Low CAR affinity enhances T-cell synapse dynamics and activation
- 1AMOLF
Abstract
Low affinity chimeric antigen receptors (CARs) can clinically outperform high-affinity variants, yet the molecular mechanisms underlying affinity-dependent activity remain unclear. Here, we show that low CAR affinity enables faster CAR cluster motion, which is central to T-cell activation. We compared low-affinity CAT and high-affinity FMC63 CARs signaling at different stages of activation on supported lipid bilayers functionalized with defined CD19 densities. At low CAR expression on dense CD19 bilayers, low-affinity CAT CARs formed more mature and dynamic synapses, recruited ZAP70 more efficiently, and showed faster microcluster motion. These proximal signaling changes were accompanied by stronger calcium flux and increased NFAT nuclear translocation. Our data support a model in which weaker, lower-affinity CAR-antigen interactions promote receptor turnover, microcluster mobility, and signaling-adaptor recruitment, thereby enhancing downstream signal propagation. Our work provides a mechanistic basis for the superior functional performance reported for affinity-tuned CD19 CAR-T cells.
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BibTeX
@misc{nguyen2026affinity,
title = {{Low CAR affinity enhances T-cell synapse dynamics and activation}},
author = {C. Nguyen and G. Castro-Linares and L. C. D. E. Gatti and Z. Sebestyen and R. A. Fernandes and S. J. Tans and M. V. Farrell and K. A. Ganzinger},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.09.757739},
url = {https://www.biorxiv.org/content/10.64898/2026.10.09.757739v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Low CAR affinity enhances T-cell synapse dynamics and activation
AU - C. Nguyen
AU - G. Castro-Linares
AU - L. C. D. E. Gatti
AU - Z. Sebestyen
AU - R. A. Fernandes
AU - S. J. Tans
AU - M. V. Farrell
AU - K. A. Ganzinger
PY - 2026
DA - 2026/10/09
PB - bioRxiv
DO - 10.64898/2026.10.09.757739
UR - https://www.biorxiv.org/content/10.64898/2026.10.09.757739v1
AB - Low affinity chimeric antigen receptors (CARs) can clinically outperform high-affinity variants, yet the molecular mechanisms underlying affinity-dependent activity remain unclear. Here, we show that low CAR affinity enables faster CAR cluster motion, which is central to T-cell activation. We compared low-affinity CAT and high-affinity FMC63 CARs signaling at different stages of activation on supported lipid bilayers functionalized with defined CD19 densities. At low CAR expression on dense CD19 bilayers, low-affinity CAT CARs formed more mature and dynamic synapses, recruited ZAP70 more efficiently, and showed faster microcluster motion. These proximal signaling changes were accompanied by stronger calcium flux and increased NFAT nuclear translocation. Our data support a model in which weaker, lower-affinity CAR-antigen interactions promote receptor turnover, microcluster mobility, and signaling-adaptor recruitment, thereby enhancing downstream signal propagation. Our work provides a mechanistic basis for the superior functional performance reported for affinity-tuned CD19 CAR-T cells.
ER -