Abstract
Antibody-drug conjugates (ADCs) have recently emerged as efficient and selective cancer treatment therapeutics. Currently, alternative forms of drug carriers that can replace monoclonal antibodies are under intensive investigation. Here, a cytotoxic conjugate of an anti-HER2 (Human Epidermal Growth Factor Receptor 2) diaffibody with monomethyl-auristatin E (MMAE) is proposed as a potential anticancer therapeutic. The anti-HER2 diaffibody was based on the ZHER2:4 affibody amino acid sequence. The anti-HER2 diaffibody has been expressed as a His-tagged protein in E. coli and purified by Ni-nitrilotriacetyl (Ni-NTA) agarose chromatography. The molecule was properly folded, and the high affinity and specificity of its interaction with HER2 was confirmed by surface plasmon resonance (SPR) and flow cytometry, respectively. The (ZHER2:4)2DCS-MMAE conjugate was obtained by coupling the maleimide group linked with MMAE to cysteines, which were introduced in a drug conjugation sequence (DCS). Cytotoxicity of the conjugate was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide MTT assay and the xCELLigence Real-Time Cell Analyzer. Our experiments demonstrated that the conjugate delivered auristatin E specifically to HER2-positive tumor cells, which finally led to their death. These results indicate that the cytotoxic diaffibody conjugate is a highly potent molecule for the treatment of various types of cancer overexpressing HER2 receptors.
Highlights
Antibody-drug conjugates (ADCs) are one of the most advanced approaches in the targeted treatment of cancers and are the leading cause of death in developed countries [1]
The diaffibody construct used in our study consists of a duplicated anti-Human Epidermal Growth Factor Receptor 2 (HER2) affibody ZHER2:4 (Affibody AB) [28,36], separated by a single glutamate residue
The characterization of this protein by circular dichroism showed that the (ZHER2:4)2DCS diaffibody retains the α-helical structure of the parental protein (Figure 2)
Summary
Antibody-drug conjugates (ADCs) are one of the most advanced approaches in the targeted treatment of cancers and are the leading cause of death in developed countries [1]. The affibody molecule adopts a three-helix bundle structure that can be engineered to bind to a large number of target proteins or peptides with high affinity. It has a number of advantages over antibodies, including a smaller size and a simple, robust, tertiary structure, which results in lower production costs. The cytotoxic effect of our conjugate on HER2-positive cancer cells was confirmed in in vitro cytotoxicity assays, demonstrating that this cytotoxic conjugate, upon further in vivo evaluation, can serve as a potential anticancer agent
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