Elsevier

Experimental Eye Research

Volume 85, Issue 4, October 2007, Pages 425-430
Experimental Eye Research

Ranibizumab inhibits multiple forms of biologically active vascular endothelial growth factor in vitro and in vivo

https://doi.org/10.1016/j.exer.2007.05.008Get rights and content

Abstract

Neovascular age-related macular degeneration (AMD) is the leading cause of blindness in older adults in the Western world. Ranibizumab (Lucentis®), a humanized antibody fragment directed against vascular endothelial growth factor (VEGF-A), was recently approved by the US Food and Drug Administration (FDA) for the treatment of neovascular AMD. The objective of this study was to characterize the binding affinity and pharmacological activity of ranibizumab for 3 biologically active forms of VEGF-A: VEGF165, VEGF121, and VEGF110. The apparent equilibrium binding affinity of ranibizumab for VEGF-A molecules was determined by Biacore® analysis; the biological activity of VEGF-A was demonstrated in a human umbilical vein endothelial cell (HUVEC) proliferation–inhibition assay. Inhibition of VEGF-A-induced vascular permeability by ranibizumab was assessed in vivo using hairless guinea pigs and a modified Miles assay. Ranibizumab was capable of binding to recombinant human VEGF165, VEGF121, and VEGF110 (KD  192 pM), inhibiting VEGF-A-induced HUVEC proliferation in a concentration-dependent manner. Ranibizumab also exerted potent dose-dependent inhibition (IC50 of 0.4–1.2 nM) of the vascular permeability-enhancing activity of VEGF165, VEGF121, and VEGF110 in the Miles assay. In conclusion, these results show that ranibizumab is capable of binding to and specifically inhibiting the activities of 3 biologically active forms of VEGF-A. As VEGF-A plays a pivotal role in the pathogenesis of neovascular AMD, ranibizumab activity, as demonstrated in this study, supports its clinical utility in the treatment of this disease.

Introduction

Neovascular (wet) age-related macular degeneration (AMD) is the most common cause of severe, irreversible visual loss in older adults in the Western world. The pathology of neovascular AMD involves choroidal neovascularization (CNV) and increased vascular permeability, ultimately resulting in permanent visual loss (Ambati et al., 2003).

Vascular endothelial growth factor (VEGF-A, also referred as VEGF) (Ferrara, 2004) plays a pivotal role in the pathogenesis of AMD. Evidence implicating VEGF in this role includes the detection of VEGF-A in CNV membranes excised from patients with neovascular AMD (Kvanta et al., 1996, Lopez et al., 1996) and increased VEGF-A expression in the macular retinal pigment epithelium cells in early-stage AMD (Kliffen et al., 1997).

In humans, at least 9 VEGF-A isoforms (ranging from 121 to 206 amino acids) are generated via alternative exon splicing (Lei et al., 1998, Robinson and Stringer, 2001, Takahashi and Shibuya, 2005). The 4 major isoforms are VEGF121, VEGF165, VEGF189, and VEGF206 (Robinson and Stringer, 2001). All of these isoforms can, in turn, be cleaved by plasmin to generate the smaller, biologically active form, VEGF110 (Robinson and Stringer, 2001). These various forms of VEGF-A possess varying degrees of angiogenic and permeability-enhancing activity (Ferrara, 2004, Robinson and Stringer, 2001). VEGF189 and VEGF206 bind to the extracellular matrix (ECM) in vivo via a heparin-binding domain, whereas VEGF110 and VEGF121 do not bind to the ECM and are readily dissociated from secreting cells (Houck et al., 1992). VEGF165 has intermediate properties: 50–70% remains bound to the cell surface and the ECM (Houck et al., 1992). The VEGF-A molecules of interest in the current study were VEGF165, VEGF121, and VEGF110 because these are the 3 major soluble forms of biologically active VEGF found in vivo.

Ranibizumab (Lucentis®, ranibizumab injection; Genentech, Inc., South San Francisco, CA) is a recombinant humanized anti-VEGF-A monoclonal Fab recently approved by the FDA for the treatment of neovascular AMD. Ranibizumab was designed to bind to all biologically active VEGF-A forms by targeting amino acid residues in the “80s loop” required for receptor binding (Chen et al., 1999). This report summarizes the results of studies characterizing ranibizumab's binding affinity for VEGF165, VEGF121, and VEGF110 as well as its ability to inhibit the biological activity of these 3 forms of VEGF.

Section snippets

Biacore® binding analysis

The apparent equilibrium binding affinities of ranibizumab for three forms of VEGF were determined by surface plasmon resonance (SPR) technology on a Biacore 3000 instrument (Biacore, Inc., Piscataway, NJ). In these experiments, purified Escherichia coli-expressed rhVEGF165 (Genentech, Inc.), rhVEGF121 (R&D Systems, Inc., Minneapolis, MN), and rhVEGF110 (Genentech, Inc.) were used. Each of the 3 forms of VEGF-A (10 μg/mL) was coupled to 1 of the 4 different flow cells on a CM5 sensor chip using

Ranibizumab binding to VEGF-A

Biacore analyses shown in Fig. 1 demonstrate that ranibizumab binds to rhVEGF165, rhVEGF121, and rhVEGF110 in a dose-dependent manner. The sensorgram profiles in Fig. 1 also illustrate the good fit of the data to the 1:1 binding model used (goodness of fit data not shown). Table 1 summarizes the apparent association rate constant (ka) of ranibizumab for each VEGF-A form. The apparent ka values for ranibizumab binding were essentially the same for all VEGF-A forms tested (Table 1) and similar to

Discussion

We have shown that ranibizumab binds to rhVEGF165, rhVEGF121, and rhVEGF110 with high affinity and is a potent inhibitor of the biological activities of these VEGF-A isoforms in vitro and in vivo.

Our studies indicate that ranibizumab dissociates from rhVEGF165, rhVEGF121, and rhVEGF110 at remarkably slow rates, confirming previously published data (Chen et al., 1999). Ranibizumab forms inactive drug–ligand complexes with VEGF-A. Based on the relative affinities of VEGF for ranibizumab versus

Acknowledgments

At the time of this study, all of the authors were employees of Genentech, Inc. We thank Lisa Damico and Bert Gunter for their help in preparing this manuscript.

References (18)

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1

Formerly at Genentech; no current affiliation.

2

Present address: F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland.

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