Chimeric antigen receptors for adoptive T cell therapy in acute myeloid leukemia
Fan et al. Journal of Hematology & Oncology
Chimeric antigen receptors for adoptive T cell therapy in acute myeloid leukemia
Mingxue Fan 0 2
Minghao Li 0 2
Lipeng Gao 0 2
Sicong Geng 1
Jing Wang 0 2
Yiting Wang 0 2
Zhiqiang Yan 0 2
Lei Yu 0 2
0 Institute of Biomedical Engineering and Technology, Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering , East China Normal University, NO. 3663 Zhongshan Road, Shanghai 200062 , People's Republic of China
1 China Novartis Institutes for Biomedical Research Co., Ltd., GDD/TRD/Chemical and Pharmaceutical Profiling , 5F, Building 3, Novartis Campus 4218 Jinke Rd, Zhangjiang Hi-Tech Park Pudong District, Shanghai 201203 , China
2 Institute of Biomedical Engineering and Technology, Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering , East China Normal University, NO. 3663 Zhongshan Road, Shanghai 200062 , People's Republic of China
Currently, conventional therapies for acute myeloid leukemia (AML) have high failure and relapse rates. Thus, developing new strategies is crucial for improving the treatment of AML. With the clinical success of anti-CD19 chimeric antigen receptor (CAR) T cell therapies against B-lineage malignancies, many studies have attempted to translate the success of CAR T cell therapy to other malignancies, including AML. This review summarizes the current advances in CAR T cell therapy against AML, including preclinical studies and clinical trials, and discusses the potential AML-associated surface markers that could be used for further CAR technology. Finally, we describe strategies that might address the current issues of employing CAR T cell therapy in AML.
Chimeric antigen receptors; Acute myeloid leukemia; Immunotherapy
Acute myeloid leukemia (AML) is a cancer of the myeloid
line of blood cells that is characterized by the clonal
expansion of abnormal myeloid progenitors in the bone
marrow and peripheral blood, which interferes with the
normal production of blood cells. AML is a rare disease,
and its incidence increases with an aging population, as
this disease is most commonly found in adults [
]. In the
past 5 years, the cure rate was 35–40% for AML patients
under 60 years old and 5–15% for patients older than 60.
The elderly, who are unable to withstand intensive
chemotherapy, have an average survival of 5–10 months
]. Despite improving our understanding of AML, the
disease still has poor outcomes due to high disease- and
Forty years ago, the combined injection of cytarabine
and anthracycline was introduced as the first standard
treatment for AML [
]. Since then, many chemotherapy
regimens have improved outcomes for some AML patients
. However, the effectiveness of traditional chemotherapy
may have hit a ceiling for treating AML, especially for
older patients and those who either tend to relapse or have
intermediate- or high-risk factors associated with AML [
In addition, allogeneic hematopoietic stem cell
transplantation (allo-HSCT) has been the most successful
immunotherapy for AML over the past decade, especially with the
advances made in using alternative donors [
Unfortunately, older and less fit patients are poor candidates for
allogeneic HSCT due to significant toxicity and a high
relapse rate [
]. The limited success and high toxicity of
the currently available strategies indicate an urgent need
for new therapeutics. It is possible that the infusion of
allogeneic chimeric antigen receptor (CAR) T cells could
enhance the efficacy of allogeneic HSCT [
possibility is supported by recent evidence that a child with
acute lymphoblastic leukemia (ALL) at the Children’s
Hospital of Philadelphia relapsed after a cord blood
transplant and then received infusions of CTL019 CAR T cells,
resulting in a remission of leukemia without
graft-versushost disease (GVHD) [
]. In addition, another recent
study showed that the treatment of allogeneic CAR T cells
is beneficial for patients with relapsed B cell malignancies
after allo-HSCT with low toxicities and complications [
Therefore, the CAR-expressing T cell technology, which
has been successfully implemented in treating acute
lymphoblastic leukemia (ALL), has been considered a
promising immunological approach for the treatment of
]. This new type of targeted
immunotherapy merges the exquisite targeting specificity of
