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. 2023 Nov 28;11:103. doi: 10.1186/s40364-023-00537-x

Table 3.

Summary of the critical findings of preclinical CAR-M studies

Targeted- cancer or cell line Genetic enhancement CAR structure Results Ref

Melanoma

Neuroblastoma

CRISPR/cas9 hPSCs-derived GD2 targeted with CD3ζ-CD28-OX40

In-vitro results showed substantial anti-tumor activity

The neuroblastoma tumor burden is reduced in the xenograft mouse model with minimal adverse effects

[121]

Ovarian cancer

Pancreatic cancer

Leukemia

Lymphoma

Lentiviral

iPSCs-derived CD19-targeted CD86-FcγRI or

iPSCs-derived mesothelin-targeted-4-1BB-CD3ζ

In-vitro CAR-iMacs showed cytokines expression and phagocytosis in an antigen-dependent manner and polarized them toward an M1 state

in-vivo result CAR-iMacs showed some anti-tumor effect on leukemia cells expressing and high mesothelin-expressing ovarian cancer cell line

[129]

Lung tumors

Plural effusion malignant cells

Lentiviral THP-1 derived-MUC1 CAR-M CD3ζ-CD28-OX40 In-vitro results showed potent anti-tumor function by phagocytosis and secretion of pro-inflammatory cytokines such as IL-1β, IL-8, and TNFα [177]
Breast cancer Lentiviral HER2-targeting-CD147 CAR-M

in-vitro, CAR-147 macrophages did not inhibit the growth of tumor cells

in-vivo significantly inhibited tumor growth, remodeled the tumor ECM, and promoted T-cell infiltration, resulting in tumor growth

[178]
Ovarian cancer Adenoviral vector (Ad5f35) THP-1 derived HER2 CAR-M

In-vitro sustainable M1 phenotype and polarize M2 toward M1 phenotype. CAR-Ms phagocyte and eliminate tumor cells in an antigen-dependent manner

Significantly decrease tumor burden and improve overall survival in xenograft mice models

[105]
Mesothelin-positive tumor cells Adenoviral vector (Ad5f35) PBMC-derived mesothelin CAR-M with CD3ζ (CT-1119)

In-vitro: high CAR expression, possesses M1 phenotype with relative resistance to M2 shift, and exhibits robust tumor cell-killing capability and pro-inflammatory cytokines

Substantially decreased tumor burden in a murine xenograft model of lung cancer

[179]
Neuroblastoma Nano complex Anti-ALK CAR-M with- CD3ζ-CD28- IFN-γ gene

In-vitro: Transfection with MPEI/pCAR-IFN-γ did not very significantly polarize macrophage toward the M1 phenotype

The MPEI/pCAR-IFN-γ injection into Neuro-2a tumor-bearing mice reduced tumor growth, decreased the Treg cell, and increased the function of activated CD8+ T cells in the tumors

[123]
Glioma Nano complex Anti-CD133 CAR-M with- CD3ζ NP-pCAR induces M1 polarization and increases the secretion of IL-1β and TNF-α of targeted macrophages in-vitro. Also, it decreases the number of CD133-positive tumor cells and causes tumor regression without traceable toxicity in the orthotropic mouse glioma model [125]
Brain stem glioma Nano complex Anti-HER2 CAR-Ms In-vitro: PCD68/PB/N/R nanoparticles produce CAR-Ms with M1 phenotype and greater phagocytic and cytotoxic ability in an antigen-dependent manner. Intratumoral injection of P/PB/N/R nanoparticles generates Anti-HER2 CAR-Ms, causes phagocyte tumor cells, enhances immune system response, and tumor regression in mice without any notable side effects [124]
EGFRvIII positive cells Lentiviral IPSCs derived anti-EGFRIII CAR M with different intracellular domains, including CD3ζ, TIR, and CD3ζ-TIR CAR-iMACs demonstrate significant anti-tumor activity, but TIR-CAR-iMACs showed more pro-inflammatory activity, killing persistence, and greater anti-tumor activity in-vivo. In addition, the CD3ζ-TIR-CAR-iMACs could increase anti-tumor activity and contribute to retaining M1 polarization of CAR-iMACs, more robust anti-tumor capability compared to alone them in-vivo [128]
4T1 tumor cells (breast cancer) Lentiviral

RAW264.7-derived anti-CCR7 CAR M with

domain derived from TLR2, TLR4, TLR6, MerTK or 4-1BB-CD3ζ

MerTK-CAR-M displays targeted anti-tumor function in both in-vitro and in-vivo and exhibits most phagocytic and anti-tumor activity against tumor cells by reducing tumor burden, increasing median survival time and creating the inflammatory environment by increasing the levels of proinflammatory cytokines in serum In-vivo [131]
Raji B cells Lentiviral

J774A.1 cell line

Anti-CD19/CD22 CAR-P with different intracellular domain including Megf10, FcRγ, Bai1, and MerTK with/without PI3K

CAR-Ms with the Megf10 or FcRγ intracellular domains demonstrate the greater phagocytic capacity of CD19-positive cells compared to others

