Identification of the residues in the extracellular region of KDR important for interaction with vascular endothelial growth factor and neutralizing anti-KDR antibodies.
The kinase domain receptor (KDR) of vascular endothelial growth factor (VEGF) is the main human receptor responsible for the angiogenic activity of VEGF. The extracellular region of KDR is comprised of seven immunoglobulin-like domains, of which the first three have been shown to be required for ligand binding. We have previously described antibodies directed against the extracellular region of KDR, including MAB383 and MAB664, which were shown to block the binding of VEGF to the receptor and to inhibit both VEGF-induced mitogenesis of human endothelial cells in vitro and tumor growth in vivo. Here we generated a series of KDR deletion mutants consisting of truncated extracellular regions and mapped out the domain(s) responsible for binding to VEGF and the neutralizing anti-KDR antibodies. All neutralizing antibodies were found to require domain 3 for efficient binding. Alanine-scanning mutagenesis of domain 3 identified two different sets of five residues, Ile(256), Asp(257), Glu(261), Leu(313), and Thr(315) and Tyr(262), Pro(263), Ser(264), Ser(265), and Lys(266), that were critical for binding to MAB383 and MAB664, respectively. Combination of alanine mutations affecting both MAB383 and MAB664 binding resulted in a variant that also lost binding to VEGF. These results suggest that the residues within this region of domain 3 are critical for VEGF binding. Our studies provide a basis for the mechanism of action of our anti-KDR antibodies and establish a functional foundation for the development of other classes of antagonists to the receptor.
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Department of Molecular Biology, ImClone Systems Incorporated, New York, New York 10014, USA.
Kussie
P
P
Pytowski
B
B
Persaud
K
K
Bohlen
P
P
Witte
L
L
Zhu
Z
Z
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D016228:Endothelial Growth Factors / Q000378:metabolism*; D018604:Epitope Mapping; D006801:Humans; D008222:Lymphokines / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009500:Neutralization Tests; D011485:Protein Binding; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D040262:Receptors, Vascular Endothelial Growth Factor; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D000906:Antibodies; D017931:DNA Primers; D016228:Endothelial Growth Factors; D008222:Lymphokines; D017978:Receptors, Growth Factor; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D000409:Alanine