A Potent D-Protein Antagonist of VEGF-A is Nonimmunogenic, Metabolically Stable, and Longer-Circulating in Vivo

Polypeptides composed entirely of D-amino acids and the achiral amino acid glycine (D-proteins) inherently have in vivo properties that are proposed to be near-optimal for a large molecule therapeutic agent. Specifically, D-proteins are resistant to degradation by proteases and are anticipated to be non immunogenic. Furthermore, D-proteins are manufactured chemically and can be engineered to have other desirable properties, such as improved stability, affinity, and pharmacokinetics. Thus, a well-designed D-protein therapeutic would likely have significant advantages over L-protein drugs. Toward the goal of developing D-protein therapeutics, we previously generated RFX001.D, a D-protein antagonist of natural vascular endothelial growth factor A (VEGF-A) that inhibited binding to its receptor. However, RFX001.D is unstable at physiological temperatures (T-m = 33 degrees C). Here, we describe RFX037.D, a variant of RFX001.D with extreme thermal stability (T-m > 95 degrees C), high affinity for VEGF-A (K-d = 6 nM), and improved receptor blocking. Comparison of the two enantiomeric forms of RFX037 revealed that the D-protein is more stable in mouse, monkey, and human plasma and has a longer half-life in vivo in mice. Significantly, RFX037.D was nonimmunogenic in mice, whereas the L-enantiomer generated a strong immune response. These results confirm the potential utility of synthetic D-proteins as alternatives to therapeutic antibodies.