuli produce a large amount of functional memory CD4. + and CD8 + Evidence of T cells, and a strong bias towards TH1 response.

[0259] Overactivated dendritic cells (DCs) can utilize complex antigen sources to stimulate T cell-mediated antitumor immunity.

[0260] Based on the above findings, it is inferred that overactivating stimulants could be particularly useful in strategies where immunity is difficult to achieve clinical (or preclinical) benefits. One aspect of interest relates to cancer immunotherapy. Current efforts to stimulate antitumor immunity include strategies to activate resident T cell populations (e.g., PD-1 blockade) or personalized cancer vaccine strategies (REF) to stimulate the generation of nascent T cell responses to tumor-specific antigens (TSAs). The latter effort is hampered by the inability to use tumor cell lysates as a source of TSAs, also known as neoantigens. Therefore, efforts are being made to improve the identification of neoantigens that can be used in their pure form to elicit T cell-mediated antitumor immunity. Despite the success of these efforts (DB Keskin, et al., Nature, vol. 565, no. 7738, pp. 234–239, Jan. 2019; Z. Hu, PAOtt, and CJ Wu, Nat. Rev. Immunol., vol. 18, no. 3, pp. 168–182, Dec. 2017; PAOtt, et al., Nature, vol. 547, no. 7662, pp. 217–221, 2017), the pathway to neoantigen identification requires channels for the discovery of mutated and aberrantly expressed TSAs (CMLaumont, et al., Sci. Transl. Med., vol. 10, no. 470, p. eaau5516, Dec. 2018), which is laborious and does not represent the natural course of events. Naturally, DCs never encounter pure antigens but can stimulate T cell responses in complex environments.

[0261] There is reason to believe that, since overactivated dendritic cells (DCs) are excellent stimulants for T cell-mediated responses, overactivation stimuli could allow us to bypass the need for neoantigen identification and permit the use of whole tumor cell lysates (WTLs) as an antigen source. WTLs are an attractive alternative source of antigens because these lysates provide a spectrum of mutated and aberrantly expressed TSAs and can generate a broad T cell repertoire specific for tumor-associated antigens.

[0262] To address the possibility that overactivating stimuli could assist WTL, mice were immunized in the right flank with WTL alone, or with a mixture of WTL and the activating stimulant LPS or the overactivating stimulants LPS+oxPAPC or LPS+PGPC. The WTL was derived from B16 melanoma cells expressing OVA (B16OVA). Fifteen days post-immunization, mice were challenged subcutaneously (sc) in the left upper back with parental B16OVA cells. Unimmunized mice or mice immunized with WTL alone showed no protection, and all mice carried large tumors and died on day 24 post-tumor inoculation. Figure 3A Similarly, WTL+LPS immunization provided minimal protection. Two of the eight mice immunized with WTL+LPS were tumor-free, but rapidly relapsed after B16OVA re-challenge. Figure 3A This indicates that stimuli that only activate DCs do not confer protective immunity. Conversely, WTL immunization in the presence of LPS and oxPAPC significantly delayed tumor growth induction and resulted in strong protection against subsequent lethal re-attacks with parental B16OVA tumor cells; 50% of immunized mice were completely protected. Figure 3A and 9A ).

[0263] To determine whether the protective response induced by the overactivating stimuli was related to T cell responses, tumors were harvested from mice immunized with the various activating stimuli. Tumors from mice immunized with the overactivating stimuli contained abundant CD4+ and CD8+ T cells. Figure 3B Under anti-CD3 and anti-CD28 stimulation, T cells enriched from these tumors secrete large amounts of IFNγ (…). Figure 3B Therefore, better restriction of tumor growth induced by overactive stimuli (LPS+oxPAPC) is consistent with inflammatory T cell infiltration into the tumor.

[0264] Note that the protective phenotype of oxPAPC is replaced by that induced by the pure oxPAPC component PGPC. WTL immunization in the presence of LPS+PGPC resulted in 100% of mice being tumor-free 150 days after tumor challenge. These mice completely rejected lethal re-challenge with B16OVA cells and remained tumor-free 300 days after the initial tumor challenge. Figure 3Aand 9A ).

[0265] Within the memory T cell subset, the T-resident memory cell (TRM) is defined by the expression of CD103 integrin and C-type lectin CD69, which contributes to their residence characteristics (REF) in peripheral tissues. CD8 + TRM cells have recently gained considerable attention because these cells accumulate at tumor sites in various human cancer tissues and are associated with more favorable clinical outcomes (JR Webb, et al. Clin. Cancer Res., vol. 20, no. 2, pp. 434–444, Jan. 2014; F. Djenidi, et al. J. Immunol., vol. 194, no. 7, pp. 3475–86, Apr. 2015; SLPark, et al. Nature, vol. 565, no. 7739, pp. 366–371, Jan. 2019). In experimental cutaneous melanoma models, CD8+ cells in the skin... + TRM cells promote durable protection against melanoma development.

[0266] The presence of TRM cells at the tumor injection site was examined, as well as immunohistochemical biopsy of surviving mice previously immunized with the overactivating stimulant LPS+PGPC. Interestingly, 200 days post-tumor inoculation, CD8+ cells were present in all surviving mice. + CD69 + CD103 + TRM cells are highly enriched at the tumor injection site, but insufficient at the immune site. Figure 3C and 10A These data are consistent with clinical and trial reports that have associated the presence of high levels of TRM with long-term tumor control, where TRM may be maintained long-term to investigate tumor injection sites (SLPark, et al., Nature, vol. 565, no. 7739, pp. 366–371, Jan. 2019; CMKoebel, et al., Nature, vol. 450, no. 7171, pp. 903–907, Dec. 2007).

[0267] CD8 + TRMs accumulate in white adipose tissue after antigen contraction and migrate to the site of infection upon a second challenge (S.-J. Han et al., Immunity, vol. 47, no. 6, p. 1154–1168.e6, 2017). T cell compartments in the subcutaneous adipose tissue surrounding the inguinal lymph nodes (LN) at the site of immune drainage were analyzed. High frequencies of CD8+ cells were observed. +T cells, and antigen-specific CD8 in adipose tissue of mice immunized with WTL and the overactivating stimulant LPS+PGPC. + TRM cells ( Figure 3D Left image and 10B, 10C). Conversely, fewer TRM cells were observed in the inguinal adipose tissue of unimmunized mice. Figure 3D (Left figure and 10B, 10C). In addition, compared with the unimmunized counterpart, circulating memory T cells from the spleen of surviving mice immunized with WTL and the overactivating stimulant LPS+PGPC contained a large number of OVA-specific T cells.

[0268] To examine the functional specificity of these T cells, we monitored the activity of cytotoxic lymphocytes (CTLs) in vitro. Circulating memory CD8 + T cells and TRM cells were isolated from the spleen or skin adipose tissue of surviving mice previously treated with an overstimulatory agent. These cells were cultured using B16OVA cells, or B16 cells that do not express OVA, or the unrelated cancer cell line CT26. As assessed by LDH release, they were found to be present only on CD8 cells. + CTL activity was observed when T cells were mixed with B16OVA or B16 cells. Figure 3E No killing effect on CT26 cells was observed. Figure 3E This demonstrates the function and antigen specificity of the T cell response induced by overactivation.

[0269] Based on antigen-specific T cell responses induced by overactivating stimuli, it was determined whether T cells were sufficient to protect against tumor progression. CD8+ T cells were transferred from surviving mice to juvenile mice, which were then challenged by parental tumor cell lines used as initial immunogens. + TRM or loop CD8 + Transfer of T cells from surviving mice to naive recipients confers strong protection against subsequent tumor attack, with TRM subsets playing a major protective role. Figure 3F One week prior to tumor inoculation, two T-cell subsets were transferred from surviving mice to juvenile mice to provide 100% protection to the recipient mice from subsequent tumor attack. Figure 3F These combined data indicate that PGPC is a major bioactive hyperactivator, which exerts its effects through inducing potent cycling and retention of antitumor CD8. + T-cell responses provide optimal protection in the B16 melanoma model.

[0270] To determine whether the benefits of PGPC-based overactivation stimuli extend to other mouse models of cancer, similar experiments were performed using parental B16-F10 melanoma cells. Unlike B16OVA melanoma, which contains a large number of neoantigens in the form of OVA (with a few exceptions, JCCastle et al., Cancer Res., vol.72, no.5, pp.1081–91, Mar. 2012; MOMohsen et al., Front. Immunol., vol.10, p.1015, 2019), the tumor-specific antigens present in B16-F10 cells are less clearly defined. Therefore, B16-F10 cells represent a common clinical situation where neoantigen identification is rare. Note that the overactivation strategy based on PGPC induces protection against B16-F10 tumor growth (…). Figure 9B ).

[0271] Similar findings emerged when we replaced LPS+PGPC with MPLA+PGPC; 100% of mice immunized in this manner remained tumor-free 90 days after tumor challenge, and 75% of the mice rejected lethal re-challenge with B16OVA cells. Figure 3G These data suggest that, unlike activating stimuli, hyperactivating stimuli can assist complex antigen mixtures (e.g., WTL) in promoting durable and protective antitumor immunity.

[0272] Overactivating stimuli induce inflammasome-dependent antitumor immunity. A typical property of overactivating stimuli is their ability to induce the secretion of IL-1β from living cells. To determine whether IL-1β and its upstream inflammasome regulators are important for the antitumor response, several procedures were performed. First, immunization was performed as described above, except for an intravenous (iv) injection of neutralizing anti-IL-1β antibodies 2 days prior to immunization, followed by three consecutive injections on days 0, 1, and 2 post-immunization to ensure chronic depletion of IL-1β. Fifteen days post-immunization, mice were challenged with parental tumor cells. Neutralizing IL-1β completely eliminated the protection against tumor growth in mice in the B16OVA melanoma model. Figure 3G and 9C Similar to the phenotype observed in the case of IL-1β neutralization, when in NLRP3 - / - Casp1 - / - / 11 - / - Or Casp11 - / - When mice are immunized, overactivating stimulants do not confer protection against tumor attack. Figure 3H Similar results were obtained using a different cancer model—the MC38 colon adenocarcinoma model. Figure 3IThe data demonstrate the ability of IL-1β to neutralize and eliminate overactivation stimuli against attack by the MC38 lineage expressing OVA (MC38OVA). These functional data establish a link between the in vitro activity of overactivation stimuli and their in vivo T-cell-promoting activity. Note that not all NLRP3 agonists confer antitumor immunity; for example, LPS+alum does not assist WTLs in protecting against tumor growth (Figure 10E). These results highlight the importance of overactivated DCs (not pyroptotic DCs) in the induction of antitumor immunity.

[0273] Overactivating stimuli can assist WTLs or neoantigens in inducing antitumor immunity. The ability of overactivating stimuli to induce protective immunity against a mixture of complex antigens raises the question of how these protective responses compare to immunization with pure neoantigens. To address this question, parallel immunizations were performed with pure OVA or OVA present in tumor lysates. Mice were injected with WTLs from MC38OVA cells in or without the activating stimuli MPLA or the overactivating stimuli MPLA+PGPC. These injections were compared to those in which pure OVA was used as the antigen instead of WTLs. Fifteen days post-immunization, mice from each group were blindly divided into two sister populations. One population received a challenge with MC38OVA cells, while the other population was sacrificed and dissected to assess CD8. + T cell response ( Figure 11A ). It was found that compared with mice immunized with WTL alone or with MPLA alone ( Figure 11B (Right figure) Overactivating stimuli induce a higher absolute number of infiltrating dLN CD8. + T cells ( Figure 11B (Left image) and a higher number of SIINFEKL-specific CD8s + T cells. CD8 cells isolated from dLNs of mice immunized with an overactivating stimulant. + T cells exhibited the highest degranulation ability when co-cultured with MC38OVA cells. Figure 11C These cells also produced higher levels of IFNγ upon in vitro restimulation with BMDCs loaded with OVA. Figure 11D In addition, CD8 stimulated by PMA and ionomycin. + Single-cell analysis of intracellular cytokine staining in T cells revealed CD8+ in mice immunized with overactive stimulants. + The T cell compartment contains highly pluripotent T cells. These CD8 cells... + T cells can simultaneously produce multiple cytokines, including IL-2, TNFα, and IFNγ. Figure 11E ).

[0274] Enhanced T cell activity in mice immunized with overactive stimulants was associated with survival for more than 150 days after challenge with MC38OVA cells. Figure 3I Note that, compared to immunization using pure OVA, WTL-based immunization conferred superior protection after tumor attack. Figure 3I The inability of a single antigen to provide strong protection against cancer is consistent with recent work demonstrating the value of using multiple neoantigens (up to 20 peptides) in individual cancer vaccines (PAOtt et al., Nature, vol. 547, no. 7662, pp. 217–221, 2017; JC Castle et al., Cancer Res., vol. 72, no. 5, pp. 1081–91, Mar. 2012).

[0275] To determine whether the antitumor response generated during overactivated immunity in the MC38 cancer model was antigen-specific, surviving mice were re-challenged with unrelated tumor cell lines. Surviving mice injected with MC38OVA tumors died rapidly after challenge with B16-F10 cells. Figure 3J Furthermore, in all cases of overactivated antitumor immunity, protection against B16-F10, B16OVA, or MC38OVA cells was conferred only when mice were immunized with the corresponding WTL. Figure 9F These findings highlight the ability of overactivating stimuli to drive antigen-specific antitumor responses, even when the experimenter is unaware (and unable to know) the identity of the neoantigen.

[0276] Overactivating stimulants (OTS) were used to protect against metastasis to the lungs. To determine whether OTS could be used as a cancer immunotherapy, antitumor responses in mice carrying growing tumors prior to any additional treatment were examined. For these studies, instead of cultured tumor cells as an antigen source, ex vivo OTS derived from syngeneic tumors from unimmunized mice was used, in which 10 mm harvested tumors were dissected and then CD45 depleted. + Tumor cells were subcutaneously (sc) injected into the left upper back of mice. When the tumor reached 3-4 mm in size, the tumor-bearing mice were placed in the untreated (unimmunized) position or in the right flank to receive a therapeutic injection consisting of ex vivo WTL and LPS plus PGPC. Two subsequent subcutaneous boosters were administered after the therapeutic injection. Figure 4A Interestingly, these overactivation-based therapeutic injections induced tumor elimination in B16OVA and B16F10 melanoma models, and in MC38OVA and CT26 colon cancer models. Figure 4B-4E In all these models, a high percentage of mice receiving immunotherapy regimens remained tumor-free long-term after tumor inoculation. Figure 4B-4EThe efficacy of immunotherapy in all experimental tumor models depends on IL-1β, because the neutralization and elimination of IL-1β is achieved through the protection conferred by the overactivating stimulant plus in vitro WTL. Figure 4B-4E In addition, CD8 + T cells are crucial for protection against immunogenic tumor models such as B16OVA or MC38OVA tumors; however, CD4... + and CD8 + Both T cells are necessary for protection against smaller immunogenic tumors, such as CT26 and B16F-10 tumors. Figure 4B-4E ).

[0277] To determine the efficacy comparison between overactivation-based immunotherapy and PD-1 blockade-based therapy, a parallel evaluation was conducted. Overactivation-based immunotherapy was as effective as anti-PD-1 therapy in the immunogenic B16OVA model, but was more effective in tumor models insensitive to anti-PD-1 therapy, such as CT26 and B16F-10 tumors. Figure 4C-4E ).

[0278] Overactivated immunotherapy not only protected mice against subcutaneously transplanted tumors, but in fact, compared with immunization using WTL alone or WTL+LPS, overactivated immunotherapy protected mice from B16 lung metastases. Figure 4F These observations suggest that overactivation-induced antitumor immunity is not limited to local responses but also contributes to protective systemic immunity.

[0279] Overactivation of inflammasome-competent dendritic cells (DCs) is sufficient to confer protective antitumor immunity. Since DCs are the primary cells responsible for stimulating de novo T cell-mediated immunity, it was sought to determine whether conditions of specifically overactivated DCs were sufficient to confer antitumor immunity. This possibility was explored by adoptively transferring BMDCs stimulated in vitro with different activating stimuli and WTLs to mice. BMDCs were chosen because these cells 1) are well-characterized as overactivated and 2) are considered a model of DCs derived from monocytes, the most commonly used APCs for DC-based immunotherapy in humans (RLSabado et al., Cell Res., vol. 27, no. 1, pp. 74–95, Jan. 2017).

[0280] BMDCs were treated with various activating stimuli, along with WTL, and then subcutaneously injected into B16OVA-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 slight protection against B16OVA-induced lethality; 25-30% of mice receiving DC transfer rejected the tumor and remained tumor-free long-term after the last / third DC transfer process. Note that in 100% of tumor-bearing mice, overactivated BMDCs induced complete rejection of B16OVA tumors. In these cells, the antitumor activity of overactivated DCs depended on inflammasomes because of NLRP3. - / - and Casp1 - / - 11 - / - BMDC metastasis induces only a smaller rejection compared to activated DCs. Therefore, these data suggest that overactivated DCs are sufficient to induce durable protective antitumor immunity, and that inflammasomes within DCs are essential for this process.

[0281] discuss

[0282] In this study, the upregulated immunological activity of dendritic cells (DCs) treated with overactivating stimuli was expanded. Not only did these stimuli elicit the release of IL-1β from living cells, but the overactivating stimuli also outperformed other activating stimuli in their ability to induce CD40 expression and IL-12p70 secretion. Furthermore, cells exposed to overactivating stimuli exhibited enhanced surface expression of MHC-peptide complexes. These shared findings highlight the overactivating nature of DCs exposed to oxPAPC or its pure component PGPC and provide evidence of their enhanced ability to stimulate adaptive immunity. It was also found that overactivated DCs actually elicited better T-cell responses than activated or pyroptotic cells, and the most important aspect of their activity was their ability to stimulate TH1- and CTL-focused responses. In fact, stimuli for overactivating DCs resulted in a 100:1 TH1:TH2 cell ratio; other strategies for DC activation do not induce such a biased T-cell response.

