title: The VD1 neutralizing antibody to vascular endothelial growth factor-D: binding epitope and relationship to receptor binding.
abstract: Vascular endothelial growth factor-D (VEGF-D) is a secreted protein that promotes tumor growth and metastatic spread in animal models of cancer. Expression of VEGF-D in prevalent human cancers was reported to correlate with lymph node metastasis and patient outcome-hence, this protein is a potential target for novel anticancer therapeutics designed to restrict tumor growth and spread. Here, we define the binding site in VEGF-D of a neutralizing antibody, designated VD1, which blocks the interaction of VEGF-D with its cell surface receptors vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 and is being used for the development of therapeutic antibodies. We show by peptide-based mapping and site-directed mutagenesis that the VD1 binding site includes the five residues (147)NEESL(151) and that immunization with a synthetic peptide containing this motif generates antibodies that neutralize VEGF-D. The tertiary structure of VEGF-D indicates that the (147)NEESL(151) epitope is located in the L2 loop of the growth factor, which is important for receptor binding. Mutation of any of these five residues influences receptor binding; for example, mutations to E148, which abolished binding to VD1, impaired the interaction with VEGFR-2 but enhanced binding to VEGFR-3. This structure/function study indicates that the VD1 binding epitope is part of the receptor binding site of VEGF-D, identifies a region of VEGF-D critical for binding of receptors and explains why VD1 does not bind other members of the VEGF family of growth factors.
authors.affiliation: Ludwig Institute for Cancer Research, Post Office Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia.
authors.forename: Natalia
authors.initials: N
authors.lastname: Davydova
authors.forename: Sally
authors.initials: S
authors.lastname: Roufail
authors.forename: Victor A
authors.initials: VA
authors.lastname: Streltsov
authors.forename: Steven A
authors.initials: SA
authors.lastname: Stacker
authors.forename: Marc G
authors.initials: MG
authors.lastname: Achen
mesh_terms: D057134:Antibodies, Neutralizing / Q000378:metabolism*; D001665:Binding Sites; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009500:Neutralization Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D042461:Vascular Endothelial Growth Factor A / Q000276:immunology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000378:metabolism
chemical_list: D057134:Antibodies, Neutralizing; D000939:Epitopes; D050505:Mutant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; D040321:Vascular Endothelial Growth Factor Receptor-3