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 ZHER2:4 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.

A Conjugate of Pentamethine Cyanine and 18F as a Positron Emission Tomography/Near-Infrared Fluorescence Probe for Multimodality Tumor Imaging

“…A dual-modality PET/NIRF synthon exploits the non-invasive and unlimited penetration of PET imaging and the high local spatial resolution of NIR fluorescence imaging to guide intraoperative resection or post-surgery pathological examination to ultimately improve clinical outcome [18,19,20,21]. …”

Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1

“…Recently, we have generated an auristatin conjugate based on the bivalent Z HER2:4 affibody referred to as (Z HER2:4 ) 2 DCS-MMAE [ 21 ]. Z HER2:4 is an earlier version of the anti-HER2 affibody and binds HER2 receptor with the affinity three orders of magnitude lower than Z HER2:2891 [ 29 , 30 ].…”

“…In this context, the Z HER2:2891 -Fc fusion provides a better platform for a cytotoxic conjugate development than single and bivalent affibodies. Interestingly, the dimeric (Z HER2:4 ) 2 DCS affibody slightly affected the SK-BR-3 cell viability during a 4-day incubation [ 21 ]. This finding is in contrast to our preliminary observation that the Z HER2:2891 affibody fused to the Fc fragment enhances viability of the HER2 positive cells (data not shown).…”

“…Recently, the feasibility of other molecules to deliver cytotoxic payloads or radionuclides to cancer cells has been addressed in an increasing number of studies [ 7 , 8 ]. Among the antibody alternatives are antibody fragments such as scFvs (28 kDa) and Fabs (55 kDa) [ 9 , 10 , 11 , 12 ], nanobodies (12–15 kDa) [ 13 , 14 , 15 , 16 ], affimers (12–14 kDa) [ 17 ], signaling molecules [ 18 ] and affibodies (6–7 kDa) [ 19 , 20 , 21 , 22 , 23 , 24 ]. Affibodies are engineered proteins based on a three-helix bundle Z domain that was derived from the IgG-binding B domain of protein A from Staphylococcus aureus [ 25 ].…”

Correction: Serwotka-Suszczak, A. M. et al. A Conjugate Based on Anti-HER2 Diaffibody and Auristatin E Targets HER2-Positive Cancer Cells. Int. J. Mol. Sci. 2017, 18, 401

“…It has been brought to our attention that the affiliation of Dr. Jerzy Pieczykolan at the time when he was responsible for the work described in the paper [ 1 ] was not mentioned in the previous published version.…”