Expression of an anti-CD3 single-chain immunotoxin with a truncated diphtheria toxin in a mutant CHO cell line

ADP-ribosylating immunotoxins are generally expressed in Escherichia coil a nd then refolded in vitro, Because the efficiency of the in vitro refolding process decreases with the number of protein domains and internal disulfid e bonds, these immunotoxins have been generally limited to single-chain mon ovalent 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, 453-46 8) that has been mutated to ADP-ribosylation insensitivity, a level of 4 mu g/ml of a truncated anti-T cell immunotoxin, DT390-scFvUCHT1, can be secre ted 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 (IC50 4.8 x 10(-10) M). Removal of the N-linked oligosa ccharides by N-glycosidase F treatment or mutations at the two N-linked gly cosylation sites results in a highly active immunotoxin with an IC50 of 4 x 10(-12) M toward CD3(+) Jurkat cells. This is a 12-fold increase in toxici ty over the same immunotoxin harvested from E. coli periplasm without refol ding. 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 glyco sylation is the major cause for the loss of toxicity. (C) 2000 Academic Pre ss.