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Induced Pluripotent Stem Cell-derived CAR-Macrophage Cells with Antigen-dependent
Anti-Cancer Cell Functions for Liquid and Solid Tumors
Li Zhang1, *, Lin Tian1, *, Xiaoyang Dai2, *, Hua Yu1, *, Jiajia Wang2, *, Anhua Lei1, Wei Zhao1,
Yuqing Zhu1, Zhen Sun1, Hao Zhang3, George M. Church4, He Huang3, Qinjie Weng2, †, Jin
Zhang1, †
1Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, &
The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang,
China. Institute of Hematology, Zhejiang University, Hangzhou 310058, China
2Institute of Pharmacology & Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer
Drug Research, College of Pharmaceutical Sciences, Center for Drug Safety Evaluation and
Research, Zhejiang University, Hangzhou 310058, China.
3The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang,
China. Institute of Hematology, Zhejiang University, Hangzhou 310058, China.
4Department of Genetics and Wyss Institute for Biologically Inspired Engineering, Harvard
Medical School, Boston, Massachusetts 02115, United States.
*These authors contributed equally to this work
†Correspondence: zhgene@zju.edu.cn, wengqinjie@zju.edu.cn
One sentence summary: We developed CAR-expressing iPSC-induced macrophage cells that
have antigen-dependent phagocytosis and pro-inflammatory functions and anti-cancer cell
activity for both liquid and solid tumor cells.
Abstract: The Chimera antigen receptor (CAR)-T cell therapy has gained great success in the
clinic. However, there are still major challenges for its wider applications in a variety of cancer
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2020. ; https://doi.org/10.1101/2020.03.28.011270doi: bioRxiv preprint
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types including lack of effectiveness due to the highly complex tumor microenvironment, and
the forbiddingly high cost due to personalized manufacturing procedures. In order to overcome
these hurdles, numerous efforts have been spent focusing on optimizing Chimera Antigen
Receptors, engineering and improving T cell capacity, exploiting features of subsets of T cell
or NK cells, or making off-the-shelf universal T cells. Here, we developed induced pluripotent
stem cells (iPSCs)-derived, CAR-expressing macrophage cells (CAR-iMac). These cells
showed antigen-dependent macrophage functions such as expression and secretion of cytokines,
polarization toward the pro-inflammatory/anti-tumor state, and phagocytosis of tumor cells, as
well as some in vivo anti-cancer cell activity for both liquid and solid tumors. This technology
platform for the first time provides an unlimited source of iPSC-derived engineered CAR-
macrophage cells which could be utilized to eliminate cancer cells or modulate the tumor
microenvironment in liquid and solid tumor immunotherapy.
Introduction
Human iPSCs derived from patients’ own cells can be differentiated to multiple cell types and
used for disease modeling, drug screening cell models and cell-based therapies1. Recently,
engineered T cells have shown great success in treating cancers in the clinic2. However, there
are a few outstanding challenges for engineering primary immune cells, including variable
transduction efficiency, highly heterogeneous genetic outcomes upon editing the genome of the
targeted cells, and limited cell resources for certain cases such as from hematological cancer
patients for which immune cell itself is transformed. iPSC-derived immune cells, due to their
flexibility of expansion and genome editing at the iPSC stage3, 4, in theory have advantages in
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dealing with those challenges above, and have been proved to be effective in treating B cell and
ovarian cancer cells in pre-clinical settings such as iPSC differentiated T cells5 and NK cells6.
Other than lymphoid lineage cytotoxic cells, a larger number of myeloid cells exist in the tumor
microenvironment7. Recently, myeloid cells such as macrophages have been utilized as a type
of effector cells to combat cancer cells by means of their phagocytosis function8. However, the
immortalized cell lines used in the previous study are not applicable to clinical settings, and
bone marrow-derived macrophages are not easily engineered, thus leaving iPSC-derived
macrophage cells as a great source for myeloid cell-based cancer immunotherapy. In addition
to phagocytosis function, it is believed that macrophages in a polarized pro-inflammatory state
can alter the tumor microenvironment through secreting cytokines to stimulate T cells9.
Moreover, it was reported that tumor macrophages transition to a pro-inflammatory polarized
state upon immune-checkpoint therapy (ICT) treatment in animal models10, and an
inflammatory signature of tumor samples predicts clinical benefits in ICT-treated patients11.
Applying the above features of macrophages to combat tumor cells is currently under intensive
investigation largely by targeting endogenous macrophages in vivo12, but not much effort has
been spent on adoptive transfer therapies using monocyte/macrophages13, partly due to the
difficulties in obtaining or expanding enough cells, and the low efficiency in engineering them
in order to harness these cells for combating cancer. Here, we used human iPSCs to introduce
a CD19 or mesothelin-targeting CAR, and differentiated them into macrophages, a product we
named as CAR-iMac. We thoroughly characterized these cells with bulk/single cell RNA-
sequencing and functional analyses, and showed they are heterogeneous and functional
macrophage cells. Upon challenge with antigen-expressing cancer cells, CAR-iMac cells show
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antigen-dependent increase of phagocytosis, strong induction of antigen-dependent cytokine
expression and secretion, and polarization toward a more inflammatory M1-like state.
Transplanting CD19 or Mesothelin CAR-iMac cells to liquid and solid tumor models showed
some in vivo anti-tumor effect. This CAR-iMac system provides a proof-of-principle platform
to use pluripotent stem cells to obtain a large amount of engineered macrophage cells with
antigen-dependent functions for adoptive transfer-based immune cell therapies.
