Biomedicine & Pharmacotherapy 139 (2021) 111605

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

CAR-macrophage: A new immunotherapy candidate against solid tumors

Yizhao Chen a, Zhiying Yu c, Xuewen Tan a, Haifeng Jiang a, Zhen Xu a, Yilong Fang a, Dafei Han a,
Wenming Hong b, Wei Wei a, *, Jiajie Tu a, c, **
a
Institute of Clinical Pharmacology, Anhui Medical University, Key Laboratory of Anti-Inflammatory and Immune Medicine, Ministry of Education, Anhui Collaborative
Innovation Center of Anti-Inflammatory and Immune Medicine, Hefei, China
b
Department of Neurosurgery, The First Affiliated Hospital of Anhui Medical University, China
c
Department of Gynecology, The First Affiliated Hospital of Shenzhen University, Health Science Center; Shenzhen Second People’s Hospital, Shenzhen, China

A R T I C L E I N F O A B S T R A C T

Keywords: Chimeric antigen receptor (CAR)-T cell therapy has been shown to be an effective treatment for hematological
CAR-M tumors, but the treatment of solid tumors still lacks effectiveness. In the tumor microenvironment, macrophages
Phagocytosis are the innate immune cells with the highest infiltration rate. Tumor-associated macrophages (TAMs) stimulate
Solid tumor
angiogenesis, increase tumor invasion, and mediate immunosuppression. Because macrophages can infiltrate
CAR-T
CAR-NK
solid tumor tissue and interact with almost all cellular components in the tumor microenvironment (including
Immunotherapy tumor cells, immune cells such as T-cells, NK cells, DCs, and other resident non-immune cells), researchers are
trying to use macrophages modified with CAR (CAR-M) against solid tumors. This review describes recent reports
of CAR-M-based tumor treatments and summarizes their shortcomings and future applications.

1. Introduction
Tumor immunotherapy is developed on the basis of the study of
tumor immune escape, by manipulating the immune system to reac­
tivate the anti-tumor immune response and overcome the pathway of
tumor immune escape [1]. The purpose of tumor immunotherapy is to
modify immune cells in order to restore their ability to attack cancer
cells [2]. The main immunotherapies which are currently available are
chimeric antigen receptor (CAR)-T cell therapy, immune checkpoint
inhibitor and cancer vaccines [3]. CAR-T cell therapy is a cellular
immunotherapy proposed earlier, which is designed to avoid tumor cells
escaping TCR（T cell receptor） recognition. It refers to the method of
transferring genetic material with specific antigen recognition domain
and T cell activation signal into T cells by gene cloning technology, so
that T cells can be directly activated by binding with specific antigen on
the surface of tumor cells [4]. CAR-T cells have been successfully used to
treat malignant tumors in the circulatory system, such as B cell-source
malignancies [5,6]. After the success of CAR-T therapy, some re­
searchers applied CAR strategy to NK cells and developed CAR NK cell
therapy, which is similar to CAR-T therapy. They all kill tumor cells
thruough CAR targeting, the difference is that this therapy relies on the
broad cytotoxicity and rapid killing ability of NK cells [7]. However, due
to the high complexity of the tumor microenvironment in solid tumors,
CAR-T cell-mediated immunotherapy has shown low success rates [8].
Currently, tumor immunotherapy has mainly focused on adaptive im­
mune systems, such as T cells and B cells [9,10]. However, innate im­
mune cells, such as macrophages, have not been widely studied in
cancer treatment [11]. Taking into account their capabilities of phago­
cytosis, antigen presentation and penetration in the tumor microenvi­
ronment, macrophages should be considered in the treatment of solid
cancer [12,13].
The two main cell types of mononuclear-phagocyte systems (MPS)
are monocytes and monocyte-derived macrophages, both of which have
phagocytic functions [14]. MPS selectively devour external targets, such

Abbreviations: CAR, chimeric antigen receptor; TAMs, tumor-associated macrophages; NK, natural killer; DCs, dendritic cells; M, macrophages; TCR, T cell re­
ceptor; MPS, mononuclear-phagocyte systems; IL, interleukin; TGF, transforming growth factor; CAR-P, chimeric antigen receptor-phagocytes; iPSCs, induced
pluripotent stem cells; ECM, extracellular matrix; MMPs, matrix metalloproteinase; TIMPs, tissue inhibitors of metalloproteinases; HER2, human epidermal growth
factor receptor 2; INF, interferon; CRS, cytokine release syndrome; PBMCs, peripheral blood monouclear cells; GVHD, graft versus host disesse; NR, not reported.
* Correspondence to: 81# Meishan Road, Shushan District, Hefei City, Anhui Province, China.
** Corresponding author at: Institute of Clinical Pharmacology, Anhui Medical University, Key Laboratory of Anti-Inflammatory and Immune Medicine, Ministry of
Education, Anhui Collaborative Innovation Center of Anti-Inflammatory and Immune Medicine, Hefei, China.
E-mail addresses: wwei@ahmu.edu.cn (W. Wei), tujiajie@ahmu.edu.cn (J. Tu).

