새로운 재조합 면역 독소 HER2(scFv)-PE26의 HER2 발현 유방암 세포주에 대한 세포 독성 효과

Abstract

Breast cancer is known to be mainly caused by the estrogen signal and the human epithelial growth factor receptor 2 (HER2) signal. Patients overexpressing HER2 have a significantly lower survival rate and overexpression of HER2 in breast tissue stimulates transformation into a malignant phenotype. In addition, HER2-overexpressing tumors are resistant to general chemotherapy treatments. The anti-HER2 antibody, trastuzumab, blocks HER2 signaling that concerned about growth of tumor cells. It has been approved for the treatment of HER2 positive early breast cancer and metastatic breast cancer. Pseudomonas exotoxin A (PE) is a bacterial toxin of Pseudomonas aeruginosa, consisting of an A-domain with enzymatic activity and a B-domain with cell binding activity. In this study, the novel recombinant immunotoxin composing of a single-chain of anti-HER2 (HER2(scFv)) from trastuzumab and 26 kDa catalytic fragment (PE26) from PE was created. The recombinant immunotoxin was fused with the maltose binding protein (MBP) at the N-terminus to increase the expression and solubility of protein expression in E. coli. After cation exchange chromatography and removal of MBP, HER2(scFv)-PE26 was stable and further immobilized metal affinity chromatography and gel filtration chromatography allowed a purity of greater than 91%. Finally, 0.25 mg of pure HER2(scFv)-PE26 was successfully obtained from 500 mL flask culture. FACS analysis with HER2(scFv)-GFP showed the highest expression of HER2 on SKBR3 and BT-474 cell lines with low expression on MDA-MB-231 cell line and little expression on MCF7 cell line. The purified HER2(scFv)-PE26 showed cytotoxicity at much lower doses than the purified HER2(scFv) or PE26 alone. IC50s of HER2(scFv)-PE26 were estimated to be 28.1 ± 2.5 pM (n = 9), 19 ± 1.4 pM (n = 9) with the Hill coefficient of 2.24 ± 0.16 and 1.87 ± 0.17, for the high HER2-expressing cell lines, SKBR3 and BT-474, respectively. On the other hand, the cytotoxicity of HER2(scFv)-PE26 on MDA-MB-231 and MCF7, was weaker than those on SKBR3 and BT-474. These results suggest the new immunotoxin, HER2(scFv)-PE26, could be a new drug candidate for the HER2-overexpressing cancer patients.