Novel Oxygen-Dependent Degradable Immunotoxin Regulated by the Ubiquitin-Proteasome System Reduces Nonspecific Cytotoxicity.

Abstract

The use of bacterial toxins as antitumor agents has received considerable attention. Immunotoxins based on antigen recognition of single-chain antibodies have been widely explored for cancer therapy. Despite their impressive killing effect on tumor cells, immunotoxins still display unspecific toxicity with undesired side effects. High levels of hypoxia-inducible factor 1α (HIF-1α) are well-known indicators of hypoxia in cancer cells. In this study, different linkers were employed to fuse the immunotoxin DAB389-4D5 scFv (DS) with the oxygen-dependent degradation domain (ODDD) of HIF-1α, a domain selectively facilitating the accumulation of HIF-1α under hypoxia, to construct the oxygen-dependent degradable immunotoxin DS-ODDD (DSO). The engineered fusion protein DSO-2 containing a linker (G4S)3 possesses the best killing effect on cancer cells under hypoxia and displayed considerably reduced nonspecific toxicity to normal cells under normoxic conditions. Flow cytometry, immunofluorescence, and immunoblot analyses demonstrated that DSO-2 was degraded via the ubiquitin-proteasome pathway regulated by the oxygen-sensitive mechanism. Western blot analysis indicated that the degradation of DSO-2 significantly decreased the activation of apoptosis-related molecules in normal cells. The engineered immunotoxin with oxygen-sensing properties developed herein is a potential therapeutic agent for cancer treatment.