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Tumors that lack pre-existing immune infiltration respond poorly to T cell checkpoint blockade immunotherapy. These cancers often surround themselves with high densities of suppressive myeloid stroma while excluding immunostimulatory dendritic cells. Tumor-resident myeloid cells and selected lymphocyte populations retain expression of Toll-like Receptors (TLR) that sense common features of pathogens and activate innate immunity in response. We explored whether agonists of TLR9 could augment innate immunity to promote tumor regression alone or in combination with T cell checkpoint blockade. In the setting of the immunogenic B16-Ova (Ovalbumin) expressing melanoma model, local injection of the CpG oligonucleotide TLR9 agonist ODN1826 combined with systemic CTLA-4 blockade cured 45% of mice of both their treated and an untreated tumor on the opposite flank demonstrating the synergistic potential of this combination. Next, in the non-immunogenic B16-F10 melanoma model, we showed that only intra-tumoral, but not systemic TLR9 activation augments the therapeutic potential of checkpoint blockade. In this setting, intra-tumoral TLR9 activation cooperated equally with either CTLA-4 or PD-1 blockade co-administered locally or given systemically; however, the uninjected tumor rarely regressed. Anti-CTLA-4 combinations were associated with improved intra-tumoral CD8 to regulatory T cell ratios, while anti-PD-1 combinations elicited improved ratios of CD8 T cells relative to suppressive myeloid stroma. Using both a TLR9 agonist (MGN1703) and a CTLA-4 antibody (9D9-IgG2a) of increased potency cured 50% of bi-lateral B16-F10 melanoma. These findings suggest that intra-tumoral TLR9 agonists can improve sensitivity of poorly immunogenic tumors to T cell checkpoint blockade, and that newer, higher potency TLR agonists and checkpoint antibodies can raise the therapeutic ceiling for this combination therapy.

BackgroundIntratumoral injection of cyclic dinucleotide (CDN) agonists of the stimulator of interferon genes (STING) pathway engages innate immune activation and priming of adaptive immune effectors to foster local and distal tumor clearance. Despite proven therapeutic efficacy in preclinical models, a thorough understanding of how CDNs reprogram suppressive myeloid stroma in mouse and man is lacking.MethodsHere, we perform deep transcript-level and protein-level profiling of myeloid-derived suppressor cells and M2 macrophages following stimulation with CDNs of ascending potency. Additionally, we leverage orthotopic Kras+/G12DTP53+/R172HPdx1-Cre (KPC) derived models of pancreatic adenocarcinoma (PDAC) to determine the capacity for locally administered CDNs to sensitize PDAC to immune checkpoint blockade. We use bioluminescent in vivo imaging and 30-parameter flow cytometry to profile growth kinetics and remodeling of the tumor stroma post-therapy.ResultsHighly potent synthetic STING agonists repolarize suppressive myeloid populations of human and murine origin in part through inhibition of Myc signaling, metabolic modulation, and antagonism of cell cycle. Surprisingly, high-potency synthetic agonists engage qualitatively unique pathways as compared with natural CDNs. Consistent with our mechanistic observations, we find that intratumoral injection of the highest activity STING agonist, IACS-8803, into orthotopic pancreatic adenocarcinoma lesions unmasks sensitivity to checkpoint blockade immunotherapy. Dimensionality reduction analyses of high parameter flow cytometry data reveals substantial contributions of both myeloid repolarization and T cell activation underlying the in vivo therapeutic benefit of this approach.ConclusionsThis study defines the molecular basis of STING-mediated myeloid reprogramming, revealing previously unappreciated and qualitatively unique pathways engaged by CDNs of ascending potency during functional repolarization. Furthermore, we demonstrate the potential for high potency CDNs to overcome immunotherapy resistance in an orthotopic, multifocal model of PDAC.

