Abstract A28: Adenoviruses up-regulate IL17F, but not IL17A, and activate NK cells, with potential impact on oncolytic adenovirus efficacy

Abstract

Abstract Oncolytic viruses (OVs) are a new class of anti-cancer agent. They infect and replicate selectively within malignant cells, whilst sparing normal cells. OVs disrupt chemokine and cytokine networks, which may influence clinical efficacy. This study investigates the role of innate immune responses, in particular IL17F and Natural Killer cells (NK), on the efficacy of oncolytic adenoviruses. A transcriptional array (Qiagen RT2 Profiler) of 84 human chemokines and cytokines revealed highly consistent changes (r=0.623, p<0.0001) following infection of ovarian cancer lines (TOV21G, OVCAR4) by the E1A CR2-deleted oncolytic adenovirus dl922-947. The most highly upregulated cytokine in both cells was IL17F (fold change=166.9 in TOV21G, p<0.0001, and 97.6 in OVCAR4, p<0.05), whilst CXCL1 and CCL2 were the most down-regulated. Similarly, an increase in IL17F transcription was seen after dl922-947 infection in HeLa cells (fold change=4.3, p<0.05) and after Ad5 wild-type infection in OVCAR4 (fold change=18.3, p<0.01). By ELISA, an increase in IL17F production was also observed after dl922-947 infection in TOV21G (mean IL17F was 4930pg/106 cells for dl922-947 versus 1167pg/106 for mock infection, n=2 in triplicates, p<0.05). We also observed increased transcription (median fold-change=91.5, p<0.05) and translation (median 2086pg/106 for dl922-947 vs 1381pg/106 for mock infection, p<0.05) of IL17F in 6 primary lines derived from ascites of patient with ovarian cancer, suggesting immediate clinical relevance of our findings. As human adenoviruses do not replicate in mice, HeLa subcutaneous xenografts in CD1 nu/nu mice were used to evaluate the infiltration of immune cells after dl922-947 infection. Tumors were harvested 48 hours following a single intra-tumoral injection of dl922-947 (1010 particles). IL17F transcription increased significantly in virus-treated tumors (mean fold-change=1 for mock-infected, n=5, vs 10.7 for dl922-947-infected, n=7), accompanied by a significant neutrophil infiltration (as detected by NIMP-R14, which binds Ly6G and Ly6C; median histoscores 2 and 16.5 for mock and dl922-947 infected tumors, respectively, p<0.05). We are currently generating TOV21G IL17F-/- cells using CRISPR-Cas9 technology, which will be used to evaluate potential changes in dl922-947 efficacy in vitro and in vivo. NK have potential impact on oncolytic virus efficacy (1). The interaction between NK and dl922-947 was evaluated in vitro using peripheral blood NK from health donors. Significant increases in CD107a+ NK, a degranulation marker, and interferon-Υ production were observed after co-culture with dl922-947-infected TOV21G, compared to co-culture with mock-infected TOV21G, by flow cytometry and ELISA, respectively (CD107a in 9.0% vs 1.8% of total NK, p<0.01 and interferon-Υ were 8559 +/-356pg/mL vs 4 +/-1pg/mL, respectively). Furthermore, Ad-CMV-GFP, an E1-deleted Ad5 vector, infects ovarian cancer lines but not NK-92 cells (92% vs 0% infected, respectively, p<0.001), suggesting the activation observed was not due to direct infection of NK by Ad5. We will investigate alteration of NK cytotoxicity against ovarian cancer cells after exposure to dl922-947 infection by calcein cytotoxicity assays, using both peripheral blood NK and NK from ascites of women with ovarian cancer. Any change in efficacy of dl922-947 with or without NK depletion in vivo will be assessed using ovarian cancer (TOV21G) intraperitoneal xenografts in CD1 nu/nu mice. To conclude, adenovirus infection leads to distinctive changes in chemokines and cytokines, as well as activation of NK. In particular, IL17F, but not IL17A, was up-regulated after adenovirus infection. The influence of these observations on oncolytic adenovirus efficacy is currently being evaluated. 1. Alvarez-Breckenridge CA, et al. Nat Med. 2012 Dec;18(12):1827-34. Citation Format: Elaine YL Leung, Melanie Weigert, Josephine Walton, Darren Ennis, Dimitris Athineos, Suzanne Dowson, Chris Hansell, Karen Blyth, Gerard Graham, Iain McNeish. Adenoviruses up-regulate IL17F, but not IL17A, and activate NK cells, with potential impact on oncolytic adenovirus efficacy. [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 A28.