A RECOMBINANT IMMUNOTOXIN CONTAINING A DISULFIDE-STABILIZED FV FRAGMENT

B3(dsFv)-PE38KDEL is a recombinant immunotoxin composed of the Fv regi on of monoclonal antibody B3 connected to a truncated form of Pseudomo nas exotoxin (PE38KDEL), in which the unstable Fv heterodimer (compose d of heavy- and light-chain variable regions) is held together and sta bilized by a disulfide bond [termed disulfide-stabilized Fv (dsFv)]. A computer modeled structure of the B3(Fv), made by mutating and energy minimizing the amino acid sequence and structure of McPC603, enabled us to identify positions in conserved framework regions that ''hypothe tically'' could be used for disulfide stabilization without changing t he structure or affecting antigen binding. This prediction was evaluat ed experimentally by constructing a disulfide-linked two-chain dsFv-im munotoxin that was produced in Escherichia coli. The activity and spec ificity of this immunotoxin was indistinguishable from its single-chai n Fv (scFv) counterpart, indicating that, as in B3(scFv), the structur e of the binding region is retained in B3(dsFv). Because we introduced the stabilizing disulfide bond in between two framework residues in a position that is conserved in most Fv molecules, this method of linka ge between the heavy- and light-chain variable regions should be gener ally applicable to construct immunotoxins and dsFv molecules using oth er antibodies. Furthermore, the finding that B3(dsFv) was much more st able at 37-degrees-C in human plasma than B3(scFv) indicates that dsFv s are possibly more versatile for therapeutic application than scFvs.