title: Expression of an anti-CD3 single-chain immunotoxin with a truncated diphtheria toxin in a mutant CHO cell line.
abstract: ADP-ribosylating immunotoxins are generally expressed in Escherichia coli and then refolded in vitro. Because the efficiency of the in vitro refolding process decreases with the number of protein domains and internal disulfide bonds, these immunotoxins have been generally limited to single-chain monovalent structures. We now show that using the hamster cell line CHO K1 RE1.22c (J. M. Moehring and T. J. Moehring, 1979, Somat. Cell Genet. 5, 453-468) that has been mutated to ADP-ribosylation insensitivity, a level of 4 microg/ml of a truncated anti-T cell immunotoxin, DT390-scFvUCHT1, can be secreted into the medium. This immunotoxin is glycosylated at the two potential N-linked glycosylation sites in the toxin moiety: positions 16-18 in the A chain and residues 235-237 in the B chain. The glycosylated immunotoxin is relatively nontoxic (IC(50) 4.8 x 10(-10) M). Removal of the N-linked oligosaccharides by N-glycosidase F treatment or mutations at the two N-linked glycosylation sites results in a highly active immunotoxin with an IC(50) of 4 x 10(-12) M toward CD3(+) Jurkat cells. This is a 12-fold increase in toxicity over the same immunotoxin harvested from E. coli periplasm without refolding. A single Asn(235) Ala mutation that removed the B chain glycosylation was nearly as toxic as the double mutant. This suggests that B chain glycosylation is the major cause for the loss of toxicity.
authors.affiliation: Section on Biophysical Chemistry, National Institute of Mental Health, Bethesda, Maryland, 28092-4034, USA.
authors.forename: Y Y
authors.initials: YY
authors.lastname: Liu
authors.forename: I
authors.initials: I
authors.lastname: Gordienko
authors.forename: A
authors.initials: A
authors.lastname: Mathias
authors.forename: S
authors.initials: S
authors.lastname: Ma
authors.forename: J
authors.initials: J
authors.lastname: Thompson
authors.forename: J H
authors.initials: JH
authors.lastname: Woo
authors.forename: D M
authors.initials: DM
authors.lastname: Neville
mesh_terms: D000818:Animals; D015153:Blotting, Western; D017252:CD3 Complex / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D004167:Diphtheria Toxin / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000378:metabolism*; D000922:Immunotoxins / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D019169:Jurkat Cells; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D014176:Protein Biosynthesis; D011487:Protein Conformation
chemical_list: D017252:CD3 Complex; D004167:Diphtheria Toxin; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins