Adoptive Transfer of Macrophages

Macrophages are phagocytes widely distributed in various tissues, they can recognize, engulf and degrade pathogens and cellular debris, and participate in innate and adaptive immune responses. There are two developmental sources of macrophages: one is developed from hematopoietic stem cells in the bone marrow to monocytes, migrates to tissues and then differentiates into tissue-specific macrophages; the other is in the yolk sac, fetal Embryonic regions near the liver or dorsal aorta develop as resident macrophages. The differentiation and function of macrophages are influenced by a variety of factors, including signaling molecules, growth factors, transcription factors, epigenetic and post-transcriptional mechanisms and changes, and niche signaling. According to their function and activation status, macrophages can be divided into two subtypes: classically activated or M1-type macrophages and alternatively activated or M2-type macrophages. M1-type macrophages mainly have pro-inflammatory, anti-bacterial and anti-tumor effects, they can be polarized by lipopolysaccharide (LPS) alone or in combination with Th1 cytokines (such as IFN-γ, GM-CSF), and produce pro-inflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, IL-12, IL-23 and TNF-α; M2 type ,acrophages mainly have anti-inflammatory, immune regulation and tissue repair effects, and they can be stimulated by Th2 cytokines IL-4 and IL-13 polarize and produce anti-inflammatory cytokines such as IL-10 and TGF-β.

Fig 1 Summary of the main macrophage polarization states of activated macrophages. (Atri, 2018)

Adoptive transfer is an experimental method in which immune cells that have been manipulated or engineered outside the body are transplanted into another individual to observe their behavior and effects in vivo. Adoptive transfer can be used to study the development, differentiation, migration, function and interaction of immune cells. Adoptive transfer of macrophages refers to transplanting in vitro cultured or transgenic macrophages into another individual to explore their mechanism of action and therapeutic potential in different disease models. Adoptive transfer of macrophages is an important research direction, because it can reveal the key role of macrophages in infection, tumor, autoimmune diseases, etc., and provide a basis for the development of new immunotherapy strategies.

Introduction of Adoptive Cell Transfer Therapy

The principle of adoptive cell transfer therapy is to take the patient's own immune cells out of the body, undergo in vitro treatment or genetic modification, so that they have a stronger ability to recognize and kill tumor cells, and then infuse them back into the patient's body to exert anti-tumor effects. This approach can overcome the mechanism by which tumor cells escape the surveillance and clearance of the immune system, enhance the activity and persistence of immune cells, and improve the therapeutic effect.

Fig.2 Schematic representation of adoptive cell transfer immunotherapy procedure. (Maria, 2015)

Currently, the following types of cells are used in Adoptive Cell Transfer Therapy:

• Tumor-Infiltrating Lymphocyte (TIL) Therapy: This method uses lymphocytes isolated from tumor tissue of patients, expanded and activated in vitro, and then reinfused inside the patient. These lymphocytes are mainly T cells with killing function. They have shown the ability to recognize tumor cells, but the number or activity is not enough to destroy tumors. Through in vitro culture, the number and activity of these T cells can be increased, so that they can exert a stronger anti-tumor effect in vitro.
• Engineered T Cell Receptor (TCR) Therapy: This method utilizes T cells isolated from the patient's peripheral blood, which are genetically engineered to express specific T cell receptors (TCR), which can recognize specific antigens on the surface of tumor cells and activate the killing function of T cells. These genetically modified T cells are then infused back into the patient, enabling them to specifically attack tumor cells.
• Chimeric Antigen Receptor (CAR) T Cell Therapy: This approach also uses T cells isolated from the patient's peripheral blood and genetically engineered to express an artificial molecule called a chimeric antigen receptor (CAR). These CARs consist of a single-chain antibody fragment that recognizes a tumor-associated antigen and a T cell co-stimulatory molecule that delivers an activation signal. These genetically modified T cells are then infused back into the patient, enabling them to directly recognize and kill tumor cells.
• Natural Killer (NK) Cell Therapy: This method uses NK cells isolated from peripheral blood or umbilical cord blood of the patient or donor, expanded and activated in vitro, and then infused back into the patient. NK cells are a type of immune cell with natural killer ability, they can recognize and destroy abnormal cells lacking major histocompatibility complex (MHC) molecules, such as tumor cells and virus-infected cells. NK cells can also recognize and kill antibody-coated tumor cells through the mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC).

Macrophages-based Adoptive Cell Transfer Therapies

Macrophage-based immunotherapeutic strategies have been developed, such as targeted clearance or reprogramming of TAMs, engineering modification, or adoptive transfer of macrophages. Macrophage adoptive transfer is a method of using cultured macrophages in vitro to treat cancer. Macrophages are immune cells that can phagocytize and kill tumor cells, but in the tumor microenvironment, they are often transformed by tumor factors into M2 macrophages that promote tumor growth and metastasis. Therefore, genetically engineering or otherwise modifying macrophages to have a persistent M1 phenotype and infusing them into patients is a potential tumor immunotherapy strategy.

