1. ¹Department of Radiation Oncology (Maastro Lab), GROW Research Institute, University of Maastricht, UNS 50/23, PO Box 616, Maastricht 6200 MD, The Netherlands
2. ²Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK
3. ³Experimental Radiobiology/LEO, Gasthuisberg-CDG, Herestraat 49, Leuven 3000, Belgium
4. ⁴Laboratory of Bacteriology, Rega Institute, KU Leuven, Minderbroedersstraat 10, Leuven 3000, Belgium
5. ⁵Enact Pharma, Porton Down Science Park, Salisbury SP4 0JG, UK
6. ⁶Centre for Applied Microbiology and Research, Porton Down, Salisbury SP4 0JG, UK
7. ⁷Mikrobiologie und Biotechnologie, University Ulm, Ulm 89069, Germany

Correspondence: Dr P Lambin, E-mail: philippe.lambin@maastro.nl

⁸These authors contributed equally to this work.

Revised 10 August 2006; Accepted 10 August 2006; Published online 3 October 2006.

Abstract

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. Therefore, we have assessed a series of nitroreductase (NTR) enzymes for their capacity to convert the innocuous CB1954 prodrug to its toxic derivative. Among the enzymes tested, one showed superior prodrug turnover characteristics. In addition, we established an efficient gene transfer procedure, based on conjugation, which allows for the first time genetic engineering of Clostridium strains with superior tumour colonisation properties with high success rates. This conjugation procedure was subsequently used to create a recombinant C. sporogenes overexpressing the isolated NTR enzyme. Finally, analogous to a clinical setting situation, we have tested the effect of multiple consecutive treatment cycles, with antibiotic bacterial clearance between cycles. Importantly, this regimen demonstrated that intravenously administered spores of NTR-recombinant C. sporogenes produced significant antitumour efficacy when combined with prodrug administration.