CAR Macrophage: Pioneering M1 Approach in Cancer Therapy

Engineering immune cells to fight cancer has led to significant breakthroughs, with CAR T cell therapy showing success in some blood cancers. However, solid tumors pose additional challenges, such as an immunosuppressive microenvironment that limits treatment efficacy. Researchers are now exploring alternative immune cells, including macrophages, which have unique tumor-fighting capabilities.

A promising strategy involves modifying macrophages to adopt a pro-inflammatory M1 phenotype, enhancing their ability to attack cancer cells. This approach leverages chimeric antigen receptor (CAR) technology to reprogram macrophages for improved tumor targeting and immune activation.

Key Characteristics Of CAR Macrophages

CAR macrophages are a novel class of engineered immune cells designed to enhance tumor clearance through their innate phagocytic and antigen-presenting functions. Unlike CAR T cells, which rely on direct cytotoxicity, CAR macrophages engulf cancer cells and modulate the surrounding environment. This dual functionality makes them well-suited for targeting solid tumors, where immune evasion mechanisms often hinder conventional therapies. By integrating CAR constructs, researchers aim to amplify their tumoricidal activity while preserving their capacity to orchestrate broader immune responses.

A key advantage of CAR macrophages is their ability to infiltrate tumor masses more effectively than T cells. Solid tumors develop dense stromal barriers and immunosuppressive signaling that limit lymphocyte penetration. Macrophages, however, possess intrinsic migratory properties that help them navigate these hostile environments. Engineered macrophages can persist within tumor sites, actively engaging malignant cells through phagocytosis. This sustained presence contrasts with the transient activity of many T cell-based therapies, which often struggle to maintain efficacy in solid malignancies.

Beyond direct tumor engagement, CAR macrophages enhance antigen presentation, stimulating adaptive immune responses. By processing and presenting tumor-associated antigens to T cells, they contribute to a sustained and systemic anti-cancer effect. This characteristic is particularly relevant in tumors that employ immune evasion strategies, as macrophages help restore immune surveillance. Preclinical models indicate that CAR macrophages not only reduce tumor burden but also promote long-term immune memory, potentially lowering the risk of recurrence.

M1 Versus M2 Polarization

Macrophages exhibit plasticity, adopting distinct functional states in response to environmental cues. These states are broadly categorized into M1 and M2 polarization, each influencing tumor progression differently. M1 macrophages, or classically activated macrophages, are pro-inflammatory and exhibit potent anti-tumor activity. They produce inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-12 (IL-12), and interferon-gamma (IFN-γ), which contribute to tumor cell apoptosis and inhibit proliferation. Their ability to generate reactive oxygen species (ROS) and nitric oxide (NO) further enhances their cytotoxic effects, making them attractive for cancer immunotherapy.

In contrast, M2 macrophages, or alternatively activated macrophages, are associated with tissue repair and immune suppression. These cells secrete anti-inflammatory cytokines such as interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β), which contribute to immune evasion by dampening pro-inflammatory responses. Within the tumor microenvironment, M2 macrophages promote angiogenesis and extracellular matrix remodeling, facilitating tumor growth and metastasis. Their abundance in many solid tumors correlates with poor prognosis, as they actively suppress anti-tumor immunity and create a protective niche for malignant cells.

Reprogramming macrophages toward an M1 phenotype has emerged as a promising strategy in cancer treatment. CAR macrophages engineered to sustain an M1-like state can maintain anti-tumor activity despite immunosuppressive signals. By incorporating genetic modifications that enhance pro-inflammatory signaling and resist M2-inducing factors, researchers aim to create macrophages that retain tumoricidal properties. Recent studies indicate that CAR M1 macrophages exhibit heightened phagocytic activity and improved persistence within the tumor microenvironment, leading to more effective tumor clearance.

Genetic Modification Methods

To enhance the tumor-fighting capabilities of macrophages, researchers employ various genetic modification techniques to introduce CARs and reinforce an M1-like phenotype. Methods such as viral vector transduction, nonviral genome editing, and CRISPR-based modifications each offer distinct advantages and challenges.

Viral Vector Transduction

Viral vector transduction is widely used for introducing CAR constructs into immune cells. Lentiviral and retroviral vectors effectively integrate genetic material into the host genome, ensuring long-term CAR expression. Lentiviral vectors are particularly effective for macrophage engineering since they can transduce non-dividing cells. Studies show that lentiviral-modified CAR macrophages exhibit sustained tumoricidal activity and persistence within the tumor microenvironment. However, concerns regarding insertional mutagenesis and potential off-target effects necessitate careful vector design and safety assessments. Additionally, viral transduction efficiency varies depending on macrophage differentiation status, requiring optimization to achieve consistent CAR expression.

