An Aflibercept-derived Mini-VEGF-Trap as a Potential Next-Generation VEGF Inhibitor

Abstract

Purpose : Aflibercept (Eylea®) is a VEGF inhibitor indicated for the treatment of patients with Wet AMD, MEfRVO, DME, and DR. Aflibercept is a fusion of the second Ig domain of VEGFR1 with the third Ig domain of VEGFR2 and that fused with to the Fc segment of human IgG, creating a chimeric protein with a very high VEGF binding affinity (K_d≈0.2-1pM). The goal of this study was to characterize a VEGF Mini-Trap generated by removing the Fc portion of the aflibercept, resulting in a smaller protein that could retain the VEGF binding and inhibitory properties of aflibercept and potentially provide higher molar dosing intravitreally. We hypothesize that this higher dosing could result in longer-lived activity.

Methods : We generated REGN7483F (VEGF Mini-Trap) using the IdeS protease derived from Streptococcus pyogenes. This enzyme cleaves just below the hinge region of the Fc. Aflibercept was incubated with agarose-immobilized IdeS for 60 mins, then the supernatant was collected and purified by protein A affinity chromatography. REGN7483F and aflibercept were characterized for binding kinetics by BiaCore, potency in a VEGFR1 bioassay, homogeneity by SDS-PAGE, thermal stability, solubility, viscosity, and complex formation with VEGF₁₆₅ by SEC-MALS and negative-stain electron microscopy.

Results : REGN7483F and aflibercept showed similar binding affinity for VEGF₁₆₅ (K_D 1.27 pM and 1.32 pM) and stoichiometric inhibition of VEGF₁₁₀ in the VEGFR1 bioassay. SDS-PAGE showed molecular weights expected for aflibercept (~65 kD) and REGN7483F (~ 32 kD). REGN7483F exhibited a similar degradation pathway as aflibercept under thermal stress analysis. In the base formulation buffer, REGN7483F showed low viscosity (< 14 cP at 120 mg/mL) and high solubility (> 160 mg/mL). SEC-MALS of aflibercept and REGN7483F in complex with VEGF₁₆₅ showed both favored a discrete 1:1 cis-complex containing one VEGF₁₆₅ dimer held between the two arms of REGN7483F or aflibercept.

Conclusions : REGN7483F has comparable binding affinity, bioassay blocking potency, stability and degradation pathway as aflibercept. Each exhibited a discrete 1:1 complex with VEGF_165. At a concentration of 110 mg/mL (viscosity ~12 centipoise), REGN7483F provides 5.5X molar equivalents of VEGF binding sites as the commercial aflibercept formulation. Therefore, REGN7483F shows favorable properties as a potential next-generation VEGF inhibitor.

This is a 2021 ARVO Annual Meeting abstract.