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 its antigen ligand. As such, antigen-multimers have broad clinical and research applications.
Keywords: antigen-multimer; chimeric antigen receptor; detection; dodecamer; enrichment; extensible; sensitivity; single-cell omics; stimulation; tetramer.