Results
Engineering a CAR-expressing iPSC line for producing macrophages
We aim to develop engineered macrophages that have the following features: (1) the
differentiated macrophages are capable of phagocytosing tumor cells and producing cytokines
that may alter the tumor microenvironment; (2) these functions should be antigen-dependent;
and (3) the engineered cells should have a high percentage of transgene expression and can be
produced in large scale (Fig. 1a). First, we started from deriving iPSCs from Peripheral blood
mononuclear cells (PBMC) of a healthy donor with non-integrable episomal vectors encoding
OCT4, SOX2, KLF4, L-MYC and LIN28A (Supplementary Fig. 1a,b). Isolated single iPSC
clones showed pluripotent markers OCT3/4 and LIN28A/B expression (Supplementary Fig.
1c), and formed teratomas when injected to immune-deficient mice (Supplementary Fig. 1d).
We originally designed a CD19 CAR supposedly more suitable for macrophages by replacing
the 4-1BB and CD3ζ intracellular domains (T-CAR) with the macrophage CD86 and FcγRI
intracellular domains (M-CAR) (Supplementary Fig. 2a), and transduced M-CAR and T-CAR
into the THP-1 human monocyte cell line with lentivirus and achieved comparable levels of
expression (Supplementary Fig. 2b,c). To our surprise, even though the M-CAR-transduced
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cells showed increased level of cytokine expression and secretion when encountering the
antigen CD19-expressing K562 cells compared with the untransduced control, the increase was
more significant for the T-CAR-transduced cells (Supplementary Fig. 2d,e), suggesting T-CAR
is capable of stimulating monocyte/macrophage intracellular signaling and activating
macrophage specific genes. Based on these results, the CD19-targeting T-CAR transgene
(named as CAR thereafter) was introduced into the iPSCs by lentiviral transduction (Fig. 1a
and Supplementary Fig. 2f). qPCR showed mRNA of the CAR transgene was highly expressed
in these iPSCs (Supplementary Fig. 2g). We thus engineered a CAR-expressing iPSC line
amenable for producing differentiated products with antigen-dependent functions.
CAR-expressing iPSCs can differentiate to macrophage cells
Next, we established a protocol of myeloid/macrophage differentiation to induce iPSCs toward
myeloid cell lineages (Fig. 1b). After a period of mesoderm induction, hematopoietic
specification, myeloid expansion, and macrophage maturation, differentiated cells showed
typical macrophage marker gene expression such as CD14, CD11b, CD163 and CD86 (Fig. 1c),
as well as CAR expression on cell surface detected with an antibody against CD19 CAR
(Supplementary Fig. 2h). Consistently, key macrophage genes were induced such as AIF1,
CSF1R and SPI1, whereas pluripotent marker genes disappeared such as LIN28A and POU5F1
(Fig. 1d). RNA-sequencing using undifferentiated cells, early-day precursor cells and 18-day,
28-day 38-day differentiated cells showed that iPSCs clustered with precursor cells, and late-
day differentiated cells clustered with primary macrophage cells, or iPSC-differentiated
macrophage cells from previous studies14, 15 (Fig. 2a,b). However, it is challenging to classify
iMac to M0, M1 or M2 types of macrophages, likely because of the heterogeneous feature of
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the primary and differentiated cells from the published bulk RNA-sequencing data. Similarly,
selected M1 and M2 marker genes both showed high expression determined by qRT-PCR
(Supplementary Fig. 3a, b). GO and KEGG pathway analyses showed strong enrichment of
innate immunity-related functions in 18 or 28-day differentiated cells such as: “positive
regulation of cytokine production”, “regulation of innate immune responses” and “regulation
of phagocytosis” (Fig. 2c, d). Quantitative PCR also validated that the differentiated cells
expressed high levels of Toll-like receptor, Fc receptor and cytokine receptor genes, HLA genes,
NF-κB pathway genes, and costimulatory signaling genes, etc (Supplementary Fig. 3c-h), all
of which are required for certain specific monocyte/macrophage/dendritic cell (DC) functions.
Importantly, these genes are expressed in comparable levels with primary PBMC-derived
macrophage cells (Supplementary Fig. 3i). Together, these data show CAR-expressing iPSCs
can differentiate to myeloid lineage and macrophage-like cells.
Single cell RNA-seq analysis of CAR-iMac reveals a differentiation trajectory mainly
toward M2 macrophage and dendritic cells
To dissect heterogeneity of these CAR-iMac cells, we performed single cell RNA-sequencing
analysis of differentiated cells. These cells clustered away from undifferentiated iPSCs (Fig.
3a), and they appeared to be largely homogenous with only a few sub-populations not clustered
with the main population (Fig. 3b). Blasting the differentiated single cells in a database of
human cell atlas containing single cell RNA-sequencing data of various cell types revealed that
these iMac cells mainly clustered with macrophages (C0 and C5) or DCs (dendritic cells, C1,
C2, C3 and C8) (Fig. 3b, c), with typical genes expressed in each clusters such as CD14 in
macrophages and CD1C and HLA-DRB1 in DCs (Supplementary Fig. 4a). Small populations
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of cells still retained the HSC (hematopoietic stem cell) signature (C9, C7, C4) or differentiated
toward other lineages (C6). Trajectory analysis revealed that CAR-iMac cells went through a
path from HSC to macrophage and DC cells without major branches (Fig. 3d).
In order to determine whether each of these single cells or clusters are more polarized toward
the M1 or M2 state, we examined marker gene expression and found high and relatively
homogenous expression of M2 genes such as CD206 (Supplementary Fig. 4b). Moreover, all
10 clusters of differentiated cells showed strong signatures of “oxidative phosphorylation” and
“respiratory electron transport” which are usually associated with the M2 state, and weak
glycolysis or pro-inflammatory signatures associated with the M1 state16-19 (Fig. 3e). We further
compared cells in the 10 clusters with the LPS/IFN-γ-polarized M1 cells or IL-4/IL-10-
polarized M2 cells14, 15, by examining differentially expressed cytokine and chemokine genes
or M1/M2 associated metabolism genes (Fig. 3f, g ), and found that most clusters are more
similar to the M2 state, especially when using metabolism genes as markers. Together, these
data showed that in the absence of antigen, the first generation of CAR-iMac cells stayed in a
state closer to the classically defined M2 along the plastic macrophage spectrum20-23, likely
because of the presence of M-CSF during the differentiation process24.
