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Michael Rivera, Arne Nystuen, Konrad Kauper, Alline M. A. Lelis, Lisa D. Orecchio, Sue Elliott, Paul Stabila; Analysis of Binding Affinity and Inhibitory Capacity of NT-503 Produced VEGF Antagonist Compared to Aflibercept. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):228.
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© ARVO (1962-2015); The Authors (2016-present)
NT-503 is a novel intraocular implant utilizing Neurotech’s encapsulated cell technology (ECT) platform to continuously deliver a VEGF antagonist (910-VEGFR) for a duration greater than two years in treatment of patients with neovascular AMD.<br /> <br /> We tested the hypotheses that Neurotech’s 910-VEGFR delivered from NT-503 implants, in both native and purified form, would demonstrate strong binding affinities to VEGF, and that this binding would inhibit VEGF165-mediated Human Umbilical Vein Endothelial Cell (HUVEC) proliferation; results are compared to aflibercept as a positive control.
In a VEGF solution binding assay (Study 1), rhVEGF165 was pre-incubated with serial dilutions of purified 910-VEGFR, cell conditioned media (CM), NT-503 device CM, or aflibercept. Following incubation, unbound VEGF165 was quantified by VEGF ELISA to determine VEGF165 binding.<br /> <br /> SPR-Biacore analysis (Study 2) was performed with 910-VEGFR and aflibercept against rhVEGF165, rhVEGF121, rhVEGF-D, and rabbit VEGF-A. Kinetic binding parameters were measured to determine binding affinity.<br /> <br /> A HUVEC proliferation assay was used to determine VEGF inhibition bioactivity (Study 3). In this study VEGF165 was pre-incubated with serial dilutions of purified 910-VEGFR, NT-503 device CM, or aflibercept followed by incubation with HUVECs. HUVEC proliferation was measured as a function of VEGFR concentrations using a fluorescence-based assay and a specific activity calculated (ED50).
In Study 1, purified 910-VEGFR and NT-503 device CM bound VEGF165 with an IC50 between 20-30 pM, comparable potency to that of aflibercept. In Study 2, 910-VEGFR demonstrated picomolar binding affinities (KD) to the VEGF isoforms tested, comparable or better to aflibercept. In Study 3, purified 910-VEGFR, and NT-503 device CM blocked VEGF165 induced proliferation of HUVEC cells with an ED50 between 3-7 ng/mL. This inhibition is comparable to aflibercept (ED50 of 4 ng/mL).
Our results demonstrate that purified 910-VEGFR, and native 910-VEGFR collected in media incubated with NT-503 implants, bind to rhVEGF165, rhVEGF121, rabbit VEGF-A, and inhibit VEGF165 mediated HUVEC proliferation. Clinically, NT-503 implants offer intravitreal delivery of a VEGF antagonist with potency (IC50), affinity (KD), and inhibitory abilities (HUVEC- ED50) similar or better to that of aflibercept.
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