Chemistry and Synthesis Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Rockville, Maryland 20892-0001, United States.
Molecular Imaging Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United States.
Bioconjug Chem. 2024 Sep 18;35(9):1335-1342. doi: 10.1021/acs.bioconjchem.4c00264. Epub 2024 Aug 22.
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 (F) is routinely used for clinical imaging, it is an attractive radioisotope for Nbs. We report a novel sortase-based, site-specific F-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[F]FHRESCA)-NH) or the unlabeled chelator (H-GGGK((±)-HRESCA)-NH) followed by labeling with Al[F]F, respectively. The overall radiochemical yields were 15-43% ( = 22, decay-corrected) in 70 min (indirect labeling) and 23-58% ( = 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 ( = 22) for each of the three Nbs tested and exhibited excellent stability profiles under physiological conditions. This simple, site-specific, reproducible, and generalizable F-labeling method to prepare nanobodies (Nb-Al[F]F-RESCA) or other low molecular weight biomolecules can easily be adopted in various settings for preclinical and clinical studies.
单域抗体，又称纳米抗体（Nbs），由于其亲和力、特异性高，在血液中清除速度快，是分子成像中很有前途的生物分子。鉴于其血液半衰期短，将 Nbs 与短寿命的成像放射性同位素结合使用是理想的。由于氟-18（F）常用于临床成像，因此它是 Nbs 的一种有吸引力的放射性同位素。我们报告了一种新的基于 sortase 的、位点特异性的 F 标记方法，应用于三种纳米抗体。标记的纳米抗体是通过两步间接放射性标记一锅法或通过一步直接标记法合成的，分别使用 sortase 介导的放射性标记螯合剂（H-GGGK((±)-Al[F]FHRESCA)-NH）或未标记的螯合剂（H-GGGK((±)-HRESCA)-NH）的缀合，然后用 Al[F]F 进行标记。间接标记法的总体放射化学产率为 15-43%（ = 22，衰减校正），70 分钟内完成；直接标记法的总体放射化学产率为 23-58%（ = 12，衰减校正），50 分钟内完成。两种方法制备的标记纳米抗体的放射化学纯度均>98%，每个测试的三种 Nb 的比活度为 400-600 Ci/mmol（ = 22），在生理条件下表现出极好的稳定性。这种简单、位点特异性、可重复且可推广的 F 标记方法可用于制备纳米抗体（Nb-Al[F]F-RESCA）或其他低分子量生物分子，可轻松应用于临床前和临床研究的各种环境。
