A Novel Therapeutic Approach - CAR-M-CAR-T Molecule Discovery Topic-Tekbiotech-Yeast and Phage Display CRO, Expert in Nano-body and Antibody Drug Development

A Novel Therapeutic Approach - CAR-M

Chimeric antigen receptor (CAR) T-cell immunotherapy has achieved remarkable success in clinical cancer treatment, raising hopes for applying the CAR strategy to other clinical scenarios. Cao et al. developed a CAR macrophage (CAR-M) that recognizes the key inflammatory molecule tumor necrosis factor (TNF) and activates the intracellular IL-4 signaling pathway, thereby programming engineered macrophages into anti-inflammatory functional cells. CAR-M therapy demonstrated efficacy in both acute and chronic inflammatory disease mouse models. In renal ischemia-reperfusion injury (IRI), infused CAR-Ms transitioned to an anti-inflammatory phenotype in the inflamed kidney and alleviated renal IRI. The anti-inflammatory phenotype of infused CAR-Ms disappeared during the recovery phase of renal IRI, coinciding with the clearance of TNF. In doxorubicin-induced nephropathy (a chronic inflammatory disease model), infused CAR-Ms maintained their anti-inflammatory phenotype under persistently high TNF levels and improved renal function and structure. CAR-Ms also effectively mitigated tissue damage in another organ, the liver. A human anti-TNF chimeric antigen receptor (CAR) exhibited anti-inflammatory phenotypes and functions in response to TNF. This signal-switching CAR-M technology holds promise for treating various acute and chronic inflammatory diseases.

Mechanism of Action of Chimeric Antigen Receptor Macrophages (CAR-Ms)

The chimeric antigen receptor designed in this study incorporates a single-chain variable fragment (scFv) targeting tumor necrosis factor (TNF) as the extracellular domain, fused to the intracellular domain of IL-4Rα. This design captures TNF and converts its signal into an IL-4 signal, thereby promoting an anti-inflammatory response in macrophages. Upon TNF capture, CAR-Ms display an M2-like macrophage phenotype, characterized by elevated p-STAT6 expression and increased M2 markers (CD206, Arginase1). In vivo experiments demonstrated that CAR-Ms alleviate inflammation and injury in both acute and chronic nephritis (renal ischemia-reperfusion injury and anti-nuclear antibody models), showing significant therapeutic potential. CAR-Ms transition to an M2-like phenotype only at sites of inflammation and revert upon inflammation resolution, ensuring a targeted and timely anti-inflammatory response. CAR-Ms also protected the liver from ischemia-reperfusion injury, indicating potential applications in inflammatory diseases across multiple organs.

Figure 1: Chimeric antigen receptor macrophages (CAR-Ms) as an immunotherapy for inflammatory diseases.

CAR-Ms Attenuate Renal Ischemia-Reperfusion Injury

This study utilized an acute kidney injury model, namely renal ischemia-reperfusion injury (IRI), to test the protective effect of CAR-Ms. Six hours after renal IRI induction, mice were treated with CD45.1? UTD-Ms, CARΔ-Ms, CAR-Ms, or M2 macrophages (Figure 2a).

Figure 2: Evaluation of the protective effect of CAR-Ms in an acute kidney injury model.

CAR-Ms Protect the Liver from Ischemia-Reperfusion Injury

This study evaluated the role of CAR-Ms in hepatic ischemia-reperfusion injury (IRI) (Figure 3a). Compared to mice treated with UTD-Ms or CAR-Δ-Ms, mice receiving CAR-M or M2 treatment exhibited significantly reduced liver damage, as evidenced by markedly smaller areas of hepatocyte necrosis and decreased serum levels of alanine aminotransferase and aspartate aminotransferase (Figure 3b-d). Furthermore, CAR-M and M2 treatment significantly reduced neutrophil infiltration and the production of pro-inflammatory cytokines/chemokines in the liver, including TNF, IL-1β, IL-6, CXCL1, and CXCL2 (Figure 3e, f). However, CAR-Ms provided stronger protection against hepatocyte necrosis, liver dysfunction, and neutrophil infiltration compared to M2 macrophages in mice with hepatic IRI. Immunofluorescence co-staining revealed that the majority of CD45.1? CAR-Ms in liver sections co-expressed the M2 marker CD206 at 48 hours post-hepatic IRI, whereas UTD-Ms and CAR-Δ-Ms did not (Figure 3g).

Figure 3: Evaluation of the protective effect of CAR-Ms in a liver injury model.

Generation and Characterization of Human Chimeric Antigen Receptor Cells

To determine the translational potential of CAR-Ms, a human anti-TNF CAR was constructed, comprising an anti-human TNF single-chain antibody (scFv) as the extracellular domain and the intracellular domain of human IL-4Rα (Figure 4a). This CAR was efficiently expressed in human macrophages via lentiviral transduction (Figure 4b, c). TNF stimulation specifically induced downstream IL-4 signaling pathways, shown by increased p-STAT6 and p-AKT expression in CAR-Ms, whereas in control macrophages it triggered only TNF downstream pathways (Figure 4d, e). CAR-M.TNF displayed significant upregulation of M2 markers, including CD206 and CD200R (Figure 4f, g). However, TNF stimulation of control macrophages led to upregulation of M1-associated markers (CD80 and CD86). Compared to IL-4 stimulation, TNF preferentially activated p-JAK3 (associated with the IL-2Rγc chain) but not p-TYK2 (associated with the IL-13Rα1 chain) on CAR-Ms, suggesting TNF primarily engages the type I IL-4 receptor complex via the CAR on human macrophages (Figure 4d, e). CAR-M cells produced equivalent amounts of IL-10 in response to TNF or IL-4 (Figure 4h), indicating their anti-inflammatory capacity. CAR-M.TNF significantly inhibited T cell proliferation in vitro compared to control macrophages (Figure 4i). CAR-M.TNF also demonstrated superior clearance of dead cells compared to control macrophages, suggesting enhanced wound healing capabilities (Figure 4j). These findings indicate that the anti-TNF CAR redirects human macrophage responses from endogenous TNF signaling towards immunosuppressive and tissue repair pathways.

Figure 4: Generation and characterization of human chimeric antigen receptor cells.

This study developed CAR macrophages (CAR-Ms) designed to respond to inflammatory cytokines for treating inflammatory diseases in various organs. Unlike conventional CARs that enhance immunity by potentiating immune cell function, this type of CAR is unique in converting immune effector responses into immunosuppressive ones. When triggered by inflammatory cytokines within damaged tissues, CAR-Ms induce anti-inflammatory and wound healing responses, effectively resolving inflammation and reducing tissue damage. This provides a novel approach for treating inflammatory diseases as well as other conditions characterized by excessive immune responses.

Tek Biotech (Tianjin) Co., Ltd. specializes in phage display and yeast display antibody discovery services as its core business. We are committed to providing high-quality CAR-M design and targeting validation services to scientists worldwide. Additionally, we offer supporting services including antibody discovery, antibody in vitro expression validation, antibody humanization, antibody affinity maturation, and bispecific antibody development, providing robust technical support for our partners' research projects and drug development endeavors.

References

[1] Cao, Q., Wang, Y., Chen, J. et al. Targeting inflammation with chimeric antigen receptor macrophages using a signal switch. Nat. Biomed. Eng (2025).

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