Abstract 2275: Characterization of the mechanistic basis of Apo2L/TRAIL-AMG 655-dr5 interactions and cooperative signaling in cancer cells

Clinical studies with multiple death receptor (DR) agonists targeting DR4 and/or DR5 s have failed to advance beyond phase 2 due to limited efficacy. Unexpectedly, treatment with DR5 agonist antibody AMG 655 markedly enhanced Apo2L/TRAIL-induced killing in cancer cells. Strong synergism observed between AMG 655 and Apo2L/TRAIL in multiple cancer cell lines suggests that additional cross-linking provided by AMG 655 can enhance the ability of soluble ligand to initiate an apoptotic signal. Since the Apo2L/TRAIL related TNF family members FasL and TNF are more potent apoptotic agonists in their membrane-bound forms, the synergy observed between soluble Apo2L/TRAIL and AMG 655 suggests that the combination may mimic a membrane bound form of the ligand. In this study, we used biochemical approaches to characterize the Apo2L/TRAIL-AMG 655-DR5 interactions and cooperative signaling. We found membrane-bound Apo2L/TRAIL induced potent apoptotic signaling against cancer cell lines that are resistant to soluble Apo2L/TRAIL. Cooperativity between Apo2L/TRAIL and AMG 655 but not single agent alone promotes caspase 8 activation and DISC complex assembly, suggesting that the combination represents a qualitative change in apoptotic signaling. To better understand the molecular interactions between Apo2L/TRAIL, DR5 and AMG 655, we determined the crystal structure of the ternary complex of Apo2L/TRAIL, the DR5 ectodomain, and the Fab fragment of AMG 655 at 3.3 A resolution. The Apo2L/TRAIL-DR5-AMG 655-Fab complex is composed of an Apo2L/TRAIL homotrimer with three DR5 monomers bound diagonally along the crevices between two neighboring Apo2L/TRAIL molecules, and three AMG 655 Fabs bound to the three DR5 monomers individually. We then modeled the positioning of additional ternary complexes, which results in an array of DR5 molecules on the cell surface in a hexagonal honeycomb pattern, with three DR5 molecules in each node. Using different AMG 655 fragments, we demonstrated that AMG 655-Fab92 but not AMG 655-Fab fragment is sufficient to cooperate with Apo2L/TRAIL suggesting that the two DR5 binding moieties of AMG 655 mediate the effect. These observations illustrate how co-administration of trimeric Apo2L/TRAIL and bivalent AMG 655 may promote super-clustering of DR5, thereby leading to efficient DISC assembly and enhanced apoptotic signaling. The enhanced agonism generated by combining Apo2L/TRAIL and AMG 655 provides insight into the limited efficacy observed in previous clinical trials and suggests testable hypotheses to reconsider death receptor agonism as a therapeutic strategy. Citation Format: Tzu-Hsuan Huang, Wesley Chang, Alexander Long, Xin Huang, Pamela Holland. Characterization of the mechanistic basis of Apo2L/TRAIL-AMG 655-dr5 interactions and cooperative signaling in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2275. doi:10.1158/1538-7445.AM2014-2275