KLRG1-deficient CAR-T cells exhibit superior antitumor efficacy against colorectal cancer cell lines by modulating the ZAP70/ERK signaling axis

The incidence of colorectal cancer (CRC) is on the rise globally, necessitating new treatment options. CRC cells highly express cadherins, which can suppress CAR-T cell function by engaging the inhibitory receptor KLRG1. The current study aimed to enhance CAR-T cell therapy by targeting the inhibitory axis mentioned. To achieve this, a KLRG1-downregulated CAR-T cell product (shK1-CAR-T) was developed by incorporating a KLRG1 short hairpin RNA (shRNA) into an NKG2D-based CAR backbone. Compared to wild-type CAR-T cells, KLRG1-downregulated CAR-T cells demonstrated superior activation, proliferation, cytokine production, and cytotoxicity against E-cadherin-positive target cells. Additionally, KLRG1 depletion decreased the expression of exhaustion markers, while the CAR-T cell proliferation and survival were enhanced, and the cells differentiated more towards the T central memory phenotype. Mechanistically, KLRG1 knockdown augmented T cell signaling by enhancing phosphorylation of ZAP70 and ERK, and inhibiting this pathway reversed the functional gains. In a human cell line-derived mouse xenograft model, KLRG1-deficient CAR-T cells achieved significantly better tumor control. Our findings establish KLRG1 interference as a potent strategy to augment CAR-T cell function against CRC by modulating the ZAP70/ERK pathway.