PI3K CAR-P induced some whole-cell phagocytosis, comparable to the CAR-P-FcRγ. Also, CAR-P tandem macrophages notably decreased the number of tumor cells. However, the CAR-P tandem was much more efficient at whole-cell phagocytosis than the CAR-P-FcRγ

[106]
CT26-HER2 cell line Adenoviral vector (Ad5f35) BM-derived anti-HER2 CAR M cells CAR M cells showed anti-tumor activity and phagocyte HER2-overexpressing tumor cells and boosted the cytotoxicity of CD8+ T cells by inducing MHC-I expression on tumor cells. In-vivo, CAR-M causes significant tumor regression, improves overall survival, increases infiltration rate of CD4+ and CD8+ T, NK, and DC cells in the TME, and ameliorates epitope spreading, improving T-cell reaction to TAA [180]
Raji cells Lentiviral BM-derived anti-CD19 CAR M with different intracellular domains, including Megf10, PI3K, and FcRγ CAR-M PI3K and CAR-M FcRγ exhibit more phagocytic capacity; CAR-M FcRγ possesses more substantial cytotoxic and phagocytic ability. Also, the utilization of CAR-M FcRγ and CAR-T cells together showed substantially greater cytotoxic power than CAR-M FcRγ or CAR-T cells alone, and inflammatory factors such as IL-1β, IL-6, IFN-γ, CXCL1, MCP-1, and MIP-2 have notably increased in-vitro. CAR-T cells Secrete Inflammatory factors like IFN-γ and GM-CSF, which increase CD80/86 expression and probably induce M1 polarization of CAR-Ms. Upregulated CD80/86 on CAR-Ms may ameliorate CAR-T cell activation and fitness [130]
Lung cancer brain metastasis cell (H2030BrM) - Anti-MSLN CAR-M with MyD88 signaling molecule In the humanized mouse model, CAR-Ms penetrated BBB and significantly reduced brain metastasis growth. The CAR-Ms exhibit antigen-specific phagocytosis activity against MSLN-positive tumor cells. Also, CAR-M demonstrates much fewer neuron toxicities and liver compared to CAR-T [181]
Mammary gland squamous carcinoma cell line (HCC-1806) -

RAW 264.7 derived Anti-MSLN CAR-M with

TLR4 and TLR2-based toll-like receptor signaling domains

In-vitro, MOTO-CARs effectively kill cancer cells and secrete notable levels of TNF-α, which displays that MOTO-CARs polarize toward the M1 phenotype upon target recognition

In-vivo, the MOTO-CARs can traffic effectively to the tumor site and substantially reduce tumor burden compared to the mock control

[182]
Non-small cell lung carcinoma NCI-H460 and A549 cell lines

lentiviral or adenoviral

(Ad5f35)

Human monocyte-derived anti-TK1 CAR M with TIR signaling In-vitro, after transduction, MOTO-CARs exhibit sustainable M1 phenotype and express low levels of CD163 and high levels of CD14, CD80, and CD206. MOTO-CARs demonstrated a nearly fourfold increase in killing activity against cancer cells compared with the controls [183]

HT1080

cells

Lentiviral CB-HSPCs, THP-1 and MONO-MAC-6 anti-CEA CAR-M with different intracellular domains including 2B4-DAP12, CD28-CD3ζ

Ex vivo, the CAR gene was transduced to CB-derived HSPCs successfully and generated CAR-Ms have stable CAR expression. Also, any evidence of that

CAR expression might alter CD34 positive cell-derived macrophage morphology, phenotype, or basic

anti-bacterial phagocytic function, but it was not observed. Both anti-CEA-CARs showed antigen-specific function by increased cytokine secretion, and CD3ζ CAR-expressingTHP-1-derived macrophages showed enhanced antigen-specific phagocytosis of target cells

[143]

Abbreviations: TK1 Anti-Thymidine Kinase1, hPSCs Human pluripotent stem cells, GD2 Disialoganglioside, iPSCs Induced pluripotent stem cells, MUC1 Mucin short variant S1, TNFα Tumor necrosis factor, HER2 Human epidermal growth factor receptor 2, ECM Extracellular matrix, PBMC Peripheral blood mononuclear cells, ALK Anaplastic lymphoma kinase, MPEI, NP-pCAR Nano particle-plasmid chimeric antigen receptor, Epidermal Growth Factor Receptor (EGFRvIII), TLR Toll-like receptors, MerTK MER Proto-Oncogene, Tyrosine Kinase, Megf10 Multiple EGF-like-domains 10, FcRγ Fc receptor γ-chain, Bai1 Brain angiogenesis inhibitor 1, MHC-I major histocompatibility complex, TME Tumor microenvironment, NK Natural killer, DC Dendritic cell, TAA Tumor associated antigen, PI3K Phosphatidylinositol-3 kinase, MIP-2 Macrophage inflammatory protein 2, MCP-1 Monocyte chemoattractant protein-1, CXCL1 CXC motif chemokine ligand 1, GM-CSF Granulocyte macrophage colony-stimulating factor, MSLN Mesothelin, BBB Blood brain barrier, MyD88 Myeloid differentiation primary response protein, CB Cord blood, CEA Carcinoembryonic antigen, DAP12 DNAX activating protein of 12 kDa