Regulatory T cells (Tregs) are highly attractive targets for immunotherapy development, however, there remains a critical need for clinically actionable targeting strategies that specifically inhibit human tumor-infiltrating Tregs (TI-Tregs) while preserving function of cytotoxic effectors and peripheral non-tumor Tregs (P-Tregs). To this end, we sought to identify, validate, and target novel master regulators of TI-Tregs by leveraging a suite of next-generation bioinformatic tools and rigorous ex vivo and in vivo screening and validation methodologies. Specifically, we performed VIPER (Virtual Inference of Protein Activity) analysis on a large dataset of T cell transcriptional profiles from matched peripheral blood and tumors of 36 human patients, yielding 17 master regulator (MR) proteins predicted to uniquely drive the TI-Treg phenotype across cancers. To identify putative therapeutics that modulate TI-Treg MRs, we performed a systematic ex vivo drug screen with an unbiased panel of 1,554 FDA-approved and experimental compounds coupled to RNA sequencing (PLATE-Seq) on human TI-Tregs and P-Tregs. Drug candidates with preferential cytotoxic activity on TI-Tregs versus P-Tregs that also reversed MR transcriptional activity were thoroughly validated in vivo in the MC38 tumor model. In parallel, we performed a pooled in vivo CRISPR/Cas9 screen via the CHIME (CHimeric IMmune Editing) system to identify candidate MRs that regulate TI-Treg recruitment to and/or retention within MC38 tumors. By these approaches, we successfully validated an as-yet poorly described TI-Treg MR, TRPS1 (Transcriptional Repressor GATA Binding 1), which after genetic deletion across the hematopoietic compartment diminished TI-Tregs while preserving P-Tregs in MC38-bearing mice, and led to enhanced spontaneous control of MCA205 sarcomas. In addition, we found the widely used nucleoside analog chemotherapeutic Gemcitabine exhibits preferential inhibitory activity against human TI-Tregs versus P-Tregs, and validated in mice that at sub-clinical dose levels Gemcitabine exhibits immune-dependent therapeutic activity that significantly potentiates checkpoint blockade control of late-stage MC38 tumors. By single cell RNA sequencing of TI- and P-Tregs flow-sorted from Gemcitabine-treated mice, we observe specific depletion of a TI-Treg subset enriched for expression of TI-Treg MRs, including TRPS1. Together, these studies reveal new putative regulators of human TI-Tregs and identify a readily available clinical therapeutic that exhibits inhibitory activity against them. These findings specifically warrant future investigation into the role of TRPS1 in TI-Treg activity and clinical evaluation of low-dose Gemcitabine as a Treg-targeting strategy capable of enhancing checkpoint blockade immunotherapy. Citation Format: Casey R. Ager, Aleksandar Obradovic, Mikko Turunen, Mohsen Khosravi-Maharlooei, Charles Karan, Andrea Califano, Charles G. Drake. Elucidating and targeting master regulators of tumor infiltrating regulatory T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3612.

Despite the clinical success of T-cell checkpoint blockade, most patients with cancer still fail to have durable responses to immunotherapy. The molecular mechanisms driving checkpoint blockade resistance, whether preexisting or evolved, remain unclear. To address this critical knowledge gap, we treated B16 melanoma with the combination of CTLA-4, PD-1, and PD-L1 blockade and a Flt3 ligand vaccine (≥75% curative), isolated tumors resistant to therapy, and serially passaged them in vivo with the same treatment regimen until they developed complete resistance. Using gene expression analysis and immunogenomics, we determined the adaptations associated with this resistance phenotype. Checkpoint resistance coincided with acquisition of a “hypermetabolic” phenotype characterized by coordinated upregulation of the glycolytic, oxidoreductase, and mitochondrial oxidative phosphorylation pathways. These resistant tumors flourished under hypoxic conditions, whereas metabolically starved T cells lost glycolytic potential, effector function, and the ability to expand in response to immunotherapy. Furthermore, we found that checkpoint-resistant versus -sensitive tumors could be separated by noninvasive MRI imaging based solely on their metabolic state. In a cohort of patients with melanoma resistant to both CTLA-4 and PD-1 blockade, we observed upregulation of pathways indicative of a similar hypermetabolic state. Together, these data indicated that melanoma can evade T-cell checkpoint blockade immunotherapy by adapting a hypermetabolic phenotype.

Despite the success of immune checkpoint blockade against melanoma, many "cold" tumors like prostate cancer remain unresponsive. We found that hypoxic zones were prevalent across preclinical prostate cancer and resisted T cell infiltration even in the context of CTLA-4 and PD-1 blockade. We demonstrated that the hypoxia-activated prodrug TH-302 reduces or eliminates hypoxia in these tumors. Combination therapy with this hypoxia-prodrug and checkpoint blockade cooperated to cure more than 80% of tumors in the transgenic adenocarcinoma of the mouse prostate-derived (TRAMP-derived) TRAMP-C2 model. Immunofluorescence imaging showed that TH-302 drives an influx of T cells into hypoxic zones, which were expanded by checkpoint blockade. Further, combination therapy reduced myeloid-derived suppressor cell density by more than 50%, and durably reduced the capacity of the tumor to replenish the granulocytic subset. Spontaneous prostate tumors in TRAMP transgenic mice, which completely resist checkpoint blockade, showed minimal adenocarcinoma tumor burden at 36 weeks of age and no evidence of neuroendocrine tumors with combination therapy. Survival of Pb-Cre4, Ptenpc-/-Smad4pc-/- mice with aggressive prostate adenocarcinoma was also significantly extended by this combination of hypoxia-prodrug and checkpoint blockade. Hypoxia disruption and T cell checkpoint blockade may sensitize some of the most therapeutically resistant cancers to immunotherapy.

