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

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 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 triple-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 conjugate. 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.

A Novel Monoclonal Antibody Targeting Cancer-Specific Plectin Has Potent Antitumor Activity in Ovarian Cancer

“…Future studies will continue to decipher 1H11′s mechanism of action, differentiating between signaling cascades and phenotypic changes directly modulated by 1H11 from those resulting from signaling crosstalk. Importantly, the induction of inhibitory effects with 1H11 is within the reported therapeutic ranges of targeting conventional functional proteins such as HER2 and EGFR with clinically approved drugs [ 53 , 54 , 55 , 56 ]. Overall, not only are we the first to report that therapeutic targeting of CSP is a robust anticancer strategy, thus expanding our anticancer repertoire, but we reveal that targeting a non-conical cell surface protein has molecular consequences.…”

Smooth Muscle Cell Phenotypic Switch Induced by Traditional Cigarette Smoke Condensate: A Holistic Overview

“…After 72 h, cells in control dishes were counted with a Coulter Counter (Beckman Coulter, Life Scientific, Milan, Italy) and this was considered the “basal” number of cells at T0. Then, medium was removed and replaced with medium containing 30 μg/mL CSC and 10% of FBS for 24 and 48 h. Cell number was measured and compared to the zero time-point [ 126 ].…”

PURPL and NEAT1 Long Non-Coding RNAs Are Modulated in Vascular Smooth Muscle Cell Replicative Senescence

“…At different time points (up to 96 h), cell number was measured and compared to the T0. Results were also used to calculate the doubling time [23].…”