Paula W. Allan

Antitumor activity of 2-fluoro-2′-deoxyadenosine against tumors that express Escherichia coli purine nucleoside phosphorylase

The selective expression of Escherichia coli purine nucleoside phosphorylase (PNP) in solid tumors has been successfully used to activate two purine nucleoside analogs [9-(2-deoxy-β-D-ribofuranosyl)-6-methylpurine (MeP-dR) and 9-β-D-arabinofuranosyl-2-fluoroadenine (F-araA)] resulting in lasting tumor regressions and cures. E. coli PNP also cleaves 2-fluoro-2′-deoxyadenosine (F-dAdo) to 2-F-adenine, which is the toxic purine analog liberated from F-araA that has high bystander activity and is active against nonproliferating tumor cells. As F-dAdo is 3000 times better than F-araA as a substrate for E. coli PNP, we have evaluated its antitumor activity against D54 gliomas that express E. coli PNP and have characterized its in vivo metabolism in order to better understand its mechanism of action with respect to the other two agents. Like MeP-dR and F-araA-5′-monophosphate (F-araAMP, a prodrug of F-araA), treatment of mice bearing D54 tumors that express E. coli PNP with F-dAdo resulted in excellent antitumor activity. Although F-dAdo was as active as MeP-dR and better than F-araAMP, it was not dramatically better than either compound because of its short plasma half-life and the limited activation of F-adenine to toxic metabolites. Regardless, these results indicated that F-dAdo was also an excellent prodrug for use with gene vectors that deliver E. coli PNP to tumor cells.

CELL TO CELL CONTACT IS NOT REQUIRED FOR BYSTANDER CELL KILLING BY ESCHERICHIA COLI PURINE NUCLEOSIDE PHOSPHORYLASE

Expression of Escherichia coli purine nucleoside phosphorylase (PNP) activates prodrugs and kills entire populations of mammalian cells, even when as few as 1% of the cells express this gene. This phenomenon of bystander killing has been previously investigated for herpes simplex virus-thymidine kinase (HSV-TK) and has been shown to require cell to cell contact. Using silicon rings to separate E. coli PNP expressing cells from non-expressing cells sharing the same medium, we demonstrate that bystander cell killing by E. coli PNP does not require cell-cell contact. Initially, cells expressing E. coli PNP convert the non-toxic prodrug, 6-methylpurine-2′-deoxyriboside (MeP-dR) to the highly toxic membrane permeable toxin, 6-methylpurine (MeP). As the expressing cells die, E. coli PNP is released into the culture medium, retains activity, and continues precursor conversion extracellularly (as determined by reverse phase high performance liquid chromatography of both prodrug and toxin). Bystander killing can also be observed in the absence of extracellular E. coli PNP by removing the MeP-dR prior to death of the expressing cells. In this case, 100% of cultured cells die when as few as 3% of the cells of a population express E. coli PNP. Blocking nucleoside transport with nitrobenzylthioinosine reduces MeP-dR mediated cell killing but not MeP cell killing. These mechanisms differ fundamentally from those previously reported for the HSV-TK gene.

Excellent In vivo Bystander Activity of Fludarabine Phosphate against Human Glioma Xenografts that Express the Escherichia coli Purine Nucleoside Phosphorylase Gene

Escherichia coli purine nucleoside phosphorylase (PNP) expressed in tumors converts relatively nontoxic prodrugs into membrane-permeant cytotoxic compounds with high bystander activity. In the present study, we examined tumor regressions resulting from treatment with E. coli PNP and fludarabine phosphate (F-araAMP), a clinically approved compound used in the treatment of hematologic malignancies. We tested bystander killing with an adenoviral construct expressing E. coli PNP and then more formally examined thresholds for the bystander effect, using both MuLv and lentiviral vectoring. Because of the importance of understanding the mechanism of bystander action and the limits to this anticancer strategy, we also evaluated in vivo variables related to the expression of E. coli PNP (level of E. coli PNP activity in tumors, ectopic expression in liver, percentage of tumor cells transduced in situ, and accumulation of active metabolites in tumors). Our results indicate that F-araAMP confers excellent in vivo dose-dependent inhibition of bystander tumor cells, including strong responses in subcutaneous human glioma xenografts when 95 to 97.5% of the tumor mass is composed of bystander cells. These findings define levels of E. coli PNP expression necessary for antitumor activity with F-araAMP and demonstrate new potential for a clinically approved compound in solid tumor therapy.

Effect of expression of adenine phosphoribosyltransferase on the in vivo anti-tumor activity of prodrugs activated by E. coli purine nucleoside phosphorylase

The use of E. coli purine nucleoside phosphorylase (PNP) to activate prodrugs has demonstrated excellent activity in the treatment of various human tumor xenografts in mice. E. coli PNP cleaves purine nucleoside analogs to generate toxic adenine analogs, which are activated by adenine phosphoribosyl transferase (APRT) to metabolites that inhibit RNA and protein synthesis. We created tumor cell lines that encode both E. coli PNP and excess levels of human APRT, and have used these new cell models to test the hypothesis that treatment of otherwise refractory human tumors could be enhanced by overexpression of APRT. In vivo studies with 6-methylpurine-2′-deoxyriboside (MeP-dR), 2-F-2′-deoxyadenosine (F-dAdo) or 9-β-D-arabinofuranosyl-2-fluoroadenine 5′-monophosphate (F-araAMP) indicated that increased APRT in human tumor cells coexpressing E. coli PNP did not enhance either the activation or the anti-tumor activity of any of the three prodrugs. Interestingly, expression of excess APRT in bystander cells improved the activity of MeP-dR, but diminished the activity of F-araAMP. In vitro studies indicated that increasing the expression of APRT in the cells did not significantly increase the activation of MeP. These results provide insight into the mechanism of bystander killing of the E. coli PNP strategy, and suggest ways to enhance the approach that are independent of APRT.

Development of drug targeting based on recombinant expression of the chicken avidin gene

The chemistry required for covalent biotinylation of drugs, radiopharmaceuticals and other ligands is highly developed, and a large number of biotinylated reagents can be readily synthesized. In order to investigate whether expression of avidin cDNA in mammalian cells might be useful as part of a drug targeting strategy, we transiently expressed the avidin gene in two human tumor cell lines (the cervical carcinoma cell line, HeLa, and the liver derived line, Hep G2). Avidin protein as detected by either immunohistochemistry or binding of streptavidin-biotin complexes was present and functional following transient expression. This result indicated that the mechanisms underlying avidin oligomerization which are necessary for proper protein folding are present within mammalian carcinoma cell lines. Next, we generated a producer cell line (derived from psi2) capable of releasing a recombinant retrovirus encoding chicken avidin, and a tumorigenic murine breast cancer cell line (16/C) with stable avidin expression. We show that these cell lines are suitable for conferring functional expression of avidin in vitro. These experiments establish a means by which avidin gene expression can be explored as a mechanism for targeted gene delivery of biotin-derivitized drugs in vitro, and have important implications for utilization of this strategy in vivo.