Antoine Tanne

Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function

Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates many functions of these organelles that allow phagosomes to participate in processes that are essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3 inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3 inflammasome and caspase-1 in host defense.

Dectin-1 Activation Controls Maturation of β-1,3-Glucan-containing Phagosomes

Background: Dectin-1 is able to recognize and phagocytose the fungal carbohydrate, β-1,3-glucan, but its contribution to phagosomal maturation has not been explored. Results: Dectin-1-dependent Syk activation promotes phagolysosomal fusion and acidification. Conclusion: Dectin-1-dependent Syk-activation permits egress of early phagosomes to mature phagolysosomes. Significance: The surface recognition receptor, Dectin-1 shapes anti-fungal responses by controlling fungal phagosome maturation. Elimination of fungal pathogens by phagocytes requires phagosome maturation, a process that involves the recruitment and fusion of intracellular proteins. The role of Dectin-1, a β-1,3-glucan receptor, critical for fungal recognition and triggering of Th17 responses, to phagosomal maturation has not been defined. We show that GFP-Dectin-1 translocates to the fungal phagosome, but its signal decays after 2 h. Inhibition of acidification results in retention of GFP-Dectin-1 to phagosome membranes highlighting the requirement for an acidic pH. Following β-1,3-glucan recognition, GFP-Dectin-1 undergoes tyrosine phosphorylation by Src kinases with subsequent Syk activation. Our results demonstrate that Syk is activated independently of intraphagosomal pH. Inhibition of Src or Syk results in prolonged retention of GFP-Dectin-1 to the phagosome signifying a link between Syk and intraphagosomal pH. β-1,3-glucan phagosomes expressing a signaling incompetent Dectin-1 failed to mature as demonstrated by prolonged Dectin-1 retention, presence of Rab5B, failure to acquire LAMP-1 and inability to acidify. Phagosomes containing Candida albicans also require Dectin-1-dependent Syk activation for phagosomal maturation. Taken together, these results support a model where Dectin-1 not only controls internalization of β-1,3-glucan containing cargo and triggers proinflammatory cytokines, but also acts as a master regulator for subsequent phagolysosomal maturation through Syk activation.

Dectin-1–Dependent LC3 Recruitment to Phagosomes Enhances Fungicidal Activity in Macrophages

Autophagy has been postulated to play role in mammalian host defense against fungal pathogens, although the molecular details remain unclear. Here, we show that primary macrophages deficient in the autophagic factor LC3 demonstrate diminished fungicidal activity but increased cytokine production in response to Candida albicans stimulation. LC3 recruitment to fungal phagosomes requires activation of the fungal pattern receptor dectin-1. LC3 recruitment to the phagosome also requires Syk signaling but is independent of all activity by Toll-like receptors and does not require the presence of the adaptor protein Card9. We further demonstrate that reactive oxygen species generation by NADPH oxidase is required for LC3 recruitment to the fungal phagosome. These observations directly link LC3 to the inflammatory pathway against C. albicans in macrophages.

Distinguishing the immunostimulatory properties of noncoding RNAs expressed in cancer cells

Significance Using an approach derived from statistical physics, we quantify transcriptome-wide motif usage in human and murine noncoding RNAs (ncRNAs), determining that most have motif usage consistent with the coding genome. However, an outlier subset of tumor-associated ncRNAs comprises repetitive elements whose motif usage patterns are more typically associated with the genomes of inflammatory pathogens. We demonstrate that a key subset of these elements directly activates the cellular innate immune response. We propose that the innate response in tumors partially originates from direct interaction of immunogenic ncRNAs preferentially expressed in cancer cells with innate pattern recognition receptors. Recent studies have demonstrated abundant transcription of a set of noncoding RNAs (ncRNAs) preferentially within tumors as opposed to normal tissue. Using an approach from statistical physics, we quantify global transcriptome-wide motif use for the first time, to our knowledge, in human and murine ncRNAs, determining that most have motif use consistent with the coding genome. However, an outlier subset of tumor-associated ncRNAs, typically of recent evolutionary origin, has motif use that is often indicative of pathogen-associated RNA. For instance, we show that the tumor-associated human repeat human satellite repeat II (HSATII) is enriched in motifs containing CpG dinucleotides in AU-rich contexts that most of the human genome and human adapted viruses have evolved to avoid. We demonstrate that a key subset of these ncRNAs functions as immunostimulatory “self-agonists” and directly activates cells of the mononuclear phagocytic system to produce proinflammatory cytokines. These ncRNAs arise from endogenous repetitive elements that are normally silenced, yet are often very highly expressed in cancers. We propose that the innate response in tumors may partially originate from direct interaction of immunogenic ncRNAs expressed in cancer cells with innate pattern recognition receptors, and thereby assign a previously unidentified danger-associated function to a set of dark matter repetitive elements. These findings potentially reconcile several observations concerning the role of ncRNA expression in cancers and their relationship to the tumor microenvironment.

