Engineering dual-specific M-CSF antagonists that inhibit c-FMS and α_vβ₃ 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 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 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 integrin 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. We engineered novel proteins based on the M-CSF scaffold with the RGD motif introduced on 2 loops of the scaffold. 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 proteins were shown to significantly inhibit osteoclasteogensis on murine bone marrow derived monocytes (BMMs) compared to control group in a dose dependent manner. Moreover, M-CSF_RGD proteins were shown to inhibit osteoclasts function in vivo on overiactomized mice compared to mice that were injected with PBS. Following this proof of concept, we conjugated the M-CSF_RGD proteins with human albumin (HSA) in order to conduct prolong in vivo studies. These proteins were shown to keep a high bone mass in vivo and accumulate in the femur for over a week. Novel methods of protein engineering allowed us to create dual-specific proteins that will be tight binders, cell specific antagonists for osteoporosis treatment.