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Molecular Biotechnology

, Volume 61, Issue 7, pp 513–520 | Cite as

Human Recombinant VEGFR2D4 Biochemical Characterization to Investigate Novel Anti-VEGFR2D4 Antibodies for Allosteric Targeting of VEGFR2

  • Rossella Di Stasi
  • Lucia De Rosa
  • Donatella Diana
  • Roberto Fattorusso
  • Luca D. D’AndreaEmail author
Original paper

Abstract

VEGF-A/VEGFR2 complex is the major signaling pathway involved in angiogenesis and the inhibition of this axis retards tumor growth and inflammatory disorders progression, reducing vessel sprouting. Signaling by VEGFR2 requires receptor dimerization and a well-defined orientation of monomers in the active dimer. The extracellular portion of receptor is composed of seven Ig-like domains, of which D2–3 are the ligand binding domains, while D4 and D7, establishing homotypic contacts, allosterically regulate receptor activity. The allosteric targeting of VEGFR2 represents a promising alternative to study neovascular disorders overcoming drawbacks related to competition with VEGF. In this work, we expressed in bacterial host domain 4 of VEGFR2 (VEGFR2D4). After protein refolding, we characterized the purified domain and administered it in mice for monoclonal antibodies production. One of them, mAbD4, was tested in ELISA assays, showing a nanomolar affinity for VEGFR2D4. Finally, the methodology here described could contribute to the development of antibodies which can allosterically bind VEGFR2 and therefore to be used for imaging purposes or to modulate receptor signaling.

Keywords

VEGFs VEGFRs Anti-angiogenic agents Allosteric binders Monoclonal antibodies Extracellular domain 

Abbreviations

TEMED

Tetramethylethylenediamine

APS

Ammonium persulfate

dNTPs

Deoxynucleotide triphosphates

IPTG

Isopropyl β-D-1-thiogalactopyranoside

O.D.600

Optical density at 600 nm

Ni–NTA resin

Nickel charged-nitrilotriacetic resin

TEV protease

Tobacco etch virus protease

EDTA

Ethylenediaminetetraacetic acid

DTT

Dithiothreitol

RP-HPLC

Reversed-phase high-performance liquid chromatography

ESI-Tof

Electrospray ionization time-of-flight

TFA

Trifluoroacetic acid

Balb c mouse

Albino laboratory-bred strain

ELISA

Enzyme-linked immunosorbent assay

PBS

Phosphate-buffered saline buffer

BSA

Bovine serum albumin

HRP

Horseradish peroxidase

OPD

o-Phenylenediamine dihydrochloride

VEGFR

Vascular endothelial growth factor receptor

Notes

Acknowledgements

We would like to thank Leopoldo Zona and Maurizio Amendola for technical assistance.

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Istituto di Biostrutture e BioimmaginiCNRNapoliItaly
  2. 2.Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e FarmaceuticheUniversità della Campania “L. Vanvitelli”CasertaItaly
  3. 3.Istituto di Biostrutture e BioimmaginiCNRTorinoItaly

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