Purpose: Agonist antibodies targeting the T-cell costimulatory receptor 4-1BB (CD137) are among the most effective immunotherapeutic agents across preclinical cancer models. In the clinic, however, development of these agents has been hampered by dose-limiting liver toxicity. Lack of knowledge of the mechanisms underlying this toxicity has limited the potential to separate 4-1BB agonist–driven tumor immunity from hepatotoxicity. Experimental Design: The capacity of 4-1BB agonist antibodies to induce liver toxicity was investigated in immunocompetent mice, with or without coadministration of checkpoint blockade, via (i) measurement of serum transaminase levels, (ii) imaging of liver immune infiltrates, and (iii) qualitative and quantitative assessment of liver myeloid and T cells via flow cytometry. Knockout mice were used to clarify the contribution of specific cell subsets, cytokines, and chemokines. Results: We find that activation of 4-1BB on liver myeloid cells is essential to initiate hepatitis. Once activated, these cells produce interleukin-27 that is required for liver toxicity. CD8 T cells infiltrate the liver in response to this myeloid activation and mediate tissue damage, triggering transaminase elevation. FoxP3+ regulatory T cells limit liver damage, and their removal dramatically exacerbates 4-1BB agonist–induced hepatitis. Coadministration of CTLA-4 blockade ameliorates transaminase elevation, whereas PD-1 blockade exacerbates it. Loss of the chemokine receptor CCR2 blocks 4-1BB agonist hepatitis without diminishing tumor-specific immunity against B16 melanoma. Conclusions: 4-1BB agonist antibodies trigger hepatitis via activation and expansion of interleukin-27–producing liver Kupffer cells and monocytes. Coadministration of CTLA-4 and/or CCR2 blockade may minimize hepatitis, but yield equal or greater antitumor immunity. Clin Cancer Res; 24(5); 1138–51. ©2018 AACR.

Coordinated manipulation of independent immune regulatory pathways in the tumor microenvironment—including blockade of T-cell checkpoint receptors and reversal of suppressive myeloid programs—can render aggressive cancers susceptible to immune rejection. Elevated toxicity associated with combination immunotherapy, however, prevents translation of the most efficacious regimens. We evaluated T-cell checkpoint–modulating antibodies targeting CTLA-4, PD-1, and 4-1BB together with myeloid agonists targeting either STING or Flt3 in the TRAMP-C2 model of prostate cancer to determine whether low-dose intratumoral delivery of these agents could elicit systemic control of multifocal disease. Intratumoral administration of the STING agonist cyclic di-GMP (CDG) or Flt3 Ligand (Flt3L) augmented the therapeutic effect of systemic triple checkpoint modulation and promoted the cure of 75% of mice with bilateral TRAMP-C2; however, when all agents were administered locally, only CDG mobilized abscopal immunity. Combination efficacy correlated with globally enhanced ratios of CD8+ T cells to regulatory T cells (Treg), macrophages, and myeloid-derived suppressor cells, and downregulation of the M2 marker CD206 on tumor-associated macrophages. Flt3L improved CD8+ T-cell and dendritic cell infiltration of tumors, but was diminished in efficacy by concomitant Treg expansion. Although intratumoral CDG/checkpoint therapy invokes substantial ulceration at the injection site, reduced CDG dosing can preserve tissue integrity without sacrificing therapeutic benefit. For high-order combinations of T-cell checkpoint antibodies and local myeloid agonists, systemic antibody administration provides the greatest efficacy; however, local administration of CDG and antibody provides substantial systemic benefit while minimizing the potential for immune-related adverse events. Cancer Immunol Res; 5(8); 676–84. ©2017 AACR.