https://doi.org/10.1016/j.biopha.2021.111605
Received 22 December 2020; Received in revised form 30 March 2021; Accepted 12 April 2021
Available online 23 April 2021
0753-3322/© 2021 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y. Chen et al. Biomedicine & Pharmacotherapy 139 (2021) 111605

as microorganisms, rather than devouring the body’s own cells or the
extracellular matrix [15]. However, because of the mutated genome and
powerful "domestication" ability of tumor cells, macrophages are unable
to identify and attack tumors. Even within the tumor tissues, macro­
phages do not play a role in phagocytosis or antigen presentation, but
instead transition into an immunosuppressive M2 type that prevents the
immune system from removing tumor cells [16]. M2 macrophages can
repair tissue damage and build new blood vessels, and are also known as
alternative active macrophages. Secreted cytokines (such as interleukin
[IL]− 6 and transforming growth factor [TGF]-β) and the low-oxygen
environment of tumor tissues lead macrophages to mistakenly recog­
nize them as tissues which need to be repaired. Macrophages subse­
quently start repairing tumor tissues, building new blood vessels which
lead to the further development of cancer [17,18]. In addition to
phagocytosis, the high plasticity and polarization of macrophages are
also worthy of attention. Diversity and plasticity are significant char­
acteristics of monocyte macrophage lineage cells [19]. Macrophages
produce different phenotypes in different tissues to different microen­
vironment stimuli and signals, and may undergo M1 or M2 activation. In
immune response, macrophage polarization can reflect Th1-Th2 polar­
ization of T cells [20]. Moreover, macrophages polarized into M1 or M2
phenotypes can be reversed to some extent. In short, M1 phenotype is
highly expressed in inflammatory cytokines and has strong anti micro­
bial and tumor activity, while M2 is considered to promote tissue
remodeling and tumor growth [21]. It is of great significance to reverse
the M1-M2 phenotype in the treatment of tumor.
Attempts to combat cancer using macrophages began several de­
cades ago [22]. However, the evaluation of efficacy in these early clin­
ical trials showed that macrophage transplantation therapy had little
effect. This suggests that macrophages need additional signals to guide
their activity to fight tumors [23]. It was later discovered that in addi­
tion to the positive signals for macrophage phagocytosis, there were
opposite signals that prevented macrophages from devouring the target
tumor cells [24]. Self-labeling is common in cancer cells and is used as a
general mechanism to avoid being swallowed by macrophages. Labels
include the "don’t eat me" signal between CD47 and the macrophage
membrane receptor SIRPα [25]. These signals have become a popular
research area since their discovery.
Given the current success of CAR-T cell therapy and the potential for
development of CAR-natural killer cells, researchers have paid great
interest in developing CAR-macrophages (CAR-M) for tumor immuno­
therapy. The emergence of CAR-M opens up a new possibility for
treating solid tumors: modifying human macrophages with specific
CARs to improve phagocytic activity and antigen presentation of mac­
rophages against tumors [26]. CAR-M therapy refers to the transfer of
the edited specific CAR gene into macrophages in order to equip them
with the ability to bind to the tumor cell surface via specific antigen
identification and subsequently activate macrophage activity against
tumor cells [27].
2. Current attempts
The current attempts to use CAR-M to treat cancer are quite diverse
(Fig. 1 and Table 1). Researchers have designed chimeric antigen
receptor-phagocytes (CAR-P) which can guide macrophages to devour
specific targets. They demonstrated that CAR-P, which express the
intracellular domain of Megf10 or FcRv, promote the phagocytic po­
tential of target antigens. A series of experiments have shown that CAR-

