In Vitro Engineering Chimeric Antigen Receptor Macrophages and T Cells by Lipid Nanoparticle-Mediated mRNA Delivery

Abstract

Chimeric antigen receptor (CAR)-engineered adoptive cell therapy marks a revolution in cancer treatment based on the highly successful responses to CAR T cell therapy in the treatment of blood cancers. Due to the versatile structure of CARs, this technology can be easily adapted to other immune cell types, including macrophages and NKs, and applied in the treatment of many other cancers. However, high costs and fatal adverse effects represent significant concerns for future development. In vitro transcribed (IVT) mRNA therapeutics, which possess a high safety profile and straightforward production methods, could provide a useful alternative for CAR cell construction. However, the low stability and transfection efficiency of IVT-mRNA in immune cells limit further applications. In this work, we successfully engineered CAR macrophages (CAR-Ms) and CAR T cells with CAR mRNA using lipid nanoparticles (LNPs). Both the LNP formulations and mRNA modifications were optimized for in vitro mRNA transfection. More importantly, the CAR macrophages and CAR T cells both demonstrated significant cytotoxic effects on B lymphoma in vitro, underscoring the great potential of mRNA-engineered adoptive cell therapy.

Lipid Nanoparticle (LNP) Enables mRNA Delivery for Cancer Therapy

“…Reproduced with permission. [ 177 ] Copyright 2022, American Chemical Society. C) CD5‐targeted LNPs achieved in vivo production of CAR‐T cells.…”

“…Similarly, Ye et al successfully generated CAR macrophages (CAR-Ms) and CAR-T cells ex vivo using the mRNA-LNP platform, both of which showed significant toxic effects for B lymphoma (Figure 10B). [177] Due to their ease of handling and comparable or higher efficacy, LNPs offer a more convenient approach to generating mRNA-based CAR cells.…”

“…B) Schematic illustration of the CAR mRNA-LNP production and the evaluation of ex vivo produced CAR-T cells and CAR-Ms (the key lipids used in these LNPs and their synthetic routes are also included). Reproduced with permission [177]. Copyright 2022, American Chemical Society.…”

Nanodrugs Targeting T Cells in Tumor Therapy

“…However, also mRNA transfection of T cells by electroporation bears limitations with regard to method-associated cytotoxicity and low transfection efficacy. As an alternative especially lipid NP that complex mRNA and contain helper lipids for stabilization have been tested for ex vivo T cell transfection with mRNA ( 75 – 78 ) and plasmid DNA ( 79 ). Based on library screening, Billingsley and coworkers identified an ionizable lipid which induced comparable mRNA transfection efficiency at lower cytotoxicity on Jurkat T cells ( 75 ), and subsequently optimized this vector by varying the molar ratios of DOPE, cholesterol and lipid-anchored PEG ( 76 ).…”

“…The optimized formulation conferred CAR mRNA transfection of primary T cells at similar efficacy as electroporation and viral transfer, yielding comparable CAR-T-mediated target cell killing. By screening a library encompassing chemically differing lipid-like formulations, termed lipidoids, Zhao and coworkers showed that lipidoids with an imidazole group yielded stronger reporter mRNA transfection efficacies of primary human CD8 + T cells in vitro ( 77 ), and was suitable for transfer of CAR-T mRNA into CD8 + T cells as reflected from potent killing activity of lymphoma cells ( 78 ). Furthermore, lipoids also mediated efficient in vivo transfection of T cells as assessed by applying Cre recombinase-encoding mRNA into a reporter mouse strain yielding dTomato expression in response to Cre recombinase-mediated recombination ( 77 ).…”