Antibody-drug conjugates (ADCs) have recently emerged as efficient and selective cancer\ntreatment therapeutics. Currently, alternative forms of drug carriers that can replace monoclonal\nantibodies are under intensive investigation. Here, a cytotoxic conjugate of an anti-HER2 (Human\nEpidermal Growth Factor Receptor 2) diaffibody with monomethyl-auristatin E (MMAE) is proposed\nas a potential anticancer therapeutic. The anti-HER2 diaffibody was based on the ZHER2:4 affibody\namino acid sequence. The anti-HER2 diaffibody has been expressed as a His-tagged protein in\nE. coli and purified by Ni-nitrilotriacetyl (Ni-NTA) agarose chromatography. The molecule was\nproperly folded, and the high affinity and specificity of its interaction with HER2 was confirmed\nby surface plasmon resonance (SPR) and flow cytometry, respectively. The (ZHER2:4)2DCS-MMAE\nconjugate was obtained by coupling the maleimide group linked with MMAE to cysteines, which\nwere introduced in a drug conjugation sequence (DCS). Cytotoxicity of the conjugate was evaluated\nusing the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide MTT assay and the\nxCELLigence Real-Time Cell Analyzer. Our experiments demonstrated that the conjugate delivered\nauristatin E specifically to HER2-positive tumor cells, which finally led to their death. These results\nindicate that the cytotoxic diaffibody conjugate is a highly potent molecule for the treatment of\nvarious types of cancer overexpressing HER2 receptors.
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