A Dual Specific Macrophage Colony Stimulating Factor Antagonist of C-FMS and αvβ3 Integrin for Osteoporosis Therapy

Osteoclasts are multi–nucleated, tissue specific cells, which differentiate from monocyte/macrophage lineage in a process called osteoclasteogenesis. One of the most important factors mediating osteoclasteogenesis is the signaling cascade of the tyrosine kinase receptor, c-FMS and its ligand, macrophage colony stimulating factor (M-CSF). This cascade initiates monocyte proliferation, survival and differentiation. Osteoclasts express integrins which are a family of membrane proteins that induce cell–cell and cell–extracellular matrix (ECM) interactions. Integrin α_vβ₃ is expressed on osteoclasts, platelets and endothelia and it binds to extra cellular proteins including vitronectin, fibronectin and osteopontins, all containing amino acid motif of Arg-Gly-Asp (RGD) that is crucial for integrin binding. Several studies show that c-FMS and α_vβ₃ cross-interact with each other making them a quality target for osteoporosis therapy. Therefore we developed dual-specific antagonists by combining rational and random protein design methods. We engineered novel proteins based on the M-CSF scaffold with the RGD motif introduced on 2 loops of the scaffold (M-CSF_RDG). Using the yeast surface display (YSD) system, we enriched the α_vβ₃ integrin and c-FMS binding population resulting in several unique dual-specific protein variants with high affinity and specificity to both targets. The purified M-CSF_RGD proteins were shown to significantly inhibit osteoclasteogensis on murine primary cells (BMMs) compared to control group in a dose dependent manner. Moreover, M-CSF_RGD proteins, namely 4.22 and 5.6, were shown to inhibit osteoclasts function in vivo in ovariectomized female mice compared to mice that were injected with PBS. Novel methods of protein engineering allowed us to create dual-specific proteins that will be tight binders, cell specific antagonists for osteoporosis treatment.