An ovine atadenovirus encoding E. coli purine nucleoside phosphorylase (PNP) with prodrug activating activity. Under the control of a prostate-directed promoter, PNP-expressing atadenovirus vaccine FP253 expresses PNP in prostate tissue only after intraprostatic administration; this enzyme catalyzes systemically administered fludarabine prodrug into the active agent, 2-fluoroadenine. Localized prodrug activation provides prostate-targeted chemotherapy, potentially reducing systemic side effects.