title: Functional characterization of a VEGF-A-targeting Anticalin, prototype of a novel therapeutic human protein class.
abstract: Human tear lipocalin (Tlc) was utilized as a protein scaffold to engineer an Anticalin that specifically binds and functionally blocks vascular endothelial growth factor A (VEGF-A), a pivotal inducer of physiological angiogenesis that also plays a crucial role in several neovascular diseases. Starting from a naive combinatorial library where residues that form the natural ligand-binding site of Tlc were randomized, followed by affinity maturation, the final Anticalin PRS-050 was selected to bind all major splice forms of VEGF-A with picomolar affinity. Moreover, this Anticalin cross-reacts with the murine ortholog. PRS-050 efficiently antagonizes the interaction between VEGF-A and its cellular receptors, and it inhibits VEGF-induced mitogenic signaling as well as proliferation of primary human endothelial cells with subnanomolar IC50 values. Intravitreal administration of the Anticalin suppressed VEGF-induced blood-retinal barrier breakdown in a rabbit model. To allow lasting systemic neutralization of VEGF-A in vivo, the plasma half-life of the Anticalin was extended by site-directed PEGylation. The modified Anticalin efficiently blocked VEGF-mediated vascular permeability as well as growth of tumor xenografts in nude mice, concomitantly with reduction in microvessel density. In contrast to bevacizumab, the Anticalin did not trigger platelet aggregation and thrombosis in human FcγRIIa transgenic mice, thus suggesting an improved safety profile. Since neutralization of VEGF-A activity is well known to exert beneficial effects in cancer and other neovascular diseases, including wet age-related macular degeneration, this Anticalin offers a novel potent small protein antagonist for differentiated therapeutic intervention in oncology and ophthalmology.
authors.affiliation: Pieris Pharmaceuticals GmbH, Lise-Meitner-Strasse 30, 85354, Freising, Germany.|Silence Therapeutics GmbH, Robert-Rössle-Str. 10, 13125, Berlin, Germany.
authors.forename: Hendrik
authors.initials: H
authors.lastname: Gille
authors.affiliation: Pieris Pharmaceuticals GmbH, Lise-Meitner-Strasse 30, 85354, Freising, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Hülsmeyer
authors.affiliation: Pieris Pharmaceuticals GmbH, Lise-Meitner-Strasse 30, 85354, Freising, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Trentmann
authors.affiliation: Pieris Pharmaceuticals GmbH, Lise-Meitner-Strasse 30, 85354, Freising, Germany. Matschiner@pieris.com.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Matschiner
authors.affiliation: Pieris Pharmaceuticals GmbH, Lise-Meitner-Strasse 30, 85354, Freising, Germany.
authors.forename: Hans Jürgen
authors.initials: HJ
authors.lastname: Christian
authors.affiliation: Center for Thrombosis Research, Florida Hospital, Orlando, FL, USA.
authors.forename: Todd
authors.initials: T
authors.lastname: Meyer
authors.affiliation: Center for Thrombosis Research, Florida Hospital, Orlando, FL, USA.
authors.forename: Ali
authors.initials: A
authors.lastname: Amirkhosravi
authors.affiliation: Pieris Pharmaceuticals GmbH, Lise-Meitner-Strasse 30, 85354, Freising, Germany.
authors.forename: Laurent P
authors.initials: LP
authors.lastname: Audoly
authors.affiliation: Pieris Pharmaceuticals GmbH, Lise-Meitner-Strasse 30, 85354, Freising, Germany.
authors.forename: Andreas M
authors.initials: AM
authors.lastname: Hohlbaum
authors.affiliation: Lehrstuhl für Biologische Chemie, Technische Universität München, 85350, Freising-Weihenstephan, Germany. skerra@tum.de.
authors.forename: Arne
authors.initials: A
authors.lastname: Skerra
mesh_terms: D000818:Animals; D000936:Antigen-Antibody Complex / Q000378:metabolism; D000068258:Bevacizumab / Q000494:pharmacology / Q000627:therapeutic use; D001813:Blood-Retinal Barrier / Q000473:pathology; D002199:Capillary Permeability; D049109:Cell Proliferation / Q000187:drug effects; D005260:Female; D006207:Half-Life; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D054834:Lipocalins / Q000493:pharmacokinetics / Q000494:pharmacology* / Q000627:therapeutic use; D008819:Mice, Nude; D008822:Mice, Transgenic; D008934:Mitogens / Q000378:metabolism; D058990:Molecular Targeted Therapy; D009369:Neoplasms / Q000188:drug therapy / Q000473:pathology; D011092:Polyethylene Glycols / Q000737:chemistry; D015202:Protein Engineering; D011817:Rabbits; D017452:Receptors, IgG / Q000378:metabolism; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D013927:Thrombosis / Q000473:pathology; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D000936:Antigen-Antibody Complex; C080568:Fc gamma receptor IIA; D054834:Lipocalins; D008934:Mitogens; D017452:Receptors, IgG; D042461:Vascular Endothelial Growth Factor A; D000068258:Bevacizumab; D011092:Polyethylene Glycols
keywords: Angiogenesis; Cancer; Lipocalin; Protein engineering; Protein scaffold