The functionalized human serine protease granzyme B/VEGF₁₂₁ targets tumor vasculature and ablates tumor growth.

The serine protease granzyme B (GrB) induces apoptosis through both caspase-dependent and -independent multiple-cascade mechanisms. VEGF₁₂₁ binds to both VEGF receptor (VEGFR)-1 and VEGFR-2 receptors. We engineered a unique GrB/VEGF₁₂₁ fusion protein and characterized its properties in vitro and in vivo. Endothelial and tumor cell lines showed varying levels of sensitivity to GrB/VEGF₁₂₁ that correlated closely to total VEGFR-2 expression. GrB/VEGF₁₂₁ localized efficiently into VEGFR-2-expressing cells, whereas the internalization into VEGFR-1-expressing cells was significantly reduced. Treatment of VEGFR-2(+) cells caused mitochondrial depolarization in 48% of cells by 48 hours. Exposure to GrB/VEGF₁₂₁ induced apoptosis in VEGFR-2(+), but not in VEGFR-1(+), cells and rapid caspase activation was observed that could not be inhibited by treatment with a pan-caspase inhibitor. In vivo, GrB/VEGF₁₂₁ localized in perivascular tumor areas adjacent to microvessels and in other areas in the tumor less well vascularized, whereas free GrB did not specifically localize to tumor tissue. Administration (intravenous) of GrB/VEGF₁₂₁ to mice at doses up to 40 mg/kg showed no toxicity. Treatment of mice bearing established PC-3 tumor xenografts with GrB/VEGF₁₂₁ showed significant antitumor effect versus treatment with GrB or saline. Treatment with GrB/VEGF₁₂₁ at 27 mg/kg resulted in the regression of four of five tumors in this group. Tumors showed a two-fold lower Ki-67-labeling index compared with controls. Our results show that targeted delivery of GrB to tumor vascular endothelial cells or to tumor cells activates apoptotic cascades and this completely human construct may have significant therapeutic potential.