Ann-Charlott Steffen

Effects of HER2-Binding Affibody Molecules on Intracellular Signaling Pathways

Background: HER2, which is overexpressed in 25–30% of human breast cancers, is a tyrosine kinase receptor critical for the signal transduction network that regulates proliferation, migration and apoptosis of cells. Method: We report the effects of two novel HER2-binding affibody molecules (Affibody^®), (Z_HER2:₄)₂ and Z_HER2:₃₄₂, on intracellular signal transduction pathways (Erk1/2, Akt and PLCγ1) using quantitative immunoblotting techniques and their biological effects in cell culture. The clinically approved antibody trastuzumab (Herceptin^®) was used as reference substance. Results: Our data showed that, although all substances target HER2, the effects on the receptor and signaling molecules differed. For example, HER2 phosphorylation was induced by trastuzumab and (Z_HER2:₄)₂ but inhibited by Z_HER2:₃₄₂. The effects these substances had on signal transduction correlated to some degree with changes in growth and migration, e.g. (Z_HER2:₄)₂ stimulated phosphorylation of Erk1/2 and PLCγ1, as well as growth and migration, while Z_HER2:₃₄₂ did not. Z_HER2:₃₄₂ even inhibited phosphorylation of PLCγ1 and migration. Conclusion: Our data suggest that Z_HER2:342 is a promising small agent (7 kDa) that may be used as an alternative, or complement, to trastuzumab. If radiolabelled, it can hopefully also be used for HER2 imaging and radionuclide therapy.

Cellular retention of radioactivity and increased radiation dose. Model experiments with EGF-dextran

Targeting of tumor cells with radiolabeled biomolecules is a possible approach to inactivate disseminated tumor cells. However, rapid degradation of the biomolecules after cellular internalization and subsequent excretion of the radioactivity is a problem. We studied the possibility of using dextran as a carrier of radionuclides to improve the intracellular retention. An EGF-dextran conjugate, aimed for targeting of tumor cells overexpressing the EGF-receptor, was used as model. Retention tests were performed with (125)I on different parts: [(125)I]-EGF-dextran-[(125)I], [(125)I]-EGF-dextran and EGF-dextran-[(125)I]. Comparisons were made with [(125)I]-EGF. The radiolabeled compounds were incubated with cultured glioma cells for different times. The cellular retention of radioactivity was then measured for up to 24 h. Expected radiation doses at the cellular level were calculated assuming that (131)I, instead of (125)I, was coupled to EGF and EGF-dextran. The results indicated that the EGF-part of the conjugate was degraded and the EGF-attached radioactivity was rapidly excreted, whereas radioactivity on dextran was retained intracellularly to a high degree, i.e. 70-80% of the radioactivity bound to dextran was still cell-associated after 24 h. The retention after 24 h was significantly higher (p < 0.001) when the radioactivity was on the dextran instead of the EGF-part. The radiolabeled EGF-dextran had a notably high specific radioactivity; up to 11 MBq/microg. There was potential for at least hundred times increased radiation dose per receptor interaction when the radioactivity was on the dextran part. The advantage with radioactivity on the dextran part was the high cellular retention and the high specific radioactivity (higher than previously reported for other residualizing labels) without severe loss of receptor specific binding. Thus, dextran seems suitable as a carrier of radionuclides aimed for therapy and gives potential for a highly increased radiation dose.

Differences in radiosensitivity between three HER2 overexpressing cell lines

PurposeHER2 is a potential target for radionuclide therapy, especially when HER2 overexpressing breast cancer cells are resistant to Herceptin® treatment. Therefore, it is of interest to analyse whether HER2 overexpressing tumour cells have different inherent radiosensitivity.MethodsThe radiosensitivity of three often used HER2 overexpressing cell lines, SKOV-3, SKBR-3 and BT-474, was analysed. The cells were exposed to conventional photon irradiation, low linear energy transfer (LET), to characterise their inherent radiosensitivity. The analysis was made with clonogenic survival and growth extrapolation assays. The cells were also exposed to alpha particles, high LET, from 211At decays using the HER2-binding affibody molecule 211At-(ZHER2:4)2 as targeting agent. Assays for studies of internalisation of the affibody molecule were applied.ResultsSKOV-3 cells were most radioresistant, SKBR-3 cells were intermediate and BT-474 cells were most sensitive as measured with the clonogenic and growth extrapolation assays after photon irradiation. The HER2 dependent cellular uptake of 211At was qualitatively similar for all three cell lines. However, the sensitivity to the alpha particles from 211At differed; SKOV-3 was most resistant, SKBR-3 intermediate and BT-474 most sensitive. These differences were unexpected because it is assumed that all types of cells should have similar sensitivity to high-LET radiation. The sensitivity to alpha particle exposure correlated with internalisation of the affibody molecule and with size of the cell nucleus.ConclusionThere can be differences in radiosensitivity, which, if they also exist between patient breast cancer cells, are important to consider for both conventional radiotherapy and for HER2-targeted radionuclide therapy.

Targeting EGFR and HER2 with 211At-Labeled Molecules: Unexpected and Expected Dose-Effect Relations in Cultured Tumor Cells

This article is based on results from four published experimental studies. I: Three HER2 overexpressing cell lines were exposed to α-particles from the HER2 binding affibody molecule 211At- (ZHER2:4)2. The sensitivity differed; SKOV3 was resistant, SKBR3 intermediate and BT474 sensitive. The differences were unexpected since it is assumed that different types of cells should have a similar sensitivity to high-LET radiation. II: Effects of 211At-EGF in combination with the lysosomotropic base ammonium chloride were studied in A431 cells. The therapy effects of 211At-EGF increased by co-treatment with ammonium chloride, which was expected. III: Therapy effects of combined 211At-EGF and gefitinib treatment were studied using gefitinib sensitive A431 and resistant U343MG cells. The combined treatment reduced the survival of the resistant cells but in the gefitinib sensitive cells, the combined treatment increased survival, which was highly unexpected. IV: SKBR3 cells were exposed to the antibody trastuzumab that was labeled with 211At. A limited number of astatine decays per cell gave low survival, which was expected. Conclusion: The well-known high effectiveness of alpha particles to kill tumor cells was confirmed in all four studies. However, the dose-effect relations were complicated and more research is needed to evaluate factors of importance, such as internalization rate of the targeting agent, subcellular distribution and retention time of 211At.