Abstract 865: Engineering a next-generation dual-knockout TIL product with AaCas12b nuclease editing

Advancing cellular therapies, particularly allogeneic and functionally enhanced treatments using gene editing technologies, necessitates rigorous safety and quality assessments to ensure clinical success. Among CRISPR nucleases, Cas12b—a type V CRISPR enzyme—stands out for its high editing fidelity, owing to its stringent mismatch intolerance between single guide RNA (sgRNA) and target DNA, surpassing SpCas9 in precision. An engineered variant of Cas12b from Alicyclobacillus acidiphilus, termed AaCas12bMax, has demonstrated exceptional on-target editing efficiency with minimal off-target activity, making it a promising tool for complex therapeutic gene editing. This study employed AaCas12bMax to develop GT300, a next-generation tumor-infiltrating lymphocyte (TIL) product incorporating a dual-gene knockout. Candidate sgRNAs for the target genes were identified through in silico analysis to minimize genome-wide mismatches. Top-performing guides, validated in HEK293 cells and human PBMC T cells, were further evaluated in TIL products and compared with SpCas9-mediated editing. The AaCas12bMax system achieved comparable knockout efficiency and target protein reduction to SpCas9, with functional evaluations showing similar cytotoxicity, cytokine secretion in vitro, and antitumor efficacy in a mouse PDX model using autologous TILs. Notably, TIL products edited with AaCas12bMax exhibited 3-5 times greater expansion and enhanced viability during large-scale production compared to those edited with SpCas9. These cells demonstrated improved stemness, reduced exhaustion, and apoptosis, alongside superior expansion post-infusion in vivo, suggesting reduced genetic stress during editing. Comprehensive off-target and chromosomal translocation analyses using SITE-seq, GUIDE-seq, and PEM-seq, validated by Amp-seq and hybrid capture assays, confirmed a superior safety profile for AaCas12bMax-edited GT300 compared to SpCas9. RNA-seq analyses at 6 and 24 hours post-electroporation revealed diminished activation of apoptosis and exhaustion pathways in AaCas12bMax-edited TILs. In conclusion, GT300 edited with AaCas12bMax demonstrated efficient gene editing, a superior safety profile, and significant improvements in cell expansion, viability, and fitness. A first-in-human clinical trial of GT300 is currently underway in China. Citation Format: Jingwei Sun, Yao Sheng, Hongwei Zhang, Xi Zhu, Jingman Wang, Ke Liu, Shin-Shay Tian, Pin Wang, Xiaoping Zhao, Yarong Liu. Engineering a next-generation dual-knockout TIL product with AaCas12b nuclease editing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 865.