LAUSR

&NA; Recombinant immunotoxins are chimeric proteins composed of a targeting peptide that binds to a specific tumor antigen and a toxin protein killing target cells. Recombinant immunotoxin exhibits potent cancer inhibiting effects both in vivo and in vitro. However, the non‐specific toxicity causes severe syndromes limiting their clinical application. To reduce toxicity caused by recombinant immunotoxins in general, we divided an immunotoxin into two nontoxic segments that may restore toxic bioactivity on tumor cell surface based on the intein mediated trans‐splicing reaction. Both split and reconstituted immunotoxins were tested for their biological activities. We found that the reconstituted immunotoxin retained antigen specificity and affinity toward cancer cells overexpressing HER2/neu. After being internalized into HER2/neu positive cells, the reconstituted immunotoxin showed comparable cytotoxicity as the original immunotoxin, while the split immunotoxin fragments showed no toxic activity to cells with or without HER2/neu expression. This approach can potentially be used under clinical settings to reduce non‐specific toxicity by administering patients with inactive immunotoxin fragments. Cytotoxic effect only occurs at tumor sites where the inactive fragments bind, trans‐splice and become active toxin. HighlightsAn immunotoxin was divided to designed two‐segments to eliminat toxic activity. The cytotoxicity can be recovered when they meet on cancer cell surface.It is a facile method to prepare the split immunotoxin and to recover its cytotoxicity through intein mediated trans‐splicing reaction.This new approach can be applied to reduce non‐specific toxicity of immunotoxic molecules and to expand their clinical usage.