Purification and In Vitro Evaluation of an Anti-HER2 Affibody-Monomethyl Auristatin E Conjugate in HER2-Positive Cancer Cells

SIMPLE SUMMARY: Antibody-drug conjugates (ADCs) represent an innovative class of anticancer agents specifically aimed at targeting cancer cells, reducing damage to healthy tissues but showing some weaknesses. A promising approach for the development of high-affinity tumor targeting ADCs is the use of engineered protein drugs, such as affibody molecules. Our aim was to develop a more efficient purification method for the cytotoxic conjugate Z(HER2:2891)DCS-MMAE that targets human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells. The conjugate is based on Z(HER2:2891) affibody and a drug conjugation sequence (DCS), which allowed for site-specific conjugation of the cytotoxic auristatin E molecule (MMAE) to the affibody. We tested the in vitro efficacy of Z(HER2:2891)DCS-MMAE on several parameters, such as cell viability, proliferation, migration, and apoptosis. Our results confirmed that the cytotoxic conjugate efficiently interacts with high affinity with HER2 positive cancer cells, allowing the selective and specific delivery of the cytotoxic payload. ABSTRACT: 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 Z(HER2:2891)DCS 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 Z(HER2:2891)DCS-MMAE were compared with the clinically approved monoclonal antibody trastuzumab (Herceptin(®)). To demonstrate that Z(HER2:2891)DCS-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 triple-negative breast cancer cell line MDA-MB-231. MTT assay showed that Z(HER2:2891)DCS-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 (IC(50) of 80.2 nM). On the contrary, MDA-MB-231 cells, which express basal levels of HER2, were not affected by the conjugate. The cytotoxic effect of the Z(HER2:2891)DCS-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.