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Quantification of internalization of EGFR-binding Affibody molecules: Methodological aspects
Lovisa Göstring1, Ming Tsuey Chew, Anna Orlova
1Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden. lovisa.gostring@bms.uu.se
International Journal of Oncology
|March 4, 2010
Summary
Quantitative fluorescence methods effectively measure tumor targeting agent internalization, crucial for radionuclide imaging and therapy. The Alexa488-quenching assay provides reliable internalization data for Affibody molecules and cetuximab in A431 cells.
Area of Science:
- Biomedical imaging
- Molecular imaging
- Radiopharmaceutical science
Background:
- Tumor cell internalization of targeting agents impacts radionuclide retention, affecting PET/SPECT imaging and therapy.
- Quantitative fluorescence techniques offer alternatives to nuclear methods for studying cellular internalization.
Purpose of the Study:
- To evaluate and compare fluorescence-based methods (CypHer and Alexa488-quenching) for studying cellular internalization of targeting agents.
- To quantify the internalization of EGFR-binding Affibody molecules (monomer Z1907 and dimer (Z1907)2) in A431 cells.
Main Methods:
- Application of fluorescence microscopy and flow cytometry using CypHer and Alexa488-quenching assays.
- Analysis of Affibody molecules (Z1907, (Z1907)2), EGF, and cetuximab in A431 cells.
- Comparison with the acid wash assay for 111In-labeled Z1907 internalization.
Main Results:
- The Alexa488-quenching method is preferred due to its independence from the dye-to-protein ratio.
- Internalization within one hour: ~45% for EGF, 19-24% for Affibody molecules and cetuximab.
- The Alexa488-quenching method successfully quantified internalization for agents resistant to acid wash, unlike the acid wash assay itself.
Conclusions:
- The fluorescence-based Alexa488-quenching method is well-suited for quantitative analysis of targeting agent internalization, including acid-resistant agents.
- Both monomeric and dimeric Affibody molecules are expected to exhibit good uptake and radionuclide retention, leading to favorable tumor-to-background ratios.
