Abstract B04: Intralesional vaccination with Ad-MAVS alters the immunosuppressive tumor microenvironment and elicits robust anti-tumor immunity in non-immunogenic cancers
Authors: Leon Gabriel De , Wang Tao , Agarwal Pankaj , Lyerly H. Kim , Hartman Zachary 
Published: March 2017

Abstract
Background and Hypothesis: While the use of immune checkpoint blockade has gained significant traction as a viable therapy for certain cancers, many tumors remain refractory to these immunotherapies due to their highly immunosuppressive microenvironment. As a novel approach to boost local anti-tumor immunity and sensitize cancers to these therapies, we constructed an adenoviral (Ad) vector to overexpress the innate signaling adaptor molecule, Mitochondrial Antiviral Signaling protein (MAVS). Overexpression of MAVS results in innate immune signaling downstream of the RIG-I like receptors (RLRs) resulting in the activation of multiple signaling pathways as well as the release of a Th1 cytokine milieu and type I interferons. We therefore hypothesized that adenoviral delivery of MAVS into the tumor microenvironment would produce robust inflammatory signaling which would alter the tumor microenvironment to both efficiently suppress tumor growth and stimulate anti-tumor immune responses.
Methods: In order to determine the possible effect MAVS would have on different cell types within the tumor microenvironment we infected a range of primary and cancer cells types (Bone-marrow derived dendritic cells (BMDCs), primary fibroblasts, as well as 5 different murine tumor cell lines representing breast-4T1, colon-CT26CL25, bladder-MB49, melanoma-B16F10, and prostate-TRAMPC2) with Ad-MAVS or a control Ad. Innate immune responses were measured via luminex ELISA, microarray, and RT-qPCR. The impact on tumor growth was assessed by MTT and expression of PD-L1 by FACS. Lastly, we measured the anti-tumor effect and adaptive immune responses of animals treated with various combinations of Ad-MAVS, a control Ad, and PD-L1 targeting antibodies in vivo utilizing various immunocompetent and immune-compromised mouse models.
Results: In vitro infection of BMDCs, MEFs, and murine tumor cell lines demonstrated that MAVS expression elicited widespread production of cytokines and chemokines indicative of a type I immune response (such as IL-12, TNF-alpha, RANTES, etc.) and interferon beta. These profound responses also triggered immune regulatory feedback expression of PD-L1, enabling their targeting by PD-L1 antibodies. Additionally, we found that Ad-MAVS infection of tumor cell lines significantly inhibited their proliferation in vitro, likely via interferon pathways. In vivo, treatment with Ad-MAVS also elicited robust stimulation of innate immune responses and numerous type I inflammatory cytokines. Moreover, in multiple types of tumor bearing animals, we found that a single intralesional treatment of Ad-MAVS significantly prolonged overall survival and was capable of eliciting systemic anti-tumor adaptive immunity to non-immunogenic tumors.
Conclusion: Our study identifies that MAVS expression can elicit the production of a significant number of Th1-type cytokines, chemokines, and interferon-beta in multiple cell types within the tumor microenvironment. We also demonstrate that overexpression of MAVS results in the direct suppression of tumor cell growth and stimulation of tumor PD-L1 expression. These activities were also observed in vivo, as intralesional injection of Ad-MAVS elicited Th1 cytokines and anti-tumor immunity in multiple tumor types, which correlated with enhanced systemic T-cell responses to tumor-specific epitopes. Collectively, these data demonstrate that intralesion injection of Ad-MAVS alters the tumor microenvironment to stimulate anti-tumor immunity and potentially sensitizes non-immunogenic tumors to PD-L1 therapies. As such, Ad-MAVS represents a novel approach of stimulating anti-tumor immunity via activation of innate adaptor molecules to improve immunogenic anti-tumor responses and potentially the therapeutic efficacy of PD-L1 immune checkpoint blockade.
Citation Format: Gabriel De Leon, Tao Wang, Pankaj Agarwal, H. Kim Lyerly, Zachary Hartman. Intralesional vaccination with Ad-MAVS alters the immunosuppressive tumor microenvironment and elicits robust anti-tumor immunity in non-immunogenic cancers. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B04.