ANTI-CD3 SINGLE-CHAIN IMMUNOTOXIN WITH A TRUNCATED DIPHTHERIA-TOXIN AVOIDS INHIBITION BY PREEXISTING ANTIBODIES IN HUMAN BLOOD

Diphtheria toxin (DT) is often used in the construction of immunotoxin s. One potential problem using DT-based immunotoxins is the pre-existi ng anti-DT antibodies present in human blood due to vaccination. The p resent study examined the effect of human serum with pre-existing anti -DT antibodies on the toxicity of UCHT1-CRM9, an immunotoxin directed against CD3 molecules on T-lymphocytes. Sera with detectable anti DT a ntibodies at 1:100 or greater dilutions inhibited the immunotoxin toxi city. Experiments with radiolabeled UCHT1-CRM9 indicate that anti-DT a ntibodies partially block its binding to the cell surface as well as i nhibit the translocation from the endosome to the cytosol. The inhibit ory effect could be adsorbed using a full-length DT mutant or B-subfra gment. A C-terminal truncation mutant could not adsorb the inhibitory effect, suggesting that the last 150 amino acids contain the epitope(s ) recognized by the inhibitory antibodies. Therefore, an anti-CD3 sing le-chain immunotoxin, sFv-DT390, was made with a truncated DT. The IC5 0 of sFv-DT390 was 4.8 x 10(-11) M, 1/16 the potency of the divalent U CHT1-CRM9. More importantly, sFv-DT390 toxicity was only slightly affe cted by the anti-DT antibodies in human sera.