CAR-iMac Cells Show Antigen-dependent Functions When Co-cultured with Leukemia
and Lymphoma Cells in Vitro
In order to test whether iPSC-derived CAR-expressing macrophages assumed the capability to
phagocytose tumor cells in an antigen-dependent manner, we incubated the CAR-iMac cells
labeled by a green dye with CD19-expressing K562 cells or K562 cells (K562 cell itself does
not express CD19) labeled by a red dye. Compared with K562 alone, K562-CD19 cells were
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more likely to be phagocytosed by CAR-iMac indicated by an increase from 51.9% to 76.4%
double positive cells which are the macrophages in the process of phagocytosing tumor cells
(Fig. 4a, b). Intracellular signaling such as phosphorylation of ERK and NF-κB P65 proteins
were increased in CAR-iMac co-cultured with CD19-expressing K562 cells compared to K562
cells, or to CAR-iMac cells cultured alone (Fig. 4c), indicating antigen engagement promotes
activation of macrophage intracellular signaling pathways.
One important role of macrophages is to modulate the tumor microenvironment by producing
cytokines that either stimulate or suppress T cell activity9. In order to harness these features,
we first examined cytokine gene expression in CAR-iMac cells, and found antigen-dependent
increase of pro-inflammatory cytokine expression, such as IL1B, IL21, IL12A and IL6, when
CAR-iMac cells were incubated with CD19-expressing K562 cells (Fig. 4d), as well as antigen-
dependent increase of cytokine secretion in the culture medium such as IL-6 and IL-12
determined by ELISA from the same condition (Fig. 4e). Moreover, transcriptional analysis
showed that CAR-iMac cells co-cultured with K562-CD19, in comparison to those with K562,
showed strong enrichment of up-regulated genes in GO or KEGG terms of “positive regulation
of cytokine secretion”, “antigen processing and presentation” and “Toll-like receptor signaling
pathway”, indicating these cells are more wired toward the pro-inflammatory state, when they
encounter the antigen (Fig. 4f, g). We also examined another leukemia cell line Nalm6
expressing CD19, and consistently, the CAR-expressing iMac cells showed increased
production of pro-inflammatory cytokines and increased level of phosphorylation of ERK,
compared to CAR-iMac cells alone (Supplementary Fig. 5a, b), again demonstrating the role
of CAR-antigen engagement in conferring the stimulated macrophage activity.
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We next investigated the effects of CAR-iMac or iMac cells on a CD19-expressing B cell
lymphoma cell line Raji. There is mild enhancement of phagocytosis conferred by the CAR
(Supplementary Fig. 5c, d). Moreover, CAR-iMac also had significantly increased pro-
inflammatory cytokine gene expression such as IL18, IL6 and IL1B, and other macrophage
function-specific genes compared to iMac without a CAR (Supplementary Fig. 5e). When we
examined intracellular signaling pathways, we found phosphorylation of AKT, SRC, STAT5
and ERK were all up-regulated when CAR-iMac cells encountered Raji cells (Supplementary
Fig. 5f). Taken together, the above data demonstrate that CAR-mediated signaling promotes
iMac phagocytosis and tuning toward the pro-inflammatory M1-like state upon encountering
the antigen on multiple types of leukemia and lymphoma cells (Fig. 4h).
CAR-iMac Cells Show Antigen-dependent Functions When Co-cultured with Solid
Tumor Cells in Vitro
To test iMac cells functions against solid tumors, we replaced the CD19 CAR scFv with a
mesothelin scFv in the lentiviral construct, and transduced the new construct to iPSCs followed
by differentiating them to CAR (meso)-iMac cells (Fig. 5a). We incubated the CAR (meso)-
iMac cells or control iMac cells with mesothelin-expressing OVCAR3 ovarian cancer cells.
Compared with control cells, CAR (meso)-iMac showed increased phagocytosis activity
against OVCAR3 (Fig. 5b, c). Moreover, transcriptome analysis showed that CAR (meso)-
iMac cells also had significantly up-regulated inflammatory and cytokine activity signatures
compared to the control when encountering OVCAR3 cells (Fig. 5d, e), which were validated
by increased pro-inflammatory M1 cytokine gene expression such as IL6, IL1A, IL1B, and TNF
(Fig. 5f). We also used a mesothelin-expressing pancreatic cancer line ASPC1, and found the
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same trends of up-regulated phagocytosis, M1 cytokine gene expression, and global
inflammatory signatures (Fig. 5g-j). Together, these data demonstrate CAR confers enhanced
phagocytosis and pro-inflammatory immune activities when CAR-iMacs are stimulated by
antigen-bearing solid tumor cells.
