Tumor targeting and molecular imaging of protein markers specific for or overexpressed in tumors can add useful information in deciding upon treatment and assessing the response to treatment for a cancer patient. The epidermal growth factor receptor (EGFR) is one such tumor-associated receptor, which expression is abnormal or upregulated in various cancers and associated with a poor patient prognosis. It is therefore considered a good target for imaging and therapy. Monoclonal antibodies and recently also antibody fragments have been investigated for in vivo medical applications, like therapy and imaging. In molecular imaging a small sized targeting agent is favorable to give high contrast and therefore, antibody fragments and lately also small affinity proteins based on a scaffold structure constitute promising alternatives to monoclonal antibodies. Affbody molecules are such affinity proteins that are developed by combinatorial protein engineering of the 58 amino acid residue Z-domain scaffold, derived from protein A. In this thesis, novel Affibody molecules specific for the EGFR have been selected from a combinatorial library using phage display technology. Affibody molecules with moderate high affinity demonstrated specific binding to native EGFR on the EGFR-expressing epithelial carcinoma A431 cell line. Further cellular assays showed that the EGFR-binding Affibody molecules could be labeled with radiohalogens or radiometals with preserved specific binding to EGFR-expressing cells. In vitro, the Affibody molecule demonstrated a high uptake and good retention to EGFR-expressing cells and was found to internalize. Furthermore, successful imaging of tumors in tumor-bearing mice was demonstrated. Low nanomolar or subnanomolar affinities are considered to be desired for successful molecular imaging and a directed evolution to increase the affinity was thus performed. This resulted in an approximately 30-fold improvement in affinity, yielding EGFR-binding Affibody molecules with KD´s in the 5-10 nM range, and successful targeting of A431 tumors in tumor-bearing mice. To find a suitable format and labeling, monomeric and dimeric forms of one affinity matured binder were labeled with 125I and 111In. The radiometal-labeled monomeric construct, 111In-labeled-ZEGFR:1907, was found to provide the best tumor-to-organ ratio due to good tumor localization and tumor retention. The tumor-to-blood ratio, which is often used as a measure of contrast, was 31±8 at 24 h post injection and the tumor was clearly visualized by gamma-camera imaging. Altogether, the EGFR-binding Affibody molecule is considered a promising candidate for further development of tumor imaging tracers for EGFR-expressing tumors and metastases. This could simplify the stratification of patients for treatment and the assessment of the response of treatment in patients. / QC 20100723