Advances in our understanding of tumor immune biology and development of cancer immunotherapies have led to improved outcomes for patients who suffer from aggressive cancers such as melanoma. Despite the clinical success of immune checkpoint blockade, a majority of patients still fail to respond, and the underlying mechanisms that drive resistance remain unclear. To understand why a subset of tumors fail to respond to immunotherapy, we established a novel murine model of melanoma that is fully resistant to immune checkpoint blockade. By in vivo passaging nonresponding B16 melanoma tumor cells, we selected for a resistant variant that fails to respond to the combination of CTLA-4, PD-1, and PD-L1 blockade. In comparing gene expression of parental versus resistant tumor cells and analyzing the corresponding immune infiltrate, we determined the adaptations associated with resistance to therapy. We found that evasion of immunotherapy was associated with a “hypermetabolic” phenotype, characterized by an upregulation of glycolytic, oxidoreductase, and mitochondrial oxidative phosphorylation pathways to establish a hypoxic, metabolically hostile microenvironment. Enforced expression of two key genes associated with these pathways in parental tumor cells was sufficient to mediate resistance to triple checkpoint blockade. Flow cytometry assays determined that T cells infiltrating resistant tumors had diminished glycolytic capacity and effector function, indicating a metabolic disadvantage. Consistent with our findings, melanoma patients who failed dual checkpoint blockade exhibited similar metabolic alterations as seen in our resistant variant. Using a novel MRI-based imaging approach, we observed distinct metabolic changes that stratified responding versus nonresponding tumors in live mice. Applying this method to patients could provide insight into predicting response rates to checkpoint modulation. Overall, our data indicate that resistant melanoma tumor cells acquire a “hypermetabolic” phenotype to establish a hostile microenvironment that is capable of inhibiting the antitumor immune response. This abstract is also being presented as Poster A24. Citation Format: Ashvin R. Jaiswal, Arthur J. Liu, Shivanand Pudakalakatti, Prasanta Dutta, Priyamvada Jayaprakash, Todd Bartkowiak, Casey Ager, Zhiqiang Wang, Alex Reuben, Zachary Cooper, Cristina Ivan, Zhenlin Ju, Felix Nwajei, Jing Wang, Michael A. Davies, R. Eric Davis, Jennifer A. Wargo, Pratip K. Bhattacharya, David S. Hong, Michael A. Curran. Melanoma evolves complete immunotherapy resistance through acquisition of a hypermetabolic phenotype [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR02.

Activation of the stimulator of interferon genes (STING) pathway by both exogenous and endogenous cytosolic DNA results in the production of interferon beta (IFN-β) and is required for the generation of cytotoxic T-cell priming against tumor antigens. In the clinical setting, pharmacological stimulation of the STING pathway has the potential to synergize with immunotherapy antibodies by boosting anti-tumor immune responses. We report the discovery of two highly potent cyclic dinucleotide STING agonists, IACS-8803 and IACS-8779, which show robust activation of the STING pathway in vitro and a superior systemic anti-tumor response in the B16 murine model of melanoma when compared to one of the clinical benchmark compounds.

Immune checkpoint blockade is effective in “hot” tumors like melanoma with pre-existing immune infiltrates; however, “cold” tumors like prostate cancer fail to respond. We found that prostate cancers harbor regions of hypoxia that resist T cell infiltration even in the context of anti-CTLA-4 (cytotoxic T lymphocyte associated protein-4) and anti-PD-1 (programmed cell death protein 1) blockade. These hypoxic zones serve as islands of immune privilege through the recruitment and suppressive polarization of immature myeloid cells into myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM). We found that targeted hypoxia ablation using TH-302, a hypoxia-activated prodrug, sensitized both transplantable and spontaneous models of prostate cancer to checkpoint blockade, coincident with enhanced T cell infiltration and effector function and loss of MDSC recruitment and suppressive function. Tumors treated with the combination of TH-302 and checkpoint blockade showed a reduced capacity to suppressively polarize new myeloid immigrants, implying a durable reconditioning of the tumor microenvironment (TME) into an immune-infiltrated, pro-inflammatory milieu. T cells infiltrating combination-treated tumors exhibited increased mitochondrial respiration, consistent with creation of a metabolically favorable milieu for T cell function. Based on these findings, we hypothesized that other approaches capable of metabolically rewiring the TME should promote anti-tumor immunity and sensitize checkpoint blockade-resistant tumors to immunotherapy. With this in mind, we performed a longitudinal study comparing a panel of different mitochondrial respiration inhibitors and a glutaminase inhibitor for their efficacy in reducing hypoxia, improving T cell infiltration and decreasing myeloid cell recruitment and suppressive polarization using immunofluorescence staining and confocal microscopy. Our preliminary data suggests that inhibitors targeting mitochondrial respiration, rather than those targeting glutamine metabolism synergize with checkpoint blockade and exhibit the highest efficacy in increasing T cell recruitment. We continue to characterize the dynamics of hypoxia reduction, duration of normalization following drug withdrawal, and impact on the immune microenvironment of these diverse approaches to metabolic reconditioning. Citation Format: Priyamvada Jayaprakash, Midan Ai, Arthur Liu, Pratha Budhani, Todd Bartkowiak, Jie Sheng, Casey Ager, Courtney Nicholas, Ashvin Jaiswal, Yanqiu Sun, Krishna Shah, Sadhana Balasubramanyam, Nan Li, Guocan Wang, Jing Ning, Anna Zal, Tomasz Zal, Michael Curran. Targeting hypoxia-induced immune suppression to overcome immunotherapy resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5011.

