Tuning Sensitivity of CAR to EGFR Density Limits Recognition of Normal Tissue While Maintaining Potent Antitumor Activity

Abstract

Many tumors over express tumor-associated antigens relative to normal tissue, such as epidermal growth factor receptor (EGFR). This limits targeting by human T cells modified to express chimeric antigen receptors (CARs) due to potential for deleterious recognition of normal cells. We sought to generate CAR+ T cells capable of distinguishing malignant from normal cells based on the disparate density of EGFR expression by generating two CARs from monoclonal antibodies which differ in affinity. T cells with low affinity Nimo-CAR selectively targeted cells over-expressing EGFR, but exhibited diminished effector function as the density of EGFR decreased. In contrast, the activation of T cells bearing high affinity Cetux-CAR was not impacted by the density of EGFR. In summary, we describe the generation of CARs able to tune T-cell activity to the level of EGFR expression in which a CAR with reduced affinity enabled T cells to distinguish malignant from non-malignant cells.

Architecture of a minimal signaling pathway explains the T-cell response to a 1 million-fold variation in antigen affinity and dose

“…As a result of the bell-shaped doseresponse, we find that low-affinity receptors can actually outperform high-affinity receptors at high antigen doses. Our model provides a rationale for optimizing the affinity of therapeutic receptors based on the target antigen dose, as recently proposed for a CAR (30). We provide a tool that can be used to examine the predicted T-cell response for antigens of different affinities presented at different doses (SI Appendix, Applet S1).…”

“…A specific example is the NY-ESO-1 cancer antigen, for which both high-affinity TCRs and CARs have been produced (26,27). The optimizing of these therapies has focused, in part, on trying to determine the optimal receptor affinity for clinical efficacy (20,22,(28)(29)(30); reviewed in ref. 31).…”

Chimeric antigen receptor-modified T cells for the immunotherapy of patients with EGFR-expressing advanced relapsed/refractory non-small cell lung cancer

“…EGFR expression has been observed in both epithelium-derived malignancies and most epithelial cells, raising great concerns in the target-mediated toxicity on EGFR-expressing normal tissues. However, the lower affinity of CAR molecule with EGFR antigen may discriminate the damage activities between normal cells with relatively lower level expression of EGFR and tumors with higher expression (Caruso et al, 2015;Chmielewski et al, 2014;Liu et al, 2015). In this study, tolerable and controllable EGFR targeting-related toxicities were just observed in 11 NSCLC cases as shown in Table 2, implying an appropriate affinity of EGFR-CAR epitope we adopted.…”

Structural basis for cancer immunotherapy by the first-in-class checkpoint inhibitor ipilimumab

“…40). Notable examples are provided by recent reports of chimeric antigen receptor (CAR) T cells engineered to present scFv modules spanning a range of affinities for the target antigen (41,42). These studies demonstrate that scFvs with reduced affinities had increased selectivity for solid tumors, relative to normal cells, due to the requirement for higher surface density of target antigens to support productive engagement, a state often afforded by the targeted tumor cells.…”