Abstract 2185: Development and characterization of the fully human chimeric protein GrB-TWEAK targeting Fn14-positive solid tumor cells.

Abstract

Abstract The TNFR superfamily member Fn14, the receptor for TNF-like weak inducer of apoptosis (TWEAK), has emerged as a potentially clinically valuable target for cancer therapy. Specifically, Fn14 expression is relatively low in normal tissues but elevated in various human solid tumor types, including brain, breast, lung and melanoma, and can be a negative prognostic indicator. To target Fn14-overexpressing cancer cells, we constructed via PCR a novel chimeric protein designated granzyme B (GrB)-TWEAK, using the human TWEAK receptor-binding domain as the targeting moiety and the human pro-apoptotic serine protease GrB as the cytotoxic agent. The fusion construct was expressed in mammalian cells and purified to homogeneity from conditioned media. BiaCore analysis of TWEAK and GrB-TWEAK binding to the Fn14 extracellular domain indicated that these proteins bound to Fn14 with similar affinity, with Kds of ∼ 3 nM and ∼ 8 nM, respectively. Confocal immunofluoresence studies showed that GrB-TWEAK specifically and rapidly (within 3 hrs) internalized into Fn14-expressing MDA-MB-231 breast cancer and HT-29 colorectal adenocarcinoma cells. GrB-TWEAK activity was assessed using a panel of 26 human cancer cell lines and it demonstrated impressive and selective cytotoxicity to Fn14-expressing cells in the low nanomolar range (IC50 ranged from 0.4 nM-330 nM), which was 6- to 1117-fold more potent than free GrB. Treatment of cells expressing the multidrug resistance protein MDR1 showed no cross-resistance to the fusion construct in vitro. Mechanistic studies demonstrated that GrB-TWEAK activated caspase cascades and cytochrome C-related pro-apoptotic mechanisms consistent with the known intracellular functions of GrB in target cells. Preliminary mouse xenograft tumor model studies are ongoing. This work was conducted, in part, by the Clayton Foundation for Research (MGR) and supported by NIH grant NS055126 (JAW). Citation Format: Hong Zhou, Mary Migliorini, Yu Cao, Lawrence H. Cheung, Walter N. Hittelman, Jeffrey A. Winkles, Michael G. Rosenblum. Development and characterization of the fully human chimeric protein GrB-TWEAK targeting Fn14-positive solid tumor cells. [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 2185. doi:10.1158/1538-7445.AM2013-2185