(47f) Enzyme Prodrug Therapy Targeted to Breast Tumor Vasculature Using a Recombinant Fusion Protein
Invasive breast cancer will appear in almost a quarter of a million people in the United States this year, with a current mortality rate of almost 25%. Limited efficacy and severe side effects of current breast cancer treatments have created a demand for improved and innovative therapeutic strategies. Targeted therapeutics is a promising approach for the development of new cancer treatments that seek to address these challenges. A targeted enzyme prodrug therapy has been developed to localize cytotoxicity to the site of the tumor using a protein fusion of annexin V (AV) and bacterial purine nucleoside phosphorylase (PNP). The high affinity, calcium-dependent binding of annexin V acts as the tumor targeting component of the fusion, as it has been shown to bind to phosphatidylserine (PS) expressed externally almost exclusively on cancer cells and the endothelial cells of the tumor vasculature. The enzymatic component of the fusion, PNP, converts the prodrug fludarabine into a more cytotoxic form, 2-fluoroadenine. This conversion allows for increased impact at the site of the tumor, while reducing side effects on normal tissue.
Recombinant production and purification of the fusion protein has yielded an active enzyme and promising in vitro results. Binding strength and stability were determined for breast cancer cell lines MDA-MB-231 and MCF-7, as well as non-confluent endothelial cells HAAE-1 that mimic tumor vasculature with externalized phosphatidylserine. These studies reveal that the dissociation constant for each cell line is less than 100 pM and the fusion protein remains bound for a minimum of three days. Cytotoxicity tests indicated susceptibility of both cancer cell lines to prodrug and prodrug with fusion protein at concentrations as low as 1.0 µM fludarabine. Non-confluent endothelial cells showed a dose dependent cytotoxic effect of fludarabine with PNP-AV with a reduced impact of fludarabine alone.
This novel enzyme-prodrug therapy warrants in vivo studies, which will be performed with SCID mice and MDA-MB-231/GFP breast cancer xenografts. PEGylation of the fusion protein will allow for future studies in immunocompetent mice. Combinatory treatments with immunotherapeutics are also exciting possible future directions for vasculature targeted strategies such as this enzyme prodrug therapy.
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