monoclonal antibodies with the potent cytotoxicity and long-term
persistence provided by cytotoxic T cells. CAR is an
artificial antigen receptor that mediates
antibodytargeted recognition. The binding between CAR and its
antigen on tumor cells triggers a signal transduction
cascade through signaling domains and then activates T
cells to kill the target directly or through other
components of the immune system (Fig. 1) [
]. At the
beginning of the in vitro expansion stage, CAR can be
transferred to the patient’s selected T cells using either
viral vectors or non-viral approaches [
]. The viral
vectors include retroviruses (including lentivirus),
adenovirus and adeno-associated virus. Among them,
γ-retroviral and lentiviral vectors have been the most
useful carriers for long-term gene expression because
of their ability to integrate into the host genome and
their low intrinsic immunogenicity [
]. In contrast
to γ-retroviral vectors, lentiviral vectors can deliver
larger DNA sequences and integrate into non-dividing cells,
which are less susceptible to silencing by host restriction
factors . Lentiviral vectors are more commonly used
in clinical trials because of their safer integration site
]. Non-viral systems, including nude DNA, mRNA,
liposomes, etc., are very effective in gene delivery because
of their higher efficiency, non-infectiousness, unlimited
carrier capacity, controlled chemical constitution and
generous production. For instance, mRNA electroporation
in clinical trials induced the transient expression of CAR
for approximately one week and prevented the potential
toxicity of CRS [
The adoptive cell therapy of CAR-expressing T cells is
a new but promising approach in the field of cancer
immunotherapy. The development of CAR T cells can be
divided into four generations based on the different
characteristics of intracellular domains (Fig. 2). The
CAR prototype consists of an extracellular domain that
serves as the targeting moiety (which is usually a single
chain variable fragment (scFv) formed from a
monoclonal antibody, mAb), a transmembrane domain, and an
intracellular signaling domain(s) [
]. CARs with the
typical structure of “scFv-spacer-CD3z” design are called
“1st-generation CARs”. In contrast to the T cell receptor
(TCR), “1st-generation CAR” recognizes the targets
independent of major histocompatibility complex (MHC)
restriction, therefore making it highly specific for various
surface antigens on tumor cells [
]. However, further
research has gradually shown that “1st-generation CARs”
exhibit the problems of deficient secreted cytokines,
inadequate proliferation, and low persistence in vivo. To
overcome these weaknesses, CD3ζ as well as
costimulatory signaling domains, such as 41BB and CD28,
were incorporated into the intracellular domain to form
the so-called “2nd-generation CAR” [
]. The added
co-stimulatory signal can help complete the activation of
T cells and avoid apoptosis by promoting the IL-2
synthesis. The CD28ζ CAR T cells primarily caused
constitutive stimulation, proliferation and growth. In contrast,
the 41BBζ CAR T cells induced early exhaustion, thereby
limiting the antitumour efficacy [
the CD3ζ plus two co-stimulatory signaling domains,
41BB- and CD28, were introduced into the intracellular
domain of “3rd-generation CARs” to augment cytokine
production and cancer-killing ability [
Unfortunately, the latest studies have shown that the
“3rd-generation CARs” did not produce more desirable outcomes
compared with the “2nd-generation CARs”. Because the
stronger stimulation may produce potential side effects,
such as cytokine release syndrome (CRS), further studies
should be performed to explore the safety of
“3rd-generation CARs”. Notably, recent studies have indicated that
CAR reached its limit when targeting tumors with a
remarkable phenotypic heterogeneity. Subsequently, the
“4th-generation CARs”, i.e., so-called TRUCK T cells,
were proposed, which are formed by an additional
modification with an inducible expression cassette for a
transgenic protein. For example, a cytokine such as IL-12 can
be released by CAR T cells to modulate the T cell
response, which can help keep the local therapeutic
concentration and avoid systemic toxicity by releasing a
variety of therapeutic proteins . We anticipate that
the future new generations of CAR will overcome some
of the corresponding problems of CAR T cell therapy.
