Abstract C231: Novel, fully-human GrB-containing constructs targeting the Fn14 receptor for TWEAK on 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. Fn14 expression was relatively low in normal tissues but was found to be elevated in various human solid tumor types, including brain, breast and melanoma, and can be a negative prognostic indicator. TMAs of melanoma patient tumors found elevated Fn14 expression in 173/190(92%) of primary specimens and 86/150(58%) of metastases. Studies are underway examining expression vs survival. Studies are also underway examining TNBC microarrays for expression as well. To target the Fn14 receptor, we constructed a novel chimeric protein designated granzyme B (GrB)-TWEAK, using the human TWEAK receptor-binding domain as a targeting moiety and the human Granzyme B as a cytotoxic domain. In addition, we developed a high mw(160kDa) fusion protein composed of a single-chain anti-Fn14 scFv and GrB(designated scIGB). Both fusion construct was expressed in mammalian cells and purified to homogeneity. BiaCore analysis of TWEAK, GrB-TWEAK and scIGB binding to the Fn14 extracellular domain indicated that these proteins bound to Fn14 with similar affinities(Kds of ∼ 3-8 nM). Confocal immunofluoresence studies showed that GrB-TWEAK and scIGB specifically and rapidly (within 3 hrs) internalized into Fn14-expressing MDA-MB-231 breast cancer and HT-29 colorectal adenocarcinoma cells. Against a panel of 26 human cancer cell lines in culture, GrB-TWEAK demonstrated impressive and selective cytotoxicity to Fn14-expressing cells in the low nanomolar range (IC50 ranged from 0.4 nM-330 nM) and were 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 in keeping with the known intracellular functions of GrB in target cells. Preliminary mouse xenograft tumor model studies are ongoing. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C231. Citation Format: Hong Zhou, Mary Migliorini, Walter Hittelman, Suhendan Ekmekcioglu, Nhan Tran, Janine LoBello, Jeffrey A. Winkles, Michael G. Rosenblum. Novel, fully-human GrB-containing constructs targeting the Fn14 receptor for TWEAK on solid tumor cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C231.