Abstract
Purpose :
Angiopoietin-2 (Ang2) along with VEGF signaling have been implicated in abnormal angiogenesis of ocular diseases, including wet age-related macular degeneration (AMD). Ang2 antagonizes Angiopoietin-1 (Ang1) stabilizing effects while promoting vascular destabilization. Antibody therapies targeting both Ang2 and VEGF lead to reduced vascular permeability with noninferior outcomes compared to anti-VEGF therapy alone. Here, we engineered affibodies, high-affinity proteins that are 10-fold smaller than antibodies, to test the hypothesis that inhibiting the binding of Ang2 to its receptor, Tie2, modulates their interaction and signaling.
Methods :
Using yeast surface display, we sorted through a library with 108 unique affibody-bearing S. cerevisiae via streptavidin magnetic and fluorescence activated cell sorting (FACS), and identified 20 candidate colonies. Colonies were sequenced, inserted into expressing vector pET-28b+, expressed in BL21(DE)3 E. coli, and purified using Cobalt resin/His-Tag affinity chromatography. The identity of the affibodies were confirmed on protein immunoblot. Binding and inhibitory kinetics of affibodies on the yeast surface and purified affibodies were assessed with FACS and ELISA, respectively. Structure binding were computationally predicted and validated.
Results :
DNA sequencing showed distinct sequences for all 20 colonies. Western blot confirmed the successful expression of affibody proteins in E. coli. Out of the 20 candidates, 2 colonies showed high affinity to Ang2 proteins on yeast-surface and ELISA. Colony 2 had high affinity (Kd: 8.2 nM) to Ang2 and partial affinity to Ang1 (Kd: 4.4 nM), with IC50 values equal to 65 and 87 nM in inhibiting Ang1/Tie2 and Ang2/Tie2 interactions, respectively. Colony 4 can bind only to Ang2 (Kd: 12.4 nM), with IC50 value equals to 124 nM. Top docking poses from each program showed points of contact with the active site of Ang2.
Conclusions :
We demonstrated the binding and inhibitory effects of affibodies that modulates Ang2/Tie2 interactions on yeast-surface, in ELISA, and in silico, which potentially eliminates the Ang2-antagonism on Ang1 signaling and enhances Ang1 stabilizing effects. These findings will be confirmed on cell cultures and animals. With the ability to prevent Ang2-induced permeability and angiogenesis of AMD, affibody is a potential small therapeutic agent for future treatment of AMD.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.