Fig. 1. Structure diagram of CAR-M (A) Schematics show the structure of CAR-P. An αCD19 (orange) scFv directs CAR specificity, Intracellular signaling domains
from Megf10(green), FcRγ(blue) or tandem connection of fcrv and p85 subunit of PI3K(orange) activate engulfment. All constructs include a transmembrane domain
from CD8 and a C-terminal GFP. (B) An αHER2(purple) scFv directs CAR specificity, Intracellular signaling domains from CD3ζ(wathet blue) activate engulfment. (C)
CAR-147 is composed of a single-chain antibody fragment targeting human HER2, the hinge region of mouse IghG1, and the transmembrane and intracellular regions
of the mouse CD147 molecule. The HER2-scFv sequence was designed by Sangon Biotech based on reports in the NCBI database (PDB: 3H3B_D). (D) Schematics show
the structure of CAR-iMac, An αCD19 (orange) scFv directs CAR specificity, Intracellular signaling domains from FcγRI(grey) or CD3ζ activate engulfment. Compared
with FcγRI, the expression and secretion of cytokines in CD3ζ is higher.
All strucutures are modified from [22].

2
Y. Chen et al.
Table 1
Summary of the common CAR constructs used in CAR-M cell.
Target antigen Target cells
CD19 K562 cell
HER2 SKOV3 ovarian cancer cell line
HER2 4T1 cells overexpressing human HER2-EGFP
CD19 CD19 cancerous Raji B cells
Table 2
CAR-M-based clinical trials.
NO.NCT Star year Stage Tumors
03608618 2018 I Advanced Ovarian and Peritoneal Mesothelioma
04660929 2020 I HER2 Overexpressing Solid Tumors
The data used in the table are from https://clinicaltrials.gov/.
PMegf10 specifically triggered the phagocytic effect of the target ligand.
During the process of phagocytosis, local signaling cascades initiate
phagocytosis via tyrosine phosphorylation. The TCR-CD3ζ chain may
promote the phagocytic effect of CAR-P through the recruitment of syk
kinase. Whole-cell phagocytosis is rare, while biting or nibbling of target
cells is more frequent, suggesting that the interaction between CAR-P
macrophages and target cells is not sufficient to trigger direct whole-
cell phagocytosis. Previous studies have found that PI3K signals play
an important role in the internalization of large targets and promote
phagocytosis by macrophages. Researchers have linked the PI3K p85
subunit to CAR-P-FcRv to form a "tandem" CAR (CAR-Ptandem). CAR-
Ptandem has better whole-cell phagocytosis, implying that assembling
a motif of phagocytic effectors could increase the whole-cell phagocytic
activity of CAR-P [28].
Zhang et al. [29] used induced pluripotent stem cells (iPSCs) to ex­
press CAR structure and differentiate into macrophages, named
CAR-iMac. Experiments have shown that iPSCs, which express CAR, can
differentiate into macrophage-like cells. In the absence of antigen,
CAR-iMac is closer to the M2 polarization state. However, in the pres­
ence of specific antigens, such as leukemia and lymphoma cells,
CAR-mediated signals promote phagocytosis in CAR-iMac and lead to
transition of CAR-iMac to the inflammatory M1 subtype. Moreover,
stimulation by solid tumor cells expressing antigens also promoted the
phagocytosis and immune activity of CAR-iMac. Although CAR-iMac
exhibits anti-tumor activity, its efficiency can be further improved by
designing a more effective CAR structure or gene modification to keep
them closer to the M1 state. In general, iPSC-derived macrophages may
become an important source of cells for myeloid-based cancer
immunotherapy.
An important reason why CAR-T is not effective for solid tumors is
that T cells have difficulty entering into the tumor tissues. This is
because the physical barriers formed by the extracellular matrix (ECM)
of solid tumor and the consequent high tissue pressure prevent T cells
from entering the tumor tissue. ECMs are produced by highly organized
fiber molecules, glycoproteins, and other large molecules. Its synthesis
and degradation are mainly regulated by matrix metalloproteinase
(MMPs) and tissue inhibitors of metalloproteinases (TIMPs). MMP de­
grades almost all ECM and substrate membranes, and TIMP is an
important enzyme family that inhibits MMP activity. Macrophages are
an important source of MMP, and studies have shown that injecting bone
marrow-derived macrophages into mice can significantly reduce
cirrhosis and improve liver function [30].
Researchers designed a CAR, named CAR-147, consisting of a single-
strand antibody fragment targeting human HER2, a hinger of IghG1, and
a trans-membrane and intracellular region of CD147 molecules in mice.
Biomedicine & Pharmacotherapy 139 (2021) 111605
CAR construction Macrophage source Reference
CD19 + 4–1BB+CD3ζ induced pluripotent stem cells (iPSC) Li Zhang et al. [29]
HER2 + CD3ζ Primary human monocytes Michael Klichinsky et al. [32]
HER2 +CD147 Raw264.7 macrophages Wenlong Zhang et al. [31]
CD19 + Megf10 J774A.1 murine macrophage Meghan A Morrissey et al. [28]
