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

We attempted to develop a novel method for the chemical modification of cyt okines with synthetic polymers to increase in vivo therapeutic efficacy. A pH-reversible amino-protective reagent, dimethylmaleic anhydride (DMMAn), w as used for polymer conjugation of tumor necrosis factor-alpha TNF-alpha wi th polyethylene glycol (PEG). The novel PEGylated TNF-alpha, PEG-TNF-alpha( +), which was pretreated with DMMAn before PEGylation, had 20% to 40% highe r specific activity than PEG-TNF-alpha(-) (not treated with DMMAn) in vitro . Moreover, PEG-TNF-alpha(+) more potently caused tumor necrosis in Meth-A solid tumors in mice than did PEG-TNF-alpha(-). The middle fraction (M) of PEG-TNF-alpha(+), which was of the optimal degree of modification among PEG -TNF-alpha(+)s with different molecular weights, caused the highest degree of tumor hemorrhagic necrosis: 30-fold higher than native TNF-alpha and 2-f old higher than the most potent MPEG-TNF-alpha(-) 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-alpha caused a dose-dependent body weight loss in mice, whereas no obvious side effects were observed in any PEG-TNF-alpha-treated mice. T hese results suggest that PEGylation using DMMAn is a useful for clinical c ytokine delivery.