The Characterization of the Ang-2 Arm of a Novel Anti-VEGF-A and Ang-2 Bispecific Protein (RO-101) in Comparison to Faricimab-svoa

Abstract

Purpose : To characterize the binding of Angiopoietin-2 (Ang-2) to a novel bispecific Surrobody, RO-101, that binds to both Ang-2 and VEGF-A, through investigating the half maximal inhibitory concentration (IC50) and the half maximal effective concentration (EC50) of RO-101 in comparison to Faricimab-svoa. These findings may have important implications for treatment of retinal disease including diabetic macular edema and exudative macular degeneration.

Methods : An Enzyme-Linked Immunoassay (ELISA) was utilized to determine the IC50 of RO-101 to inhibit the interaction of Ang-2 and its receptor Tie-2. In this inhibitory ELISA, the binding of Ang-2 to Tie-2 was detected with an anti-histidine tag. In the second assay, target-binding ELISA was utilized to determine the EC50 of the bispecific RO-101 and Faricimab-svoa to human Ang-2 analytes, which was detected by HRP-conjugated donkey anti-human IgG Fc antibody. SgG1 or IgG1 were tested in duplicates starting at 100nM; 1:3 serial dilutions. After adding the TMB substrate and stop solution, both ELISA results were visualized at OD450 nM by Victor 3 (PerkinElmer 1420 Multilabel Counter).

Results : The IC50 of RO-101 to inhibit Ang-2 binding of Tie-2 was found to be 0.7868 nM, the EC50 of RO-101 binding to Ang-2 was found to be 0.118 nM. On the other hand, the IC50 of Faricimab-svoa was found to be 13.55 nM and the EC50 of Faricimab-svoa was found to be 0.7868 nM.

Conclusions : RO-101 demonstrates 17-fold greater inhibition of Ang-2 binding to the tie-2 receptor than Faricimab-svoa and reaches its half maximal effective concentration at 53 times lower concentration than Faricimab. These data suggest that RO-101 has several desirable characteristics as compared to Faricimab-svoa and warrants further development as a therapeutic.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Figure 1: EC50 of RO-101 to Ang-2 (red square) vs. Faricimab (blue triangle).

 

Figure 2: IC50 of RO-101 (blue square) vs. Faricimab (green square): inhibition of Ang-2 and Tie-2 binding.