Biophysical and biological characterization of PEGylated recombinant human endostatin

Abstract

Endostatin, a 20 kDa C-terminal fragment digested from collagen XVIII, is an endogenous angiogenesis inhibitor, which inhibits the migration and proliferation of endothelial cells, and potently suppresses tumour growth without inducing toxicity and acquired drug resistance. [1][2][3] Our studies proved that endostatin inhibits angiogenesis, lymphangiogenesis, and tumour progression, and also demonstrated that the molecular mechanism of endostatin is dependent on the internalization through the cell surface receptor nucleolin and integrins. [4][5][6] Thus, endostatin appears to be an ideal candidate for tumour therapy; then, the anti-tumour activity of endostatin was assessed in preclinical and clinical trials. However, there are some obstacles for getting endostatin with high quality and activity, which affects the application of endostatin for extensive clinical evaluation. 7 ZBPendostatin, an N-terminal modified recombinant human endostatin, was approved by the State Food and Drug Administration of China in 2005 for the treatment of non-small cell lung cancer. 8 Our studies Abstract Recombinant human endostatin (MES), showing potent inhibition on angiogenesis and tumour growth, has great potential as a therapeutic agent for tumours. The aim of this study was to evaluate the biophysical and biological characterization of PEGylated recombinant human endostatin (M 2 ES). Recombinant human endostatin was mono-PEGylated by conjugation with methoxy polyethylene glycol aldehyde (mPEG-ALD), and the modification site was identified by digested peptide mapping and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The purity was assessed by SDS-PAGE, high-performance liquid chromatography (HPLC), and capillary zone electrophoresis. The physicochemical property was analyzed through fluorescence spectroscopy, and circular dichroism. The bioactivity and anti-tumour efficacy of M 2 ES were evaluated using an in vitro endothelial cell migration model and a null-mouse xenograft model of a prostatic cancer, respectively. M 2 ES molecules contain a single 20 kDa mPEG-ALD molecule conjugated at the N-terminal portion of MES. The purity of M 2 ES was greater than 98%. The physicochemical analysis demonstrated that PEGylation does not change the secondary and tertiary structure of MES. Notably, M 2 ES retards endothelial cell migration and tumour growth when compared to control group. These biophysical and biological characterization study data contribute to the initiation of the ongoing clinical study. K E Y W O R D S M 2 ES, PEGylation, recombinant human endostatin Capillary zone electrophoresis (CZE) was performed on a P/ ACE 5000 instrument (Beckman). Samples were injected using nitrogen pressure and peaks were detected on-column with a UV detector operating at 214 nm. Data were analyzed using the P/ACE Gold data analysis software. CZE electropherogram obtained on a fused-silica capillary at 15 kV, 20°C, 10 seconds pressure injection with detection at 214 nm. The electrophoretic buffer ...

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