Translating the success of CAR T cell therapy to other
malignancies that have an unmet medical need, such as
AML, is currently underway. For instance, CAR T cell
therapy has been developed in patients with AML, as
many detailed studies have been published. This review
summarizes the recent application of CAR T cell therapy
in AML and focuses on AML-associated cell surface
antigens that could be potential target candidates for CAR
T cell therapy. Finally, we discuss the common issues of
CAR T cell therapy in AML and summarize the
strategies of building CAR T cells with improved safety and
The application of CAR-modified T cells in AML
Despite the enormous challenge of developing CAR T
cells for multiple diseases, several potential CAR T cell
targets have been actively explored in preclinical studies
and clinical trials over the past decade (Fig. 3). From the
evolution of CAR T cell therapy for treating AML, it
may be observed that we have gradually shifted the focus
of our research on creating safer and more effective
CAR-modified T cells for the past two years using
Lewis Y antigen
One of the remarkable advantages of CAR T cell therapy
is the ability to recognize a broad variety of targets such
as non-protein antigens. The Lewis Y antigen (LeY) is an
example of this situation; LeY is an oligosaccharide that
is overexpressed on many epithelial cancers and
hematological malignancies (including AML) [
but has limited expression on normal healthy tissues
]. The LeY - CAR T cell trial was the first CART
therapy clinical trial targeting AML (ClinicalTrials.gov
number, NCT01716364), evaluating the effect of an
autologous second-generation anti-LeY CAR T cell therapy
in 4 patients with relapsed AML. Following fludarabine
preconditioning, the patients were administered up to
1.3 × 109 of total T cells (14–38% CAR T cells). The
results showed that two patients achieved protracted
remission, one patient achieved cytogenetic remission, and
the fourth patient with active leukemia presented a
reduction in peripheral blood (PB) blasts. Incredibly, no
grade 3 or 4 toxicity was observed. The most notable
finding from this study was the lack of toxicity and the
durable in vivo persistence after infusion [
addition, LeY is the first antigen that was successfully
implemented in CAR T cell therapy to target AML.
Retroviral transduction of anti-LeY-CD28ζ into CAR T
cells has exhibited potent cytotoxicity against LeY+
epithelial tumor cell lines in vitro and animal models in
vivo without affecting normal tissues [
The hyaluronate receptor CD44 is a type I
transmembrane glycoprotein commonly used as a marker to
identify cancer stem/initiating cells. CD44 variant domain 6
(CD44v6) is a CD44 variant isoform expressed in AML
] and multiple myeloma (MM) [
], correlating with
a poor prognosis. Importantly, CD44v6 is absent in
hematopoietic stem cells (HSCs) and expressed at low
levels on normal cells, which may provide a therapeutic
window. The Italy San Raffaele Scientific Institute
designed a second-generation CD28-CD3ζ CAR and
derived the scFv from a mutated sequence of the
humanized CD44v6-specific mAb (bivatuzumab). This CAR
exerted a significantly positive effect in targeting cancer
cells in vitro and in vivo. However, these
anti-CD44v6CD28ζ CAR T cells caused an unexpected and
doselimiting toxicity (DLT), monocytopenia. Subsequently,
this group focused their attention on co-expressing
clinical-grade suicide genes [
] to control these
adverse events .
Natural killer group 2D (NKG2D) ligands contain six
members of the UL16-binding protein, or the retinoic
acid early transcript (ULBP/RAET) family and two
members of the MHC class I-related chain (MIC) family
], all of which are either absent or minimally
expressed on healthy tissues but widely expressed on
numerous malignancies (including ovarian cancer [
and AML [
]). Several different variants of
NKG2D-directed CAR have been developed and tested for their
cytotoxicity and the ability of achieving complete
]. From April 2015 to July 2016, a phase
I (ClinicalTrials.gov number, NCT02203825)
doseescalation study was performed to establish the
feasibility and safety of NKG2D-DAP10-CD3ζ CAR T cells
(CM-CS1 T cells) in treating AML and was completed
ahead of schedule. A total of 11 subjects were infused
with 1 × 106 to 3 × 109 (8 cohorts) of CM-CS1 T cells
based on a 3 + 3 design. The results showed that 9
subjects treated in the first 3 cohorts completed their
28day evaluation period without any DLTs. It is worth
mentioning that there was no case of cell-related neurotoxicity,
cytokine release syndrome (CRS), autoimmunity or CAR
T cell-related death during treatment [
Folate receptor β
The folate receptor β (FRβ) is a member of the
folatebinding protein receptors family, which is primarily
expressed on myeloid-lineage hematopoietic cells and
frequently up-regulated in AML blasts (~70%) [
]. Preclinical models using anti-FRβ-CD28ζ CAR T
cells presented potent and targeted killing of leukemia
cells while preserving healthy CD34+ cells.
