The clinical use of the potent antitumor activity of TNF-alphahas been limited by the proinflammatory side effects including fever, dose-limiting hypotension, hepatotoxicity, intravascular thrombosis, and hemorrhage. Designing clinically applicable TNF-alphamutants with low systemic toxicity has been an intense pharmacological interest. Human TNF-alpha, which binds to the murine TNF-R55 but not to the murine TNF-R75, exhibits retained antitumor activity and reduced systemic toxicity in mice compared with murine TNF-alpha, which binds to both murine TNF receptors. Based on these results, many TNF-alphamutants that selectively bind to TNF-R55 have been designed. These mutants displayed cytotoxic activities on tumor cell lines in vitro, and exhibited lower systemic toxicity in vivo.