Pronounced antitumor efficacy by extracellular activation of a doxorubicin-glucuronide prodrug after adenoviral vector-mediated expression of a human antibody-enzyme fusion protein.

ABSTRACT

Tumor-specific activation of the glucuronide prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate (DOX-GA3), by beta-glucuronidase present in necrotic tumor areas might be improved after transduction of tumor cells to secrete a targeted form of beta-glucuronidase. To that end, we constructed an adenovirus vector, designated Ad/C28-GUSh, encoding human beta-glucuronidase fused to a human single-chain Fv (scFv) against the epithelial cell adhesion molecule (EpCAM), C28, and preceded by a signal sequence for secretion. Antibody specificity and enzyme activity were retained in the fusion protein secreted by tumor cells infected with Ad/C28-GUSh. Diffusion of fusion protein from transduced tumor cells within MCF-7 multicellular spheroids was visualized by immunohistochemistry. Treatment of spheroids with Ad/C28-GUSh and DOX-GA3 resulted in growth inhibition comparable to treatment with doxorubicin alone. Treatment of well-established FMa human ovarian cancer xenografts with intravenous injection of DOX-GA3 (500 mg/kg) resulted in a tumor volume-doubling time of 23.8 days compared to 8.0 days for phosphate-buffered saline (PBS)-treated mice. Intratumoral administration of Ad/C28-GUSh before DOX-GA3 enhanced the growth inhibition and increased the tumor volume-doubling time to 43.1 days (p < 0.01), while virus alone had no effect. Thus, we have successfully shown that an adenovirus vector encoding a secreted, targeted form of human beta-glucuronidase can further improve DOX-GA3 monotherapy.