Abstract
Chimeric antigen receptor macrophages (CAR-M) are emerging as a next-generation cellular modality for therapies ranging from viral infection to solid tumors, leveraging innate phagocytic and antigen-presenting functions. Here, we compared CAR constructs incorporating intracellular signaling domains (ICDs) derived from CD3ζ, Fc gamma receptor IIa (CD32a), complement receptor 3 (CR3), and Toll-like receptor 4 (TLR4) in THP-1-derived monocytes and macrophages. Using an anti-viral SARS-CoV-2 model as a screening platform, we subsequently validated key findings in an anti-tumor mesothelin (MSLN) model. Results indicated that CARCD32a exhibited superior phagocytic capacity compared with CARCD3ζ in both monocytes and macrophages. While combining CR3 (CD11b and CD18) and CD32a domains did not enhance phagocytosis, it significantly increased the expression of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α). The incorporation of TLR4 signaling domain reduced surface CAR expression and phagocytic capacity but markedly increased inflammatory cytokine induction, suggesting that TLR4-driven cytokine production can be enhanced despite diminished phagocytosis in this setting. Furthermore, following phagocytosis, CAR-monocytes induced antigen-specific CD8+ T cell activation via antigen presentation. Collectively, these findings highlight CD32a-based and combinatorial ICD designs as a framework for functionally tuned CAR-M platform for solid tumor immunotherapy and anti-viral applications.
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All data that support the findings of this study are available from the corresponding author upon reasonable request.
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Funding
This work was supported in part by the Creative-Pioneering Researchers Program through Seoul National University (to C.-H. L. and H.-R. Kim) and the Bio & Medical Technology Development Program of the National Research Foundation (NRF) & funded by the Korean government (MSIT) (RS-2021-NR056559 to H.-R. Kim, RS-2024-00440679 to C.-H.L.).
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C.-H.L. and J.S.H. conceived and designed the project. J.S.H., S.J.L., and Y.J.K. performed the experiments. C.-H.L., H.R.K., and J.S.H. analyzed the data. C.-H.L., H.R.K., J.S.H., C.K.K., W.B.P., and H.M.S. wrote the manuscript. All authors have reviewed the manuscript and approved its submission.
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Hong, J., Lee, S., Kim, Y. et al. Optimization of THP-1-CAR monocytes utilizing CD32a signaling phagocytosis for antigen-specific T cell activation. Sci Rep (2026). https://doi.org/10.1038/s41598-026-39406-6
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DOI: https://doi.org/10.1038/s41598-026-39406-6