TPS2669 Background: Drugs targeting pathogen associated molecular patterns are an attractive strategy to stimulate the immune system. Toll-like receptors (TLR) have generated significant interest as an effective means of stimulating the immune system that result in the killing of tumor cells. TLR-9 agonists can function to promote an early immune response and are an appealing partner for combination with checkpoint blockade to improve immune activation. Lefitolimod (MGN1703) is a covalently closed dumbbell-shaped DNA molecule that functions as a TLR-9 agonist. We developed a clinical trial combining lefitolimod with ipilimumab (anti-CTLA4) in patients with advanced malignancies. In the dose-escalation phase 19 patients were enrolled and no DLTs were encountered. Safety data and maximum planned dose level of lefitolimod at 120mg weekly and ipilimumab 3mg/kg every 3 weeks was previously presented. Adverse events related to the combination included fatigue, appetite loss, rash, and anemia. Methods: This trial (NCT02668770) was designed to evaluate the safety profile and maximum tolerated dose of lefitolimod with ipilimumab. A 3+3 trial design was used to establish safety of the combination at each dose level and guide the decision to escalate dose. Lefitolimod is administered via subcutaneous (SC) injection weekly while ipilimumab is given at 3mg/kg intravenous on day 8 of each 3 week cycle. Lefitolimod starting dose was 15mg SC weekly with 3 dose level escalations up to 120mg SC weekly. Patients receive treatment for 4 cycles (total 12 weeks) with the combination, and those with stable disease or response were eligible to remain on lefitolimod therapy for up to 1 year. Eligible patients have a metastatic or unresectable solid tumor refractory to standard therapies, ECOG ≤ 2, and normal organ and bone marrow function. Patients are allowed to have received prior checkpoint blockade agents. Enrollment in expansion cohorts in ongoing. To better understand relevant immunologic changes associated with treatment, paired pre- and post-treatment biopsies of target lesions and peripheral blood collection during treatment is required for target expansion cohort patient populations. In addition to evaluating target patient populations at the combination dose established during escalation, an expansion cohort for patients with cutaneous metastases involves combination treatment with intratumoral delivery of lefitolimod. Clinical trial information: NCT02668770.

Immunosuppressive myeloid populations including tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) are abundant within pancreatic adenocarcinoma (PDAC) tumors and play critical roles in constraining cytotoxic T-cell function in the tumor microenvironment. We hypothesized that intratumoral engagement of innate pathogen recognition receptors such as Stimulator of Interferon Genes (STING) could induce proinflammatory polarization of the myeloid stroma and liberate the antitumor T-cell response to regress refractory PDAC tumors in the presence of checkpoint blockade.We developed and characterized a novel cyclic dinucleotide (CDN) STING agonist IACS-8803, and found that 8803 activates downstream STING signaling in both human (THP-1) and murine (J774) myeloid reporter cells with over 10-fold greater potency than ML-RR-S2-CDA, the first-in-class CDN currently undergoing clinical evaluation (NCT02675439, NCT03172936). Intratumoral delivery of 8803 into subcutaneous B16 melanoma and PDAC tumors additionally revealed a greater capacity to induce tumor regression relative to ML-RR-S2-CDA. In order to evaluate the specific effects of 8803 on the phenotype and function of suppressive myeloid populations, we generated in vitro polarized human M2 macrophages and murine bone marrow-derived MDSC. Upon exposure to 8803, we observe downregulation of M2 markers CD163, LAP/TGF-β, and Arginase on human M2 macrophages, concomitant with upregulation of M1 markers CD86, CD80, and IL-6. Additionally, 8803-stimulated murine MDSC exhibit reduced T-cell suppressive capacity compared to unstimulated MDSC. In these studies, we consistently observe that the magnitude of phenotypic and functional repolarization by 8803 is superior to that of ML-RR-S2-CDA as well as other known CDN, 2’3’-cGAMP and c-di-GMP. Therefore, we describe IACS-8803 as a novel, highly potent STING agonist with the capacity to induce inflammatory repolarization in suppressive myeloid cells of both human and murine origin. We next investigated the capacity for intratumoral delivery of IACS-8803 to sensitize murine pancreatic cancer to checkpoint blockade and to mobilize systemic immunity against disseminated lesions. We utilized mT4-2D, a novel pancreatic cancer cell line from Kras+/LSL-G12D Tp53+/LSL-R172H Pdx1-Cre tumor organoids. We isolateda single cell clone of mT4-2D with reduced in vivo growth kinetics (termed mT4-LS), as well as a clone that maintains the aggressive nature of the parental line (termed mT4-LA). Mice bearing 10-day established orthotopic and subcutaneous mT4-LS tumors received standard regimens of αCTLA-4, αPD-1, or combined αCTLA-4/αPD-1 in the presence or absence of 8803 CDN injected into the orthotopic pancreatic tumor. We find single-agent treatment with 8803, αCTLA-4, αPD-1, or αCTLA-4/αPD-1 can cure 40-60% of mice of both orthotopic and subcutaneous tumors in this system; however, combining 8803 with checkpoint blockade completely eradicates both injected and distal mT4-LS tumors in all mice. We replicated these studies using the highly aggressive and refractory mT4-LA model, and found that combination therapy with intra-pancreatic 8803 and systemic αCTLA-4/αPD-1 significantly extends survival compared to 8803 or αCTLA-4/αPD-1 alone (p=0.001, p=0.0086, respectively). Analysis of treated mT4-LA tumors by flow cytometry reveals that combination therapy enhances the cytotoxic potential of CD8 T-cells at both injected and uninjected lesions, and promotes dendritic cell proliferation within the pancreatic milieu. These studies provide a preclinical rationale for pursuing the use of STING-activating CDN as a localized approach to sensitize refractory PDAC tumors to checkpoint blockade immunotherapy. Citation Format: Casey R. Ager, Maria E. Di Francesco, Philip Jones, Michael A. Curran. Intratumoral delivery of a novel STING agonist synergizes with checkpoint blockade to regress multifocal pancreatic cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A050.

