Enzyme/prodrug therapy for head and neck cancer using a catalytically superior cytosine deaminase

The use of cytosine deaminase (CD) in conjunction with 5-fluorocytosine (5- FC) has been studied for cancer gene therapy as a means of achieving tumor- specific generation of the toxic metabolite 5-fluorouracil (5-FU), Since 5- FC is frequently used as an antifungal agent, and because it has little or no efficacy as an antibacterial agent, we hypothesized that yeast CD (YCD) might be more efficient at utilizing 5-FC as a substrate and hence be a bet ter choice for a CD/5-FC gene therapy strategy than the typically utilized bacterial CD (BCD), To that end Saccharomyces cerevisiae CD was cloned from yeast genomic DNA and expressed in vitro. Functional analysis of BCD and Y CD expressed in COS-1 cells indicated that BCD and YCD both utilized cytosi ne with equal efficacy; however, 5-FC was an extremely poor substrate for B CD, with an apparent catalytic efficiency 280-fold lower than that observed for YCD, Retroviral infection of tumor cell lines in vitro indicated that the IC50 of 5-FC was 30-fold lower in PCD-infected cultures as compared wit h cultures infected with BCD retrovirus, In addition, when SCCVII murine sq uamous cell carcinoma cells were infected in vitro at low rates of infectio n (less than or equal to 10%) there was no significant cytotoxicity toward BCD-expressing cells while there was potent cytotoxicity to both YCD-expres sing cells and "bystander cells" even at this low level of expression. Fina lly, stable BCD- or YCD-expressing SCCVII clones were developed and used in an orthotopic immune-competent model of head and neck cancer. Subsequent t reatment with 5-FC followed by monitoring of tumor growth by noninvasive ma gnetic resonance imaging (MRI) and survival of animals indicated a growth d elay during the course of 5-FC treatment for BCD-expressing tumors, which q uickly regrew at the end of treatment. In contrast, YCD-expressing tumors e xhibited not only a growth delay, which was of longer duration, but also in some cases frank tumor regression and complete cures occurred.