CAR affinity modulates the sensitivity of CAR-T cells to PD-1/PD-L1-mediated inhibition

Abstract

Abstract CAR-T cell therapy for solid tumors faces significant hurdles, including T-cell inhibition mediated by the PD-1/PD-L1 axis. The effects of disrupting this pathway on T-cells are being actively explored and controversial outcomes have been reported. Here, we hypothesize that CAR-antigen affinity may be a key factor modulating T-cell susceptibility towards the PD-1/PD-L1 axis. We systematically interrogated CAR-T cells targeting HER2 with either low (LA) or high affinity (HA) in various preclinical models. Our results revealed an increased sensitivity of LA CAR-T cells to PD-L1-mediated inhibition when compared to their HA counterparts by using in vitro models of tumor cell lines and supported lipid bilayers (SLB) modified to display varying PD-L1 densities. CRISPR/Cas9-mediated knockout (KO) of PD-1 enhanced LA CAR-T cell cytokine secretion and polyfunctionality in vitro and antitumor effect in vivo and resulted in downregulation of gene signatures related to T-cell exhaustion. By contrast, HA CAR-T cell features remained unaffected following PD-1 KO. This behavior held true for CD28 and ICOS but not 4-1BB co-stimulated CAR-T cells, which were less sensitive to PD-L1 inhibition. Our findings may inform CAR-T therapies involving disruption of PD-1/PD-L1 pathway tailored in particular for effective treatment of solid tumors.