Abstract 3654: AGEN1884, an IgG1 anti-CTLA-4 antibody, combines effectively with PD-1 blockade in primary human T cell assays and in a non-human primate pharmacodynamic (PD) model

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) play important non-redundant roles in negatively regulating T cell immune responses. Therapeutic blockade of CTLA-4 or PD-1 pathways has been demonstrated to enhance T cell reactivity to tumor-specific antigens, translating to a significant improvement in overall survival. This anti-tumor effect can be further augmented when PD-1 and CTLA-4 antagonist antibodies are co-administered. The therapeutic impact of this combination is exemplified by the approval of this combination for advanced melanoma, as well as clinical benefit of the combination observed in NSCLC, mRCC, and most recently, mUC. AGEN1884, a human IgG1 antibody directed against CTLA-4, potently inhibits CTLA-4 binding to CD80 and CD86, resulting in enhanced T cell responsiveness in vitro, as well as in a vaccination model in non-human primates. A Phase 1 clinical study (NCT02694822) is currently ongoing to evaluate the safety and pharmacokinetic (PK)/pharmacodynamic (PD) relationships in patients with advanced solid tumors. AGEN2034 is a human IgG4 antibody that binds selectively to PD-1 with high affinity and potentiates T cell responsiveness via the blockade of PD-L1 and PD-L2 binding to PD-1. Here we evaluated the pharmacologic effect of combining AGEN1884 with AGEN2034, and other molecules targeting the PD-1/PD-L1 pathway, on primary human T cell immune responses. AGEN1884 combined effectively with AGEN2034, and other antibodies targeting the PD1/PD-L1 pathway, to promote superior T cell immune responses compared to monotherapies. Consistent with these in vitro findings, the co-administration of AGEN1884 with an anti-PD-1 antibody in cynomolgus monkeys (Macaca fascicularis) induced a dynamic PD effect, including a proliferative T cell response in peripheral blood, as compared to animals receiving either antibody alone. Finally, co-administration of an anti-mouse CTLA-4 antibody together with Agenus’ tumor-specific neo-epitope-based vaccine (AutoSynVax™) in mice induced effective amplification of vaccine-driven T cell responses, compared to animals that received the vaccine alone. These data further exemplify the versatility of harnessing antibody-mediated CTLA-4 blockade to influence apical events involved in T cell priming by antigen presenting cells. Taken together, these in vitro and in vivo findings demonstrate that the combination of AGEN1884 with PD-1 pathway blockade or with neo-epitope-based vaccines has the potential to provide therapeutic activity that is superior to that of either checkpoint- or vaccine-based monotherapies. Citation Format: Elise E. Drouin, David Savitsky, Ana M. Gonzalez, Randi Gombos, Dhan Chand, Jeremy Waight, Xia Yang, Mithun Khattar, Benjamin Morin, Mark Findeis, David Schaer, Taha Merghoub, Gerd Ritter, Antoine Tanne, Marc van Dijk, John M. Goldberg, Daniel Levey, John Castle, Jean-Marie Cuillerot, Jennifer S. Buell, Robert Stein, Nicholas S. Wilson. AGEN1884, an IgG1 anti-CTLA-4 antibody, combines effectively with PD-1 blockade in primary human T cell assays and in a non-human primate pharmacodynamic (PD) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3654. doi:10.1158/1538-7445.AM2017-3654

Abstract A028: Pre-clinical development of a first-in-class fully synthetic heat shock protein-based personalized cancer vaccine

