Abstract 5479: The fully human pro-apoptotic fusion toxin Granzyme B/VEGF121 targets vasculature and ablates tumor growth.
Authors: Mohamedali Khalid A. , Cheung Lawrence H. , Cao Yu , Hittelman Walter N. , Rosenblum Michael G. 
Published: August 2014

Abstract
Vascular endothelial growth factor 121 (VEGF121) is a naturally-occurring splice variant that binds to both VEGFR-1 and VEGFR-2 receptors over-expressed on the endothelium of tumor vasculature but not normal vasculature. The serine protease granzyme B (GrB) is capable of inducing apoptosis through both caspase-dependent and caspase-independent multiple-cascade mechanisms. We expressed GrB/VEGF121 in HEK293T cells and harvested the fusion protein under serum-free conditions. The specific activity of the GrB moiety, as assessed by cleavage of a synthetic chromogenic Granzyme B substrate, was comparable to that of human GrB. Cytotoxicity of GrB/VEGF121 indicated specific targeting into PAE/hVEGFR-2 (IC50 ∼ 10 nM), but not PAE/hVEGFR-1 cells (IC50 &gt; 1 μM). The targeting efficacy of GrB/VEGF121 against a panel of cells expressing a range of VEGFR receptor levels demonstrated varying levels of sensitivity to GrB/VEGF121. Internalization of GrB/VEGF121 into PAE/hVEGFR-2 and PAE/hVEGFR-1 cells was assessed by immunofluorescence and confocal microscopy 24 h after treatment. GrB/VEGF121 localized efficiently into PAE/hVEGFR-2 cells while the internalization into PAE/hVEGFR-1 cells was significantly lower. Forty-eight percent of PAE/VEGFR-2 cells underwent mitochondrial depolarization by 48 h, compared to 13% of untreated cells and 14% of cells treated with GrB. A significant degree of mitochondrial depolarization (34%) occurred within the first four hours of treatment. GrB/VEGF121 induced apoptosis in PAE/hVEGFR-2, but not in PAE/hVEGFR-1 cells, as determined by Annexin V staining. Thirty-five percent of GrB/VEGF121-treated PAE/VEGFR-2 cells were found to have mobilized into early apoptosis (Annexin V+ Propidium Iodide−), compared to 4% of control cells. The loss of plasma membrane integrity, as assessed by propidium iodide exclusion, was not observed for these cells over the 24 hour period, indicating that necrosis is not a major mechanism of cell death over the observed period of time. In vivo localization studies in tissues harvested four hours after GrB/VEGF121 or GrB i.v. injection detected GrB/VEGF121 in the tumors by immunofluorescence microscopy. Free GrB did not localize to tumor tissue. GrB/VEGF121 treatment appeared to be well tolerated by mice and significantly delayed tumor growth compared to treatment with saline or GrB alone. There was impressive antitumor efficacy at doses below the estimated Maximum Tolerated Dose (MTD). Our results suggest that targeted delivery of Granzyme B to tumor vascular endothelial cells or to tumor cells 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 pro-apoptotic fusion toxin Granzyme B/VEGF121 targets vasculature and ablates tumor growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5479. doi:10.1158/1538-7445.AM2013-5479