T-cell inhibition within the tumor microenvironment is a barrier for CAR-T cell therapy efficacy in solid tumors. Disruption of PD-1 signaling by CAR-T cells has been postulated as a strategy to overcome PD-L1 mediated T-cell suppression. The effects of ablating this pathway in different CAR constructs have been reported with contradictory results. Here, we hypothesize that CAR affinity may be a yet unexplored factor modulating T-cell sensitivity to PD-1/PD-L1 axis.

In this work, we aim at interrogating the sensitivity of CAR-T cells targeting HER2 with low (LA) or high (HA) affinity to inhibition by PD-L1. By using a preclinical model of tumor cell lines engineered to express different levels of PD-L1, we observed that cytokine secretion by LA HER2-CAR-T cells gradually decreased as PD-L1 densities increased. By contrast, HA HER2-CAR-T cells displayed a lower degree of inhibition. Accordingly, CRISPR-mediated knockout of PD-1 restored effector functions of LA HER2-CAR-T cells as assessed by different methods, including gene expression analysis and single-cell cytokine profiling, and improved their performance in vivo in mice bearing tumors expressing variable or wild type levels of PD-L1. PD-1 KO did not significantly impact transcriptomic profile or functionality of HA HER2-CAR-T cells neither in vitro nor in vivo.

In conclusion, CAR-T cells targeting HER2 with low affinity are more sensitive to PD-L1-mediated inhibition as compared to those recognizing the same antigen with higher affinity. This inhibition can be overcome by genetic disruption of PD-1. Mechanisms accounting for these differences are currently under investigation.