N-terminal Site-Specific PEGylation Enhances the Circulation Half-Life of Thymosin Alpha 1

Covalent attachment of polyethylene glycol (PEG) to peptides and proteins has been used to increase the clinical efficacy and circulation half-life of biopharmaceuticals. The purpose of this study was to enhance the half-life of an important immune-modulating agent, Thymosin alpha 1 (Tα1), using site-specific PEGylation. A novel N-terminal modified Tα1 (Mal-Tα1) was constituted by condensing the N-terminal amino group of Tα1 with 3-maleimidopropionic acid (Mal). Then, Mal-Tα1 was site-specifically conjugated with methoxy polyethylene glycol thiol (mPEG-SH) under mild conditions to form mPEG-Mal-Tα1. The conjugation efficiency was greater than 95%. mPEG-Mal-Tα1 was stable in the presence of 2-mercaptoethanol and exhibited significantly increased immunoactivity compared to Tα1 in mice. The terminal half-life (t1/2β) of mPEG-Mal-Tα1 was 2.5 times longer than that of mPEG-Cys-Tα1 (PEGylation product of mPEG-Mal conjugated with Cysteine-Tα1) in rats. These results demonstrate that site-specific PEGylation of N-terminal Mal-Tα1 is a promising strategy for enhancing the circulation t1/2β of Tα1 compared to Cysteine-Tα1.