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Recent Trends in Chimeric Antigen Receptor (CAR) Macrophages for Cancer Immunotherapy

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Introduction

CAR-macrophage therapy is an emerging cancer treatment strategy that engineers macrophages to express chimeric antigen receptors (CARs), redirecting their natural tumor-homing, phagocytic, and immune-activating functions against cancer. Unlike CAR-T cells, which primarily kill through cytotoxicity, CAR-macrophages are designed to infiltrate solid tumors, engulf cancer cells, and remodel the tumor microenvironment to favor immune activation. Once activated by tumor antigens, these engineered macrophages not only phagocytose tumor cells but also present tumor-derived antigens to T cells and secrete pro-inflammatory cytokines, thereby bridging innate and adaptive immunity. This multifaceted approach aims to overcome barriers that limit current immunotherapies, such as poor penetration of solid tumors and immunosuppressive microenvironments, positioning CAR-macrophages as a promising complement to existing cancer therapies.

Macrophage Role in Immunity

Macrophages are frontline defenders of the immune system, acting as both scavenger and coordinator. Their main role is to recognize, engulf, and destroy harmful microbes and dead or damaged cells, while also activating and shaping adaptive immunity. Activation stimuli:

Microorganisms: bacteria, fungi, viruses, and parasites activate macrophages through recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors
Immune cells such as helper T cells activate macrophages primarily through interferon-γ (IFN-γ), while natural killer (NK) cells and dendritic cells also contribute by releasing cytokines (e.g., IFN-γ, TNF-α).
Damaged or apoptotic cells release “danger signals” (DAMPs) that trigger macrophage activation.

Functional responses:

Phagocytosis: Activated macrophages engulf microbes, apoptotic cells, and debris into phagosomes, which fuse with lysosomes to form phagolysosomes. Reactive oxygen species (ROS), nitric oxide, and proteolytic enzymes kill and degrade the material.
Antigen presentation: Fragments of internalized pathogens are processed and loaded onto major histocompatibility complex (MHC) molecules. Macrophages display these antigens to T cells (MHC II for CD4⁺ T cells, MHC I for cross-presentation to CD8⁺ T cells), bridging innate and adaptive immunity.
Effector protein regulation: Activated macrophages upregulate both secreted and membrane-bound molecules to amplify immune responses:
Secreted proteins: pro-inflammatory cytokines (IL-1, IL-6, TNF-α), chemokines (CCL2, CXCL8) to recruit immune cells, and effector molecules like nitric oxide to kill pathogens.
Membrane proteins: increased expression of MHC molecules, costimulatory molecules (CD80, CD86), and Fc/complement receptors to enhance antigen presentation and immune cell activation.

Through these functions, macrophages act as both killers of microbes and conductors of the immune orchestra, ensuring that pathogens are eliminated while adaptive immune cells are properly engaged.

Chimeric Antigen Receptors (CAR)

A CAR is a synthetic receptor engineered into immune cells (commonly T cells or macrophages) to give them the ability to specifically recognize and attack target cells, such as cancer cells, in a way that bypasses the normal antigen recognition pathways. CARs are “chimeric” because they combine parts from different proteins into a single molecule. CARs receptors are most commonly introduced into immune cells using lentiviral vectors. These are engineered viruses that cannot replicate but can efficiently insert the CAR gene into the immune cell’s DNA. This results in stable, long-term expression of the CAR. Lentiviruses are especially useful because they can infect both dividing and non-dividing cells, making them highly effective for macrophages and other immune cells. Their efficiency and durability make lentiviral delivery the standard method in most clinical CAR therapies. A CAR typically consists of four main domains, each with a distinct role:

Domain	Source/Composition	Function
Antigen-binding domain (scFv)	Derived from the variable regions of a monoclonal antibody (single-chain variable fragment, scFv)	Recognizes and binds specifically to the target antigen on the surface of a cell, e.g., CD19 on B cells.
Hinge (or spacer) domain	Often derived from IgG Fc or CD8 molecules	Provides flexibility and proper distance between the cell membrane and the antigen-binding domain, allowing the CAR to access antigens on the target cell.
Transmembrane domain	Typically, from CD3ζ, CD8, or CD28	Anchors the CAR to the immune cell membrane and can help with receptor stability and dimerization.
Intracellular signaling domain(s)	Derived from intracellular signaling protein domains of immune receptors	Transmits activation signals into the immune cell after antigen binding, inducing cell proliferation, cytokine release, cytotoxic activity, and survival.

CAR Design for Macrophages

Chimeric antigen receptors (CARs) used to engineer CAR-macrophages are synthetic transmembrane receptors designed to redirect macrophage phagocytosis and immune activation toward specific tumor-associated antigens. At the molecular level, CARs specifically designed for macrophages, typically consist of an extracellular single-chain variable fragment (scFv) derived from an antibody, which provides antigen recognition independent of MHC presentation. This scFv is fused via a flexible hinge and a transmembrane domain, to intracellular signaling modules tailored to macrophage biology. In CAR-macrophages, the CAR has intracellular signaling domains that incorporate phagocytic signaling motifs, combined with costimulatory or activating domains to promote both engulfment and inflammatory cytokine release. This modular design enables macrophages not only to phagocytose antigen-expressing tumor cells but also to remodel the tumor microenvironment through antigen-dependent activation, bridging innate and adaptive immune responses. Several intracellular signaling domains have been used to enhance the phagocytic ability and activation of CAR-macrophages. The Fcγ receptor γ-chain (FcRγ ITAM) contains tyrosine residues that become phosphorylated upon activation, promoting the formation of phagocytic cups and enhancing the engulfment of opsonized targets. The Megf10 intracellular domain, when activated, undergoes phosphorylation of cytoplasmic tyrosines, which facilitates efficient engulfment of apoptotic cells or antibody-tagged targets. DAP12, an ITAM-containing adaptor, triggers tyrosine phosphorylation upon ligand binding, leading to both phagocytosis and the production of inflammatory cytokines. Similarly, CD3ζ ITAMs from T cells become phosphorylated after CAR engagement, promoting target engulfment and the secretion of pro-inflammatory mediators. In all cases, these domains function as intracellular “switches” that, once activated, drive macrophages to engulf targets more effectively and enhance their immune response.

Process for Generation of CAR-Macrophages

Enrichment and cell selection

Isolation of monocytes or myeloid progenitors from peripheral blood or leukapheresis products using GMP-compliant immunomagnetic or flow-based selection to ensure a highly defined and homogeneous starting population.
Selection strategy is optimized to preserve precursor viability, functional plasticity, and differentiation potential while minimizing contaminating lymphoid or granulocytic populations.

Differentiation and conditioning

Ex vivo differentiation employs cytokine cocktails (e.g., M-CSF, GM-CSF, IL-4) and culture matrices to drive monocytes toward a macrophage lineage with defined phenotypic and functional characteristics.
Polarization toward pro-inflammatory (M1-like) or immunomodulatory (M2-like) states can be incorporated to tailor cytokine secretion profiles, phagocytic activity, and antigen presentation potential.

Genetic modification

CAR transgene integration is achieved via integrating viral vectors (lentivirus, retrovirus), non-integrating viral platforms (adenovirus), or transient delivery systems (mRNA electroporation) to control expression kinetics and duration.
Vector and construct design consider promoter strength, transgene copy number, immunogenicity, and regulatory compliance to balance stable expression, biosafety, and in vitro/in vivo potency.

Post-modification recovery and characterization

Engineered macrophages are recovered under optimized culture conditions that preserve viability, adhesion, and cytoskeletal integrity, crucial for effective phagocytosis and tumor infiltration.
In-process quality control includes multiparameter phenotyping (surface markers, CAR density), functional assays (antigen-dependent phagocytosis, cytokine release), and viability metrics to ensure consistent, therapeutically relevant product attributes.

Target Molecules for CAR-Macrophages

CAR-macrophages are being engineered to recognize a wide diversity of tumor-associated antigens, reflecting the heterogeneity of cancers and the need for broad therapeutic applicability. Targets include surface proteins such as HER2, mesothelin, and GD2, as well as extracellular matrix components like integrins and phosphatidylserine exposed in the tumor microenvironment. This diversity not only enables CAR-macrophages to address a range of solid and hematologic malignancies but also leverages their innate ability to remodel the tumor microenvironment, phagocytose malignant cells, and stimulate adaptive immune responses. The breadth of antigens under investigation underscores the adaptability of CAR-macrophage platforms and their potential to complement or overcome limitations of CAR-T cell therapies. Examples of tumor antigens targeted by CAR-macrophages

