SLP-76/LAT AND-gated CAR-T cells mediate highly specific and durable antitumor activity in pancreatic ductal adenocarcinoma with reduced gastric toxicity

Background Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive human malignancies, characterized by a five-year survival rate of approximately 13 percent and limited therapeutic efficacy. Although Claudin18.2 (CLDN18.2)-directed chimeric antigen receptor (CAR) T cells have demonstrated promising antitumor activity, physiological CLDN18.2 expression in normal gastric epithelium poses a risk of severe on-target, off-tumor toxicity. Methods An AND-gated CAR platform incorporating LAT and SLP-76 signaling modules was generated to restrict T-cell activation to concurrent recognition of CLDN18.2 and Mesothelin (MSLN). Antitumor efficacy, specificity, and safety were evaluated using PDAC cell lines and murine PDAC xenograft models. Durability-associated transcriptional programs and functional stability were assessed by transcriptomic profiling with quantitative PCR validation and multi-round killing assays. Results The AND-gated CAR-T cells exhibited stringent antigen dependency, selectively eliminating dual-antigen-positive tumor cells while sparing single-antigen or low antigen density targets. In addition, transcriptomic profiling revealed enrichment of memory- and durability-associated programs in AND-gated CAR-T cells, which was supported by increased expression of C-X-C motif chemokine receptor 6 (CXCR6), interleukin-7 receptor (IL7R), and C-C motif chemokine ligand 5 (CCL5), along with reduced expression of interleukin-2 receptor alpha (IL2RA) and nuclear receptor subfamily 4 group A member 1 (NR4A1) in both CD4⁺ and CD8⁺ subsets. Consistent with this profile, AND-gated CAR-T cells maintained cytotoxic activity across repeated antigen challenges, whereas conventional CLDN18.2 CAR-T cells exhibited progressive functional decline. Importantly, in murine PDAC models, AND-gated CAR-T cells achieved pronounced tumor regression while preserving gastric tissue integrity and without overt toxicity, corroborating their translational potential. Conclusions These findings demonstrate that AND-gated CLDN18.2&MSLN CAR-T cells achieve tumor-restricted cytotoxicity in PDAC while exhibiting durable antitumor function and transcriptional programs associated with T-cell functional stability, supporting AND-gated CAR-T strategies to improve the safety and durability of CAR-T therapy in solid tumors.