Expression of
prodrug-activating
enzymes that convert non-toxic substrates to cytotoxic derivatives is a promising strategy for cancer gene
therapy. However, their catalytic activity with unnatural,
prodrug substrates is often suboptimal. Efforts to improve these
enzymes have been limited by the inability to select directly for increased
prodrug activation. We have focussed on developing variants of Escherichia coli (E. coli)
nitroreductase (NTR) with improved ability to activate the
prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (
CB1954), and describe here a novel, direct, positive selection for improved
enzymes that exploits the alternative life cycles of bacteriophage lambda. In lambda lysogens of E. coli, the activation of the
prodrug CB1954 by NTR triggers the SOS response to DNA damage, switching integrated lambda prophages into lytic cycle. This provides a direct, positive selection for phages encoding improved NTR variants, as, upon limiting exposure of lysogenized E. coli to
CB1954, only those encoding the most active
enzyme variants are triggered into lytic cycle, allowing their selective recovery. We exemplify the selection by isolating highly improved 'turbo-NTR' variants from a library of 6.8 x 10(5) clones, conferring up to 50-fold greater sensitivity to
CB1954 than the wild type.
Carcinoma cells infected with adenovirus expressing T41Q/N71S/F124T-NTR were sensitized to
CB1954 concentrations 40- to 80-fold lower than required with WT-NTR.