Dr. Peter Searle
Senior Lecturer, School of Cancer Sciences, University of Birmingham
Telephone +44 (0)121 414 4487
Dr. Searle established the Gene Therapy Research Group in the School of Cancer Sciences, leading the development of prodrug activation gene therapy using the enzyme nitroreductase (E. coli NfsB) in combination with the prodrug CB1954. The group developed both retroviral and adenoviral vectors for delivery of nitroreductase, using cell cultures and mouse models to evaluate the system. Through collaboration with ML Laboratories plc, these studies led to a series of clinical trials of CB1954 alone, the CTL102 adenovirus alone, and finally both in combination. The trials established the safety and tolerability of the treatment, and provided encouraging evidence of anti-tumour activity in some patients.
Stimulation of tumour-specific immune responses could allow a localised gene therapy to have systemic benefit. In one approach, we have investigated the ability of viruses expressing co-stimulatory proteins (including CD80 and 4-1BBL) to enhance immune responses. The cytokine GM-CSF has been widely investigated in immune-stimulatory gene therapies, and we found that combining GM-CSF expression with nitroreductase-mediated CB1954 activation increased the anti-tumour efficacy in mouse models. The combination of Ad-NRGM (a replication-defective adenovirus co-expressing nitroreductase and human GM-CSF) with CB1954 is currently being investigated in a phase I clinical trial in men with locally relapsed prostate cancer.
The maximum achievable concentration of CB1954 in patients is far below the KM of the NfsB nitroreductase, thus limiting the rate of CB1954 activation in vivo. Potentially greater clinical efficacy could be achieved through enzyme engineering for improved catalytic properties. In collaboration with Drs Eva Hyde and Scott White in the School of Biosciences at Birmingham, we have undertaken site-directed mutagenesis of active site residues of nitroreductase, developing a series of single, double, and — with the help of a direct positive selection for enzymes with improved CB1954 activation — triple mutants showing significantly improved efficiency of CB1954 activation (up to ~100-fold greater kcat/KM). These could be candidates for use in future clinical trials.
Some of the most exciting developments in cancer gene therapy involve tumour-selective, conditionally replicating (oncolytic) viruses. The group demonstrated that expressing nitroreductase from an oncolytic adenovirus modelled on the ONXY015 (dl1520) adenovirus showed much increased nitroreductase expression and improved antitumour activity in mice; however there was less apparent benefit from combining prodrug activation with more less attenuated oncolytic viruses. The current focus of research, in collaboration with Dr Simon Afford (School of Immunology & Infection, Birmingham) and Oncos Therapeutics, has turned towards the use of oncolytic adenoviruses expressing immune-stimulatory proteins in the context of liver cancer.
Patel P, Young JG, Mautner V, Ashdown D, Bonney S, Pineda RG, et al. A phase I/II clinical trial in localized prostate cancer of an adenovirus expressing nitroreductase with CB1954. Mol Ther 2009;17(7):1292-9.
Young JG, Green NK, Mautner V, Searle PF, Young LS, James ND. Combining gene and immunotherapy for prostate cancer. Prostate Cancer Prostatic Dis 2008;11(2):187-93.
Chen MJ, Green NK, Reynolds GM, Flavell JR, Mautner V, Kerr DJ, et al. Enhanced efficacy of Escherichia coli nitroreductase/CB1954 prodrug activation gene therapy using an E1B-55K-deleted oncolytic adenovirus vector. Gene Ther 2004;11(14):1126-36.
Guise CP, Grove JI, Hyde EI, Searle PF. Direct positive selection for improved nitroreductase variants using SOS triggering of bacteriophage lambda lytic cycle. Gene therapy 2007;14(8):690-8.
Jarrom D, Jaberipour M, Guise CP, Daff S, White SA, Searle PF, et al. Steady-state and stopped-flow kinetic studies of three Escherichia coli NfsB mutants with enhanced activity for the prodrug CB1954. Biochemistry 2009;48(32):7665-72.
Trials and tribulations: Prodrug activation gene therapy using E. coli nitroreductase and CB1954
Expression of prodrug-activating enzymes in cancer cells enables them to be killed by concentrations of prodrug that are harmless to cells that lack the enzyme. This provides a new, experimental approach to cancer treatment, using viral vectors administered to the patient to express the enzyme in their cancer cells.
The prodrug CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide) is a weak, mono-functional alkylating agent that can be converted to a much more reactive, bi-functional DNA-crosslinking agent which is highly cytotoxic, by the NAD(P)H-dependent activity of bacterial nitroreductase enzymes. We have developed replication-defective adenovirus vectors that express the NfsB nitroreductase of E. coli, and tested these in early phase, prodrug activation gene therapy clinical trials, focusing on men with localised prostate cancer.
In collaboration with ML Laboratories plc, the NfsB-expressing adenovirus CTL102 was injected under ultrasound guidance directly into cancer-containing prostates via the transrectal route. Safety of the virus alone (at doses escalating from 1×1010 to 1×1012 virus particles), and expression of nitroreductase, were first demonstrated in men with operable disease, in whom radical prostatectomy was performed a few days after virus injection. Subsequently, men with inoperable, locally recurrent prostate cancer received similar intraprostatic injections of CTL102, followed after 2 days by intravenous CB1954 (24 mg/m2). Patients had rising blood levels of the tumour marker, prostate specific antigen (PSA) at recruitment. Several patients showed marked reductions in their PSA levels following treatment; by 6 months after treatment, 9/19 patients had progressive disease (>10% increase in PSA), 4/19 showed stable disease (PSA within 10% of baseline value), while 6/19 showed >10% reduction in PSA, including 2/19 whose PSA was still >50% below baseline. These reductions in PSA suggest that the treatment successfully killed a proportion of the cancer burden in these patients. Interestingly, the extended duration of the responses suggests the treatment may induce some immune-mediated tumour control, although attempts to measure T-cell responses to tumour antigens were inconclusive.
Another, ongoing phase I clinical trial incorporates two important changes designed to improve both proposed mechanisms of action. Firstly, the virus is being delivered to many more locations throughout the prostates, via template-guided stereotactic injection, aiming to achieve saturation coverage with delivery of virus to all tumour cells. Secondly, the virus AdNRGM has been engineered to co-express the immune-stimulatory cytokine GM-CSF, in addition to nitroreductase. This is expected to recruit antigen-presenting cells, which would then be able to take up debris from tumour cells killed by the activated prodrug, helping to activate stronger immune responses targeting tumour antigens. This trial is currently recruiting patients.
Additional approaches to improve the efficiency of prodrug activation gene therapy include the use of replicating vectors, the development of improved prodrugs, and engineering of the enzyme for more efficient prodrug activation. I will summarise our engineering of the E. coli NfsB nitroreductase, which has improved its efficiency of CB1954 activation by ~100-fold.
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