A Non-immunogenic Bivalent D-Protein Potently Inhibits Retinal Vascularization and Tumor Growth

We report a general approach to engineering multivalent D-proteins with antibody-like activities in vivo. Mirror-image phage display and structure-guided design were utilized to create a D-protein that uses receptor mimicry to antagonize vascular endothelial growth factor A (VEGF-A). Selections against the D-protein form of VEGF-A using phage-displayed libraries of two different domain scaffolds yielded two proteins that bound distinct receptor interaction sites on VEGF-A. X-ray crystal structures of the D-protein/VEGF-A complexes were used to guide affinity maturation and to construct a heterodimeric D-protein VEGF-A antagonist with picomolar activity. The D-protein VEGF-A antagonist prevented vascular leakage in a rabbit eye model of wet age-related macular degeneration and slowed tumor growth in the MC38 syngeneic mouse tumor model with efficacies comparable to those of approved antibody drugs, and in contrast with antibodies, the D-protein was non-immunogenic during treatment and following subcutaneous immunizations.