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 Dprotein 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.

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis 1 . Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

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