Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo.

The unique properties of the tumour microenvironment can be exploited by using recombinant anaerobic clostridial spores as highly selective gene delivery vectors. Although several recombinant Clostridium species have been generated during the past decade, their efficacy has been limited. Our goal was to substantially improve the prospects of clostridia as a gene delivery vector.

Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954.

A nitroreductase with distinct properties that can activate the prodrug 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) was isolated from Bacillus amyloliquefaciens. The encoding gene was identified as a homologue of the ywrO of Bacillus subtilis, and was obtained as a PCR product by reverse genetics, cloned and the entire nucleotide sequence determined. The gene was found to reside between homologues of the B.

Sensitization of colorectal and pancreatic cancer cell lines to the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) by retroviral transduction and expression of the E. coli nitroreductase gene.

Expression of genes encoding prodrug-activating enzymes can increase the susceptibility of tumor cells to prodrugs, and may ultimately achieve a better therapeutic index than conventional chemotherapy. CB1954 is a weak, monofunctional alkylating agent which can be activated by Escherichia coli nitroreductase to a potent dysfunctional alkylating agent which crosslinks DNA. We have inserted the nitroreductase gene into an LNCX-based retroviral vector, to allow efficient gene transfer and expression in colorectal (LS174T) and pancreatic (SUIT2, BxPC3, and AsPC1) cancer cell lines.

Bioactivation of dinitrobenzamide mustards by an E. coli B nitroreductase.

A nitroreductase isolated and purified from Escherichia coli B has been demonstrated to have potential applications in ADEPT (antibody-directed enzyme prodrug therapy) by its ability in vitro to reduce dinitrobenzamides (e.g. 5-aziridinyl 2,4-dinitrobenzamide, CB 1954 and its bischloroethylamino analogue, SN 23862) to form cytotoxic derivatives.

Virtual cofactors for an Escherichia coli nitroreductase enzyme: relevance to reductively activated prodrugs in antibody directed enzyme prodrug therapy (ADEPT).

A nitroreductase enzyme has been isolated from Escherichia coli that has the unusual property of being equally capable of using either NADH or NADPH as a cofactor for the reduction of its substrates which include menadione as well as 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954). This property is shared with the mammalian enzyme, DT diaphorase. The nitroreductase can, like DT diaphorase, also use simple reduced pyridinium compounds as virtual cofactors.