Hyperactivated dendritic cells enable long-lasting antitumor immunity via adoptive cell transfer

Abstract

The present application relates to cancer immunotherapy, such as T cell stimulation-mediated anti-tumor therapy.

Description

[0001] Priority declaration

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 937,075, filed on November 18, 2019. The entire contents of the foregoing are incorporated herein by reference.

[0003] Federally Sponsored Research or Development

[0004] This invention was made with U.S. Government support under Grant No. AI116550 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. Technical Field

[0005] The present application relates to cancer immunotherapy, for example, anti-tumor therapy mediated by T cell stimulation. Background Art

[0006] The core of the understanding of protective immunity to infection and cancer is dendritic cells (DC), 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). DC monitors environmental threats to the host, most common signs of infection or tissue damage. This monitoring is achieved by 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 PRRs are classified as pathogen-associated molecular patterns (PAMPs), while host-derived PRR ligands are damage-associated molecular patterns (DAMPs) (P. Matzinger, Science, vol. 296, no. 5566, pp. 301-5, Apr. 2002).

[0007] Upon detection of PAMPs, PRRs initiate signaling pathways that fundamentally alter the physiology of DCs expressing these receptors (A. Iwasaki and R. Medzhitov, Nat. Immunol., vol. 16, no. 4, pp. 343–53, Apr. 2015; O. Joffre, et al. Immunol. Rev., vol. 227, no. 1, pp. 234–247, Jan. 2009). For example, before PRR activation, DCs are generally considered to be non-inflammatory cells. Upon encountering extracellular PAMPs, PRRs stimulate rapid and powerful upregulation of many inflammatory mediators including cytokines, chemokines, and interferons. Concurrent with the expression of these genes is the migration of DCs to draining lymph nodes (dLNs) and the upregulation of factors important for T cell activation such as MHC and co-stimulatory molecules. Therefore, the PRR signaling process causes DC activity to shift 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 RM Steinman, Cell, vol. 106, no. 3, pp. 255-8, Aug. 2001). Summary of the invention

[0008] It is necessary to diversify current cancer immunotherapy. Therefore, the present invention relates to a method for producing a colony of therapeutic dendritic cells, a method for inducing an immune response in a subject, a method for treating cancer, and a method for overactivating dendritic cells (DC) that induce type I T helper cells (TH1) and cytotoxic T lymphocytes (CTL) responses in the absence of TH2 immunity. Overactivated stimuli drive T cell responses that protect against tumors that are sensitive or resistant to PD-1 inhibition. These protective responses rely on inflammasomes in DC and can be produced using tumor lysates as immunogens.

[0009] In certain embodiments, the method of inducing a protective immune response to an immunogen in a subject comprises obtaining dendritic cells, culturing the dendritic cells ex vivo with an effective amount of non-classical inflammasome-activated lipids, and administering an effective amount of live dendritic cells to the subject to enhance the protective immune response, thereby inducing a protective immune response. In some embodiments, the therapeutically effective amount of non-classical inflammasome-activated lipids overactivates dendritic cells.

[0010] In certain embodiments, dendritic cells are optionally cultured ex vivo with an immunogen. In certain embodiments, dendritic cells are optionally cultured ex vivo with a cytokine.

[0011] In certain embodiments, the non-classical inflammasome-activating lipids include: 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), oxPAPC species, components thereof, or combinations thereof.

[0012] In certain embodiments, the method further comprises administering a chemotherapeutic agent.

[0013] In certain embodiments, the treatment methods disclosed herein may also be combined with any of the following therapies: radiation, chemotherapy, surgery, therapeutic antibodies, immunomodulators, proteasome inhibitors, pan-deacetylase (DAC) inhibitors, histone deacetylase (HDAC) inhibitors, checkpoint inhibitors, adoptive cell therapy including CAR T and NK cell therapy, and vaccines.

[0014] Preferably, the methods described herein inhibit the growth or progression of a cancer, such as a tumor, or a viral infection in a subject. For example, the methods described herein inhibit the growth of a tumor by at least 1%, for example, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%. In other cases, the methods described herein reduce the size of a tumor by at least 1 mm in diameter, e.g., at least 2 mm in diameter, at least 3 mm in diameter, at least 4 mm in diameter, at least 5 mm in diameter, at least 6 mm in diameter, at least 7 mm in diameter, at least 8 mm in diameter, at least 9 mm in diameter, at least 10 mm in diameter, at least 11 mm in diameter, at least 12 mm in diameter, at least 13 mm in diameter, at least 14 mm in diameter, at least 15 mm in diameter, at least 20 mm in diameter, at least 25 mm in diameter, at least 30 mm in diameter, at least 40 mm in diameter, at least 50 mm in diameter, or more. In some cases, the subject has had a majority of the tumor removed.

[0015] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains. Methods and materials for use with the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification including definitions shall prevail.

[0016] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figures 1A-1H is a series of graphs showing that in the absence of evidence of TH2 immunity, hyperactivated DCs are excellent antigen presenting cells and drive a TH1 biased immune response. Figures 1A-1F : WT BMDCs were left untreated (None) or treated with LPS alone, Alum alone, or oxPAPC or PGPC alone for 24 hours, or BMDCs were primed with LPS for 3 hours and then treated with the indicated stimuli for 21 hours. Figure 1A : IL-1β, and TNFα cytokine release were monitored by ELISA. Figure 1B : The percentage of cell death was measured by LDH release in the cell supernatant. Figure 1C :Use live-dead violet kit, CD11c and CD40 to Figure 1A BMDCs were stained with the indicated stimuli. Surface CD40 (CD11c + The mean fluorescence intensity (MFI) in living cells. Figure 1D : BMDCs pretreated with the indicated stimuli were transferred to CD40-coated plates and cultured for 24 h. IL-12p70 cytokine release was measured by ELISA. Figure 1E :Will use Figure 1A BMDCs pretreated with the indicated 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 panel). Data are presented as OVA-associated CD11c + Percentage of BMDC and normalized to OVA-associated CD11c at 4°C + BMDC. OVA peptide presentation on MHC-I was monitored using a PE-conjugated antibody against H-2Kb that binds to the OVA peptide SIINFEKL (SEQ ID NO: 1) (right panel). Data are presented as CD11c + Frequency of SIINFEKL (SEQ ID NO: 1)-associated DCs in living cells. Means and SD from three replicates are shown, and data are representative of at least three independent experiments. Figure 1F :Will use Figure 1A BMDCs treated with the indicated stimuli were loaded (or not) with OVA protein or OVA peptide SIINFEKL for 1 hour and then incubated with naive spleen OT-II ( )CD4 +T cells or OT-I naive CD8 + T cells were incubated for 4 days. Figure 1F : Supernatants were collected on day 4 and IFNγ, IL-2, IL-10, TNFα, and IL-13 cytokine release was measured by ELISA. Figure 1G BMDCs were left untreated (none), treated with LPS for 24 hours, or sensitized with LPS for 3 hours and then treated with PGPC or Alum for 21 hours. Figure 1F BMDCs were cultured with spleen OT-I or OT-II T cells in splenic culture medium. Four days after co-culture, CD4 + and CD8 + T cells for 5 h. Measurement of CD4 by intracellular staining + TH1 cells such as TNFα + IFNγ + , and TH2 cells such as Gata3 + IL-4 + IL-10 + The data are expressed as the ratio of TH1 / TH2 cells (left graph). CD8 + IFNγ in T cells + The frequencies are shown in the right graph. Means and SD from three replicates are shown, and each graph is representative of at least two independent experiments. Figure 1H As shown in the figure, C57BL / 6 mice were injected subcutaneously in the right flank with 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 plus OxPAPC or PGPC, both emulsified in IFA. CD4+ T cells were isolated from the skin draining lymph nodes (dLN) 40 days after immunization. + and CD8 + T cells. Then, T cells were cultured for 5 days with naive BMDC loaded (or not loaded) with OVA or SIINFEKL peptide. IFNγ, IL-10, and IL-13 secretion were measured by ELISA. Mean values ​​and SD from four mice are shown, and each figure represents two independent experiments. *P<0.05; **P<0.01; ***P<0.005.

[0018] Figure 2 C57BL / 6WT mice were subcutaneously inoculated with 5x10 57, 14 and 21 days after tumor challenge, mice were injected subcutaneously in the right flank with 5x10 6 Untreated WT BMDC (DC 幼稚 ), or activated WT BMDCs treated with LPS for 23 h and then pulsed with B16OVAWTL for 1 h (DC LPS ), or WT sensitized with LPS for 3 h, treated with PGPC for 20 h, and then pulsed with B16OVA WTL for 1 h, or NLRP3 - / - , or casp1 / 11 - / - BMDC(DC LPS+PGPC ). Survival was monitored daily (n=5 mice per group).

[0019] Figures 3A-3E is a series of graphs showing that hyperactivated DCs are excellent antigen presenting cells and drive TH1 biased immune responses in the absence of evidence of TH2 immunity. Figure 3A , 3B : BMDCs generated with GMCSF were left untreated (None), or treated with MPLA alone, Alum alone, or OxPAPC or PGPC alone, or BMDCs were sensitized with MPLA for 3 hours and then treated with the indicated stimuli for 21 hours. Figure 5 A: IL-1β, and TNFα cytokine release was monitored by ELISA. Figure 3B : The percentage of cell death was measured by LDH release in the cell supernatant. Figure 3C , 3D :Splenic CD11c + DCs were sorted and left untreated (None), treated with LPS alone, Alum alone, or PGPC alone, or DCs were sensitized with LPS for 3 hours and then treated with the indicated stimuli for 21 hours. Figure 3C : IL-1β, and TNFα cytokine release were monitored by ELISA. Figure 3D : The percentage of cell death was measured by LDH release in the cell supernatant. Figure 3E -3F: GMCSF-derived BMDCs treated with the indicated stimuli in A were stained with Live-Dead Purple kit, anti-CD11c, anti-CD80, anti-CD69, and anti-H2kb antibodies. Figure 3E : Measurement of CD11c by flow cytometry + Mean fluorescence intensity (MFI) of surface CD80 (left panel) and CD69 (middle panel) and H2Kb (right panel) in living cells. Means and SD from three replicates are shown, and all graphs represent at least three independent experiments. *P<0.05.

[0020] Figures 4A-4C is a graph and a series of plots showing that hyperactivated DCs are excellent antigen presenting cells and drive a TH1 biased immune response in the absence of evidence of TH2 immunity. Figures 4A-4C : WT BMDCs were left untreated (None), or treated with LPS alone, Alum alone, or OxPAPC or PGPC alone for 24 hours, or BMDCs were sensitized with LPS for 3 hours and then treated with the indicated stimuli for 21 hours. Figure 4A : BMDCs were cultured with fixable FITC-labeled-OVA for 45 minutes at 37°C or 4°C. BMDCs were then stained with a live-dead purple kit. Gating strategy was used to determine the frequency of OVA-FITC-associated BMDCs at 37°C compared to OVA-associated BMDCs at 4°C by flow cytometry. Figure 4B : BMDCs were cultured with endofit-OVA protein for 2 hours. Gating strategy was used to determine the frequency of SIINFEKL (SEQ ID NO: 1) peptide bound to H2bk on the surface of live BMDCs measured by flow cytometry using PE-conjugated antibodies against H-2Kb bound to the OVA peptide SIINFEKL. Each figure represents three replicates of one of three experiments. Figure 4C : As shown, C57BL / 6 mice were injected subcutaneously in the right flank with 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 plus OxPAPC or PGPC, both emulsified in IFA. CD4+ T cells were isolated from skin draining lymph nodes (dLN) 40 days after immunization. + T cells. T cells were then cultured with naive BMDC loaded (or not loaded) with OVA for 5 days. IL-4 secretion was measured by ELISA. Mean values ​​and SD of four mice are shown, and each figure represents two independent experiments. ***P<0.005.

[0021] Figure 5 is a series of plan views showing that hyperactivated DCs are excellent antigen presenting cells and drive a TH1 biased immune response in the absence of evidence of TH2 immunity. BMDCs were left untreated (None), or treated with LPS for 24 hours, or BMDCs were sensitized with LPS for 3 hours and then treated with PGPC or Alum for 21 hours. The treated BMDCs were then cultured with splenic OT-II T cells at a ratio of 1:5 (BMDC:T cells). Four days after co-culture, CD4 +T cells were stimulated with PMA and ionomycin for 5 h. Gating strategy was used to determine viable CD4 + TH2 cells such as IL-4 in T cells + IL-10 + Each graph represents three replicates of one of three experiments.

[0022] Figures 6A-6D is a series of images and immunostaining showing that cDC1 and cDC2 cells achieve a hyperactivated state in vitro. Figure 6A-6B ) Splenic DCs (left) or FLT3-derived DCs (right) were sorted into cDC1 (CD11c + CD24 + ) or cDC2(CD11c + Sirpa + DCs were left untreated (none), treated with LPS alone, Alum alone, or OxPAPC or PGPC alone for 24 hours, or DCs were sensitized with LPS for 3 hours and then treated with the indicated stimuli for 21 hours. Fig. 6A ) Cell death was measured by LDH release in the supernatant. Figure 6B ) IL-1β and TNFα cytokine release was monitored by ELISA. Means and SD of 3 independent experiments are shown. ( Figure 6C ) FLT3-DCs treated with the indicated stimuli were stained with Phalloidin-FITC and DAPI. Images were acquired on a Zeiss confocal microscope using a 40X oil immersion lens. ( Fig.6D ) FLT3-DCs treated with the indicated stimuli were stained with a live-dead purple kit, CCR7 PE, and CD11c-APC. The mean fluorescence intensity (MFI) of CCR7 (for CD11c + Live cells were gated).

[0023] Figure 7 Schematic diagram and plan view showing that hyperactivated cDC1 controls tumor rejection induced by hyperactivation-based immunotherapy. WT mice were inoculated subcutaneously on the back with 3×10 5 7, 14, and 21 days after tumor challenge, mice were left untreated or injected subcutaneously in the right flank with 1×10 6 untreated WT cDC1 or WT cDC1 treated with LPS for 23 h (cDC1 活化 ), or WT cDC1 cells sensitized with LPS for 3 h and then treated with PGPC for 20 h (cDC1 过度活化All DCs were pulsed with tumor lysate for 1 hour prior to injection. Survival was monitored daily (n=5 mice / group).

[0024] Figures 8A-8C is a series of graphs, plans and schematics showing that hyperactivated cDC1 controls tumor rejection and enhances tumor infiltration of anti-tumor specific T cells. Figures 8A-8B Batf3- / - mice were subcutaneously inoculated with 3×10 5 7, 14, and 21 days after tumor challenge, mice were injected subcutaneously in the right flank with 1×10 6 Untreated WT cDC1 or WT cDC1 treated with LPS for 23 h and then pulsed with B16OVA tumor lysate for 1 h (cDC1 活化 ), or WT cDC1 cells sensitized with LPS for 3 h, then treated with PGPC for 20 h, and then pulsed with tumor lysate for 1 h (cDC1 过度活化 ). Figure 8C ) Survival was monitored daily (n=5 mice / group). Figure 8B ) 15 days after tumor inoculation, skin draining lymph nodes (dLN), tumors, and spleen tissues were excised from immunized mice. Antigen-specific CD8 + and CD4 + Percentage of T cells (n = 5 mice / group). Figure 8C ) SIINFEKL in tumors and dLNs of treated mice + CD8 + Representative images of T cells.

[0025] Fig. 9A and 9B is a series of schematic diagrams and plan views showing that overactivated cDC1 controls tumor rejection in an inflammasome-dependent manner. Fig. 9A )Casp1 / 11 - / - Mouse, ( Fig. 9B )NLRP3 - / - Mice were inoculated subcutaneously in the back with 3.105 live B16OVA cells. 7, 14 and 21 days after tumor challenge, mice were injected subcutaneously in the right flank with 1.106 untreated WT cDC1 (cDC1 幼稚 ), or WT cDC1 treated with LPS for 23 h and then pulsed with B16OVA tumor lysate for 1 h (cDC1 活化 ), or WT or Casp1 / 11 sensitized with LPS for 3 h and then treated with PGPC for 20 h - / - cDC1 过度活化All DCs were pulsed with tumor lysate for 1 hour prior to injection. Fig. 9A )Casp1 / 11 - / - Mice and ( Fig. 9B )NLRP3 mice were monitored daily for survival (n=5 mice / group).

[0026] Fig. 10A and 10B The mass spectra of synthetic lipids are shown. Non-oxidized PAPC ( Fig. 10A )、oxPAPC( Fig. 10B ), PEIPC-rich oxPAPC ( Fig. 10C ) and biotinylated oxPAPC( Fig. 10D ) by mass spectrometry.

[0027] Fig.11A -B. Oxidized phospholipids induce hyperactivated cDC1 and cDC2 cells that exhibit a highly migratory phenotype. (A) Wild-type or NLRP3 - / - or Casp1 / 11 - / - BMDC is placed untreated (none), or treated with independent LPS, or independent Alum, or independent PGPC for 24 hours, or BMDC is sensitized with LPS for 3 hours, and then treated with specified stimulus for 21 hours. IL-1β and TNFα are released by ELISA monitoring. The percentage of cell death is measured by LDH release in the cell supernatant. Mean values ​​and SD from three repetitions are shown, and data represent at least three independent experiments. (B) Wild-type BMDC produced using FLT3L is sorted as cDC1 or cDC2 cells, and then treated with the specified stimulus in A. IL-1β and TNFα are released by ELISA monitoring. The percentage of cell death is measured by LDH release in the cell supernatant. Mean values ​​and SD are from three independent experiments completed in two different laboratories.

[0028] Fig. 12A -B. Hyperactivated DCs induce strong CTL responses and long-term antitumor immunity that are dependent on CCR7 expression and inflammasome activation. (AB) Wild-type BMDCs generated with FLT3L were left untreated (DC 幼稚 ), or treated with LPS alone (DC 活化 ) for 18 hours, or BMDCs were sensitized with LPS for 3 hours and then with PGPC (DC 过度活化 ) or Alum(DC 细胞焦亡 ) for 15 hours. - / - or CCR7 - / -The BMDC of mouse was sensitized with LPS for 3 hours, and then treated with PGPC for 15 hours. 1.10e6 BMDC was cultured with OVA protein for 1 hour, and then injected subcutaneously into wild-type mice. The BMDC without OVA protein was injected as a control group. After 7 days of BMDC injection, the skin draining lymph nodes were excised and stained with live-dead purple test kit, OVA peptide tetramer antibody, anti-CD45, anti-CD3, anti-CD8a, and anti-CD4. (A) The percentage of SIINFEKL+CD8+T cells (upper figure) and AAHAEINEA+CD4+ live T cells was measured by flow cytometry. (B) The absolute number of SIINFEKL+CD8+T cells (upper figure) and AAHAEINEA+CD4+ live T cells was measured by flow cytometry using CounterBright beads.

