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


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.


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





Ammonium persulfate


Deoxynucleotide triphosphates


Isopropyl β-D-1-thiogalactopyranoside


Optical density at 600 nm

Ni–NTA resin

Nickel charged-nitrilotriacetic resin

TEV protease

Tobacco etch virus protease


Ethylenediaminetetraacetic acid




Reversed-phase high-performance liquid chromatography


Electrospray ionization time-of-flight


Trifluoroacetic acid

Balb c mouse

Albino laboratory-bred strain


Enzyme-linked immunosorbent assay


Phosphate-buffered saline buffer


Bovine serum albumin


Horseradish peroxidase


o-Phenylenediamine dihydrochloride


Vascular endothelial growth factor receptor



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


  1. 1.
    Karkkainen, M. J., & Petrova, T. V. (2000). Vascular endothelial growth factor receptors in the regulation of angiogenesis and lymphangiogenesis. Oncogene, 19, 5598–5605.CrossRefGoogle Scholar
  2. 2.
    Patel-Hett, S., & D’Amore, P. A. (2011). Signal transduction in vasculogenesis and developmental angiogenesis. International Journal of Developmental Biology, 55, 353–363.CrossRefGoogle Scholar
  3. 3.
    Moens, S., Goveia, J., Stapor, P. C., Cantelmo, A. R., & Carmeliet, P. (2014). The multifaceted activity of VEGF in angiogenesis: Implications for therapy responses. Cytokine and Growth Factor Reviews, 25, 473–482.CrossRefGoogle Scholar
  4. 4.
    Shibuya, M. (2014). VEGF-VEGFR signals in health and disease. Biomolecules and Therapeutics, 22, 1–9.CrossRefGoogle Scholar
  5. 5.
    Fong, G. H., Rossant, J., Gertsenstein, M., & Breitman, M. L. (1995). Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature, 376, 66–70.CrossRefGoogle Scholar
  6. 6.
    Shalaby, F., Rossant, J., Yamaguchi, T. P., Gertsenstein, M., Wu, X. F., Breitman, M. L., et al. (1995). Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature, 376, 62–66.CrossRefGoogle Scholar
  7. 7.
    Veikkola, T., Jussila, L., Makinen, T., Karpanen, T., Jeltsch, M., Petrova, T. V., et al. (2001). Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice. The EMBO J., 20, 1223–1231.CrossRefGoogle Scholar
  8. 8.
    Lemmon, M. A., & Schlessinger, J. (2010). Cell signaling by receptor tyrosine kinases. Cell, 141, 1117–1134.CrossRefGoogle Scholar
  9. 9.
    Shibuya, M. (2013). Vascular endothelial growth factor and its receptor system: Physiological functions in angiogenesis and pathological roles in various diseases. Journal of Biochemistry, 153, 13–19.CrossRefGoogle Scholar
  10. 10.
    Abhinand, C. S., Raju, R., Soumya, S. J., Arya, P. S., & Sudhakaran, P. R. (2016). VEGF-A/VEGFR2 signaling network in endothelial cells relevant to angiogenesis. Journal of Cell Communication and Signaling, 10, 347–354.CrossRefGoogle Scholar
  11. 11.
    Lania, G., Ferrentino, R., & Baldini, A. (2015). TBX1 represses Vegfr2 gene expression and enhances the cardiac fate of VEGFR2+ cells. PLoS ONE, 10, e0138525.CrossRefGoogle Scholar
  12. 12.
    Dosch, D. D., & Ballmer-Hofer, K. (2010). Transmembrane domain-mediated orientation of receptor monomers in active VEGFR-2 dimers. FASEB Journal, 24, 32–38.CrossRefGoogle Scholar
  13. 13.
    Leppanen, V. M., Prota, A. E., Jeltsch, M., Anisimov, A., Kalkkinen, N., Strandin, T., et al. (2010). Structural determinants of growth factor binding and specificity by VEGF receptor 2. Proceedings of the National Academy of Sciences of the United States of America, 107, 2425–2430.CrossRefGoogle Scholar
  14. 14.
    Brozzo, M. S., Bjelic, S., Kisko, K., Schleier, T., Leppanen, V. M., Alitalo, K., et al. (2012). Thermodynamic and structural description of allosterically regulated VEGFR-2 dimerization. Blood, 119, 1781–1788.CrossRefGoogle Scholar
