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

Abstract

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 the T cell 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 ε subunit (also known as a TRuC) and fully restored by exchanging the TCR α β 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.

Engineered yeast cells simulating CD19+ cancers to control CAR T cell activation

“…5F). This antigen-dependent up-and down-modulation of CAR surface levels has previously been shown to be dependent on CAR stimulation levels, and reported to be caused by at least CAR expression regulation and CAR internalization 32,34,41,50,76 . The % CAR+ of CD3+ T cells dropped with an increase in target cell numbers (p<0.0001), indicating a loss of CAR+ T cells (Fig.…”

“…CD19 modulation reshapes the patients' total antigen burden and can occur through a variety of mechanisms 21,[25][26][27][28][29][30][31][32] . Regardless of the targeted antigen, even a gradual decrease in antigen density alone provides a mechanism of reduced efficiency and increased resistance to CAR T cell therapy [32][33][34][35][36][37][38][39][40][41] .…”

“…Despite the clinical importance of understanding antigen densities and the hundreds of new CAR designs and therapies for new target antigens and cancers being developed or undergoing clinical trials 47 , only a few options exist to assess antigen-density thresholds for CAR T cell responses, such as material-fixed antigens or antigen multimers 41,48,49 . Additionally, cell lines with varying antigen densities, which more closely imitate in vivo cell-to-cell interactions, are also used, albeit with a lack of orthogonality and limited control of antigen densities [32][33][34][35][36][37][38][39][40]50,51 .…”

Innate receptors with high specificity for HLA class I–peptide complexes

“…The relative contribution of KIR ligand abundance and of ligand affinity to functional NK cell responses has yet to be determined. To what extent signaling by KIR engaged with MHC-I can be modulated by co-activation and co-inhibition receptors, as is the case for TCRs 64 , is not clear. Understanding how NK cells integrate KIR signals with the many other signals they receive from target cells to mount a response could also be a key to parsing KIR-HLA association with disease.…”

Programming CAR T Cell Tumor Recognition: Tuned Antigen Sensing and Logic Gating

“…The ε-TRuC receptor is formed via the incorporation of an overexpressed scFv–CD3ε fusion into the endogenous TCR–CD3 complex ( 54 ). The ε-TRuC receptor is the only of these to have been investigated for its antigen sensitivity, which showed sensitivity to be superior to a 28ζ CAR but inferior to the STAR receptor, all bearing the same antigen specificity ( 55 ). Altogether, the above studies suggest that some CD3-based receptors have superior sensitivity to canonical CARs, which increases their ability to lyse targets with low antigen densities, on the order of a few hundred in the case of HIT receptors.…”