Affibody-mediated controlled release of fibroblast growth factor 2.

Protein therapeutics possess high target affinity and specificity, yet short residence times, which limit their broad utility. To overcome this challenge, we used affinity interactions to modulate protein release from a hydrogel delivery vehicle thereby prolonging therapeutic availability. Specifically, we designed an affibody-modified hyaluronan (HA)-based hydrogel as a delivery platform for fibroblast growth factor 2 (FGF2), a neuroprotective and neuroregenerative factor in the central nervous system (CNS). We identified a highly specific affibody binding partner with moderate affinity for FGF2 using yeast surface display and flow cytometry-based screening. Importantly, we demonstrated controlled release of bioactive FGF2 from the hydrogel by varying the ratio of affibody to protein and showed increased thermal stability of FGF2 in the presence of affibody. This versatile delivery platform will allow the distinct, simultaneous release of multiple proteins based on specific affinity interactions.

Directed Evolution Enables Simultaneous Controlled Release of Multiple Therapeutic Proteins from Biopolymer-Based Hydrogels.

With the advent of increasingly complex combination strategies of biologics, independent control over their delivery is the key to their efficacy; however, current approaches are hindered by the limited independent tunability of their release rates. To overcome these limitations, directed evolution is used to engineer highly specific, low affinity affibody binding partners to multiple therapeutic proteins to independently control protein release rates. As a proof-of-concept, specific affibody binding partners for two proteins with broad therapeutic utility: insulin-like growth factor-1 (IGF-1) and pigment epithelium-derived factor (PEDF) are identified. Protein-affibody binding interactions specific to these target proteins with equilibrium dissociation constants (KD ) between 10-7 and 10-8 m are discovered. The affibodies are covalently bound to the backbone of crosslinked hydrogels using click chemistry, enabling sustained, independent, and simultaneous release of bioactive IGF-1 and PEDF over 7 days. The system is tested with C57BL/6J mice in vivo, and the affibody-controlled release of IGF-1 results in sustained activity when compared to bolus IGF-1 delivery. This work demonstrates a new, broadly applicable approach to tune the release of therapeutic proteins simultaneously and independently and thus the way for precise control over the delivery of multicomponent therapies is paved.