Radiolabeling of tetrazines has gained increasing attention due to their important role in pretargeted imaging or therapy. The most commonly used radionuclide in PET imaging is fluorine-18. For this reason, we have recently developed a method which enables the direct aromatic ¹⁸F-fluorination of tetrazines using stannane precursors through copper-mediated fluorinations. Herein, we further optimized this labeling procedure. 3-(3-fluorophenyl)-1,2,4,5-tetrazine was chosen for this purpose because of its high reactivity and respective limited stability during the labeling process. By optimizing parameters such as elution conditions, precursor amount, catalyst, time or temperature, the radiochemical yield (RCY) could be increased by approximately 30%. These conditions were then applied to optimize the RCY of a recently successfully developed and promising pretargeting imaging agent. This agent could be isolated in a decay corrected RCY of 14 ± 3% and Am of 201 ± 30 GBq/µmol in a synthesis time of 70 min. Consequently, the RCY increased by 27%.

Background: Peptides that selectively bind to cancer-associated proteins are attractive candidates for molecular imaging and targeted therapeutic applications. The urokinase plasminogen activator receptor (uPAR) represents a promising molecular target as it is highly expressed in several cancers and has been linked to cancer aggressiveness, invasion, and metastasis. This study applied an NGS-informed biopanning strategy against uPAR, combined with multi-assay downstream validation, to identify and prioritize candidate peptides. Methods: An optimized biopanning protocol incorporating additional selection controls was combined with next-generation sequencing to identify candidate uPAR-binding peptides from a phage display library. Selected peptides were evaluated using complementary in vitro assays, including flow cytometry and a quantitative plate assay. Results: Three peptide candidates were identified and selected for further evaluation with complementary in vitro assays. The optimized workflow incorporates measures to reduce, but not eliminate, enrichment of target-unrelated peptides, and includes complementary validation of the peptides in in vitro assays. Differences observed between the validation methods highlighted the importance of evaluating peptide candidates using complementary assays. Conclusions: This study demonstrates the value of combining an optimized phage display selection with next-generation sequencing and complementary validation assays for identifying candidate peptides. The identified peptides represent promising candidates for further optimization toward molecular imaging and targeted therapeutic applications.