Background: Resistance of pancreatic ductal adenocarcinoma (PDAC) to checkpoint blockade therapy is commonly attributed to its status as an immunologically cold tumor. However, it remains to be determined whether the lack of endogenous immunity to PDAC is predominantly due to a paucity of targetable neoantigens or to the unique tolerogenic environment of malignant pancreatic tissue. Using an implantable model derived from the Kras+/LSL-G12DTrp53+/LSL-R172HPdx1-Cre (KPC) mouse, we investigated how tissue implantation site and exogenous antigen presentation influenced immune surveillance of KPC tumors and response to combination immunotherapy. Methods: The KPC organoid-derived cell line mT4-2D was stably transduced with various expression constructs including luciferase (mT4-Luc), ovalbumin (mT4-OVA), or mCherry (mT4-Cherry). Each line was implanted subcutaneously (2.5x105 cells) or orthotopically at indicated doses to assess effects of exogenous antigen expression on engraftment efficiency. To evaluate how tissue environment governs response to therapy, mice were co-implanted with mT4-Luc at both orthotopic and subcutaneous sites and were exposed to combination therapy consisting of intratumoral STING agonist ML-RR-CDA (10ug/mouse) and checkpoint antagonists αCTLA-4 and αPD-1 beginning on day 14 post-implantation. Tumor growth at both sites was monitored weekly by IVIS bioluminescent imaging and mice were followed for survival, or tumors were harvested on day 25 for analysis of immune infiltrates by flow cytometry. Results: mT4 tumors grew aggressively at both subcutaneous and orthotopic sites, with doses as low as 3.5x104 causing mortality within 3 weeks post-implantation. However, expression of either OVA or mCherry resulted in spontaneous clearance in 100% of mice, regardless of implantation site. Luciferase expression yielded progressing tumors, and ex vivo peptide restimulation failed to identify Luciferase-specific T cells within mT4-Luc challenged mice; suggesting that Luciferase is a minimally immunogenic protein in C57BL/6 mice. Despite the poor immunogenicity of the mT4 model, we found that intratumoral injections of ML-RR-CDA delayed growth of subcutaneously-implanted mT4, and that concomitant administration of αCTLA-4/αPD-1 induced complete clearance in ~40% of mice. In contrast, intratumoral delivery of ML-RR-CDA was ineffective against orthotopic mT4 tumors, and combination therapy with αCTLA-4/αPD-1 achieved only modest delays in tumor progression. Flow cytometric analysis of tumor infiltrating immune populations in mice with co-implanted orthotopic and subcutaneous lesions revealed dichotomies in the density of CD8 T cells, CD4 effector T cells, and dendritic cell populations between tissue sites in response to local therapy. Conclusions: Although implantable cell lines isolated from KPC tumors are highly aggressive and resistant models of PDAC, we found that engraftment and growth of mT4-2D was completely negated upon forced expression of a single foreign protein antigen. This suggests that the lack of targetable mutanome products may explain the resistance of KPC and potentially human PDAC tumors to immunotherapy. However, when implanted subcutaneously, mT4-2D was sensitive to in situ STING activation in combination with checkpoint blockade, which indicates that immune responses against tissue-specific antigens are potentially sufficient to control growth of KPC tumors when removed from the suppressive pancreatic environment. Flow analysis revealed a greater capacity of CD8 T cells, CD4 T cells, and dendritic cells to infiltrate subcutaneous vs orthotopic tumors following intratumoral therapy, reinforcing the theory that therapies which enhance infiltration of select immune populations are needed in order to sensitize PDAC tumors to therapy. Citation Format: Casey R. Ager, Michael A. Curran. Effects of tissue site and antigenicity on KPC-derived pancreatic tumor growth and response to combination immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B52.

