Vol. 290, Issue 1, 368-372, July 1999

Enhanced Antitumor Potency of Polyethylene Glycolylated Tumor Necrosis Factor-: A Novel Polymer-Conjugation Technique with a Reversible Amino-Protective Reagent¹

Shinichi Tsunoda, Tomoyoshi Ishikawa, Yoko Yamamoto, Haruhiko Kamada, Keiichi Koizumi, Junji Matsui, Yasuo Tsutsumi, Takashi Hirano and Tadanori Mayumi

Department of Biopharmaceutics, School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan (S.T., T.I., Y.Y., H.K., K.K., J.M., Y.T., T.M.); and Department of Molecular Biology, National Institute of Bioscience and Human Technology, Tsukuba, Ibaraki, Japan (T.H.)

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- (TNF-) with polyethylene glycol (PEG). The novel PEGylated TNF-, PEG-TNF-(+), which was pretreated with DMMAn before PEGylation, had 20% to 40% higher specific activity than PEG-TNF-() (not treated with DMMAn) in vitro. Moreover, PEG-TNF-(+) more potently caused tumor necrosis in Meth-A solid tumors in mice than did PEG-TNF-(). The middle fraction (M) of PEG-TNF-(+), which was of the optimal degree of modification among PEG-TNF-(+)s with different molecular weights, caused the highest degree of tumor hemorrhagic necrosis: 30-fold higher than native TNF- and 2-fold higher than the most potent MPEG-TNF-() 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- caused a dose-dependent body weight loss in mice, whereas no obvious side effects were observed in any PEG-TNF--treated mice. These results suggest that PEGylation using DMMAn is a useful for clinical cytokine delivery.