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Novel muteins of human tumor necrosis factor with potent antitumor activity and less lethal toxicity in mice

Eight muteins of recombinant human tumor necrosis factor‐α (rhTNF; SSSRTP … 29RR … 155L), in which 29Arg was replaced by another amino acid, were prepared and their anti‐tumor effects in BALB/c mice bearing Meth A fibrosarcoma were evaluated. The therapeutic indices, which mark the extent of the therapeutically effective dose, of V29 (29Arg ± Val) and D29 (± Asp) were 3.5 and 3.2, respectively, whereas that of rhTNF was 1.4. Clearly, the therapeutically effective range of these muteins was extended along with a decrease in lethal toxicity. V29 did not produce hypotension in the rat system, but D29 did. In addition, V29 showed potent anti‐tumor activity (Tumor Volume Inhibition Rate = 81% on day 15 after implantation) in 3 consecutive injection schedules despite the decreases in toxicity compared with rhTNF. The relative receptor binding constant was determined using HEp‐2 cells (expressing mainly 55‐kDa‐TNF receptor; p55R) and HL60 cells (expressing mainly 75‐kDa‐TNF receptor; p75R), and revealed that the reduced toxicity of V29 in mice was due to the reduced binding to p55R (34% of rhTNF). On the other hand, the ratio of the constants HEp‐2/HL60 of V29 was 11 in comparison with the value of 1.0 for rhTNF, suggesting that this mutein binds preferentially to p55R. The biological activities in human cell lines (HEp‐2 and HL60 cells) correlated well with the binding activities to each receptor in vitro. Therefore, the much lower toxicity and the potent anti‐tumor activity of this mutein suggest that V29 merits further investigation in pre‐clinical and clinical trials.