[0029] Fig.13A -E. Overactive stimuli induce strong CTL responses in an inflammasome-dependent manner. (A) C57BL / 6 mice were injected subcutaneously in the right flank with OVA alone, OVA and LPS, OVA and PGPC, or OVA and LPS plus oxPAPC or PGPC, all emulsified in incomplete Freund's adjuvant (IFA). 7 or 40 days after immunization, T cells were isolated from skin draining lymph nodes (dLN) by magnetic enrichment using anti-CD8 beads. (A) Among the CD3+CD8+ live cells, T effector cells (Teff) such as CD44 low CD62L low , T effector memory cells (TEM) such as CD44 hi CD62L low , and the percentage of T central memory cells (TCM) such as CD44hiCD62Lhi. (B) 7 days after immunization, CD8+T cells were sorted from dLN and then treated with PMA and ionomycin, or co-cultured with B16OVA cells (target cells) at a ratio of 1:3 (effector cells: target cells) for 5 hours. The degranulation of CD8+T cells 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 with OVA alone or OVA and LPS, or OVA and LPS plus oxPAPC or PGPC, or OVA and LPS plus Alum, all emulsified in IFA. Optionally, NLRP3 - / -Mice were injected with OVA emulsified in IFA with LPS plus PGPC. Seven days after immunization, CD8+ T cells were sorted from the skin dLN of immunized mice and co-cultured with BMDC loaded (or not loaded) with OVA at a ratio of 1:10 (DC:T cells) for 7 days. The percentage of SIINFEKL+IFNγ+ in CD8+ live T cells was measured using OVA peptide tetramer staining followed by intracellular IFNγ staining. (DE) CD45.1 mice were irradiated and then treated with bone marrow ZBTB46DTR mice plus WT or NLRP3 - / - or Casp1 / 11 - / - or CCR7 - / - (ratio 5:1) reconstruction, all on a CD45.2 C57BL / 6 background. Six weeks after reconstruction, mouse chimeras were injected with tamoxifen every other day for 7 days. Chimeric mice were then immunized subcutaneously in the right flank with OVA emulsified in IFA and LPS plus PGPC. Seven days after immunization, CD8+T cells were isolated from skin draining lymph nodes (dLN) or spleen by magnetic enrichment, using anti-CD8 beads. (D) The percentages of Teff, TEM, TCM, and T naive cells in skin dLN were measured by flow cytometry. (E) The percentage of SIINFEKL+ in CD8+ live T cells in dLN (left figure) or spleen (right figure) was measured by flow cytometry using OVA peptide tetramer staining. Total CD8+ T cells were sorted from dLN and co-cultured with untreated BMDCs loaded (or not loaded) with OVA at a ratio of 1:10 (DC:T cells) for 7 days.

[0030] Fig.14A -D. Immunization with hyperactivating stimulants eliminates immunogenic tumors from hot tumors to cold tumors. (A) C57BL / 6 mice were inoculated subcutaneously in the left upper back with 5x10 5 14 days later, mice were left untreated (unimmunized) or injected subcutaneously in the right flank with syngeneic MC38OVA whole tumor lysate (WTL), plus LPS and PGPC, with or without intravenous (iv) injection of neutralizing anti-IL-1β, or with or without intraperitoneal injection of anti-CD4, or anti-CD8a. Mice received 2 booster injections of WTL and LPS plus PGPC on days 37 and 55 after tumor inoculation. Tumors were allowed to reach 20 mm in diameter. The percentage of survival is shown (n = 10 mice per group). (B) C57BL / 6 mice were inoculated subcutaneously in the left upper back with 3X10 5live B16OVA cells. After 10 days, the mice were left untreated (unimmunized) or injected intraperitoneally with anti-PD1 antibodies. Alternatively, mice were injected subcutaneously in the right flank with homologous B16OVA WTL, plus LPS and PGPC, with or without intravenous (iv) injection of neutralizing antibody anti-IL-1β, or with or without intraperitoneal injection of anti-CD4, or anti-CD8a. On days 17 and 24 after tumor inoculation, mice received 2 booster injections of B16OVA WTL, plus LPS and PGPC. The percentage of survival is shown (n=10 mice / group). (C) C57BL / 6 mice were inoculated subcutaneously in the upper left back with 3X10 5 7 days later, mice were left untreated (unimmunized) or injected intraperitoneally with anti-PD1 antibody. Alternatively, mice were immunized subcutaneously in the right flank with syngeneic B16-F10 WTL, plus LPS and PGPC, with or without intravenous (iv) injection of neutralizing antibody anti-IL-1β, or with or without intraperitoneal injection of anti-CD4, or anti-CD8a. Mice received 2 booster injections on days 14 and 21 after tumor inoculation. The percentage of survival is shown (n=10 mice per group). (D) BALB / c WT mice were inoculated subcutaneously in the left back with 3X10 5 Live CT26 cells. After 7 days, mice were left untreated (unimmunized) or injected intraperitoneally with anti-PD1 antibodies. Alternatively, mice were injected subcutaneously in the right flank with syngeneic CT26 WTL, plus LPS and PGPC, with or without intravenous (iv) injection of neutralizing antibody anti-IL-1β, or intraperitoneal injection of or without anti-CD4, or anti-CD8a. Mice received 2 booster injections on days 14 and 21 after tumor inoculation. The percentage of survival is shown (n=10 mice per group).

[0031] Fig.15A -F. Overactivated cDC1 can use complex antigen sources to stimulate T cell-mediated antitumor immunity. (A) Zbtb46DTR mice were injected subcutaneously with B16OVA cells. Mice were injected with diphtheria toxin (DTx) every other day for 4 consecutive injections, or mice were injected with PBS. 7 days after tumor injection, all mice were immunized with B16OVA WTL plus LPS and PGPC, followed by 2 booster injections. The percentage of mouse survival is shown (n=10 mice per group). (B) CD45.1 mice were irradiated and then immunized with cells from Zbtb46DTR mice plus WT or Nlrp3 - / - or Casp1 / 11 - / - or Ccr7 - / -Mixed BM reconstitution of mice. Six weeks after reconstitution, mouse chimeras were injected subcutaneously with B61OVA cells, and then all mice received DTx three times a week for a total of 12 consecutive injections. Seven days after tumor inoculation, chimeric mice were immunized with B16OVA WTL and LPS plus PGPC and received 2 booster injections. The percentage of mouse survival is shown (n=5 mice per group). (CD) WT or Batf3 - / - Mice were injected subcutaneously with B16OVA cells. Seven days after tumor inoculation, mice were left untreated or treated with WT and Batf3 - / - Mice were immunized with B16OVA WTL and LPS plus PGPC, followed by 2 booster injections. (C) Shows the percentage of mouse survival (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 injected with B16OVA cells subcutaneously in the right flank. 7 days after tumor inoculation, mice were placed untreated (no cDC1 injection), or mice were injected with FLT3-derived naive cDC1 or activated cDC1 treated with LPS or over-activated cDC1 pretreated with LPS plus PGPC in the left flank. Before injection, all cDC1 were loaded with B16OVA WTL for 1 hour. (E) The percentage of mouse survival is shown (n=5 mice per group). (F) 21 days after tumor inoculation, tetramer staining was used to assess OVA-specific CD8+T cells and CD4+T cells (n=5 mice per group).

[0032] Fig.16A-C. Oxidized phospholipids induce inflammasome-dependent IL-1β secretion of cDC1 and cDC2 cells, and promote high migration DC phenotype. (A) Wild-type BMDC produced using FLT3L was placed untreated (none), or treated with a single CpG 1806 or a single PGPC for 24 hours, or BMDC was sensitized with CpG1806 for 3 hours and then treated with a 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. Mean values ​​and SD from three repetitions are shown, and the data represent at least three independent experiments. (B) Gate strategy is to separate cDC1 or cDC2 from BMDC produced from FLT3L- or from the spleen of wild-type mice. Purity post-sorting is shown for spleen cDC or FLT3L DC. (C) Spleen cDC1 or cDC2 were left untreated (none), or treated with LPS alone, Alum alone, or oxPAPC or PGPC alone for 18 hours, or BMDC were sensitized with LPS for 3 hours and then treated with the specified stimulants 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 values ​​and SD are from three independent experiments performed in two different laboratories.

[0033] Fig.17A -C. Hyperactivated DCs induce strong CTL responses and long-term antitumor immunity that are dependent on CCR7 expression and inflammasome activation. Wild-type BMDCs generated with FLT3L were left untreated (DC 幼稚 ), or treated with LPS alone (DC 活化 ) for 18 hours, or BMDCs were sensitized with LPS for 3 hours and then PGPCs (DCs 过度活化 ) or Alum(DC 细胞焦亡 ) was added to the culture medium for 15 hours. - / - or CCR7 - / - BMDCs from mice were sensitized with LPS for 3 hours, and then PGPCs were 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 that binds to the OVA peptide SIINFEKL. Data are expressed as the frequency of SIINFEKL-associated DCs in CD11c+ live cells. Means and SDs from three replicates are shown, and the data represent three independent experiments. (B) Wild-type or NLRP3 produced with FLT3L were stimulated as in A. - / - or CCR7 - / -BMDC. BMDC were washed and then stained with Live-Dead Purple Kit, CD11c and CD40. The mean fluorescence intensity (MFI) of surface CD40 (in CD11c+ living cells) was measured by flow cytometry. (C) CCR7 produced by FLT3L - / - BMDC is placed untreated (none), or treated with independent LPS, or independent Alum, or independent PGPC for 24 hours. Alternatively, BMDC is sensitized with LPS for 3 hours, and then treated with specified stimulants for 21 hours. IL-1β and TNFα are released by ELISA monitoring. The percentage of cell death is measured by LDH release in the cell supernatant. Mean values ​​and SD from three repetitions are shown, and data represent at least three independent experiments.

[0034] Fig.18A -D. Hyperactivating stimuli increase memory T cell production and enhance antigen-specific IFNγ effector responses in an inflammasome-dependent manner. C57BL / 6 mice were injected subcutaneously in the right flank with OVA alone, OVA with LPS, OVA with PGPC, or OVA with LPS plus oxPAPC or PGPC, all emulsified in incomplete Freund's adjuvant (IFA). Seven days after immunization, T cells were isolated from skin draining lymph nodes (dLN) by magnetic enrichment using anti-CD8 beads. (A) Gating strategy to determine T effector cells (Teff) such as CD44 low CD62L low , T effector memory cells (TEM) such as CD44 high CD62L low , and T central memory cells (TCM) such as CD44 high CD62L high Percentage. (B) The absolute number of Teff or TEM cells in the skin dLN of each mouse was assessed by flow cytometry in total CD3+ live cells. (C) Seven days after immunization, CD8+T cells were sorted from dLN and then cultured with untreated BMDCs loaded (or not loaded) with a serial dilution of OVA protein (starting at 1000ug / ml). IFNγ cytokine secretion was measured by ELISA. The mean and SD of five mice are shown. (D) Seven days after immunization, CD8+T cells were sorted from dLN and then treated with PMA and ionomycin or co-cultured with B16OVA cells (target cells) at a ratio of 1:3 (effector cells: target cells) for 5 hours. The gate strategy was set to determine the percentage of CD107a+ in live CD8+T cells by flow cytometry. Each figure represents five mice. *P<0.05; **P<0.01.

[0035] Fig.19A-B. Hyperactivating stimuli increase memory T cell production and enhance antigen-specific IFNγ effector responses in an inflammasome-dependent manner. (AB) CD45.1 mice were irradiated and then treated with bone marrow from ZBTB46DTR mice plus WT or NLRP3 - / - or Casp1 / 11 - / - or CCR7 - / - (ratio 5: 1) reconstruction, all on CD45.2 C57BL / 6 background. Six weeks after reconstruction, mouse chimeras were injected with tamoxifen every other day for 7 days. Then, chimeric mice were immunized subcutaneously in the right flank with OVA emulsified in IFA and LPS plus PGPC. Seven days after immunization, CD8+T cells were isolated from skin draining lymph nodes (dLN) or spleen by magnetic enrichment, using anti-CD8 beads. (A) The percentages of Teff, TEM, TCM and T naive cells in skin dLN were measured by flow cytometry. Each figure represents five mice. (B) The percentage of SIINFEKL+ in CD8+ live T cells in dLN (upper figure) or spleen (lower figure) was measured by flow cytometry, using OVA peptide tetramer staining.

[0036] Fig. 20A -C. Mice were injected subcutaneously (sc) in the right flank with PBS (unimmunized), B16OVA cell lysate alone (none), B16OVA cell lysate and LPS, or B16OVA lysate plus LPS and oxPAPC or PGPC. 15 days after immunization, 3x10 5 The mice were challenged subcutaneously with 10 surviving B16OVA cells in the left upper back. 5 1 surviving B16OVA cells were re-challenged in the back subcutaneously without tumor mice. (A) Tumor growth was monitored every 2 days (upper figure). For survival experiments (lower figure), mice were made to reach 20 mm in diameter (n=8-15 mice per group). (BC) Tumors were harvested at the end of tumor growth and separated 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 was measured by ELISA (lower figure) (n=4 mice per group).

[0037] Fig.21A-D. (AB) The absolute number of CD8+T cells and CD69+CD103+T resident memory CD8+T cells was assessed at the immunization or tumor injection site of surviving mice and measured by flow cytometry (n=4 mice). (CD) Circulating memory CD8+T cells (TCM) were isolated from the spleen of surviving mice or age-matched non-immunized tumor-bearing mice, and T resident memory CD8+ cells (TRM) were isolated from skin inguinal adipose tissue. (C) TCM and TRM from surviving mice were co-cultured with B16OVA or B16-F10 or CT26 tumor cells at a ratio of 1:5 (tumor cells: T cells) for 5 hours. Cell death caused by cytolytic CD8+T cells was measured by LDH release in the supernatant. (D) Mice were left untreated (no Tx), or 5x10 5 CD8+ TCM cells and / or intradermal (id) inoculation of 5x10 5 CD8+ TRM cells isolated from surviving mice or age-matched non-immunized tumor-bearing mice were used. 5 All mice were challenged with 100 surviving B16OVA cells. The percentage of survival was monitored every 2 days. Mice were allowed to reach 20 mm in diameter (n=5 mice per group). DETAILED DESCRIPTION

[0038] The innate immune system is generally considered to work in an all-or-nothing manner, with DCs acting to elicit inflammatory responses that promote adaptive immunity, or not. Therefore, it is believed that Toll-like receptors (TLRs) expressed by DCs play an important role in determining the immunogenic potential of these cells. The mammalian immune system is responsible for detecting microorganisms and activating protective responses that limit infection. The key to this task is dendritic cells, which sense microorganisms and subsequently promote T cell activation. It has been shown that dendritic cells can measure the threat of any infection and indicate a proportional response (Blander, JM (2014). Nat Rev Immunol 14, 601-618; Vance, RE et al., (2009) Cell hostµbe 6, 10-21), but it is still unclear what mechanism these immunomodulatory activities can occur.

[0039] PRRs function to directly or indirectly detect molecules common to a wide range of microbial classes. 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.

[0040] The important property of PRR as an immune regulator is their ability to recognize specific microbial products. In this way, PRR-mediated signaling events can provide clear indications of infection. It is assumed that the PRRs expressed on DCs activate the "GO" signals that promote inflammation and T cell-mediated immunity. Interestingly, several groups have recently proposed that DCs do not only work 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 trigger a proportional response. The most commonly discussed method for measuring virulence is based on the ability of toxic pathogens to activate more different PRRs than non-pathogens. However, not all microorganisms have a common set of PRR activators, and not all PRR activators have considerable effectiveness. Therefore, the number of PRRs activated during infection is not an ideal measure of virulence. In addition, increasing the number of PRRs activated during infection will generally lead to a greater inflammatory response, which can indirectly promote a greater T cell response. It has been previously shown that conditions that enhance the activation state of DC (e.g., by using toxic pathogens as stimuli) are also expected to enhance the activation state of MΦ (Vance, RE et al., (2009) Cellhostµbe 6, 10-21). Therefore, it is unclear whether the immune system (i.e., DC) really has a mechanism to specifically measure the threat of infection.

[0041] One possible method for assessing the threat of infection is through a known coincidence detection process, in which independent inputs result in responses different from those triggered by any single input. In the case of PRR, regardless of the threat of virulence, one such input must be a microbial product that is an indicator of infection. In order to measure the threat of virulence, a second input must be present. Without wishing to be limited by theory, it is currently believed that the hypothetical second input is a molecule produced at the site of tissue damage, because cell damage is generally a feature associated with highly pathogenic microorganisms. Candidate molecules that can provide a second stimulus to DC are molecules of different families called damage-associated molecular patterns (DAMPs), which are 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 infectious or non-infectious tissue damage and are thought to regulate immune responses, although their mechanism of action remains unclear. Representatives of one class of such DAMPs are oxidized phospholipids derived from 1-palmitoyl-2-arachidonoyl-sn-glycero-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 Opin Lipidol 14, 421-430), where local concentrations can be as high as 10-100 μM (Oskolkova, OV et al. (2010) J Immunol 185, 7706-7712). The association between oxPAPC and dying cells raises the possibility that these lipids can be used as general indicators of tissue health. Therefore, in the presence of microbial products (one or more), oxPAPC may indicate an increased threat of infection.

[0042] Due to the aforementioned characteristics of activated DC, these cells have the ability to stimulate antigen-specific T cell responses and many strategies have been adopted to promote DC activation to drive protective immunity. These strategies generally involve the use of synthetic or natural microbial products that stimulate the PRR of the Toll-like receptor (TLR) family, and a notable example is the molecule monophosphoryl lipid A (MPLA, a TLR4 ligand approved by the FDA for assisting more and more vaccines) (J. Paavonen, Lancet, vol. 374, no. 9686, pp. 301–314, Jul. 2009; M. Kundi, Expert Rev. Vaccines, vol. 6, no. 2, pp. 133–140, Apr. 2007; A. M. Dierlaurent, et al. J. Immunol., vol. 183, no. 10, pp. 6186–6197, Nov. 2009). Note that TLR alone cannot promote all molecular signals required for 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 generation, and effector function (SZ Ben-Sasson, et al. Proc. Natl. Acad. Sci. USA, vol. 106, no. 17, pp. 7119–24, Apr. 2009; SZ Ben-Sasson, et al. J. Exp. Med., vol. 210, no. 3, pp. 491–502, Mar. 2013; A. Jain, et al. Nat. Commun., vol. 9, no. 1, pp. 1–13, 2018). Expression of IL-1β, a well-characterized family member, is highly induced by TLR signals, but the cytokine lacks an N-terminal secretion signal and is therefore not released from cells through conventional biosynthetic pathways. However, IL-1β accumulates in an inactive state in the cytoplasm of DCs that have been activated by TLR ligands (C. Garlanda, et al. Immunity, vol. 39, no. 6, pp. 1003–1018, Dec. 2013). The lack of IL-1β release from activated DCs raises the possibility that TLR signaling alone is insufficient to maximally stimulate T cell responses and protective immunity.

