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Updated: Sep 27, 2026
Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries
Published on: January 17, 2015
Selection of high affinity human neutralizing antibodies to VEGFR2 from a large antibody phage display library for
Dan Lu1, Xenia Jimenez, Haifan Zhang
1Department of Molecular and Cell Biology, ImClone Systems Incorporated, 180 Varick Street, New York, NY 10014, USA.
Abstract:
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.
Insights
Researchers developed human antibody fragments targeting kinase insert domain-containing receptor (KDR) to inhibit tumor angiogenesis. These human anti-KDR antibodies show potential for cancer therapy by blocking vascular endothelial growth factor (VEGF) signaling.
Area of Science:
- Oncology
- Immunology
- Molecular Biology
Background:
- Vascular endothelial growth factor (VEGF) and its receptors are crucial for tumor angiogenesis and metastasis.
- VEGF signals through Flt-1 (VEGFR1) and KDR (VEGFR2) tyrosine kinase receptors.
- Previous murine antibodies against KDR showed therapeutic potential but risked immunogenicity in humans.
Purpose of the Study:
- To develop human antibody fragments targeting KDR.
- To evaluate the efficacy of these human anti-KDR antibodies in blocking VEGF signaling and cellular functions.
- To identify the binding epitope of these neutralizing antibodies on KDR.
Main Methods:
- Construction of an antibody phage display library from human B lymphocytes.
- Isolation and characterization of high-affinity human Fab antibody fragments against KDR.
- In vitro assays to assess KDR/VEGF binding inhibition, HUVEC mitogenesis, and leukemia cell migration.
- Epitope mapping to determine the antibody binding site on KDR.
Main Results:
- Several high-affinity human Fab fragments targeting KDR were isolated.
- These fragments bind KDR with nanomolar affinity and inhibit VEGF binding (IC50: 2-20 nM).
- The human antibodies effectively inhibited VEGF-stimulated HUVEC mitogenesis and leukemia cell migration.
- Neutralizing antibodies bound to the N-terminal immunoglobulin-like domains of KDR, overlapping the VEGF binding site.
Conclusions:
- Human anti-KDR Fab fragments effectively neutralize VEGF signaling and inhibit angiogenesis-related cellular processes.
- These human antibodies represent promising candidates for therapeutic applications in cancer and other diseases driven by pathologic angiogenesis.
- The identified binding site provides insights for further antibody engineering and drug development.
