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Functional characterization of a VEGF-A-targeting Anticalin, prototype of a novel therapeutic human protein class

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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.

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Acknowledgments

We are indebted to all current and previous members of the Pieris team, in particular Steffen Schlehuber and Antje Walz, for their contributions and support. We are also grateful to Shane Olwill and Ulrich Moebius for critical reading of the manuscript.

Author contributions

H. G., A. M. H., G. M., M. H., L. P. A. and A. S. designed experiments. H. G., M. H., S. T., G. M., H. J. C. and S. T. performed experiments and analyzed the data. T. M. and A. A. designed, performed and analyzed the experiments in FcγIIa transgenic mice. H. G., A. M. H., L. P. A. and A. S. wrote the manuscript.

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  1. Hendrik Gille

    Present address: Silence Therapeutics GmbH, Robert-Rössle-Str. 10, 13125, Berlin, Germany

Authors and Affiliations

  1. Pieris Pharmaceuticals GmbH, Lise-Meitner-Strasse 30, 85354, Freising, Germany

    Hendrik Gille, Martin Hülsmeyer, Stefan Trentmann, Gabriele Matschiner, Hans Jürgen Christian, Laurent P. Audoly & Andreas M. Hohlbaum

  2. Lehrstuhl für Biologische Chemie, Technische Universität München, 85350, Freising-Weihenstephan, Germany

    Arne Skerra

  3. Center for Thrombosis Research, Florida Hospital, Orlando, FL, USA

    Todd Meyer & Ali Amirkhosravi

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Correspondence to Gabriele Matschiner or Arne Skerra.

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G. M. is a full-time employee at Pieris Pharmaceuticals GmbH. H. G., M. H., S. T., H.-J. C., A. M. H. and L. P. A. were full-time employees of Pieris AG. A. S. is founder of Pieris AG and shareholder of Pieris Pharmaceuticals, Inc.

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All experiments described in this manuscript comply with the laws of the USA and the European Union. All animal experiments were reviewed and approved by the responsible animal ethics committees.

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Gille, H., Hülsmeyer, M., Trentmann, S. et al. Functional characterization of a VEGF-A-targeting Anticalin, prototype of a novel therapeutic human protein class. Angiogenesis 19, 79–94 (2016). https://doi.org/10.1007/s10456-015-9490-5

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