Fibronectin Type III Domain Based Monobody with High Avidity

Abstract

Significant efforts have been made to improve the target-binding strength of numerous affinity molecules that are widely used in biomedical research and clinical applications. While many antibody-like fragments have been successfully optimized through affinity maturation, the process is time-consuming and restricted by the stability, solubility, and expression level of the protein sequences. To generate a fibronectin type III domain (FN3) based monobody that binds to the tumor-related biomarkers with exceptional high strength and stability, we developed a multivalent strategy by fusing an alphavbeta3-binding FN3 monobody with a short COMP pentamerization domain through a linker that facilitates appropriate display of multivalent FN3 domains. The fusion protein was highly expressed in the soluble fraction of Escherichia coli and efficiently self-assembled into a pentameric molecule that could be readily purified. Compared to the monomeric form, the pentameric monobody bound to alphavbeta3 integrin much more tightly with significantly slower off-rate, while still maintaining its excellent specificity toward alphavbeta3 when tested by using purified integrins and integrin-expressing cell lines. The multivalent strategy we describe here could be applied to engineer other FN3 monobodies to acquire significantly improved targeting-binding strengths.