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Characterization of a new potent, in vivo neutralizing monoclonal antibody to human vascular endothelial growth
J M Schlaeppi1, G Siemeister, K Weindel
1Novartis Pharmaceuticals, Core Technology Department, Novartis Limited, Basel, Switzerland. jean-marc.schlaeppi@pharma.novartis.com
Abstract:
Vascular endothelial growth factor (VEGF) is an important mediator of tumor-induced angiogenesis and represents a potential target for anticancer therapy. Therefore, we prepared a panel of monoclonal antibodies (mAb) against both the VEGF121 and VEGF165 isoforms. Three of them completely neutralized the mitogenic stimulation by VEGF of human umbilical vein endothelial cells at mAb concentrations below 0.1 microg/ml. The most potent one, with a dissociation constant (Kd) of 8 pM, inhibited, in a dose-dependent manner, VEGF-induced angiogenesis in a growth factor implant model in mice. A complete inhibition of the angiogenic response was obtained by daily intraperitoneal injections of 10 microg mAb/mouse. Angiogenesis induced by basic fibroblast growth factor was not inhibited by the mAb. Epitope mapping of the mAb, performed by competitive enzyme-linked immunosorbent assay and Western blot analysis, showed that it did not bind to the reduced and denatured monomer of VEGF. Substitutions of three residues (Q87R, G88K, Q89K), located on the major surface loop beta5 to beta6 of VEGF, resulted in the complete loss of binding (more than 400-fold reduction). The results suggest that the mAb binds primarily to a conformation-dependent epitope on the VEGF dimeric form, encompassing one of the loop regions involved in KDR receptor binding. The mAb with its strong neutralizing properties represents a useful agent for effective blocking of VEGF-mediated tumor neovascularization.
Insights
Researchers developed potent monoclonal antibodies (mAbs) that neutralize vascular endothelial growth factor (VEGF), effectively inhibiting tumor-induced angiogenesis. These antibodies show promise for anticancer therapy by blocking new blood vessel formation in tumors.
Area of Science:
- Oncology
- Immunology
- Molecular Biology
Background:
- Vascular endothelial growth factor (VEGF) drives tumor angiogenesis, making it a key target for cancer therapies.
- Monoclonal antibodies (mAbs) offer targeted approaches to inhibit disease-mediating factors.
Purpose of the Study:
- To develop and characterize monoclonal antibodies (mAbs) targeting VEGF isoforms (VEGF121 and VEGF165).
- To evaluate the efficacy of these mAbs in neutralizing VEGF-driven endothelial cell proliferation and angiogenesis.
- To elucidate the binding epitope and mechanism of action of the most potent anti-VEGF mAb.
Main Methods:
- Generation of a panel of mAbs against VEGF121 and VEGF165.
- In vitro assays measuring mitogenic stimulation of human umbilical vein endothelial cells.
- In vivo mouse model assessing VEGF-induced angiogenesis in growth factor implants.
- Epitope mapping using competitive ELISA and Western blot analysis.
- Site-directed mutagenesis to identify critical binding residues.
Main Results:
- Three mAbs demonstrated potent neutralization of VEGF-induced mitogenic activity at low concentrations (<0.1 microg/ml).
- The most effective mAb (Kd = 8 pM) significantly inhibited VEGF-induced angiogenesis in mice, with complete blockage at 10 microg/day.
- This mAb did not inhibit angiogenesis induced by basic fibroblast growth factor.
- Epitope mapping indicated binding to a conformation-dependent epitope on the VEGF dimer, involving the beta5-beta6 loop crucial for KDR receptor interaction.
Conclusions:
- A highly potent mAb targeting a specific conformation of VEGF was developed.
- This mAb effectively blocks VEGF-mediated tumor neovascularization.
- The findings support the therapeutic potential of this anti-VEGF mAb for anticancer strategies targeting angiogenesis.