Development of antigen multimers for detection and evaluation of CAR T cells.

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 dextran multimers by conjugating multiple CAR-specific antigens to a dextran backbone. The multimers 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. We tested the staining and functional properties of the multimers across a range of CAR constructs with different affinities, using flow cytometry and microscopy.

resultsThe dextran multimers demonstrated high specificity and sensitivity in staining CAR T cells, with adjustable antigen density to optimize binding. Dextran multimers also enabled effective clustering and subsequent activation of CARs, showing their utility as both a staining and functional assessment tool. The multimers revealed that CARs with different affinities and clustering tendencies displayed varied binding and activation in response to different antigen densities.

conclusionDextran multimers 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.