title: Mutants of lymphotoxin-α with augmented cytotoxic activity via TNFR1 for use in cancer therapy.
abstract: The cytokine lymphotoxin-α (LTα) is a promising candidate for use in cancer therapy. However, the instability of LTαin vivo and the insufficient levels of tumor necrosis factor receptor 1 (TNFR1)-mediated bioactivity of LTα limit its therapeutic potential. Here, we created LTα mutants with increased TNFR1-mediated bioactivity by using a phage display technique. We constructed a phage library displaying lysine-deficient structural variants of LTα with randomized amino acid residues. After affinity panning, we screened three clones of lysine-deficient LTα mutant, and identified a LTα mutant with TNFR1-mediated bioactivity that was 32 times that of the wild-type LTα (wtLTα). When compared with wtLTα, the selected clone showed augmented affinity to TNFR1 due to slow dissociation rather than rapid association. In contrast, the mutant showed only 4 times the TNFR2-mediated activity of wtLTα. In addition, the LTα mutant strongly and rapidly activated caspases that induce TNFR1-mediated cell death, whereas the mutant and wtLTα activated nuclear factor-kappa B to a similar extent. Our data suggest that the kinetics of LTα binding to TNFR1 play an important role in signal transduction patterns, and a TNFR1-selective LTα mutant with augmented bioactivity would be a superior candidate for cancer therapy.
authors.affiliation: Laboratory of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Morishige
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yoshioka
authors.forename: Shogo
authors.initials: S
authors.lastname: Narimatsu
authors.forename: Shinji
authors.initials: S
authors.lastname: Ikemizu
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Tsunoda
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Tsutsumi
authors.forename: Yohei
authors.initials: Y
authors.lastname: Mukai
authors.forename: Naoki
authors.initials: N
authors.lastname: Okada
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Nakagawa
mesh_terms: D000595:Amino Acid Sequence; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007526:Isoelectric Point; D007700:Kinetics; D008233:Lymphotoxin-alpha / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D016328:NF-kappa B / Q000378:metabolism; D009369:Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology*; D011485:Protein Binding / Q000187:drug effects; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008233:Lymphotoxin-alpha; D050505:Mutant Proteins; D016328:NF-kappa B; D047888:Receptors, Tumor Necrosis Factor, Type I; D020169:Caspases