[0043] The DC activation state is not the only cell fate that DC can achieve under PRR signaling. In fact, different PRRs stimulate different fates of these cells. One such fate is a commitment to an inflammatory form of cell death called pyroptosis. Pyroptosis is a regulated process caused by the activity of inflammasomes, which are supramolecular organizing centers (SMOCs) assembled in the cytoplasm of DCs and other cells (A. Lu, et al. Cell, vol. 156, no. 6, pp. 1193–1206, Mar. 2014; JC Kagan, et al. Nat. Rev. Immunol., vol. 14, no. 12, pp. 821–826, Dec. 2014). Inflammasome assembly is usually stimulated when PAMPs or DAMPs are detected in the cytoplasm of host cells. Thus, cytoplasmic PRRs are responsible for associating threat assessment in the cytoplasm with inflammasome-dependent cell pyroptosis (KJ Kieser and JCKagan, Nat. Rev. Immunol., vol. 17, no. 6, pp. 376–390, May 2017; M. Lamkanfi and VM Dixit, Cell, vol. 157, no. 5, pp. 1013–22, May 2014). The process of cell pyroptosis causes IL-1β and other IL-1 family members to be released from cells, thereby providing T cells with signals that TLRs cannot provide. In addition to this increase in activity, pyroptotic cells die in a manner that promotes IL-1β release, thereby losing the ability to participate in the days-long process required to stimulate and differentiate naive T cells in dLNs (TRMempel, et al. Nature, vol. 427, no. 6970, pp. 154-159, Jan. 2004). Indeed, stimulants that promote pyroptosis, such as the commonly used vaccine adjuvant alum (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), which is not suitable for the elimination of many microbial infections or cancers.

[0044] Adoptive cell therapy (ACT), including allogeneic and autologous hematopoietic stem cell transplantation (HSCT) and recombinant cell (i.e., CAR T) therapy, is a treatment option for many malignant conditions (for reviews of HSCT and adoptive cell therapy approaches, see, Rager & Porter, Ther Adv Hematol (2011) 2(6): 409–428; Roddie & Peggs, Expert Opin. Biol. Ther. (2011) 11(4): 473-487; Wang et al. Int. J. Cancer. (2015) 136, 1751–1768; and Chang, YJ and XJ Huang, Blood Rev, 2013. 27(1): 55-62). Such adoptive cell therapy includes, but is not limited to, allogeneic and autologous hematopoietic stem cell transplantation, donor leukocyte (or lymphocyte) infusion (DLI), adoptive transfer of tumor-infiltrating lymphocytes, or adoptive transfer of T cells or NK cells (including recombinant cells, i.e., CAR T, CAR NK, gene-edited T cells or NK cells, see Hu et al. Acta Pharmacologica Sinica (2018) 39: 167–176, Irving et al. Front Immunol. (2017) 8: 267). In addition to the necessity of donor-derived cells to reconstruct hematopoiesis after radiation and chemotherapy, immune reconstitution from transferred cells is important for eliminating residual tumor cells. The efficacy of ACT as a treatment option for malignancies is influenced by many factors, including the source, composition and phenotype (lymphocyte subsets, activation state) of the donor cells, the underlying disease, the pre-transplant conditioning regimen and post-transplant immune support (i.e., IL-2 therapy), and the graft-versus-tumor (GVT) effect mediated by the donor cells within the graft. In addition, these factors must be balanced with transplant-related mortality, which is often caused by the conditioning regimen and / or excessive immune activity of the donor cells within the host (i.e., graft-versus-host disease, cytokine release syndrome, etc.).

[0045] The present application is based in part on the following discovery: stimulants that activate dendritic cells (DCs), or promote DC pyroptosis, induce a mixed T cell response consisting of type I and type 2 T helper (Th) cells. In contrast, stimulants that overactivate DCs selectively stimulate TH1 and cytotoxic T lymphocyte (CTL) immune responses, with no signs of TH2-induced immunity. Even when using a complex antigen source (e.g., tumor cell lysate), the TH1-biased immunity produced by overactivated DCs confers these cells with a unique ability to mediate long-term protective anti-tumor immunity. As described herein, overactivated DCs can be generated ex vivo and used, for example, in adoptive cell therapy.

[0046] Therefore, provided herein is a method for producing a population of therapeutic dendritic cells, the method comprising obtaining living dendritic cells from a cell donor, sensitizing the dendritic cells ex vivo with a TLR ligand, culturing the sensitized dendritic cells ex vivo with lipids that activate non-classical inflammasomes, and loading the dendritic cells with an immunogen, thereby producing a population of therapeutic dendritic cells.

[0047] Also provided herein is a method of inducing an immune response in a subject, the method comprising obtaining live dendritic cells from a cell donor, sensitizing the dendritic cells ex vivo with a TLR ligand, culturing the sensitized dendritic cells ex vivo with lipids activated by non-classical inflammasomes, loading the dendritic cells with an immunogen, thereby producing a population of therapeutic dendritic cells, and administering the population of therapeutic dendritic cells to a subject, thereby inducing an immune response in the subject.

[0048] Also provided herein is a method for treating cancer, comprising obtaining living dendritic cells from a cell donor, sensitizing the dendritic cells ex vivo with a TLR ligand, culturing the sensitized dendritic cells ex vivo with lipids that activate non-classical inflammasomes, loading the dendritic cells with an immunogen, thereby generating a population of therapeutic dendritic cells, and administering the population of therapeutic dendritic cells to a subject, thereby treating cancer in the subject.

[0049] Dendritic cells

[0050] The methods disclosed herein involve obtaining live dendritic cells from a cell donor. The dendritic cells obtained from the cell donor may be immature or mature. The dendritic cells may be differentiated in vivo or in vitro.

[0051] In some embodiments, obtaining live dendritic cells from a cell donor includes harvesting progenitor cells from the cell donor and culturing the progenitor cells in vitro under conditions effective to induce differentiation, thereby obtaining dendritic cells from the cell donor. Methods for differentiating progenitor cells into dendritic cells in vitro are known in the art. See, for example, Ardavin et al., "Origin and Differentiation of Dendritic Cells," TRENDS in Immunol. 22(12): 691–700 (2001).

[0052] In some embodiments, the progenitor cells are lymphoid progenitor cells. In some embodiments, the progenitor cells are myeloid progenitor cells. In some embodiments, the progenitor cells are blood monocytes.

[0053] In some embodiments, the progenitor cells are derived from bone marrow. In some embodiments, the progenitor cells are derived from blood. In some embodiments, the progenitor cells are derived from peripheral blood mononuclear cells. In some embodiments, the progenitor cells are derived from umbilical cord blood.

[0054] In some embodiments, culturing the progenitor cells ex vivo under conditions effective to induce differentiation comprises culturing the progenitor cells in the presence of one or more cytokines.

[0055] In some embodiments, culturing the progenitor cells ex vivo under conditions effective to induce differentiation comprises culturing the progenitor cells in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4), tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), interleukin 7 (IL-7), stem cell factor (SCF), fms-like tyrosine kinase 3 ligand (FLT3-L), interleukin 1 (IL-1), or a combination thereof.

[0056] In some embodiments, the progenitor cells are cultured ex vivo for between about 1 and about 48 hours. In some embodiments, the progenitor cells are cultured for about 6 to about 48, about 12 to about 48, about 18 to about 48, about 24 to about 48, about 30 to about 48, about 36 to about 48, about 42 to about 48, about 1 to about 42, about 6 to about 42, about 12 to about 42, about 18 to about 42, about 24 to about 42, about 30 to about 42, about 36 to about 42, about 1 to about 36, about 6 to about 36, about 12 to about 36, about 18 to about 36, about 24 to about 36, about 30 to about 36, about 1 to about 30, about 6 to about 30, about 12 to about 30, about 18 to about 30, about 24 to about 30, about 1 to about 24, about 6 to about 24, about 12 to about 24, about 18 to about 24, about 1 to about 18, about 6 to about 18, about 12 to about 18, about 1 to about 12, about 6 to about 12, or about 1 to about 6 hours.

[0057] In some embodiments, the progenitor cells are blood monocytes.In some embodiments, the blood monocytes are cultured in the presence of GM-CSF and / or IL-4.

[0058] In some embodiments, obtaining dendritic cells from a cell donor comprises harvesting dendritic cells differentiated in vivo from a cell donor. In some embodiments, the dendritic cells differentiated in vivo are immature dendritic cells. In some embodiments, the dendritic cells differentiated in vivo are mature dendritic cells.

[0059] In some embodiments, the in vivo differentiated dendritic cells are harvested from the spleen of the cell donor. In some embodiments, the in vivo differentiated dendritic cells are harvested from the lymph nodes of the cell donor. In some embodiments, the in vivo differentiated dendritic cells are harvested from the thymus of the cell donor. In some embodiments, the in vivo differentiated dendritic cells are harvested from the blood of the cell donor. In some embodiments, the in vivo differentiated dendritic cells are harvested from the skin of the cell donor.

[0060] In some embodiments, obtaining dendritic cells from a subject comprises freezing progenitor cells and / or dendritic cells differentiated in vivo.

[0061] The methods disclosed herein include sensitizing dendritic cells ex vivo with TLR ligands. Suitable TLR ligands are described herein. Sensitizing dendritic cells can include culturing progenitor cells, dendritic cells differentiated in vitro, and / or dendritic cells differentiated in vivo in the presence of TLR ligands.

[0062] In some embodiments, the dendritic cells are sensitized with the TLR ligand ex vivo for about 1 to about 24 hours. In some embodiments, dendritic cells are sensitized ex vivo with a TLR ligand for about 3 to about 24, about 6 to about 24, about 9 to about 24, about 12 to about 24, about 15 to about 24, about 18 to about 24, about 21 to about 24, about 1 to about 21, about 3 to about 21, about 6 to about 21, about 9 to about 21, about 12 to about 21, about 15 to about 21, about 18 to about 21, about 1 to about 18, about 3 to about 18, about 6 to about 18, about 9 to about 18, about 12 to about 18, about 15 to about 18, about 1 to about 15, about 3 to about 15, about 6 to about 15, about 9 to about 15, about 12 to about 15, about 1 to about 12, about 3 to about 12, about 6 to about 12, about 9 to about 12, about 1 to about 9, about 3 to about 9, about 6 to about 9, about 1 to about 6, about 3 to about 6, or about 1 to about 3 hours.

[0063] In embodiments of in vitro differentiation of progenitor cells, dendritic cells may be sensitized before in vitro culturing of progenitor cells under conditions effective to induce differentiation. In some embodiments, dendritic cells may be sensitized after in vitro culturing of progenitor cells under conditions effective to induce differentiation. Sensitizing dendritic cells may occur simultaneously with in vitro culturing of progenitor cells under conditions effective to induce differentiation.

[0064] The methods disclosed herein include culturing sensitized dendritic cells in vitro with lipids activated by non-classical inflammasomes. Suitable lipids activated by non-classical inflammasomes are described herein. Cultivating sensitized dendritic cells may include culturing progenitor cells, dendritic cells differentiated in vitro, and / or dendritic cells differentiated in vivo in the presence of lipids activated by non-classical inflammasomes.

[0065] In some embodiments, the sensitized dendritic cells are cultured ex vivo with non-classical inflammasome activating lipids for between about 1 to about 48 hours. In some embodiments, the progenitor cells are cultured for about 6 to about 48, about 12 to about 48, about 18 to about 48, about 24 to about 48, about 30 to about 48, about 36 to about 48, about 42 to about 48, about 1 to about 42, about 6 to about 42, about 12 to about 42, about 18 to about 42, about 24 to about 42, about 30 to about 42, about 36 to about 42, about 1 to about 36, about 6 to about 36, about 12 to about 36, about 18 to about 36, about 24 to about 36, about 30 to about 36, about 1 to about 30, about 6 to about 30, about 12 to about 30, about 18 to about 30, about 24 to about 30, about 1 to about 24, about 6 to about 24, about 12 to about 24, about 18 to about 24, about 1 to about 18, about 6 to about 18, about 12 to about 18, about 1 to about 12, about 6 to about 12, or about 1 to about 6 hours.

[0066] In some embodiments, the dendritic cells sensitized with lipids activated by non-classical inflammasomes are cultured ex vivo and the dendritic cells are sensitized ex vivo with TLR ligands simultaneously. In some embodiments, the dendritic cells sensitized with lipids activated by non-classical inflammasomes are cultured ex vivo after the dendritic cells are sensitized ex vivo with TLR ligands.

[0067] The methods disclosed herein include loading dendritic cells with an immunogen. Suitable immunogens are described herein. Loading dendritic cells with an immunogen may include culturing dendritic cells with the immunogen.

[0068] In some embodiments, loading the dendritic cells with the immunogen can be performed for about 1 to about 24 hours. In some embodiments, loading the dendritic cells with the immunogen can be performed for about 3 to about 24, about 6 to about 24, about 9 to about 24, about 12 to about 24, about 15 to about 24, about 18 to about 24, about 21 to about 24, about 1 to about 21, about 3 to about 21, about 6 to about 21, about 9 to about 21, about 12 to about 21, about 15 to about 21, about 18 to about 21, about 1 to about 18, about 3 to about 18, about 6 to about 18, about 9 to about 18, about 12 to about 18, about 15 to about 18, about 1 to about 15, about 3 to about 15, about 6 to about 15, about 9 to about 15, about 12 to about 15, about 1 to about 12, about 3 to about 12, about 6 to about 12, about 9 to about 12, about 1 to about 9, about 3 to about 9, about 6 to about 9, about 1 to about 6, about 3 to about 6, or about 1 to about 3 hours.

[0069] In some embodiments, loading dendritic cells with an immunogen is performed simultaneously with culturing dendritic cells ex vivo with lipids that activate non-classical inflammasomes. In some embodiments, loading dendritic cells with an immunogen is performed after culturing dendritic cells ex vivo with lipids that activate non-classical inflammasomes.

[0070] In some embodiments of the method of producing a colony of therapeutic dendritic cells and / or the method of inducing an adaptive immune response, the dendritic cells and / or progenitor cells are frozen. In some embodiments, the dendritic cells and / or progenitor cells are frozen before being loaded with an immunogen. In some embodiments, the dendritic cells are frozen after being loaded with an immunogen.

[0071] Methods of obtaining dendritic cells and loading dendritic cells with, for example, cancer immunogens are described in the art, for example, in US20060134067A1, US9694059B2, US9962433B2, US20080254537A1, US9701942B2, US20060057129A1, US20160263206A1, US20150352200A1, US20070292448A1, US6251665B1, WO2003010292A3, US2017036325A1, US20040197903A1, and US10731130B2.

[0072] TLR ligands

[0073] 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 receptors (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 diAMP, and cyclic diGMP.

[0074] In some embodiments, the TLR ligand is selected from a TLR1 ligand, a TLR2 ligand, a TLR3 ligand, a TLR4 ligand, a TLR5 ligand, a TLR6 ligand, a TLR7 ligand, a TLR8 ligand, a TLR9 ligand, a TLR10 ligand, a TLR11 ligand, a TLR12 ligand, a TLR13 ligand, and combinations thereof.

[0075] In some embodiments, the TLR ligand is a TLR4 ligand. In some embodiments, the TLR4 ligand is LPS. In some embodiments, the TLR4 ligand is MPLA.

[0076] Oxidized phospholipids

[0077] As used herein, the term "non-classical inflammasome-activating lipid" refers to a lipid that can cause an inflammatory response in the caspase 11-dependent inflammasome of a cell. Exemplary "non-classical inflammasome-activating lipids" include PAPC, oxPAPC, and oxPAPC-like (e.g., HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC, POVPC, PGPG), and Rhodo LPS (LPS-RS or LPS from Rhodobacter sphaeroides).

[0078] As used herein, the term "oxPAPC" or "oxidized PAPC" refers to a lipid produced by oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), which produces a mixture of oxidized phospholipids containing fragmented or full-length, oxidized sn-2 residues. Well-characterized oxidative fragmentation species include five-carbon sn-2 residues carrying ω-aldehyde or ω-carboxyl groups. Oxidation of arachidonic acid residues also produces phospholipids containing esterified isoprostane. oxPAPC includes HOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC classes, as well as other oxidation products present in oxPAPC.

[0079] In some embodiments, non-classical inflammasome activating lipids include a class of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC).

[0080] The oxPAPC class is known and described in the art, see, e.g., 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.

[0081] In some embodiments, non-classical inflammasome-activating 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-hydroxyhept-6-enoyl]oxy-3-hexadecanoyloxypropyl]-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-octenoyl)-sn-glycero-3-phosphocholine (HOOA-PC), 2-[[(2R)-2-[(E)-5,8-dioxooct-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylammonium (KOOA-PC), [(2R)-3-hexadecanoyloxy [(2R)-2-(4-carboxybutyryloxy)-3-hexadecanoyloxypropyl]2-(trimethylammonio)ethyl phosphate (PGPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[2-[(Z)-oct-2-enyl]-5-oxocyclopent-3-ene-1-ylidene] ... [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentylidene]methyl]oxiran-2-yl]butyryloxy]propyl]2-(trimethylammonio)ethyl phosphate (PECPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentylidene]methyl]oxiran-2-yl]butyryloxy]propyl]2-(trimethylammonio)ethyl phosphate (PEIPC), or a combination thereof.

[0082] In some embodiments, the non-classical inflammasome activating lipid comprises [(2R)-2-(4-carboxybutanoyloxy)-3-hexadecanoyloxypropyl] 2-(trimethylammonio)ethyl phosphate (PGPC).

[0083] In some embodiments, the oxPAPCs are oxPAPCs listed in Table 1, or a combination thereof.