Tumors accumulate spontaneous somatic mutations which can give rise to neo-epitopes (neoantigens) recognizable as non-self by T-cells. Advances in next-generation sequencing and bioinformatics make it possible to analyze individual patients9 tumor genomes and predict immunogenic mutations, which can be synthesized as short peptides, RNA or DNA for use in personalized cancer vaccines. However, these approaches need optimization for safe and effective induction of anti-tumor T-cell responses. We addressed this challenge by complexing synthetic long peptides comprising predicted tumor neo-epitopes to recombinant Hsc70, creating personalized vaccine candidates, AutoSynVax™ (ASV™), administered with QS-21 Stimulon® adjuvant. The same platform comprising Hsc70 plus synthetic long viral peptides was previously validated in a Phase-2 clinical trial, demonstrating effective T-cell responses and reduced viral load in HSV-2+ subjects. ASV immunization is intended to facilitate antigen processing and presentation of neo-epitopes to T-cells, resulting in robust immune responses. The innate immune modulating properties of the Hsc70 protein as well as that of QS-21 Stimulon adjuvant are also under investigation at the vaccine injection site, in draining lymph node, and in cultures of antigen presenting cell populations exposed to the two agents. Finally, ASV is being explored in combination with various agonistic and antagonistic immune modulating monoclonal antibodies with the aim of increasing the duration of immunological memory and durability of tumor control. With an acceptable safety profile associated with Hsc70 and QS-21 Stimulon exposure in humans and demonstrated preclinical anti-tumor activity of ASV, we are poised to begin a first-in-human clinical trial by the first half of 2017. Citation Format: Mithun Khattar, Antoine Tanne, Benjamin Morin, Nicholas Wilson, Mohamed Uduman, Justin Zelin, Robert Stein, Mark Exley, John Castle, Daniel L. Levey. Pre-clinical development of a first-in-class fully synthetic heat shock protein-based personalized cancer vaccine [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A028.

Abstract A087: Quantifying the landscape of immunostimulatory tumoral RNA

Recent studies demonstrate an unexpected connection between aberrant transcription of noncoding RNA in tumors and innate immune system activation in the tumor microenvironment. Such RNA is often of unknown function and may consist of typically silenced interspersed elements, satellite repeats, and endogenous retroviruses. For instance, satellite RNA from the pericentromere (particularly HSATII) is abundantly transcribed in several solid tumors - such as pancreatic ductal adenocarcinoma - yet it is virtually silent in normal tissue. The genomic DNA repetitive regions this RNA is transcribed from frequently expand during tumorigenesis. Using novel quantitative methods, we have shown that a set of such repetitive elements, abundantly expressed in tumors, display sequence patterns typically associated with viruses. We therefore predicted they are immunogenic, particularly HSATII. In a novel, theory-experiment collaboration between the laboratories of Professors Benjamin Greenbaum and Nina Bhardwaj, the most significant set of these RNA have been validated as immunostimulatory (HSATII and murine GSAT), capable of activating antigen presenting cells - HSATII stimulated production of IL-6, IL-12 and TNFalpha (Tanne, et al., PNAS, 2015). At the same time a set of recent papers has shown that ERV transcription may be a predictor of immunotherapy response in melanoma. Hence, it is critical to profile key immunostimulatory endogenous RNA in the tumor microenviroment, understand which immune pathways different sets of such RNA activate, and assess the link between the specific pathways activated, prognosis, and immunotherapy. We further profile the landscape of activation and expression for endogenous elements. We have currently demonstrated a key set of noncoding RNAs preferentially expressed in cancer cells have sequence features that are immunostimulatory in humans, and have characterized the range and breadth of the expression of such elements in several solid tumors. We present several new results on the topic and the potential consequences of the aberrant expression of endogenous RNA that mimic pathogen features in cancer. Citation Format: Alexander Solovyov, Antoine Tanne, Luciana Muniz, Simona Cocco, Remi Monasson, Arnold Levine, David T. Ting, Nina Bhardwaj, Benjamin Greenbaum. Quantifying the landscape of immunostimulatory tumoral RNA [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A087.

