Abstract 4143: The novel hexavalent human GITR agonist HERA-GITRL promotes anti-tumor efficacy independent of Fc-functionality and shows superior activity compared with the monoclonal anti-GITR antibody TRX518
Authors: Schröder Matthias , Marshall Viola , Thiemann Meinolf , Richards David M. , Merz Christian , Sykora Jaromir , Sefrin Julian P. , Redondo-Müller Mauricio , Heinonen Karl , Billian-Frey Katharina , Hill Oliver , Gieffers Christian 
Published: August 2019

Abstract
Glucocorticoid-induced TNFR-related protein (GITR, TNFRSF18, CD357), a TNFR-SF member, is a co-stimulatory receptor that increases anti-tumor T cell activation. Based on Apogenix TNFR-SF agonist HERA-technology, we created a fully human hexavalent GITR ligand fusion protein - HERA-GITRL - intended for T cell costimulatory approaches in immuno-oncology (IO) therapy. HERA-GITRL is composed of a trivalent single chain GITRL-receptor-binding-domain fused to an IgG1-derived silenced Fc-domain serving as dimerization scaffold. The unique design that combines a molecular mimic of the endogenous GITRL with a silenced Fc-domain, allows the study of pure GITR agonism in contrast to Fc-mediated mixed modes of action. Here we report in vitro and in vivo properties of our novel HERA-GITRL construct. For functional characterization of HERA-GITRL in vitro, human immune cells isolated from healthy-donor blood were profiled by multicolor flow cytometry and real-time cell analysis. Stimulation of unfractionated human T cells or purified naïve CD4+ T cells by anti-CD3 antibody was further augmented by HERA-GITRL. This effect was accompanied by increased proliferation, differentiation and elevated levels of TNF-α and IFN-γ. Importantly, HERA-GITRL-mediated T cell activation increases tumor cell killing by PBMCs in vitro and showed in vivo anti-tumor efficacy as a single agent in a subcutaneous syngeneic colon cancer model (CT26wt) in mice. This anti-tumor effect is independent of its Fc functionality, as murine HERA-GITR ligands with functional Fc- or silenced Fc-domains show similar tumor growth inhibition. TRX518, an anti-human GITR monoclonal antibody currently investigated in a clinical Phase I study, was used in a direct in vitro comparison with trivalent GITRL and our hexavalent HERA-GITRL. Without crosslinking HERA-GITRL showed superior agonistic activity over trivalent GITRL and TRX518. Crosslinking increased the activity of trivalent GITRL while the residual activity of TRX518 was even decreased. We constructed a CHO cell line stably expressing fully human HERA-GITRL with high purity and yield. The resulting research cell bank is ready to be used for subsequent GMP process development. By clustering the receptor chains in a spatially well-defined manner, HERA-GITRL induces potent agonistic activity without being dependent on additional Fc-mediated crosslinking. A comparison of HERA-GITRL with the anti-GITR antibody TRX518 showed superior agonistic activity of our HERA construct in vitro with and without cross-linking. The HERA-ligand concept has also been successfully translated to HERA-TRAIL (now in Phase I), -CD40L, -CD27L,-LIGHT and -4-1BBL.
Citation Format: Matthias Schröder, Viola Marshall, Meinolf Thiemann, David M. Richards, Christian Merz, Jaromir Sykora, Julian P. Sefrin, Mauricio Redondo-Müller, Karl Heinonen, Katharina Billian-Frey, Oliver Hill, Christian Gieffers. The novel hexavalent human GITR agonist HERA-GITRL promotes anti-tumor efficacy independent of Fc-functionality and shows superior activity compared with the monoclonal anti-GITR antibody TRX518 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4143.