Target-binding proteins based on the 10th human fibronectin type III domain (¹⁰Fn3)

We describe concepts and methods for generating a family of engineered target-binding proteins designed on the scaffold of the 10th human fibronectin type III domain ((10)Fn3), an extremely stable, single-domain protein with an immunoglobulin-like fold but lacking disulfide bonds. Large libraries of possible target-binding proteins can be constructed on the (10)Fn3 scaffold by diversifying the sequence and length of its surface loops, which are structurally analogous to antibody complementarity-determining regions. Target-binding proteins with high affinity and specificity are selected from (10)Fn3-based libraries using in vitro evolution technologies such as phage display, mRNA display, or yeast-surface display. (10)Fn3-based target-binding proteins have binding properties comparable to those of antibodies, but they are smaller, simpler in architecture, and more user-friendly; as a consequence, these proteins are excellent building blocks for the construction of multidomain, multifunctional chains. The ease of engineering and robust properties of (10)Fn3-based target-binding proteins have been validated by multiple independent academic and industrial groups. In addition to performing well as specific in vitro detection reagents and research tools, (10)Fn3-based binding proteins are being developed as therapeutics, with the most advanced candidate currently in Phase II clinical trials.