Abstract A077: Unraveling predicted immunomodulatory effects of novel cancer-associated noncoding RNAs

Cellular noncoding RNA (ncRNA) transcription and regulation has only recently been systematically investigated. Extended transcriptomic analysis from recent studies has demonstrated abundant transcription of a set of ncRNAs preferentially within tumors, as opposed to normal tissue. Many of those ncRNA are associated with repeated regions of the genomes that in steady state are prone to regulation by epigenetic silencing. The contribution of the abnormal expression of this component of the dark matter genome to the oncogenic or antitumoral process remains unknown. In some cases, these tumor-associated ncRNAs have been associated with specific inflammatory transcriptomic regulation such as type I-IFN signatures, suggesting that the cell can sense these ncRNA and that they have potential immunostimulatory qualities. Derived from Eukaryotic cells possess a large panel of innate immune nucleic acid sensors, allowing the recognition of exogenous nucleic acids associated with microbial infections, but also of endogenous nucleic acids. Nucleic acid recognition is strongly associated with the antiviral innate immune response, autoimmune diseases development such as SLE and RA, but also inflammatory cancer development and anti-cancer immunity priming. The goal of this project was to identify novel endogenous immune modulators derived from tumor-associated ncRNAs that have immune stimulatory properties. Using mathematical methods adapted from physics statistics we previously analyzed the viral genome versus their host genomes and identified abnormal nucleotide motifs patterns. Using this novel approach to quantify transcriptome-wide motif usage in human and murine ncRNAs, we observed that, whereas most of the ncRNA are similar to the coding genome, an outlier subset of tumor-associated ncRNAs, typically of recent evolutionary origin, has motif usage more associated with pathogen derived RNA. Previous extended analysis of viral genomes highlighted that such motif patterns are strongly associated with immunogenic properties and absence of host-pathogen adaptation. We have now demonstrated that those ncRNA function directly as immunostimulatory “self-agonists” and can be classified as danger associated molecular patterns (DAMPS). A key subset of these ncRNA directly activate cells of the mononuclear phagocytic system (MPS), inducing pro-inflammatory cytokines in a MYD88 and UNC93b-dependent and possibly TLR8-dependant manner. Some of these ncRNA are also upregulated in specific tumor cell lines when they proliferate in spheroids. We have shown that the high level of ncRNA expression also correlates with immunogenic properties related to the RNA extracted from those tumor spheroids versus their 2D counterparts. We therefore propose that a part of the innate response in tumors could initiate from direct, likely extrinsic, interaction of immunogenic ncRNAs expressed in cancer cells, with innate pattern recognition receptors on APC. We are extensively characterizing the innate immune pathways involved in their sensing using forward genetic strategies, biochemistry and cellular biology techniques. We are also characterizing their intrinsic functions at the cellular level and to understand their function in the tumor microenvironment. The pattern of expression in different cancer models is under evaluation to establish their relevancy as prognostic and therapeutic strategies, or as biomarkers in addition to characterizing their relevancy as new immunotherapeutic adjuvants. In conclusion, we are characterizing an epigenome guardian function related to the newly discovered immunogenic properties of those non-coding RNA. Deeper analysis and refinement of our computational analysis will allow us to associate specific RNA features to their immunogenic properties in cancer. Citation Format: Antoine Tanne, Luciana Muniz, David Ting, Arnold Levine, Nina Bhardwaj, Benjamin Greenbaum. Unraveling predicted immunomodulatory effects of novel cancer-associated noncoding RNAs. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A077.

Abstract A037: The Agenus Immunogenic Mutation platform (AIM™) generates synthesis-ready blueprints for the AutoSynVax™ vaccine patient-specific neo-antigen vaccine

