Diphtheria toxin-based efficient target-switchable anticancer drug

Abstract

Recombinant immunotoxins (RITs) have been extensively utilized in the field of targeted cancer therapy. RITs are composed of toxins derived from natural bacterial strains and antibodies for target specific killing of cancer cells. The conjugated antibodies have specificities against the surface receptors of target cancer cells, facilitate the toxin internalization and consequently induce fatal effects to the target cell. However, low intracellular penetration and endosomal escape efficiency has limited the application of RITs. Diphtheria toxin is a candidate toxin which has potential of overcoming those limitation of RIT. The diphtheria toxins are produced by Corynebacterium diphtheriae and belong to AB toxin families which consist of catalytic (A) domain, binding (B) domain, and translocation(T) domain. Once binding domain of the diphtheria toxin bind to the specific target receptors, it facilitates the receptor-mediated endocytosis. Translocation domain delivers the catalytic domain from endosome to cytosol, which helps its catalytic domain to escape endosome. By substituting the binding domain with recombinant affinity molecules and using the diphtheria toxin, it is expected to overcome the low endosomal escape efficiency of the conventional RITs. In this study, we designed the diphtheria toxin-based target-switchable modular anticancer drugs by using protein ligation system, killing target cells specifically. To create module toxin and module affibody, the catalytic and translocation domain of diphtheria toxin (DTA), and cancer specific affibodies were prepared, genetically fused with protein ligation modules. By simple mix of module diphtheria toxin and module affibodies, we can easily introduce the target-specific diphtheria toxin (DTA-Afb) within an hour. Finally, we confirmed the targeted toxicity against the specific cancer in vitro as well as in vivo. DTA-Afb shows much higher toxicity than doxorubicin, which is a general anticancer drug. The diphtheria toxin-based RIT is promising method for targeted anticancer drug.