Protein production was successful, and the binding stability and cytotoxicity of the PNP-AV enzyme prodrug therapy were characterized for estrogen receptor-positive breast cancer MCF-7 cells, estrogen receptor-negative breast cancer MDA-MB-231 cells (a triple-negative breast cancer), and endothelial HAAE-1 cells grown under conditions to mimic the tumor vasculature. This evidence concerns the gene ESR1 and breast carcinoma.