Abstract 35: In situ CAR T cell engineering using lipid nanoparticles for B cell lymphoma therapy
Authors: Zhang Nisi , Robinson Elise , Rivera-Rodriguez Angelie , Wang James , Guo Yutong , Wu Bo , Tumbale Spencer K. , Raie Marina , Inayathullah Mohammed , Czerwinski Debra K. , Levy Ronald , Ferrara Katherine W. 
Published: March 2024

Abstract
Chimeric antigen receptor (CAR)-T cell therapy represents a breakthrough in immunotherapy, offering promising outcomes in the management of hematological malignancies. Current CAR-T cell in vitro manufacturing routes involve T cell isolation, purification, activation, gene transduction and cell expansion. The process duration and the costs present significant barriers. Therefore, there is a need for innovative strategies to address these concerns. Herein, we developed lipid nanoparticles (LNPs) to deliver CAR-mRNA efficiently and safely to T cells in vivo, thereby enabling in situ CAR-T cell engineering. By optimizing their composition and introducing anti-CD5 as the targeting ligand, we were able to enhance mRNA delivery and T cell transfection efficiency while maintaining T cell function. Furthermore, to augment T cell responsiveness to exogenous genetic elements, we investigated the pretreatment of T cells with interleukin (IL-7), thereby modulating T cell homeostasis by enhancing survival and proliferation, and rendering the cells more receptive to exogenous genetic modifications. We tracked firefly luciferase (FLuc) in naïve Balb/c mice using in vivo and ex vivo bioluminescence imaging after intravenous injection of anti-CD5 FLuc-mRNA LNPs. We observed positive mRNA expression in lymphoid organs, particularly in the spleen. Additionally, we harvested the lymphoid organs, and performed immunophenotyping analysis using flow cytometry after the administration of anti-CD5 LNPs encapsulating mRNA encoded CD19-CAR with a flag tag. We showed enhanced LNP binding in 65.2% of T cells and CAR expression in 11.64% of T cells as compared with non-treated controls, anti-CD5 FLuc-mRNA LNPs, or IgG CAR-mRNA LNPs. In a CD19+ A20 tumor-bearing mouse model, treatment with CAR-LNPs resulted in a significant increase in chemoattractants (CCL2, CCL4, CCL5, CCL7 and CXCL10) and pro-inflammatory cytokines (TNF-α, IFN-γ, IL6, IL8 and IL15) in blood, and infiltration of T cells and other immune cells in tumors, compared to the non-treated control group over 48 hours post treatment. In vivo engineering allows simultaneous CAR T cell engineering and delivery, reducing the time between manufacturing and treatment, minimizing contamination risks, eliminating complex ex vivo processes, and reducing the need for high-dose preconditioning chemotherapy. In conclusion, the adoption of in situ CAR-T cell production holds promise for improving this therapeutic approach. The authors acknowledge the funding support from NIHR01CA253316 and Leukemia and Lymphoma Society (LLS, Grant ID: 8022-20)
Citation Format: Nisi Zhang, Elise Robinson, Angelie Rivera-Rodriguez, James Wang, Yutong Guo, Bo Wu, Spencer K. Tumbale, Marina Raie, Mohammed Inayathullah, Debra K. Czerwinski, Ronald Levy, Katherine W. Ferrara. In situ CAR T cell engineering using lipid nanoparticles for B cell lymphoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 35.