ABE-mediated dual targeting of TOX and ARHGEF1 synergistically enhances antitumor activity of tumor-infiltrating lymphocytes in PDAC

Tumor-infiltrating lymphocytes (TILs) represent a promising immunotherapeutic modality in solid tumors, yet their clinical efficacy in pancreatic ductal adenocarcinoma (PDAC) remains severely limited by inefficient expansion and functional exhaustion. TILs were isolated from pancreatic cancer surgical specimens and were found to exhibit progressive functional impairment under sustained tumor exposure. Accordingly, we employed adenine base editing (ABE) to selectively target multiple exhaustion-associated genes in TILs. We found that concurrent knockdown of TOX and ARHGEF1 exerted a pronounced synergistic effect, characterized by significantly enhanced cytokine production, reduced expression of exhaustion markers such as PD-1 and TIM-3, and improved T-cell proliferative capacity. Functionally, dual TOX/ARHGEF1 knockdown increased the cytotoxic activity of TILs against matched tumor organoids and resulted in superior antitumor efficacy in vivo. Mechanistic investigations further indicated that this functional enhancement was closely associated with augmented IL-21 signaling and elevated STAT3 phosphorylation in gene-edited TILs. Collectively, our findings demonstrate that ABE-mediated co-targeting of TOX and ARHGEF1 effectively alleviates TIL functional exhaustion and enhances antitumor activity in PDAC, providing mechanistic insight and translational rationale for optimizing gene-engineered TIL-based immunotherapies.