title: Protein engineering of diphtheria-toxin-related interleukin-2 fusion toxins to increase cytotoxic potency for high-affinity IL-2-receptor-bearing target cells.
abstract: We have used site-directed insertion and point mutagenesis in an attempt to increase the cytotoxic potency and receptor-binding affinity of the diphtheria-toxin-related interleukin-2 (IL-2) fusion toxins. Previous studies have demonstrated that both the DAB486-IL-2 and DAB389-IL-2 forms of the fusion toxin consist of three functional domains: the N-terminal fragment-A-associated ADP-ribosyltransferase, the hydrophobic-membrane-associating domains, and the C-terminal receptor-binding domain of human IL-2. By insertion mutagenesis we have increased the apparent flexibility of the polypeptide chain between the membrane-associating domains and the receptor-binding domain of this fusion toxin. In comparison to DAB486-IL-2, the cytotoxic potency of the insertion mutants was increased by approximately 17-fold for high-affinity IL-2-receptor-bearing cell lines in vitro. Moreover, competitive displacement experiments using [125I]rIL-2 demonstrate that the increase in cytotoxic potency correlates with an increase in receptor-binding affinity for both the high and intermediate forms of the IL-2 receptor.
authors.affiliation: Evans Department of Clinical Research, University Hospital, Boston, MA 02118.
authors.forename: T
authors.initials: T
authors.lastname: Kiyokawa
authors.forename: D P
authors.initials: DP
authors.lastname: Williams
authors.forename: C E
authors.initials: CE
authors.lastname: Snider
authors.forename: T B
authors.initials: TB
authors.lastname: Strom
authors.forename: J R
authors.initials: JR
authors.lastname: Murphy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004167:Diphtheria Toxin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D015375:Receptors, Interleukin-2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D012984:Software
chemical_list: D004167:Diphtheria Toxin; D007376:Interleukin-2; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins