Nanoscale CAR Organization at the Immune Synapse Correlates with CAR-T Effector Functions

Abstract

T cells expressing chimeric antigen receptors (CARs) are at the forefront of clinical treatment of cancers. Still, the nanoscale organization of CARs at the interface of CAR-Ts with target cells, which is essential for TCR-mediated T cell activation, remains poorly understood. Here, we studied the nanoscale organization of CARs targeting CD138 proteoglycans in such fixed and live interfaces, generated optimally for single-molecule localization microscopy. CARs showed significant self-association in nanoclusters that was enhanced in interfaces with on-target cells (SKOV-3, CAG, FaDu) relative to negative cells (OVCAR-3). CARs also segregated more efficiently from the abundant membrane phosphatase CD45 in CAR-T cells forming such interfaces. CAR clustering and segregation from CD45 correlated with the effector functions of Ca++ influx and target cell killing. Our results shed new light on the nanoscale organization of CARs on the surfaces of CAR-Ts engaging on- and off-target cells, and its potential significance for CAR-Ts’ efficacy and safety.

Functional avidity of anti-B7H3 CAR-T constructs predicts antigen density thresholds for triggering effector function

“…The CAR-T to tumor interaction encompasses a CAR immune synapse, wherein the initial contact between T cell and tumor is stabilised by the CAR antibody to target antigen binding interaction, which develops into a cluster of engaged CAR constructs 25,26 . Although the biophysical properties of the CAR-T to antigen synapse are not as well characterized as native TCR immune synapses, it is likely that the strength of this interaction as well as the number of target antigens will determine whether the synapse becomes functional; i.e.…”

“…Although the biophysical properties of the CAR-T to antigen synapse are not as well characterized as native TCR immune synapses, it is likely that the strength of this interaction as well as the number of target antigens will determine whether the synapse becomes functional; i.e. triggers death of the target cell through release of cytolytic granules concomitant with T cell signalling to promote proliferation and cytokine secretion 25,26 .…”

Broadening the horizon: potential applications of CAR-T cells beyond current indications

“…Additionally, CD138 has recently emerged as a potentially identifying feature of WM tumor cells, associated with IgM peaks and MYD88 mutations ( 378 ). Recent research has shown the role of nanoscale organization of CARs and TCRs for CAR-T cells targeting CD138 with yet unknown consequences ( 379 ). Future investigations can determine whether these structures are suitable targets for CAR-T-cell therapy in WM.…”