[0084] Table 1. Oxidized PAPC molecular classes identified in Ni et al., and the 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 represents a phosphatidylcholine lipid containing 36 carbons and 4 double bonds. When the fatty acid characteristics and sn positions are known, as in our case, a slash delimiter is used (e.g., PC 16:0 / 20:4). Since there is no unified nomenclature available for oxidized lipids, the shorthand notation provided by the LPPtiger tool is used

[28] . Short-chain oxidized lipids are represented by the corresponding termini enclosed within angle brackets (e.g., “<” and “>”), and the truncation site represented by the number of carbon atoms (e.g., <COOH@C9> and <CHO@C12). For long-chain products, our recommendation is to indicate the number of oxygen additions after the fully identified parent lipid when the type of addition is unknown (e.g., PC16:0 / 20:4+1O), or to indicate the known functional groups within parentheses (e.g., PC16:0 / 20:4[1xOH@C11]). Ni et al., “Evaluation of Air Oxidized PAPC: A MultiLaboratory Study by LC-MS / MS,” Free Radical Biology and Medicine 144:156–66 (2019) at 2.7.

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094] Immunogen

[0095] "Immunogen" and "antigen" are used interchangeably and refer to any compound to which a cellular or humoral immune response is directed. Non-living immunogens include, for example, inactivated immunogens, subunit vaccines, recombinant proteins or peptides, etc. The adjuvants disclosed herein can be used with any suitable immunogen. Exemplary immunogens of interest include those composed of or derived from viruses, mycoplasmas, parasites, protozoa, or prions, etc. Thus, the immunogen of interest may be derived from, but is 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, bacteria of the genus Mycobacterium, amoeba, Plasmodium, and / or Trypanosoma cruzi.

[0096] The immunogen of interest is expressed by diseased target cells (e.g., neoplastic cells, infected cells) and expressed or not expressed in lower amounts in other tissues. Examples of target cells include cells from neoplastic diseases, including, but not limited to, sarcomas, lymphomas, leukemias, carcinomas, melanomas, breast cancer, prostate cancer, ovarian cancer, cervical cancer, colon cancer, lung cancer, glioblastomas, and astrocytomas. Alternatively, target cells can be infected by, for example, viruses, mycoplasmas, bacteria, parasites, protozoa, and prions. Thus, the immunogen of interest may be derived from, but is 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 virus, rhinovirus, respiratory syncytial virus, cytomegalovirus, adenovirus, Mycoplasma pneumoniae, Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, bacteria of the genus Mycobacterium, amoeba, Plasmodium, and Trypanosoma cruzi.

[0097] In some embodiments, infection with an infectious agent is associated with the development of cancer. See, e.g., Kuper et al., "Infections as a Major Preventable Cause of Human Cancer," Journal of International Medicine 249(S741):61-74 (2001).

[0098] In addition to tumor antigens and antigens of infectious agents, tumor suppressor gene products including but not limited to p53, BRCA1, BRCA2, retinoblastoma, and TSG101, or mutants of oncogene products such as but not limited to RAS, WT, MYC, ERK, and TRK can also provide target antigens for use according to the present disclosure. The target antigen may be a self-antigen, such as an antigen associated with a cancer or neoplastic disease. In one embodiment, the immunogen is a peptide of a heat shock protein (hsp)-peptide complex from a diseased cell, or the hsp-peptide complex itself.

[0099] 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, for example, leukemias such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia, AIDS-related cancers such as Kaposi's sarcoma (Kaposi's sarcoma); breast cancer; bone cancer such as osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, giant cell tumor, adamantinomas, and chordomas; brain cancer such as meningioma, glioblastoma, low grade astrocytoma, oligodendroglioma, pituitary tumor, Schwannomas, and metastatic brain cancer; head and neck cancer including various lymphomas such as mantle cell lymphoma, non-Hodgkin's lymphoma, adenoma, squamous cell carcinoma, laryngeal cancer, gallbladder and bile duct cancer, retinal cancer such as retinoblastoma, esophageal cancer, gastric cancer, multiple Myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, lung cancer, bladder cancer, prostate cancer, lung cancer (including non-small cell lung cancer), pancreatic cancer, sarcoma, Wilms' tumor, cervical cancer, head and neck cancer, skin cancer, nasopharyngeal cancer, liposarcoma, epithelial cancer, renal cell carcinoma, gallbladder adenocarcinoma, parotid tumors, endometrial sarcoma, multidrug-resistant cancers; 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.

[0100] Immunogens such as cancer immunogens and their use, for example, in loading dendritic cells are known and described in the art. See, for example, Michael J. P. Lawman and Patricia D. Lawman (eds.) "Cancer Vaccines, Methods and Protocols" Methods in Molecular Biol. 1136 (2014); Chiang et al., "Whole Tumor Antigen Vaccines: Where Are We?" Vaccines (Basel) 3 (2): 344–72 (2015); Thumann et al., "Antigen Loading of Dendritic Cells with Whole Tumor Cell Preparations," J. Immunol. Methods 277: 1–16 (2003); Kamigaki et al., "Immunotherapy of Autologous Tumor Lysate-Loaded Dendritic Cell Vaccines by a Closed-Flow Electroporation System for Solid Tumors," Anticancer Immunology 2003. Res. 33:2971–6 (2013); US3823126A; US3960827A; and US4160018A.

[0101] In some embodiments, the immunogen is a cancer antigen. In some embodiments, the cancer antigen is selected from tumor lysates, apoptotic bodies, peptides, tumor RNA, tumor-derived exosomes, tumor-DC fusions, or a combination thereof.

[0102] In some embodiments, the immunogen is a whole tumor lysate.

[0103] In some embodiments, whole tumor lysates are prepared by irradiation, boiling, and or freeze-thaw lysis.

[0104] In some embodiments, the immunogen is autologous. In some embodiments, the immunogen is allogeneic.

[0105] In some embodiments of the methods of inducing an immune response in a subject, the immunogen is a tumor lysate derived from a cell donor.

[0106] Cell donors and subjects

[0107] The terms "patient" and "individual" or "subject" are used interchangeably herein and refer to a mammalian subject to be treated, preferably a human patient. In some cases, the methods disclosed herein can be used in experimental animals, veterinary applications, and for the development of animal models of disease, including but not limited to rodents including mice, rats, hamsters, and primates.

[0108] In some embodiments, the cell donor and / or the subject is a mammalian subject.The term "mammal" as used herein is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and farm animals.

[0109] In some embodiments, the cell donor and / or the subject is a human subject.

[0110] In some embodiments of the method of inducing an immune response in a subject, the cell donor is a subject. In some embodiments, the cell donor is not a subject. In some embodiments, the progenitor cells and / or the dendritic cells differentiated in vivo are autologous. In some embodiments, the progenitor cells and / or the dendritic cells differentiated in vivo are allogenic.

[0111] Application

[0112] In the methods disclosed herein, a population of therapeutic dendritic cells is administered to a subject. In some embodiments, a therapeutically effective amount of live dendritic cells is administered to a subject.

[0113] The dosage of the population of therapeutic dendritic cells disclosed herein will vary depending on the nature of the immunogen and the condition of the dendritic cells, but should be sufficient to enhance the efficacy of the live dendritic cells in eliciting an immunogenic response. For both therapeutic and prophylactic treatments, the amount of live dendritic cells administered may range from 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1x10 10 or 1×10 11 The dendritic cells of the present disclosure are generally non-toxic and are generally administered in relatively large amounts as living cells without causing life-threatening side effects.

[0114] In some embodiments, the method comprises isolating cells from a subject, preparing, processing, culturing, and reintroducing them into the same patient before and after cryopreservation as described herein.

[0115] The administration of the population of therapeutic dendritic cells disclosed herein is by any suitable means to produce a cell concentration effective to improve, reduce, or stabilize cancer. The population of therapeutic dendritic cells can be provided in a dosage form suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, intravascular, intratumoral or intraperitoneal) administration routes.

[0116] Human doses are initially determined by extrapolating from the amount of a population of therapeutic dendritic cells disclosed herein for use in mice or non-human primates, as will be appreciated by those skilled in the art, and it is routine practice in the art to modify doses for humans as compared to animal models. For example, doses may be about 1×10 3 , 1×10 4 , 1×10 5 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 About 1×10 11 cells or more.

[0117] "Suitable dosage level" refers to a dosage level that provides a therapeutically reasonable balance between pharmacological efficacy and deleterious effects (e.g., sufficient immunostimulatory activity conferred by administration of the dendritic cells disclosed herein, and sufficiently low levels of macrophage stimulation). For example, the dosage level can be related to the peak or average serum level of anti-immunogen antibodies produced in a subject, for example, after administration of a specific dosage level of an immunogenic composition (including dendritic cells disclosed herein).

[0118] As defined herein, a "therapeutically effective" amount (i.e., an effective dose) of a compound or agent refers to an amount sufficient to produce a therapeutically (e.g., clinically) desired result. The composition may be administered from one or more times per day to one or more times per week; including once every other day. Those skilled in the art will appreciate that certain factors may affect the dose and time required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatments, the overall health and / or age of the subject, and the presence of other diseases. In addition, treatment of a subject with a therapeutically effective amount of live dendritic cells disclosed herein may include a single treatment or a series of treatments.

[0119] The population of therapeutic dendritic cells disclosed herein is administered parenterally by injection, infusion or transplantation (subcutaneous, intravenous, intramuscular, intratumoral, intravascular, intraperitoneal), in a dosage form, formulation or by a suitable delivery device or implant containing a conventional non-toxic pharmaceutically acceptable carrier. The formulation and preparation of such carriers are well known to those skilled in the art of pharmaceutical formulations. The formulation can be found in Remington: The Science and Practice of Pharmacy, as described above.

[0120] As used herein, "pharmaceutically acceptable" components / carriers, etc. are components / carriers that are suitable for use in humans and / or animals without undue adverse side effects (e.g., toxicity, irritation, and allergic response) and are commensurate with a reasonable benefit / risk ratio.

[0121] Provided herein are methods for treating cancer or a symptom thereof, the methods comprising administering a population of therapeutic dendritic cells. Thus, one embodiment is a method for treating a subject suffering from or susceptible to cancer. The method comprises the steps of administering to a subject a therapeutic amount of a population of therapeutic dendritic cells disclosed herein at a dose sufficient to treat the disease or disorder or a symptom thereof, under conditions that treat the disease or disorder.

[0122] As used herein, an "effective amount" refers to an amount that provides a therapeutic or prophylactic benefit.

[0123] As used herein, the term "treating" a disease refers to reducing the frequency or severity of at least one sign or symptom of a disease or disorder, such as cancer, experienced by a subject.

[0124] "Treatment" is an intervention intended to prevent the development of a disease or to alter the pathology or symptoms of a disease. Thus, "treatment" refers to both therapeutic treatment and prophylactic or preventive measures. "Treatment" may also refer to palliative care. Patients in need of treatment include patients who already have a disorder as well as patients for whom the disorder is to be prevented. Thus, "treating" or "treatment" of a state, disorder, or condition may include: (1) preventing or delaying the onset of clinical symptoms of a state, disorder, or condition that develops in a human or other mammal that may be subject to or susceptible to the state, disorder, or condition but has not yet experienced or exhibited clinical or subclinical symptoms of the state, disorder, or condition; (2) inhibiting a state, disorder, or condition, i.e., preventing, reducing, or delaying the development of the disease or its recurrence (in the case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) alleviating the disease, i.e., causing regression of the state, disorder, or condition or at least one clinical or subclinical symptom thereof. The benefit to the individual to be treated is statistically significant or at least perceptible to the patient or physician.

[0125] Thus, in the context of cancer, "treatment" may include: (1) reducing tumor size and / or number; (2) reducing the number of circulating tumor cells; (3) reducing the risk of metastasis; (4) reducing the risk of cancer development and / or recurrence.

[0126] "Modulation" of, for example, symptoms, levels or molecular biological activities, etc., refers to, for example, detectably increased or decreased symptoms or activities, etc. Such increases and decreases are observed in treated subjects compared to subjects not treated with hyperactivated DCs, wherein untreated subjects (e.g., subjects administered immunogens in the absence of adjuvant lipids) have or have developed the same or similar disease or infection as treated subjects. Such increases and decreases may 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. Adjustment can be determined subjectively or objectively, for example, by the self-assessment of the subject, by the assessment of a clinician, or by performing suitable tests or measurements, including, for example, the assessment of the degree and / or quality of immunostimulation in the subject realized by administering the dendritic cells disclosed herein. Adjustment can be instantaneous, long-term or permanent, or it is variable at a relevant time during or after the administration of the dendritic cells disclosed herein to the subject or for the test or other method described herein or in the references cited, for example, within the time described below, or from about 12 hours to about 24 or 48 hours after administering or using the adjuvant lipid disclosed herein to about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28 days, or 1, 3, 6, 9 months or more after the subject has received such immunostimulatory composition / treatment.

[0127] The present disclosure includes methods of inducing an immune response. In some embodiments, the immune response is an adaptive immune response.

[0128] In some embodiments, the immune response is a therapeutic immune response. As used herein, the term "therapeutic immune response" refers to an increase in humoral and / or cellular immunity directed against a target antigen as measured by standard techniques. Preferably, the level of immunity induced directly against the target antigen is at least 4 times, and preferably at least 5 times, the level before administration of the immunogen. The immune response can also be measured qualitatively, where it is believed that stagnation or alleviation of the progression of a neoplastic or infectious disease in a subject indicates the induction of a therapeutic immune response by means of a suitable in vitro or in vivo assay.

[0129] The methods herein include administering to a subject (including a subject identified as needing such treatment) an effective amount of a population of therapeutic dendritic cells disclosed herein to produce such an effect. Identifying a subject in need of such treatment can be a judgment of the subject or a health care professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method).

[0130] The methods of treatment disclosed herein (including prophylactic treatment) generally include administering a therapeutically effective amount of a population of therapeutic dendritic cells disclosed herein to a subject (e.g., animal, human) in need thereof, including mammals, particularly humans. Such treatment will be appropriately administered to subjects, particularly humans, who suffer from, are susceptible to cancer or its symptoms, or are at risk of cancer or its symptoms. Determining that these subjects are "at risk" is made by any objective or subjective determination of diagnostic tests or the opinions of subjects or health care providers (e.g., genetic testing, enzyme or protein markers, markers (Marker, as defined herein), and family history, etc.).

[0131] The present disclosure also provides a method for monitoring the course of treatment. The method includes determining a diagnostic marker (Marker) (e.g., any target, protein, or indicator thereof described herein, etc., regulated by a compound described herein) or a diagnostic measurement (e.g., screening, testing) level in a subject suffering from or susceptible to a cancer-related condition or its symptoms, wherein the subject has been administered a therapeutic amount of a compound described herein sufficient to treat the disease or its symptoms. The level of the marker determined in the method can be compared with the known marker level in a healthy normal control or other sick patient to determine the disease state of the subject. In some cases, a second level of the marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of the disease or the efficacy of the therapy. In some methods, before starting treatment according to the method disclosed herein, the pre-treatment level of the marker in the subject is determined; then, the pre-treatment level of the marker is compared with the level of the marker in the subject after the start of treatment to determine the efficacy of the treatment.

[0132] In some embodiments, a population of therapeutic dendritic cells disclosed herein is administered as part of a pharmaceutical composition.

[0133] In some embodiments, the pharmaceutical composition is systemically administered, for example, formulated in a pharmaceutically acceptable buffer such as normal saline. Preferred routes of administration include, for example, bladder instillation, subcutaneous, intravenous, intraperitoneal, intramuscular, intratumoral or intradermal injection of a composition providing a continuous, lasting or effective level in a patient. The therapeutic agent identified herein in a physiologically acceptable carrier in a therapeutically effective amount is used to treat a human patient or other animal. Suitable carriers and their preparations are described in, for example, Remington's Pharmaceutical Sciences by EWMartin. The amount of the therapeutic agent to be administered varies according to the method of administration, the age and weight of the patient, and the clinical symptoms of cancer. Typically, the amount will be within the range of the amount used by other medicaments in the treatment of other diseases associated with cancer, although in some cases, due to the increased specificity of the compound, a lower amount will be required. As determined by methods known to those skilled in the art, live dendritic cells are administered in a dose that enhances the immune response of the subject, or reduces the proliferation, survival, or invasion of neoplastic or infected cells.

[0134] Compositions comprising a population of therapeutic dendritic cells disclosed herein can be administered transdermally, subcutaneously, intravenously, intramuscularly, parenterally, intrapulmonarily, intravaginally, rectally, nasally or topically. Compositions can be delivered by injection, orally, by aerosol, or particle bombardment.

[0135] The pharmaceutical compositions of the populations of therapeutic dendritic cells disclosed herein can be included in a kit, container, pack, or dispenser together with instructions for administration.

[0136] Combination therapy

[0137] As used herein, the term "in combination" in the context of administering a therapy to a subject refers to the use of more than one therapy for therapeutic benefit. The term "in combination" in the context of administration also refers to the preventive use of a therapy to a subject when used with at least one additional therapy. The use of the term "in combination" does not limit the order in which the therapies (e.g., the first and second therapies) are administered to a subject. Therapies can be administered prior to (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), concurrently with, or after (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the second therapy is administered to a subject who had, has, or is susceptible to cancer. The therapies are administered to the subject sequentially and within time intervals so that the therapies can work together. In specific embodiments, the therapies are administered to the subject sequentially and within time intervals so that they provide increased benefit over administration otherwise. Any additional therapy may be administered in any order with the other additional therapies.

[0138] 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, for example, surgery, chemotherapeutic agents, immunotherapy, growth inhibitors, cytotoxic agents, agents used for radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin 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), HER1 / EGFR inhibitors (e.g., erlotinib (TARCEVA TM )), platelet-derived growth factor inhibitors (eg, GLEEVEC TM (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGF receptors, TRAIL / Apo2, and other biologically active and organic chemical agents, etc. Combinations thereof are also contemplated for use with the methods disclosed herein.

[0139] Some embodiments of the method of inducing an immune response in a subject include administering an anticancer agent to the subject. In some embodiments, the anticancer agent is a chemotherapeutic agent. In some embodiments, the anticancer agent is an immune checkpoint regulator.

[0140] Anticancer agent: In certain embodiments, the method further comprises administering an anticancer agent. In some embodiments, the anticancer agent is a chemotherapeutic agent or a growth inhibitory agent, a T cell expressing a chimeric antigen receptor, an antibody or antigen-binding fragment thereof, an antibody drug conjugate, an angiogenesis inhibitor, and combinations thereof.

[0141] In some embodiments, the anticancer agent is a chemotherapeutic agent or a growth inhibitor. For example, the chemotherapeutic agent or growth inhibitor may include an alkylating agent, an anthracycline, an antihormone, an aromatase inhibitor, an antiandrogen, a protein kinase inhibitor, a lipid kinase inhibitor, an antisense oligonucleotide, a ribozyme, an antimetabolite, a topoisomerase inhibitor, a cytotoxic agent or an antitumor antibiotic, a proteasome inhibitor, an antimicrotubule agent, an EGFR antagonist, a retinoid, a tyrosine kinase inhibitor, a histone deacetylase inhibitor, and a combination thereof.

[0142] "Chemotherapeutic agents" are compounds used in the treatment of cancer. Examples of chemotherapeutic agents may include erlotinib (TARCEVA™, Genentech / OSI Pharm.), bortezomib (VELCADE™, Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEXT™, AstraZeneca), sunitinib (SUTENT™, Pfizer / Sugen), letrozole (FEMARA™, Novartis), imatinib mesylate (imatinib), fulvestrant (FASLODEXT™, AstraZeneca), sunitinib (SUTENT™, Pfizer / Sugen), letrozole (FEMARA™, Novartis), imatinib mesylate (imatinib), fulvestrant (FASLODEXT™, AstraZeneca), fulvestrant (FASLODEXT™, AstraZeneca), fulvestrant (FASLODEXT™, AstraZeneca), fulvestrant (FEMARA™, Novartis ... mesylate (GLEEVECTM, Novartis), finasunate (VATALANIBTM, Novartis), oxaliplatin (ELOXATINTM, Sanofi), 5-FU (5-fluorouracil), folinic acid, rapamycin (Sirolimus, RAPAMUNETM, Wyeth), lapatinib (TYKERBTM, GSK572016, Glaxo Smith Kline), lonafamib (SCH 66336), sorafenib (NEXAVARTM, Bayer Labs), gefitinib (IRESSATM, AstraZeneca), AG1478, alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredepa; redopa); ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trihydroxymethylmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan); bryostatin; callystatin;CC-1065 (including its synthetic analogs of adozelesin, carzelesin, and bizelesin); candidiasis (particularly candidiasis 1 and candidiasis 8); adrenocorticosteroids (including prednisone and prednisolone); cyproterone acetate; 5α-reductases (including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat, dolastatin; aldesleukin, talc, duocarmycin, tadalafil, valproic acid ... duocarmycin) (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma 1I and calicheamicin omega 1I (Angew Chem. Intl. Ed. Engl. 1994 33: 183-186); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomysin, actinomycin, authramycin, azaserine, bleomycins, actinomycin C, carabicin, caminomycin, carzinophilin, chromomycinis, actinomycin D , daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, noramycin galamycin, 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, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, e.g., calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folinic acid; aceglatone; aldophosphamide glycosides, e.g., leucovorin; glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate 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™ 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; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANETM (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Oncology, Princeton, NJ). Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE™ (docetaxel, doxetaxel; Sanofi-Aventis); chloranmbucil; GEMZAR™ (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINETM (vinorelbine); mitoxantrone hydrochloride; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODATM); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; and any pharmaceutically acceptable salts, acids and derivatives thereof. ;

[0143] In some embodiments, chemotherapeutic agents may include alkylating agents (including monofunctional and bifunctional alkylating agents), such as thiotepa, CYTOXAN™ cyclophosphamide, nitrogen mustards, such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, oxychloride mustard, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, mustard); nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; temozolomide; and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0144] In some embodiments, the chemotherapeutic agent may include an anthracycline, such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0145] In some embodiments, chemotherapeutic agents may include antihormones, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX™; tamoxifen citrate), raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON™ (toremifine citrate); and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0146] In some embodiments, chemotherapeutic agents may include aromatase inhibitors that inhibit aromatase (regulating estrogen production in the adrenal glands), such as 4(5)-imidazoles, aminoglutethimide, MEGASE™ (megestrol acetate), AROMASIN™ (exemestane; Pfizer), formestanie, fadrozole, RIVISORT™ (vorozole), FEMARA™ (letrozole; Novartis), and ARIMIDEX™ (anastrozole; AstraZeneca); and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0147] In some embodiments, chemotherapeutic agents may include anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all-trans retinoic acid, fenretinide, and troxacitabine (1,3-dioxolane nucleoside cytosine analog); and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0148] In some embodiments, chemotherapeutic agents may include protein kinase inhibitors, lipid kinase inhibitors, or antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC-α, Ralf, and H-Ras.

[0149] In some embodiments, chemotherapeutic agents may include ribozymes such as VEGF expression inhibitors (eg, ANGIOZYME™) and HER2 expression inhibitors.

[0150] In some embodiments, chemotherapeutic agents may include cytotoxic agents or antitumor antibiotics, such as actinomycin D, dactinomycin, bleomycin, plicamycin, mitomycins such as mitomycin C, and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0151] In some embodiments, chemotherapeutic agents may include proteasome inhibitors such as bortezomib (VELCADE™, Millennium Pharm.), epoxomicins such as carfilzomib (KYPROLIST™, Onyx Pharm.), marizomib (NPI-0052), MLN2238, CEP-18770, oprozomib, and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0152] In some embodiments, chemotherapeutic agents may include anti-microtubule agents such as vinca alkaloids, including vincristine, vinblastine, vindesine, and vinorelbine; taxanes, including paclitaxel and docetaxel; podophyllotoxin; and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0153] In some embodiments, the chemotherapeutic agent may include an "EGFR antagonist," which refers to a compound that binds to EGFR or otherwise directly interacts with EGFR and prevents or reduces its signaling activity, or is referred to as "EGFR i." Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBUTIX) and reshaped human 225 (H225) (see, WO 96 / 40210, Imclone Systems Inc.); IMC-11F8, a fully human EGFR targeting antibody (Imclone); antibodies that bind to type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind to EGFR as described in U.S. Pat. No. 5,891,996; human antibodies that bind to EGFR, such as ABX-EGF or panitumumab (see, WO98 / 50433, Abgenix / Amgen); EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab), a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF-α for EGFR binding (EMD / Merck); a human EGFR antibody, HuMax-EGFR (GenMab); fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E7.6.3 and described in U.S. Pat. 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 generate immunoconjugates (see, eg, EP659439A2, Merck Patent GmbH).EGFR antagonists include small molecules, such as the compounds described in U.S. Pat. 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 publications: WO 98 / 14451, WO 98 / 50038, WO 99 / 09016, and WO 99 / 24037.Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA™ Genentech / OSI Pharmaceuticals); PD 183805 (CI 1033, 2-propenoic acid amine, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholino)propoxy]-6-quinazolinyl]-dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA™, 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4--d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol)-; (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine); CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(-dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU 5271; Pfizer); dual EGFR / HER2 tyrosine kinase inhibitors such as lapatinib (TYKERB™, GSK572016 or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[2-methylsulfonyl)ethyl]amino]methyl]-2-furyl]-4-quinazolinamine).

[0154] In some embodiments, chemotherapeutic agents may include tyrosine kinase inhibitors, including the EGFR targeted 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 kinase (Pfizer and OSI); dual HER inhibitors such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits both HER2 and EGFR overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as HER2 inhibitors available from ISIS Pharmaceuticals' antisense drug ISIS-5132, which inhibits Raf-1 signaling; non-HER-targeted TK inhibitors such as imatinib mesylate (GLEEVECTM, available from Glaxo SmithKline); multi-target tyrosine kinase inhibitors such as sunitinib (SUTENTTM, available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787 / ZK222584, available from Novartis / Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferuloyl methane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines containing a nitrothiophene moiety; PD-0183805 (Warner-Lamber); antisense molecules (e.g., those that bind to HER-encoding nucleic acids); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis / Schering AG); pan-HER inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis / Lilly);Imatinib mesylate (GLEEVECTM); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone), sirolimus (RAPAMUNETM); or as described in any of the following patent publications: U.S. Pat. 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). ;

[0155] In some embodiments, chemotherapeutic agents may include retinoids such as retinoic acid and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0156] In some embodiments, the chemotherapeutic agent may include antimetabolites. Examples of antimetabolites include folic acid analogs and folic acid antagonists (antifolate), such as dimethoate, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs, such as 5-fluorouracil (5-FU), cyclosporine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, deoxyfluridine, enocitabine, fluorodeoxyuridine; nucleoside analogs; and nucleotide analogs.

[0157] In some embodiments, the chemotherapeutic agent may include a topoisomerase inhibitor. Examples of topoisomerase inhibitors may include topoisomerase 1 inhibitors such as LURTOTECANTM and ABARELIXTM rmRH; topoisomerase II inhibitors such as doxorubicin, epirubicin, etoposide, and bleomycin; and topoisomerase inhibitor RFS 2000.

[0158] In some embodiments, chemotherapeutic agents may include histone deacetylase (HDAC) inhibitors such as vorinostat, romidepsin, belinostat, moxistat, valproic acid, panobinostat, and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0159] Chemotherapeutic agents may also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortolone pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetate, acetonide), betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, acetonide, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasone-17-propionate, fluocortolone hexanoate, fluocortolone pivalate, and flupredniidine acetate; immunoselective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and its D-isomerized form (feG) (IMULAN Bio Therapeutics, LLC); antirheumatic drugs, such as azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide, minocycline, sulfasalazine, tumor necrosis factor alpha (TNFα) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), golimumab (Simponi), interleukin 1 (IL-1) blockers such as anakinra (Kineret), T cell costimulation blockers such as abatacept (Orencia), interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERATM); interleukin 13 (IL-13) blockers such as levofloxacin; interferon alpha (IFN) blockers such as rontalizumab; β7 integrin blockers such as rhuMAb Beta7; IgE pathway blockers such as anti-M1 prime; secretory homotrimeric LTa3 and membrane-bound heterotrimeric LTa1 / β2 blockers such as anti-lymphotoxin alpha (LTa); radioisotopes (e.g., 211At, 131I, 125I, 90Y, 186Re, 188Re, 212Bi, 32P, 212Pb, and radioisotopes of Lu);Other investigational drugs include thioplatin, PS-341, phenyl butyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavins, flavanols, procyanidins, betulinic acid and its derivatives; autophagy inhibitors such as chloroquine; Δ9-tetrahydrocannabinol (dronabinol, MARINOLTM); β-lapachone; flavonoids; colchicines; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin); podophyllotoxin; tegafur (UFTORALTM); bexarotene (TARGRETINTM); bisphosphonates. acid salts such as clodronate (e.g., BONEFOS™ or OSTAC™), etidronate (DIDROCAL™), NE-58095, zoledronic acid / zoledronate (ZOMETA™), alendronate (FOSAMAX™), pamidronate (AREDIA™), tiludronate (SKELID™), or risedronate (ACTONEL™); and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE™ vaccine; perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteasome inhibitors (e.g., PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors such as oblimersen sodium (GENASENSET™); pixantrone; farnesyl transferase inhibitors such as lonafarnib (SCH 6636, SARASAR; TM ); any of the above pharmaceutically acceptable salts, acids and derivatives; and combinations of two or more of the above, such as CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, oxaliplatin (ELOXATIN TM ) in combination with 5-FU and folinic acid.

[0160] Chemotherapeutic agents may also include nonsteroidal anti-inflammatory drugs which have analgesic, antipyretic and anti-inflammatory effects. NSAIDs include non-selective inhibitors of cyclooxygenase. Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid and fenamic acid. NSAIDs are used for symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthritis, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headache and migraine, postoperative pain, mild to moderate pain due to inflammation and tissue damage, fever, intestinal obstruction, and renal colic.

[0161] Immune checkpoint modulation: In certain embodiments, immune checkpoint modulators are co-administered with hyperactivated dendritic cells. Immune checkpoints refer to inhibitory pathways of the immune system that are responsible for maintaining self-tolerance and regulating the duration and magnitude of physiological immune responses.

[0162] Certain cancer cells thrive by utilizing immune checkpoint pathways as a primary mechanism of immune resistance, particularly with respect to T cells that are specific for tumor antigens. For example, certain cancer cells may overexpress one or more immune checkpoint proteins that are responsible for suppressing cytotoxic T cell responses. Thus, immune checkpoint regulators may be administered to overcome inhibitory signals and allow and / or enhance immune attacks against cancer cells. Immune checkpoint regulators may promote immune cell responses against cancer cells by reducing, inhibiting, or eliminating signal transduction with the aid of negative immune response regulators (e.g., CTLA4), or may stimulate or enhance signal transduction of positive regulators (e.g., CD28) of immune responses.

[0163] Immunotherapeutics targeting immune checkpoint regulators can be administered to promote immune attacks on targeted cancer cells. Immunotherapeutics can be or include antibody agents targeting immune checkpoint regulators (e.g., specific for immune checkpoint regulators). Examples of immunotherapeutics include targeting CTLA-4, PD-1, PD-L1, GITR, OX40, LAG-3, KIR, TIM-3, CD28, CD40; and one or more antibody agents in CD137. Specific examples of antibody agents may include monoclonal antibodies. Certain monoclonal antibodies targeting immune checkpoint regulators are available. For example, ipilimumab targets CTLA-4; tremelimumab targets CTLA-4; pembrolizumab targets PD-1, etc.

[0164] The programmed death 1 (PD-1) protein is an inhibitory member of the extended CD28 / CTLA-4 family of T cell regulators (Okazaki et al. (2002) Curr Opin Immunol 14:391779-82; Bennett et al. (2003) J. Immunol. 170:711-8). Other members of the CD28 family include CD28, CTLA-4, ICOS, and BTLA. Two cell surface glycoprotein ligands for PD-1 have been identified, programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2). PD-L1 and PD-L2 have been shown to downregulate T cell activation and cytokine secretion when bound 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).

[0165] PD-L1 (also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1)) is a 40kDa type 1 transmembrane protein. PD-L1 binds to its receptor PD-1 found on activated T cells, B cells, and myeloid cells to regulate activation or inhibition. Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not to CD28 or CTLA-4 (Blank et al. (2005) Cancer Immunol Immunother. 54: 307-14). The binding of PD-L1 to its receptor PD-1 on T cells delivers signals of TCR-mediated activation that inhibit IL-2 production and T cell proliferation. The mechanism involves inhibition of ZAP70 phosphorylation and its binding to CD3.zeta (Sheppard et al. (2004) FEBS Lett. 574: 37-41). PD-1 signaling attenuates the PKC-θ activation loop phosphorylation caused by TCR signaling, 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 CD86 (B7-2) (Butte et al. (2008) Mol Immunol. 45: 3567-72).

[0166] It has been shown that PD-L1 expression on the cell surface is upregulated by IFN-γ stimulation. PD-L1 expression has been found in many cancers, including human lung cancer, ovarian cancer and colon cancer and various myeloma, 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). It has been shown that PD-L1 plays a role in tumor immunity by increasing apoptosis of antigen-specific T cell clones (Dong et al. (2002) Nat Med 8: 793-800). It has also been suggested that PD-L1 may be involved in intestinal mucosal inflammation, and inhibition of PD-L1 suppresses wasting disease associated with colitis (Kanai et al. (2003) J Immunol 171:4156-63).

[0167] Exemplary anti-PD1 antibodies include pembrolizumab (MK-3475, Merck), nivolumab (BMS-936558, Bristol-Myers Squibb), and pidilizumab (CT-011, Curetech LTD.). Anti-PD1 antibodies are commercially available, for example, from ABCAMTM (AB137132), BIOLEGEND TM (EH12.2H7, RMP1-14) and Affymetrix Ebioscience (J105, J116, MIH4).

[0168] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture, and transgenic biology, which are within the skill of the art. See, e.g., 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); Paper, Methods In Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and MP Calos, ed., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vol. 154 and 155 (Wu et al., ed.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, ed., Academic Press, London, 1987); Handbook Of Experimental Immunology, Vol. I-IV (DM Weir and CC Blackwell, ed., 1986); Riott, Essential Immunology, 6th edition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986); Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), (4th edition, Univ. of Oregon Press, Eugene, 2000). .

[0169] Gene: All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species to which the compositions and methods disclosed herein are applicable. It should be understood that when genes or gene products from a particular species are disclosed, the disclosure is intended to be illustrative only and is not to be construed as limiting unless the context in which it appears clearly indicates otherwise. Thus, for example, for genes or gene products disclosed herein, homologous and / or orthologous genes and gene products from other species are intended to be encompassed.

[0170] Range: In the present disclosure, various aspects of the present disclosure may be presented in the form of a range. It should be understood that the description in the form of a range is only for convenience and brevity, and should not be interpreted as an unchangeable limitation on the scope of the present invention. Therefore, the description of a range should be considered to specifically disclose all possible sub-ranges and individual numerical values ​​within the range. For example, a description of a range such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and individual numerical values ​​within the range such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0171] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0172] Unless otherwise noted, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, suitable methods and materials are described below. It should be understood and anticipated that those skilled in the art may make changes to the principles of the invention disclosed herein, and it is expected that such modifications are included within the scope of the present invention.

[0173] Example

[0174] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0175] Example 1: Hyperactivated dendritic cells stimulate persistent anti-tumor immunity to complex antigen mixtures

[0176] The ideal strategy to stimulate protective immunity is to combine the benefits of activated and pyroptotic DCs, so that activated cells will have the ability to release IL-1β while maintaining vitality. The inventors have recently identified a new activation state of DCs that exhibit these characteristics. When DCs are exposed to PAMPs (e.g., TLR ligands) and some oxidized phospholipids (DAMPs) released from dying cells, cells achieve a long-term "over-activation" state (I. Zanoni, et al. Science, vol. 352, no. 6290, pp. 1232–1236, 2016; I. Zanoni, et al. Immunity, vol. 47, no. 4, p. 697–709. e3, 2017). These oxidized lipids are referred to as oxPAPC (oxidized 1- palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine). Overactivated DCs exhibit the activity of activated DCs in terms of cytokine (e.g., TNFα) release, but they acquire the ability to also release IL-1β over the course of several days. Consistent with their role as "hyperactivated" DCs, these cells were superior to their activated counterparts in their ability to stimulate T cell responses to model antigens.

[0177] The underlying mechanism of the hyperactivated state of DCs has been defined, as discussed DAMPs (oxPAPC) can bind and stimulate the 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 lead to cell pyroptosis, but leads to the release of IL-1β from living cells. The release of IL-1β from overactivated cells is mediated by the pore-forming protein gasdermin D, which serves 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; RA 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). It is believed that repairing the plasma membrane removes the sclerotin D pore in a way that ensures cell viability (S. Rühl, et al. Science, vol. 362, no. 6417, pp. 956–960, Nov. 2018), whereas in other cases (alum stimulation), the membrane repair pathway may be overwhelmed and pyroptosis may occur. Despite the understanding of the mechanism of how IL-1β is released from living cells, the physiological benefits of the overactivated cell state in guiding adaptive immunity remain poorly defined.

[0178] Materials and methods

[0179] 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) and OT-II (Jax004194) and BALB / c (Jax 000651) mice were purchased from Jackson Labs. For syngeneic tumor models in C57BL / 6J, two melanoma cell lines were used. Parental cell line: B16.F10 and OVA expressing cell line: B16.F10OVA. For syngeneic colorectal models, the OVA expressing MC-38 cell line derived from C57BL6 murine 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.