Background: Despite the success of T-cell checkpoint blockade antibodies in treating an array of cancers, the majority of patients still fail to respond to these therapies, or respond transiently and then relapse. The molecular mechanisms that drive lack of response to checkpoint blockade, whether pre-existing or evolved on therapy, remain unclear. Materials and Methods: To address this critical gap in clinical knowledge, we established a mouse model of melanoma designed to elucidate the molecular mechanisms underlying immunotherapy resistance. Through multiple in vivo passages, we selected a B16 melanoma tumor line that evolved complete resistance to combination blockade of CTLA-4, PD-1, and PD-L1, which cures ~80% of mice of the parental tumor. Using gene expression analysis, proteomics, and immunogenomics, we determined the adaptations engaged by this melanoma to become completely immunotherapy resistant. NMR spectroscopy, Seahorse XF analysis, flow cytometry, confocal microscopy, and Western blot analysis provided further insight into the mechanisms driving checkpoint blockade resistance. Results: Acquisition of immunotherapy resistance by these melanomas was driven by coordinate upregulation of the glycolytic and aldose reductase pathways to create a metabolically hostile microenvironment in which T-cell function is profoundly suppressed. When reintroduced into the parental tumor, the genes most closely associated with these metabolic adaptations confer enhanced immunotherapy resistance. We have validated upregulation of these pathways in a unique cohort of melanoma patients who failed dual checkpoint blockade. Additionally, we employed MRI imaging to visualize metabolic changes acquired by resistant tumors in live mice. Clinical application of this technique could provide a much-needed noninvasive tool to predict immunotherapeutic sensitivity of patients. Conclusion: Upregulation of glycolytic metabolism and the aldose reductase pathway by melanoma tumor cells cripples T cells in the microenvironment and confers resistance to checkpoint blockade. This abstract is also being presented as Poster A71. Citation Format: Ashvin R. Jaiswal, Shivanand Pudakalakatti, Prasanta Dutta, Arthur Liu, Todd Bartkowiak, Casey Ager, Cristina Ivan, Richard Eric Davis, Michael A. Davies, Jennifer Wargo, James P. Allison, Pratip K. Bhattacharya, David Hong, Michael A. Curran. Metabolic adaptations establish immunotherapy resistance in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr PR08.

Background: Despite the success of T-cell checkpoint blockade antibodies in treating an array of cancers, the majority of patients still fail to respond to these therapies, or respond transiently and then relapse. The molecular mechanisms that drive lack of response to checkpoint blockade, whether pre-existing or evolved on therapy, remain unclear. Materials and Methods: To address this critical gap in clinical knowledge, we established a mouse model of melanoma designed to elucidate the molecular mechanisms underlying immunotherapy resistance. Through multiple in vivo passages, we selected a B16 melanoma tumor line that evolved complete resistance to combination blockade of CTLA-4, PD-1, and PD-L1, which cures ~75% of mice of the parental tumor. Using gene expression analysis, proteomics, and immunogenomics, we determined the adaptations engaged by this melanoma to become completely immunotherapy resistant. NMR spectroscopy, Seahorse XF Analysis, flow cytometry, confocal microscopy and Western blot analysis provided further insight into the mechanisms driving checkpoint blockade resistance. Results: Acquisition of immunotherapy resistance by these melanomas was driven by acquisition of a hypermetabolic state through coordinate upregulation of both glycolysis and oxidative phosphorylation (OxPhos). Depletion of essential nutrients from the tumor microenvironment by these resistant tumors functionally compromises infiltrating T cells to an extent that cannot be rescued by even triple checkpoint blockade. The glycolysis and OxPhos-associated genes most highly induced by these resistant melanomas were capable of conferring enhanced resistance to checkpoint blockade when reintroduced monogenically into the parental tumor. We have validated upregulation of these pathways in a unique cohort of melanoma patients who failed dual checkpoint blockade. Additionally, we employed a novel MRI imaging technique to visualize metabolic changes acquired by resistant tumors in live mice. Clinical application of this technique could provide a much-needed noninvasive tool to predict immunotherapeutic sensitivity of patient cancers. Conclusion: Acquisition of a hypermetabolic state by melanoma tumor cells cripples T cells in the microenvironment and confers complete resistance to checkpoint blockade. Citation Format: Ashvin R. Jaiswal, Shivanand Pudakalakatti, Dutta Prasanta, Arthur Liu, Todd Bartkowiak, Casey Ager, Michael A. Davies, Richard E. Davis, Jennifer Wargo, Pratip K. Bhattacharya, David S. Hong, Michael A. Curran. Metabolic adaptations confer immunotherapy resistance in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1700.

