A dual-action humanized DKK-1 antibody that activates the Wnt/β-catenin signaling pathway and inhibits the RANKL signaling pathway for osteoporosis therapy.

Introduction: Osteoporosis remains a major global health challenge, and current single-pathway therapies often fail to achieve coordinated bone remodeling. Dickkopf-1 (DKK-1) is a potent inhibitor of the Wnt/β-catenin signaling pathway and plays a critical role in osteoporotic bone loss. Neutralization of DKK-1 represents a promising therapeutic strategy to promote bone formation while simultaneously inhibiting bone resorption. Methods: A humanized anti-DKK-1 monoclonal antibody (IgG4) was generated via CDR grafting combined with structure-guided back-mutations. Its binding affinity and specificity were assessed by ELISA and surface plasmon resonance. The antibody's ability to restore Wnt/β-catenin signaling was evaluated in human bone marrow mesenchymal stem cells (hMSCs) by Western blotting. Osteogenic differentiation and mineralization were assessed by ALP activity assay and Alizarin Red S staining, respectively. An osteoblast-osteoclast Transwell co-culture model was established to evaluate the antibody's effects on osteoclastogenesis via the OPG/RANKL axis, with osteoclast differentiation assessed by TRAP staining and qPCR. Developability properties, including stability and immunogenicity, were also characterized. Results: The humanized antibody bound to DKK-1 with high affinity (EC Discussion: These findings demonstrate that the humanized anti-DKK-1 antibody exerts dual anabolic and anti-resorptive effects through the Wnt/β-catenin/OPG/RANKL axis. This dual mechanism of action positions the antibody as a promising therapeutic candidate for osteoporosis, warranting further preclinical and clinical evaluation.