Nonviral Genome Editing

Nonviral genome editing techniques reduce the risk of insertional mutagenesis while allowing precise genetic modifications. Electroporation of plasmid DNA or mRNA encoding CAR constructs enables transient expression, which can be beneficial for applications requiring controlled CAR activity. Transposon-based systems, such as Sleeping Beauty or PiggyBac, facilitate stable genomic integration without the risks associated with viral vectors. These methods show promise in preclinical models, demonstrating efficient CAR expression and functional macrophage activation. However, challenges such as low transfection efficiency and potential cytotoxicity must be addressed. Advances in nanoparticle-based delivery systems are also being explored to enhance stability and uptake of genetic material.

CRISPR-Based Approaches

CRISPR-Cas9 technology enables precise genome modifications. In CAR macrophages, CRISPR can knock out inhibitory genes that promote M2 polarization or enhance pro-inflammatory factors that sustain an M1 phenotype. CRISPR-mediated knock-in strategies allow targeted insertion of CAR constructs into safe harbor loci, ensuring stable expression. Recent studies indicate that CRISPR-edited macrophages exhibit enhanced tumor infiltration and prolonged anti-tumor activity. Despite its potential, challenges such as off-target effects, delivery efficiency, and immune responses to CRISPR components must be managed. Ongoing research focuses on optimizing guide RNA design and employing high-fidelity Cas9 variants to improve specificity.

Intracellular Signaling Domains

The functionality of CAR macrophages depends on their intracellular signaling domains, which dictate how these cells respond upon antigen engagement. Unlike CAR T cells, which rely on CD3ζ and co-stimulatory domains such as 4-1BB or CD28, CAR macrophages require distinct signaling modules that align with their phagocytic and inflammatory nature.

A promising approach involves incorporating Fc receptor gamma (FcRγ) or DAP12 signaling motifs, which are naturally involved in macrophage-mediated phagocytosis. These domains activate spleen tyrosine kinase (Syk), a key regulator of actin remodeling and phagosome formation. Studies show that CAR macrophages equipped with FcRγ or DAP12 demonstrate increased engulfment of tumor cells, enhancing tumor clearance. Additionally, these signaling domains amplify pro-inflammatory mediator secretion, reinforcing the macrophage’s tumoricidal profile.

Further refinements have explored hybrid signaling constructs that integrate elements from classical macrophage activation pathways. For example, chimeric receptors incorporating Toll-like receptor (TLR) adaptor proteins, such as MyD88, enhance inflammatory signaling upon CAR activation. This design sustains macrophage activation and circumvents immunosuppressive cues in the tumor microenvironment. Researchers continue to evaluate which combinations yield the most durable anti-tumor responses while minimizing unintended hyperactivation.

Interaction With Tumor Microenvironment

The tumor microenvironment (TME) significantly influences CAR macrophage therapy, as it is characterized by immunosuppressive signals, dense extracellular matrices, and hypoxic conditions. Unlike CAR T cells, which struggle to persist in these conditions, macrophages naturally adapt to the biochemical and structural barriers present within solid tumors. Their ability to migrate through fibrotic tissue and respond to local cues allows them to infiltrate tumor masses more effectively.

Once inside the tumor, CAR macrophages directly target malignant cells for phagocytosis while influencing other immune populations through cytokine secretion. By releasing pro-inflammatory mediators such as IL-12 and TNF-α, they counteract immunosuppressive effects and shift the local immune balance toward an anti-tumor state. Additionally, CAR macrophages can degrade extracellular matrix components, reducing physical barriers that limit immune cell infiltration. Recent studies indicate that CAR macrophages engineered to express matrix-degrading enzymes show improved tumor clearance.

Distinctions From CAR T Cells

While both CAR macrophages and CAR T cells use engineered antigen receptors to recognize and eliminate cancer cells, their mechanisms differ. CAR T cells rely on direct cytotoxicity, using perforin and granzymes to induce apoptosis in target cells. This approach has proven effective in hematologic malignancies but faces limitations in solid tumors due to poor infiltration and exhaustion. In contrast, CAR macrophages employ phagocytosis, allowing them to function even in environments where T cell-mediated cytotoxicity is impaired.

Beyond direct tumor elimination, CAR macrophages enhance antigen presentation and modulate the immune landscape. Their ability to recruit and activate other immune cells provides a broader and more sustained response than CAR T cells alone. Additionally, macrophages are less susceptible to exhaustion, making them a promising alternative or complement to CAR T therapy, particularly in cancers where T cell-based strategies have struggled.