[0283] It is noteworthy that the well-defined inflammasome stimulus alum does not exhibit the same activity as oxPAPC or PGPC. In fact, alum is well-known to induce TH2 immunity. These findings were confirmed in this study, as alum or alum+LPS treatment induced strong TH2 immunity. One possible reason for the lack of TH1-focused immunity in alum-treated cells is based on the findings presented in this paper: 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 levels of expression of these factors may render the pyroptosis stimulus a weak inducer of TH1 response and subsequent antitumor immunity. Not wishing to be limited by theory, it is proposed that TH1-focused immunity induced by overactivated DCs is caused by inflammasome activity and several other characteristics of these cells. These additional characteristics include enhanced antigen-presenting capacity, CD40 expression, IL-12p70 expression, and increased activity. Each of these enhanced activities may be important for the function of DCs as APCs and may contribute to the strong TH1-focused immune response observed under conditions of DC overactivation.

[0284] These results help explain why certain chemotherapeutic agents (e.g., oxaliplatin) induce tumor cell death and inflammasome-dependent antitumor T-cell immunity (F. Ghiringhelli et al., Nat. Med., vol. 15, no. 10, pp. 1170–1178, 2009). Oxaliplatin is a strong stimulant of reactive oxygen species (ROS) production, which oxidizes biomembranes and produces a complex mixture of various oxidized phospholipids, including PGPC. Therefore, the protective immunity induced by oxaliplatin may be caused by the activity of overactivated dendritic cells (DCs) that trigger an antitumor T-cell response.

[0285] This study discovers that overactivated stimuli can be used as a complex mixture of antigens for immunotherapy. WTLs are an attractive antigen source for several reasons, the most important being from a practical standpoint. A significant benefit of WTL-based approaches is their reduction of the need for neoantigen recognition. While WTL-based immunotherapies offer potential benefits, previous work in this field has yielded mixed results. The finding that overactivated stimuli uniquely assist WTLs in evoking strong anti-tumor immunity explains the lack of success in previous work, as the DC activation strategy discovered in this paper had not been considered before. Notably, the DC overactivation strategy protected mice from lethality associated with both PD-1-sensitive and PD-1-resistant tumors. The full spectrum of tumors treatable with overactivated stimuli has not yet been determined, but these studies provide indications of the value of further exploring DC-centric strategies in cancer immunotherapy.

[0286] Example 2: cDC1 control of tumor rejection induced by over-activated immunotherapy

[0287] Based on our previous finding that overactivated DCs are excellent stimulants for antigen-specific Th1 and CTL responses, we hypothesized that overactivated stimulants could be used as an immunotherapy strategy for cancer. To test the efficacy of overactivated stimulant injections against tumor immunity, tumor-bearing mice (carrying a 4 mm tumor in the right flank) received subcutaneous injections of whole tumor lysate (WTL) and the overactivated stimulant LPS+PGPC in the left flank. Two subsequent subcutaneous booster injections of WTL and LPS+PGPC were performed. Based on the fact that overactivated injections induced tumor rejection in B16OVA, and that a high percentage of WT mice receiving the immunotherapy regimen remained tumor-free (for more than 40 days) after tumor inoculation, the protection induced by overactivated DCs was dependent on cDC1 cells, as were Batf3 cells in the immunization regimen. - / - In mice lacking cDC1 cells, tumor control could not be induced. Furthermore, tumor-specific CD4 cells exhibited a high frequency. + and CD8 + In contrast to WT mice, which showed T-cell infiltration at the tumor injection site, Batf3... - / - The mice lacked OVA-specific CD8+ T cells and exhibited a low frequency of OVA-specific CD4+ cells in the tumor microenvironment. + T cells. In summary, these data suggest that: 1) the overactivation stimulant (LPS+PGPC) uniquely assists WTLs in triggering potent antitumor immunity, and 2) cDC1 cells are the primary antigen-presenting cells that induce overactivation-induced antitumor immunity in vivo.

[0288] Example 3: Oxidized phospholipids induce overactivated cDC1 and cDC2 cells

[0289] Almost all studies assessing the state of cell-over-activation have focused on the ability of bone marrow-derived dendritic cells (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 state of over-activation, we used BMDCs generated using DC-derived erythropoietin Fms-like tyrosine kinase 3 ligand (Flt3L). To assess over-activation, FLT3-DCs were sensitized with LPS and then treated with a pure lipid fraction of oxidized phospholipid oxPAPC or oxPAPC called PGPC

[36] . Alternatively, FLT3-DCs were stimulated with conventional activating stimuli such as LPS alone, or FLT3-DCs were sensitized with LPS and then treated with pyroptosis stimuli such as alum. Unlike conventional activating stimuli that do not induce the release of IL-1β from dendritic cells (DCs), pyroptosis-induced DCs promote the release of IL-1β into extracellular mediators. Figure 14A As assessed by the release of the cytoplasmic enzyme lactate dehydrogenase (LDH), IL-1β secretion is consistent with cell death in pyroptotic dendritic cells (DCs). Figure 14B Interestingly, stimulation with the overactivating stimulant LPS+PGPC, or with a smaller degree of stimulation with LPS+oxPAPC, induced the secretion of IL-1β from DCs, which occurred in the absence of LDH release. Figure 14A All DCs sensitized or stimulated with LPS promote the secretion of the cytokine TNFα. Figure 14A In both cases, IL-1β secretion in pyroptotic or overactivated dendritic cells depends on inflammasome components NLRP3 and caspase 1 / 11. Figure 14A These findings are consistent with previous work defining the underlying mechanism of the hyperactivated state of DCs, in which oxPAPC binds to and stimulates cytoplasmic PRR caspase-11, leading to NLRP3 activation and assembly of non-pyroptotic inflammasomes, which results in the release of IL-1β from living cells

[24] . Similar behavior of DCs was observed when they were sensitized with other TLR agonists, such as the TLR9 agonist CpG. Figure 19ATherefore, these data suggest that FLT3 DCs can achieve an overactivated state. DCs are divided into two main subgroups, called cDC1 and cDC2. cDC1 is a classic DC that can cross-present tumor-associated antigens and sensitize CD8+ T cells