CAR-iMac Cells Show in Vivo Phagocytosis and Anti-Cancer Cell Function
In order to detect phagocytosis functions of CAR-iMac cells in vivo, we injected GFP-
expressing Raji cancer cells and CAR-iMac cells to NSG mice intravenously, and after three
days, the sorted human CD11b+ (from CAR-iMac cells) and GFP+ (from cancer cells) double
positive cells were examined with Giemsa staining (Fig. 6a). The macrophages showed multiple
nuclei, suggesting in vivo phagocytosis took place in the injected mice (Fig. 6a). To evaluate
the anti-cancer cell function of these CAR-iMac cells in vivo, we intraperitoneally injected
leukemia cells expressing a luciferase gene into NSG mice, and after tumors formed, mice
bearing comparable tumor loads were separated into two groups for treatment and controls. In
order to achieve better anti-cancer cell outcome, we further polarized the CAR-iMac cells
toward M1 by treating them with IFN-γ for 24 hours (Supplementary Fig. 6a), and then one
group of mice was injected intraperitoneally with the polarized CAR-iMac cells for treatment
(Fig. 6b). The treated mice showed smaller tumor size on day 10, with two mice having
complete remission (Fig. 6c). For solid tumors, we inoculated a high mesothelin-expressing
ovarian cancer cell line HO8910 intraperitoneally to NSG mice, followed by intraperitoneally
injecting IFN-γ-treated CAR (meso)-iMac cells (Fig. 6b). Two out of the five treated mice
showed tumor remission (Fig. 6d), and four out of five mice in the treated group lived for three
weeks but only two mice in the control group (Fig. 6d). The treatment did not work to the same
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extent in all mice, likely because not all the plastic iMac cells preserved in the same polarized
state in the complex and sometime immunosuppressive in vivo microenvironment in all mice.
Nevertheless, these data demonstrate CAR-iMac cells showed some anti-cancer cell functions
in vivo, but their efficiency needs to be further improved by designing more effective CARs or
further modifying the CAR-iMac cells to stay constitutively in M1 state.
Discussion
Recently, iPSC-differentiated CAR-expressing T cells and NK cells have been reported to have
potent cytotoxic activity against cancer cells, and they represent a new family of engineered
stem cell-derived immune cells for CAR therapies5, 6 A new member of this family, CAR-iMac
has three advantages. First, CAR-iMac can be utilized to specifically “eat” tumor cells or to
alter the tumor microenvironment, especially when its function is antigen-dependent, providing
a tool to directly kill tumor cells or to modulate a specific niche at the interface of tumor and
immune cells. Second, increasingly complicated genome engineering approaches can be
applied to iPSCs, followed by single clone expansion and thorough characterization to obtain a
clonal and genetically homogenous population with minimal off-target risk. Third, the seed
iPSC clone can be expanded in large scale, and when combined with the recently reported
techniques in making immune-tolerant universal iPSCs25-27, CAR-iMac can be developed into
an off-the-shelf product with unlimited sources. The last two points are particularly useful when
the effector cells from primary cell sources are difficult to engineer and to expand which is the
case for PBMC-derived macrophage cells. Thus, CAR-iMac is an excellent platform for
engineering-friendly and expandable macrophage cells, and a valuable addition to other iPSC
differentiated immune cells for further cancer immunotherapies.
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There are several general challenges of using iPSC differentiated cells for clinical applications,
such as undifferentiated cell can form teratomas, yield of differentiation and heterogeneity of
differentiated cells. We have achieved high differentiation efficiency with a yield of ~10-100
myeloid cells per starting iPSC. Even though certain cells remained some gene expression
signature of stem/precursor cells, we have not seen teratomas formation when we transplanted
the bulk differentiated cells into mice. In terms of heterogeneity, differentiated cells collected
from the floating shedded cells are primarily positive for macrophage markers such as CD14
and CD11b (Fig. 1c). More careful examination with single cell RNA-sequencing data revealed
a clear trajectory of differentiation toward macrophages and dendritic cells (Fig. 3c), which by
themselves share overlapping transcriptome signatures and functions, and can be named
altogether as “mononuclear phagocyte system (MPS)” cells28. Notably, in CD19 CAR-T cell
therapies or immune checkpoint therapies, combinations of CD8+ and CD4+ T cells have
superior anti-cancer cell activity29-31. It is thus conceivable that MPS cells may achieve
synergistic effects from different subsets of cells by simultaneously phagocytosing tumor cells
or presenting antigens to T cells. Therefore, it will be important to compare the purified versus
mixed myeloid products for their efficacy, persistence and toxicity in future studies. It will also
be of high interest to use humanized mouse models to study the role of differentiated DC-like
cells in tumor antigen presentation and T cell stimulation.
Another challenge to solve in order to take our findings to the clinic is to balance the anti-cancer
cell functions from pro-inflammatory cytokines and the toxicity induced by the same cytokines
such as IL-1 and IL-632, 33. In our system, we achieved antigen-dependent induction of these
cytokines, so that they can only be released in large amount within a restricted niche of antigen
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exposure. Indeed, our first generation of iMac cells were in a state closer to the immune-
suppressive M2 state and were tuned toward M1 after exposing to antigens, which suggests that
the cells may travel safely in the blood vessel, and may not become inflammatory until it
reaches to the tumor microenvironment (Fig. 4h). Another potential solution is to engineer
molecular switches to enable CAR or cytokine expression under control of endogenous
promoters34or exogenously administered small molecules35 so that inflammatory cytokine
expression is controlled by the cell state or controlled in a temporal way. Moreover, further
engineering the CAR intracellular domain to confer phagocytosis function without eliciting
inflammation -stimulating cytokines, such as using intracellular domain of FcγR36, or knocking
out endogenous inflammation-stimulating cytokine genes, are all possible solutions to explore.
Besides the safety issues, we explored the anti-tumor effect in animal models of both liquid and
solid tumors. The data showed that our first generation of CAR-iMac had some effect. While
our paper is being reviewed, Klichinsky et. al reported PBMC-derived macrophage transduced
with a CAR through adenovirus showed strong in vivo anti-tumor effect, and one key reason is
the adenoviral vector Ad5f35 used imparted a sustained pro-inflammatory M1 phenotype37.
