B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which the Fv portion of the B3 antibody in a single-chain form, which serves as the targeting moiety, is fused to PE38, a truncated form of Pseudomonas exotoxin A, which serves as the cytotoxic moiety. B3(Fv)-PE38 is specifically cytotoxic to many human cancer cell lines and is currently evaluated in a clinical trial. Monoclonal antibodies B3 (IgG1k) and B5 (IgMk) recognize related carbohydrate epitopes on human carcinoma cells. The Fv regions of these antibodies were previously cloned and expressed as the single-chain Fv-immunotoxins B3(Fv)-PE38 and B5(Fv)-PE38, respectively. The B3(Fv)-PE38 immunotoxin binds to antigen-positive cancer cells with a higher affinity than B5(Fv)-PE38 and is a more potent cytotoxic agent than B5(Fv)-PE38. However, it is less stable and rapidly aggregates upon incubation at 37 degrees C. The VL domains of the two Fvs are very similar, differing by only three residues, the fourth and seventh Fr1 residues and the fifth CDR1 residue. The VH domains of the two Fvs vary considerably. To investigate whether any of the different VL residues may influence the stability of the B3(Fv), we constructed a chimeric immunotoxin containing the B3VH and the B5VL. This chimera had an improved stability and a higher apparent antigen binding affinity and cytotoxic activity when compared with B3(Fv)-PE38. Site-specific mutagenesis was used to show that the VL M4L mutation has an important role in stabilizing B3(Fv), although residues VL Ser-7 and VL Ile-28 also play a role in the increased stability. When tested in an in vivo model system, the chimera containing the B3VH and the B5VL had an improved antitumor activity in a human xenograft mouse model. These studies indicate that the common use of degenerate ("family-specific") primers to clone Fv fragments may introduce destabilizing mutations.