Abstract 592: Improving therapeutic activity of agonistic DR5 antibodies by inducing target binding-dependent hexamer formation

Abstract

Abstract Death receptor 5 (DR5) is a highly interesting tumor target based on the enhanced sensitivity of cancer cells for DR5-dependent apoptosis. In recent years, multiple therapeutic DR5 antibodies have been evaluated in the clinic for which results however have been disappointing. IgG molecules against membrane-bound targets have shown an ability to form ordered hexameric structures upon antigen binding, a process that is dependent on Fc-Fc interactions between IgG molecules. We identified specific mutations in the human IgG1 Fc domain that enhance such antigen-dependent hexamerization while retaining solution-monomericity and developability characteristics of regular IgG1 molecules (HexaBody technology). We hypothesized that antibody-mediated hexamerization, when applied to DR5-specific antibodies, would enhance DR5 signaling and apoptosis, resulting in strongly improved therapeutic potential. The technology was applied to two non-crossblocking DR5-specific IgG1 antibodies, IgG1-DR5-01 and IgG1-DR5-05, by mutating a glutamic acid residue at position 430 in the Fc domain to glycine (the HexaBody mutants were designated Hx-DR5-01 and Hx-DR5-05). Cytotoxicity of the DR5 antibodies was explored in vitro using the CellTiter-Glo luminescent cell viability assay and the Caspase-Glo 3/7 assay in a broad panel of cancer cell lines, and in vivo in xenograft models. Both Hx-DR5-01 and Hx-DR5-05 induced increased cytotoxicity compared to their wild type (WT) IgG1 counterparts. Moreover, the combination of Hx-DR5-01 and Hx-DR5-05 (referred to as Hx-DR5-01/05) was found to be more potent than either Hx-DR5-01 or Hx-DR5-05 alone, or than the combination of the WT antibodies (IC50 in BxPC3 cells 0.5 and 1.5 μg/ml; maximal cytotoxicity 91% and 25% for Hx-DR5-01/05 and WT IgG1-DR5-01/05 respectively). In contrast to wild type agonistic DR5 antibodies, tumor cell killing by Hx-DR5-01/05 was independent of secondary crosslinking. Potent anti-tumor activity was observed in seven xenograft models for multiple indications, with Hx-DR5-01/05 consistently showing significantly better efficacy than the WT DR5 comparator antibody conatumumab. The cytotoxic activity of DR5 antibodies was significantly enhanced by the introduction of a hexamer-enhancing mutation in the IgG1 Fc domain. Maximal killing activity was obtained by combining two non-crossblocking DR5 antibodies carrying this mutation (Hx-DR5-01 and Hx-DR5-05). The strong cytotoxicity of Hx-DR5-01/05 was completely dependent on target binding but, in contrast to WT antibodies, did not require secondary crosslinking. These promising pre-clinical results support the selection of Hx-DR5-01/05 for clinical development for the treatment of cancer. Citation Format: Marije B. Overdijk, Kristin Strumane, Antonio Ortiz Buijsse, Claudine Vermot-Desroches, Andreas Lingnau, Esther C.W. Breij, Janine Schuurman, Paul W.H.I. Parren. Improving therapeutic activity of agonistic DR5 antibodies by inducing target binding-dependent hexamer formation. [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 592.