Enhanced antitumor potency of polyethylene glycolylated tumor necrosis factor-α: A novel polymer-conjugation technique with a reversible amino- protective reagent

We attempted to develop a novel method for the chemical modification of cytokines with synthetic polymers to increase in vivo therapeutic efficacy. A pH-reversible amino-protective reagent, dimethylmaleic anhydride (DMMAn), was used for polymer conjugation of tumor necrosis factor-α (TNF-α) with polyethylene glycol (PEG). The novel PEGylated TNF-α, PEG-TNF-α(+), which was pretreated with DMMAn before PEGylation, had 20% to 40% higher specific activity than PEG-TNF-α(-) (not treated with DMMAn) in vitro. Moreover, PEG- TNF-α(+) more potently caused tumor necrosis in Meth-A solid tumors in mice than did PEG-TNF-α(-). The middle fraction (M) of PEGTNF-α(+), which was of the optimal degree of modification among PEG-TNF-α(+)s with different molecular weights, caused the highest degree of tumor hemorrhagic necrosis: 30-fold higher than native TNF-α and 2-fold higher than the most potent MPEG-TNF-α(-) that also had nearly the same molecular weight. Significantly, improvements in antitumor activity in vivo were more marked than were changes in specific activity. Furthermore, native TNF-α caused a dose-dependent body weight loss in mice, whereas no obvious side effects were observed in any PEG-TNF-α-treated mice. These results suggest that PEGylation using DMMAn is a useful for clinical cytokine delivery.