Abstract 4512: Construction and characterization of novel, human serine protease granzyme B-based cancer therapeutics targeting the TNFR family member Fn14

Abstract

Abstract The TNF superfamily cytokine TWEAK and its receptor Fn14 have emerged as potentially clinically valuable targets for cancer therapy. The expression of Fn14 is comparatively low in normal tissues but elevated in various human solid tumor types, including brain, breast, lung and melanoma, and has been shown to be a negative prognostic indicator. We generated Granzyme B (GrB) containing, Fn14-targeted constructs using either the extracellular domain (ECD) of the human TWEAK ligand (GrB-TWEAK) or an engineered anti-Fn14 humanized single-chain antibody (GrB-Fc-IT4) as the targeting moieties. Both fusion constructs were expressed in mammalian HEK293T cells and purified to homogeneity from conditioned media. Both GrB-TWEAK and GrB-Fc-IT4 constructs were designed to be relatively large in molecular weight (150 kDa and 160 kDa respectively) to allow prolonged circulation in vivo. BiaCore analysis of GrB-TWEAK and GrB-Fc-IT4 binding to the Fn14 ECD indicated that these proteins bound to Fn14 with Kds of ∼8 nM and ∼18 nM, respectively. Confocal immunofluorescence analysis showed that both proteins specifically and rapidly (within 3 hrs) internalized into Fn14-expressing MDA-MB-231 breast cancer and HT-29 colorectal adenocarcinoma cells. GrB-TWEAK and GrB-Fc-IT4 demonstrated impressive and selective cytotoxicity to a panel of Fn14-expressing human tumor cell lines in the low nanomolar range. The endogenous presence of the GrB proteinase inhibitor 9 (PI-9) had no impact on the cytotoxic effect of either construct. Treatment of cells expressing the multidrug resistance protein MDR1 showed no cross-resistance to the fusion constructs in vitro. Mechanistic studies demonstrated that GrB-TWEAK and GrB-Fc-IT4 activated caspase cascades and cytochrome C-related pro-apoptotic mechanisms consistent with the known intracellular functions of GrB in target cells. Treatment of mice bearing well-established HT-29 xenografts with GrB-TWEAK (at 20 or 40 mg/kg total doses) showed significant tumor growth inhibition compared to controls (P< 0.05). GrB-TWEAK and GrB-Fc-IT4 were administered to mice bearing MDA-MB-231 orthotopic human breast xenograft tumors and both constructs displayed significant tumor growth inhibition. Treatment with GrB-Fc-IT4 displayed a more pronounced and prolonged tumor growth inhibition and longer survival than GrB-TWEAK treatment. IHC analysis demonstrates excellent tumor uptake of both agents. These data suggest that Fc-containing fusions with GrB may have a design advantage with respect to efficacy and may form the basis of novel, highly effective and nontoxic constructs for targeted therapeutic applications. Research conducted, in part, by the Clayton Foundation for Research (MGR) and National Institutes of Health grant R01 NS055126 (JAW). Citation Format: Hong Zhou, Khalid A. Mohamedali, Yu Cao, Mary Migliorini, Lawrence H. Cheung, Walter N. Hittelman, Jeffrey A. Winkles, Michael G. Rosenblum. Construction and characterization of novel, human serine protease granzyme B-based cancer therapeutics targeting the TNFR family member Fn14. [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 4512. doi:10.1158/1538-7445.AM2014-4512