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.
Highlights
Biotechnology potentially offers unconventional routes to new cancer therapies, and research in this area has focused on using viruses as vectors to deliver therapeutic genes to tumors
Each gene encoding a candidate CB1954-activating enzyme was cloned in an E. coli expression vector
Crude cell lysates were used in initial screens for functional expression, and to test for CB1954 nitroreductase activity using an HPLC assay
Summary
Biotechnology potentially offers unconventional routes to new cancer therapies, and research in this area has focused on using viruses as vectors to deliver therapeutic genes to tumors. Issues with the safety of viral vectors have been encountered [1, 2] and only recently has a virus system showing good specificity and tumor infiltration been described [3]. Spores of some species of Clostridium, which are strictly anaerobic bacteria, naturally target tumors with high specificity following intravenous administration, because the dormant spores can germinate and grow only in the hypoxic/ necrotic cores of solid tumors [6,7,8] which are difficult www.impactjournals.com/oncotarget to target using viral vectors [9, 10]. The growing bacteria secrete proteases inside the tumor, rapidly digesting the tumor mass. This approach is especially interesting because it directly targets the hypoxic cells in poorly vascular regions which are refractory to conventional treatments. Any Clostridium cells entering normal tissue from a colonized tumor would be poisoned by oxygen and die
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