title: A Potent d-Protein Antagonist of VEGF-A is Nonimmunogenic, Metabolically Stable, and Longer-Circulating in Vivo.
abstract: 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 nonimmunogenic. 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 (Tm = 33 °C). Here, we describe RFX037.D, a variant of RFX001.D with extreme thermal stability (Tm > 95 °C), high affinity for VEGF-A (Kd = 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.
authors.affiliation: Banting and Best Department of Medical Research and Department of Molecular Genetics, the Donnelly Centre, University of Toronto , Toronto, Ontario, Canada M5S 3E1.
authors.forename: Maruti
authors.initials: M
authors.lastname: Uppalapati
authors.affiliation: Department of Chemistry; Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago , Chicago, Illinois 60637, United States.
authors.forename: Dong Jun
authors.initials: DJ
authors.lastname: Lee
authors.affiliation: Department of Chemistry; Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago , Chicago, Illinois 60637, United States.
authors.forename: Kalyaneswar
authors.initials: K
authors.lastname: Mandal
authors.affiliation: Pharmacokinetics & Drug Metabolism, Amgen, Inc. , Thousand Oaks, California 91320, United States.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Li
authors.affiliation: Therapeutic Discovery, Amgen, Inc. , Thousand Oaks, California 91320, United States.
authors.forename: Les P
authors.initials: LP
authors.lastname: Miranda
authors.affiliation: Antibody Solutions , Sunnyvale, California 94089, United States.
authors.forename: Joshua
authors.initials: J
authors.lastname: Lowitz
authors.affiliation: Antibody Solutions , Sunnyvale, California 94089, United States.
authors.forename: John
authors.initials: J
authors.lastname: Kenney
authors.affiliation: Banting and Best Department of Medical Research and Department of Molecular Genetics, the Donnelly Centre, University of Toronto , Toronto, Ontario, Canada M5S 3E1.
authors.forename: Jarrett J
authors.initials: JJ
authors.lastname: Adams
authors.affiliation: Reflexion Pharmaceuticals, Inc. , San Francisco, California 94104, United States.
authors.forename: Dana
authors.initials: D
authors.lastname: Ault-Riché
authors.affiliation: Department of Chemistry; Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago , Chicago, Illinois 60637, United States.
authors.forename: Stephen B H
authors.initials: SB
authors.lastname: Kent
authors.affiliation: Banting and Best Department of Medical Research and Department of Molecular Genetics, the Donnelly Centre, University of Toronto , Toronto, Ontario, Canada M5S 3E1.
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
mesh_terms: D002138:Calibration; D002942:Circular Dichroism; D006801:Humans; D013058:Mass Spectrometry; D012015:Reference Standards; D020349:Surface Plasmon Resonance; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors*
chemical_list: D042461:Vascular Endothelial Growth Factor A