Optimizing radiocobalt labeled HER3-targeting affibody molecules for next day PET-imaging of HER3 expression

445 Objectives: Overexpression of the human epidermal growth factor receptor type 3 (HER3) in cancer is related to disease progression and therapy resistance. Imaging of HER3 expression could improve patient management. However, imaging contrast is limited by low target expression and natural expression, particularly in the liver. We previously demonstrated the feasibility of radiolabeled anti-HER3 affibody molecules for PET imaging of HER3 expression in preclinical models. We observed that increased negative charge of the radiometal-chelator complex, conjugation of a hydrophilic (HE)3-tag and delayed imaging (up to 24 h pi) could reduce hepatic uptake and improve the tumor-to-liver contrast of anti-HER3 affibody molecules. Cobalt-55 is a positron-emitting isotope with an intermediate half-life of 17.5 h allowing for later time point imaging compared to conventional, shorter-lived isotopes for PET, such as 68Ga. Aim of this study was to first, investigate the influence of different radiocobalt-chelator complexes on the anti-HER3 affibody molecule (HE)3-ZHER3 and then to compare the most favorable radiocobalt-labeled variant with the recently optimized 68Ga-labeled variant to evaluate the potential benefit of later time point PET-imaging of HER3 expression. Methods: (HE)3-ZHER3-X (X=NOTA, NODAGA, DOTA, DOTAGA) was labeled with [57Co]Co as a surrogate for 55Co. Stability was tested in PBS and human serum. Binding specificity and cellular processing were investigated in two HER3-expressing cancer cell lines. Binding kinetics were studied in living BxPC3 cells in real-time. In vivo specificity and biodistribution were studied in Balb/c nu/nu mice with BxPC-3 xenografts 3 h and 24 h pi and compared to [68Ga]Ga-(HE)3-ZHER3-NODAGA. microSPECT/CT imaging was performed at 3 h and 24 h pi. Results: (HE)3-ZHER3-X was stably labeled with radiocobalt. In vitro, specific binding to HER3 was preserved after labeling. Overall, internalization of all conjugates was slow, with an internalized fraction below 5% of cell-associated activity after 24 h in both cell lines. Binding affinities were in picomolar range. All conjugates except [57Co]Co-(HE)3-ZHER3-DOTAGA demonstrated specific uptake in tumors and mErbB3 expressing organs. Clearance of [57Co]Co-(HE)3-ZHER3-X from blood was rapid (