Tumor antigen	Cancer type(s) targeted	Preclinical results
HER2	HER2-overexpressing solid tumors (breast, gastric, others)	Induced antigen-specific phagocytosis, reduced tumor burden and prolonged survival in xenograft models; also remodeled TME with increased CD8+/CD4+ T cell infiltration in preclinical studies.
Mesothelin	Mesothelin-positive ovarian, pancreatic, mesothelioma	Mesothelin-CAR-macrophages showed targeted phagocytosis, tumor growth control in mouse models and induction of systemic anti-tumor immunity in preclinical studies.
MUC1	MUC1-expressing epithelial tumors (breast, ovarian, pancreatic)	Lipid-nanoparticle and ex vivo approaches to generate MUC1-CAR macrophages demonstrated efficient CAR expression in macrophages and tumor cell clearance in vitro and reduced tumor growth in preclinical models.
GD2	Neuroblastoma and other GD2+ solid tumors	Anti-GD2 CARs integrated into macrophage/ iPSC-derived macrophage platforms showed robust phagocytosis and antitumor activity in vitro and in vivo proof-of-concept studies.
PSMA	Prostate cancer (PSMA+)	Preclinical PSMA-CAR-macrophage constructs mediate PSMA-dependent tumor cell phagocytosis and slowed tumor growth in xenograft models.
PSCA	Pancreatic, prostate (PSCA+)	PSCA-targeted CAR-iMac (iPSC-derived CAR macrophage) platforms exhibited potent in vitro killing, good in vivo activity in pancreatic cancer models, and appeared safe in preclinical testing.
FLT3	Acute myeloid leukemia (FLT3+ AML)	FLT3-directed CAR-macrophages restored phagocytosis of FLT3+ leukemic cells and reduced leukemic burden / improved survival in AML xenograft models in preclinical reports.
CD26	Chronic myeloid leukemia (CD26+ CML / LSCs)	Recent preclinical work reports CD26-CAR-macrophages efficiently phagocytized CD26+ CML cells and reduced disease burden in preclinical models.
Claudin-18.2 (CLDN18.2)	Gastric, pancreatic and other CLDN18.2+ solid tumors	Groups have generated CLDN18.2-directed CAR constructs for macrophages showing antigen-dependent engulfment and tumor control in preclinical assays.
B7-H3 (CD276)	Glioblastoma, ovarian and other B7-H3+ tumors	B7-H3 CAR constructs adapted for macrophages have been described in preclinical development, showing target-dependent tumor cell clearance and potential to convert the TME.

CAR-macrophages in Clinical Development

Chimeric antigen receptor- macrophage therapies are engineered to combine the innate phagocytic and tumor-penetrating properties of macrophages with the antigen-specific targeting ability of CARs. Unlike CAR-T cells, CAR-macrophages are particularly attractive for solid tumors because of their ability to infiltrate tumor tissue, phagocytose malignant cells, and remodel the immunosuppressive tumor microenvironment. Although the field is still in its infancy, several early-phase clinical studies have been initiated against distinct tumor-associated antigens, providing the first signals of feasibility and safety. Representative CAR-macrophage clinical trials

Clinical trials	Target	Cancer	Status
NCT04660929	HER2	HER2-overexpressing advanced / metastatic solid tumors	Phase 1 first-in-human study of autologous adenovirally transduced anti-HER2 CAR-macrophages. Active/ongoing; preliminary safety & feasibility data reported.
NCT03608618	Mesothelin (MSLN)	Advanced ovarian cancer / peritoneal mesothelioma	Phase 1 intraperitoneal mesothelin-targeting CAR. Registered as an early clinical program
NCT05138458	CD5	Relapsed / refractory CD5-positive peripheral T-cell lymphomas (PTCL)	Phase 1/2 (Active; first-in-human dosing reported and FDA fast-track designation granted.
NCT04405778	Glypican-3 (GPC3)	GPC3-expressing solid tumors	Phase 1 study listed for a GPC3-directed CAR program in solid tumors. Status listed as early phase / recruiting in source summaries.

CAR-macrophage therapy has now advanced into first-in-human studies across multiple distinct tumor antigens. The most clinically mature is HER2, which has reported the first signs of safety and activity in patients. These trials underscore the potential of CAR-macrophages to directly attack cancer cells and reprogram the tumor microenvironment, offering a complementary strategy to existing T cell–based immunotherapies. Further clinical development will be critical to establish durability, efficacy, and combinatorial potential of CAR-macrophages in solid tumors.

