**10. Strategies to Maximize Cell Killing by Oncolytic Viruses**

Oncolysis is an HLA independent killing mechanism that should be exploited in cancers with severe immunological defects. Several hallmarks of cancer, including immune deregulation, appear to provide an advantageous niche for virus activity [90]. These also include the intrinsic resistance of cancer cells to apoptosis and their reprogrammed energy metabolism that provides biosynthetic intermediates to support macromolecular synthesis. When this is combined with hard HLA defects and little to no prospect of a functional cytotoxic T lymphocyte response, a virus might be expected to remain on station for as long as there are substrate cancer cells to infect. In xenografts, efficacy through oncolysis can be demonstrated without help or hindrance from T-cells [91–93].

The combination of virotherapy with chemotherapy can also lead to enhanced virus spread and cell lysis. Synergy with a wide range of chemotherapeutic agents, including platinum drugs, taxanes and topoisomerase inhibitors, among others, have been reported and reviewed in detail elsewhere [94]. Arguably, the most convincing anticancer efficacy in solid carcinomas with oncolytic viruses was in combination with chemotherapy [95]. In this study, patients refractory to several lines of previous treatment were injected intratumorally with ONYX-015 concurrently with intravenous cisplatin and 5FU. Only one of the tumor nodules (the largest) was injected, with uninfected tumors acting as internal controls. In 19 evaluable patients, eight had a complete response in the injected tumor and the remainder had a partial response with signs of extensive lysis. That this effect was mediated by direct oncolysis without adaptive immunity was evidenced by the lack of any effect on non-injected tumors.

Arming viruses to treat HLA compromised tumors can be fully focused on promoting intratumoral virus spread and direct oncolysis. This is made easier because virus gene products that evade premature CTL killing like E3 19k in adenovirus or ICP47 in HSV-1 become redundant in this setting and removing them makes space for additional transgenes. Matrix degrading enzymes or cell fusion peptides have both demonstrated enhanced spread of oncolytic viruses in vitro and in vivo. Enzyme prodrug therapy using thymidine kinase (TK) or cytosine deaminase (CD) [96] is an alternative approach that combines many of the benefits of targeted chemotherapy with the anatomical selectivity of a virus. An innocuous prodrug given systemically that is activated into a cytotoxic entity only by the enzyme within the tumor restricts cytotoxicity to the environs of the infected tumor cells and spares normal tissues, including the bone marrow. In the case of TK, the prodrug is usually ganciclovir, which is metabolized to yield a potent DNA polymerase inhibitor, whereas CD metabolism 5-fluorocytosine to become 5-fluorouracil (5FU), which is a widely used inhibitor of thymidylate synthase. In particular, 5FU is an attractive therapeutic for this approach because it diffuses widely and will mediate a considerable bystander toxicity against cells that are not directly virus infected [97]. Future strategies to enhance virus spread or cytotoxicity could involve transgene products, including proteins or nucleic acids packaged into exosomes and other membrane vesicles that are shed from the infected cell, and thereby allow their transfer into the cytosol of uninfected cancer cells nearby [98].
