Engineering dual-specific M-CSF antagonists that inhibit c-FMS and α_vβ₃ integrin for osteoporosis therapy

Bone resorbing osteoclasts are multi–nucleated, tissue specific cells, which differentiate from monocyte/macrophage lineage. One of the most important factors mediating osteoclasts differentiation is the tyrosine kinase receptor, c-FMS and its ligand, macrophage colony stimulating factor (M-CSF). This cascade initiates monocyte proliferation, survival and differentiation towards osteoclasts. Moreover, osteoclasts express the integrin α_vβ₃ that binds the extra cellular proteins vitronectin, fibronectin and osteopontins, all containing amino acid motif of Arg-Gly-Asp that is crucial for binding. Several studies show that c-FMS and α_vβ₃ integrin cross-interact with each other making them a quality target for osteoclast inhibition and osteoporosis therapy.

Therefore, we developed dual-specific antagonists by combining rational and random protein design. We engineered novel proteins based on the M-CSF scaffold with the RGD motif and using the yeast surface display system, we enriched the α_vβ₃ integrin and c-FMS binding population resulting in several protein variants with high affinity to both targets (M-CSF_RGD). We show that these proteins significantly inhibit osteoclasts differentiation and function in vitro in a dose dependent manner. Moreover, we show that M-CSF_RGD proteins inhibit bone resorption without effecting bone formation and increase the bone mass of ovariectomized mice, an established model for postmenopausal osteoporosis.