Abstract 2391: DR5 agonist activity of HexaBody®-DR5/DR5 (GEN1029) is potentiated by C1q and independent of Fc-gamma receptor binding in preclinical tumor models (2019)

Hyperclustering of Death Receptor 5 (DR5) after binding of its ligand TRAIL induces apoptosis. Targeting DR5 with agonistic antibodies has been evaluated for the treatment of cancer, however clinical efficacy of conventional DR5-targeting monoclonal antibodies (mAbs) has been disappointing. We applied the HexaBody® technology to improve antibody-mediated DR5 clustering on cancer cells. This technology is based on the natural concept that, upon binding to antigens on a cell surface, immunoglobulin G (IgG) molecules can organize into ordered hexamers through intermolecular Fc-Fc interactions. HexaBody molecules are IgG1 molecules with a single point mutation in the Fc domain that enhances these Fc-Fc interactions upon binding to membrane-bound targets, while retaining solution-monomericity.HexaBody-DR5/DR5 (Hx-DR5-01/05) is a 1:1 mixture of two humanized non-competing DR5-specific mAbs, each carrying an E430G hexamerization-enhancing mutation. We previously demonstrated that both dual epitope targeting and enhanced hexamerization through Fc-Fc interactions are required for DR5 agonist activity of Hx-DR5-01/05 in vitro.Here, we confirmed that Hx-DR5-01/05 showed superior anti-tumor activity compared to the single HexaBody molecules or a 1:1 mixture of their wild type counterparts in vivo, using a mouse xenograft model. Furthermore, we screened the potency of Hx-DR5-01/05 in vitro in a broad panel of human tumor cells lines using a cell viability assay, and in vivo in xenograft models. As IgG hexamers are known to provide an optimal docking site for complement component C1q, we studied if there was a role for C1q in Hx-DR5-01/05-dependent DR5 agonist activity.Hx-DR5-01/05 induced potent cytotoxicity in 104 tumor cell lines and in more than ten xenograft models representing many solid cancer lineages. For optimal cytotoxicity in vitro, Hx-DR5-01/05 required the presence of serum or purified C1q. In contrast, polyclonal IgG crosslinking, a mimic for FcγR-mediated antibody crosslinking, did not enhance potency. These data were confirmed in vivo. A Hx-DR5-01/05 variant deficient for both C1q and FcγR binding, showed significantly reduced anti-tumor activity in a colon cancer xenograft model, while a Hx-DR5-01/05 variant deficient in FcγR but not C1q binding showed anti-tumor activity comparable to Hx-DR5-01/05.In summary, Hx-DR5-01/05 is a mixture of two DR5-specific HexaBody molecules that shows potent DR5 agonist activity in a multitude of preclinical models through enhanced IgG hexamerization upon binding to two different DR5 epitopes on the cell surface. Cytotoxicity of Hx-DR5-01/05 was most optimal in the presence of C1q and completely independent of FcγR-mediated antibody crosslinking or effector functions in vitro and in vivo. A clinical trial to assess clinical safety of Hx-DR5-01/05 in patients is currently ongoing.Citation Format: Marije B. Overdijk, Kristin Strumane, Antonio Ortiz Buijsse, Claudine Vermot-Desroches, Thessa Kroes, Bart de Jong, Naomi Hoevenaars, Frank J. Beurskens, Rob N. de Jong, Andreas Lingnau, Paul W. Parren, Ulf Forssmann, A Kate Sasser, Janine Schuurman, Esther C. Breij. DR5 agonist activity of HexaBody®-DR5/DR5 (GEN1029) is potentiated by C1q and independent of Fc-gamma receptor binding in preclinical tumor models [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 2391.