Abstract 4509: The fully human, proapoptotic fusion protein Granzyme B/VEGF121 targets tumor vasculature and inhibits tumor growth
Authors: Mohamedali Khalid A. , Cheung Lawrence H. , Cao Yu , Hittelman Walter N. , Rosenblum Michael G. 
Published: May 2015

Abstract
Angiogenesis is a critical process in numerous diseases, and targeting neovascularization has therapeutic value in several disease states including tumor progression and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) and its receptors play a central role in the control of neovascularization. Within the VEGF-A family, VEGF121 is a naturally-occurring splice variant that binds to receptors (VEGFR-1 and VEGFR-2) that are over-expressed on the endothelium of tumor vasculature but not normal vasculature. Granzyme B (GrB) is a serine protease that plays a critical role in the body's defense against viral infection and tumor development by initiating the apoptotic cascade via both caspase-dependent and -independent mechanisms. We expressed GrB/VEGF121 in HEK-293T cells and harvested the secreted fusion protein under serum-free conditions. Endothelial and tumor cells lines showed varying levels of sensitivity to GrB/VEGF121 that correlated closely to total VEGFR-2 expression. IC50 levels were found to be in the nanomolar range. GrB/VEGF121 internalized rapidly into VEGFR-2 expressing cells while the internalization into VEGFR-1 expressing cells was significantly reduced. Forty percent of VEGFR-2+ cells underwent mitochondrial depolarization within 24 h of exposure to GrB/VEGF121, compared to 13% of controls. Over this same time period, 35% of GrB/VEGF121-treated cells mobilized into early apoptosis, compared to 4% of control cells. In vivo, intravenous administration of GrB/VEGF121 to non-tumor bearing mice at doses up to 40 mg/kg had no observed toxicity. GrB/VEGF121 localized specifically in xenografts of non-VEGFR-expressing PC-3 and MDA-MB-231/Luc cells, primarily in perivascular regions and in other areas of the tumor less well-vascularized, whereas free GrB did not localize to tumor tissue. Treatment of mice bearing established prostate PC-3 tumor xenografts 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. Treatment of mice bearing orthotopic triple negative breast (MDA-MB-231/Luc) tumors with GrB/VEGF121 resulted in statistically significant differences in tumor volumes by Day 39 when compared to controls (saline vs 10 mg/kg GrB/VEGF121: P &lt; 0.04; saline vs 25 mg/kg GrB/VEGF121: P &lt; 0.03). In both tumor models, the vascular area as a percent of tumor area was significantly lower in tumor samples from mice treated with GrB/VEGF121 vs saline (PC-3: 14-fold, P &lt; 0.0001; MDA-MB-231/Luc: 5-fold, P &lt; 0.01) indicating that GrB/VEGF121 treatment directly impacted tumor vasculature. Our results show that targeted delivery of granzyme B to tumor vascular endothelial cells or to tumor cells activates apoptotic cascades and this completely human construct may have significant therapeutic potential. Research conducted, in part, by the Clayton Foundation for Research.
Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Yu Cao, Walter N. Hittelman, Michael G. Rosenblum. The fully human, proapoptotic fusion protein Granzyme B/VEGF121 targets tumor vasculature and inhibits tumor growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4509. doi:10.1158/1538-7445.AM2014-4509