Moderate-Affinity Affibodies Modulate the Delivery and Bioactivity of Bone Morphogenetic Protein-2.

Uncontrolled bone morphogenetic protein-2 (BMP-2) release can lead to off-target bone growth and other adverse events. To tackle this challenge, we used yeast surface display to identify unique BMP-2-specific protein binders known as affibodies that bound to BMP-2 with different affinities. Biolayer interferometry revealed an equilibrium dissociation constant of 10.7 nM for the interaction between BMP-2 and high-affinity affibody and 34.8 nM for the interaction between BMP-2 and the low-affinity affibody. The low-affinity affibody-BMP-2 interaction also exhibited an off-rate constant that was an order of magnitude higher. Computational modeling of affibody-BMP-2 binding predicted that the high- and low-affinity affibodies bind to two distinct sites on BMP-2 that function as different cell-receptor binding sites. BMP-2 binding to affibodies reduced expression of the osteogenic marker alkaline phosphatase (ALP) in C2C12 myoblasts. Affibody-conjugated polyethylene glycol-maleimide hydrogels increased uptake of BMP-2 compared to affibody-free hydrogels, and high-affinity hydrogels exhibited lower BMP-2 release into serum compared to low-affinity hydrogels and affibody-free hydrogels over four weeks. Loading BMP-2 into affibody-conjugated hydrogels prolonged the ALP activity of C2C12 myoblasts compared to soluble BMP-2. This work demonstrates that affibodies with different affinities can modulate BMP-2 delivery and activity, creating a promising approach for controlling BMP-2 delivery in clinical applications. This article is protected by copyright. All rights reserved.