CAR Macrophages in Tumor Immunotherapy: A New Era

Cell-based immunotherapies have transformed cancer treatment, with CAR-T cells leading the way in targeting hematologic malignancies. However, solid tumors present unique challenges, such as an immunosuppressive microenvironment and limited T cell infiltration. To overcome these barriers, researchers are exploring alternative immune cells, including macrophages.

CAR macrophages (CAR-Ms) harness the innate phagocytic abilities of macrophages while enhancing their tumor-targeting specificity. Their ability to reshape the tumor microenvironment and improve therapeutic outcomes is driving significant interest.

Macrophage Biology

Macrophages are highly adaptable immune cells that play a central role in tissue homeostasis, pathogen clearance, and inflammatory regulation. Their function is shaped by environmental signals, broadly classifying them into pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. M1 macrophages, activated by interferon-gamma (IFN-γ) and microbial stimuli, exhibit tumoricidal properties by producing cytokines like tumor necrosis factor-alpha (TNF-α) and interleukin-12 (IL-12). M2 macrophages, induced by interleukin-4 (IL-4) and interleukin-13 (IL-13), support tissue repair and immune suppression. These states are not fixed, as macrophages can transition in response to changing conditions.

Their origin also influences function. While tissue-resident macrophages, such as Kupffer cells in the liver and microglia in the brain, arise from embryonic progenitors and self-renew, others derive from circulating monocytes that differentiate upon tissue infiltration. Monocyte-derived macrophages often exhibit heightened inflammatory responses, making them particularly relevant in pathological conditions. Recruitment and differentiation of these cells are regulated by chemokines like C-C motif chemokine ligand 2 (CCL2) and growth factors like macrophage colony-stimulating factor (M-CSF).

Metabolic reprogramming further defines macrophage function. M1 macrophages rely on glycolysis to sustain their pro-inflammatory activity, while M2 macrophages favor oxidative phosphorylation and fatty acid metabolism. This metabolic divergence actively shapes macrophage behavior, with metabolites such as succinate and itaconate influencing polarization.

Key Features of Chimeric Antigen Receptors

Chimeric antigen receptors (CARs) are synthetic fusion proteins engineered to direct immune cells toward specific targets. Their design integrates antigen recognition with intracellular signaling to trigger a tailored response. The fundamental structure consists of an extracellular antigen-binding domain, a transmembrane region, and intracellular signaling motifs.

The extracellular domain, typically derived from a single-chain variable fragment (scFv) of a monoclonal antibody, allows CAR-expressing cells to recognize surface antigens independently of major histocompatibility complex (MHC) presentation. This is advantageous for targeting tumors that evade immune detection by downregulating MHC. Optimizing the binding affinity of the scFv is crucial, as excessive affinity can cause off-target toxicity, while inadequate affinity reduces efficacy. Researchers are also exploring alternative binding domains, such as natural receptor ligands and nanobody-based constructs, to expand target specificity.

The transmembrane domain anchors the CAR within the cell membrane and influences receptor stability. Some CAR designs use transmembrane sequences from native immune receptors, such as CD8α or CD28, while others incorporate modified domains to enhance clustering and signal propagation.

Intracellular signaling domains dictate CAR activation strength and persistence. First-generation CARs contained only the CD3ζ signaling motif, providing activation but lacking durability. Later designs incorporated co-stimulatory domains such as CD28 or 4-1BB to enhance survival and function. Emerging CAR constructs integrate additional pathways, such as Toll-like receptors or cytokine receptors, to refine cellular behavior.

Generating CAR Macrophages in the Lab

CAR macrophages (CAR-Ms) are typically derived from human monocyte-derived macrophages (MDMs) or induced pluripotent stem cell (iPSC)-derived macrophages. MDMs are commonly used due to their accessibility and ability to be differentiated ex vivo using macrophage colony-stimulating factor (M-CSF). iPSC-derived macrophages provide a more homogeneous population with controlled differentiation potential.

Gene transfer methods are essential for equipping macrophages with CAR constructs. Lentiviral and adenoviral vectors are frequently used due to their high transduction efficiency. Lentiviral transduction ensures stable CAR expression, while adenoviral vectors provide transient expression, which may be preferable for short-term applications. Non-viral approaches, such as electroporation and nanoparticle-mediated delivery, are also being explored to improve safety by avoiding risks associated with viral vectors.

Following gene transfer, CAR expression and function must be validated. Flow cytometry confirms surface expression, while quantitative PCR and Western blotting assess transcript and protein levels. Functional validation includes phagocytic assays, where CAR-Ms are co-cultured with antigen-expressing target cells to evaluate their tumor-engulfing ability. Cytokine profiling ensures that CAR-Ms maintain their engineered phenotype without excessive inflammatory activation.

Role in the Tumor Microenvironment

The tumor microenvironment (TME) presents a major challenge to effective cancer therapies, characterized by stromal cells, extracellular matrix components, and biochemical signals that promote tumor survival and immune evasion. CAR macrophages (CAR-Ms) can alter the TME to enhance anti-tumor activity. Unlike conventional immune cells that struggle to infiltrate solid tumors, macrophages naturally migrate to inflammatory and hypoxic regions, making them well-suited for therapeutic intervention.

Once within the TME, CAR-Ms leverage their phagocytic and remodeling functions to degrade tumor-supportive structures and disrupt pro-tumor signaling. Their ability to digest extracellular matrix components, such as collagen and fibronectin, facilitates immune cell infiltration. Additionally, CAR-Ms secrete pro-inflammatory mediators that counteract immunosuppressive factors like transforming growth factor-beta (TGF-β) and interleukin-10 (IL-10), making the environment less conducive to tumor survival.

Distinguishing Factors From T Cell-Based Approaches

CAR-T cell therapies have shown success in hematologic malignancies but face challenges in solid tumors due to limited infiltration, antigen heterogeneity, and immune suppression. CAR macrophages (CAR-Ms) offer a distinct approach, leveraging their ability to navigate these barriers through mechanisms fundamentally different from T cell-based strategies. Unlike T cells, which rely on antigen recognition through MHC, macrophages engage in direct phagocytosis and antigen-independent tumor targeting, allowing them to attack malignancies that evade T cell-mediated immunity.

Beyond their targeting mechanisms, CAR-Ms contribute to tumor destruction in ways that extend beyond cytotoxicity. While CAR-T cells eliminate tumor cells through perforin and granzyme-mediated apoptosis, CAR-Ms remodel the tumor stroma, degrade extracellular matrix components, and modulate the immune landscape. This activity facilitates deeper tumor penetration and recruits additional immune effectors by altering cytokine and chemokine gradients.

Macrophages also exhibit greater resilience in harsh tumor conditions, where T cells often succumb to exhaustion due to persistent antigen stimulation and metabolic constraints. These differences position CAR-Ms as a complementary or alternative strategy to T cell-based therapies, particularly in cases where CAR-T cells have faced significant limitations.