Trastuzumab‐monomethyl auristatin E conjugate exhibits potent cytotoxic activity in vitro against HER2‐positive human breast cancer

Abdollahpour‐Alitappeh, Meghdad and Lotfinia, Majid and Bagheri, Nader and Sineh Sepehr, Koushan and Habibi‐Anbouhi, Mahdi and Kobarfard, Farzad and Balalaie, Saeed and Foroumadi, Alireza and Abbaszadeh‐Goudarzi, Ghasem and Abbaszadeh‐Goudarzi, Kazem and Abolhassani, Mohsen (2019) Trastuzumab‐monomethyl auristatin E conjugate exhibits potent cytotoxic activity in vitro against HER2‐positive human breast cancer. Journal of Cellular Physiology, 234 (3). pp. 2693-2704. ISSN 0021-9541

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Targeted therapy using specific monoclonal antibodies (mAbs) conjugated to chemotherapeutic agents or toxins has become one of the top priorities in cancer therapy. Antibody–drug conjugates (ADCs) are emerging as a promising strategy for cancer-targeted therapy. In this study, trastuzumab, a humanized monoclonal anti-HER2 antibody, was reduced by dithiothreitol and conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) through a valine-citrulline peptide linker (trastuzumab-MC-Val-Cit-PABC-MMAE [trastuzumab-vcMMAE]). After conjugation, ADCs were characterized by using UV–vis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and flow cytometry. The antitumor activity of the ADC was evaluated in breast cancer cells in vitro. In addition, ADCs were further characterized using purification by the protein A chromatography, followed by assessment using apoptosis and MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assays. Hydrophobic interaction chromatography was used to determine drug-to-antibody ratio species of ADCs produced. Our finding showed that approximately 5.12 drug molecules were conjugated to each mAb. H2L2, H2L, HL, H2, H, and L forms of ADCs were detected in nonreducing SDS-PAGE. The binding of trastuzumab-vcMMAE to HER2-positive cells was comparable with that of the parental mAb. The MTT assay showed that our ADCs induced significant cell death in HER2-positive cells, but not in HER2-negative cells. The ADCs produced was a mixture of species, unconjugated trastuzumab (14.147%), as well as trastuzumab conjugated with two (44.868%), four (16.886%), six (13.238%), and eight (10.861%) molecules of MMAE. These results indicated that MMAE-conjugated trastuzumab significantly increases the cytotoxic activity of trastuzumab, demonstrating high affinity, specificity, and antitumor activity in vitro. Trastuzumab-vcMMAE is an effective and selective agent for the treatment of HER2-positive breast tumors.

Item Type: Article
Subjects: WH Hemic and Lymphatic System
QZ pathology-Neoplasms
Divisions: Reserach Vice-Chancellar Department > Cellular and Molecular Research Center
Depositing User: zeynab . bagheri
Date Deposited: 17 May 2020 07:39
Last Modified: 17 May 2020 07:39

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