1:IDIBAPS; 2:Hospital Clínic de Barcelona

CAR-T cell therapy of solid tumours faces several hurdles, including dysfunction of the therapeutic T-cells. While inhibition of the PD-1/PD-L1 axis is being actively investigated, there is still controversy about the effects of long-lasting PD-1 genetic deletion on CAR-T cell function. Here, we hypothesized that consequences of the PD1-PDL1 axis on CAR-T cell therapy may depend on the CAR design and PD-L1 densities in the tumour. To test this, we generated a model of cancer cell lines expressing varying PD-L1 densities. PDCD1 was knocked out on CAR-T cells targeting HER2 or mesothelin. We show that even low expression of PD-L1 in tumour cells could significantly impair CAR-T cell effector functions in vitro, and that cytokine secretion was improved by genetic or pharmacological inhibition of the PD1-PD-L1 axis. In vivo, we observed that while CAR-T cells could induce complete responses (CR) in 87% of mice bearing PD-L1-negative ovarian tumours (PD-L1 KO), the percentages of CR in animals with PD-L1 expressing tumours (PD-L1 low, high or wild-type) were below 25%. PDCD1 ablation in HER2-CAR-T cells showed a significantly enhanced antitumour effect compared to CAR-T cells alone or in combination with checkpoint inhibitors, with > 87% of animals with PD-L1-expressing tumours exhibiting CR. We confirmed our observations using CAR-T cells against other cancer types expressing HER2 or mesothelin. Our results show that, in a context where PD-L1 is expressed by tumour cells, the deletion of PD-1 in CAR-T cells can restore CAR-T cell functions, suggesting an overall beneficial effect on CAR-T cell therapy