(5ak) Fibronectin Domain Engineering: Improved Agents for Biotechnology and Tumor Targeting

Molecular recognition, the ability to provide high affinity, high specificity binding to a target of interest, is of critical importance to biology and biotechnology. Binding reagents enable selective detection and purification for laboratory research and biotechnology applications as well as potent delivery of therapeutic or diagnostic agents for clinical applications. The natural solution to molecular recognition, the antibody, has historically dominated binding reagent selection because of its broad binding capacity and the ability to generate binders by immunization. However, antibodies are large, complex, multi-domain proteins of moderate stability; thus alternative protein scaffolds can provide a multitude of advantages. In particular, the fibronectin type III domain provides facile cysteine- and amine-based chemical conjugation; small size for in vivo imaging and proteomics, a cysteine-free fold for intracellular use; single-domain architecture for protein fusions; production in bacterial culture; and improved stability and solubility.

Yet, native fibronectin binds a single target and must be engineered to provide broad binding capacity akin to antibodies. To robustly engineer high affinity binders we have developed a platform in which a combinatorial protein library is screened for binders and lead clones are improved by directed evolution. Multiple studies of synthetic combinatorial library design elucidated the critical elements including loop length diversity, stability bias, and a tailored amino acid repertoire; these elements were combined to produce a vastly improved synthetic library. A magnetic bead technique was developed to exploit avidity for selection of low affinity binders prior to affinity maturation. To improve the efficiency and efficacy of the search of combinatorial sequence space during clone evolution, a novel dual-mutagenesis method was developed and validated. Techniques and insights generated during development of the fibronectin platform are directly applicable to protein engineering in general.

This platform has enabled us to generate high affinity binders to myriad targets including immunoglobulin G for biotechnology applications and the tumor targets carcinoembryonic antigen, CD276, and epidermal growth factor receptor. Of multiple tumor targeting directions, particular focus was placed on downregulation of epidermal growth factor receptor by novel bispecific fibronectin constructs. Unique heterobivalent constructs were developed that robustly and swiftly decrease surface receptor levels resulting in reduced proliferation and migration of cells thereby presenting potential for therapeutic use.