605. ABSTRACT WITHDRAWN

CANCER-ONCOLYTIC VIRUSES II imaging of virus treated animals demonstrated an intensifying tumor signal during the rst 24-48 hours after virus administration followed by loss of signal during the subsequent 48 hours, after which the tumors rapidly regressed. Tc99m is a gamma-emitting radioisotope known to be concentrated by NIS-expressing cells. In subsequent experiments designed to elucidate the process of intratumoral virus spread, tumors were harvested from virus treated mice at 24, 48 and 72 hours after virus infusion. In each case Tc99m was administered intraperitoneally 4 hours prior to tumor harvest. Serial sections from these explanted tumors were subjected to autoradiography to localize radioactivity, immunohistochemistry to detect VSV-infected cells, TUNEL staining to detect apoptotic cells, and DAPI staining to detect intact cell nuclei. Autoradiographs revealed multiple discrete foci of radioisotope concentration throughout the parenchyma of treated tumors whose average diameter increased between the 24 and 48 hour timepoints. Immunohistochemical staining for VSV proteins in sections adjacent to those used for autoradiography revealed discrete foci of virally infected cells corresponding precisely to sites of radioisotope concentration. By 24 hours the VSV infected cells at the center (not at the periphery) of each focus were strongly TUNEL positive and had lost their nuclear DAPI staining. Overall, the data elucidate a model for successful systemic oncolytic virotherapy in which intravenously administered viral particles extravasate from tumor blood vessels (probably by EPR effect) establishing multiple discrete foci of virus infection in the tumor parenchyma which rapidly expand through centripetal virus spread. Cells in the centers of expanding foci amplify the virus, undergo apoptosis and release progeny which apparently migrate only as far as the adjacent rim of uninfected cells whereupon the cycle is repeated. Provided there are a sufcient number of infectious centers distributed at an appropriate density throughout the tumor, the centripetally expanding foci of virus infection eventually coalesce and the tumor is destroyed.

605. ABSTRACT WITHDRAWN

Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy

606. Combination of Anti Angiogenic Cancer Therapy with Systemic Oncolytic Virotherapy To Treat Established Tumors

Timothy Kottke,1 Geoff Hall,2 Jose Pulido,1,3 Rosa Diaz,1 Jill Thompson,1 Debabrata Mukhopadhyay,4 Heung Chong,5 Peter Selby,2 Matt Coffey,6 Hardev Pandha,7 John Chester,2 Alan Melcher,2 Kevin Harrington,8 Richard Vile.1,2 1 Department of Molecular Medicine, Mayo Clinic, Rochester, MN; 2Cancer Research UK Clinical Centre, St. James’ University Hospital, Leeds, United Kingdom; 3Department of Ophthalmology and Ocular Oncology, Mayo Clinic, Rochester, MN; 4Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN; 5St George’s Hospital Medical School, Tooting, London, United Kingdom; 6Oncolytics Biotech, Inc., Calgary, Canada; 7 Department of Oncology, University of Surrey, Guildford, United Kingdom; 8The Institute of Cancer Research, London, United Kingdom. Clinical trials of oncolytic virotherapy have shown low toxicity and encouraging signs of efcacy. However, it remains critically important to develop clinically implementable methods for systemic delivery to treat established tumors. In this respect, inhibitors of VEGF165 signaling are already widely used to treat several different cancers. We have developed novel protocols in which these inhibitors are combined with systemic delivery of oncolytic viruses to treat metastatic tumors. Using non-VEGF-expressing tumors in immunocompetent mice, we achieved long-term cures in mice treated with VEGF165 followed, after a specic interval, by intravenous reovirus or VSV. Therapeutic effects derived mainly from VEGF165-mediated stimulation of endothelial cells transiently to support viral replication. Appropriately timed systemic virus delivery led to replication in, and lysis of, tumor-associated endothelial cells and innate immunemediated anti-vascular effects with subsequent vascular collapse. By extending this principle to tumors over-expressing VEGF165, we combined clinically-approved VEGF165 inhibitors (Sunitinib and Avastin) with virus delivery to achieve long-term cures. Using molecular constructs in which the extracellular binding domain of the EGF receptor is ligated to the intracellular signaling domains of either VEGFR1 or VEGFR2, we observed that a) the ability to induce Reovirus replication in endothelial cells is dependent upon VEGFR2 signaling; b) that endothelial cell sensitivity to NK cells upon Reovirus infection is dependent upon VEGFR1 signaling; and c) that NK activation by Reovirus exposed endothelial cells does not require ongoing viral replication (VEGFR2-dependent) but simple virus infection (VEGFR1-dependent). We also observed that, although the VEGF165+Reovirus combination was still surprisingly effective in Reovirus-immune mice, combination with cyclophosphamide signicantly improved the efcacy of the treatment possibly through CPA-mediated effects on NK cell activation and virus-infected endothelium. Therefore, we have developed here a novel method by which clinically approved inhibitors of VEGF165 can be combined S235