CAR-macrophage therapies undesired effects

CAR-macrophage therapies are an emerging immunotherapy approach with strong potential for treating solid tumors. However, their safety profile reflects both the natural biology of macrophages and the added complexity of engineered receptors. The table below outlines the main risks associated with CAR-macrophage therapies. Undesired clinical effects of CAR-macrophages

Undesired Effect	Mechanism / Cause	Potential Consequence
Cytokine Release Syndrome (CRS)	Activated macrophages secrete IL-6, TNF-α, IL-1β	Systemic inflammation, fever, organ stress
On-target, Off-tumor Toxicity	Antigen expressed on normal cells	Phagocytosis of healthy tissue
Excessive Pro-inflammatory Polarization	Over-activation toward M1 phenotype	Bystander tissue injury
Tumor-driven Reprogramming	Tumor-derived IL-10, TGF-β convert CAR-M to M2-like cells	Loss of anti-tumor activity, possible tumor promotion
Non-tumor Tissue Infiltration	Migration into sensitive tissues (e.g., brain)	Local inflammation, risk of neuroinflammation
Persistence-related Risks	Limited survival reduces efficacy; engineered persistence may prolong activity	Either poor tumor clearance or chronic inflammation

Conclusion

Despite these challenges, CAR-macrophages show immense promise as a novel cancer therapy. They can directly phagocytose tumor cells while also reshaping the immunosuppressive tumor microenvironment into one that supports immune activation. Importantly, they can function as local “immune stimulators,” releasing cytokines and antigen fragments that recruit and activate endogenous T cells. Advances in CAR design, generation methods, antigen targeting, and mechanistic understanding have collectively positioned CAR-macrophages as strong candidates for next-generation cancer therapies. Early preclinical data suggest that CAR-macrophages can overcome barriers that have limited CAR-T cell efficacy in solid tumors, making them a powerful and versatile addition to the cell therapy toolkit. As preclinical findings transition into clinical testing, CAR-macrophage therapy may overcome some of the central challenges facing cancer cell therapy, making them a powerful and versatile addition to the anti-cancer toolkit. Disclaimer: This blog post is intended solely for educational and scientific informational purposes. Any mention of therapeutic drug names, including FDA-approved medications, is for the purpose of accurate reporting and discussion of biomedical research and does not constitute medical advice, endorsement, or promotion. Readers should not interpret the content as a recommendation for any specific treatment. Always consult a qualified healthcare professional for medical advice or treatment decisions.

About Marin Biologic Laboratories

Our Recent Publication/Meeting Presentation on Gene Therapy

1. Development of VNX-101, an Adeno-Associated Virus with Less Immunogenicity and Efficient Long-Term Expression of a CD19 T-Cell Engager. Molecular Therapy Methods & Clinical Development, published online July 24, 2025.

2. Development of a Pharmacokinetic (PK) Mouse Serum GLP ELISA for an Anti–CD19–AntiCD3 Diabody bioRxiv 2025.03.19.644217; doi: https://doi.org/10.1101/2025.03.19.644217.

3. Cell-Based Potency Assay for Anti-CD3-Anti-CD19 Diabody. bioRxiv 2025.04.15.648836v1 https://www.biorxiv.org/content/10.1101/2025.04.15.648836v1.

4. American Society of Hematology (ASH) Annual Meeting 2024. Abstract link: Using Gene Therapy to Solve Challenges with CAR-T Cell Immunotherapy: Lead Selection and Preclinical Development of an Adeno-Associated Virus with Reduced Immunogenicity Exhibiting Efficient and Long-Term Expression of an Anti-CD19 T-Cell Engager.

Comprehensive Assay Solutions for In Vitro and Cell Based Potency Assays and Pharmacokinetics Studies- Our Expertise

With 30 years of expertise in cell culture, cell-based assays, and preclinical/clinical PK/PD analysis, we specialize in offering assay services essential for a wide variety of therapeutic drug development programs, preclinical studies, IND/BLA applications, and commercialization. Our comprehensive services include both preclinical non-GLP and GLP assays, as well as non-GMP and GMP assays, providing critical support throughout the entire development pipeline. Watch the following video and explore our latest presentation on the development and validation of potency and pharmacokinetic (PK) assays for AAV vectors, highlighting innovative methodologies and industry-leading expertise.