A Novel Chimeric Antigen Receptor (CAR) - Strategy to Target EGFRVIII-Mutated Glioblastoma Cells via Macrophages
Posted on: 21 January 2026 , updated on: 22 January 2026
Preprint posted on 23 December 2025
Outsmarting Brain Tumors with Engineered Immune Cells: ''CAR‑Macrophages on the Frontline”
Selected by Dina Kabbara
Categories: bioengineering, cancer biology, cell biology, immunology
What I like about this preprint
This study shows how CAR-macrophages can be used to treat solid tumors. It is truly interesting that THP-1 monocytes could be engineered to express an EGFRvIII-specific CAR while still developing into functional M1 macrophages. With remarkable antigen specificity, these CAR-macrophages selectively phagocytose tumor cells, trigger a targeted pro-inflammatory response, and spare non-target cells. The transcriptomic analysis was particularly informative, which provided mechanistic understanding of antigen-driven macrophage activation. All things considered, these findings are highly encouraging, and I would like to learn more about the mechanisms and potential therapeutic applications of CAR-macrophages.
Background
CAR-T cells have been very successful in treating blood cancer. Their use in solid tumors is still limited due to poor tumor infiltration and immune suppression. (Klichinsky et al., 2020; Alrehaili et al., 2025). Macrophages, by contrast, naturally infiltrate solid tumors and possess potent phagocytic and antigen-presenting capacities, making them an attractive platform for next-generation CAR-based therapies (Alrehaili et al., 2025). However, for this purpose, EGFRvIII is an ideal target, as it is a tumor-specific and constitutively active mutation of EGFR that is commonly found in glioblastoma tumor cells but not present in normal cells, thereby reducing off-target toxicity. To this end, the authors of this preprint engineered a macrophage-specific CAR by combining an EGFRvIII-specific scFv with the intracellular domain of the MEGF10 receptor, which promotes phagocytosis of apoptotic cells rather than signaling pathways associated with T cell-mediated cytotoxicity. They produced CAR-expressing monocytes and M1-polarized CAR-macrophages using the THP-1 monocytic cell line. These CAR-macrophages were much more effective at engulfing EGFRvIII-expressing DK-MG glioblastoma cells than wild-type macrophages. They also showed higher levels of IL-6 and LDH, and gene expression patterns consistent with active tumor cell uptake, supporting their tumor-killing activity. Overall, this study provides a strong proof-of-concept for using CAR-macrophages as a potential therapy against EGFRvIII-positive solid tumors.
Figure: Representative confocal image (Figure 4A) showing CAR-macrophages selectively engulfing EGFRvIII⁺ glioblastoma cells. Yellow arrows indicate phagocytosed tumor cells.
Key Findings
- M1 polarization is unaffected by CAR expression
M1 macrophages are considered anti-tumor macrophages. The canonical M1 marker profiles of CAR-THP-1 monocytes and CAR-M1 macrophages were similar to those of wild-type cells, suggesting that CAR integration does not alter basal phenotypic identity or differentiation capacity (preprint Figure 1).
- Antigen-specific phagocytosis and cytotoxicity are displayed by CAR-macrophages
EGFRvIII⁺ DK-MG glioma cells were effectively taken up by CAR-M1 macrophages (>65%), whereas EGFRvIII⁻ U87 or A549 cells were only slightly taken up (<20%) (preprint Figure 4). This selective phagocytosis confirmed antigen-specific cytotoxicity and inflammatory activation, which was accompanied by increased LDH release and pro-inflammatory cytokines (IL-6, TNF-α).
- CAR-Macrophages undergo transcriptional reprogramming upon target engagement
RNA-seq analysis revealed upregulation of 239 genes, including CD8A and MEGF10, linked to cell division, DNA replication, and mitotic processes, whereas 100 genes were specifically repressed, highlighting selective transcriptional responses triggered by phagocytosis of EGFRVIII+ targets (preprint Figure 5).
Future directions
By thoroughly assessing efficacy and safety in vivo and refining CAR design to optimize selective phagocytosis and anti-tumor immune signaling, future research should push CAR-macrophage therapy past in vitro proof-of-concept. The immunosuppressive glioblastoma microenvironment may be overcome by combining CAR-macrophages with combinatorial techniques like checkpoint blockade or targeted therapies. In the end, applying this strategy to additional tumor-specific antigens may create a flexible, precisely engineered macrophage platform for solid tumor immunotherapy in the future.
Questions for the authors
Q1. Glioblastoma tumors are highly heterogeneous, and EGFRvIII is expressed only in a subset of tumor cells. Because antigen loss or mutation can give rise to EGFRvIII⁻ subpopulations, could the authors comment on how CAR-macrophages might overcome this limitation? Furthermore, would dual antigen targeting or combination therapies enhance their effectiveness in the context of tumor heterogeneity?
Q2: Given that CAR-macrophages may have limited survival in the immunosuppressive tumor microenvironment, could the authors discuss strategies to enhance their in vivo persistence, tissue infiltration, and functional durability, and how these improvements can influence therapeutic efficacy?
Q3: THP-1 cells are immortalized monocytes; primary human macrophages often differ in activation thresholds, cytokine production, and phagocytic kinetics. Could the authors comment on how these differences might affect translation of CAR-macrophages findings to patient-derived cells?
Q4: Klichinsky et al. showed that HER2-specific CAR-macrophages can reprogram the TME toward a pro-inflammatory (M1-like) phenotype and promote T cell recruitment. Could the authors comment on whether their EGFRvIII-directed CAR-macrophages exhibit similar TME reprogramming and T cell engagement, and how differences in tumor antigen or microenvironment might affect these outcomes?
Q5: The study shows transcriptomic changes in CAR-macrophages after antigen engagement, but it is unclear which of these changes directly contribute to cytotoxic activity. Could the authors clarify the causal link between gene expression alterations and functional phagocytosis, and discuss whether machine learning approaches could be applied to identify predictive gene signatures or key regulatory networks that drive CAR-macrophages function?
References
- Klichinsky, M., Ruella, M., Shestova, O., Lu, X. M., Best, A., Zeeman, M., Schmierer, M., Gabrusiewicz, K., Anderson, N. R., Petty, N. E., Cummins, K. D., Shen, F., Shan, X., Veliz, K., Blouch, K., Yashiro-Ohtani, Y., Kenderian, S. S., Kim, M. Y., O’Connor, R. S., … Gill, S. (2020, March 23). Human chimeric antigen receptor macrophages for cancer immunotherapy. Nature News. https://www.nature.com/articles/s41587-020-0462-y
- Alrehaili, M., Couto, P. S., Khalife, R., & Rafiq, Q. A. (2025, November 27). Car-macrophages in solid tumors: Promise, progress, and prospects. Nature News. https://www.nature.com/articles/s41698-025-01170-7
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