Abstract 575: Human serine protease therapeutics- potent constructs targeting Fn14 in multiple tumor types
Authors: Alvarez-Cienfuegos Ana , Cheung Lawrence H. , Mohamedali Khalid A. , Zhao Yunli , Ruisaard Kathryn E. , Winkles Jeffrey A. , Rosenblum Michael G. 
Published: July 2016

Abstract
The cytokine TWEAK, a TNF superfamily member, and its cognate receptor, Fn14, have emerged as potentially valuable targets for cancer therapy since their expression has been frequently identified as independent negative prognostic indicators in a variety of tumor types. The serine protease Granzyme B (GrB) is a highly cytotoxic component of human immune effector cells and induces multiple, intense pro-apoptotic signals when delivered to the cytoplasm of target cells. This human protein has been well-studied, operating through multimodal pathways which are both caspase-dependent and caspase-independent. Our laboratory has designed series of new Fn14 targeted fusion constructs containing an engineered GrB payload. The GrB-Fc-IT4 containing either WTCYS or EACYS mutants of GrB, and a human scFv targeting Fn14 were expressed in HEK293E and suspension CHO cells, harvested under serum-free conditions and purified to homogeneity. Both constructs display high-level protein production (&gt;50 mg/L), enhanced serum stability and impressive cytotoxic activity (IC50 &lt; 20 nM) against Fn14 positive cells. The specific activity of the GrB moiety, as assessed by cleavage of a synthetic chromogenic GrB substrate, was comparable to that of human GrB for the WTCYS while the EACYS variant showed a 50% reduction in specific activity. On the other hand, in vitro stability studies showed that EACYS was more stable after 48h incubation in human serum, retaining over 60% of its initial specific activity. Against a panel of 20 human cell lines expressing Fn14, both constructs showed high affinity and selective cytotoxicity within the nanomolar range (IC50 ranged from 4 to 284 nM) and were two to over a hundred times more potent than free GrB. Mechanistic studies demonstrated that GrB-Fc-IT4 constructs activated caspase cascades and cytochrome C related pro-apoptotic mechanisms in keeping with the known intracellular functions of GrB in target cells. Pharmacokinetic studies in mice revealed that GrB-Fc-IT4 fusion proteins exhibited a bi-exponential clearance from plasma with a rapid initial clearance (t ½ α = 0.36 hours) followed by a prolonged terminal-phase plasma half-life (t 1/2 β = 35 hours). Toxicity studies in mice demonstrated that the MTD is above 100 mg/kg total dose. Murine orthotropic tumor model efficacy and bio-distribution studies are ongoing and will be presented. Research conducted, in part, by the Clayton Foundation for Research.
Citation Format: Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Yunli Zhao, Kathryn E. Ruisaard, Jeffrey A. Winkles, Michael G. Rosenblum. Human serine protease therapeutics- potent constructs targeting Fn14 in multiple tumor types. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 575.