Molekulare in vivo Fluoreszenzbildgebung zur Darstellung von ErbB/Her2- und CCK2-rezeptorpositiven Tumoren im Tiermodell
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Abstract
The great aim of molecular imaging is to visualize biochemical processes at the cellular level. An interesting method for this purpose is in vivo near-infrared fluorescence imaging. However, the strong UV-VIS-absorption of blood and tissue due to hemoglobin and its derivatives makes it necessary to move to fluorescent dyes which absorb in near-IR and therefore reach much higher fluorescence emission intensities in tissue.
Exploiting this methodology, near-IR fluorescent dyes were coupled to peptide ligands for CCK2 and ErbB/Her2 to image tumors expressing these receptors in an animal model. As ligand for the CCK2 receptor, we chose D-Glu1-minigastrin, a peptide which had already been employed for the scintigraphic imaging of this receptor. D-Glu1-minigastrin could be coupled with the activated dye Cy5.5-NHS, but in vitro binding studies showed that the construct does not represent a high-affinity ligand to the CCK2 receptor. A possible rationale for this is that the receptor binding of the peptide is negatively affected by the covalently bound hydrophobic dye. In vivo studies supported the data collected in the binding assays.
Herceptin, an antibody used in the therapy of metastasizing Her2-positive breast cancer, was chosen as a suitable ligand for imaging the ErbB/Her2 receptor. In addition, the F(ab’)2 - and F(ab’)-fragments of herceptin were studied as possible ligands. We expected them to show a more favorable signal to noise ration due to their lower molecular weight and therefore faster blood clearance. The ligands could be successfully prepared, coupled to Cy5.5-NHS. The IC50 values obtained from binding assays with herceptin and its antibody fragments indicate a high-affinity interaction with the ErbB/Her2 receptor. Encouraged by these results, we turned to microscopic investigations with ErbB/Her2-positive SKOV3 cells. For this purpose, the cells were incubated for up to 24 h with either herceptin, its F(ab’)2 - or its F(ab)’ fragment. After 9 h in case of the antibody fragments and 24 h in case of herceptin, distinct fluorescence signals could be observed inside the cells, indicating efficient cellular internalization of the receptor-ligand complex.
After having shown in vitro as well as in cell culture assays that the Cy5.5-labeled antibodies and their fragments efficiently bind to ErbB/Her2 receptors, we tried in vivo imaging of mice carrying Her2-positive tumors by NIR-fluorescence reflectance techniques. The ErbB2-positive tumors could be successfully imaged with our dye-labeled ligands Cy5.5-herceptin and Cy5.5-F(ab’). In case of the Cy5.5-F(ab’)2 fragment however, only poor contrast between tumor and surrounding tissue could be obtained, possibly due to less favorable pharmacokinetic properties of the F(ab’)2 fragment.
Possible clinical applications of these results may arise in the non-invasive diagnostics of ErbB/Her2neu-positive breast tumors. Once the receptor-status of a diagnosed tumor is established using the previously discussed imaging techniques, a rational therapy using herceptin can be envisaged. Further applications of fluorescence imaging with herceptin derivatives are the intraoperative or laparoscopic identification of abdominal metastases of ErbB/Her2neu-positive ovarial, cervical, colon or breast tumors, using existing endoscopic fluorescence imaging apparatus.
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Created: 2008Issued: 2008-03-17Updated: 2011-08-10
Faculty
Medizin
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Philipps-Universität Marburg
Language
ger
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DoctoralThesis
Keywords
Molekulare BildgebungErbB2 receptorErbB2FluorescenceHerceptinMolecular imagingHerceptinFluorezenz
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RezeptorBildgebendes VerfahrenTrastuzumab
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610
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Tischer, Nadine: Molekulare in vivo Fluoreszenzbildgebung zur Darstellung von ErbB/Her2- und CCK2-rezeptorpositiven Tumoren im Tiermodell. : Philipps-Universität Marburg 2008-03-17. DOI: https://doi.org/10.17192/z2008.0254.
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This item has been published with the following license: In Copyright