464 Development of a next-generation dual-KO TIL product GT300 with AaCas12b^Max nuclease

<h3>Background</h3> Novel cellular therapy, particularly in developing allogeneic or functionally enhanced cell therapies through gene editing technology, necessitates thorough safety and quality assessments for successful clinical translation. Cas12b, the third family of class 2 effector nucleases, shows high editing fidelity due to its minimal tolerance for mismatches between the single guide RNA (sgRNA) and target DNA sequences, surpassing SpCas9 in precision. A previous study reported that an engineered variant of Cas12b from Alicyclobacillus acidiphilus, AaCas12b^Max, has demonstrated high on-target editing efficiency with minimal off-target effects, making it ideal for complex therapeutic gene editing applications. In this study, AaCas12b^Max was employed to develop a next-generation tumor infiltrating lymphocyte (TIL) product, GT300, with double genetic knockout. <h3>Methods</h3> Candidate guides for two target genes were designed to minimize genome-wide mismatches through in silico prediction. The knockout (KO) efficiency of these guides was screened in HEK293 and human PBMC T cells. Top-performing guides were selected, and their editing efficiency and functional impact were further evaluated in TIL products compared to the SpCas9 editing system. <h3>Results</h3> The selected guides of AaCas12b^Max demonstrated similar KO efficiency and reduction in protein expression for the two target genes as SpCas9. Functional assessments of GT300 edited by AaCas12b^Max or SpCas9 showed comparable cytotoxicity, cytokine release in vitro, and anti-tumor efficacy in a mouse PDX model treated with autologous TIL. Notably, TIL products edited by AaCas12b^Max yielded 3–5 folds more total cell numbers with higher viability after large-scale expansion. Additionally, these products exhibited higher stemness, lower exhaustion and apoptosis profile, and greater expansion post-infusion in vivo, suggesting reduced genetic stress for TIL products post editing. Consistent with these findings, SITE-seq and GUIDE-seq analysis identified significantly fewer off-target sites in TIL products edited by AaCas12b^Max, which were further validated by hybrid capture assay. <h3>Conclusions</h3> Taken together, the GT300 TIL product edited by AaCas12b^Max achieved high editing efficiency and minimal off-target events, demonstrating significantly improved expansion and viability during manufacturing, and in vivo fitness post-infusion. Currently, a first-in-human clinical trial of GT300 is underway in China.