Development of bacterial nitroreductase enzymes for noninvasive imaging in cancer gene therapy

Abstract

There is strong interest in developing novel targeted cancer therapies. It has been known for over a century that certain viruses and bacteria can preferentially infect and lyse cancerous cells. Clinical utility has lagged behind the initial promise of the idea; however three therapeutic agents from the oncolytic virus field are currently in Phase IIB/Phase III clinical trials. The development path of such therapies would be substantially smoothed by an ability to nonin vasively monitor the ir location in the patient’s body post-administration. This would allay fears that viral/bacterial distribution may not be confined to the tumour and provide real time information on vector localisation and replication. This could be achieved by positron emission tomography (PET) scanning if the vector expressed a reporter protein which could activate a PET suitable imaging agent. Furthermore the potency of such therapies could be increased by if this reporter protein could also act therapeutically by converting a systemically delivered benign prodrug into a potent chemotherapeutic – thus targeting the toxicity of the prodrug specifically to cancerous cells. A promising enzyme/prodrug combination is the use of bacterial nitroreductase (NTR) enzymes to activate DNA damaging prodrugs, such as the dinitrobenzamides CB1954 and PR-104A.

This thesis presents work aimed at developing the ability to noninvasively image bacterial NTR expression so that these enzymes can act as both therapeutic and reporter proteins. The primary focus of this study was to achieve this by repurposing pre-existing 2-nitroimidazole (NI) PET imaging agents, originally developed for imaging tumour hypoxia. Microplate based screening strategies were developed to enable detection of 2-NI bioreductive activation by different bacterial NTRs over-expressed heterologously in Escherichia coli, and these technologies were used to screen a 58-membered library of nitroreductase candidates. Although the most widely studied NTR for enzyme/prodrug therapy - NfsB from E. coli - was found to lack activity with 2-NI substrates, numerous NTRs from the NfsA family were able to metabolise these molecules to the cell entrapped form required for PET imaging. Following this discovery, a directed evolution study was conducted to improve the native activity of the enzyme NfsA from E. coli. In this study targeted mutagenesis of active site residues was carried out, resulting in identification of several NfsA multi-site mutants that were substantially improved in their ability to activate a range of 2-NI imaging agents.

In addition to repurposing existing PET probes, this work sought to identify and engineer NTRs for efficient activation of a next - generation PET probe that is designed to be substantially less responsive to hypoxia and hence give a cleaner signal for NTR imaging (i.e. low to no background resulting from tumour hypoxia). SN 33623, a novel 5-NI analogue of the existing 2-NI PET probe EF5, was designed and synthesised by our University of Auckland collaborators. It was found that this novel probe was not only activated by NfsA enzymes, but also by a subset of NfsB enzymes. Although this subset did not include E. coli NfsB, sequence alignment and site-directed mutagenesis were used to identify two key mutations that can be introduced into E. coli NfsB (as well as engineered variants thereof) to confer high levels of SN 33623 activity.

Finally work was carried out, as part of a wider collaborative project, to generate NfsA mutants that retained the ability to metabolise 2-NI imaging agents while also showing increased activation of the nitroaromatic prodrug PR-104A. Ongoing evaluation of these enzymes will include assessment of their therapeutic effect in preclinical models and their ability to be noninvasively imaged (by microPET) when expressed from the tumour targeting bacterial strain Clostridium sporogenes.