[0180] Reagent: Escherichia coli (E. coli) LPS (serotype O55:B5-TLRGRADE TM) was purchased from Enzo and used at 1 μg / ml for cell culture or 10 μg / mouse for in vivo use. Monophosphoryl lipid A (MPLA) from Salmonella minnesota R595 (S.minnesotaR595) was purchased from Invivogen and used at 1 μg / ml for cell culture or 20 μg / mouse for in vivo use. OxPAPC was purchased from Invivogen, resuspended in preheated serum-free medium and used at 100 μg / ml for cell stimulation or 65 μg / mouse for in vivo use. POVPC and PGPC were purchased from Cayman Chemical. Reconstitution of commercially available POVPC and PGPC was performed as previously described (CLEvavold et al., Immunity, 2018 Jan 16; 48(1): 35-44). In brief, the ethanol solvent was evaporated using a gentle stream of nitrogen. The preheated serum-free medium was then immediately added to the dried lipids to a final concentration of 1 mg / ml. The reconstructed lipids were incubated at 37°C for 5-10 minutes and sonicated for 20 seconds before being added to the cells. POVPC or PGPC were used for cell stimulation at 100 μg / ml and for in vivo use at 65 μg / mouse. EndoFit chicken egg ovalbumin and OVA 257-264 peptides with endotoxin levels <1 EU / mg were purchased from Invivogen for in vivo use at a concentration of 200 μg / mouse or for in vitro use at a concentration 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, a squalene-water-in-oil adjuvant, was used instead of IFA at a working concentration of 1:2 (AddaVax: antigen).

[0181] Cell culture: BMDCs were generated by differentiating bone marrow in IMDM (Gibco), 10% B16-GM-CSF-derived supernatant, 2 μM 2-mercaptoethanol, 100 U / ml penicillin, 100 μg / ml streptomycin (Sigma-Aldrich), and 10% FBS. After 6 days in culture, BMDCs were washed with PBS and cultured at 1×10 6 Cells were re-plated at a concentration of 10 cells / ml in IMDM with 10% FBS in a final volume of 100 μl. + DC purity was assessed by flow cytometry using a BD Fortessa and was routinely above 80%. Splenic DC from mice injected with B16-FLT3 for 15 days were purified to CD11c + MHC+ Living cells were then cultured at 1x10 6 The cells were plated in complete IMDM at a concentration of 100 μl cells / ml. To induce overactivated or pyroptotic BMDCs, DCs were sensitized for 3 hours with LPS (1 μg / ml) in complete IMDM and then stimulated for 21 hours with OxPAPC or PGPC (100 μg / ml) or alum (100 μg / ml). In some cases, activated BMDCs were restimulated to plate-bound agonistic anti-CD40 for another 24 hours 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.

[0182] LDH detection and ELISA: After BMDC stimulation, fresh supernatant was clarified by centrifugation and then assayed for LDH release using the Pierce LDH Cytotoxicity Colorimetric Assay Kit (Life Technologies) according to the manufacturer's protocol. Absorbance readings were measured at wavelengths of 490nm and 680nm on a Tecan plate reader. To measure secreted cytokines, supernatants were collected, clarified by centrifugation and stored at -20°C. ELISAs for IL-1β, TNFα, IL-10, IL-12p70, IFNγ, IL-2, IL-13, IL-4 and IL-17 were performed using eBioscience Ready-SET-Go! (now ThermoFisher) ELISA kits according to the manufacturer's protocol.

[0183] Flow cytometry: After FcR blockade, 7-day BMDCs were resuspended in MACS buffer (PBS with 1% FCS and 2 mM EDTA) and stained with the following fluorescent-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), anti-H-2Kb (clone AF6-88.5). Single cell suspensions from tumor or draining inguinal lymph nodes, or skin inguinal adipose tissue were resuspended in MACS buffer (PBS with 1% FCS and 2 mM EDTA) and stained with the following fluorescently conjugated antibodies (BioLegend): anti-CD8α (clone 53–6.7), anti-CD4 (clone RM4-5), anti-CD44 (clone IM7), anti-CD62L (MEL-14), anti-CD3 (17A2), anti-CD103 (2E7), anti-CD69 clone (H1.2F3), anti-CD45 (A20 or 30F11). LIVE / DEAD TM Fixable Violet Dead Cell Stain Kit (Molecularprobes) was used to determine cell viability and cells were stained in PBS at 4°C for 20 minutes. Draining inguinal lymph node T cells were stained with OVA-peptide tetramers for 1 hour at room temperature. PE-conjugated H2K(b)SIINFEKL (OVA 257-264; SEQ ID NO: 1) and APC-conjugated IA(b)AAHAEINEA (OVA 329-337; SEQ ID NO: 2) were used. IA(b) and H2K(b) associated with CLIP peptide were used as isotype controls. Tetramers were purchased from the NIH Tetramer Core Facility. In some experiments, FITC anti-CD8.1 (clone Lyt-2.1CD8-E1) purchased from Accurate Chemical was used with tetramers. To determine the absolute number of cells, countBright counting beads (Molecularprobes) were used according to the manufacturer's protocol. Appropriate isotype controls were used as staining controls. Data were acquired on a BD FACS ARIA or BD Fortessa. Data were analyzed using FlowJo software.

[0184] Antigen uptake detection: In order to examine the antigen uptake and endocytosis ability of BMDC during different activation states (activated, overactivated or pyroptotic states), FITC-labeled chicken OVA (FITC-OVA) (Invitrogen-Molecular Probes) was used. Briefly, pretreated BMDCs were cultured with FITC-OVA or AF488-dextran (0.5 mg / ml) at 37°C or 4°C (as a control for surface binding of antigens) during 45 minutes. Then, BMDCs were washed and stained with a live / dead fixable purple dead cell staining kit (Molecular probes) to distinguish live cells from dead cells. Then, the cells were fixed with BD fixation solution and resuspended in MACS buffer (PBS with 1% FCS and 2mM EDTA). The FITC fluorescence of live cells was measured every 15 minutes using a Fortessa flow cytometer (Becton-Dickenson). Fluorescence values ​​of BMDCs cultured at 37 °C were reported as the percentage of OVA-FITC or dextran-AF488-associated cells, and the data were normalized to the percentage of OVA-FITC-associated cells cultured at 4 °C.

[0185] OVA antigen presentation assay: To measure the efficiency of OVA antigen presentation on MHC-I, cells (0.5 × 10 6 )BMDC were cultured for 2 hours. The cells were then washed with MACS buffer and stained with APC anti-mouse H-2K on ice. b antibody (clone AF6-88.5, BioLegend), and H-2K binding to the OVA peptide SIINFEKL (SEQ ID NO: 1; Clone 25-D1.16, BioLegend) b The bound PE-conjugated antibody was stained for 20 to 30 minutes. An appropriate isotype control was used as a staining control. Calculation of total surface H-2K b The percentage of cells expressing OVA peptides on MHC-I was calculated using the FlowJo software (Tree Star).

[0186] In vitro T cell stimulation of OT-I and OT-II: Splenic CD8 T cells were isolated from OT-I and OT-II mice by magnetic cell sorting using anti-CD8 beads or anti-CD4 beads (Miltenyi Biotech), respectively.+ and CD4 + T cells. Then, in the presence of 20.000 or 10.000 DC (5: 1 or 10: 1 ratio) pretreated with LPS (activation stimulant) or LPS+PGPC (overactivation stimulant) or LPS+Alum (pyroptosis stimulant) and pulsed (or not pulsed) for 2 hours with 100 μg / ml of OVA protein or SIINFEKL (SEQ ID NO: 1) peptide, the sorted T cells were seeded in 96-well plates at a concentration of 100.000 cells per well. After 5 days of cultivation, the supernatant was collected, clarified by centrifugation for short-term storage at -20°C, and cytokine measurements were performed by ELISA.

[0187] Intracellular staining: For intracellular cytokine staining, cells were stimulated for 4-5 hours with 50 ng / ml phorbol 12-myristate 13-acetate (PMA) and 500 ng / ml ionomycin (Sigma-Aldrich) in the presence of GolgiStop (BD) and brefeldin A. Cells were then washed twice with PBS and incubated with LIVE / DEAD in PBS at 4°C. TM Fixable purple or green dead cell staining kit (Molecular probes) staining for 20 minutes. The cells were washed with MACS buffer and stained for appropriate surface markers at 4°C for 20 minutes. After two washes, the cells were fixed and permeabilized for 20 minutes at 4°C using the BD Cytofix / Cytoperm kit according to the manufacturer's protocol, and then washed with 1X perm wash buffer (BD). Intracellular cytokine staining was performed for 20-30 minutes in 1Xperm buffer at 4°C with the following conjugated antibodies all purchased from BioLegend: anti-Ki67 (clone 16A8), anti-IFN-γ (clone XMG1.2), anti-TNFα (clone MP6-XT22), anti-Gata3 (16E10A23), anti-IL4 (11B11), anti-IL10 (clone JES5-16E3). Data were obtained on BD FACSARIA or BD Fortessa. Data were analyzed using FlowJo software.

[0188] CD107a degranulation assay: To evaluate CD8 + Effector antitumor activity of T cells was assessed by flow cytometry for surface exposure of the lysosomal-associated protein CD107a. Briefly, CD8 T cells from skin-draining lymph nodes of immunized mice were isolated by magnetic cell enrichment using anti-CD8 beads and columns (Miltenyi Biotech). + T cells were then sorted for CD3 on a FACS ARIA (BD) +CD8 + Live cells. Freshly sorted CD8 + T cells were expressed at 1x10 6 The cells were resuspended in complete RPMI at a concentration of 1 μg / 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 100,000 cells per well onto 10,000 MC38OVA or B16OVA tumor cells in a 96-well plate. + T cells were plated individually and stimulated with 50 ng / ml phorbol 12-myristate 13-acetate (PMA) and 500 ng / ml ionomycin (Sigma-Aldrich). After 5 hours of incubation, cells were washed with MACS buffer and incubated with LIVE / DEAD TM Fixable purple dead cell staining kit (Molecular probes), and APC anti-CD8 (clone 53-6.7, BioLegend) staining. Then, cells were fixed with BD fixative for 20 minutes at 4°C and resuspended in MACS buffer. CD107a was determined by flow cytometry on a Fortessa flow cytometer (BD). + The percentage of cells.

[0189] In vitro cytotoxicity assay: CD8 cytokines were isolated from spleen or skin inguinal adipose tissue of viable mice using anti-CD8 MACS beads and columns (Miltenyi Biotech). + Then, the enriched T cells were sorted as live CD45 + CD3 + CD8 + Cells. Purity after sorting was >97%. Tumor cell lines such as B16OVA, B16F-10 or CT26 cells were seeded onto 96-well plates (2x10 4 cells / well). 5 CD8 + T cells were inoculated onto tumor cells for 12 hours, and cytotoxicity was then assessed by LDH release assay using the Pierce LDH Cytotoxicity Colorimetric Detection Kit (Life Technologies) according to the manufacturer's protocol.

[0190] Preparation of whole tumor cell lysate: To prepare whole tumor cell lysate (WTL) for immunization, tumor cell lines were cultured in complete DMEM for 4-5 days. When cells were confluent, supernatants were collected, cells were washed and dissociated with trypsin-EDTA (Gibco). Then, tumor cell lines were plated at 5×10 6 Cells / ml were resuspended in their pooled culture supernatant and then lysed by 3 cycles of freeze-thaw.

[0191] Tumor Infiltration: To assess the frequency of tumor infiltrating lymphocytes (TILs) in immunized mice, tumors were harvested when they reached 1.8-2 cm in size. Tumors were dissociated using a tumor dissociation kit (Milteny Biotec) and a gentleMACS dissociator according to the manufacturer's protocol. After digestion, tumors were washed with PBS and passed through 70-μm and 30-μm filters. CD45 positive selection was performed using CD45 microbeads (Milteny Biotec). + Tumor-infiltrating T cells were cultured with dynabeads mouse T-Activator CD3 / CD28 (Gibco) for T cell activation and expansion.

[0192] Adoptive cell transfer: For T cell transfer, CD8 T cells were isolated from spleen or skin inguinal adipose tissue of surviving mice using anti-CD8 MACS beads and columns (Miltenyi Biotech). + Then, the enriched T cells were sorted as live CD45 + CD3 + CD8 + 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 The sorted T cells were stimulated for 24 hours in 5x10 5 activated circulating spleen or skin inguinal fat resident CD8 + T cells were transferred into naive recipient mice either intravenously or intradermally (id). Some mice received both T cell subsets.

[0193] For DC transfer, BMDCs were harvested on day 6 and 5x10 6Cells were seeded into 6-well plates. DC activation was induced by culturing with over-activation stimulants (LPS+PGPC) or activation stimulants (LPS). Tumor lysates were added to DC culture plates at a ratio of 1 DC to 2 tumor cell equivalents (i.e. 1:2) for 1 hour. Non-loaded naive DCs were used as negative controls.

[0194] Statistical analysis: Statistical significance for experiments with more than two groups was tested using two-way ANOVA with Tukey multiple comparison test correction. Adjusted p values ​​calculated with Prism (Graphpad) are marked by asterisks: <0.05 (*); <0.0005 (***); ≤0.0001 (****).

[0195] result

[0196] Overactivating stimuli upregulate several activities important for DCs to stimulate T cell immunity.

[0197] Almost all studies of DC overactivation focus on the ability of these cells to release IL-1β while maintaining viability. The spectrum of DC functions 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 pure lipid component 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). In contrast to activation stimuli that are not expected to induce the release of IL-1β, pyroptotic or overactivated stimuli promote the release of IL-1β into the extracellular medium ( Figure 1A All stimuli tested promoted the secretion of the cytokine TNFα ( Figure 1A These findings are consistent with previous work that determined that LPS-activated BMDC release TNFα but not IL-1β (I. Zanoni et al., 2016). IL-1β secretion is consistent with cell death in pyroptotic DCs as assessed by the release of cytosolic lactate dehydrogenase (LDH) ( Figure 1B ). In contrast, IL-1β occurs in hyperactivated cells in the absence of LDH release ( Figure 1B Similar BMDC behavior was observed when LPS was replaced with MPLA ( Figure 3A , 3B), MPLA is an FDA-approved TLR4 ligand used in vaccines against human papillomavirus (HPV) and hepatitis B virus (HBV). To determine whether the behavior of hyperactivated BMDCs extends to differentiated DCs in vivo, CD11c isolated from the spleens of mice injected with B16-FLT3 were examined. + Similar to the behavior of GMCSF-derived BMDC, as assessed by LDH release, treatment with LPS and PGPC resulted in the removal of TNFα and IL-1β from splenic CD11c + Release of DC ( Figure 3C , 3D ). These results indicate that the hyperactivated stimulator PGPC can be used to induce the release of IL-1β from live DC that have been differentiated in vitro or in vivo.

[0198] Several signals important for T cell differentiation were examined, for example, expression of the co-stimulatory molecules CD80, CD69, and CD40, and secretion of the p70 subunit of IL-12. CD80 surface expression was similar in DCs responding to all activation stimuli ( Figure 3E In contrast, CD40 expression is highly affected by activation stimuli. Compared with the activation stimulus LPS, the over-activation stimulus induces more CD40 expression ( Figure 1C Pyroptotic stimuli are very weak inducers of CD40 and CD69, even in the 20-30% of viable cells remaining after LPS-alum treatment ( Figure 1C and 3E When cultured on agonistic anti-CD40-coated plates, differential expression of CD40 was associated with hyperactivated DCs having the greatest capacity to secrete IL-12p70 ( Figure 1D ).

[0199] Hyperactivated BMDCs were no better than their activated counterparts at antigen capture as assessed by equivalent internalization of fluorescent ovalbumin (OVA-FITC) ( Figure 4A , 6B ), but the former cell population showed a greater abundance of OVA-derived SIINFEKL peptide on the cell surface MHC-I molecules ( Figure 1E and 4C Total surface MHC-I abundance did not differ between activated and hyperactivated cells ( Figure 3E ). In summary, compared with other stimulators of DC, overactivation stimulators show enhancement of several activities important for T cell differentiation.

[0200] Hyperactivated DCs stimulate a TH1-focused immune response with no evidence of TH2 immunity.

[0201] To evaluate the effect of DC activation state on T cell guidance, BMDCs were treated as described above and then loaded with OVA. These cells were exposed to naive OT-II or OT-I T cells. OT-II cells express T cell receptors (TCRs) specific for MHC-II restricted OVA peptides (OVA323-339), while OT-I cells express TCRs specific for 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 to TH1 response (IFNγ production) or TH2 response (IL-10, IL-4 or IL-13 production). OVA-treated BMDCs stimulated IFNγ production from OT-II T cells regardless of DC activation state. The extent of IFNγ production varied moderately between the activation stimuli tested ( Figure 1F Similarly, TNFα production by responding OT-II cells was comparable when comparing all DC activation states ( Figure 1F ). These results indicate that TH1 responses are generally induced in vitro regardless of the activation state of antigen presenting cells (APCs). In contrast, TH2 responses differ significantly when comparing DC activation states. Stimuli that induce BMDC activation (LPS) or pyroptosis (LPS+alum) promote the release of large amounts of IL-10, and IL-13, whereas overactivating stimuli result in minimal production of these TH2-associated cytokines ( Figure 1F Intracellular staining of single cells for TH1 (IFNγ and TNFα) and TH2 (IL-4 and IL-10) cytokines and the TH2 lineage-restricted transcription factor GATA3 made it possible to calculate the proportion of TH1 and TH2 cells generated by different DC activation stimuli. This analysis showed that overactivated BMDCs induced a strong bias of single T cells towards the TH1 lineage producing IFNγ ( Figure 1G and 5 ). The ratio of TH1 to TH2 cells under over-activated conditions is greater than 100:1 ( Figure 1G In contrast, all other activating stimuli induced mixed T cell responses, with pyroptotic stimuli resulting in a near 1:1 ratio of TH1 to TH2 cells ( Figure 1G ).

[0202] Use CD8 + Similar studies were performed with OT-I T cells, which showed that stimuli that overactivated BMDCs resulted in a slight enhancement of IFNγ production compared with activating or pyroptotic stimuli ( Figure 1F IL-2 production by responding OT-I cells was comparable when all DC activation states were compared ( Figure 1F ). These combined results indicate that in vitro, the hyperactivated BMDC state results in a highly TH1-biased T cell response and a slightly enhanced CD8 T cell response. Conversely, activation or pyroptosis stimuli produce mixed TH1 and TH2 responses.

