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Oncolytic viruses that are replication competent in tumor but not in normal cells represent a novel approach for treating neoplastic diseases. However, the oncolytic potency of replicating agents is determined directly by their capability of infecting target cells. Most adenoviruses used for gene therapy or virotherapy have been based on serotype 5 (Ad5). Unfortunately, expression of the primary receptor for Ad5 (the coxsackie-adenovirus receptor, or CAR) is highly variable on ovarian and other cancer cells. By performing genetic fiber pseudotyping, we created Ad5/3-Delta24, a conditionally replicating adenovirus that does not bind CAR but facilitates entry into and killing of ovarian cancer cells. We show replication of Ad5/3-Delta24 and subsequent oncolysis of ovarian adenocarcinoma lines. Replication was also analyzed with quantitative PCR on three-dimensional primary tumor cell spheroids purified from patient samples. Moreover, in a therapeutic orthotopic model of peritoneal carcinomatosis, dramatically enhanced survival was noted. Finally, Ad5/3-Delta24 achieved a significant antitumor effect as assessed by noninvasive, in vivo bioluminescence imaging. Therefore, the preclinical therapeutic efficacy of Ad5/3-Delta24 is improved over the respective CAR- and integrin-binding controls. Taken together with promising biodistribution and toxicity data, this approach could translate into successful clinical interventions for ovarian cancer patients.

Adenovirus serotype 5 (Ad5) displays unparalleled gene transfer efficacy to cells with high coxsackie-adenovirus receptor (CAR) expression. Unfortunately, cells isolated from clinical human cancers, both ovarian and other types, express highly variable and often low levels of CAR. Fortunately, native Ad5 tropism can be modified to circumvent CAR deficiency and to enhance infectivity. Ad5/3luc1 incorporates the serotype 3 fiber knob and binds to a receptor distinct from CAR, while the fiber of Ad5lucRGD is modified with an RGD-4C motif, allowing CAR-independent binding to integrins. We studied the liver tropism and blood clearance of these viruses after intravenous (i.v.) injection, and biodistribution after intraperitoneal (i.p.) injection to tumor-bearing mice. To estimate efficacy, we assessed gene transfer to purified human primary ovarian cancer cells, and in a mouse model of ovarian cancer. Ad5/3luc1 achieved improved gene transfer over Ad5lucRGD, and both infectivity-enhanced viruses were superior to the isogenic control with an unmodified Ad5 capsid. In the presence of malignant ascites, gene transfer was improved with both Ad5/3luc1 and Ad5lucRGD. Thus, retargeting to the Ad3 receptor enhances gene transfer to clinically relevant ovarian cancer substrates, while the mouse toxicity and biodistribution profile of both fiber-modified Ad vectors is comparable to Ad5., (c)2002 Elsevier Science (USA).)