Abstract 6119: Efficient generation of active CAR-T cells from healthy donors and lymphoma patients

Abstract CAR T-cells have set a new standard of clinical activity in patients with hematologic malignancies but there are several barriers to broader patient access. Currently, the genetic modification of patients' T cells to produce CAR-T cell therapies is carried out ex vivo before infusing the cells back into the patient, using methods that are complex and hinder widespread use. Here we share a targeted lipid nanoparticle (LNP) encapsulating mRNA to reprogram circulating human T-cells in vivo, designed to overcome the significant limitations of current CAR T therapy. The LNP consists of a proprietary ionizable lipid formulation, developed for efficient transfection of T cells and improved tolerability, containing an mRNA encoding a second-generation CD22 CAR. Transfection of human primary T cells was only observed with the addition of a NANOBODY® VHH targeting CD8 to the surface of the LNP. CAR expression was measured for multiple days in up to 80% of CD8+ T cells, with minimal loss of viability and no nonspecific activation. In a whole-blood assay, CAR expression was limited to CD8+ cells (T cells and NK cells), with no CAR expression observed in CD4+ T cells, B cells, or granulocytes. LNP transfected T cells are capable of antigen specific killing. In a serial transfection and rechallenge experiment, cultured T cells were able to maintain cytotoxicity against repeat challenges of Nalm 6 cells but only with additional LNP treatments. The repeated transfected and cellular challenge did not induce exhaustion or disfunction. The in vivo CAR LNP system also efficiently transfected T cells from lymphoma patient PBMCs. Overall levels of in vitro transfection were generally increased in lymphoma patients and the resulting CAR expressing cells were active. CAR expression was greatest in central memory and effector memory T cell phenotypes and decreased in naïve T cells. High-dimensional Spectral Flow profiling identified increased populations of Tem cells in both DLBCL and follicular lymphoma patients, potentially explaining the increased levels of CAR expression. In both healthy donors and lymphoma patient samples, treatment with CAR LNPs significantly reduced B cells, showing the activity of the transfected cells. The capabilities of the LNP system have also been demonstrated in a PBMC humanized mouse model. LNP dosing resulted in expression of CAR in over 80% of circulating human CD8 T cells 6 hours after a single dose. CAR expression was also observed after 4 serial doses, with no substantial loss of CAR expression. Expression of a CAR eliminated any remaining B cells from the PBMC population without severe systemic toxicities. Taken together, we have demonstrated that this targeted LNP system is capable of specific, re-dosable expression of an active CAR that is tolerable for multiple transfections. These are the necessary capabilities to fundamentally alter patient access to CAR-T therapies. Citation Format: Andrew J. Sawyer, Viktor Lemgart, William Kuhlman, Aaron Griset, Mir Ali, Jennifer Richards, Angela Hadjipanayis, Emiko Desvaux, Seth Garren, Chris Hoefler, Haley Nguyen, Laura Strauss, Jing Jiao, Brandon Quido, Laura Powers, Allison Caron, Eyoung Shin, Virna Cortez-Retamozo, Austin Boesch, Dharini Shah, Tiffany Le, Rasika Kunden, Samantha Stewart, Fazila Nasimi, Sampa Maiti, Olga Lihoradova, Christian Mueller, Christopher Borges, Carla Lawendowski, Ulrik Nielsen, Valeria Fantin, Daryl Drummond, Donald Shaffer. Efficient generation of active CAR-T cells from healthy donors and lymphoma patients [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 6119.