17AAG-induced internalisation of HER2-specific Affibody molecules.

The geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) is known to induce internalisation and degradation of the otherwise internalisation-resistant human epidermal growth factor receptor 2 (HER2) receptor. In the present study, 17-AAG was used to increase internalisation of the HER2-specific Affibody molecule ABY-025. The cellular redistribution of halogen-labelled 211At-ABY-025 and radiometal-labelled 111In-ABY-025 following treatment with 17-AAG was studied. 17-AAG treatment of SKOV-3 human ovarian carcinoma and SKBR-3 human breast carcinoma cells to some extent shifted the localisation of 111In-ABY-025 from the cell surface to intracellular compartments in the two cell lines. ABY-025 labelled with the high-linear energy transfer α emitter 211At was also internalised to a higher degree; however, due to its physiological properties, this nuclide was excreted faster. The results indicate that 17-AAG may be used to facilitate cell-specific intracellular localisation of a suitable cytotoxic or radioactive agent coupled to ABY-025 in HER2-overexpressing cells.

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.

Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules.

Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.

Quantification of internalization of EGFR-binding Affibody molecules: Methodological aspects.

Tumor cell internalization of targeting agents is of interest, since internalization influences the local retention time of a radionuclide and thereby imaging quality in PET and SPECT and effects of radionuclide therapy. In cases where nuclear methods are not applicable at the cellular level, quantitative fluorescent techniques are useful as described in this article. Two fluorescence-based methods to study cellular internalization were applied: the CypHer and the Alexa488-quenching methods, both utilized in fluorescence microscopy and flow cytometry. Two EGFR-binding Affibody molecules were analyzed in A431 cells: the monomer Z1907 and the dimer (Z1907)2. EGF, cetuximab and non-specific Affibody molecules were used as controls. For comparison, internalization of 111In-labeled Z1907 was studied with the acid wash internalization assay. The Cypher method is straightforward, but requires equal labeling of all compounds for accurate quantification. The Alexa488-quenching method is preferable since it is independent of the dye-to-protein ratio. According to this method, about 45% of EGF and 19-24% of the bound Affibody molecules and cetuximab were internalized within one hour. Similar results were seen with 111In-Z1907 in the acid wash method, while (Z1907)2 was not removed by acid and thus could not be studied this way. The fluorescence-based Alexa488-quenching method is well suited to quantitatively analyze internalization of targeting agents, also those that resist acid wash. The internalized fraction showed that both the monomeric and dimeric Affibody molecules are expected to give good uptake and thereby good retention of metallic radionuclides which will render good tumor to background values.

Differences in radiosensitivity between three HER2 overexpressing cell lines.

PURPOSE: HER2 is a potential target for radionuclide therapy, especially when HER2 overexpressing breast cancer cells are resistant to Herceptin(R) treatment. Therefore, it is of interest to analyse whether HER2 overexpressing tumour cells have different inherent radiosensitivity. METHODS: The 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 (211)At decays using the HER2-binding affibody molecule (211)At-(Z(HER2:4))(2) as targeting agent. Assays for studies of internalisation of the affibody molecule were applied. RESULTS: SKOV-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 (211)At was qualitatively similar for all three cell lines. However, the sensitivity to the alpha particles from (211)At 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. CONCLUSION: There 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.

Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule.