CD19 + FcRV
CD19 +FcRV+PI3K
M source CAR structure Gene transfer
PBMC(including T cell, NK cell and monocyte) NR mRNA transfection
Primary human macrophages NR Adenovirus
transfection
After CAR-147 was co-cultured with HER2 + human breast cancer cells,
the expression of multiple MMPs in CAR-147 macrophages was induced,
proving that CAR-147 can specifically identify the antigen HER2 and
effectively activate the expression of MMP in macrophages. CAR-147
macrophages did not inhibit tumor cell proliferation in vitro, but
intravenous injection of CAR-147 macrophages significantly inhibited
tumor growth in 4T1 breast cancer mouse models. It was also found that
the proportion of T cells in tumors treated with CAR-147 macrophages
was much higher than that in tumors treated with controlled macro­
phages, demonstrating that CAR-147 macrophages can destroy the tu­
mor’s extracellular matrix and promote T cell infiltration into tumors. In
addition, it was found that CAR-147 macrophages can significantly in­
crease the levels of IL-12 and IFNγ in tumor tissue, thus playing an anti-
tumor role [31].
Researchers at the University of Pennsylvania have designed
adenovirus-induced CAR-M using an anti-HER2 CAR containing the
CD3ζ intracellular domain. First, the specificity of antigen-specific
phagocytosis of HER2 + tumor cells was validated in vitro. In two
ectopic mice models of solid tumor transplantation, a single injection of
anti-HER2 CAR-M reduced tumor load and prolonged the survival time
of mice. In a humanized mouse model, it was also found that HER2 CAR-
M transduced by adenovirus was able to transform M2 macrophages into
M1 macrophages, induce an inflammatory tumor microenvironment,
and enhance anti-tumor cytotoxicity of T-cells. In addition, it was also
found that HER2 CAR-M may produce epitope diffusion, which provides
new ideas for avoiding tumor immune escape [32,33].
3. The clinical applications of CAR-macrophages for the
treatment of solid tumors
Until November 2020, two clinical trials based on the CAR-M strat­
egy have been approved by the FDA. The first is a drug candidate from
CARISMA Therapeutics, CT-0508, which treats tumor patients with
relapsed/refractory HER2 over-expression with anti-HER2 CAR macro­
phages (Phase I clinical trial). The study recruited 18 patients with HER2
overexpressed solid tumors for the first time to study the effects of
adenovirus transduction CAR-M in humans.The other is MaxCyte’s
MCY-M11, which uses mRNA-targeted PBMCs(including CAR-M) to
express mesothelin-CAR, treating patients with relapsed/refractory
ovarian cancer and peritoneal mesothelioma. Volunteers were recruited
for Phase I clinical trial (Table 2).
As for the clinical translation of CAR-M, there are several aspects that
need attention: first, the safety and effectiveness of CAR-M is the basis of
this therapy, although it has been verified by animal experiments, the
safety and effectiveness of CAR-M in human body still need to be
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Y. Chen et al.
verified; second, reliable source and expansion are the necessary con­
ditions for the clinical application of CAR-M, which can be prepared by
PBMC or by iPSCs. In addition, unlike T cells, macrophage has a low risk
of GVHD, which means that products can be made in advance for pa­
tients to use on demand.Third, there is also a problem that has to be
considered. At present, CAR-M gene transfer using viral transfection,
which may induce insertion mutations and have an uncontrollable effect
on treatment. CRISPR/Cas9 genome targeting system provides a new
possibility to solve this problem, which takes only a week to complete
CAR-T gene editing [34].
4. Discussion and prospective
Similar to CAR-T and CAR-NK cells, CAR-M cells consist of extra­
cellular signaling domains that identify specific tumor antigens, trans-
membrane regions, and intracellular domains. At present, the study of
the extracellular signal domain identifies several common tumor targets,
such as CD19 and HER2. CAR-M research focuses on the activation and
enhancement of the phagocytic effect via different intracellular do­
mains. Beyond the structure of CAR-M, the method to transfer the CAR
gene also needs to be considered, as researchers at the University of
Pennsylvania have identified the M1 polarization induced by the
chimeric adenovirus vector Ad5f35.
Unlike CAR-T cells, CAR-M cells present the following three advan­
tages. 1) While T cells cannot enter the tumor environment because of
the physical barriers formed by the matrix around the tumor cells,
macrophages can significantly immerse in the tumor environment [35].
TAM plays an essential role in tumor invasion, metastasis, immuno­
suppression, and angiogenesis [36]. CAR-M can reduce the ratio of TAM
and affect the cellular phenotype of TAM, which has positive effects on
