Process Biochemistry, Vol.80, 26-34, 2019
High level expression and characterization of the recombinant immunotoxin DAB389-4D5 scFv targeting HER2/neu-positive ovarian carcinoma cells
Third generation recombinant immunotoxins are the hotspot in targeted antitumor therapy. Since the late 1970s, diphtheria toxin has been widely studied, and selected preferentially to prepare immunotoxins. In the current study, we fused the N-terminal 389 amino acids of diphtheria toxin to humanized monoclonal anti-HER2/neu single-chain variable fragment (scFv) 4D5, yielding the immunotoxin DAB389-4D5 scFv that targets HER2/neu-overexpressing carcinoma cells. Because of the low expression level of DAB389-4D5 scFv in Escherichia coli, synonymous codon optimization was conducted in the secondary structure of the translation initiation region (TIR) of the DAB389 gene. After optimization, the expression level reached 36.7% of the total soluble proteins and the yield of purified target protein was 30 mg/L. Subsequently, the results of cytotoxicity experiments indicated that DAB389-4D5 scFv exerted a high proliferation inhibition effect on the HER2/neu-overexpressing ovarian cancer line, SK-OV-3. The results of immunofluorescence analysis showed that DAB389-4D5 scFv exhibited specific binding to SK-OV-3 cells rather than human embryonic kidney 293 cells (HEK-293). Moreover, flow cytometry analysis demonstrated that DAB389-4D5 scFv induced apoptosis and necrosis of SK-OV-3 cells but not HEK-293 cells. Thus, we developed a novel immunotoxin for the treatment of HER2/neu-overexpressing cancer cells.