Thus, there are many directions to improve the efficacy of our first generation of CAR-iMac
along this line. Our current iMac differentiation protocol contains M-CSF which is not only
required to support myeloid cell survival, but also unavoidably biases the differentiation toward
M2 state. Even though we added IFN-γ to polarize it to M1 state and CAR signaling tuned it
toward M1, but the epigenetic memory inherited may influence the ultimate state, particularly
for the plastic macrophage in the complicated in vivo microenvironment. Thus, a constitutive
M1 signaling is required to guarantee the cells to be constantly pro-inflammatory and anti-
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tumor. With the iMac system, this can be achieved by inserting certain M1 cytokine genes in
the genome of iPSC seed cells, or by further optimizing the intracellular domain of CAR to
confer M1 signaling, similar as the second signal provided by the “co-stimulatory” domain
from CD28 and 4-IBB in the second generation of CAR-T cells38, 39.
Together, our data provided a proof-of-principle platform of iPSC-based, engineering-friendly
macrophage for adoptive transfer immune cell therapy, and suggest that further development
and optimization of CAR-iMac is warranted. Its kaleidoscopic applications in various contexts
of liquid and solid tumors, and in combination of genome engineering or in combination with
other types of immunotherapies are full of great promises for further immune cell therapies.
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23. Martinez, F.O., Sica, A., Mantovani, A. & Locati, M. Macrophage activation and polarization. Front Biosci 13, 453-461 (2008).
24. Lachmann, N. et al. Large-scale hematopoietic differentiation of human induced pluripotent stem cells provides granulocytes or macrophages for cell replacement therapies. Stem Cell Reports 4, 282-296 (2015).
25. Deuse, T. et al. Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. Nat Biotechnol 37, 252-258 (2019).
26. Xu, H. et al. Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility. Cell Stem Cell 24, 566-578 e567 (2019).
27. Han, X. et al. Generation of hypoimmunogenic human pluripotent stem cells. Proc Natl Acad Sci U S A 116, 10441-10446 (2019).
28. Guilliams, M. et al. Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny. Nat Rev Immunol 14, 571-578 (2014).
29. Sommermeyer, D. et al. Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo. Leukemia 30, 492-500 (2016).
30. Turtle, C.J. et al. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. The Journal of clinical investigation 126, 2123-2138 (2016).
31. Alspach, E. et al. MHC-II neoantigens shape tumour immunity and response to immunotherapy. Nature 574, 696-701 (2019).
32. Giavridis, T. et al. CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade. Nat Med 24, 731-738 (2018).
33. Norelli, M. et al. Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells. Nat Med 24, 739-748 (2018).
34. Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113-117 (2017).
35. Sakemura, R. et al. A Tet-On Inducible System for Controlling CD19-Chimeric Antigen Receptor Expression upon Drug Administration. Cancer Immunol Res 4, 658-668 (2016).
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36. Ruffell, B. & Coussens, L.M. Macrophages and therapeutic resistance in cancer. Cancer Cell 27, 462-472 (2015).
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28
Figure legends
Fig. 1. Differentiation of CAR–iPSCs into macrophages. a, Schematics showing the
procedures of PBMC reprogramming, introduction of chimera antigen receptor and
differentiation to macrophage cells. b, Overview of the differentiation protocol to derive
macrophages from iPSCs. Representative microscopic pictures showing cells at different
stages during differentiation. The last picture is a magnified filed for matured macrophages
attached at the bottom of the plate at day 28. c, Flow cytometry analysis of iPSC-derived cells
at different stages of differentiation with stage specific markers. d, qRT-PCR showing
pluripotent marker gene and key macrophage marker gene expression at different stages of
differentiation. n=3, error bar: standard error of the mean.
Fig. 2. Transcriptional profiling of iPSC-derived CAR-iMac cells. a, Hierarchical clustering
of transcriptomes of iPSCs, differentiated cells, and primary and stem cell-differentiated
macrophages in different states. b, Principle component analysis (PCA) of the same samples as
in a. c, Top GO terms enriched in genes up-regulated on day 18 differentiated cells compared
with iPSCs. Right panel is an example of GSEA analysis of one GO term “Positive regulation
of cytokine production”. NES: normalized enrichment score. P=0: p-value is a very small
number. d, Top GO terms enriched in genes up-regulated on day 28 compared with iPSCs, and
GSEA analysis of “Positive regulation of cytokine production”.
Fig. 3. Single cell RNA-sequencing analysis shows CAR-iMac cells are closer to the M2 state
on the macrophage polarization spectrum. a, UMAP plot showing separation between human
iPSCs and CAR-iMac cells. b, UMAP plot showing sub-population clustering of CAR-iMac
cells. Ten clustered C0-C9 were identified and labeled as 0-9 with different colors. c, Heatmap
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29
showing blasting the C0-C9 clusters of cells illustrated in b against a human single cell atlas
database containing single cell RNA-seq data of hundreds of cell types including macrophages
(http://scibet.cancer-pku.cn). d, Trajectory analysis of differentiated cells along a pseudotime
axis. Left panel includes all clusters on the same trajectory map, and right panel is the
breakdown of each cluster on the same trajectory. The numbers on the trajectory mean branch
points or bifurcating events revealed by the trajectory analysis. MAC/DC means the cluster
matches both Macrophage and Dendritic cells. Cluster 6 is not assigned to a specific cell type.
e, Heatmap showing averaged expression of M1 or M2 signature pathway genes in different
clusters of cells illustrated in b. f, g, Heatmaps to compare (benchmark) the 10 clusters (C0-C9)
of CAR-iMac cells against previously published M1 or M2 polarized macrophages using either
top differentially expressed cytokine and chemoattractant genes f or using metabolism genes g
as signature genes. IPS_M1: Human iPS cells differentiated macrophages polarized by IFN-γ
and LPS; IPS_M2: Human iPS cells differentiated macrophages polarized by IL-4; HM_M1:
Human PBMC-derived macrophages polarized by IFN-γ and LPS; HM_M2: Human PBMC-
derived macrophages polarized by IL-4.