Agenus is clinically testing Prophage™, an individualized tumor vaccine which uses heat-shock proteins endogenously complexed to neo-antigens harvested from the patient9s own tumor. Agenus9 genomics and bioinformatics unit rapidly identifies potentially immunogenic neo-antigens in the patient9s tumor for use in immunomonitoring. However, in cases such as when the tumor mass is insufficient to generate the Prophage vaccine, we plan to treat patients with an individualized synthetic neo-antigen vaccine, AutoSynVax™ vaccine, combined with QS21 Stimulon® adjuvant and optionally immunomodulatory antibodies. In a murine model, we have demonstrated tumor control after treatment with our synthetic vaccine. The majority of mutations in human tumors are unique to the individual tumor, necessitating the rapid identification of mutations using next generation sequencing and bioinformatics. Further, the number of candidate immunogens presented on a tumor can range from a handful to hundreds. Accurately identifying tumor mutations; determining their impact, expression and translation; and predicting ability to bind patient-specific HLA class I and II molecules and elicit an immune response (immunogenicity) is complex, particularly in the context of clinical trials. The AutoSynVax™ vaccine platform leverages AIM™, the Agenus Immunogenic Mutation workflow, to generate a synthesis-ready blueprint for optimal immunogenic patient-specific neo-antigen vaccines. AIM™ comprises a robust and efficient approach to computational vaccinology designed to generate an immunogenic candidate blueprint, agnostic to vaccine format, followed by a format-specific blueprint ready for vaccine synthesis and manufacture. Citation Format: Mohamed Uduman, Armen Karapetyan, Mithun Khattar, Antoine Tanne, Benjamin Morin, Justin Zelin, Sandra Craig, Shiwen Shiwen, Bishnu Joshi, Mark A. Findeis, Nicholas Wilson, Elise Drouin, Amy Yang, Jeffrey Raizer, John Goldberg, Jennifer Buell, Robert Stein, John Castle, Daniel L. Levey. The Agenus Immunogenic Mutation platform (AIM™) generates synthesis-ready blueprints for the AutoSynVax™ vaccine patient-specific neo-antigen vaccine [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A037.

Abstract 531: The landscape of immunostimulatory RNA transcription

Recent studies have demonstrated an unexpected connection between aberrant transcription of noncoding RNA (ncRNA) in tumors and innate immune system activation in the tumor microenvironment (TME). Such ncRNA are often of unknown function and may consist of typically silenced interspersed elements, satellite repeats, and endogenous retroviruses (ERVs). For instance, satellite RNA from the pericentromere (particularly HSATII) has been shown to be abundantly transcribed in several solid tumors, including the often lethal pancreatic ductal adenocarcinoma, yet is virtually silent in normal tissue. The genomic DNA repetitive regions this RNA is transcribed from frequently expand during tumerigenesis. Using a novel computational approach, we have shown that, unlike ncRNA expressed under normal conditions, a set of such repetitive elements, abundantly expressed in tumors, display patterns typically associated with viruses. We therefore predicted they are immunogenic, particularly HSATII. In a novel, theory-experiment collaboration between the laboratories of Professors Benjamin Greenbaum and Nina Bhardwaj, the most significant set of these ncRNA have been validated as immunogenic (HSATII and murine GSAT), capable of activating antigen presenting cells - HSATII stimulated production of IL-6, IL-12 and TNFalpha (Tanne, et al., PNAS, 2015). At the same time a set of recent papers has shown that ERV transcription may be a predictor of immunotherapy response in melanoma. Hence, it is critical to profile key “immunogenic” ncRNA (i-ncRNA) in the tumor microenviroment, understand which immune pathways different sets of i-ncRNA activate, and assess the link between the specific pathways activated, prognosis, and immunotherapy. Our preliminary work on melanoma transcriptomes has shown that activation of endogenous elements implies better prognosis. We further profile the landscape of activation for i-ncRNA candidates. We have currently demonstrated a key set of ncRNAs preferentially expressed in cancer cells have sequence features that are immunostimulatory in humans. Quantitatively, our analysis uses a new, unique set of computational methods to study the innate immune system in the TME, originally designed to find motifs in viruses targeted by innate immune receptors (Greenbaum, et al., PNAS, 2014). Our methods for motif characterization utilize a novel mathematical approach we created based on transfer matrix approaches in statistical physics, and are here utilized for the purpose of discovering immunogenic nucleic acids in the tumor microenvironment. They offer several advantages over previous approaches. In particular they are far more computationally efficient, allowing the discovery of anomalous, potentially immunostimulatory patterns of motif usage in far larger sequence datasets than was previously practical. Citation Format: Alexander Solovyov, Antoine Tanne, David Ting, Arnold Levine, Nina Bhardwaj, Benjamin Greenbaum. The landscape of immunostimulatory RNA transcription. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 531.