The use of Trichomonas vaginalis purine nucleoside phosphorylase to activate fludarabine in the treatment of solid tumors

Treatment with fludarabine phosphate (9-β- D -arabinofuranosyl-2-F-adenine 5′-phosphate, F-araAMP) leads to regressions and cures of human tumor xenografts that express Escherichia coli purine nucleoside phosphorylase ( Ec PNP). This occurs despite the fact that fludarabine (F-araA) is a relatively poor substrate for Ec PNP, and is cleaved to liberate 2-fluoroadenine at a rate only 0.3% that of the natural E. coli PNP substrate, adenosine. In this study, we investigated a panel of naturally occurring PNPs to identify more efficient enzymes that may be suitable for metabolizing F-araA as part of experimental cancer therapy. We show that Trichomonas vaginalis PNP ( Tv PNP) cleaves F-araA with a catalytic efficiency 25-fold greater than the prototypic E. coli enzyme. Cellular extracts from human glioma cells (D54) transduced with lentivirus stably expressing Tv PNP (D54/ Tv PNP) were found to cleave F-araA at a rate similar to extracts from D54 cells expressing Ec PNP, although much less enzyme was expressed per cell in the Tv PNP transduced condition. As a test of safety and efficacy using Tv PNP, human head and neck squamous cell carcinoma (FaDu) xenografts expressing Tv PNP were studied in nude mice and shown to exhibit robust tumor regressions, albeit with partial weight loss that resolved post-therapy. F-araAMP was also a very effective treatment for mice bearing D54/ Tv PNP xenografts in which approximately 10% of tumor cells expressed the enzyme, indicating pronounced ability to kill non-transduced tumor cells (high bystander activity). Moreover, F-araAMP demonstrated activity against D54 tumors injected with an E1, E3 deleted adenoviral vector encoding Tv PNP. In that setting, despite higher F-araA cleavage activity using Tv PNP, tumor responses were similar to those obtained with Ec PNP, indicating factors other than F-Ade production may limit regressions of the D54 murine xenograft model. Our results establish that Tv PNP is a favorable enzyme for activating F-araA, and support further studies in combination with F-araAMP for difficult-to-treat human cancers.