Purification and in vitro evaluation of an anti-HER2 affibody-2 monomethyl auristatin E conjugate in HER2-positive cancer cells

A promising approach for the development of high-affinity tumor-targeting ADCs is the use of engineered protein drugs, such as affibody molecules, which represent a valuable alternative to monoclonal antibodies (mAbs) in cancer-targeted therapy. We developed a method for a more efficient purification of the ZHER2:2891DCS affibody conjugated with the cytotoxic antimitotic agent auristatin E (MMAE), and its efficacy was tested in vitro on cell viability, proliferation, migration, and apoptosis. The effects of ZHER2:2891DCS-MMAE were compared with the clinically approved monoclonal antibody trastuzumab (Herceptin®). To demonstrate that ZHER2:2891DCS-MMAE can selectively target HER2 overexpressing tumor cells, we used three different cell lines: the human adenocarcinoma cell lines SK-BR-3 and ZR-75-1, both overexpressing HER2, and the tri-ple-negative breast cancer cell line MDA-MB-231. MTT assay showed that ZHER2:2891DCS-MMAE induces a significant time-dependent toxic effect in SK-BR-3 cells. A 30% reduction of cell viability was already found after 10 min exposure at a concentration of 7 nM (IC50 of 80.2 nM). On the contrary, MDA-MB-231 cells, which express basal levels of HER2, were not affected by the con-jugate. The cytotoxic effect of the ZHER2:2891DCS-MMAE was confirmed by measuring apoptosis by flow cytometry. In SK-BR-3 cells, increasing concentrations of conjugated affibody induced cell death starting from 10 min of treatment, with the strongest effect observed after 48 h. Overall, these results demonstrate that the ADC, formed by the anti-HER2 affibody conjugated to monomethyl auristatin E, efficiently interacts with high affinity with HER2 positive cancer cells in vitro, allowing the selective and specific delivery of the cytotoxic payload.