Sortase-Mediated Site-Specific Conjugation to Prepare Fluorine-18-Labeled Nanobodies

Single-domain antibodies, or nanobodies (Nbs), are promising biomolecules for use in molecular imaging due to their excellent affinity, specificity, and fast clearance from the blood. Given their short blood half-life, pairing Nbs with short-lived imaging radioisotopes is desirable. Because fluorine-18 (¹⁸F) is routinely used for clinical imaging, it is an attractive radioisotope for Nbs. We report a novel sortase-based, site-specific ¹⁸F-labeling method applied to three nanobodies. Labeled nanobodies were synthesized either by a two-step indirect radiolabeling method in one pot or by a one-step direct labeling method using a sortase-mediated conjugation of either the radiolabeled chelator (H-GGGK((±)-Al[¹⁸F]FH₃RESCA)-NH₂) or the unlabeled chelator (H-GGGK((±)-H₃RESCA)-NH₂) followed by labeling with Al[¹⁸F]F, respectively. The overall radiochemical yields were 15-43% (<i>n</i> = 22, decay-corrected) in 70 min (indirect labeling) and 23-58% (<i>n</i> = 12, decay-corrected) in 50 min (direct labeling). The radiochemical purities of the labeled nanobodies prepared by both methods were >98% with a specific activity of 400-600 Ci/mmol (<i>n</i> = 22) for each of the three Nbs tested and exhibited excellent stability profiles under physiological conditions. This simple, site-specific, reproducible, and generalizable ¹⁸F-labeling method to prepare nanobodies (Nb-Al[¹⁸F]F-RESCA) or other low molecular weight biomolecules can easily be adopted in various settings for preclinical and clinical studies.