Development of antigen-dextramers for detection and evaluation of CAR T cells

Abstract

BackgroundChimeric antigen receptor (CAR) T cell therapy has transformed the treatment landscape of hematologic cancers by engineering T cells to specifically target and destroy cancer cells. Monitoring CAR T cell activity and function is essential for optimizing therapeutic outcomes, but existing tools for CAR detection are often limited in specificity and functional assessment capability. MethodsWe developed antigen-dextramers by conjugating multiple CAR-specific antigens to a dextran backbone. The dextramers were compared to previously reported antigen-tetramers for their ability to stain and detect CAR T cells. Because these multimers incorporate the CAR target antigen, they uniquely enable assessment of CAR T cell functionality by facilitating binding and activation analyses. We tested the staining and functional properties of the multimers across a range of CAR constructs with different affinities, using flow cytometry, microscopy, and NFAT-luciferase reporter assays. ResultsThe antigen-dextramers demonstrated high specificity and sensitivity in staining CAR T cells, with adjustable antigen density to optimize binding. Antigen-dextramers also enabled effective clustering and subsequent activation of CARs, showing their utility as both a staining and functional assessment tool. The dextramers revealed that CARs with different affinities and clustering tendencies displayed varied binding and activation in response to different antigen densities. ConclusionAntigen-dextramers offer a dual advantage as versatile reagents for both staining and functional analysis of CAR T cells. Their capacity to engage CARs with the specific antigen provides a valuable platform for evaluating CAR functionality, informing CAR design improvements, and enhancing therapeutic precision.