Real-Time Monitoring of CAR-T Cell Efficiency through a Biorthogonal Cycloaddition Labeling Strategy

Chimeric antigen receptors (CARs) recognizing tumor-associated antigens (TAAs) effectively target tumor cells without using the major histocompatibility complex (MHC). However, CARs have inaccurate dose determination in clinical practice, and the methods that can solve this problem often produce cytotoxic substances, such as green fluorescent protein (GFP) insertion. Therefore, in this study, we tried to anchor harmless fluorescent labels on CAR-T cell membranes using highly biologically compatible strain-promoted alkyne-azide cycloaddition (SPAAC) without any byproducts. Our conjugated fluorescent label was stable on the CAR-T cell surface for at least two weeks, with excellent light stability and metrology. Also, this method enabled the rapid quantification of the living CAR-T cells without affecting their activity. Thus, this method is a promising reliable strategy for accurately diagnosing and treating cancer.