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-β-glucuronyl carbamate (DOX-GA3), by β-glucuronidase present in necrotic tumor areas might be improved after transduction of tumor cells to secrete a targeted form of β-glucuronidase. To that end, we constructed an adenovirus vector, designated Ad/C28-GUSh, encoding human β-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 β-glucuronidase can further improve DOX-GA3 monotherapy.