376126 Targeting Cystathionine-Gamma-Lyase to the Tumor Vasculature in Enzyme Prodrug Therapy of Breast Cancer in an Immune-Competent Mouse Model
Enzyme prodrug systems attempt to circumvent the issues created by the systemic administration of current cancer drugs by localizing cytotoxic compounds to the tumor. An enzyme converts a nontoxic prodrug to a cytotoxic drug at the tumor. This study utilizes a targeted approach to enzyme delivery by capitalizing on the externalization of phosphatidylserine on cancer cells and tumor vasculature. The enzymes are fused to annexin V (AV), a protein with strong binding affinity for phosphatidylserine. Fusions with annexin I (AI) are also under development to examine the effect of different internalization properties. Three fusions have been evaluated using bacterial purine nucleoside phosphorylase (PNP), yeast cytosine deaminase (CD), or bacterial methionine-γ-lyase (MET). The most efficient system, MET-AV, has been selected for transition to immune-competent models using mammalian cystathionine-γ-lyase (CGL) engineered to have MET activity.
Active fusion proteins were recombinantly 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 confocal 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 or BALB/c mice with 4T1 mammary tumors.
In vitro results show successful binding and killing of breast cancer and endothelial cells representative of tumor vasculature. All three fusion proteins were cleared from circulation in SCID mice within 8 hours. Binding to tumor vasculature was confirmed with immunohistochemistry. The CD system yielded unsatisfactory in vivo results; however both the PNP and MET systems achieved tumor growth suppression for the duration of the treatment period with the strongest effect shown with MET. Preliminary studies combining rapamycin (Rap) with MET-AV yielded a >80% reduction in tumor volume. Mouse CGL (mCGL) was mutated in three positions to impart MET activity and fused to mouse AV and AI, resulting in mCGL-AV and mCGL-AI fusion proteins with activity equivalent to MET-AV. Neither fusion triggered adverse immunological effects in BALB/c mice. mCGL-AV effectively inhibited tumor growth during treatment and extended the median survival compared to untreated mice.