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

Abstract

Uncontrolled bone morphogenetic protein‐2 (BMP‐2) release can lead to off‐target bone growth and other adverse events. To tackle this challenge, yeast surface display is used to identify unique BMP‐2‐specific protein binders known as affibodies that bind to BMP‐2 with different affinities. Biolayer interferometry reveals 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 exhibits an off‐rate constant that is an order of magnitude higher. Computational modeling of affibody‐BMP‐2 binding predicts 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 reduces expression of the osteogenic marker alkaline phosphatase (ALP) in C2C12 myoblasts. Affibody‐conjugated polyethylene glycol‐maleimide hydrogels increase uptake of BMP‐2 compared to affibody‐free hydrogels, and high‐affinity hydrogels exhibit 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 prolongs 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.

Statistical optimization of hydrazone-crosslinked hyaluronic acid hydrogels for protein delivery

“…As proof-of-concept of affinity-controlled protein delivery from our HA hydrogels, we modified HA-Ox to contain a norbornene functional group for bioconjugation of a previously discovered BMP-2-specific affibody with high affinity for BMP-2 (dissociation constant, K D = 10.7 nM). 41 Affibodies are small proteins with 58 amino acids folded into a bundle of three α-helices. 61,62 Affibodies can be engineered to bind to different proteins with a range of affinities and can be used to release proteins at various rates by tuning the strength of the affinity interactions.…”

“…61,62 Affibodies can be engineered to bind to different proteins with a range of affinities and can be used to release proteins at various rates by tuning the strength of the affinity interactions. 41,63,64 HA was functionalized with norbornene through BoC 2 O activated coupling (Fig. 5A).…”

“…Release of BMP-2 was evaluated over 28 days from unmodified HA hydrogels or hydrogels conjugated with affibodies with high affinity and specificity for BMP-2 (dissociation constant, K D = 10.7 nM). 41 Hydrogels were formed with BMP-2 and incubated in 1% w/v BSA in PBS. Aliquots were taken from the supernatant over 28 days to evaluate a release profile of BMP-2 from the HA hydrogel.…”

“… 65 In other work, we have demonstrated that BMP-2 release from polyethylene glycol (PEG) hydrogels can be tuned over a range of release rates and amounts by changing the affinity between the affibodies and BMP-2. 41 In the future, we plan to further explore how changes to the HA hydrogel formulation, affibody amount, and affibody affinity strength can influence BMP-2 release to enhance bone regeneration.…”

“…BMP-2-specific affibodies were expressed in E. coli and purified as previously described. 41 HA-Nor-Ox (14.5 mg) was dissolved in 1.5 mL of 0.71 mg mL −1 affibody in PBS (1.45 × 10 −4 mmol) to create a solution with 1 : 1 ratio of affibody to HA-Nor-Ox. 15 μL of 10% w/v Irgacure 2595 in methanol was added to the reaction vial, stirred, and illuminated with 365 nm light for 10 minutes.…”