Metabolic glycoengineering enables fluorine-18 radiolabeling of T lymphocytes via dual-bioorthgonal chemistry

The ability to track therapeutic cells is critical for advancing adoptive cell therapy (ACT). Positron emission tomography (PET) offers sensitive and quantitative imaging, yet improved cell radiolabeling strategies are sorely needed. We report a metabolic glycoengineering (MGE) approach that installs azide moieties on Jurkat T lymphocyte surface using the canonical derivative Ac₄ManNAz, enabling bioorthogonal radiolabeling via strain-promoted azide– alkyne cycloaddition (SPAAC) and inverse electron-demand Diels–Alder (IEDDA) chemistry. A dual-clickable sulfo-DBCO-PEG₄-TCO intermediate was synthesized to bridge these functionalities, allowing radiolabeling with aluminum [¹⁸F]fluoride-tetrazine (Al[¹⁸F]F-Tz) through cell-surface TCOs. Optimization of the labeling conditions yielded robust in vitro radiolabeling without compromising cell viability. Both Al[¹⁸F]F-Tz and in vitro fluorine-18-labeled Jurkat cells were subsequently evaluated in vivo in athymic nude mice, alongside preliminary pretargeting studies with TCO-modified cells. In vitro fluorine-18-labeled cells exhibited predictable trafficking and biodistribution, supporting the suitability of this radiolabeling strategy for cell tracking with PET, whereas pretargeted imaging requires further optimization, revealing mostly the hepatobiliary excretion of the Al[¹⁸F]F-Tz tracer. Overall, these findings underscore the potential of MGE-mediated bioorthogonal radiolabeling for noninvasive tracking of immune cells and provide a foundation for further development of pretargeted imaging approaches for cell transplants.