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Functionalization of Tumor Necrosis Factor- Using Phage Display Technique and PEGylation Improves Its Antitumor Therapeutic Window

¹ Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan; ² National Institute of Health Sciences, Tokyo, Japan; ³ Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan; ⁴ Laboratory of Molecular Biology, Division of Basic Science, National Cancer Institute, NIH, Bethesda, Maryland; ⁵ Department of Cell Therapeutics, Graduate School of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan; and ⁶ National Institute of Health Sciences, Osaka Branch Fundamental Research Laboratories for Development of Medicine, Osaka, Japan

Purpose: In this study, the optimization of antitumor therapy with tumor necrosis factor- (TNF-) was attempted.

Experimental Design: Using the phage display technique, we created a lysine-deficient mutant TNF- (mTNF-K90R). This mutant had higher affinities to both TNF receptors, despite reports that certain lysine residues play important roles in trimer formation and receptor binding.

Results: The mTNF-K90R showed an in vivo therapeutic window that was 13-fold higher than that of the wild-type TNF- (wTNF-). This was due to the synergistic effect of its 6-fold stronger in vitro bioactivity and its 2-fold longer plasma half-life derived from its surface negative potential. The reason why the mTNF-K90R showed a higher bioactivity was understood by a molecular modeling analysis of the complex between the wTNF- and TNF receptor-I. The mTNF-K90R, which was site-specifically mono-PEGylated at the NH₂ terminus (sp-PEG-mTNF-K90R), had a higher in vitro bioactivity and considerably longer plasma half-life than the wTNF-, whereas the randomly mono-PEGylated wTNF- had 6% of the bioactivity of the wTNF-. With regard to effectiveness and safety, the in vivo antitumor therapeutic window of the sp-PEG-mTNF-K90R was 60-fold wider than that of the wTNF-.

Conclusions: These results indicated that this functionalized TNF- may be useful not only as an antitumor agent but also as a selective enhancer of vascular permeability in tumors for improving antitumor chemotherapy.