title: A Conjugate Based on Anti-HER2 Diaffibody and Auristatin E Targets HER2-Positive Cancer Cells.
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 ZHER2:4affibody amino acid sequence. The anti-HER2 diaffibody has been expressed as a His-tagged protein inE. coliand 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.
authors.affiliation: Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, 50-137 Wroclaw, Poland. aniaserwotka@wp.pl.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Serwotka-Suszczak
authors.affiliation: Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, 50-137 Wroclaw, Poland. alicja.sochaj-gregorczyk@uj.edu.pl.
authors.forename: Alicja M
authors.initials: AM
authors.lastname: Sochaj-Gregorczyk
authors.affiliation: Preclinical Development Department, R&D Celon Pharma Inc., 05-092 Lomianki/Kielpin, Poland. jerzy.pieczykolan@celonpharma.com.
authors.forename: Jerzy
authors.initials: J
authors.lastname: Pieczykolan
authors.affiliation: Department of Protein Biotechnology, Faculty of Biotechnology, University of Wroclaw, 50-137 Wroclaw, Poland. daniel.krowarsch@uwr.edu.pl.
authors.forename: Daniel
authors.initials: D
authors.lastname: Krowarsch
authors.affiliation: Research and Development Department, Pure Biologics Ltd., 54-427 Wroclaw, Poland. filip@purebiologics.com.
authors.forename: Filip
authors.initials: F
authors.lastname: Jelen
authors.affiliation: Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, 50-137 Wroclaw, Poland. jacek.otlewski@uwr.edu.pl.
authors.forename: Jacek
authors.initials: J
authors.lastname: Otlewski
mesh_terms: D062365:Aminobenzoates / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D000918:Antibody Specificity; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004195:Disease Models, Animal; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry / Q000494:pharmacology*; D058990:Molecular Targeted Therapy; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D055550:Protein Stability; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013816:Thermodynamics; D023041:Xenograft Model Antitumor Assays
chemical_list: D062365:Aminobenzoates; D000911:Antibodies, Monoclonal; D018796:Immunoconjugates; D009842:Oligopeptides; D018719:Erb-b2 Receptor Tyrosine Kinases; C543533:auristatin
keywords: HER2; diaffibody; monomethyl auristatin E (MMAE); targeted therapy