Site-specific PEGylation of recombinant protein SAC-TRAIL and characterization of the effect on antitumor activity

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent with selective cytotoxicity across a broad spectrum of tumor cell lines. In previous studies, we engineered a recombinant protein drug, SAC-TRAIL, which significantly enhanced the antitumor activity of TRAIL without exhibiting toxicity to normal cells. However, its application in cancer therapy is restricted due to poor resistance to proteolytic degradation and a limited in vivo half-life.

To address these limitations, we designed a site-specific PEGylation method by conjugating methoxy-polyethylene glycol maleimide (mPEG-MAL) to the thiol group of specific cysteine residues on SAC-TRAIL. In this study, we optimized the PEGylation conditions for SAC-TRAIL, evaluated the in vitro activity and stability of mPEG-MAL-SAC-TRAIL, and conducted in vivo studies to assess its antitumor efficacy. It was shown that approximately 95% of SAC-TRAIL was PEGylated by mPEG-MAL within 30 min, exhibiting improved in vitro stability and antitumor activity. Furthermore, mPEG-MAL-SAC-TRAIL demonstrated enhanced anti-tumor activity and stability in an animal tumor model.

In summary, site-specific PEGylation at Cys-SH residues offers a promising strategy for extending the effective duration of SAC-TRAIL.