Mechanical Properties Measured by Atomic Force Microscopy Help Evaluate Different Constructions of Re-engineered Chimeric Antigen Receptor-T Models.

Abstract

Despite the success of chimeric antigen receptor-T (CAR-T) in hematological malignancies, challenges persist, including limited efficacy in solid tumors, on-off tumor toxicity, and CAR-T cell persistence. Cellular mechanics profoundly influence cell behavior and function, yet the biophysical aspects of CAR-T cells remain underexplored. Here, we investigate various CAR molecules incorporating CD19 or CD123 recognition domains. We assess their <i>in vitro</i> cytotoxicity against cancer cells expressing CD19 and/or CD123 and evaluate their <i>in vivo</i> efficacy in mouse models. Notably, single-specific CAR-T cells targeting CD19 or CD123 exhibit potent cytotoxicity, while dual-target CAR-T cells─arranged in parallel or in crossing series─yield optimal outcomes in animal experiments. Through atomic force microscopy (AFM), we uncover a negative correlation between the binding forces of CAR-T cells and antigens and the efficacy of CAR-T therapy in animal experiments in our five dual CAR-expressing CAR-T cells. We proposed that lower binding forces lead to a faster CAR-T cell effect and detachment, enhancing killing efficiency. Our findings underscore the significance of binding forces in CAR-T cell function, highlighting the role of cellular mechanics in guiding the design and evaluation of CAR-T therapies.