Radiolabeling of modified recombinant epidermal growth factor (EGF) by Huisgen-Click reaction for multimodal imaging and therapy of tumors

Introduction
The epidermal growth factor receptor (EGFR) plays a critical role in epithelial tumors and, therefore, is a promising target for both tumor imaging and therapy. In order to develop novel EGFR-targeted radiotracers/radiotherapeutics the present work aims at synthesis of structurally modified recombinant EGF. One strategy implies incorporation of azidohomoalanine instead of methionine using supplementation-based incorporation by E. coli. Consequently, the biomolecule can be labeled site-selectively by Click reaction (Huisgen 1,3-dipolar cycloaddition) with fluorescence and radiolabeled building blocks, respectively.

Method and Results
The EGF gene motif was successfully ligated into vectors containing either a GST-tag or His6-tag. Resulting plasmids were transformed into methionine-auxotrophic E. coli (B834(DE3)pLysS), protein synthesis under addition of azidohomoalanine and protein purification were established. Purification of the GST fusion protein only resulted in the unmodified protein, but not in the azido-functionalized protein. In contrast, purification of His6-tagged azido-functionalized EGF was successfully established. This could be demonstrated by reaction with various alkynylated fluorescent dyes. Moreover, the modified EGF was radiolabeled with 1-(but-3-inyl)-4-(3-[18F]fluoropropyl)piperazine ([18F]BFP) as the alkyne with a radiochemical yield of about 0.5 % using Click reaction. First cell binding studies of [18F]BFP-labeled EGF demonstrated cellular association in EGFR-overexpressing A431 human epidermoid carcinoma cells. Cellular uptake amounted to about 25 % of injected dose/mg protein after 1 hour at 37°C.

Conclusion
Ongoing work focuses on the optimization of site-selective radiolabeling of EGF by Click reaction with higher radiochemical yield and specific activity. Afterwards, the radiolabeled protein will be radiopharmacologically tested concerning its properties as potential radiotracer.