Metabolic Glycoengineering Enables Fluorine-18 Radiolabeling of T Lymphocytes via Dual-Bioorthogonal Chemistry

The ability to track therapeutic cells is critical for advancing adoptive cell therapy. Positron emission tomography (PET) offers sensitive, quantitative imaging, but improved strategies for cell labeling remain needed. Here, we report a metabolic glycoengineering approach that installs azide groups onto the Jurkat T lymphocyte surface using the canonical tetraacetylated N-azidoacetylmannosamine (Ac₄ManNAz). Azide-bearing cells were functionalized via strain-promoted azide-alkyne cycloaddition (SPAAC-based ligation) with a dual-clickable trancyclooctene (TCO)-bearing dibenzocyclooctyne (DBCO) derivative (sulfo-DBCO-PEG₄-TCO), enabling subsequent inverse electron-demand Diels-Alder (IEDDA, TCO-tetrazine ligation) radiolabeling at cell-surface TCO moieties using an aluminum [¹⁸F]fluoride-tetrazine (Al[¹⁸F]F-Tz) tracer. Labeling conditions were optimized to achieve suitable cell-associated activity while maintaining good viability. We evaluated Al[¹⁸F]F-Tz pharmacokinetics, in vitro fluorine-18-labeled Jurkat cells, and a proof-of-concept pretargeting strategy in athymic nude mice. In vitro fluorine-18-labeled cells exhibited predictable trafficking and biodistribution over the imaging period, supporting the feasibility of MGE-based bioorthogonal radiolabeling for PET cell tracking. In contrast, pretargeted imaging requires further optimization and was dominated by the hepatic and intestinal signal consistent with hepatobiliary clearance of Al[¹⁸F]F-Tz. Overall, these findings underscore the potential of MGE-mediated bioorthogonal radiolabeling as a nongenetic platform for PET tracking of immune cells and provide a foundation for further development of pretargeted approaches for adoptively transferred cells.