(270f) Evaluation of Three Vascular-Targeted Enzyme Prodrug Systems for Breast Cancer Therapy

The use of targeted therapeutics is a promising approach for the development of new cancer treatments that seek to reduce the devastating side effects caused by the systemic administration of current drugs.  This study evaluates three fusion proteins developed as enzyme prodrug therapies targeted to tumor vasculature.  Cytotoxicity is localized to the site of the tumor using a protein fusion of annexin V and one of three enzymes: bacterial purine nucleoside phosphorylase (PNP), yeast cytosine deaminase (CD), or bacterial methionine-γ-lyase (MT).  Annexin V acts as the tumor targeting component of the fusion protein as it has been shown to bind to phosphatidylserine expressed externally only on cancer cells and the endothelial cells of tumor vasculature.  The enzymatic component of the fusion converts a non-toxic prodrug (fludarabine, 5-fluorocytosine, methylselenol) into a cytotoxic drug (2-fluoroadenine, 5-fluorouracil, selenomethionine).  This conversion allows for increased impact at the site of the tumor, while decreasing side effects that would otherwise result from the high systemic concentrations necessary for non-targeted treatments.

Fusions of the three enzymes and human annexin V were produced and purified in Escherichia coli.  Binding strength and stability studies were performed with human endothelial cells HAAE-1 and breast cancer cell lines, MCF-7 and MDA-MB-231.  Results were qualitatively confirmed with fluorescence microscopy.  The same cell lines were used for a cytotoxicity analysis of the enzyme prodrug treatment.  In vivo studies were conducted using SCID mice and MDA-MB-231/GFP xenografts.  A pharmacokinetic profile was obtained for each fusion protein.   The PNP and MT studies examined possible synergism with a non-toxic level of docetaxel, a chemotherapeutic shown to increase externalization of phosphatidylserine in vivo for endothelial cells in the tumor vasculature.

Recombinant production and purification of the fusion proteins yield active proteins and promising in vitro results for all three enzyme prodrug systems.  Our in vitro results show successful binding and killing of breast cancer and endothelial cells representative of tumor vasculature.   The CD system yielded unsatisfactory in vivo results; however both the PNP and MT systems achieved tumor growth suppression for the duration of the treatment period with the strongest effect shown with MT.  The results suggest that this treatment has a positive clinical therapeutic potential with minimal side effects.