Engineered B7-H3 Binding in Modular Gp2 Miniproteins.

B7-H3, an important immune checkpoint modulator of T-cell function, is a tumor vasculature biomarker and is overexpressed in a variety of cancers. Its expression is associated with tumor growth, metastasis, and poor clinical prognosis, which makes B7-H3 an appealing target for diagnostics and therapeutics. High-affinity, specific, modular ligands are needed to achieve the various modes of molecular targeting strategies. A designed combinatorial library of the small (45 amino acid) Gp2 scaffold was sorted for binders to B7-H3 via yeast surface display with magnetic and flow cytometric cell sorting. Select variants were sequenced, characterized for binding affinity and specificity to B7-H3, and assessed for modularity in a protein-protein fusion. Protein ligands achieved single-digit nanomolar affinities and retained binding affinity upon conjugation, via a glycine-rich linker, to an enzyme. Directed evolution resulted in a potent, 0.7 nM binder with increased stability as assessed through the apparent midpoint of denaturation of 67°C. The engineered ligands provide small, modular, high-affinity B7-H3 binders for molecularly targeted therapeutics and diagnostics.