Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors in vivo

// John T. Heap 1,5,* , Jan Theys 2,* , Muhammad Ehsaan 1 , Aleksandra M Kubiak 1 , Ludwig Dubois 2 , Kim Paesmans 2 , Lieve Van Mellaert 3 , Richard Knox 4 , Sarah A. Kuehne 1 , Phillipe Lambin 2 and Nigel P. Minton 1 1 Clostridia Research Group, Centre for Biomolecular Sciences, School of Life Sciences, The University of Nottingham, University Park, Nottingham, UK. 2 Maastro Lab, Research Institute GROW, University of Maastricht, MD Maastricht, The Netherlands. 3 Molecular Bacteriology, Rega Institute for Medical Research, University Leuven, Minderbroedersstraat,Belgium. 4 Morvus Technology Limited, Ty Myddfai, Llanarthne, Carmarthen, UK. 5 Present address: Centre for Synthetic Biology and Innovation, Department of Life Sciences, Imperial College London, London, UK. * These authors contributed equally Correspondence: Nigel P Minton, email: // Keywords : Clostridia; spores; hypoxia; pro-drug converting enzyme; nitroreductase; CB1954; solid tumor Received : December 17, 2013 Accepted : January 12, 2014 Published : January 12, 2014 Abstract Spores of some species of the strictly anaerobic bacteria Clostridium naturally target and partially lyse the hypoxic cores of tumors, which tend to be refractory to conventional therapies. The anti-tumor effect can be augmented by engineering strains to convert a non-toxic prodrug into a cytotoxic drug specifically at the tumor site by expressing a prodrug-converting enzyme (PCE). Safe doses of the favored prodrug CB1954 lead to peak concentrations of 6.3 µM in patient sera, but at these concentration(s) known nitroreductase (NTR) PCEs for this prodrug show low activity. Furthermore, efficacious and safe Clostridium strains that stably express a PCE have not been reported. Here we identify a novel nitroreductase from Neisseria meningitidis , NmeNTR, which is able to activate CB1954 at clinically-achievable serum concentrations. An NmeNTR expression cassette, which does not contain an antibiotic resistance marker, was stably localized to the chromosome of Clostridium sporogenes using a new integration method, and the strain was disabled for safety and containment by making it a uracil auxotroph. The efficacy of Clostridium -Directed Enzyme Prodrug Therapy (CDEPT) using this system was demonstrated in a mouse xenograft model of human colon carcinoma. Substantial tumor suppression was achieved, and several animals were cured. These encouraging data suggest that the novel enzyme and strain engineering approach represent a promising platform for the clinical development of CDEPT.