Integrin-independent activation of Tie2 using the de novo designed protein H8mb

Abstract

The Angiopoietin–Tie2 signaling axis is a central regulator of vascular stability, yetefforts to harness it therapeutically have been limited by the poor developability and receptor promiscuity of Angiopoietin-1 (Ang1), which binds both Tie2 and α5β1 integrin. Here we report the de novo design of a compact Tie2 binding protein that overcomes these constraints. We use RFdiffusion to design a stable, high-affinity Tie2 binder that functions as a selective antagonist when monomeric, but as a potent agonist when octavalent (H8mb) that activates the Tie2/pAKT pathway without binding to α5β1 integrin. H8mb induces Tie2 activation with more rapid internalization kinetics than Ang1, suggesting that while integrin engagement is dispensable for Tie2 activation, it may modulate signaling persistence by functioning as a co-receptor for Ang1 and delaying receptor internalization. In a mouse model of Acute Respiratory Distress Syndrome (ARDS), H8mb markedly improved survival. These findings show that designed receptor binding enables dissection of co-receptor control of signaling dynamics, potentially enabling the development of more selective and effective therapeutics.