432. Characterization of a Novel Oncolytic Measles Virus Retargeted Against the Urokinase Receptor: In Vitro and In Vivo Studies
Authors: Jaime R. Merchan, Caili Tong, Takafumi Nakamura, Ianko Iankov, Stephen J. Russell
Journal: Molecular Therapy (2006)

Objective. The Edmonston vaccine strain of measles virus (MV- Edm) is a novel oncolytic agent with potent in vitro and in vivo antitumor activity. In order to improve viral tumor specificity, suitable targets need to be identified. The "ideal" target for an oncolytic virus should be biologically relevant, overexpressed in tumor compared to normal cells, and preferably overexpressed by tumor stroma, to amplify the virus' antitumor effects. To test the retargeted virus' effects in appropriate syngeneic models, it should be relevant for murine tumors. The urokinase receptor (uPAR) is a biologically validated tumor and stromal target that has been associated with an aggressive tumor phenotype in several cancer types. Our goals are to determine if uPAR is a suitable target for MV oncolytic virotherapy in human and mouse cancer models. Methods and Results. The receptor binding, aminoterminal fragment (ATF) of human (h) and murine (m) uPA displayed into the C-terminus of a CD46 "blind" mutant MV-H glycoprotein (Y481A, R533A, S548L, F549S-Nakafumi et al., 2005-). Co-transfection of human and murine cells with uPAR retargeted H and F genes demonstrated potent in vitro fusion in uPAR overexpressing tumor cells, but not in uPAR non-overexpressing tumor or normal cells. CD46-ablated, eGFP expressing uPAR retargeted MV-Edm [MV-ATF (h) and (m)] were successfully rescued and expanded. Expression of chimeric H-ATF was successfully demonstrated by RT-PCR of MV-ATF-infected VERO-His cells, and by Western Blot of viral particles. Viruses retargeted to (h) and (m) uPAR entered cells selectively through their respective targeted receptors and induced cell fusion, syncitia formation and cytotoxicity. Since uPA/ uPAR binding is species specific, evidence of uPAR target specificity was further demonstrated by the absence of cell fusion of human uPAR(+) cells after infection with MV-ATF (murine) and vice versa. Successful uPAR dependent viral replication of MV-ATF (m) was demonstrated in uPAR (+) murine tumorigenic cells. Murine mammary cancer cells (4T1) were implanted in immunocompetent Balb/C and SCID mice. Mice received intratumor (IT) injections of MV-GFP retargeted against (m) uPAR and sacrificed 48-96 hours after the last dose. IT administration of MV-ATF (m) resulted in successful infection and syncytia formation in tumor cells as well as tumor vasculature in vivo in immunocompetent and SCID tumor bearing mice, as demonstrated by confocal laser microscopy. Conclusions: uPAR mediated cell fusion is feasible in human and murine cancer cells. uPAR retargeted MV-Edm specifically and efficiently induces fusion and cytotoxicity in human and murine cancer cells in a uPAR dependent manner. Our results demonstrate for the first time successful replication of a fully retargeted measles virus in murine tumor cells in vitro and in vivo, and suggest targeting of tumor vasculature. MV-ATF is a novel, promising viral oncolytic agent, which can be used in syngeneic-immunocompetent tumor models. Experiments assessing the antitumor effects of this novel virus in syngeneic and xenograft models are underway.