Abstract 4816: Enhancing the persistence of universal CAR-T against DLL3 positive small cell lung cancer using the epigenetic regulation system mediated by CRISPR

Background: Chimeric antigen receptor T-cell (CAR-T) therapy faces challenges in solid tumors. Long-term exposure of CAR-T to antigens and the immunosuppressive tumor microenvironment leads to T cell exhaustion, which impairs T cell antitumor persistence. Moreover, universal CAR-T (UCAR-T) based on knockout of TRAC, HLA or CD52 genes often has the problem of diminished function. Previous studies have shown that stem-like T cells are essential for offsetting undesirable effects of exhaustion, and both exhaustion and stemness-related genes are epigenetic regulated, which offer a safer alternative to genetic modifications in CAR-T therapy. In this study, we designed a system to turn off or turn on epigenetic regulation genes by CRISPR, named as Epi-off and Epi-on, to enhance the persistence of CAR-T and UCAR-T therapy. And the exhaustion-related epigenetic regulation gene ARID1A was chosen to test the system function. Methods and Results: The ARID1A KO DLL CAR-T cells were constructed by an Epi-off system mediated by CRISPR using lentiviral and electrical transduction. Continuous repetitive chronic stimulation was performed to induce T cell exhaustion in vitro. The ARID1A KO DLL CAR-T cells exhibited better antitumor effects and less exhaustion, with a less exhausted T cells population (TIM3+ or LAG3+). Meanwhile, they maintained higher proportion of TCF-1+, CD62L+Slamf6+ subsets and faster expansion, which indicated ARID1A KO led to more stem-like characteristics and longer persistence. In vivo, ARID1A KO DLL CAR-T exhibited more effective and long-lasting antitumor effect. The longer survival, smaller tumor volume and more tumor infiltrating lymphocytes (TILs) were observed in tumor mice who received ARID1A KO DLL CAR-T. Moreover, we also assessed the anti-tumor effect between ARID1A KO and SUV39H1 KO CAR-T which was another epigenetic regulation gene against exhaustion. ARID1A KO CAR-T showed a comparable anti-tumor effect at reinfusion dose of 1×107 CAR-T cells per mouse but better than SUV39H1 KO CAR-T at comparatively low reinfusion of 5×106. Furthermore, ARID1A KO was implemented on UCAR-T. In vivo, ARID1A KO improved DLL UCAR-T therapy (like CAR-T) with a limited toxicity (like conventional UCAR-T) in SCLC mouse model. In addition, we also established an Epi-on system to enhance the persistence and safety of CAR-T, which had a gated structure so that the CRISPR activation component would be released into nucleus by protease to up-regulate expression of certain gene when CAR-T recognized DLL3+ SCLC. Conclusion: We established an epigenetic regulation system (Epi-off and Epi-on) to enhance CAR-T/UCAR-T function. And the ARID1A KO DLL3 CAR-T and UCAR-T based on this system exhibited better persistence, reliable anti-tumor effects and low toxicity in both in vitro and vivo. Citation Format: Zichong Peng, Zhuoran Yao, Linglu Yi, Kai Kang, Ren Luo, Shanghai Liu, Yufeng Zhang, Shuangsi Liao, Feifei Na, Lisa Xiang, Ruizhan Tong, You Lu. Enhancing the persistence of universal CAR-T against DLL3 positive small cell lung cancer using the epigenetic regulation system mediated by CRISPR [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 4816.