Analysis of Binding Affinity and Inhibitory Capacity of NT-503 Produced VEGF Antagonist Compared to Aflibercept

Abstract

Purpose: 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.

Methods: 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).

Results: 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 (K_D) 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).

Conclusions: 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 (K_D), and inhibitory abilities (HUVEC- ED₅₀) similar or better to that of aflibercept.