In this project, a panel of ten individual anti-Kv10.1 nanobodies was generated by phage display and subsequent screening on the fusion protein H1X. Molecular
4 Discussion 91
characterization of the binding properties revealed two clones, C4 and D9, detecting specifically Kv10.1 transfected HEK cells and the fusion protein H1X.
For a later in vivo tumor visualization applying the anti-Kv10.1 nanobodies C4 and D9, a clinical SPECT system was set up and the infrastructure for the handling and housing of radioactive animals was established. This allowed the in vivo tumor visualization on EGFR expressing tumors with the clinically applied anti-EGFR antibody 99mTc-Cetuximab and demonstrated as proof of principle the successful application of multi-pinhole SPECT. Tumors expressing high levels of EGFR and a volume above 100 mm3 were reliably detected by in vivo SPECT, whereas tumors with a relatively low EGFR expression showed a tumor uptake of 99mTc-Cetuximab similar to the unspecific accumulation of the 99mTc-isotype antibody.
The anti-EGFR nanobody D10 was used in vivo for the first time. It could be shown that
99mTc-D10 represents a versatile tool for the specific detection of small EGFR overexpressing tumor lesions and the assessment of EGFR expression in tumors by in vivo SPECT. The identification of tumors at the earliest stage with 99mTc-D10 allowed the acquisition of high contrast images with a high tumor to background background ratio shortly after probe administration, i.e. allowing imaging at the same day.
Therefore, 99mTc-D10 might be suitable in a clinical setting as non-invasive diagnostic tracer to not only detect small tumors but also to obtain information on the expression level of EGFR in heterogenic tumors at the time of diagnosis and during disease progression.
5 Outlook 92
5 Outlook
The anti-Kv10.1 nanobodies C4 and D9, that were generated in the course of this project, show very promising binding characteristics to this challenging target, but specific detection of the native ion channel remains to be validated. The successful application as in vivo tracers will depend on the tumor entity and the number of expressed potassium channels on the tumor cells as well as affinity of an engineered anti-Kv10.1 nanobody to its target protein. The application of the nanobodies as unmodified monomers might not lead to a successful tumor detection. However, genetically engineered bi- or trivalent nanobodies could facilitate this task.
The impact of the anti-Kv10.1 nanobodies on the potassium channel was not investigated yet. A possible impaired signaling due to the binding anti-Kv10.1 nanobodies C4 or D9 could refer to a therapeutic effect and an establishment of a specific Kv10.1 targeted therapy in future.
With the generation of the anti-Kv10.1 nanobodies C4 and D9, a versatile toolkit consisting of the two nanobodies with different fluorophores and enzymes for the detection of Kv10.1 are soon available for the integration in various laboratory methods like flow cytometry, immunofluorescence microscopy, histology, ELISA or Western blotting.
The anti-EGFR nanobody D10, which was applied in an in vivo SPECT approach for the first time, allowed a tumor visualization of small tumors with a high contrast shortly after administration. However, the high kidney uptake caused by the 6xHis tag remains problematic for further in vivo studies. Therefore, the nanobody needs to be reengineered and the use of different chelators might help to reduce the kidney uptake to continue the translation of D10 to larger tumor animal models.
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