Inhibition of VEGFA induced HUVEC migration via RO-104, a novel trispecific protein

Abstract

Purpose : Recognizing that neovascular age-related macular degeneration (nAMD) is a multifactorial disease process, we have developed RO-104, a novel, first-in-class trispecific against VEGF-A, VEGF-C and Ang-2 to address these clinically validated targets. The purpose of this study is to evaluate the in vitro efficacy of RO-104 against VEGF-A via human umbilical vein endothelial cells (HUVECs) wound closure.

Methods : VEGF-A inhibitor potency was determined via HUVEC scratch wound cell migration assay and analyzed by Incucyte Cell Migration Scratch Wound Analysis. Huvec cells (Lonza) were seeded at 50,000/well to achieve near 100% confluence in EBM-2 and incubated overnight. The next day, the Incucyte WoundMaker was used to create uniform scratch wounds in all wells, followed by three PBS washes. HUVEC cells were then treated with 4 nM VEGF-A (Peprotech) in the presence and absence of RO-104. The response and diminution of HUVEC migration by RO-104 was monitored using the IncuCyte live-cell analysis system. Percent confluence and relative wound density occupied by VEGF-A stimulated HUVEC migration were calculated.

Results : Wound measurements at 8, 10, and 12 hours post-treatment were analyzed. At these timepoints, RO-104 and aflibercept both achieved similar relative inhibitory maximums, allowing for comparative analysis of wound density and wound confluence. Analysis of wound density at these timepoints revealed an IC50 of 5.65nM for RO-104, comparing favorably to aflibercept’s IC50 of 14.97nM. Wound confluence inhibition via RO-104 yielded an IC50 of 1.44nM, demonstrating comparable results to aflibercept’s IC50 of 1.37nM.

Conclusions : This data shows that the trispecific RO-104 is highly effective at inhibiting VEGF-A. Stimulated HUVEC wound closure by wound density was superior to that of aflibercept while wound confluence measured noninferior to aflibercept.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Wound density (left panel) and wound confluence (right panel) of VEGF-A induced in vitro wound closure of VEGF treated HUVEC cells in presence of either RO-104 or aflibercept.

 

Wound closure of HUVEC cells treated with 4 nM VEGF and 30 nM aflibercept versus RO-104.