Potency-enhancing mutations in E3-19K and i-leader increase the cytolytic activity of the PH20/SPAM1-armed oncolytic adenovirus Ad5Δ24RGD.

The oncolytic adenovirus Ad5-delta-24-RGD (Ad5Δ24RGD) exhibits suboptimal therapeutic efficacy in high-grade glioma. Here, we show that combining two specific potency-enhancing mutations in E3-19K and i-leader (designated 19K(SS)-iL(Q125Ter)) markedly increases the cytolytic efficiency and the size of vital dye-stained plaques. Unexpectedly, these mutations do not enhance actual spread efficiency, measured as the size of fluorescent plaques, in most tested cell lines. In contrast, the type of fiber modification (F5RGD4C, F5/3, or F5/35) strongly influences actual spread efficiency, with F5RGD4C generally conferring the greatest enhancement. In some cell lines, the fiber modification F5RGD10(2C) or verapamil treatment further improves actual spread efficiency. Nelfinavir inhibits spread and plaque formation of oncolytic adenoviruses with the 19K(SS)-iL(Q125Ter) modifications, irrespective of adenovirus death protein (ADP) expression. Expression of the reporter transgenes EGFP and Fluc or the human hyaluronidase PH20/SPAM1 from the E1B-55K region or downstream of the L3-23K or L5-Fiber region differentially affects the oncolytic potency of Ad5-delta-24-RGD-19K(SS)-iL(Q125Ter). We identified an insertion site downstream of the L3-23K region that supports relatively high hPH20 activity while preserving the enhanced oncolytic potency of the virus. Combining 19K(SS)-iL(Q125Ter) with hPH20 expression may potentially improve therapeutic benefit in glioma.