Engineering tumor-infiltrating lymphocytes (TILs) via the T-Editor platform to enhance antitumor activity.

2643 Background: Tumor-infiltrating lymphocyte (TIL) therapy has demonstrated clinical potential in melanoma, cervical cancer, and non-small cell lung cancer following failure of standard-of-care therapies, achieving objective response rates of 25-40%. However, two major challenges remain: the limited understanding of biological mechanisms underlying differential patient responses, and the absence of robust engineering technologies for manipulating fragile TIL populations. These limitations have constrained the application of genetic modification strategies to improve TIL therapeutic efficacy. Methods: By comparing transcriptomic profiles of infused TILs from responders (R) and non-responders (NR) in a Phase I investigator-initiated trial evaluating TIL therapy for solid tumors, we identified more than 10 differentially expressed genes as candidate therapeutic targets. We established the T-Editor platform, a CRISPR-mediated gene editing system optimized for TIL engineering, through systematic optimization of stimulation conditions, electroporation parameters, and CRISPR/Cas9 component dosing. Functional validation was performed by generating knockout (KO) constructs for each candidate gene using T-Editor, assessing their impact on TIL expansion, cytokine secretion, and cytolytic activity. The most promising target was selected for advanced cytosine base editing (CBE) studies, employing sgRNA designs to minimize Cas9-induced double-strand break risks. Base-edited TILs were comprehensively compared with Cas9-KO counterparts in vitro, while in vivo efficacy was evaluated using patient-derived xenograft (PDX) mouse models. Results: The T-Editor platform we established achieved optimal gene editing efficiency with defined stimulation condition, electroporation program and CRISPR/Cas9 doses. Functional screening revealed four critical regulatory genes, with FAM84B emerging as the most significant target where knockout demonstrated an increase in cytolytic activity compared to controls. CBE-mediated C·G→T·A conversion at FAM84B exon achieved high editing efficiency with minimal insertion-deletion events. Base-edited TILs showed comparable expansion kinetics, phenotypic stability, and cytokine production to Cas9-KO counterparts. In vivo studies demonstrated increased tumor growth inhibition in PDX models with FAM84B-edited TILs, which exhibited enhanced memory phenotype, superior effector function and elevated IFN-γ secretion. Conclusions: The T-Editor platform represents a rapid, efficient, and safe CRISPR-based system for TIL engineering, enabling both gene knockout and precise base editing applications. Our discovery of FAM84B as a potential novel inhibitory regulator of TIL function establishes a promising therapeutic target for improving adoptive cell therapy outcomes in solid tumor treatment.