In Situ Bioorthogonal Labeling for Non-Invasive, Dynamic In Vivo Nirf/PET Imaging of CAR-T Cells

Objective: With the rapid advancement of chimeric antigen receptor T cell (CAR-T) immunotherapy, CAR-T products have demonstrated significant efficiencies in refractory and relapsed hematologic neoplasms. As “living cellular drugs”, the absorption, distribution, metabolism, and excretion of CAR-T cells do not conform to the pharmacokinetics of traditional drugs. In order to enhance its therapeutic efficacy, further investigation is required into the migration, survival, and biological distribution of CAR-T cells within the body, as well as their impact on target sites, quantity, persistence, and adverse reactions. However, traditional tracking methods are often limited by technical constraints and fail to provide sufficient information to monitor cell dynamics within the body, and there is an increasing demand for tracking CAR-T cells dynamically in vivo. In this study, we employed a novel approach utilizing glycometabolic engineering and in situ biorthogonal chemistry labeling technology for in vivo tracking of CAR-T cells in mouse models of subcutaneous lymphoma.

Methods: Azide motifs are anchored on the CAR-T cell surface via the intrinsic glycometabolism of exogenous azide-glucose, serving as an artificial ligand for imaging probes binding, confirmed by flow cytometry and confocal laser microscopy. In vitro activity and cytotoxicity of the chemically engineered anti-CD19-CAR-T (denoted as N₃-CART) cells were determined. Then, N₃-CART cells were intravenously infused into NOD-scid-IL2Rγnull (NSG) mice subcutaneously engrafted with Raji-luc cells. The feasibility of monitoring proliferation profiles and tracking N₃-CART was verified by introducing the complementary functional moiety dibenzocyclooctyl (DBCO)-conjugated near-infrared fluorescence (NIRF) and nuclide probes (eg. DBCO-ICG and DBCO-DOTA-⁶⁸Ga) into the mice, and the NIRF and positron emission tomography (PET) imaging were performed. In addition, the pharmacodynamics and pharmacokinetics of the N₃-CART cells in mouse subcutaneous lymphoma models were simultaneously assessed using bioluminescence imaging and DBCO-DOTA-⁶⁸Ga PET imaging.

Results: The azide labeling based on biological orthogonal chemistry would not affect the activity and killing function of anti-CD19-CAR-T cells in vitro. We achieved dynamic tracking of them by NIRF in vivo for two weeks, and PET images revealed that the uptakes of in CD19+ Raji subcutaneous tumors showed an increasing tendency from 30 min to 120 h p.i. after administration of N₃-CART cells. It confirmed that the N₃-CART cells could effectively inhibit the growth of Raji hematologic subcutaneous tumors.

Conclusion: This study presents a novel approach utilizing bioorthogonal labeling technology for imaging of CAR-T cells. We achieved dual-modal, non-invasive and dynamic tracking of CAR-T cells, providing a comprehensive view of their biodistribution and kinetics of infiltration within the body. This method provides a new means for monitoring and optimizing CAR-T therapy and is expected to further advance the field of CAR-T cell therapy.