Lipid nanoparticle-mediated mRNA transfection enables efficient and safe generation of CAR macrophages.

Abstract With the development of Chimeric Antigen Receptor (CAR) T-cells and its subsequent clinical trials and FDA approval, CAR T-cells have since provided an additional therapeutic modality for hematological malignancies. Despite this success, CAR T cells have shown to be less effective against solid tumors, which account for the majority of cancers. Failure of CAR-T cells can often be attributed to the characteristics typically found within the tumor microenvironment such as its immunosuppressive milieu and dense extracellular matrices (Du et al, Cancer Cell 2025). In face of these challenges, recent studies have investigated the therapeutic value of transfecting macrophages with a CAR construct. As of now, preclinical studies on CAR macrophages (CAR-M) have shown some promising results on CAR-M's capacity to target solid tumors (Klichinsky et al., Nat. Biotechnol 2020). In addition, an initial CAR-M phase 1 clinical trial reported that CAR-Ms were well tolerated in comparison to the potential adverse effects of CAR-T therapy such as cytokine release syndrome (Reiss et al., Nat Med 2025). Nevertheless, current CAR therapies and studies continue to use lentiviral transduction methods to introduce the CAR constructs. Yet, recent case reports have suggested the possibility of potential secondary malignancies due to lentiviral-related insertional oncogenesis (Harrison et al., N. Engl. J. Med. 2025). To mitigate this risk while leveraging macrophages as CAR-Ms, our study utilizes a non-viral method of transfection of CAR-Ms through lipid nanoparticle (LNP) delivery of messenger RNA. We have conducted transfection experiments with an eGFP mRNA construct on THP-1-derived macrophages which has a transfection efficiency of 80-90% and viability of 60-70%. Afterwards, we utilized a previously characterized anti-B-cell maturation antigen (BCMA) CAR construct on THP-1-derived macrophages. Similar to eGFP, our data showed around 75-80% transfection of THP-1-derived macrophages with a viability of 60-70%. In addition, the kinetics of the anti-BCMA CAR expression demonstrates stable expression up to 4 days in vitro. Our current preliminary results suggest that relative to other models of inducing CAR expression such as lentiviral transduction, LNP-mediated transfection of macrophages may be an adequate and safer alternative form of gene transfer towards developing new CAR-M therapies. Our future studies aim to design and assess the functionality and efficacy of macrophage-specific CAR constructs.

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