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

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.

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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

References

  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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google Scholar 

  4. 4.

    Shibuya, M. (2014). VEGF-VEGFR signals in health and disease. Biomolecules and Therapeutics, 22, 1–9.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google Scholar 

  8. 8.

    Lemmon, M. A., & Schlessinger, J. (2010). Cell signaling by receptor tyrosine kinases. Cell, 141, 1117–1134.

    Article  CAS  PubMed  PubMed Central  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  PubMed  PubMed Central  Google 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.

    Article  CAS  PubMed  PubMed Central  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  PubMed  PubMed Central  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  PubMed Central  Google 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.

    Article  CAS  PubMed  Google 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.

    CAS  PubMed  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.

    Article  CAS  PubMed  Google Scholar 

  23. 23.

    Ellis, L. M. (2005). Bevacizumab. Nature Reviews Drug Discovery, 4, S8–S9.

    Article  Google 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.

    CAS  PubMed  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.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    PubMed  Google Scholar 

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Acknowledgements

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

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Correspondence to Luca D. D’Andrea.

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Di Stasi, R., De Rosa, L., Diana, D. et al. Human Recombinant VEGFR2D4 Biochemical Characterization to Investigate Novel Anti-VEGFR2D4 Antibodies for Allosteric Targeting of VEGFR2. Mol Biotechnol 61, 513–520 (2019). https://doi.org/10.1007/s12033-019-00181-7

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Keywords

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