Monoclonal antibodies to vascular endothelial growth factor-D block its interactions with both VEGF receptor-2 and VEGF receptor-3.
Vascular endothelial growth factor-D (VEGF-D), the most recently discovered mammalian member of the VEGF family, is an angiogenic protein that activates VEGF receptor-2 (VEGFR-2/Flk1/KDR) and VEGFR-3 (Flt4). These receptor tyrosine kinases, localized on vascular and lymphatic endothelial cells, signal for angiogenesis and lymphangiogenesis. VEGF-D consists of a central receptor-binding VEGF homology domain (VHD) and N-terminal and C-terminal propeptides that are cleaved from the VHD to generate a mature, bioactive form consisting of dimers of the VHD. Here we report characterization of mAbs raised to the VHD of human VEGF-D in order to generate VEGF-D antagonists. The mAbs bind the fully processed VHD with high affinity and also bind unprocessed VEGF-D. We demonstrate, using bioassays for the binding and cross-linking of VEGFR-2 and VEGFR-3 and biosensor analysis with immobilized receptors, that one of the mAbs, designated VD1, is able to compete potently with mature VEGF-D for binding to both VEGFR-2 and VEGFR-3 for binding to mature VEGF-D. This indicates that the binding epitopes on VEGF-D for these two receptors may be in close proximity. Furthermore, VD1 blocks the mitogenic response of human microvascular endothelial cells to VEGF-D. The anti-(VEGF-D) mAbs raised to the bioactive region of this growth factor will be powerful tools for analysis of the biological functions of VEGF-D.
Achen
M G
MG
Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria, Australia. marc.achen@ludwig.edu.au
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Makinen
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D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D001483:Base Sequence; D002455:Cell Division / Q000276:immunology; D002478:Cells, Cultured; D017931:DNA Primers; D016228:Endothelial Growth Factors / Q000276:immunology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000276:immunology; D018604:Epitope Mapping; D006801:Humans; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism; D040262:Receptors, Vascular Endothelial Growth Factor; D042643:Vascular Endothelial Growth Factor D; D040321:Vascular Endothelial Growth Factor Receptor-3
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D016228:Endothelial Growth Factors; D011956:Receptors, Cell Surface; D017978:Receptors, Growth Factor; D042643:Vascular Endothelial Growth Factor D; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D040321:Vascular Endothelial Growth Factor Receptor-3