Structural insights into the clustering and activation of Tie2 receptor mediated by Tie2 agonistic antibody

Gyunghee Jo,Ho Min Kim,Do-Kyun Kim,Ah-Reum Han,Jung A Kim,Sangkyu Lee,Gou Young Koh,Ho Jeong Hong,Jeomil Bae,Seon Pyo Hong

doi:10.1038/s41467-021-26620-1

Abstract

Angiopoietin (Angpt)-Tie receptor 2 (Tie2) plays key roles in vascular development and homeostasis as well as pathological vascular remodeling. Therefore, Tie2-agonistic antibody and engineered Angpt1 variants have been developed as potential therapeutics for ischemic and inflammatory vascular diseases. However, their underlying mechanisms for Tie2 clustering and activation remain elusive and the poor manufacturability and stability of Angpt1 variants limit their clinical application. Here, we develop a human Tie2-agonistic antibody (hTAAB), which targets the membrane proximal fibronectin type III domain of Tie2 distinct from the Angpt-binding site. Our Tie2/hTAAB complex structures reveal that hTAAB tethers the preformed Tie2 homodimers into polygonal assemblies through specific binding to Tie2 Fn3 domain. Notably, the polygonal Tie2 clustering induced by hTAAB is critical for Tie2 activation and are resistant to antagonism by Angpt2. Our results provide insight into the molecular mechanism of Tie2 clustering and activation mediated by hTAAB, and the structure-based humanization of hTAAB creates a potential clinical application.

Highlights

Angiopoietin (Angpt)-Tie receptor 2 (Tie2) plays key roles in vascular development and homeostasis as well as pathological vascular remodeling
We immunized BALB/c mice with a recombinant protein encompassing Ig3–Fn3 of human Tie[2] twice weekly for 6 weeks and fused B lymphocytes isolated from the spleens with cultured myeloma cells (SP2/0) (Fig. 1a)
We designated the antibody produced by the 3H7 clone as a human Tie2-activating antibody

Summary

Introduction

Angiopoietin (Angpt)-Tie receptor 2 (Tie2) plays key roles in vascular development and homeostasis as well as pathological vascular remodeling. Tie2-agonistic antibody and engineered Angpt[1] variants have been developed as potential therapeutics for ischemic and inflammatory vascular diseases. Their underlying mechanisms for Tie[2] clustering and activation remain elusive and the poor manufacturability and stability of Angpt[1] variants limit their clinical application. We develop a human Tie2-agonistic antibody (hTAAB), which targets the membrane proximal fibronectin type III domain of Tie[2] distinct from the Angpt-binding site. Angpt[2] primarily forms low-order oligomers, mainly dimers, and acts context dependently as a weak Tie[2] agonist or antagonist, despite having ~60% amino acid sequence identity with Angpt[1]

Methods

Results

Conclusion