BPS2026 – Chimeric antigen receptors transmit costimulatory domain-dependent piconewton forces to their target

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable clinical success in B cell malignancies; however, outcomes remain variable, and CARs are less effective in myeloid malignancies and solid tumors. CARs are synthetic receptors typically composed of an extracellular single-chain variable fragment (scFv) for antigen recognition, a hinge and transmembrane domain, one or more intracellular costimulatory domains, and the CD3ζ signaling domain. One critical but understudied dimension of CAR T cell immunotherapy is the mechanobiology of CAR T cells. Native T cells are mechanically active; their T cell receptor (TCR) transmits and senses piconewton (pN) forces through bonds with peptide-MHC, regulating antigen discrimination, immunological synapse organization, and downstream signaling. Emerging evidence suggests that CARs may also be mechanosensitive, yet direct measurements of CAR-mediated forces have been lacking. Here, we employ CAR ligand conjugated DNA-based molecular tension probes (MTPs), which report pN forces via fluorescence, to directly quantify forces exerted by CAR T cells. Our data demonstrate that CARs transmit actin polymerization-dependent pN-scale forces to their targets. These forces were observed across three protein targets—CD123, CD33, and CD19. Importantly, we find that costimulatory domains are major drivers of force generation. Second-generation CARs containing CD28 or 4-1BB exert robust mechanical forces exceeding 19pN to their target protein, whereas first-generation CARs lacking costimulatory domains fail to transmit detectable forces. Furthermore, we establish a link between mechanics and early signaling. CAR forces precede calcium flux, correlate with phosphorylated Zap70 recruitment, and may correlate with cytokine secretion. Together, these findings introduce force and mechanobiology as previously unappreciated axes of CAR function that may inform the engineering of next-generation therapies with improved efficacy and durability.