PROJECT SUMMARY CAR T cell therapy offers new hope to patients with relapsed/refractory B cell malignancies and has become another weapon in the war on cancer. Despite the success and excitement around CAR therapies, sensitive and universal CAR-labeling reagents are required for accurate CAR T cell detection and profiling in research, industry, and in the clinic. To improve CAR T cell therapy efficacy, scientists, clinicians, and the pharmaceutical industry need accurate and multifunctional CAR-staining reagents to perform downstream assays, conduct preclinical studies, profile patient biospecimens, and develop new CARs. To overcome this technology gap, we developed antigen-multimers – high-avidity CAR detection reagents. Akin to conventional MHC multimers, our preliminary data demonstrate that antigen-multimers sensitively and specifically detect CD19-directed CAR T cells in commercial infusion products, in peripheral blood, and in tumor biopsies of diffuse large B cell lymphoma patients. Furthermore, we show that CD19-multimers can be readily employed to isolate CAR T cells from patient samples at different treatment stages for downstream flow cytometry and single-cell multi-omics analyses. In our application, based on our exciting preliminary observations, we propose to test the overarching hypothesis that antigen-multimers will enable CAR T cell detection and isolation and facilitate downstream single-cell analyses of CAR T cells from patient biospecimens. Our antigen-multimers can be used to generate new knowledge regarding CAR T cell function and in vivo efficacy. In Specific Aim 1, we propose to develop antigen-multimers for high-sensitivity CAR T cell detection and isolation. In addition to demonstrating the flexibility of our technology by generating 4 different CAR-detecting antigen-multimers, we will also develop approaches for simultaneously isolating and restimulating CAR T cells from patient samples in order to enable downstream phenotyping and functional analyses. In Specific Aim 2, we propose to develop antigen-multimer-enabled single CAR T cell multicolor flow cytometry and multi-omics. We will first use CD19-multimers to determine CAR expression, cell number, differentiation, functional phenotype, and persistence of CAR T cells by multicolor flow cytometry. We will next use CD19-multimers to isolate CAR T cells from responder and non-responder patients for subsequent single- cell multi-omics analyses. Leveraging the experience of two investigators with complementary expertise in immunoengineering (Huang) and translational/clinical CAR T cell therapy research (Kline), as well as the UChicago Cell Therapy Biobank which contains hundreds of lymphoma and myeloma patient samples treated with CD19- or BCMA-directed CAR T cell therapy, the proposed experiments will establish antigen-multimers as a new class of CAR-staining reagents with broad research and clinical applications.