title: Novel muteins of human tumor necrosis factor with potent antitumor activity and less lethal toxicity in mice.
abstract: Eight muteins of recombinant human tumor necrosis factor-alpha (rhTNF; 1SSSRTP...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.
authors.affiliation: Medicinal Research Laboratory, Ishihara Sangyo Kaisha, Ltd., Shiga, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Kuroda
authors.forename: K
authors.initials: K
authors.lastname: Miyata
authors.forename: H
authors.initials: H
authors.lastname: Shikama
authors.forename: T
authors.initials: T
authors.lastname: Kawagoe
authors.forename: K
authors.initials: K
authors.lastname: Nishimura
authors.forename: K
authors.initials: K
authors.lastname: Takeda
authors.forename: N
authors.initials: N
authors.lastname: Sakae
authors.forename: M
authors.initials: M
authors.lastname: Kato
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000009:adverse effects / Q000737:chemistry*; D001483:Base Sequence; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000009:adverse effects / Q000737:chemistry; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*
chemical_list: D000970:Antineoplastic Agents; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha