Abstract B88: Discovery, characterization, and engineering of bacterial nitroreductases for gene-directed enzyme prodrug therapy.

Abstract

Abstract Tumor-targeting viruses and bacteria hold great promise as anti-cancer agents. They kill cells by entirely different mechanisms to radio- and chemotherapies, and have potential to synergize with these treatments without overlapping toxicities. Furthermore, these agents can be ‘armed’ with genes that encode enzymes that activate prodrugs - compounds that are deactivated in their administered form, but become highly toxic upon metabolic activation. This not only improves killing of infected cells, but also neighboring non-infected cells, as the prodrug metabolites can diffuse locally and exert a bystander effect. A highly efficient activating enzyme in partnership with a prodrug that has a strong bystander effect can address some of the historical limitations of cancer gene therapy including the inability of biological vectors to reach every target cell. Phase I/II trials of the first-generation nitroaromatic prodrug CB1954 in conjunction with the prototype gene therapy nitroreductase, Escherichia coli NfsB, have been conducted in prostate and liver cancer. These trials provided some evidence of anti-tumor activity but, due to dose-limiting hepatotoxicity, the highest achievable plasma concentration of CB1954 was less than 1% of NfsB's Km. As well as highlighting a need for more efficient nitroreductase enzymes, this has fuelled a search for superior nitroaromatic prodrugs. The next-generation dinitrobenzamide mustard prodrug PR-104A is not only 5–50 fold more dose-potent upon activation, but also better tolerated in humans (MTD 1100 mg/m2 vs 24 mg/m2; q3w, iv). However, E. coli NfsB also has relatively poor (millimolar) affinity for this substrate. To discover more efficient nitroreductases we developed screens for genotoxic prodrug activation, based on their ability to induce reporter genes linked to the E. coli SOS (DNA damage repair) response. We used these to screen a large library of candidate enzymes for DNBM activity, and selected E. coli NfsA as a top candidate for further improvement by random and targeted mutagenesis. High throughput screening of large error prone PCR libraries coupled with medium throughput screening of targeted mutagenesis libraries revealed 10 individual mutations that significantly increased NfsA activity. These mutations were then combined in a synthetic “smart” library, from which eight poly-mutant enzymes were selected for kinetic analysis. Relative to wild type the engineered variants display an 18–40 fold improvement in PR-104A Km with respect to E. coli NfsA, and are 860–1700 fold better than NfsB. Importantly they also retain, or are improved in, their ability to co-metabolize preferred 2-nitroimidazole probes with PET-imaging capabilities (see abstract: Patterson et al, “Molecular imaging using bacterial nitroreductase reporter genes by repurposing the clinical stage hypoxia PET probe EF5”). The enhanced prodrug activation and in vivo imaging potential of these enzymes is now being evaluated in human gene therapy models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B88.