The role of adoptive transfer of macrophages mainly includes the following aspects. Macrophages directly phagocytize and kill tumor cells and release cytotoxic factors such as NO and ROS. Macrophages, as antigen-presenting cells, activate and expand specific T cells to form an adaptive immune response. Macrophages secrete pro-inflammatory and anti-tumor cytokines, such as IL-12, TNF-α, etc., regulate the immune microenvironment, and recruit other immune effector cells, such as NK cells and DC cells. Macrophages remodel the phenotype and function of TAMs from M2 to M1.

The mechanism of adoptive transfer of macrophages mainly involves the following aspects. Source and differentiation of macrophages, such as bone marrow, peripheral blood, spleen, etc. Activation and polarization of macrophages, such as IFN-γ, LPS, IL-4, etc. Gene modification and expression of macrophages, such as CAR, TRAIL, CD40, etc. Interaction and signal transduction between macrophages and tumor cells or other immune cells, such as FcγR, TLR, CD47-SIRPα, etc.

The advantages of the adoptive transfer of macrophages are as follows:

• Macrophages can be isolated from peripheral blood mononuclear cells for easy acquisition and expansion.
• Macrophages can efficiently infiltrate into tumor tissues and are not restricted by tumor vascular and stromal barriers compared with other immune cells such as T cells.
• Macrophages can directly kill tumor cells, and can also activate other immune cells through antigen presentation and secretion of pro-inflammatory factors to exert a synergistic anti-tumor effect.
• Macrophages can recognize and target tumor-associated antigens by expressing specific receptors or ligands, such as CAR or TIM-3 (T cell immunoglobulin and mucin domains) or immune checkpoints to improve the selectivity and effectiveness of treatments.

Fig.3 Schematic of diagnostic adoptive cell transfer. (Aalipour, 2019)

CAR-Macrophages

CAR-macrophage is a method of using genetic engineering technology to make macrophages express chimeric antigen receptors (CAR), which allows Macrophages to directly recognize and phagocytize tumor cells. The principle of CAR-macrophage is to remove the Macrophages of the patient or donor from the body, and through gene transfection, make it express a CAR molecule consisting of a single-chain antibody fragment that can recognize tumor-associated antigens and an Fc receptor that can transmit activation signals. These genetically modified Macrophages are then infused back into the patient, enabling them to directly recognize and phagocytose tumor cells.

Currently, the following types of CAR-macrophages are used for adoptive transfer of macrophages:

• HER2-CAR-macrophages: This type of CAR-macrophages can recognize and phagocytize tumor cells expressing human epidermal growth factor receptor 2 (HER2) antigens, such as breast cancer, ovarian cancer, and gastric cancer. In mouse models, HER2-CAR-macrophages exhibited strong tumor phagocytosis without causing severe side effects such as cytokine storm or neurotoxicity. Currently, HER2-CAR-macrophages are undergoing clinical trials for the treatment of HER2-positive solid tumors (NCT03390308).
• PSMA-CAR-macrophages: This type of CAR-macrophages can recognize and phagocytize tumor cells expressing PSMA antigens, such as prostate cancer. In mouse models, PSMA-CAR-macrophages exhibited efficient phagocytosis of tumors without causing side effects such as immune-related toxicity or renal impairment. Currently, PSMA-CAR-macrophages are in clinical trials for the treatment of metastatic castration-resistant prostate cancer (NCT04402788).
• EGFRvIII-CAR-macrophages: This type of CAR-macrophages can recognize and engulf tumor cells expressing EGFRvIII variant antigens, such as glioblastoma, etc. In mouse models, EGFRvIII-CAR-macrophages exhibited significant phagocytosis of tumors without causing side effects such as autoimmune toxicity or neurotoxicity. Currently, EGFRvIII-CAR-macrophages are undergoing clinical trials for the treatment of relapsed or refractory glioblastoma (NCT04185038).

In addition to the above-mentioned types, there are other CAR-Macrophages targeting different tumor-associated antigens being developed and tested, such as CEA-CAR-macrophages, MUC1-CAR-macrophages, CD19-CAR-macrophages, etc. At present, CAR-macrophage, as a new type of immune cell therapy, has great potential and prospects, but it also faces some challenges and problems, such as the plasticity of macrophages, the inhibitory effect of tumor microenvironment, and the control of side effects. Therefore, further research and optimization are needed to improve the safety and efficacy of CAR-macrophage.

Related Section

• Overview of Macrophage-based Immunotherapy
• Monoclonal Antibodies Targeting Macrophages
• Targeting Macrophages for Tumor Immunotherapy

What We Do

Empowered by our state-of-the-art CellRapeutics™ Chimeric Antigen Receptor (CAR) Technology, Creative Biolabs offers world-leading CAR-macrophage development services, aiming to improve the ability of CAR cells to attack solid tumors. Our one-stop services covering Target Identification & Selection, High-Affinity Antigen Binder Generation, CAR-MA Design & Construction, CAR-MA Preparation, Macrophages Activation and Expansion, In Vitro Assessments, Preclinical Tests and Clinical Trials. We also provide CAR-MA Vector Products, CAR-MA Cell Products, and a proprietary online system for customizing CAR and CAR cell products, which offers full options to meet all unique needs.

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