Affibody molecules constitute a new class of probes for radionuclide tumor targeting. The small size of Affibody molecules is favorable for rapid localization in tumors and clearance from circulation. However, high renal reabsorption of Affibody molecules prevents the use of residualizing radiometals, including several promising low-energy β-and α-emitters, for radionuclide therapy. We tested a hypothesis that Affibody-based pretargeting mediated by a bioorthogonal interaction between trans-cyclooctene (TCO) and tetrazine would provide higher accumulation of radiometals in tumor xenografts than in the kidneys. Methods: TCO was conjugated to the anti-human epidermal growth factor receptor 2 (HER2) Affibody molecule Z 2395 . DOTA-tetrazine was labeled with 111 In and 177 Lu. In vitro pretargeting was studied in HER2-expressing SKOV-3 and BT474 cell lines. In vivo studies were performed on BALB/C nu/nu mice bearing SKOV-3 xenografts. Results: 125 I-Z 2395 -TCO bound specifically to HER2-expressing cells in vitro with an affinity of 45 ± 16 pM. 111 In-tetrazine bound specifically and selectively to Z 2395 -TCO pretreated cells. In vivo studies demonstrated HER2-specific 125 I-Z 2395 -TCO accumulation in xenografts. TCO-mediated 111 In-tetrazine localization was shown in tumors, when the radiolabeled tracer was injected 4 h after an injection of Z 2395 -TCO. At 1 h after injection, the tumor uptake of 111 In-tetrazine and 177 Lu-tetrazine was approximately 2-fold higher than the renal uptake. Pretargeting provided more than a 56-fold reduction of renal uptake of 111 In in comparison with direct targeting. Conclusion: The feasibility of Affibody-based bioorthogonal chemistry-mediated pretargeting was demonstrated. The use of pretargeting provides a substantial reduction of radiometal accumulation in kidneys, creating preconditions for palliative radionuclide therapy. Radi onuclide targeting of tumor-specific overexpressed cell surface proteins has a potential to improve cancer therapy. Radionuclide imaging might help to identify patients with tumors expressing a particular target and therefore would be likely to respond to a particular therapy based on antibodies or tyrosine kinase inhibitors (1). Delivery of cytotoxic radionuclides (e.g., b-or a-emitters) might have strong palliative or even curative effects (2).Two classes of substances have commonly been exploited for radionuclide targeting: monoclonal antibodies (and their derivatives) and peptide ligands to receptors overexpressed in tumors. Engineered scaffold proteins (ESPs) constitute a new type of targeting agents for radionuclide tumor targeting (3). ESPs contain a robust framework providing conformational stability and variable surface amino acids. Randomization of variable amino acids enables selection of high-affinity binders to different proteins using molecular display technology. Several ESPs, such as Affibody (Affibody AB) molecules (4), designed ankyrin repeat proteins (5), fibronectin domains (6), anticalins (7), cysteine-knot peptides (8), and albu...