Development and partial characterization of fused human endostatin

Angiogenesis plays a central role in the progression of numerous pathological conditions, making its modulation an important therapeutic strategy. Endostatin, a naturally occurring endogenous inhibitor of angiogenesis, exhibits potent anti-angiogenic activity however, its broader clinical application has been constrained by reported limitations such as structural instability and rapid systemic clearance. Fusion protein engineering represents a rational strategy to potentially enhance the therapeutic applicability of bioactive proteins. In this study, we aimed to design, produce, and partially characterize the fused human endostatin (FHE) molecule through in vitro and in vivo studies.

FHE was engineered by fusing recombinant human endostatin (rhEs) with a fragment of human transferrin through a flexible peptide linker and expressed using the Pichia pastoris expression system. The purified protein was evaluated for its biological activity using in vitro proliferation assays with primary human retinal microvascular endothelial cells (HRMECs) and A549 human lung carcinoma cells. In vivo anti-angiogenic efficacy was further assessed using the chick chorioallantoic membrane (CAM) assay.

FHE demonstrated potent, dose- and time-dependent inhibition of HRMEC proliferation, indicating strong endothelial-specific anti-proliferative activity. Notably, no anti-proliferative effect was observed in A549 non-endothelial tumor cells, confirming its target specificity. In vivo assessment using the chick chorioallantoic membrane (CAM) assay confirmed significant, dose-dependent anti-angiogenic activity, comparable to native rhEs.

The FHE fusion protein retains the biological activity of endostatin while maintaining specificity for angiogenic processes, highlighting its potential as a candidate for further preclinical and therapeutic development as an anti-angiogenic agent.