Development and Preclinical Evaluation of Enzyme Prodrug Therapies Targeted to the Tumor Vasculature

Abstract

Breast cancer is a global health concern of high prevalence that lacks safe and effective therapies for advanced cases. A targeted enzyme prodrug therapy aims to address this issue using an enzyme localized to the tumor to convert a systemically administered nontoxic prodrug into a toxic anticancer agent exclusively in the tumor. The target of the presented enzyme prodrug systems, phosphatidylserine, exists on cancer cells and the cells of the tumor vasculature. Annexin V binds to phosphatidylserine with high affinity and was successfully fused to three enzymes for the targeted delivery of the enzyme prodrug systems to the tumor. Development of the purine nucleoside phosphorylase fusion with annexin V is described, and results showing strong in vitro binding and promising cytotoxicity are presented. This system is compared in vivo with targeted cytosine deaminase and targeted methioninase enzyme prodrug systems. The methioninase system produced the strongest antitumor results showing tumor regression for the duration of treatment. Further engineering of the system resulted in the generation of a mammalian cystathionine-γ-lyase protein with methioninase activity to prevent the immune response anticipated against foreign methioninase. Successful transition to immune competent models without incurring an immune response led to studies with combination therapies to achieve an enhanced therapeutic effect. Antitumor synergism was observed when the enzyme prodrug therapy was combined with rapamycin to address the hypoxic response. Combination with immunostimulatory levels of cyclophosphamide produced an anti-metastatic response and enhanced survival. Combination of the enzyme prodrug therapy with both rapamycin and cyclophosphamide effectively reduced tumor volumes, inhibited metastatic progression, and enhanced survival.