[37] ,

[38] . On the other hand, cDC2 controls 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 spleen of wild-type juvenile mice. Figure 19B Similar to the behavior of FLT3-derived DCs, as assessed by LDH release, treatment with LPS and PGPC, and treatment with LPS and oxPAPG to a lesser extent, resulted in the release of TNFα and IL-1β from FLT3-derived cDC1 and cDC2 in the absence of cell death. Figure 14A These data indicate that PGPC is the bioactive component of oxPAPC that induces overactivation of cDC1 and cDC2. We also observed similar behavior in spleen cDC2, which responded to the pyroptosis stimuli LPS and alum to produce IL-1β accompanied by pyroptosis and cell death, and also responded to the overactivation stimuli LPS and PGPC to produce IL-1β in the absence of cell death. Figure 19C Conversely, splenic cDC1 cells respond to pyroptosis or hyperactivation stimuli by producing very small amounts of IL-1β because these cells are highly sensitive to post-sorting cell death and cannot be sensitized by LPS. Figure 19C In summary, these results suggest that overactivating stimuli can be used to induce the release of IL-1β from live dendritic cells differentiated in vitro or in vivo. For practical reasons, we continue to use FLT3-derived dendritic cells as the source of dendritic cells in this paper.

[0290] Example 4: Overactivated DCs enhance CTL responses in an inflammasome-dependent manner

[0291] IL-1β is a key regulator of T cell differentiation, long-lived memory T cell generation, and effector function

[12] –

[14] . We wanted to know whether overactivated DCs that produce IL-1β over a period of several days in the dLN could enhance CD8+ T cell stimulation. To test this idea, we attempted subcutaneous (sc) adoptive transfer of DCs loaded with OVA protein and then measured OVA-specific CD8+ T cells in the dLN. First, we tested the ability of different DC states to take up 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 degree, as confirmed by internalization of the fluorescent ovalbumin (OVA-FITC) equivalent. However, we found that both activated and overactivated DCs sensitized with LPS or CpG showed enhanced SIINFEKL cross-presentation when loaded with OVA protein 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 capacity of DCs, indicating that pyroptotic DCs are not optimal for T cell stimulation. Therefore, when 1,106 DCs loaded with naive, activated, pyroptotic, or hyperactivated DCs from OVA were injected into WT mice, we observed that hyperactivated DCs induced the highest frequency and absolute number of SIINFEKL+CD8+ T cells in the recipient mouse dLN compared to naive, activated, or pyroptotic DCs. Figure 15A and 20B Since injection of NLRP3- / - DCs treated with LPS+PGPC induces a weak OVA-specific T cell response, the enhanced CD8+ T cell response mediated by overactivated DCs depends on inflammasome activation.

[0292] Example 5: Overactivating stimuli enhance memory T cell production and strengthen antigen-specific IFNγ effector responses in an inflammasome-dependent manner.

[0293] We hypothesized that the overactivation stimulant could represent a potent adjuvant that summarizes the effects of overactivated DC injection. To test this possibility, mice were subcutaneously immunized with OVA alone, or OVA plus an activating stimulant (LPS), or OVA plus an overactivation stimulant (LPS + oxPAPC or PGPC). At 7 and 40 days post-immunization, the production of memory and effector T cells in the dLN was assessed by flow cytometry using CD44 and CD62L markers that differentiate T effector cells (Teff) such as CD44lowCD62Llow, T effector memory cells (TEM) such as CD44hiCD62Llow, and T central memory cells (TCM) such as CD44hiCD62Lhi

[47] . At 7 days post-immunization, the overactivation stimulant was superior to the activating stimulant in inducing CD8+ Teff cells. Figure 16A The above image and Figures 21A-21B Furthermore, at this early time point, the overactivating stimulant induced the highest abundance of CD8+ TEM (…). Figure 16A Intermediate image and Figures 21A-21B Forty days post-immunization, abundant TCM cells were observed in mice exposed to the overactivating stimulant; however, these cells were less abundant in mice immunized with OVA alone or with OVA and LPS. Figure 16A (See figure below). Conversely, at 40 days post-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 LPS. Therefore, these data suggest that the overactivating stimulants oxPAPC and PGPC enhance the effects and the extent of memory T cell production. Furthermore, when restimulated in vitro in the presence of naive BMDCs loaded with OVA, the increased frequency of Teff cells at 7 days post-immunization was associated with an enhanced IFNγ response in CD8+ T cells isolated from dLNs of mice immunized with OVA plus overactivating stimulants. Figure 21C Furthermore, when all CD8+ T cells were isolated from mice immunized with an overactive stimulant and co-cultured with the B16 tumor cell line expressing OVA (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 16B and 21D This indicates that overactivating stimulants enhance CTL function.

[0294] To assess the antigen specificity of T cells induced by subcutaneous immunization with different activating stimuli, mice were injected with OVA alone, or OVA and an activating stimulant (LPS), or OVA and a pyroptosis stimulant (LPS + alum), or OVA and an overactivating stimulant (LPS + oxPAPC or PGPC). Optionally, mice were subcutaneously immunized with LPS + PGPC without the OVA antigen. Seven days post-immunization, CD8+ T cells were isolated from the skin dLN of immunized mice and restimulated for 7 days in vitro with naive BMDCs loaded (or unloaded) with OVA to enrich OVA-specific T cell subsets. T cell effector function of OVA-specific T cells was assessed by intracellular staining for IFNγ. TCR specificity was assessed by staining for MHC-restricted OVA peptide tetramers. H2kb-restricted SIINFEKL (OVA 257-264) peptide tetramers were used. The frequency of tetramer + IFNγ+ double-positive cells was measured for both CD4+ and CD8+ T cell subsets. Significantly, OVA and overactivating stimulants were superior in inducing antigen-specific T cells because, upon restimulation of CD8+ T cells with OVA antigen, immunization based on oxPAPC or PGPC resulted in the highest frequency of tetramer + IFNγ+ responses. Figure 16C Conversely, pyroptosis stimulants (LPS+ alum) are the weakest inducers of antigen-specific IFNγ responses. Figure 16C These results are consistent with previous studies that have shown alum to be an effective adjuvant for promoting humoral immunity and Th2 responses, but not Th1 or CTL responses [39,42,43].