Alzheimer's disease (AD), a progressive neurodegenerative disease characterized by the accumulation of amyloid-beta protein and neuronal loss, is the leading cause of age-related dementia in the world today. The disease is also associated with neuroinflammation, robust activation of astrocytes and microglia, and evidence of activation of the complement system, localized with both fibrillar amyloid-beta (fAbeta) plaques and tangles. The observations are consistent with a complement-dependent component of AD progression. We have previously shown that inhibition of the major complement receptor for C5a (CD88) with the antagonist PMX205 results in a significant reduction in pathology in two mouse models of AD. To further characterize the role of complement in AD-related neuroinflammation, we examined the age- and disease-associated expression of CD88 in brain of transgenic mouse models of AD and the influence of PMX205 on the presence of various complement activation products using flow cytometry, western blot, and immunohistochemistry. CD88 was found to be up-regulated in microglia, in the immediate vicinity of amyloid plaques. While thioflavine plaque load and glial recruitment is significantly reduced after treatment with PMX205, C1q remains co-localized with fAbeta plaques and C3 is still expressed by the recruited astrocytes. Thus, with PMX205, potentially beneficial activities of these early complement components may remain intact, while detrimental activities resulting from C5a-CD88 interaction are inhibited. This further supports the targeted inhibition of specific complement mediated activities as an approach for AD therapy.

Tumor hypoxia contributes to chemotherapy and radiotherapy resistance, and helps foster metastasis. Hypoxia likely also supports tumor immune resistance by supporting development of suppressive myeloid and T cell populations by activating immunosuppressive signaling pathways in the tumor and stroma, and by creating a metabolically hostile environment for immune effector cells. TH-302 is a hypoxia-specific chemotherapeutic drug which is activated only in the hypoxic cores of tumors and remains harmless in the periphery. We hypothesized that concurrent combination therapy with TH-302 and T cell checkpoint blockade would promote inside-out tumor destruction with the drug killing at the core, releasing antigen, and diminishing suppression, while the antibodies helped expand and protect the T cells activated as a result. In the TRAMP-C2 prostatic adenocarcinoma model, we found that antibody blockade of CTLA-4 and PD-1, used in conjunction with TH-302 administration, promoted tumor rejection in a significantly larger percentage of mice than either therapy alone. The combination therapy promoted uniquely advantageous ratios of effector T cells to MDSC within the prostate cancer microenvironment. This novel combination of immunotherapy and hypoxia-specific chemotherapy has the potential to offer profound anti-tumor responses to a much greater percentage of patients than either chemotherapy or immunotherapy alone with the same or fewer side effects. Citation Format: Midan Ai, Todd Bartkowiak, Ashvin R. Jaiswal, Krishna Shah, Casey Ager, Beatrisa Lerman, Michael A. Curran. Combination hypoxia-specific chemotherapy and immunotherapy of prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 629. doi:10.1158/1538-7445.AM2014-629

Tumor hypoxia predicts poor outcomes across all cancers and has long been recognized as a critical source of resistance to both chemotherapy and radiotherapy. Despite the success of T cell immune checkpoint blockade in treating melanoma, aggressive adenocarcinomas of the prostate and pancreas are largely resistant to CTLA-4 and PD-1 antibody therapy in the mouse and in man. We find that hypoxic zones of these tumors resist infiltration by T cells even in the context of robust infiltration of T cells in normoxic areas of the same tumor (e.g. in the context of T cell checkpoint blockade). Beyond this lack of accessibility to tumor-specific T cells, hypoxia drives the establishment of a highly interdependent network of immunosuppressive stromal cells. Among these, we find myeloid-derived suppressor cells and myofibroblasts to be the critical populations which act together to suppress T cell responses and mediate immunotherapy resistance. Evofosfamide is a hypoxia-specific chemotherapeutic pro-drug which is activated only in the hypoxic cores of tumors and thus can be co-administered with immunotherapy. We find that Evofosfamide-driven disruption of hypoxia zones sensitizes prostate cancer to antibody blockade of CTLA-4 and PD-1 in both transplantable and genetically-engineered murine models of prostate cancer. Co-administration of Evofosfamide and α-CTLA-4/α-PD-1 promotes tumor rejection in a significantly larger percentage of mice than either therapy alone. Mechanistic studies reveals that loss of immune resistance is a consequence of re-oxygenation of hypoxia zones which results in 1) loss of active myeloid suppressor cells, 2) reduced suppressive capacity of new myeloid immigrants, 3) loss of suppressive activation of myofibroblasts, and 4) enhanced infiltration of effector T cells. Therefore, this combination of hypoxia disruption and T cell checkpoint blockade has immense potential to render some of the most therapeutically resistant cancers sensitive to immunotherapy.