An anti-CD3 single-chain immunotoxin with a truncated diphtheria toxin avoids inhibition by pre-existing antibodies in human blood.
Diphtheria toxin (DT) is often used in the construction of immunotoxins. One potential problem using DT-based immunotoxins is the pre-existing anti-DT antibodies present in human blood due to vaccination. The present 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 antibodies at 1:100 or greater dilutions inhibited the immunotoxin toxicity. Experiments with radio-labeled UCHT1-CRM9 indicate that anti-DT antibodies partially block its binding to the cell surface as well as inhibit the translocation from the endosome to the cytosol. The inhibitory effect could be adsorbed using a full-length DT mutant or B-subfragment. 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 single-chain immunotoxin, sFv-DT390, was made with a truncated DT. The IC50 of sFv-DT390 was 4.8 x 10(-11) M, 1/16 the potency of the divalent UCHT1-CRM9. More importantly, sFv-DT390 toxicity was only slightly affected by the anti-DT antibodies in human sera.
D000907:Antibodies, Bacterial / Q000097:blood* / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology*; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004167:Diphtheria Toxin / Q000276:immunology / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000378:metabolism; D000939:Epitopes / Q000032:analysis; D006801:Humans; D000922:Immunotoxins / Q000276:immunology / Q000633:toxicity*; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D014176:Protein Biosynthesis / Q000187:drug effects*; D011994:Recombinant Proteins / Q000276:immunology / Q000633:toxicity; D013601:T-Lymphocytes / Q000276:immunology*; D014611:Vaccination
D000907:Antibodies, Bacterial; D017252:CD3 Complex; D004167:Diphtheria Toxin; D000939:Epitopes; D000922:Immunotoxins; D011994:Recombinant Proteins; D007930:Leucine