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Adenovirus vector–mediated delivery of the prodrug-converting enzyme carboxypeptidase G2 in a secreted or GPI-anchored form: High-level expression of this active conditional cytotoxic enzyme at the plasma membrane

Cancer Gene Therapy volume 9, pages 897–907 (2002)Cite this article

Abstract

Carboxypeptidase G2 (CPG2) is a powerful prodrug-converting enzyme. Without a requirement for endogenous enzymes or cofactors, it can directly activate mustard alkylating prodrugs to cytotoxic species, killing both quiescent and dividing cells. This paper provides the first report of its use in the context of a clinically relevant delivery vehicle using adenovirus vectors. To strengthen the efficacy of the prodrug-activating system, the enzyme has been engineered to be secreted or glycosylphosphatidylinositol (GPI) anchored to the extracellular membrane of tumor cells, resulting in an enhanced bystander effect by facilitating diffusion of the active drug through extracellular, rather than intracellular, activation. Using the vectors, we have achieved expression of functional secreted or GPI-anchored CPG2 in a panel of tumor cell lines demonstrating no loss in efficacy as a result of GPI anchor retention. Despite variable transduction efficiencies inherent to these vectors, greater than 50% cell kill was achievable in all of the cell lines tested following only a single exposure to the prodrug ZD2767P. Even in cell lines refractive to infection with the vectors, substantial cell death was recorded, indicative of the enhanced bystander effect generated following extracellular prodrug activation. A direct evaluation of the efficacy of our system has been made against adenoviral delivery of herpes simples virus thymidine kinase plus ganciclovir (GCV), a suicide gene therapy approach already in the clinic. In a short-term human glioma culture (IN1760) resistant to the clinical chemotherapeutic drug CCNU (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea), thymidine kinase/GCV effected no cell killing compared to 70% cell killing with our system.

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Acknowledgements

We acknowledge funding from the Molecular Medicine and Gene Therapy Unit received from The Wellcome Trust, BBSRC, REMEDI, Parkinson's Disease Society, UK, MRC/UK, EU-Biomed programs (contract nos. B104-CT98-0297, BMH4-CT98-3277, QLK3-CT-1999-00364). RLC was funded by a joint BBSRC-AstraZeneca CASE studentship. PRL was a Research Fellow of The Lister Institute of Preventive Medicine. We also acknowledge the expert technical and secretarial assistance of Tricia Maleniak and Ros Poulton, respectively. Pedro R Lowenstein and Maria G Castro acknowledge the support for the GTRI from the Board of Governors at Cedars-Sinai Medical Center, Shlomo Melmed for his support and encouragement, and Juanita Gutierrez for excellent editorial assistance.

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Authors and Affiliations

  1. Molecular Medicine and Gene Therapy Unit, School of Medicine, University of Manchester, Manchester, UK

    Rachel L Cowen, Judith C Williams, Pedro R Lowenstein & Maria G Castro

  2. AstraZeneca, Alderley Park, Macclesfield, Cheshire, UK

    Steve Emery & David Blakey

  3. Department of Neurological Surgery, Institute of Neurology, University College-London, London, UK

    John L Darling

  4. Cedars-Sinai Medical Center and Department of Medicine, Gene Therapeutics Research Institute, University of California-Los Angeles, Los Angeles, California, USA

    Pedro R Lowenstein & Maria G Castro

Authors
  1. Rachel L Cowen
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  2. Judith C Williams
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  3. Steve Emery
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  4. David Blakey
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  5. John L Darling
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  6. Pedro R Lowenstein
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  7. Maria G Castro
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Correspondence to Pedro R Lowenstein or Maria G Castro.

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Cowen, R., Williams, J., Emery, S. et al. Adenovirus vector–mediated delivery of the prodrug-converting enzyme carboxypeptidase G2 in a secreted or GPI-anchored form: High-level expression of this active conditional cytotoxic enzyme at the plasma membrane. Cancer Gene Ther 9, 897–907 (2002). https://doi.org/10.1038/sj.cgt.7700514

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