Abstract LT01: Identifying Optimal Tumour-specific Promoters for CRISPR/Cas9 Engineering of Armoured CAR T Cells with Enhanced Safety and Efficacy

Abstract Background: The tremendous success of chimeric antigen receptor-T (CAR-T) cell therapy in haematological malignancies has not been recapitulated in solid tumours, owing to tumour-induced immunosuppression, tumour heterogeneity and inefficient tumour trafficking. One promising solution includes “armouring” CAR-T cells with therapeutic transgenes. Indeed, we demonstrated that CAR-T cells engineered to express dendritic cell growth factor Flt3L could effectively engage host anti-tumour immunity crucial for overcoming antigen-negative relapse1. However, synthetic promoters have demonstrated insufficiencies in driving tumour-restricted cytokine expression, which had caused systemic toxicities and trial termination2. The advent of CRISPR/Cas9 gene-editing tool has enabled the precise engineering of CAR-T cells for safety and efficacy enhancements. We previously showed that CRISPR/Cas9-mediated knock-out (KO) of immunosuppressive gene A2AR enhanced CAR-T cell function3. Now, we aim to exploit a CRISPR/Cas9-mediated knock-in (KI) strategy to leverage endogenous gene regulatory elements to restrict transgene expression to tumour for enhanced safety and efficacy. Methods: Genome-wide RNA sequencing was performed on CAR-T cells isolated from tumours and spleens of mice. 27 genes upregulated in intratumoural relative to splenic CAR-T cells were identified as potential KI sites. As KI disrupts target gene expression, the impact of each gene KO on CAR-T cell function/phenotype was first assessed. 7 genes without adverse impact following KO had GFP knocked in. Results: NR4A2 and RGS16 emerged as tumour-specific promoters upon KI. While NR4A2 was highly tumour-restricted and could deliver highly toxic cytokines (e. g. , IL-12) without inducing toxicities in mice, RGS16 had high intratumoural expression and could mediate the efficacy of less potent cytokines (e. g. , IL-2). Conclusions: Endogenous tumour-specific promoters enabled the generation of IL-12- and IL-2-expressing CAR-T cells with enhanced safety and efficacy in syngeneic and xenogeneic mouse models that was concomitant with improved CAR-T cell polyfunctionality and activation of host anti-tumour immunity. Notably, this CRISPR-KI strategy was applicable using patient-derived CAR-T cells, demonstrating its clinical translatability. Citation Format: Kah Min Yap, Amanda X. Y. Chen, Imran G. House, Phillip K. Darcy and Paul A. Beavis. Identifying Optimal Tumour-specific Promoters for CRISPR/Cas9 Engineering of Armoured CAR T Cells with Enhanced Safety and Efficacy abstract. In: Proceedings of Frontiers in Cancer Science 2024; 2024 Nov 13-15; Singapore. Philadelphia (PA): AACR; Cancer Res 2025;85 (15Suppl): Abstract nr LT01.