Fig. 4. CAR-iMac cells assume antigen- and CAR-dependent macrophage phagocytosis
function and pro-inflammatory signatures when co-cultured with liquid cancer cells. a,
Confocal microscopy pictures showing phagocytosis of K562 or K562-CD19 cells (red) by
CAR-iMac cells (green). iMac cells labeled by CMFDA were co-incubated with cancer cells
labeled by CTDR for 24 hours before imaging. b, Flow cytometry showing phagocytosis of
K562 or K562-CD19 cells by CAR-iMac cells. Labeled iMac cells were co-incubated with
labeled cancer cells and collected for FACS analysis. Double positive cells represent the iMac
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30
cells that engulfed cancer cells. c, Western blotting showing phosphorylation of ERK and NF-
κB P65 in CAR-iMac cells in the indicated conditions. d, qRT-PCR showing cytokine gene
mRNA expression when CAR-iMac cells were incubated with K562 or K562-CD19 cancer
cells for 24h, cancer cells were washed off before collecting CAR-iMac. n=3, error bar:
standard error of the mean. e, ELISA assay showing cytokine secretion in the medium when
CAR-iMac cells were incubated with K562 or K562-CD19 cancer cells. n=3, error bar: standard
error of the mean. **: p < 0.01, one-way ANOVA. f, Top GO terms enriched in genes up-
regulated in CAR-iMac cells co-cultured with K562-CD19 cells compared with CAR-iMac
cells co-cultured with K562 cells without the CD19 antigen. Right panel is GSEA analysis of
“positive regulation of cytokine production”. g, Top KEGG pathways enriched in genes up-
regulated in CAR-iMac cells co-cultured with K562-CD19 cells compared with CAR-iMac
cells co-cultured with CD19 cells. Right panel is GSEA analysis of “antigen processing and
presentation”. h, Schematic diagram showing CAR-iMac cells are more skewed toward M1-
like pro-inflammatory state upon encountering the antigen.
Fig. 5. iMac cells assume antigen- and CAR-dependent macrophage phagocytosis
functions and pro-inflammatory signatures when co-cultured with solid tumor cells. a, A
schematic picture to show the lentivirus construct of the CAR against mesothelin CAR (meso),
and the procedures to obtain CAR (meso)-iMac cells. b, Confocal microscopic images showing
phagocytosis of OVCAR3 cells (red) by iMac or CAR (meso)-iMac cells (green). c, Flow
cytometry showing phagocytosis of OVCAR3 ovarian cancer cells by iMac or CAR (meso)-
iMac cells. Labeled iMac cells were co-incubated with labeled cancer cells and collected for
FACS analysis. Squared parts represent the cancer cells engulfed by iMac cells. n=3. d, GO
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31
term analysis with RNA-seq data showing the up-regulated genes in CAR (meso)-iMac cells
compared with iMac cell when both cells were co-cultured with OVCAR3 cancer cells. e,
GSEA analysis of representative cytokine activity gene set showing it is up-regulated in CAR
(meso)-iMac cells compared with iMac cell when both cells were co-cultured with OVCAR3
cells. NES: normalized enrichment score. f, qRT-PCR showing cytokine gene mRNA
expression when iMac or CAR (meso)-iMac cells were incubated with OVCAR3 cells for 24h.
Cancer cells were washed off before collecting iMac or CAR-iMac cells. n=3. Error bar:
standard error of the mean. g, Flow cytometry showing phagocytosis of ASPC1 pancreatic
cancer cells by iMac or CAR (meso)-iMac cells. n=3. h, GO term analysis showing the up-
regulated genes in CAR (meso)-iMac cells compared with iMac cell when both cells were co-
cultured with ASPC1 cells. i, GSEA analysis of representative acute inflammatory response
gene set showing it is up-regulated in CAR (meso)-iMac cells compared with iMac cell when
both cells were co-cultured with ASPC1 cells. j, qRT-PCR showing cytokine gene mRNA
expression when iMac or CAR (meso)-iMac cells were incubated with ASPC1 cells for 24h.
Cancer cells were washed off before collecting CAR-iMac. n=3, error bar: standard error of the
mean.
Fig. 6. CAR-iMac cells showed in vivo phagocytosis function and anti-tumor effect. a,
Giemsa staining of FACS-sorted human CD11b and GFP double positive cells showing that
CAR-iMac cells phagocytosed Raji cells in vivo. 4x105 GFP-labeled cancer cells were
injected intravenously, and 6 hours later, 1x106 CAR-iMac were injected intravenously. At
day 3, cells were sorted for staining. Black arrows point to the multiple nucleus in the isolated
macrophages. b, A schematic picture of in vivo anti-tumor studies using luciferase
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32
(luc)-labeled CD19-expressing Nalm6 leukemia cells or mesothelin-expressing
HO8910 ovarian cancer cells in NSG mice treated with CAR-iMac cells. c,
Bioluminescent imaging pictures showing tumor development on the indicated days in mice
inoculated with Nalm6 leukemia cells and treated with iMac cells or PBS as described above.
d, Bioluminescent imaging pictures showing tumor development on the indicated days in
mice inoculated with HO8910 ovarian cancer cells and treated with iMac cells or PBS.
