Abstract PR-02: CRISPR/Cas9 Engineering of Next-Generation Armoured CAR T Cells
Authors: Beavis Paul 
Published: October 2024

Abstract
Chimeric antigen receptor (CAR) T cell therapy, while highly efficacious for the treatment of certain haematological malignancies, remains ineffective in solid cancers. This can be attributed to various factors, including immunosuppression and heterogeneous expression of targetable tumor antigens. Our previous work (Lai et al. 2020, Nature Immunology) demonstrated that engineering CAR T cells to engage host immunity is key to effectively treating heterogenous tumors. “Armouring” CAR T cells to express proinflammatory cytokines is a promising strategy to engage endogenous anti-tumor immunity. However, conventional armouring approaches that utilize a synthetic promoter to drive cytokine expression (e.g. NFAT promoter) lead to systemic expression of transgenes, which can cause severe clinical toxicities when potent cytokines such as IL-12 are employed. In the current work, we engineered CAR T cells to express anti-tumor factors under the transcriptional control of tumor-specific promoters, and hypothesised that this would enhance the safety and efficacy of armoured CAR T cells (Chen et al. in revision, Cell). Genome-wide RNA-sequencing was used to identify genes in CAR T cells with tumor-specific expression. A novel CRISPR/Cas9-mediated homology directed repair strategy was then employed to knock in (KI) anti-cancer factors into these gene loci. NR4A2 was identified as the promoter with the highest degree of tumor-site restriction and T cells were subsequently engineered to express cytokines from this promoter. This approach was applied to syngeneic CAR T and OT-I murine models that enable clinically relevant assessment of efficacy and safety. CRISPR-engineered primary murine and human CAR T cells exhibited gene KI efficiency of ∼50%. Integrating proinflammatory cytokines such as IL-12 into the NR4A2 promoter led to antigen-specific induction of transgene expression in tumor cocultures, and tumor-restricted expression in vivo. Importantly, this strategy achieved significantly improved tumor-site specific expression of transgenes relative to the NFAT promoter system. Tumor-specific induction of IL-12 improved anti-tumor efficacy leading to eradication of established tumors and activation of host immunity/epitope spreading that enabled effective treatment of heterogenous tumors. Importantly, this approach did not lead to significant systemic expression of IL-12, and provided a favourable safety profile. We were able to demonstrate that this approach was equally scalable as conventional CAR T cell generation protocols and could be applied to either autologous patient-derived CAR T cells or through a CRISPR Knock-In approach to generate CAR T from allogenic health donor-derived PBMCs. We therefore posit that this technology has the potential to enable the armoring of CAR T cells with cargos that are highly potent and require exquisite tumor-site specific expression of transgenes.
Citation Format: Paul Beavis. CRISPR/Cas9 Engineering of Next-Generation Armoured CAR T Cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr PR-02.