Asparagine-linked (N-linked) glycosylation is ubiquitous and can stabilize immune inhibitory PD-1 protein. Reducing N-linked glycosylation of PD-1 may decrease PD-1 expression and relieve its inhibitory effects on CAR-T cells. Considering that the codon of Asparagine is aac or aat, we wondered if the adenine base editor (ABE), which induces a·t to g·c conversion at specific site, could be used to reduce PD-1 suppression by changing the glycosylated residue in CAR-T cells. Our results showed ABE editing altered the coding sequence of N74 residue of PDCD1 and downregulated PD-1 expression in CAR-T cells. Further analysis showed ABE-edited CAR-T cells had enhanced cytotoxic functions in vitro and in vivo. Our study suggested that the single base editors can be used to augment CAR-T cell therapy.

Chimeric antigen receptor T (CAR-T) cell therapy is not satisfying in solid tumors. PD-1-mediated suppression greatly hinders CART cells in the microenvironment. It has been shown that PD-1 blockade improves the effectiveness of CART cells. Herein, we designed CART cells than could secret α-PD-1 scFv by themselves. To obtain optimal secretions of scFv, we screened several signal peptides. And the segment from human increased the extracellular production of PD-1-neutralizing proteins. The secreted neutralizing scFv efficiently blocked PD-1 and enhanced T cell activation when PD-L1 was present. Further analysis showed that CART cells themselves could secret α-PD-1 scFv with bioactivity. In contrast to the prototype, the scFv-producing CART cells demonstrated decreased PD-1 but increases expansion and toxicity against solid tumor cells. In the subcutaneous and orthotopic xenograft models, the self-delivered α-PD-1 scFv increased CART cell functionalities and tumor-suppressions. Our work suggested that engineering T cells to co-express antigen-responsive receptors and checkpoint inhibitors is effective to optimize CART cell therapy for solid tumors.

Highlights CD276 is homogeneously overexpressed in ESCC and EAC. CD276-directed CAR-T cells demonstrate remarkable anti-tumor effects in ESCC PDX model. CD276-targeting CAR-T cells are successfully generated with patients T cells and show potent cytotoxicity against autologous tumor cells.