Masami Fukuoka

Characterization of a Novel Human Tumor Necrosis Factor‐α Mutant with Increased Cytotoxic Activity

Various novel recombinant human tumor necrosis factor‐α (TNF) mutants were prepared using protein engineering techniques, and their cytotoxic activity was compared with that of the intact form of TNF (intact TNF). Mutant 471 (a TNF mutant molecule with the deletion of 7 amino acids at the amino‐terminal and the substitution of Pro8Ser9Asp10 by ArgLysArg) had a 6‐fold higher cytotoxic activity against murine L929 cells. The mutant TNF had an increased ability to bind to TNF receptor on murine L929 fibroblasts cells. A cross‐linking study revealed that mutant 471 had an increased ability to form an active trimer. Mutant 471 also showed higher cytotoxic activity against human KYM myosarcoma cells and human MIA PaCa‐2 pancreatic carcinoma cells. The possible cachectin activity of the mutant was almost the same as that of intact TNF. These results suggest that mutant 471 might be a more promising candidate as an anticancer agent than intact TNF.

Hyperactive TNF-α derivatives with combinational mutations in the amino and carboxyl terminal regions

SummaryWe prepared various TNF-α derivatives by protein engineering techniques. Mutant 471, in which 7 N-terminal amino acids were deleted and Pro8Ser9Asp10 was replaced by ArgLysArg, had a 8-fold higher antitumor activity against mouse L929 cells than wild-type TNF-α. The additional substitution of Ala156 or Leu157 by more hydrophobic amino acids enhanced the activity of mutant 471. These results suggested that the combinational mutations in the N- and C-terminal regions of TNF-α are effective for the improvement of antitumor activity.