Dual-target anti-vascular endothelial growth factor/tumor necrosis factor-α nanobody precisely intervenes in hypoxia-induced retinal neovascularization formation and inflammation activation.

OBJECTIVE

This study aims to develop and evaluate a dual-target anti-VEGF/TNF-α nanobody (V5-3), exploring its dual role and mechanism in the treatment of Retinal neovascularization (RNV).

METHODS

The V5-3 was first constructed and its effects on hypoxia-induced ARPE-19 cells and HUVEC cells were evaluated using Western blot, EdU assays, wound healing assays, among other methods. The role of V5-3 in suppressing angiogenesis and inflammation was further explored in a co-culture model designed to mimic RNV pathology.

RESULTS

The study found that V5-3 significantly inhibited TNF-α-induced apoptosis in L929 cells and suppressed proliferation, migration, and inflammatory cytokine expression of HUVEC cells stimulated by hVEGF165, compared to the model group, with the most significant effect observed at high doses (all p < 0.01). Specifically, V5-3 effectively reduced angiogenesis through the VEGFR2 signaling pathway and significantly inhibited the expression of inflammatory cytokines in the NF-κB/TNF-α pathway by Western blot and RT-qPCR (all p < 0.01). Furthermore, V5-3 inhibited abnormal angiogenesis and inflammation in the RNV model under hypoxic conditions by reducing VEGFR2 phosphorylation and the activation of related downstream signaling molecules (all p < 0.01).

CONCLUSION

This study demonstrates that the V5-3, through dual-target inhibition of VEGF and TNF-α, shows more significant therapeutic effects in RNV compared to single-target antibodies.