Epidermal growth factor receptor targeting enhances adenoviral vector based suicide gene therapy of osteosarcoma. Despite improvements in the treatment of osteosarcoma (OS) there are still too many patients who cannot benefit from current treatment modalities. Therefore, new therapeutic approaches are warranted. Here we explore the efficacy of targeted adenoviral based suicide gene therapy.Immunohistochemistry and FACS analysis detected low or absent expression levels of the primary adenovirus receptor CAR on human primary OS and human OS cell lines. These results predict a low infection efficiency and thus a reduced therapeutic effect. Targeting the adenoviruses to another receptor highly expressed on OS could overcome this limitation. We found epidermal growth factor receptor (EGFR) to be widely expressed on primary OS. Immunohistochemistry on primary tumor samples and FACS analysis on primary short-term cultures and four OS cell lines showed that EGFR was consistently expressed. The recombinant bispecific single-chain antibody 425-s11 redirects adenoviral vectors towards the EGFR. Adenovirus transduction experiments in the presence or absence of 425-s11 showed significantly enhanced gene transfer with the targeted adenoviral vector compared with the native vector (OS cell lines 2.5 to 7.2 times enhanced gene transfer and OS primary short term cultures 1.7 to 10 times enhanced gene transfer). On this basis, targeted suicide gene therapy experiments with AdCMVHSV-TK in combination with ganciclovir were performed. These experiments demonstrated up to 3.5-fold enhanced kill of OS cell lines and primary short-term cultures by the EGFR targeted vector.Suicide gene therapy with adenovirus targeted towards EGFR may have favorable therapeutic characteristics for future gene therapy applications in OS.Copyright 2002 John Wiley & Sons, Ltd.