INTRODUCTION: The cellular binding and processing of an epidermal growth factor receptor (EGFR) targeting affibody molecule, (Z(EGFR:955))(2), was studied. This new and small molecule is aimed for applications in nuclear medicine. The natural ligand epidermal growth factor (EGF) and the antibody cetuximab were studied for comparison. METHODS: All experiments were made with cultured A431 squamous carcinoma cells. Receptor specificity, binding time patterns, retention and preliminary receptor binding site localization studies were all made after (125)I labeling. Internalization was studied using Oregon Green 488, Alexa Fluor 488 and CypHer5E markers. RESULTS: [(125)I](Z(EGFR:955))(2) and [(125)I]cetuximab gave a maximum cellular uptake of (125)I within 4 to 8 h of incubation, while [(125)I]EGF gave a maximum uptake already after 2 h. The retention studies showed that the cell-associated fraction of (125)I after 48 h of incubation was approximately 20% when delivered as [(125)I](Z(EGFR:955))(2) and approximately 25% when delivered as [(125)I]cetuximab. [(125)I]EGF-mediated delivery gave a faster (125)I release, where almost all cell-associated radioactivity had disappeared within 24 h. All three substances were internalized as demonstrated with confocal microscopy. Competitive binding studies showed that both EGF and cetuximab inhibited binding of (Z(EGFR:955))(2) and indicated that the three substances competed for an overlapping binding site. CONCLUSION: The results gave information on cellular processing of radionuclides when delivered with (Z(EGFR:955))(2) in comparison to delivery with EGF and cetuximab. Competition assays suggested that [(125)I](Z(EGFR:955))(2) bind to Domain III of EGFR. The affibody molecule (Z(EGFR:955))(2) can be a candidate for EGFR imaging applications in nuclear medicine.

Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts.

A high stringency protocol, suitable for systematic purification of polyclonal antibodies, is described. The procedure is designed to allow the generation of target protein-specific antibodies suitable for functional annotation of proteins. Antibodies were generated by immunization with recombinantly produced affinity-tagged target proteins. To obtain stringent recovery of the antibodies, a two-step affinity chromatography principle was devised to first deplete the affinity tag-specific antibodies followed by a second step for affinity capture of the target protein-specific antibodies. An analytical dot-blot array system was developed to analyze the cross-reactivity of the affinity-purified antibodies. The results suggest that the protocol can be used in a highly parallel and automated manner to generate mono-specific polyclonal antibodies for large-scale, antibody-based proteomics efforts, i.e. affinity proteomics.

Cell studies of the DNA bis-intercalator Delta-Delta [mu-C4(cpdppz)(2)-(phen)(4)Ru(2)](4+): toxic effects and properties as a light emitting DNA probe in V79 Chinese hamster cells.

Coordination complexes of type [Ru(L)(3)](2+), where L is a nitrogen-containing aromatic bidentate ligand, can often be photolytically reduced, making them useful in studies of DNA- or protein-mediated electron transfer and in artifical photosynthesis model systems. Upon binding to DNA some Ru(L) complexes have been found to display strongly increased fluorescence compared with when free in solution, making those compounds interesting to test as DNA probes. Thus, they are becoming widely used in the chemistry community. Here, asynchronous cultures of V79 Chinese hamster cells were exposed to the DNA bis-intercalator Delta-Delta [mu-C4(cpdppz)(2)-(phen)(4)Ru(2)](4+) at 10(-10)-10(-4) M. The extraordinarily strong binding of the compound to DNA was the reason for testing its possible interference with DNA metabolism in intact mammalian cells. Exposure for 1 h to 10(-10)-10(-4) M did not significantly decrease DNA synthesis. Cells exposed to 10(-5) M for 27 h showed no staining of the nucleus, while DNA was stained in cells electroporated in the presence of the compound. However, the Ru dimer was probably taken up by pinocytosis, because numerous minute precipitates could be observed in the cytoplasm. Treatment for 24 h at concentrations of 10(-10)-10(-5) M did not inhibit growth, as indicated by cell density and mitotic activity. Neither did it affect chromosomal arrangements during mitosis. However, at 10(-4) M the density of cultures was reduced by approximately 45% and apoptotic cells were frequent, as opposed to mitoses. We also investigated the properties of the Ru dimer as a fluorescent DNA stain. The compound appears attractive as a red DNA stain when broad excitation in the visible range is desirable and extremely low background staining is essential. The low toxicity of the compound is a favourable trait in this context.