Cellular Effects of HER3-Specific Affibody Molecules

Recent studies have led to the recognition of the epidermal growth factor receptor HER3 as a key player in cancer, and consequently this receptor has gained increased interest as a target for cancer therapy. We have previously generated several Affibody molecules with subnanomolar affinity for the HER3 receptor. Here, we investigate the effects of two of these HER3-specific Affibody molecules, Z05416 and Z05417, on different HER3-overexpressing cancer cell lines. Using flow cytometry and confocal microscopy, the Affibody molecules were shown to bind to HER3 on three different cell lines. Furthermore, the receptor binding of the natural ligand heregulin (HRG) was blocked by addition of Affibody molecules. In addition, both molecules suppressed HRG-induced HER3 and HER2 phosphorylation in MCF-7 cells, as well as HER3 phosphorylation in constantly HER2-activated SKBR-3 cells. Importantly, Western blot analysis also revealed that HRG-induced downstream signalling through the Ras-MAPK pathway as well as the PI3K-Akt pathway was blocked by the Affibody molecules. Finally, in an in vitro proliferation assay, the two Affibody molecules demonstrated complete inhibition of HRG-induced cancer cell growth. Taken together, our findings demonstrate that Z05416 and Z05417 exert an anti-proliferative effect on two breast cancer cell lines by inhibiting HRG-induced phosphorylation of HER3, suggesting that the Affibody molecules are promising candidates for future HER3-targeted cancer therapy

Cellular Studies of HER-family Specific Affibody Molecules

The human epidermal growth-factor like receptor (HER) family of receptor tyrosine kinases are important targets for cancer therapy. The family consists of four members - EGFR, HER2, HER3 and HER4 - that normally transfer stimulatory signals from extracellular growth factors to the intracellular signalling network. Over-activation of these receptors leads to uncontrolled cell proliferation and is seen in several types of tumours. The aim of the studies reported in this thesis was to study the uptake and effects of affibody molecules against EGFR, HER2 and HER3 in cultured cells. Affibody molecules are affinity proteins originally derived from one of the domains of protein A, and their small size and robust structure make them suitable agents for tumour targeting and therapy. Papers I and II of this thesis concern EGFR-specific affibody molecules, which were shown to be more similar to the antibody cetuximab than the natural ligand EGF in terms of cellular uptake, binding site and internalisation rate. In addition, fluorescence-based methods for the quantification of internalisation were evaluated. In the studies reported in papers III and IV, HER2-specific affibody molecules were utilised as carriers of radionuclides. Paper III reports that different cell lines exhibit different radiosensitivities to 211At-labelled affibody molecules; radiosensitivity was found to correlate with cell geometry and the rate of internalisation. Paper IV discusses the use of 17-AAG, an agent that induces HER2 internalisation and degradation, to force the internalisation of 211At- and 111In-labelled affibody molecules. Papers V and VI describe the selection and maturation of HER3-specific affibody molecules, which were found to compete with the receptor’s natural ligand, heregulin, for receptor binding. These affibody molecules were demonstrated to inhibit heregulin-induced HER3 activation and cell proliferation. The studies summarised in this paper will hopefully contribute to a better understanding of these affibody molecules and bring them one step closer to being helpful tools in the diagnosis and treatment of cancer

Analysis of cellular growth inhibitory effects of the HER3-specific Affibody molecules.

<p>Mean absorbance values at 450 nm ± SD, which is proportional to the number of living cells, is given on the y-axis. A. Proliferation of MCF-7 and SKBR-3 cells grown in a dilution series of HRG. B. Proliferation of MCF-7 and SKBR-3 cells grown in 40 pM HRG and a dilution series of Affibody molecules Z05416, Z05417 or ZTaq. C. Proliferation of cells grown in medium containing 40 nM Affibody molecules, 0.04 nM HRG. Results are compared to unstimulated cells (no Affibody molecules or HRG added).</p

Western blot analysis of phosphorylated Akt and Erk upon addition of heregulin and/or HER3-specific Affibody molecules.

<p>Phospho-Akt and phospho-Erk detected by western blot of cell lysates from MCF-7 and SKBR-3 cells treated with (+) or without (−) 0.05 nM heregulin (HRG) and 100 nM Affibody molecules (Z). As a control, β-actin was detected to show that the protein concentrations of the different lysates were equivalent.</p

Immunofluorescent staining of human cancer cell lines.

<p>Images showing AU565, SKBR-3, MCF-7 and SKOV-3 cells stained with HER3-specific Affibody molecules Z05416 and Z05417, respectively. The polyclonal anti-HER3 antibody A234 and ZTaq were used as positive and negative staining controls, respectively. Affibody molecules and antibodies binding to cells are shown in green while nuclear staining by DAPI is given in blue. AU565 and SKOV-3 images were acquired on the same day using the same detection gain and laser power, enabling comparison between staining intensities. Cell staining of MCF-7 and SKBR-3 was analysed on different days, using different detection gains for optimal image acquisition. Additionally, MCF-7 images were acquired using increased laser power.</p

Cell binding of HER3-specific Affibody molecules analysed by flow cytometry.

<p>Binding of Alexa Fluor® 488-labelled Affibody molecules (150 nM) to: A. MCF-7, B. SKBR-3 and the HER3-negative cell line SKOV-3. “Bl”  =  blocking with 15 μM of the corresponding, non-labelled Affibody. MCF-7 cells were stained in a separate experiment, whereas SKBR-3 and SKOV-3 were stained simultaneously.</p

Analysis of receptor phosphorylation of MCF-7 and SKBR-3 cells.

<p>Histograms showing ELISA absorbance results for detection of: A. Phospho-HER2 in MCF-7 cells, B. Phospho-HER3 in MCF-7 cells, C. Phospho-HER2 in SKBR-3 cells and D. Phospho-HER3 in SKBR-3 cells. Cells were incubated without HRG (grey bars) or with 0.05 nM HRG (black bars), in combination with the Affibody molecules (100 nM) before being lysed and analysed in an ELISA.</p

Competitional binding between the natural ligand HRG and Affibody molecules to the HER3-positive breast cancer cell line AU565 visualised by confocal microscopy.

<p>AU565 cells were pretreated with Affibody molecules (Z05416, Z05417 or ZTaq) or PBS only (HRG) before addition of HRG in conjugation with a fluorophore. HRG binding to cells is shown in green while nuclear staining by DAPI is shown in blue.</p