cancer treatment. 2) In addition to the phagocytosis of tumor cells,
CAR-M is also able to promote the ability of antigen presentation and
enhance the cytotoxic effects of T cells. 3) Compared to CAR-T, CAR-M
has a limited time in circulation and less non-tumor toxicity [37].
Cytokine release syndrome (CRS) is an inflammatory syndrome caused
by multiple cytokines that are released by CAR-T cells, including IL-1,
IL-2, IL-4, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF) α [38].
However, some studies have suggested that CRS is also related to IL-6
released by macrophages. Interestingly, while investigating CAR-M,
two different results were found: IL-6 levels in mice treated with
CAR-147 macrophages were lower than in the control group, while
CAR-iMac cells showed an increase in the production of IL-6 in an
antigen-dependent manner [39]. This may be related to the structure
and import method of CAR-M, or the different tumor types, which re­
quires further study.
Although CAR-M has great potential to become a powerful cancer
immunotherapy, many problems need to be overcome in order to ach­
ieve the desired results. The first is the limitation number of cells:
macrophages do not proliferate either in vitro or after injection in vivo.
Patients can accept macrophages only in limited amounts, which may
affect the effectiveness of treatment [39]. The second is related to the
migration characteristics of macrophages in vivo. After injection,
exogenous macrophages pass through the lung, and then most of them
remain in the liver, which is not conducive to the treatment of cancer
[40]. The third is the complex tumor microenvironment. Although
CAR-M has achieved good results in the mouse model, the actual tumor
microenvironment in humans is much more complex than the animal
model [41]. Finally, due to the high heterogeneity of tumor cells, the
expression of target antigens may not be sufficient. This problem has
been very prominent in CAR-T therapy. Clinical studies have found that
most of the tumor cells removed by CAR-T cells have high expression
levels of the target antigen [42]. Predictably, this will also be a major
obstacle to the development of CAR-M therapy.
As mentioned above, CAR-M therapy has shown its effective anti-
tumor ability in animal experiments. Compared with CAR-T and CAR-
NK, CAR-M has its unique advantages as a new cell immunotherapy,
4
Biomedicine & Pharmacotherapy 139 (2021) 111605
but it also has many shortcomings to be overcome. Therefore, attention
should be given to maximize the effectiveness and safety of CAR-M in
clinical treatment in the future. At first, the original structure of CAR
was designed for T cells, but there is no CAR structure specifically for
macrophages. However, in CAR-NK related studies, it has been proved
that the location of car binding epitope and its distance from the cell
surface affect antigen binding and cell activation [43]. Therefore,
starting from the structure of CAR-M, we can consider adjusting the
structure of car by associating different domains, to explore the most
suitable car structure for macrophages to enhance the phagocytic effect
of CAR-M on cancer cells. Secondly, by using genetic engineering
technology, CAR-M can also express anti-tumor cytokines or
co-stimulators to enhance antigen-presenting ability. The development
of CAR-M exerts other anti-tumor functions, such as secreting MMPs to
degrade the extracellular matrix or inducing epitope spreading capa­
bility [44]. Thirdly, combining CAR-M therapy with other immuno­
therapy is also a potential attempt. By blocking the "don’t eat me" signal
CD47-SIRPα may enhance CAR-M’s phagocytosis of targeted cancer
cells. In addition, for patients with heavy burden of solid tumors, it’s
worth to try to combine CAR-M with CAR-T. CAR-M can repress the
release of cytokines throughout the treatment process, which may
reduce the risk of CRS and neurotoxicity from CAR-T therapy [45].
Finally, the mechanisms and regulatory factors that affect phagocytosis
and antigen-presenting of macrophages, such as the physical properties
of the phagocytic target, should be further clarified [46].
Funding
This study was supported by the National Natural Science Founda­
tion of China (31900616, 81673444, 82003795), Natural Science
Foundation of Anhui Province for young scholars (1908085QH379),
China Postdoctoral Science Foundation (2020M672831), Sanming
Project of Medicine in Shenzhen (SZSM201812041) and Clinical
Research Funding from Shenzhen Second People’s Hospital (4001023).
Author contributions
YC and ZY drafted the manuscript. XT, HJ, ZX, YF, DH, WH, WW and
JT revised the manuscript.
Conflict of interest statement
The authors have declared no conflicts of interest.
Data availability
No new data generated.
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