[0203] Hyperactivation of inflammasome-competent DCs is sufficient to confer protective antitumor immunity.

[0204] Since DC is the main cell responsible for stimulating newborn (de novo) T cell-mediated immunity, it is attempted to determine whether the conditions of specific over-activation of DC are sufficient to confer anti-tumor immunity. This possibility is solved by adoptively transferring BMDC stimulated with different activation stimuli and WTL in vitro to mice. BMDC was selected because these cells 1) are well characterized as over-activated and 2) are considered to be a model of monocyte-derived DC, which is the most commonly used APC for DC-based immunotherapy in humans (RLSabado et al., Cell Res., vol. 27, no. 1, pp. 74–95, Jan. 2017).

[0205] BMDCs were treated with various activation stimuli, along with WTLs, and then injected subcutaneously into B16OVA tumor-bearing mice every 7 days for 3 consecutive weeks. BMDCs activated with LPS and pulsed with B16OVA WTLs provided slight protection against B16OVA-induced lethality compared to mice injected with naive BMDCs; 25-30% of mice receiving DC transfers rejected tumors and remained tumor-free for a long time after the last / third DC transfer process ( Figure 2 Note that hyperactivated BMDCs induced complete rejection of B16OVA tumors in 100% of tumor-bearing mice ( Figure 2 In these cells, the antitumor activity of hyperactivated DCs depends on inflammasomes because NLRP3 - / - and Casp1 - / - 11 - / - BMDC transfer induced only minor rejection compared to activated DC ( Figure 2). Thus, these data suggest that hyperactivated DCs are sufficient to induce durable protective antitumor immunity and that inflammasomes within DCs are essential for this process.

[0206] discuss

[0207] In this study, the immunological activity that is upregulated in the case of DCs treated with over-activation stimulants was expanded. Not only are these stimulants able to trigger IL-1β release from live cells, but over-activation stimulants also exceed other activation stimuli in their ability to induce CD40 expression and IL-12p70 secretion. In addition, cells exposed to over-activation stimuli show enhanced surface expression of MHC-peptide complexes. These common findings emphasize the over-activation nature of DCs exposed to oxPAPC or its pure component PGPC and provide evidence of enhanced ability to stimulate adaptive immunity. It was also found that over-activated DCs are actually better stimulators of T cell responses than activated or pyroptotic cells, and the most important aspect of their activity is their ability to stimulate TH1- and CTL-focused responses. In fact, stimulants that over-activate DCs result in a 100:1 ratio of TH1:TH2 cells; other strategies for DC activation do not induce such biased T cell responses.

[0208] It is noteworthy that the well-defined inflammasome stimulator alum does not show the same activity as oxPAPC or PGPC. In fact, it is well known that alum induces TH2 immunity. These findings were confirmed in this study, because alum or alum+LPS treatment induced strong TH2 immunity. A possible reason for the lack of TH1-focused immunity in alum-treated cells is based on the findings herein: alum is a poor inducer of several signals required for TH1 differentiation, such as CD40 expression and IL-12p70 secretion. Note that even when testing DCs that do not undergo pyroptosis in response to alum+LPS, CD40 expression is significantly low. The lack of high-level expression of these factors may make pyroptosis stimulators a weak inducer of TH1 response and subsequent anti-tumor immunity. Without wishing to be limited by theory, it is proposed that the TH1-focused immunity induced by overactivated DCs is caused by the activity of inflammasomes and several other characteristics of these cells. These additional features include enhanced antigen presentation ability, CD40 expression, IL-12p70 expression and increased vitality. It is possible that each of these enhanced activities is important for the function of DC as APCs and may contribute to the strong TH1-focused immune responses observed under conditions of DC hyperactivation.

[0209] These results may help explain why certain chemotherapeutic agents (e.g., oxaliplatin) induce tumor cell death and inflammasome-dependent anti-tumor T cell immunity (F. Ghiringhelli et al., Nat. Med., vol. 15, no. 10, pp. 1170–1178, 2009). Oxaliplatin is a strong stimulator of reactive oxygen species (ROS) production, which can oxidize biomembranes and produce a complex mixture of different oxidized phospholipids including PGPC. Therefore, it is possible that the protective immunity induced by oxaliplatin is caused by the activity of overactivated DCs that trigger anti-tumor T cell responses.

[0210] It was found that overactivated stimulants can be used as immunotherapy using complex mixtures of antigens. WTL is an attractive source of antigens for several reasons, the most important of which is from a practical point of view. A significant benefit of WTL-based approaches is that they alleviate the need for new antigen recognition. Although WTL-based immunotherapy offers potential benefits, previous work in the field has produced mixed results. The discovery herein that overactivated stimulants are uniquely able to assist WTL in eliciting strong anti-tumor immunity may explain the lack of success in previous work, as the strategy for DC activation discovered herein has not been considered before. On the latter, it is noteworthy that the DC overactivation strategy protects mice from lethality associated with those tumors that are sensitive to PD-1 blockade and those that are resistant to PD-1 blockade. The full spectrum of tumors that can be treated by overactivated stimulants has not yet been determined, but these studies provide indications of the value of further exploring the DC-centric strategy for cancer immunotherapy.

[0211] Example 2: Overactivated cDC1 controls tumor rejection in an inflammasome-dependent manner

[0212] Conventional dendritic cells (cDCs) are good at presenting exogenous and endogenous antigens to T cells and regulating T cell proliferation, survival, and effector functions. cDCs are divided into two major subsets, called cDC1 and cDC2. Resident cDC1 in the spleen and lymph nodes (LN) express CD8α, CD24, and XCR1, while cDC2 express CD4 and Sirpα. cDC1 is a cross-presentation of tumor-associated antigens and triggers Th1 immunity and anti-tumor CD8 + cDC2s, on the other hand, control type 2 immune responses against parasites in which Th2 immunity is activated.

[0213] To investigate whether resident cDCs can achieve a hyperactivated state, cDC1 or cDC2 from the spleen of WT mice were sorted and left untreated, treated with LPS for 20 h, or sensitized with LPS for 3 h and then treated with the hyperactivation stimuli oxPAPC or PGPC, or the pyroptosis stimuli Alum for 21 h. Splenic cDC1 died rapidly after isolation compared to splenic cDC2 as measured by their LDH release ( Fig. 6A Thus, cDC1 cells cannot be sensitized with LPS and fail to produce TNFa cytokine in response to activating stimuli (LPS), hyperactivating stimuli (LPS+OxPAPC / PGPC), or pyroptotic stimuli (LPS+Alum). Figure 6B In response to the hyperactivating stimulus (LPS+PGPC), a small amount of IL-1β release was observed ( Figure 6B Left panel). In contrast, splenic cDC2 cells were efficiently sensitized with LPS and achieved a hyperactivated state, as confirmed by their ability to produce IL-1β without undergoing cell death ( Figure 6A-6B Left figure). Since resident cDC1 is highly sensitive to ex vivo isolation and in vitro stimulation, we alternatively use the cytokine FLT3 ligand (FLT3L) to generate cDC from bone marrow (BM) progenitor cells. After 9 days in culture, cDC1 and cDC2 produced by FLT3L were sorted and then treated with activation stimuli, overactivation stimuli or cell pyroptosis stimuli as described above. As measured by their release of large amounts of IL-1β and TNFa while maintaining their viability, cDC1 and cDC2 produced by FLT3L are effectively sensitized with LPS relative to resident cDC1 cells isolated from the spleen and respond to their stimulation with LPS+PGPC, rather than with LPS+oxPAPC to achieve a hyperactivated state ( Figure 6A-6B (Right panel). These data suggest that the pure form of the oxidized phospholipid PGPC can hyperactivate both cDC1 and cDC2 subsets.

[0214] cDC1s generated by hyperactivated FLT3L exhibit more stellate dendrites than their naive, activated or pyroptotic counterparts, suggesting a higher migratory potential. Indeed, hyperactivated cDC1s and cDC2s upregulate the chemokine receptor CCR7 (CCR7), which directs migrating DCs to lymph nodes for T cell stimulation. Figure 6C-6D Together, these results suggest that cDC1 and cDC2 subsets achieve a hyperactivated state in vitro and exhibit unique properties compared to their classically activated counterparts.

[0215] The unique function of cDC1 cells is crucial in the case of cancer, where cDC1 takes up tumor antigens and cross-presents them to T cells within the tumor microenvironment (TME) or after migrating to draining lymph nodes. The fact that cDC1 becomes hyperactivated in vitro provides an indication for further exploring the value of the hyperactivated state of cDC for cancer immunotherapy. Therefore, to study the role of the hyperactivated state of cDC1 in controlling tumor rejection, mice were inoculated subcutaneously (sc) in the left back with B16OVA cells. 7, 14 and 21 days after tumor challenge, mice were left untreated or injected subcutaneously with 1×10 6 Untreated WT cDC1 (cDC1 naive), or WT cDC1 treated with LPS for 23 hours (cDC1 activated), or WT cDC1 sensitized with LPS for 3 hours and then treated with PGPC for 20 hours (cDC1 hyperactivated). All cDCs were pulsed with B16OVA tumor lysate for 1 hour before injection. Surprisingly, adoptive transfer of hyperactivated cDC1 conferred strong and lasting protection against tumor growth in mice, while naive or activated cDC1 transfer induced only minor tumor rejection ( Figure 7 ). These data present the first evidence for a superior role of the hyperactivated state of cDC1 in persistent tumor rejection.

[0216] To further confirm the crucial role of overactivated cDC1 in tumor control, we used cells lacking CD8 + cDC1 and cross-presentation defective Batf3 - / - Mouse, therefore Batf3 - / - Mice lacking anti-tumor antigen-specific CD8 + Therefore, Batf3 mice inoculated with B16OVA cells showed a significant decrease in T cell responses compared with WT mice. - / - Mice were unable to control tumor growth ( Fig. 8A ). However, when the tumor-bearing Baft3 - / - When mice were supplemented with WT cDC1 by subcutaneous injection on days 7, 14, and 21 after tumor challenge, we found that only hyperactivated cDC1, as opposed to naive or activated cDC1, could completely eliminate tumors. This protection was consistent with the Batf3 inhibitory effect when WT hyperactivated cDC1, but not WT activated or naive cDC1 cells, were injected. - / - Restored, higher frequency of tumor-infiltrating OVA-specific CD8 + and CD4 + T cell related Figures 8B-8C Together, these data provide strong evidence that overactivated cDC1s control tumor rejection by enhancing tumor infiltration of antitumor-specific T cells.

[0217] The mechanisms underlying the hyperactivated state of DCs have been well defined, as discussed oxidized phospholipids (oxPAPC / PGPC) can bind and stimulate the cytosolic pathogen recognition receptor (PRR) caspase-11. Caspase-1 / 11 stimulation leads to the activation and assembly of the NLRP3 inflammasome, which results in the release of IL-1β from living cells through the cleaved cortex D pore. To evaluate the role of IL-1β in the antitumor activity of hyperactivated cDC1, Casp1 / 11 cells defective in IL-1β secretion were used. - / - Mouse or NLRP3 - / - Mouse. Casp1 / 11 - / - NLRP3 - / - Mice were inoculated with B16OVA cells on the left back. On days 7, 14, and 21 after tumor challenge, mice were left untreated (no DC injection) or inoculated subcutaneously in the right flank with 1.10 6 Untreated WT cDC1 (cDC1 幼稚 ) or WT cDC1 treated with LPS for 23 h (cDC1 活化 ), or WT or Casp1 / 11 sensitized with LPS for 3 h and then treated with PGPC for 20 h - / - cDC1 过度活化 ). All DCs were pulsed with B16OVA tumor lysate for 1 h prior to injection. Interestingly, we found that adoptive transfer of WT hyperactivated cDC1 to Casp1 / 11 induced only minor protection compared to WT naive or activated cDC1. - / - and NLRP3 - / - Recipient mice had complete ablation of tumor growth in 100% of tumor-bearing mice. This protection was dependent on the inflammasome mechanism, as casp1 / 11β was unable to induce IL-1β secretion in response to hyperactivating stimuli (LPS+PGPC). - / - NLRP3 - / - cDC1s were unable to induce tumor rejection. In conclusion, adoptive transfer of hyperactivated DCs was sufficient to induce durable antitumor responses and recapitulated the protection observed with hyperactivation-based vaccines.

[0218] Taken together, our data have the potential to shift the paradigm in DC-based immunotherapy regarding the activation state employed in adoptive cell transfer-based immunotherapy, thereby reinvigorating attempts to “tune” DCs in vitro to generate effective cancer immunotherapies.

[0219] Example 3: Oxidized phospholipids induce over-activated cDC1 and cDC2 cells

[0220] Almost all studies evaluating the state of cell-hyperactivation have focused on the ability of bone marrow-derived DC (BMDC) 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 indicate that monocyte-derived macrophages, rather than DC, are responsible for inflammasome activation and IL-1β secretion

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

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

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

[37] ,

[38] . On the other hand, cDC2 control type 2 immune responses against parasites that activate Th2 immunity. To determine whether the behavior of hyperactivated DCs extends to cDC1 or cDC2, we isolated cDC1 or cDC2 from the spleens of FLT3-DCs or from wild-type naive mice ( Fig. 16B Similar to the behavior of FLT3-derived DCs, treatment with LPS and PGPC, and to a lesser extent with LPS and oxPAPG, resulted in the release of TNFα and IL-1β from FLT3-derived cDC1 and cDC2 in the absence of cell death, as assessed by LDH release ( Fig.11A ). These data suggest that PGPCs are the bioactive components of oxPAPCs that induce hyperactivation of cDC1 and cDC2. We also observed similar behavior of splenic cDC2s, which produced IL-1β in response to the pyroptotic stimuli LPS and alum accompanied by pyroptotic cell death, and also produced IL-1β in response to the hyperactivation stimuli LPS and PGPCs in the absence of cell death ( Fig. 16C ). In contrast, splenic cDC1s produce minimal amounts of IL-1β in response to pyroptotic or hyperactivating stimuli, as these cells are very sensitive to postsort cell death and cannot be sensitized by LPS ( Fig. 16C ). Taken together, these results indicate that hyperactivating stimuli can be used to induce the release of IL-1β from live DCs that have been differentiated in vitro or in vivo. For practical reasons, we continue to use FLT3-derived DCs as a source of DCs in this article.

[0221] Example 4: Hyperactivated DCs enhance CTL responses in an inflammasome-dependent manner

[0222] IL-1β is a key regulator of T cell differentiation, generation of long-lived memory T cells, and effector function

[12] –

[14] . We wondered whether hyperactivated DCs that produce IL-1β over the course of several days in the dLN could enhance CD8+ T cell stimulation. To test this idea, we sought to adoptively transfer DCs loaded with OVA protein subcutaneously (sc) and then measure 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 H2kb molecules. We found that all DCs in different states took up OVA to a similar extent, as demonstrated by internalization of equivalents of fluorescent ovalbumin (OVA-FITC). However, we found that both activated DCs and hyperactivated DCs sensitized with LPS or with CpG exhibited enhanced cross-presentation of SIINFEKL when loaded with OVA protein compared to their naive counterparts. This is consistent with previous work showing that DC maturation enhances their antigen-presenting capacity. Surprisingly, the pyroptotic stimulus alum strongly reduced the cross-presentation capacity of DCs, suggesting that pyroptotic DCs are not suitable for optimal T cell stimulation. Thus, when 1.106 DCs of OVA-loaded naive, activated, pyroptotic, or hyperactivated DCs were injected into WT mice, we observed that hyperactivated DCs induced the highest frequency and absolute number of SIINFEKL+CD8+ T cells in the dLN of recipient mice compared with naive, activated, or pyroptotic DCs ( Fig. 12A and 17B ). Since injection of NLRP3- / - DCs treated with LPS+PGPC induced weak OVA-specific T cell responses, the enhanced CD8+ T cell responses mediated by overactivated DCs were dependent on inflammasome activation.

[0223] Example 5: Hyperactivating stimuli enhance memory T cell generation and increase antigen-specific IFNγ effector responses in an inflammasome-dependent manner

[0224] We hypothesized that hyperactivation stimulants may represent potent adjuvants that recapitulate the effects of hyperactivated DC injection. To test this possibility, mice were immunized subcutaneously with OVA alone, or OVA plus activation stimulants (LPS), or OVA plus hyperactivation stimulants (LPS+oxPAPC or PGPC). 7 and 40 days after immunization, the generation of memory and effector T cells in dLN was assessed by flow cytometry using CD44 and CD62L markers that distinguish T effector cells (Teff) as CD44lowCD62Llow, T effector memory cells (TEM) as CD44hiCD62Llow, and T central memory cells (TCM) as CD44hiCD62Lhi

[47] . 7 days after immunization, hyperactivation stimulants were superior to activation stimulants ( Fig.13A Above and Figures 18A-18B ). In addition, at this early time point, hyperactivating stimuli induced the highest abundance of CD8+TEM ( Fig.13A The middle picture and Figures 18A-18B ). 40 days after immunization, abundant TCM cells were observed in mice exposed to the hyperactivating stimulus, however, these cells were less abundant in mice immunized with OVA alone or with OVA and LPS ( Fig.13A (Figure 2). Conversely, 40 days after immunization, Teff and TEM cells were more abundant in mice immunized with OVA alone or with OVA and LPS compared to mice immunized with OVA plus. Thus, these data suggest that the hyperactivating stimuli oxPAPC and PGPC enhance the extent to which effector and memory T cells are generated. Furthermore, the increase in the frequency of Teff cells 7 days after immunization was associated with an enhanced IFNγ response of CD8+ T cells isolated from dLNs of mice immunized with OVA plus hyperactivating stimuli when restimulated ex vivo in the presence of naive BMDC loaded with OVA ( Fig. 18C Furthermore, when total CD8+ T cells were isolated from mice immunized with an overactivating stimulant and co-cultured with the B16 tumor cell line expressing OVA (B16OVA), CD8+ T cells exhibited enhanced degranulation activity compared to CD8+ T cells isolated from mice immunized with OVA alone or OVA plus LPS ( Fig. 13B and 18D ), indicating that overactivation stimuli enhance CTL function.

