Abstract B84: Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition
Authors: Speranza Maria Carmela , Ricklefs Franz , Passaro Carmela , Klein Sarah R. , Kasai Kazue , Kaufmann Johanna , Nakashima Hiroshi , Agnieszka Bronisz , Aguilar-Cordova Estuardo , Guzik Brian W. , Freeman Gordon J. , Reardon David A. , Wen Patrick , Chiocca E. Antonio , Lawler Sean E. 
Published: March 2017

Abstract
Early clinical trial data show that blockade of PD-1 signaling leads to significant anticancer responses in a subset of patients in certain cancer types. While the brain has traditionally been considered to be an immune-privileged site, evidence supporting the use of immunotherapeutics in brain tumors has been rapidly accumulating. Given that virus-based cancer therapies can be immunostimulatory and immune checkpoint inhibitors block the body's natural checkpoint response, the combination of these two approaches offers a potentially advantageous interaction. One of the molecular underpinnings of T-cell exhaustion is the expression of Programmed Death-1 (PD-1) on T-cells that recognizes its ligand PD-L1. AdV-TK is an immunostimulatory virus-based approach, known as Gene-Mediated Cytotoxic Immunotherapy (GMCI), that involves the intra-tumoral delivery of a non-replicating adenoviral vector carrying the Herpes virus thymidine kinase gene (TK) followed by administration of an anti-herpetic prodrug (ganciclovir GCV) and recently showed encouraging results in a Phase II trial in glioblastoma (Wheeler et al., 2016). The immunological component results from the delivery vehicle being a virus, the mode of cell death, through both necrosis and apoptosis, and the pro-immunogenic properties of the TK protein. We confirm that this approach induces glioblastoma cell death and a consistent anti-tumor immune stimulation. Not surprisingly, however, this immune stimulation also leads to increase in cell surface of immune checkpoint inhibitory ligands on tumor cells, including PD-L1, detected by flow cytometry and immunohistochemistry. We show that GMCI induces a type-I interferon response, and using IFN decoy we demonstrated that the release of IFNβ in vitro is at least partially responsible for autocrine/paracrine PD-L1 up-regulation both in human and mouse glioblastoma cell lines. In vivo studies using an intracranial GL261 model showed high numbers of long term survivors in the GMCI/PD-1 combination (11/14), compared with GMCI (6/16), anti-PD-1 (5/12) and untreated (0/11). In addition, long term survival mice were no longer able to form tumors after rechallenge indicating the establishment of anti-tumor immunity. Finally, tumor infiltrating lymphocytes after GMCI showed an increase in CD8+, CD8+/GranzymeB+, and IFNγ+ cells suggestive of cytotoxic T-cell activation. However, there was also a significant increase in CD4+, CD4+/FoxP3+, and IL-10 indicating a significant infiltration by Tregs, releasing immunosuppressive cytokines. Additionally, there was a significant increase in PD-1+ /TIM3+ T-cells, indicative of an immunosuppressive microenvironment. Overall, our data show that GMCI/anti-PD-1 combinatorial therapy is effective in a syngeneic tumor model, and strongly support clinical trials of GMCI/checkpoint inhibitor combinations in glioblastoma patients.
Citation Format: Maria Carmela Speranza, Franz Ricklefs, Carmela Passaro, Sarah R. Klein, Kazue Kasai, Johanna Kaufmann, Hiroshi Nakashima, Bronisz Agnieszka, Estuardo Aguilar-Cordova, Brian W. Guzik, Gordon J. Freeman, David A. Reardon, Patrick Wen, E. Antonio Chiocca, Sean E. Lawler. Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition. [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 B84.