title: Isolation of a novel neutralizing antibody fragment against human vascular endothelial growth factor from a phage-displayed human antibody repertoire using an epitope disturbing strategy.
abstract: Following the clinical success of Bevacizumab, a humanized monoclonal antibody that blocks the interaction between vascular endothelial growth factor (VEGF) and its receptors, the search for new neutralizing antibodies targeting this molecule has continued until now. We used a human VEGF variant containing three mutations in the region recognized by Bevacizumab to direct antibody selection towards recognition of other epitopes. A total of seven phage-displayed antibody fragments with diverse binding properties in terms of inter-species cross-reactivity and sensitivity to chemical modifications of the antigen were obtained from a human phage display library. All of them were able to recognize not only the selector mutated antigen, but also native VEGF. One of these phage-displayed antibody fragments, denominated 2H1, was shown to compete with the VEGF receptor 2 for VEGF binding. Purified soluble 2H1 inhibited in a dose dependent manner the ligand-receptor interaction and abolished VEGF-dependent proliferation of human umbilical vein endothelial cells. Our epitope disturbing strategy based on a triple mutant target antigen was successful to focus selection on epitopes different from a known one. Similar approaches could be used to direct phage isolation towards the desired specificity in other antigenic systems.
authors.affiliation: Recombinant Antibodies Laboratory, Cancer Research Department, Center for Genetic Engineering and Biotechnology, La Habana, CP 10600, Cuba. humberto.lamdan@cigb.edu.cu
authors.forename: Humberto
authors.initials: H
authors.lastname: Lamdan
authors.forename: Marta
authors.initials: M
authors.lastname: Ayala
authors.forename: Gertrudis
authors.initials: G
authors.lastname: Rojas
authors.forename: Yasmiana
authors.initials: Y
authors.lastname: Munoz
authors.forename: Yanelys
authors.initials: Y
authors.lastname: Morera
authors.forename: Osmany
authors.initials: O
authors.lastname: Guirola
authors.forename: Glay
authors.initials: G
authors.lastname: Chinea
authors.forename: Jorge V
authors.initials: JV
authors.lastname: Gavilondo
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry; D061067:Antibodies, Monoclonal, Humanized; D057134:Antibodies, Neutralizing / Q000737:chemistry*; D000941:Antigens / Q000737:chemistry; D000068258:Bevacizumab; D042783:Endothelial Cells / Q000166:cytology; D000939:Epitopes / Q000737:chemistry*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry; D009154:Mutation; D018919:Neovascularization, Physiologic; D019151:Peptide Library; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D057134:Antibodies, Neutralizing; D000941:Antigens; D000939:Epitopes; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D042461:Vascular Endothelial Growth Factor A; D000068258:Bevacizumab