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Use of a direct, positive selection strategy to generate improved prodrug-activating enzymes for cancer gene therapy

Baker, Shelley Louise (2011)
Ph.D. thesis, University of Birmingham.

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Abstract

E. coli NfsB nitroreductase (NTR) is currently being studied in combination with the prodrug CB1954, as a gene directed enzyme prodrug therapy. NTR reduces CB1954 at either the 2- or 4-nitro groups to produce highly cytotoxic hydroxylamine derivatives, using either NADH or NADPH as cofactor. Initial clinical trials suggest activity, with reduction in PSA and apparent delayed progression in some patients. This thesis is concerned with engineering the NTR enzyme to improve the efficiency of CB1954 activation, since this would be expected to improve the potential clinical efficacy. Site directed mutagenesis has been used to generate four libraries of NTR mutants with three small libraries having a maximum diversity of 6.4 x101 – 8.2 x 103 and a large library of 6.3 x 106 possible nucleotide combinations. A direct positive selection strategy, which utilises bacteriophage lambda and the SOS response, was used to select NTR variants with increase sensitivity for CB1954 from each library. NTR mutants with greatest sensitivity were analysed by bacterial IC50 assays. Despite considerable work to generate and optimise the large library, only one NTR enzyme was improved over WT. However the best mutant (T41G N71S) came from a small library, with an IC50 of 8 µM. The most promising NTR enzyme variants were column purified and their kinetic parameters and activity determined. T41G N71S showed good activity for CB1954 (0.52 µM s-1), being the second best mutant to date behind T41L N71S (0.90 µM s-1). Adenoviral vectors were generated and their ability to sensitise SKOV3 cancer cells tested. T41G N71S was ~2-fold improved relative to WT NTR. Purified enzymes were also tested with SKOV3 cells, with T41G N71S being ~2.6-fold improved relative to WT NTR. My best enzyme should be considered when planning future clinical trials using prodrug activation gene therapy with NTR.

Type of Work:Ph.D. thesis.
Supervisor(s):Searle, Peter and Hyde, Eva
School/Faculty:Colleges (2008 onwards) > College of Medical & Dental Sciences
Department:School of Cancer Studies
Subjects:Q Science (General)
Institution:University of Birmingham
ID Code:1443
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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