De novo Design of a Highly Specific Inhibitor of Integrin Αvβ6 for Pulmonary Fibrosis

We describe the de novo design of hyperstable inhibitory proteins that bind to human αvβ6 with sub-nanomolar affinity and with >1000x specificity over other RGD (Arg-Gly-Asp)-binding integrins. The crystal structure of the inhibitor closely matches the design model, with affinity and specificity arising not only from an RGDLXX(L/I)-containing loop but also a second loop that contacts the β6 subunit. The designed inhibitor blocks αvβ6 -mediated TGF-β signalling in vitro and specifically targets αvβ6 (+) tumors in vivo. When administered via intraperitoneal injection or as an inhaled therapeutic, the inhibitor reduces fibrotic burden in bleomycin induced lung fibrosis in mice. Taken together, these results illustrate the power of de novo protein design in creating highly specific integrin inhibitors with therapeutic potential for immuno-oncology and the treatment of pulmonary fibrosis, and highlight direct respiratory system delivery of small stable high affinity binding proteins as a new therapeutic modality.