  15. 15.
    Barleon, B., Totzke, F., Herzog, C., Blanke, S., Kremmer, E., Siemeister, G., et al. (1997). Mapping of the sites for ligand binding and receptor dimerization at the extracellular domain of the vascular endothelial growth factor receptor FLT-1. Journal of Biological Chemistry, 272, 10382–10388.CrossRefGoogle Scholar
  16. 16.
    Shinkai, A., Ito, M., Anazawa, H., Yamaguchi, S., Shitara, K., & Shibuya, M. (1998). Mapping of the sites involved in ligand association and dissociation at the extracellular domain of the kinase insert domain-containing receptor for vascular endothelial growth factor. Journal of Biological Chemistry, 273, 31283–31288.CrossRefGoogle Scholar
  17. 17.
    Ruch, C., Skiniotis, G., Steinmetz, M. O., Walz, T., & Ballmer-Hofer, K. (2007). Structure of a VEGF-VEGF receptor complex determined by electron microscopy. Nature Structural and Molecular Biology, 14, 249–250.CrossRefGoogle Scholar
  18. 18.
    Kisko, K., Brozzo, M. S., Missimer, J., Schleier, T., Menzel, A., Leppanen, V. M., et al. (2011). Structural analysis of vascular endothelial growth factor receptor-2/ligand complexes by small-angle X-ray solution scattering. FASEB Journal, 25, 2980–2986.CrossRefGoogle Scholar
  19. 19.
    Hyde, C. A., Giese, A., Stuttfeld, E., Abram, Saliba J., Villemagne, D., Schleier, T., et al. (2012). Targeting extracellular domains D4 and D7 of vascular endothelial growth factor receptor 2 reveals allosteric receptor regulatory sites. Molecular and Cellular Biology, 32, 3802–3813.CrossRefGoogle Scholar
  20. 20.
    Thieltges, K. M., Avramovic, D., Piscitelli, C. L., Markovic-Mueller, S., Binz, H. K., & Ballmer-Hofer, K. (2018). Characterization of a drug-targetable allosteric site regulating vascular endothelial growth factor signaling. Angiogenesis, 21, 533–543.CrossRefGoogle Scholar
  21. 21.
    Mendel, D. B., Laird, A. D., Xin, X., Louie, S. G., Christensen, J. G., Li, G., et al. (2003). In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship. Clinical Cancer Research, 9, 327–337.Google Scholar
  22. 22.
    Ferrara, N., Hillan, K. J., Gerber, H. P., & Novotny, W. (2004). Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nature Reviews Drug Discovery, 3, 391–400.CrossRefGoogle Scholar
  23. 23.
    Ellis, L. M. (2005). Bevacizumab. Nature Reviews Drug Discovery, 4, S8–S9.CrossRefGoogle Scholar
  24. 24.
    Krupitskaya, Y., & Wakelee, H. A. (2009). Ramucirumab, a fully human mAb to the transmembrane signaling tyrosine kinase VEGFR-2 for the potential treatment of cancer. Current Opinion in Investigational Drugs, 10, 597–605.Google Scholar
  25. 25.
    Kendrew, J., Eberlein, C., Hedberg, B., McDaid, K., Smith, N. R., Weir, H. M., et al. (2011). An antibody targeted to VEGFR-2 Ig domains 4-7 inhibits VEGFR-2 activation and VEGFR-2-dependent angiogenesis without affecting ligand binding. Molecular Cancer Therapeutics, 10, 770–783.CrossRefGoogle Scholar
  26. 26.
    Zhang, S., Gao, X., Fu, W., Li, S., & Yue, L. (2017). Immunoglobulin-like domain 4-mediated ligand-independent dimerization triggers VEGFR-2 activation in HUVECs and VEGFR2-positive breast cancer cells. Breast Cancer Research and Treatment, 163, 423–434.CrossRefGoogle Scholar
  27. 27.
    Wang, W., Yin, X., Li, Y., Tian, R., Yan, J., Gao, J., et al. (2013). Prokaryotic expression, purification and antigenicity identification of mouse VEGFR2 extracellular 1-4 IgG-like domains. Journal of Southern Medical University, 33, 13–17.Google Scholar

Copyright information

© 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

Personalised recommendations