Selection of high affinity human neutralizing antibodies to VEGFR2 from a large antibody phage display library for antiangiogenesis therapy.
Compelling evidence suggests that vascular endothelial growth factor (VEGF) and its receptors play an important role in angiogenesis associated with tumor growth and metastasis. VEGF exerts its biologic activities through 2 transmembrane tyrosine kinase receptors: the fms-like tyrosine kinase receptor (Flt-1, or VEGFR1) and kinase insert domain-containing receptor (KDR or VEGFR2). We have previously produced a panel of antibodies directed against KDR from mice immunized with the recombinant form receptor. These antibodies efficiently neutralized VEGF-induced KDR activation and mitogenesis of human umbilical vascular endothelial cells (HUVEC). Murine antibodies, however, may not be suitable candidates for human therapy because of their propensity to elicit human anti-mouse antibody response. Here we isolated several high-affinity human Fab antibody fragments directed against KDR from an antibody phage display library constructed from the pooled B lymphocytes of nonimmunized healthy human donors. These human Fab fragments bind specifically to KDR with nanomolar affinity and block KDR/VEGF interaction with IC(50) of approximately 2-20 nM. Further, they effectively inhibit VEGF-stimulated mitogenesis of HUVEC and migration of human leukemia cells. Epitope mapping studies demonstrated that all neutralizing human antibodies bound the epitope(s) located within the first 3 N-terminal immunoglobulin-like domains of KDR, the same region that encompasses the binding site of VEGF. Our results suggest that these human anti-KDR antibodies may have potential application in the treatment of cancer and other diseases in which pathologic angiogenesis occurs.
Lu
Dan
D
Department of Molecular and Cell Biology, ImClone Systems Incorporated, 180 Varick Street, New York, NY 10014, USA.
Jimenez
Xenia
X
Zhang
Haifan
H
Bohlen
Peter
P
Witte
Larry
L
Zhu
Zhenping
Z
D000595:Amino Acid Sequence; D020533:Angiogenesis Inhibitors / Q000737:chemistry*; D000906:Antibodies / Q000737:chemistry*; D002460:Cell Line; D002465:Cell Movement; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D007938:Leukemia / Q000378:metabolism; D008969:Molecular Sequence Data; D009389:Neovascularization, Pathologic; D019151:Peptide Library; D011485:Protein Binding; D020794:Receptor Protein-Tyrosine Kinases / Q000276:immunology*; D017978:Receptors, Growth Factor / Q000276:immunology*; D040262:Receptors, Vascular Endothelial Growth Factor; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured; D014471:Umbilical Veins / Q000166:cytology
D020533:Angiogenesis Inhibitors; D000906:Antibodies; D019151:Peptide Library; D017978:Receptors, Growth Factor; D011994:Recombinant Proteins; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor