Abstract
Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.
Highlights
Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors, including breast cancer, prostate cancer, lung cancer, bladder cancer, and gastric cancer [1]
The VH and VL domains of trastuzumab were connected by a (GGGGS)3 linker to generate the single-chain Fv (scFv)
The furin protease recognition sequence, RHRQPRGWEQL, was inserted between the scFv and PE24B so that the toxin can be released after the immunotoxin is internalized
Summary
Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors, including breast cancer, prostate cancer, lung cancer, bladder cancer, and gastric cancer [1]. HER-2 overexpression has been observed in 20–30% of all breast tumors [2,3]. Patients displaying HER2 overexpression have a significantly worse prognosis, and overexpression of HER2 in breast tissues stimulates malignant phenotypic transformation. HER2-overexpressing tumors are more resistant to general chemotherapy treatment [4]. HER2 is a 185 kDa transmembrane tyrosine kinase receptor belonging to the epidermal growth receptor (EGFR) family 2. Phosphorylation of HER dimers results in activation of various downstream pathways related to cell proliferation, survival, differentiation, angiogenesis, invasion, and metastasis [5,6]
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