A novel Tie2xVEGF bispecific antibody fusion demonstrates enhanced therapeutic efficacy through direct Tie2 activation and VEGF neutralization.

backgroundActivation of endothelial Tie2 signaling has emerged as a potential therapeutic strategy for ameliorating vascular abnormalities and hyperpermeability in ocular diseases. Early therapeutic strategies have focused on inhibiting angiopoietin-2 (Ang2), an antagonistic ligand of Tie2, thereby indirectly promoting Tie2 activation. However, accumulating evidence indicates that indirect Tie2 activation via Ang2 blockade is insufficient for enhanced therapeutic efficacy, underscoring the need for Tie2 agonists. In addition, since it is still necessary to inhibit neovascularization induced by vascular endothelial growth factor (VEGF), appropriate therapeutic efficacy could be achieved by direct Tie2 agonism and VEGF neutralization.

methodsAn affinity-matured Tie2-activating antibody, MT-101, was identified by phage display panning using a complementarity-determining region-targeted mutagenesis library. Tie2xVEGF bispecific antibodies were generated with MT-101 fused to five different anti-VEGF modules, and their functionality in Tie2 and VEGF signaling was compared using endothelial cells. The therapeutic efficacy of the bispecific antibody fusion MT-103, comprising MT-101 and VEGFR domains, was evaluated in mouse oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (LI-CNV) models compared with anti-VEGF agent Aflibercept or Ang2xVEGF bispecific antibody.

resultsMT-101 activated the Tie2 signaling pathway, including AKT-eNOS and ERK cascades, and exhibited efficacy comparable to that of Aflibercept in OIR and LI-CNV models. By directly comparing five different Tie2xVEGF bispecifics, we selected the most potent construct, MT-103, generated by fusing VEGFR1/2 domains to MT-101. MT-103 demonstrated approximately four- and five-fold greater potency than the Ang2xVEGF bispecific antibody in inhibiting VEGF signaling and reducing permeability, respectively, in retinal endothelial cells. MT-103 further demonstrated improved efficacy, reducing neovascularization by 14% compared with Aflibercept in the LI-CNV model and suppressing vascular leakage by 20% compared with Ang2xVEGF bispecific antibody in the OIR model. Moreover, MT-103 elicited robust Tie2 activation and vessel stabilization by enhancing pericyte coverage relative to Ang2xVEGF bispecific antibody.

conclusionsThese findings demonstrate that a therapeutic strategy combining direct Tie2 activation and VEGF blockade may provide improved therapeutic potential compared to neutralizing VEGF alone or Ang2 and VEGF, representing a promising therapeutic strategy that warrants further validation for the treatment of various ocular diseases.