[0295] Previous studies have shown that co-immunization with recombinant IL-1β enhances antigen-specific T cell responses [47,13]. IL-1β is a cytokine whose biological activity is naturally controlled by inflammasomes. However, despite the fact that both hyperactivation and pyroptosis stimuli induce IL-1β secretion, how do hyperactivation stimuli, rather than pyroptosis stimuli, induce higher antigen-specific T cell activity? To determine whether inflammasome-mediated events control T cell responses induced by hyperactivation stimuli, a parallel comparison of T cell activity was performed in WT and NLRP3- / - mice. Note that we found that the enhancement of antigen-specific responses induced by hyperactivation of CD8+ T cells required NLRP3 ( Figure 16C Therefore, these data indicate that immunization with either overactivating stimuli or pyroptosis stimuli induces significantly different adaptive immune T cell control, resulting in activation of non-pyroptosis and pyroptosis inflammasomes.

[0296] Our previous results using a DC injection strategy showed that DCs stimulated with pyroptosis stimulants lost their ability to migrate to neighboring dLNs and stimulate T cell activation, while DCs exposed to overactivation stimulants over-migrated to dLNs and enhanced CTL responses. Figures 15A-15B However, it is unknown whether endogenous DCs can achieve hyperactivation in vivo after immunization with an overactivating stimulant. 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 purpose, on a CD45.2 background as previously described, 4-week-old CD45.1-irradiated mice were remodeled with a mixture of 80% Zbtb46DTR and 20% WT mice, or 20% NLRP3- / - or 20% Casp1 / 11- / - mice

[51] . Six weeks after remodeling, the efficacy of BM remodeling in all mice was evaluated by flow cytometry using cD45.1 and CD45.2 markers. Chimeric mice were treated with diphtheria toxin (DT) every other day to deplete Zbtb46+ conventional DCs, resulting in mice carrying either overactivated WT or inflammasome-deficient (NLRP3- / - or Casp1 / 11- / -) DCs. To test the effect of endogenous DC overactivation on CD8+ T cell responses, all chimeric mice were subcutaneously immunized with OVA plus LPS+PGPC after three consecutive DT injections. Seven days post-immunization, CD8+ T cell responses from dLNs were assessed. Interestingly, we found that the abundance of Teff CD8+ T cells was significantly reduced in chimeric mice carrying non-overactivated DCs, such as NLRP3- / - and Casp1 / 11- / - chimeric mice, compared to chimeric mice carrying overactivated WT DCs. Figure 16D , Figure 22A Furthermore, we found that the frequency of SIINFEKL+CD8+ T cells in the dLN or spleen was decreased in NLRP3- / - and Casp1 / 11- / - chimeric mice carrying non-overactivated DCs, while high SIINFEKL+CD8+ cell counts were observed in chimeras carrying WT DCs. Figure 16E , Figure 22B Therefore, these data clearly show that: 1) endogenous DCs can achieve a state of hyperactivation in vivo and enhance CTL responses after immunization with hyperactivating stimuli, and 2) activation of inflammasomes within endogenous DCs is crucial for hyperactivation-mediated protective CTL responses.

[0297] Example 6: Overactivated DCs entering lymphoid tissues are essential for overactivated CTL responses.

[0298] We previously showed that DCs stimulated with hyperactivating stimuli migrate excessively to the dLN and enhance CTL responses. Figures 15A-15B To assess whether overactivation-mediated CTL responses require endogenously overactivated DCs to enter the dLN, we generated mouse chimeras using Zbtb46DTR and WT or Zbtb46DTR and CCR7- / -BM as described above (Fig. 15D). In summary, endogenous delivery of overactivated DCs to the dLN is essential for overactivation-mediated CTL function.

[0299] Example 7: Overactivation stimulants can be derived from complex antigens to stimulate pyroptosis-mediated T cell-mediated antitumor immunity.

[0300] Current efforts to stimulate antitumor immunity include strategies to activate resident T cell populations (e.g., PD-1 blockade) or personalized cancer vaccine strategies to stimulate the generation of nascent 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. Therefore, efforts have been made to improve the identification of neoantigens that can be used in their pure form to elicit T cell-mediated antitumor immunity. Despite the success of these efforts [50,49,51], pathways to neoantigen identification require channels for the discovery of mutated and aberrantly expressed TSAs

[54] , which is laborious and does not represent a natural progression of events. As previously discussed, WTLs represent an attractive alternative source of antigens because these lysates provide a large number of antigens needed to elicit personalized antitumor immune responses. However, fundamental questions such as what is the most effective type of adjuvant for cancer vaccines (including the types of adjuvants associated with different types of antigens) remain unresolved.

[0301] To address the possibility that the hyperactivating stimulant could be an adjuvant to WTL, mice were immunized in the right flank with WTL alone, or with a mixture of WTL and the activating stimulant LPS or the hyperactivating stimulant LPS+oxPAPC or LPS+PGPC. The WTL was derived from B16OVA cells. Fifteen days post-immunization, mice were challenged subcutaneously in the left upper back with parental B16OVA cells. Unimmunized mice or mice immunized with WTL alone showed no protection, and all mice carried large tumors and died on day 24 post-tumor inoculation. Figure 23A Similarly, WTL+LPS immunization provided minimal protection. Two of the eight mice immunized with WTL+LPS were tumor-free, but rapidly relapsed after B16OVA re-challenge. Figure 23AThis indicates that stimuli that only activate DCs do not provide protective immunity. Conversely, WTL immunization in the presence of LPS and oxPAPC significantly delayed tumor growth and resulted in strong protection against subsequent lethal re-attacks with parental B16OVA tumor cells; 50% of immunized mice were adequately protected. Figure 23A To determine whether the protective response induced by oxPAPC is related to T cell responses, tumors were harvested from mice receiving various activating stimuli. Tumors from mice immunized with LPS+oxPAPC contained abundant CD4+ and CD8+ T cells compared to LPS immunization (Fig. S7B). Furthermore, when comparing equal numbers of T cells from these tumors, oxPAPC-based immunization resulted in the highest levels of IFNγ-secreting intratumoral T cells under anti-CD3 and anti-CD28 stimulation. Figure 23C Therefore, better limitation of tumor growth induced by overactive stimuli (LPS+oxPAPC) is consistent with the infiltration of inflammatory T cells into the tumor.

