Abstract 584: Identification of mutations in HSV-1 envelope glycoprotein B that enhance retargeted infection

Conditionally-replicating herpes simplex virus type 1 (HSV-1) vectors, including mutants deleted for the neurovirulence factor ICP34.5 currently in clinical trials, are promising agents for oncolytic virotherapy. As an alternative strategy to accomplish cytocidal HSV replication exclusively in cancer cells, we have designed modified viral envelope glycoproteins to limit virus entry uniquely to EGF receptor (EGFR)-bearing cells. HSV-1 entry is initiated by the interaction of glycoprotein D (gD) with one of its receptors, herpesvirus entry mediator (HVEM) or nectin-1. Receptor binding results in a conformational change in gD triggering activation of gB and gH as the effectors of fusion between the viral envelope and cell membranes. To target virus entry solely to EGFR-expressing cells, we mutated or deleted gD residues essential for binding to the natural entry receptors and inserted an EGFR-specific single-chain antibody (scFv). Although ablation of virus entry through HVEM and nectin-1 was successful, entry through the EGFR was detectable but inefficient. To address this flaw, we carried out in vitro evolution experiments to isolate gain-of-function mutations that could compensate for defective gD-receptor interactions. We passaged a nectin-1-binding impaired HSV-1 mutant repeatedly through cells expressing a gD-binding impaired nectin-1 until visible plaques appeared. Plaque purification and sequence analysis revealed that each isolate contained the same double mutation in gB. We transferred the gB double mutation into wild-type virus and observed entry into cells that are normally resistant to HSV infection due to the absence of authentic gD receptors. In addition, we found that the mutant-gB virus was capable of entering cells through nectins other than nectin-1, and that entry into nectin-1-bearing cells was markedly accelerated compared to wild-type virus. We combined the gB double mutation with the EGFR-retargeted gD allele and found that this combination yielded increased entry into target cells without raising off-target infection. We are currently expanding this strategy to target other cancer-specific cell-surface markers. By enhancing the efficiency of retargeted infection, the gB double mutation is likely to reduce the need for strong mutations in current oncolytic vectors that not only prevent virus replication in normal cells, but also diminish replication in tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 584.