Bone homeostasis critically relies on the RANKL-RANK-OPG axis which can be targeted by the fully human monoclonal antibody denosumab in conditions with increased bone resporption such as bone metastases. The binding site and therefore the molecular mechanism by which this antibody inhibits RANKL has not been characterized so far. Here, we used random peptide phage display library screenings to identify the denosumab epitope on RANKL. Alignments of phage derived peptide sequences with RANKL suggested that this antibody recognized a linear epitope between position T233 and Y241. Mutational analysis confirmed the core residues as critical for this interaction. The spatial localization of this epitope on a 3-dimensional model of RANKL showed that it overlapped with the major binding sites of OPG and RANK on RANKL. We conclude that denosumab inhibits RANKL by both functional and molecular mimicry of the natural decoy receptor OPG.
