Genetic Engineering of Tumor‐Infiltrating Lymphocytes (TIL) via a T‐Editor Platform to Enhance Anti‐Tumor Activity

Abstract

ABSTRACT Tumor‐infiltrating lymphocytes (TIL) therapy has demonstrated clinical potential in malignancies. However, limited understanding of why only a subset of patients respond to TIL therapy, coupled with the lack of simple and efficient methods to genetically engineer fragile TIL, has hindered efforts to enhance TIL efficacy through genetic modification. A T‐Editor platform enabling rapid and efficient CRISPR‐mediated gene editing in TIL was developed and optimized. To minimize the risk of chromosomal translocations associated with Cas9‐induced double‐strand breaks (DSBs), single‐guide RNAs (sgRNAs) were designed for cytosine base editing (CBE). The expansion capacity, phenotypic profile, cytokine production, and in vitro cytolytic activity of base‐edited TIL were compared with those of Cas9‐KO TIL. In vivo efficacy was assessed using patient‐derived xenograft (PDX) mouse models. The T‐Editor platform was optimized for TIL gene editing by refining stimulation conditions, electroporation parameters, and CRISPR/Cas9 reagent dosing. FAM84B emerged as the top candidate, with its knockout resulting in the most pronounced enhancement of TIL cytolytic activity. CBE‐mediated C·G‐to‐T·A conversion in the FAM84B exon achieved high editing efficiency with minimal insertion–deletion (indel) events. Base‐edited TIL exhibited comparable expansion, phenotype, cytokine production, and in vitro cytolytic activity relative to Cas9‐KO TIL. Compared with non‐engineered control TIL, FAM84B‐edited TIL displayed an increased CD62L+ memory subset, enhanced effector function and cytolytic activity, and improved in vivo antitumor efficacy. In conclusion, the T‐Editor platform enables rapid and efficient CRISPR‐mediated gene editing for engineering TIL to enhance its therapeutic potency. FAM84B may represent a novel potential target for improving TIL‐mediated antitumor activity.