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Fig. 1
a
b
diPSEB Day2EB Day7iMac Day18iMac Day28
C
CD45
CD34
CD11bCD11b
CD14 CD14
CD163
CD86
Blood cell marker
HSC marker
iPS cells
differentiated cells
Day 14 Day 45Day 14
101 102
0
20
40
60
80
100
103 104 105 106
Macrophage marker
101 102
0
20
40
60
80
100
103 104 105 106
101 102
0
20
40
60
80
100
103 104 105 106
101 102
0
20
40
60
80
100
103 104 105 106 101 102
0
20
40
60
80
100
103 104 105 106101 102
0
20
40
60
80
100
103 104 105 106
101 102
0
20
40
60
80
100
103 104 105 106 101 102
0
20
40
60
80
100
103 104 105 106
Mesoderm induction
Hematopoietic specification
Myeloid expansion
Macrophage maturation
Macrophagemaintainenance
20μm
D0 D2 D7 D20 D28
20μm20μm
TREM2
AIF1
CSF1RSPI1
0.1
1
10
100
1000
10000
100000
1000000
Rel
ativ
e ex
pre
ssio
n
LIN28L-MYC
CAR
Reprogramming Engineering Differentiation
Human PBMC iPSC CAR-iPSC CAR-iMac
KLF4 SOX2 OCT3/4
c
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Day 18 differentiated cells vs iPS: up-regulated genes
Day 28 differentiated cells vs iPS: up-regulated genes
c
d
day 2
iPS
day 7
day 18
day 28
day 38
non−polarized macrophages
polarized with IFN−gamma and LPS
polarized with IL−4 and IL−13
polarized with IL−10
IPSDM M1
IPSDM M2
IPSDM Mac
p=0.0
positive regulation of cytokine production
NES=1.88
up-regulated at day 28 down-regulated at day 28
NES=1.78
p=0.0
positive regulation of cytokine production
up-regulated on day 18 down-regulated on day 18
pattern recognition receptor signaling pathway
positive regulation of cytokine production
response to molecule of bacterial origin
positive regulation of defense response
cytokine secretion
cellular response to lipopolysaccharide
regulation of immune effector process
regulation of innate immune response
toll-like receptor signaling pathway
regulation of inflammatory response
regulation of adaptive immune response
regulation of phagocytosis
-log10 p value
0 10 20 30
Macrophages polarized with IFN−gamma and LPS
Macrophages polarized with IL−4 and IL−13
day 2
iPS rep_1
iPS rep_2
iPS rep_3
day 7 rep_1
day 7 rep_2
day 7 rep_3
IPSDM Mac rep_3
IPSDM Mac rep_1
IPSDM Mac rep_2
IPSDM M2 rep_3
IPSDM M2 rep_2
IPSDM M2 rep_1
Non−polarized macrophages
day 18 rep_2
day 18 rep_1
day 28 rep_2
day 28 rep_1
day 38
IPSDM M1 rep_1
IPSDM M1 rep_2
IPSDM M1 rep_3
Macrophages polarized with IL−10
day 28 rep_3
CAR-iMac
iPS and precursor cells
a b
GO terms
GO terms
Fig. 2
pattern recognition receptor signaling pathway
0 10 20 30 40 50
-log10 p value
response to lipopolysaccharide
positive regulation of cytokine production
response to interferon-gamma
positive regulation of defense response
regulation of innate immune response
regulation of inflammatory response
I-kappaB kinase/NF-kappaB signaling
regulation of immune effector process
regulation of tumor necrosis factor production
antigen processing and presentation
regulation of toll-like receptor signaling pathway
iPS and precursor cellsCAR-iMac
PC1
PC
3
En
rich
men
t sc
ore
En
rich
men
t sc
ore
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C0:MAC/DC C1:DC/MAC C2:DC/MAC C3:DC/MAC C4:HSC
C5:MAC/DC C6 C7:HSC C8:DC C9:HSC
-5 0 5 10
-5
0
5
10
UMAP1
UM
AP
2Fig. 3
M1 signatures
M2 signatures
a
b
c
gf
1.25
1.00
0.75
0.50
0.25
Skeletal muscle myoblasts
Foreskin fibroblast cells
Hepatocytes
Embryonic stem cells
Muscle
Monocyte
Microglia
Mast
Macrophage
ILC
HSCsHEK and 3T3 mix
H9 cells
Epithelial
Endothelial
DC
0.75
0.50
0.25
0.00
C1 C2 C3 C4 C5 C6 C7 C8 C9C0
HM
_M
2
IPS
_M1_rep3
HK2NDUFB3SDHANDUFV1COX5BCOX7CNDUFA5COX11COX18SDHBNDUFB2NDUFB9NDUFB6COX6NDUFS7LDHBNDUFA7NDUFS6UQCRQUQCRC1UQCRBCOX4I1LDHAUQCRHCOX20NDUFAF2COX8ANDUFB8NDUFS8COX6A1NDUFS5NDUFA11UQCR10COX7A2COX6B1NDUFV2NDUFB7
HM
_M
1
IPS
_M1_rep5
IPS
_M1_rep1
IPS
_M
1_
rep
2
IPS
_M
1_
rep
4
C9
C6
C4
C7
C8
C2
C1
C3
C0
C5
IPS
_M
2_
rep
2
IPS
_M
2_
rep
3
IPS
_M
2_
rep
1
IPS
_M
2_
rep
5
IPS
_M
2_
rep
4
2
1
0
-1
-10
UM
AP
2
-10 0 10
0
5
10
-5
UMAP1
Oxidative phosphorylation
Respiratory electron transport
Glycolysis / Gluconeogenesis
Hallmark TNF-α signaling via NF-κB
Hallmark inflammatory response