[0225] In order to evaluate the antigen specificity of T cells caused by subcutaneous immunization with different activation stimulants, mice were injected with single OVA, or OVA and activation stimulant (LPS), or OVA and pyroptosis stimulant (LPS+alum) or OVA and overactivation stimulant (LPS+oxPAPC or PGPC). Alternatively, mice were subcutaneously immunized with LPS+PGPC without OVA antigen. After immunization for 7 days, CD8+T cells were separated from the skin dLN of immune mice and stimulated in vitro for 7 days with the immature BMDC loaded (or not loaded) with OVA to enrich OVA specific T cell subsets. The T cell effector function of OVA specific T cells was evaluated by intracellular staining of IFNγ. TCR specificity was evaluated by staining with MHC restricted OVA peptide tetramers. H2kb restricted SIINFEKL (OVA257-264) peptide tetramers were used. The frequency of tetramer+IFNγ+ double positive cells was measured for CD4+ and CD8+T cell subsets. Significantly, OVA and hyperactivation stimuli were superior in inducing antigen-specific T cells, as oxPAPC- or PGPC-based immunization resulted in the highest frequency of tetramer+IFNγ+ responses in the case of restimulation of CD8+ T cells with OVA antigen ( Fig. 13C ). In contrast, the pyroptotic stimulus (LPS+alum) was the weakest inducer of the antigen-specific IFNγ response ( Fig. 13C These results are consistent with previous studies showing that alum is an effective adjuvant for promoting humoral immunity and Th2 responses, but not Th1 or CTL responses [39,42,43].

[0226] Previous studies have shown that antigen-specific T cell responses can be enhanced by co-immunization with recombinant IL-1β [47, 13], a cytokine whose biological activity is naturally controlled by the inflammasome. However, despite the fact that both hyperactivation and pyroptotic stimuli induce IL-1β secretion, how can hyperactivation stimuli, but not pyroptotic stimuli, induce higher antigen-specific T cells? To determine whether inflammasome-mediated events control T cell responses generated by hyperactivation stimuli, a side-by-side comparison of T cell activity was performed in WT and NLRP3- / - mice. Of note, we found that the enhancement of antigen-specific responses induced by hyperactivation by CD8+ T cells requires NLRP3 ( Fig. 13C ). Thus, these data suggest that non-pyroptotic and pyroptotic inflammasome activation following immunization with hyperactivating or pyroptotic stimuli, respectively, induce distinctly different adaptive immune T cell controls.

[0227] Our previous results using the DC injection strategy showed that DCs stimulated with pyroptotic stimuli lost their ability to migrate to adjacent dLNs and stimulate T cell activation, whereas DCs exposed to hyperactivating stimuli hypermigrated to dLNs and enhanced CTL responses ( Figures 12A-12B ). However, it is unknown whether endogenous DCs can achieve hyperactivation in vivo following immunization with hyperactivating stimuli. To evaluate this, we generated mouse chimeras using Zbtb46DTR and WT mice or Zbtb46DTR and NLRP3- / - mice or Zbtb46DTR and Casp1 / 11- / - mice. To this end, 4-week-old CD45.1-irradiated mice were reconstituted with mixed bone marrow of 80% Zbtb46DTR and 20% WT mice or 20% NLRP3- / - or 20% Casp1 / 11- / - mice on a CD45.2 background as previously described

[51] . Six weeks after reconstitution, the efficacy of BM reconstitution was assessed in all mice 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, generating mice carrying WT or inflammasome-deficient (NLRP3- / - or Casp1 / 11- / -) DCs that could or could not be overactivated, respectively. To test the effect of endogenous DC overactivation on CD8+ T cell responses, all chimeric mice were immunized subcutaneously with OVA plus LPS+PGPCs after 3 consecutive DT injections. 7 days after immunization, CD8+ T cell responses from dLNs were assessed. Interestingly, we found that the abundance of Teff CD8+ T cells was strongly reduced in chimeric mice carrying DCs that could not be overactivated, such as NLRP3- / - and Casp1 / 11- / - chimeric mice, compared with chimeric mice carrying WT DCs that could be overactivated ( Fig.13D , Fig.19A ). In addition, we found that the frequency of SIINFEKL+CD8+ T cells in dLN or spleen was reduced in NLRP3- / - and Casp1 / 11- / - chimeric mice carrying DCs that were incapable of hyperactivation, whereas high SIINFEKL+CD8+ cells were observed in chimeras carrying WT DCs ( Fig.13E , Fig.19B ). Thus, these data clearly show that: 1) endogenous DC can achieve a hyperactivated state in vivo and enhance CTL responses following immunization with a hyperactivating stimulus, and 2) inflammasome activation within endogenous DC is critical for hyperactivation-mediated protective CTL responses.

[0228] Example 6: Hyperactivated DC entry into lymphoid tissue is essential for hyperactivation-mediated CTL responses

[0229] We have previously shown that DCs stimulated with hyperactivating stimuli migrate excessively to dLNs and enhance CTL responses ( Figures 12A-12B To assess whether hyperactivation-mediated CTL responses require endogenous hyperactivated DCs to enter dLNs, we generated mouse chimeras as described above using Zbtb46DTR and WT or Zbtb46DTR and CCR7- / - BM ( Fig.13D In conclusion, endogenous trafficking of hyperactivated DCs to dLNs is essential for hyperactivation-mediated CTL function.

[0230] Example 7: Hyperactivation stimuli can use complex antigen sources to stimulate pyroptotic T cell-mediated anti-tumor immunity

[0231] Current efforts to stimulate anti-tumor immunity include strategies that activate resident T cell populations (e.g., PD-1 blockade) or personalized cancer vaccine strategies that stimulate the generation of new 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 are being made to improve the identification of neoantigens that can be used in pure form to elicit T cell-mediated anti-tumor immunity. Although these efforts have been successful [50, 49, 51], the path to neoantigen identification requires channels for the discovery of mutated and aberrantly expressed TSAs

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

[0232] In order to solve the possibility that over-activation stimulants can be adjuvants to WTL, mice were immunized in the right flank with single WTL or WTL mixed with activation stimulant LPS or transition activation stimulant LPS+oxPAPC or LPS+PGPC. The source of WTL is B16OVA cells. 15 days after immunization, mice were subcutaneously attacked in the upper left back with parental B16OVA cells. Unimmunized mice or mice immunized with single WTL did not show any protection, and all mice carried large tumors and died on the 24th day after tumor inoculation ( Fig. 20A ). 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 ( Fig. 20A), indicating that protective immunity is not provided by stimuli that activate DCs alone. In contrast, WTL immunization in the presence of LPS and oxPAPC induced a significant delay in tumor growth and resulted in robust protection against subsequent lethal re-challenge with parental B16OVA tumor cells; 50% of immunized mice were fully protected ( Fig. 20A To determine whether the protective response induced by oxPAPC was associated with a T cell response, tumors were harvested from mice that received each activation stimulus. Tumors from mice immunized with LPS+oxPAPC contained a high abundance of CD4+ and CD8+ T cells compared to LPS immunization (Figure S7B). Furthermore, when equal numbers of T cells from these tumors were compared, oxPAPC-based immunization resulted in the highest amounts of intratumoral T cells secreting IFNγ in the presence of anti-CD3 and anti-CD28 stimulation (Figure S7B). Fig. 20C ). Therefore, the better restriction of tumor growth induced by the overactivating stimuli (LPS+oxPAPC) is consistent with the infiltration of inflammatory T cells into the tumor.

[0233] Note that the protective phenotypes of oxPAPCs were replaced by those elicited 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 ( Fig. 20A Since these mice never relapsed, we wondered how tumor cell growth was kept under control at the tumor injection site in mice immunized with WTL plus LPS+PGPC?

[0234] Among the memory T cell subsets, T-resident memory cells (TRM) are defined by the expression of the CD103 integrin together with the C-type lectin CD69, which contributes to their resident properties 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 an experimental cutaneous melanoma model, CD8+ TRM cells in the skin promote persistent protection against melanoma development

[58] .

[0235] We examined the presence of TRM cells at the site of tumor injection and immune skin biopsies in surviving mice previously immunized with the hyperactivating stimulator LPS+PGPC. Interestingly, 200 days after tumor inoculation, CD8+CD69+CD103+TRM cells were highly enriched at the site of tumor injection in all surviving mice, but were insufficient at the site of immunization ( Figures 21A-21BThese data are consistent with clinical and experimental reports correlating high levels of TRM with long-term tumor control, where TRM may be maintained long-term to the site of tumor injection [56,57]. Thus, immunization with WTL and hyperactivating stimuli may generate TRM that keeps tumor cells under control.

[0236] In order to test the functional specificity of these T cells, we monitor cytotoxic lymphocyte (CTL) activity in vitro. Circulating memory CD8+T cells and TRM cells are isolated from spleen or skin adipose tissue of surviving mice previously receiving over-activation stimuli. These cells are cultivated with B16OVA cells or B16 cells that do not express OVA or non-related cancer cell line CT26. As assessed by LDH release, CTL activity is observed only when CD8+T cells are mixed with B16OVA or B16 cells ( Fig. 21C No killing of CT26 cells was observed ( Fig. 21C ), thus demonstrating the functional and antigen-specific nature of the hyperactivation-induced T cell responses.

[0237] Based on the antigen-specific T cell responses induced by hyperactivating stimuli, we determined whether T cells are sufficient to protect against tumor progression. CD8+ T cells were transferred from surviving mice to naive mice and subsequently challenged with the parental tumor cell line used as the initial immunogen. Transferring CD8+ TRMs or circulating CD8+ T cells from surviving mice to naive recipients conferred strong protection against subsequent tumor challenge, with the TRM subset playing a major protective role ( Fig.21D Transfer of both T cell subsets from surviving mice to naive mice one week before tumor inoculation provided 100% protection of the recipient mice from subsequent tumor challenge ( Fig.21D These combined data suggest that PGPC-based hyperactivation stimulators confer optimal protection in the B16 melanoma model by inducing strong circulating and resident anti-tumor CD8+ T cell responses.

[0238] Example 8: Hyperactivating stimulants protect against established anti-PD1 resistant tumors

[0239] In order to determine whether the over-activation stimulant can be used as cancer immunotherapy, we tested the anti-tumor response in mice carrying growing tumors before any additional treatment. For these studies, cultured tumor cells were not used as antigen sources, and ex vivo WTL was produced using syngeneic tumors from unimmunized mice, in which 10 mm harvested tumors were dissociated and CD45+ cells were depleted. Mice were inoculated with tumor cells subcutaneously (sc) in the left upper back. When the tumor reached a size of 3-4 mm, tumor-bearing mice were placed untreated (unimmunized) or received a therapeutic injection consisting of ex vivo WTL and LPS+PGPC in the right flank. Subsequently, 2 subcutaneous booster therapeutic injections ( Fig.14A Interestingly, hyperactivation-based therapeutic injections induce tumor elimination in a number of tumors, such as the B16OVA and B16F10 melanoma models, and in the MC38OVA and CT26 colon cancer tumor models ( Figures 14B-14D In all of these models, a high percentage of mice receiving the immunotherapy regimen remained tumor-free long term following tumor inoculation ( Figures 14B-14D In all tumor models tested, the efficacy of immunotherapy was dependent on IL-1β, as neutralization of IL-1β abolished the protection conferred by hyperactivating stimuli plus ex vivo WTL ( Figures 14B-14D In addition, CD8+ T cells are critical for protection against immunogenic tumor models such as B16OVA or MC38OVA tumors, while both CD4+ and CD8+ T cells are required for protection against less immunogenic tumors such as CT26, and B16F-10 ( Figures 14B-14D )

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

[0240] Example 9: Endogenous hyperactivated DCs stimulate T cell-mediated persistent anti-tumor immunity

[0241] Adoptive transfer of hyperactivated DCs into tumor-bearing mice induces strong antitumor immunity ( Figures 12A-12B). To test whether endogenous DCs can trigger a hyperactivation-mediated anti-tumor response, we used Zbtb46DTR mice in which conventional DCs were depleted by DT injection. Zbtb46DTR or WT mice were injected subcutaneously with B16OVA cells. Then, before immunization of Zbtb46DTR mice, DT was injected every other day to completely deplete resident DCs in Zbtb46DTR mice. When tumors reached 4 mm in size, Zbtb46DTR or WT mice were immunized with B16OVA WTL plus the hyperactivation stimulus LPS+PGPC. We found that in contrast to WT mice, which rejected tumors in 90% of mice, Zbtb46DTR mice (lacking DCs) were unable to reject tumors. These results confirm that DCs are the initiators of hyperactivation-mediated protection ( Fig.15A ).

[0242] Example 10: Overactivated cDC1 can use complex antigen sources to stimulate T cell-mediated anti-tumor immunity

[0243] We showed in vitro that both cDC1 and cDC2 can achieve a hyperactivated state, as these cells produce IL-1β in response to LPS+PGPC while maintaining their viability. These data provide further indications for defining specific DC subsets that trigger hyperactivation-mediated anti-tumor responses in vivo. Given the importance of the cDC1 subset in tumor rejection, we hypothesized that cDC1 plays an important role in inducing hyperactivation-mediated anti-tumor protection. To test this idea, we used Batf3- / - mice (which lack cDC1 but carry cDC2 cells)

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

[65] . Interestingly, despite the fact that immunization of Batf3- / - mice increased their survival by several days compared to non-immunized Batf3- / - mice, all Batf3- / - mice died of tumor growth 25 days after tumor inoculation. In contrast, WT mice rejected tumors in 100% of tumor-bearing mice ( Fig. 15C). Therefore, cDC1 plays a crucial role in hyperactivation-mediated antitumor immunity. In addition, while immunized WT mice induced high frequencies of antigen-specific CD8+ and CD4+ cells in TEM and in skin dLN, immunized Batf3- / - mice induced slightly reduced antigen-specific CD4+ T cells, but no significant antigen-specific CD8+ T cells in TEM ( Fig.15D ).

[0244] To further confirm the role of overactivated cDC1 in inducing long-term anti-tumor protection, we attempted to evaluate the ability of overactivated cDC1 to restore anti-tumor protection in Batf3- / -. We adoptively transferred naive, activated, or overactivated cDC1 cells to Batf3- / - mice. To this end, FLT3-derived cDC1 was sorted from C57BL / 6J mice as B220-MHC-II+CD11c+CD24+ cells as previously described. cDC1 was treated in vitro and loaded with B16OVA WTL as described above, and then 1.10e6 cells were injected subcutaneously into tumor-bearing Batf3- / - mice. We observed that in contrast to naive or activated cDC1, which provided only slightly improved mouse survival compared to uninjected mice, overactivated cDC1 induced tumor rejection in 100% of tumor-bearing mice, which remained tumor-free for more than 60 days after tumor inoculation ( Fig.15E ). Note that hyperactivated cDC1 injection restored CD8+ T cell responses in Batf3- / - as measured by SIINFEKL tetramer staining in tumors and skin dLNs ( Fig.15F In contrast, injection of naive or activated cDC1 failed to restore antigen-specific CD8+ T cells. cDC1-mediated tumor rejection depends on inflammasome activation, as injection of NLRP3- / - cDC1 treated with LPS+PGPC did not provide any antitumor protection and abolished the ability of hyperactivated cDC1 to restore CD8+ T cell responses ( Fig.15F ).

[0245] In addition to their ability to produce IL-1 from viable cells, hyperactivated DCs highly migrate to adjacent dLNs to enhance CD8+ T cell responses ( Figures 12A-12B ).

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[0315] Other embodiments

[0316] It should be understood that although the invention has been described in conjunction with the detailed description of the invention, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A method for producing a population of therapeutic dendritic cells, the method comprising: Obtaining live dendritic cells from a cell donor; sensitizing the dendritic cells ex vivo with TLR ligands; Dendritic cells were sensitized ex vivo by incubation with lipids that activate nonclassical inflammasomes; and loading the dendritic cells with an immunogen to produce a population of therapeutic dendritic cells, Wherein, the TLR ligand is a TLR4 ligand, and the TLR4 ligand is selected from monophosphoryl lipid A (MPLA), lipopolysaccharide (LPS), or a combination thereof, The non-classical inflammasome-activating lipids include oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC) or [(2R)-2-(4-carboxybutyryloxy)-3-hexadecanoyloxypropyl] 2-(trimethylammonio)ethyl phosphate (PGPC), The immunogen is a cancer antigen or a whole tumor lysate.

2. Use of a population of therapeutic dendritic cells in the preparation of an agent for inducing an immune response in a subject by a method comprising: Obtaining live dendritic cells from a cell donor; sensitizing the dendritic cells ex vivo with TLR ligands; Dendritic cells were sensitized ex vivo by incubation with lipids that activate nonclassical inflammasomes; loading the dendritic cells with an immunogen, thereby generating a population of therapeutic dendritic cells; and administering the population of therapeutic dendritic cells to the subject, thereby inducing an immune response in the subject, Wherein, the TLR ligand is a TLR4 ligand, and the TLR4 ligand is selected from monophosphoryl lipid A (MPLA), lipopolysaccharide (LPS), or a combination thereof, The non-classical inflammasome-activating lipids include oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC) or [(2R)-2-(4-carboxybutyryloxy)-3-hexadecanoyloxypropyl] 2-(trimethylammonio)ethyl phosphate (PGPC), The immunogen is a cancer antigen or a whole tumor lysate.

3. Use of a population of therapeutic dendritic cells in the preparation of an agent for treating cancer in a subject by a method comprising: Obtaining live dendritic cells from a cell donor; sensitizing the dendritic cells ex vivo with TLR ligands; Dendritic cells were sensitized ex vivo by incubation with lipids that activate nonclassical inflammasomes; loading the dendritic cells with an immunogen, thereby generating a population of therapeutic dendritic cells; and administering the population of therapeutic dendritic cells to the subject, thereby treating cancer in the subject, Wherein, the TLR ligand is a TLR4 ligand, and the TLR4 ligand is selected from monophosphoryl lipid A (MPLA), lipopolysaccharide (LPS), or a combination thereof, The non-classical inflammasome-activating lipids include oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC) or [(2R)-2-(4-carboxybutyryloxy)-3-hexadecanoyloxypropyl] 2-(trimethylammonio)ethyl phosphate (PGPC), The immunogen is a cancer antigen or a whole tumor lysate.

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

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

6. The method or use according to any one of claims 1 to 3, wherein obtaining dendritic cells from a cell donor comprises: harvesting progenitor cells from the cell donor; and The progenitor cells are cultured ex vivo under conditions effective to induce differentiation, thereby obtaining dendritic cells from the cell donor.

7. The method or use according to any one of claims 1-3, wherein obtaining dendritic cells from a cell donor comprises harvesting in vivo differentiated dendritic cells from the cell donor.

8. The method or use of any one of claims 1-3, wherein the immunogen is autologous.

9. The method or use according to any one of claims 1 to 3, wherein the sensitization and the culturing occur simultaneously.

10. The method or use according to any one of claims 1-3, wherein said sensitization occurs prior to said culturing.

11. The use according to claim 2 or 3, further comprising administering an anti-cancer agent to the subject.

12. The use according to claim 11, wherein the anticancer agent is a chemotherapeutic agent.

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