Abstract 3856: Dimerized single chain TRAIL-receptor agonists do not depend on Fc-gamma-receptor cross-linking for anti-tumor efficacy in vivo
Authors: Gieffers Christian , Kluge Michael , Thiemann Meinolf , Merz Christian , Sykora Jaromir , Fischer Carmen , Branschädel Marcus , Harald Fricke , Hill Oliver 
Published: August 2012

Abstract
APG350 is a TRAIL-receptor (TRAIL-R) agonist comprising two single-chain TRAIL-R binding domains (scTRAIL-RBD) that are dimerized via the Fc-part of a human IgG1-mutein thereby creating six receptor binding sites per molecule. This improved ability to form clusters on target cells composed of six TRAIL-Rs each, distinguishes APG350 from current clinical development candidates. In vitro, comparison of APG350 with recombinant APO2L/TRAIL and a TRAIL-R2 specific agonistic antibody revealed superior apoptosis induction for APG350 on primary and established human tumor cell-lines. Treatment of mice bearing Colo205 xenograft tumors with APG350, APO2L/TRAIL or an agonistic TRAIL-R2 specific antibody showed superior anti-tumor efficacy for APG350. Pronounced anti tumor efficacy was also shown on colon cancer stem cell (CSC) derived xenografts and successful APG350 re-treatment of relapsed CSC derived tumors demonstrated that tumors did not develop drug resistance. For most agonistic TRAIL-R antibodies effective apoptosis induction is achieved only upon additional cross-linking. SEC-based fractionation of a TRAIL-R2 specific agonistic antibody indicates a small amount of multimerized antibody that showed efficient apoptosis induction in vitro. However, the respective monomeric antibody showed poor apoptosis induction in vitro that could be enhanced upon cross-linking. In contrast apoptosis induction by APG350 was only marginally enhanced by cross-linking. Although monomeric agonistic TRAIL-R antibodies are poor apoptosis inducers in vitro, they show efficient apoptosis induction on xenograft tumors in vivo. A likely explanation for this difference is given by a recent publication showing that anti-tumor efficacy of an agonistic TRAIL-R2 specific antibody (Drozitumab) depends on cross-linking by Fcα-receptors (FcαR) in vivo. These data suggest that FcαR cross-linking might be a common requirement for the anti-tumor efficacy of agonistic TRAIL-R antibodies. To exclude that in vivo efficacy of APG350 depends on cross-linking by FcαRs we designed APG350-muteins with strongly reduced (APG808) or lacking FcαR binding (APG780). Side by side comparison of APG808, APG780 and APG350 in mice bearing Colo205-derived xenograft tumors showed identical anti-tumor efficacy for all respective proteins. Given that APG780 cannot bind to FcαRs these results suggest that the anti-tumor efficacy of APG808, APG780 and APG350 is solely based on the unique construction principle of the dimerized scTRAIL-RBD. APG350 induces superior clustering of TRAIL-Rs that in contrast to agonistic TRAIL-R antibodies does not require cross-linking via FcαRs for its potent anti-tumor efficacy. APG350 based dimerized scTRAIL-RBD formats may therefore have the capacity to bridge the current gap seen between preclinical and clinical efficacy for agonistic TRAIL-R specific antibodies.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3856. doi:1538-7445.AM2012-3856