Summary Although chimeric antigen receptor (CAR) T cell therapy has transformed cancer treatment, high-quality and universal CAR-staining reagents are urgently required to manufacture CAR T cells, predict therapy response, decipher CAR biology, and engineer new CARs. Here, we developed tetrameric and dodecameric forms of a multifunctional and extensible category of high-avidity CAR-staining reagents: antigen multimers. Antigen multimers detected CARs against CD19, HER2, and Tn-glycoside with significantly higher specificity, sensitivity, and precision than existing reagents. In addition to accurate CAR T cell detection by flow cytometry, antigen multimers also enabled ≥100-fold magnetic enrichment of rare CAR T cells, selective CAR T cell stimulation, and high-dimensional CAR T cell profiling by single-cell multi-omics analyses. Finally, antigen multimers accurately captured clinical anti-CD19 CAR T cells from patients' cellular infusion products, post-infusion peripheral blood, and tumor biopsies. Antigen multimers can be readily extended to other CAR systems by switching the antigen ligand. As such, antigen multimers have broad clinical and research applications.
