Inefficient exploitation of accessory receptors reduces the sensitivity of chimeric antigen receptors

Chimeric antigen receptors (CARs) can redirect T cells to target abnormal cells, but their activity is limited by a profound defect in antigen sensitivity, the source of which remains unclear. Here, we show that CARs have a >100-fold lower antigen sensitivity compared to theTcell receptor (TCR) when antigen is presented on antigen-presenting cells (APCs) but nearly identical sensitivity when antigen is presented as purified protein. We next systematically measured the impact of engaging important T cell accessory receptors (CD2, LFA-1, CD28, CD27, and 4-1BB) on antigen sensitivity by adding their purified ligands. Unexpectedly, we found that engaging CD2 or LFA-1 improved the antigen sensitivity of the TCR by 125- and 22-fold, respectively, but improved CAR sensitivity by only <5-fold. This differential effect of CD2 and LFA-1 engagement on the TCR vs. CAR was confirmed using APCs. We found that sensitivity to antigen can be partially restored by fusing the CAR variable domains to the TCR CD3 epsilon subunit (also known as a TRuC) and fully restored by exchanging the TCR alpha beta variable domains for those of the CAR (also known as STAR or HIT). Importantly, these improvements in TRuC and STAR/HIT sensitivity can be predicted by their enhanced ability to exploit CD2 and LFA-1. These findings demonstrate that the CAR sensitivity defect is a result of their inefficient exploitation of accessory receptors and suggest approaches to increase sensitivity.

Automated summary

Chimeric antigen receptors (CARs) have lower sensitivity to antigens presented on antigen-presenting cells (APCs) compared to T cell receptors (TCR), but nearly identical sensitivity to purified protein antigens. Engaging CD2 or LFA-1 can significantly improve TCR sensitivity, but has minimal effect on CAR sensitivity. Fusion of CAR variable domains to the TCR CD3 epsilon subunit (TRuC) or exchanging TCR alpha beta variable domains for those of the CAR (STAR or HIT) can partially or fully restore antigen sensitivity, respectively, by enhancing their ability to exploit CD2 and LFA-1.