A new recombinant immunotoxin hscFv-ETA’ demonstrates specific cytotoxicity against chronic myeloid leukemia cells in vitro

Abstract

Antibodies against cell surface antigens of tumor have attracted increasing attention in immunotherapy for tumor diagnosis and treatment. Recently, we constructed a new recombinant immunotoxin for possible clinical application in patients with chronic myeloid leukemia (CML). A functional humanized single chain variable fragment (hscFv) against CML patient cells was previously obtained from an anti-CML cell hybridoma derived monoclonal antibody. By insertion into the bacterial vector pWW20, the hscFv was fused with a deletion mutant of Pseudomonas exotoxin A (ETA’). Then the fusion fragment was inserted into the bacterial vector pET32a(+). After isopropyl β-d-thiogalactoside (IPTG) induction, the 6× His tagged hscFv-ETA’ protein was periplasmically expressed and purified by Ni2+-NTA column. The characteristics of the recombinant protein were assessed by cell membrane-ELISA, flow cytometry, and toxicity assays in CML cell lines and CML patient cells. The recombinant immunotoxin showed significant toxicity toward the CML cell lines K562 and KU812 as tested by MTT and apoptosis assay. Approximately 37% of leukemia cells of CML patients were driven into apoptosis by hscFv-ETA’ as measured by flow cytometric analysis. In conclusion, the hscFv-ETA’ is efficacious against CML in vitro, providing the basis for a novel therapeutic strategy for the treatment of CML patients.