[0302] Note that the protective phenotype of oxPAPC is replaced by that induced by the pure oxPAPC component PGPC. WTL immunization in the presence of LPS+PGPC resulted in 100% of mice being tumor-free 150 days after tumor challenge. These mice completely rejected lethal re-challenge with B16OVA cells and remained tumor-free 300 days after the initial tumor challenge. Figure 20A Since these mice never relapsed, we wanted to know how tumor cell growth at the tumor injection site remained controlled in mice immunized with WTL plus LPS+PGPC.

[0303] Among memory T cell subsets, T-resident memory cells (TRMs) are defined by the expression of CD103 integrin along with the C-type lectin CD69, which contributes to their residency characteristics in peripheral tissues

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

[58] .

[0304] We examined the presence of TRM cells at the tumor injection site and in immunized skin biopsies from surviving mice previously immunized with the overactivating stimulant LPS+PGPC. Interestingly, 200 days post-tumor inoculation, CD8+CD69+CD103+ TRM cells were highly enriched at the tumor injection site in all surviving mice, but insufficient at the immunization site. Figures 24A-24BThese data are consistent with clinical and trial reports that associate high levels of TRM with long-term tumor control, where TRM may be maintained long-term to investigate tumor injection sites [56,57]. Therefore, immunization with WTL and hyperactivating stimulants may generate TRM that keeps tumor cells under control.

[0305] To examine the functional specificity of these T cells, we monitored cytotoxic lymphocyte (CTL) activity in vitro. Circulating memory CD8+ T cells and TRM cells were isolated from the spleen or skin adipose tissue of surviving mice previously treated with an overstimulatory agent. 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 24C No killing effect on CT26 cells was observed. Figure 24C This demonstrates the functional and antigen-specific nature of the overactivation-induced T cell response.

[0306] Based on antigen-specific T cell responses induced by overactivating stimuli, we determined whether T cells were sufficient to protect against tumor progression. CD8+ T cells were transferred from surviving mice to naive mice, followed by challenge with parental tumor cell lines used as initial immunogens. Transfer of CD8+ TRMs or circulating CD8+ T cells from surviving mice to naive recipients conferred strong protection against subsequent tumor attack, with TRM subsets playing a major protective role. Figure 24D One week prior to tumor inoculation, two T-cell subsets were transferred from surviving mice to juvenile mice to provide 100% protection to the recipient mice from subsequent tumor attack. Figure 24D These combined data suggest that PGPC-based hyperactivation stimulants provide optimal protection in the B16 melanoma model by inducing strong circulating and resident anti-tumor CD8+ T cell responses.

[0307] Example 8: Protection against established anti-PD1 resistant tumors by overactivating stimuli

[0308] To determine whether hyperactivating stimulants could be used as cancer immunotherapy, we examined antitumor responses in mice carrying growing tumors prior to any additional treatment. For these studies, without using cultured tumor cells as an antigen source, ex vivo WTLs were generated from syngeneic tumors from unimmunized mice, in which 10 mm harvested tumors were dissected and CD45+ cells depleted. Mice were subcutaneously (sc) inoculated with tumor cells in the left upper back. When the tumors reached 3–4 mm in size, tumor-bearing mice were placed untreated (unimmunized) or in the right flank to receive a therapeutic injection consisting of ex vivo WTLs and LPS+PGPC. Two subsequent subcutaneous booster injections were then administered. Figure 17A Interestingly, therapeutic injections based on overactivation induced tumor elimination in many tumor models, such as B16OVA and B16F10 melanoma models, and MC38OVA and CT26 colon cancer models. Figure 17B-17D In all these models, a high percentage of mice receiving immunotherapy regimens remained tumor-free long-term after tumor inoculation. Figure 17B-17D In all tested tumor models, the efficacy of immunotherapy depended on IL-1β, because the neutralization of IL-1β eliminated the protection conferred by the overactivating stimuli plus in vitro WTL. Figure 17B-17D Furthermore, CD8+ T cells are crucial for protection against immunogenic tumor models such as B16OVA or MC38OVA tumors, while both CD4+ and CD8+ T cells are essential for protection against less immunogenic tumors such as CT26 and B16F-10. Figure 17B-17D

[63] To determine how the efficacy of overactivated immunotherapy compared to PD-1 blockade-based therapy was compared, a parallel evaluation was conducted. Overactivated immunotherapy was as effective as anti-PD-1 therapy in the immunogenic B16OVA model, but was more effective in tumor models insensitive to anti-PD-1 therapy, such as CT26 and B16F-10. Figure 17B-17D ).

[0309] Example 9: Endogenously overactivated DCs stimulate T cells to induce durable antitumor immunity

[0310] Adoptive transfer of overactivated DCs to tumor-bearing mice induces strong anti-tumor immunity. Figures 15A-15BTo test whether endogenous dendritic cells (DCs) can elicit an overactivation-mediated antitumor response, 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 the Zbtb46DTR mice, DT was injected every other day to completely deplete the resident DCs in the Zbtb46DTR mice. When the tumor reached 4 mm in size, the Zbtb46DTR or WT mice were immunized with B16OVA WTL plus the overactivation stimulant LPS+PGPC. We found that, unlike WT mice which rejected the tumor in 90% of the mice, Zbtb46DTR mice (lacking DCs) could not reject the tumor. These results confirm that DCs are an initiator of overactivation-mediated protection. Figure 18A ).

[0311] Example 10: Overactivated cDC1 can be used to stimulate T cell-mediated antitumor immunity using a complex antigen source.

