Abstract 1811: Use of lipid nanoparticles to generate CAR T cells targeting colon cancer
Authors: Pedro Pires Goulart Guimaraes, Walison Nunes da Silva, Marco Tullio Rodrigues Alves, Pedro Henrique Dias Moura Prazeres, Gabriel Henrique Costa da Silva, Gabriel Vieira Azevedo, Maria Luiza Gomes Viana
Journal: Cancer Research (2025)

Abstract Introduction: Chimeric Antigen Receptor (CAR) T cells represent a breakthrough in cancer immunotherapy. However, current methods for engineering T cells to express CARs are expensive and typically rely on viral vectors, which are integrative and have been linked to severe adverse effects resulting from the continuous expression of CARs. In contrast, non-viral vectors, particularly lipid nanoparticles (LNPs), have emerged as a promising alternative for generating CAR T cells with transient CAR expression. In this study, we developed a nanoparticle-based mRNA platform to generate CAR T cells targeting Claudin 6, a marker for various solid tumors. Methods: mRNA encoding the CAR targeting Claudin 6 was synthesized via in vitro transcription and subsequently loaded into LNPs. The LNPs were synthesized using microfluidic mixing, incorporating an ionizable lipid and excipient lipids at different molar ratios. To assess transfection efficiency, Jurkat cells and primary human T cells were transfected with CAR mRNA encapsulated in LNPs (LNP-CAR) and analyzed by flow cytometry. T cell activation was evaluated by co-culturing CAR-expressing cells with colorectal cancer cells (Colo 205) for 24 hours, followed by measurement of CD69 expression in CAR+ cells. Primary human T cells were transfected with an optimized LNP formulation and co-cultured with colorectal cancer cells at varying effector-to-target (E:T) ratios to assess specific killing by flow cytometry. Results: We developed an LNP formulation to deliver mRNA encoding the CAR to T cells. The LNPs had a size of 98 nm, a polydispersity index of 0.19, a slightly negative zeta potential of -3.4 mV, and an encapsulation efficiency of 82%. Enhanced CAR expression was observed in both Jurkat and primary human T cells treated with LNP-CAR, compared to control cells, with peak CAR expression occurring 24 hours after treatment. When co-cultured with colorectal cancer cells at a 1:1 ratio, Jurkat and primary human T cells transfected with LNP-CAR exhibited increased CD69 expression, indicating T cell activation. Importantly, primary T lymphocytes transfected with the optimized LNP-CAR formulation effectively killed colorectal cancer cells in vitro. Conclusion: In this study, we identified a lead LNP formulation that induced CAR expression in primary T lymphocytes, which were subsequently activated and capable of specific killing of colorectal cancer cells. This non-viral delivery method using LNPs offers a promising alternative for producing CAR T cells. Furthermore, this approach can be adapted to other CAR constructs, potentially broadening its applicability to the treatment of solid tumors, an area where immunotherapies currently face significant challenges. Citation Format: Pedro Pires Goulart Guimaraes, Walison Nunes da Silva, Marco Tullio Rodrigues Alves, Pedro Henrique Dias Moura Prazeres, Gabriel Henrique Costa da Silva, Gabriel Vieira Azevedo, Maria Luiza Gomes Viana. Use of lipid nanoparticles to generate CAR T cells targeting colon cancer [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 1811.