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. 2017 Feb 14;18(2):401.
doi: 10.3390/ijms18020401.

A Conjugate Based on Anti-HER2 Diaffibody and Auristatin E Targets HER2-Positive Cancer Cells

Anna M Serwotka-Suszczak  1 , Alicja M Sochaj-Gregorczyk  2 , Jerzy Pieczykolan  3 , Daniel Krowarsch  4 , Filip Jelen  5 , Jacek Otlewski  6
Affiliations

A Conjugate Based on Anti-HER2 Diaffibody and Auristatin E Targets HER2-Positive Cancer Cells

Anna M Serwotka-Suszczak et al. Int J Mol Sci. .

Erratum in

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.

Keywords: HER2; diaffibody; monomethyl auristatin E (MMAE); targeted therapy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The (ZHER2:4)2DCS diaffibody construct is composed of two ZHER2:4 units separated by a single glutamate residue (E), a 6× His-tag at the N-terminus, and a drug conjugation sequence (DCS) at the C-terminus.
Figure 2
Figure 2
Circular dichroism (CD) spectrum of the diaffibody confirms a predominant α-helical secondary structure. Inset summarizes secondary structure content of (ZHER2:4)2DCS.
Figure 3
Figure 3
Normalized thermal denaturation (black line) and renaturation (dashed line) of (ZHER2:4)2DCS monitored by ellipticity changes.
Figure 4
Figure 4
Specificity of the diaffibody-HER2 (Human Epidermal Growth Factor Receptor 2) binding analyzed by flow cytometry. (a,b) Positive staining was recorded for the HER2-positive SK-BR-3 cells with the anti-HER2 monoclonal antibody and with the fluorescently labeled diaffibody at three different concentrations: 0.03, 0.3 and 3 μM. (c) Banding is observed for the control HER2-negative U-87 MG cells.
Figure 5
Figure 5
(ZHER2:4)2DCS conjugation with MC-Val-Cit-PABC-MMAE (monomethyl-auristatin E) (vcMMAE) (a) vcMMAE is attached to cysteine(s) present in the drug conjugation sequence via a valine-citrulline linker, which is cleaved by cathepsins inside tumor cells. The cleavage site is marked in red. (b) The mass spectrometry (MS) spectrum of the conjugation products showing the unmodified diaffibody and the diaffibody modified with 1, 2, and 3 vcMMAE molecules (c) SDS-PAGE separation of the conjugation mixture. Due to the low sensitivity of Coomassie brilliant blue staining in comparison to mass spectrometry, only two bands for the conjugate species were visualized.
Figure 6
Figure 6
Surface plasmon resonance (SPR) analysis of HER2 and diaffibody interaction was performed using (a) increasing concentrations of 0.01, 0.1, and 1 μM of the diaffibody; and (b) the diaffibody-MMAE conjugate. The y axis represents the response difference in relative units (RU).
Figure 7
Figure 7
Microscopic analysis of SK-BR-3 cells treated with phosphate buffer saline (PBS), 500 nM (ZHER2:4)2DCS, and 500 nM (ZHER2:4)2DCS-MMAE after 72 h. The cells incubated with the anti-HER2 diaffibody did not show any morphological changes in comparison to the untreated control cells, whereas the viability of the cells treated with the conjugate was severely affected.
Figure 8
Figure 8
The xCELLigence experiment performed on SK-BR-3 cells treated with (ZHER2:4)2DCS-MMAE. Results are shown for the selected time points of 20, 40, 60, 80, 100, and 120 h. Results were normalized against the control cells treated with PBS at each time point. The error bars show the standard deviation.
Figure 9
Figure 9
Viability assay results for human breast cancer cell lines (a) SK-BR-3 (HER2+); and (b) MDA-MB-453 (HER2+); and T-47D (HER2+/−) (c). These cells were incubated with the indicated doses of (ZHER2:4)2DCS-MMAE, 500 nM MMAE, and 500 nM (ZHER2:4)2DCS. Cell viability was assessed after 72, 96, and 120 h incubation. The error bars show the standard deviation.
Figure 10
Figure 10
Viability assay results for the three HER2-negative cell lines (a) U-87 MG (human brain cancer); (b) SK-MES-1 (human lung cancer); and (c) MDA-MB-231 (human breast cancer). These cells were incubated with the indicated doses of (ZHER2:4)2DCS-MMAE, 500 nM MMAE, and 500 nM (ZHER2:4)2DCS. Cell viability was assessed after 72, 96, and 120 h incubation. The error bars show the standard deviation.
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