[0312] In vitro, we demonstrated that both cDC1 and cDC2 can achieve a state of overactivation as these cells respond to LPS+PGPC by producing IL-1β while maintaining their viability. These data provide indications for further defining specific DC subsets that elicit overactivation-mediated antitumor responses in vivo. Given the importance of the cDC1 subset in tumor rejection, we hypothesized that cDC1 plays a crucial role in inducing overactivation-mediated antitumor protection. To test this idea, we used Batf3- / - mice (lacking cDC1 but carrying cDC2 cells)

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

[65] . Interestingly, although immunization of Batf3- / - mice increased their survival by several days compared to unimmunized Batf3- / - mice, all Batf3- / - mice died 25 days after tumor inoculation due to tumor growth. In contrast, WT mice rejected the tumor in 100% of tumor-bearing mice ( Figure 18CTherefore, cDC1 plays a crucial role in overactivation-mediated antitumor immunity. Furthermore, although immunized WT mice induced high frequencies of antigen-specific CD8+ and CD4+ cells in TEM and skin dLN, immunized Batf3- / - induced slightly reduced antigen-specific CD4+ T cells, but no significant antigen-specific CD8+ T cells were observed in TEM. Figure 18D ).

[0313] To further confirm the role of overactivated cDC1 in inducing long-term antitumor protection, we sought to evaluate the ability of overactivated cDC1 to restore antitumor protection in Batf3- / - mice. We adopted naïve, activated, or overactivated cDC1 cells into Batf3- / - mice. For this purpose, FLT3-derived cDC1 cells were sorted from C57BL / 6J mice into B220-MHC-II+CD11c+CD24+ cells as previously described. cDC1 cells were treated in vitro and loaded with B16OVA WTL as described above, and then 1.10e6 cells were subcutaneously injected into tumor-bearing Batf3- / - mice. We observed that, unlike naïve or activated cDC1 which only provided a slight improvement in mouse survival compared to uninjected mice, overactivated cDC1 induced tumor rejection in 100% of tumor-bearing mice, maintaining tumor-free status for more than 60 days post-inoculation. Figure 18E Note that, as measured by SIINFEKL tetramer staining in tumor and skin dLN, injection of overactivated cDC1 into Batf3- / - restores CD8+ T cell responses ( Figure 18F Conversely, injection of naive or activated cDC1 fails to restore antigen-specific CD8+ T cells. cDC1-mediated tumor rejection depends on inflammasome activation, as injection of NLRP3- / -cDC1 treated with LPS+PGPC provides no antitumor protection and eliminates the ability of overactivated cDC1 to restore CD8+ T cell responses. Figure 18F ).

[0314] In addition to their ability to produce IL-1 from living cells, overactivated dendritic cells (DCs) highly migrate to neighboring dLNs to enhance CD8+ T cell responses. Figures 15A-15B ).

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[0384] Other embodiments

[0385] Although the invention has been described in conjunction with a detailed description, the foregoing description is intended to be illustrative and not to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

[0386] The patents and scientific literature cited in this document establish knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited in this document are incorporated herein by reference. All published foreign patents and patent applications cited in this document are incorporated herein by reference. All other published references, documents, manuscripts, and scientific literature cited in this document are incorporated herein by reference.

[0387] Although the invention has been particularly shown and described with reference to preferred embodiments thereof, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the scope of the invention as defined in the appended claims.

Claims

1. Use of (i) Toll-like receptor (TLR) ligands, (ii) non-classical inflammasome-activated lipids, and (iii) cancer immunogens in the preparation of reagents for inducing or enhancing an adaptive immune response against cancer in subjects by means of: Apply an effective amount of (i) the Toll-like receptor (TLR) ligand; (ii) the non-classical inflammasome-activated lipids; The cancer immunogen described in (iii) was administered to the subject. The TLR ligand is TLR4 ligand, and the non-classical inflammasome activated lipid is [(2R)-2-(4-carboxybutyryloxy)-3-hexadecanoyloxypropyl]2-(trimethylammonium)ethyl phosphate (PGPC).

2. Use of (i) Toll-like receptor (TLR) ligands, (ii) non-classical inflammasome-activated lipids, and (iii) cancer immunogens in the preparation of reagents for treating cancer in a subject by means of: Apply an effective amount of (i) the Toll-like receptor (TLR) ligand; (ii) the non-classical inflammasome-activated lipids and (iii) the cancer immunogen were administered to the subject. The TLR ligand is TLR4 ligand, and the non-classical inflammasome activated lipid is [(2R)-2-(4-carboxybutyryloxy)-3-hexadecanoyloxypropyl]2-(trimethylammonium)ethyl phosphate (PGPC).

3. The use according to claim 1, wherein the cancer immunogen is an infectious agent immunogen, wherein infection with the infectious agent is associated with cancer development.

4. The use according to claim 1 or 2, wherein the cancer immunogen originates from cancer cells.

5. The use according to claim 4, wherein the cancer immunogen is a whole tumor cell lysate or includes whole tumor cell lysates.

6. The use according to claim 1 or 2, wherein the TLR4 ligand is selected from monophospholipid A (MPLA), lipopolysaccharide (LPS), or a combination thereof.

7. The use according to claim 1 or 2, wherein the subject is a mammal.

8. The use according to claim 7, wherein the subject is a human.

9. The use according to claim 1 or 2, wherein the TLR ligand, PGPC, and cancer immunogen are administered as part of a pharmaceutical composition.

10. The use according to claim 1, wherein the immune response is a prophylactic immune response.

11. The use according to claim 1, wherein the immune response is a therapeutic immune response.

12. The use according to claim 1, wherein the adaptive immune response comprises T cell activation.

13. The use according to claim 1 or 2, wherein the method further comprises treating the subject with one or more therapeutic interventions.

14. The use according to claim 13, wherein the TLR ligand, PGPC, and cancer immunogen, and one or more therapeutic interventions are administered together or sequentially.

15. The use according to claim 13, wherein the one or more therapeutic interventions comprise: Radiation, chemotherapy, surgery, immunomodulators, proteasome inhibitors, pantothenic deacetylase (DAC) inhibitors, histone deacetylase (HDAC) inhibitors, or combinations thereof.

16. The use according to claim 13, wherein the one or more therapeutic interventions comprise: Therapeutic antibodies, checkpoint inhibitors, adoptive cell therapy, vaccines, or combinations thereof.

17. The use according to claim 16, wherein the adoptive cell therapy comprises: CAR-T cell therapy, CAR-NK cell therapy, T cells, dendritic cells, or combinations thereof.

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