Antigen multimers: Specific, sensitive, precise, and multifunctional high-avidity CAR-staining reagents

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. [Display omitted] •Antigen multimers detect CARs with high specificity, sensitivity, and precision•Antigen multimers enable magnetic enrichment and stimulation of CAR T cells•Antigen multimers can isolate and phenotype CAR T cells from patient biospecimens•Antigen multimers are readily extensible to existing and new CARs Chimeric antigen receptor (CAR) T cell therapy involves modifying a cancer patient's T cells to kill cancer cells. However, better CAR-staining tools are required to ensure CAR manufacturing quality control, analyze treatment effects, decipher biological mechanisms, and engineer new formulations. To meet this critical need, we engineered antigen multimers, reagents that specifically bind CARs with high avidity. In addition to high CAR-detection sensitivity and precision, antigen multimers broadly support many functions, including magnetic enrichment of rare CAR T cells, selective CAR T cell stimulation for phenotyping, and high-purity isolation of CAR T cells from patient biospecimens for multi-omics profiling. Since antigen multimers are readily extensible to existing and new CAR systems, antigen multimers can keep pace with the fast development of next-generation CARs. Therefore, we expect broad and sustainable applications for antigen multimers in laboratories and clinics. More effec