Development of recombinant receptor tyrosine kinase ligands for diagnostics and therapy of tumors based on radionuclides

Introduction
Several receptor tyrosine kinases, like the epidermal growth factor receptor (EGFR) play a critical role in tumors and, therefore, are promising targets for tumor imaging and tumor therapy. The present work aims at development of novel recombinant EGF analogs as potential radioligands or receptor-targeted components for functionalization of nanoparticles. This approach uses selective alteration of protein structure by unnatural amino acids, which amenables the protein for both radiolabeling and functionalization by the Click-chemistry.

Material and methods
The structural alteration of the recombinant protein is possible by supplementation based incorporation, because E. coli tRNA can not differ between methionine and azidohomoalanine. So these two amino acids can be exchanged during protein biosynthesis. Therefore, azidohomolalanine was added into methionine-free medium during cultivation of transformed bacteria. Vectors with both GST-tag and His-tag and corresponding methods for purification were tested and first experiments for the Click reaction of the structurally modified protein with biotinylated alkynes, fluorescence dye-labeled and ¹⁸F labeled alkynes were realised.

Results and conclusion
The purification of the GST fusion protein only resulted in the unmodified protein, but not in the azido-functionalised protein. Subsequently, native purification of a His-tagged EGF was established and the incorporation of azides into the target protein could be demonstrated by Click reaction with an alkyne. Ongoing work focuses on optimization of this approach. Afterwards, the purified and labeled proteins will be tested concerning their pharmacological properties as potential radioligands or targeted components of functionalized nanoparticles.