0 1 3 4 5 6 7 8 92
Hallmark interferon gamma response
Clusters
6
4
2
0
-2
-4
-6
IL18CCL15IL2RAIL15TNFSF10CD80IL6CD7CD38
IL4I1TNFSF13B
IL27IL23ACXCL11CD48CD82CCL8CXCL10CXCL2IL15RAIL1RNCD40TNFAIP2CXCL9CD83CD300EIL32TLR8IL1AIL1BIL10CD1ECD36CCL26CD209TNFRSF11ATREM2CD200R1CD93CD180CSF1RCCL13IP
S_
M1
_re
p4
HM
_M
1
IPS
_M
1_
rep
3
IPS
_M
1_
rep
5
IPS
_M
1_
rep
1
IPS
_M
1_
rep
2
HM
_M
2
IPS
_M
2_
rep
4
IPS
_M
2_
rep
2
IPS
_M
2_
rep
3
IPS
_M
2_
rep
1
IPS
_M
2_
rep
5
C4
C7
C6
C9
C1
C3
C0
C5
C2
C8
CCL19
e
C0:MAC/DC C1:DC/MAC
C2:DC/MAC C3:DC/MAC
C4:HSC C5:MAC/DC
C6 C7:HSC
C8:DC C9:HSC
d
C0C1C2C3C4C5C6C7C8C9
HM
_M
2
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NES=1.41
p=0.0
positive regulation of cytokine secretion
NES=1.66
p=0.0
antigen processing and presentation
ba
dc
Fig. 4
f
g
e
CAR-iMac Cancer cell Merge
K562
K562-CD19
20μm
p-ERK
p-P65
GAPDH
CAR-iMac
positive regulation of cytokine production
pattern recognition receptor signaling pathway
positive regulation of cell motility
cell chemotaxis
positive regulation of defense responsepositive regulation of cytokine secretion
activation of innate immune response
toll-like receptor signaling pathway
chemokine-mediated signaling pathway
regulation of inflammatory response
I-κB kinase/NF-κB signaling
positive regulation of GTPase activity
-log10 p value
0 5 10 15
CAR-iMac with K562-CD19 vs CAR-iMac with K562: up-regulated genes
GO terms
KEGG terms
CAR-iMac with K562-CD19 vs CAR-iMac with K562: up-regulated genes
pg
/ml
IL-6
IL-1
2
02468
101000
1500
2000
2500**
**
IL23
CCL2
CCL13
IL12
ATNF
IL15 IL
6IL
1B
0
2
4
6
8102030405060
Rel
ativ
e ex
pre
ssio
n
CAR-iMac+K562CAR-iMac+K562-CD19
0 2 4 6
NOD-like receptor signaling pathway
Chemokine signaling pathway
NF-kappa B signaling pathway
C-type lectin receptor signaling pathway
Fc gamma R-mediated phagocytosis
Phagosome
Cytokine-cytokine receptor interaction
Antigen processing and presentation
-log10 p value
Can
cer
cell
K562 K562-CD19
CAR-iMac
51.9% 76.4%
M1 M2 M1 M2
h
En
rich
men
t sc
ore
En
rich
men
t sc
ore
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 29, 2020. ; https://doi.org/10.1101/2020.03.28.011270doi: bioRxiv preprint
iMac OVCAR3 Merge
iMac
iMac(meso)
20μm
20.9± 0.3 27.3± 1.4
iMac
OV
CA
R3
iMac CAR (meso)-iMac
7.9± 0.7 12.8± 0.6
iMac
AS
PC
1
iMac CAR (meso)-iMac
TNFIL
1BIL
1A IL6
0
2
4
6
Rel
ativ
e ex
pre
ssio
n
iMac+OVCAR3iMac(meso)+OVACR3
IL12
BIL
1BIL
1A IL6
TNF
0
5
10
15
Rel
ativ
e ex
pre
ssio
n
iMac+ASPC1iMac(meso) +ASPC1
a
c f
d e
0 10 20 30 40 50-log10 p value
immune response
inflammatory responseresponse to cytokine
regulation of defense response
immune effector process
response to lipopolysaccharide
cellular response to chemokinecytokine secretion
response to interferon-gamma
cytokine-mediated signaling pathway
0 10 20 30 40 50-log10 p value
immune response
immune effector process
defense response
inflammatory response
cytokine-mediated signaling pathway
cytokine secretion
cellular response to chemokine
chemokine-mediated signaling pathwayregulation of MAPK cascade
response to interferon-gamma
acute inflammatory response
NES=1.58
NES=1.54
cytokine activity
g j
h i
CAR-iMac with OVCAR3 vs iMac with OVCAR3: up-regulated genes
CAR-iMac with ASPC1 vs iMac with ASPC1: up-regulated genes
Fig 5b
En
rich
men
t sc
ore
En
rich
men
t sc
ore
iMac + OVCAR3CAR (meso)-iMac + OVCAR3
iMac + ASPC1CAR (meso)-iMac + ASPC1
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a
Fig. 6
cPBS CAR-iMac
Day -1
Day 3
Day 7
Day 10
Color ScaleMin=1.72e6Max=3.77e7Radiance(p/sec/cm2/sr)
Raji CAR-iMac Phagocytosing CAR- iMac
10 μm
b
Day -4
Inject Luc+ Nalm6 cells (4×105) I.P.
-1 day
+ IFN-γ(100 ng/ml)
Day 0
Inject CAR-iMac (1.2×107) I.P.
Day 3 Day 7 Day 10
Day -4Inject Luc+ HO8910 cells (7×105) I.P.
-1 day
+ IFN-γ(100 ng/ml)
Day 0
Inject CAR-iMac (3.5×106) I.P.
Day 3 Day 6 Day 10 Day 13 Day 17
Day 0 0h
I.V. GFP+ Tumor I.V. iMac
Day 0 6h
Sort human CD14+ / GFP+ iMac cells for Giemsa staining
Day 3
d
Day -1
Day 13
Day 3
Day 10
Day 17
Day 6
PBS CAR(meso)-iMac
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