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

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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

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Acknowledgements

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

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Authors and Affiliations

  1. Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134, Napoli, Italy

    Rossella Di Stasi, Lucia De Rosa & Donatella Diana

  2. Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Università della Campania “L. Vanvitelli”, Via Vivaldi, 43, 81100, Caserta, Italy

    Roberto Fattorusso

  3. Istituto di Biostrutture e Bioimmagini, CNR, Via Nizza 52, 10126, Torino, Italy

    Luca D. D’Andrea

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  1. Rossella Di Stasi
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  2. Lucia De Rosa
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  5. Luca D. D’Andrea
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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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  • DOI: https://doi.org/10.1007/s12033-019-00181-7

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