Detection of Cytosine Deaminase in Genetically Modified Tumor Cells by Specific Antibodies

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Abstract
Bacterial cytosine deaminase (CD) converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), which is toxic for mammalian cells. Therefore, the CD gene is used in cancer gene therapy to achieve high local concentration of a toxic metabolite without significant systemic toxicity. To allow the detection of CD expression at the protein level, we raised both polyclonal rabbit antisera and a monoclonal antibody (mAb) against a histidine-tagged CD fusion protein. The specificity of the polyclonal antisera and the mAb was confirmed by immunohistochemistry, immunoblot analysis, and immunoprecipitation using CD-expressing tumor cell lines. Furthermore, the antibodies can be used for ELISA assays and flow cytometry. Finally, the CD protein could be demonstrated in frozen tissue sections of CD-modified tumors in a rat tumor model using the anti-CD serum. With these antibodies, CD expression can now be monitored throughout in vitro and in vivo gene transfer studies, including clinical protocols relying on the CD suicide gene strategy. The cytosine deaminase/5-fluorocytosine (CD/5-FC) enzyme prodrug system mediates efficient cell killing when applied to mammalian cells. Killing efficiency relies on cell types and CD expression levels. This report describes the development and characterization of rabbit antisera as well as a mouse mAb raised against the Escherichia coli CD protein. The data show specific detection of the CD protein in genetically modified tumor cells by a variety of immunological techniques. These antibodies represent a useful tool for gene therapy approaches using the CD metabolic suicide system.

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