Interestingly, the investigators also used all-trans retinoic acid
(ATRA), an FDA-approved drug for subclass M3
], to up-regulate the target antigen, which
led to an improved anti-leukemia activity [
general concept of increasing antigen expression on
diseased tissue to improve the potency of the CAR T
cell agent is very likely to be further explored in
CD38, also known as cyclic ADP ribose hydrolase, is a
glycoprotein expressed on the surface of many immune
cells. Previous studies have shown that CD38 is
expressed on the majority of AML blasts but not healthy
human hematopoietic stem cells (HSCs) [
Accordingly, one research group has focused on CD38 as a
candidate therapeutic target and developed an
antiCD38-41BBζ CAR. Remarkably, studies involving this
CAR revealed another example of ATRA-enhanced
cytotoxicity on AML cells regarding enhanced CD38
expression . Therefore, these results may provide a new
paradigm for pharmacologically inducible
immunotherapy that combines ATRA and CAR T cell therapy to
Fms-like tyrosine kinase 3 (FLT-3), also known as
CD135, is a cytokine receptor belonging to the class III
receptor tyrosine kinases. The FLT3 gene is one of the
most commonly mutated genes in AML, with internal
tandem duplications of FLT3 (FLT3-ITD) as the most
frequent mutation (25%) associated with AML. In a
recent study, researchers generated anti-FLT3-41BBζ CAR
T cells, which demonstrated potent anti-AML activity in
vitro and in vivo. Notably, compared with anti-CD33
CAR T cells, anti-FLT3 CAR T cells indicated a lower
hematological toxicity [
CD7 is an NK and T cell marker that is highly expressed
in 30% of AML cases. Its expression is associated with a
worse prognosis and chemoresistance [
CD7directed CAR T cells have been created and exhibited
potent cytotoxicity against T-ALL and AML cell lines as
well as against primary AML blasts, but there was no
observed toxicity against normal myeloid progenitors
. This finding indicates that CD7 is a potential target
for AML that should be further explored in future
CD33 is a transmembrane receptor of the SIGLEC
family and is expressed in approximately 90% of AML
patients as well as on AML stem cells [
CD33 is a notable and promising myeloid-specific target,
many groups have independently designed
CD33directed CAR T cells (in Fig. 2) and reported potent
anti-leukemia outcomes using AML tumor cells and
primary xenograft models [
]. Importantly, a phase I
study at the Chinese PLA General Hospital
(ClinicalTrials.gov number, NCT01864902) used lentivirally
transduced anti-CD33-41BBζ CAR T cells delivered in
escalating fractions to a single patient with refractory
AML, which resulted in a transient response [
However, as CD33 is expressed in healthy myeloid cells and
other tissues [
], the toxicity that occurs following
CD33-directed CAR T cell infusion must be well
controlled before further evaluation in clinical trials. One
research group proposed a novel solution to this problem
by removing CD33 from normal hematopoietic stem
progenitor cells (HSPCs) using genomic editing during
CD33-mediated CAR T cell treatment of AML, as CD33
is not essential to hematopoietic differentiation, and a
lack of CD33 in myeloid progeny does not cause any
visible functional changes [
]. Overall, recent studies were
committed to reducing the toxicity of CD33-specific
CAR T cells and proposed many strategies, which will be
further described in detail below.
As the transmembrane alpha chain of the interleukin-3
receptor, CD123 is widely expressed in the majority of
AML blasts but presents low expression levels on
normal hematopoietic cells [
anti-CD123CD28ζ CAR and anti-CD123-41BBζ CAR T cells have
demonstrated potent leukemia killing ability in vitro and
in vivo but produced incongruous results regarding their
myeloablative effect on healthy CD123+ cells [
addition, two phase I trials (ClinicalTrials.gov number,
NCT02159495, NCT02623582) for CD123-directed
CAR T cell therapy are currently underway to validate
the effect and safety profiles. Subsequently, one group
generated a novel
anti-CD123-CD28-CD137-CD27CD3ζ-iCasp9 CAR (4SCAR123) that exhibited potent
cytotoxicity against AML in vitro and then infused
4SCAR123 into a 47-year-old male patient with
AMLM2. The patient exhibited a rapid response consistent
with a controllable CRS and achieved partial remission
within 20 days without any off-target cytotoxicities .
One significant concern is that CD123-directed CAR T
cells could irreversibly increase the myeloablative impact
on normal hematopoiesis. Some strategies have been
proposed to develop safer CD123-directed CAR T cells,
one of which involves using the irreversible
myeloablation of CD123-directed CAR T cells in conjunction with
allogenic HSCT, such as the chemotherapy
preconditioning prior allo-HSCT, to reduce the risk of AML
relapse and pave the way to further explore CAR T cell
combination therapies [
CLEC12A (also known as CLL1) has been previously
described as selectively overexpressed in leukemia stem
cells (LSCs). One group confirmed that CLEC12A is
heterogeneously expressed on AML blasts and
overexpressed on AML LSCs. Lentivirally transduced
antiCLEC12A-41BBζ CAR T cells can successfully target
CLEC12A+ cells, which are resistant to chemotherapy.
Hence, anti-CLEC12A CAR T cells can potentially be
used as a consolidation regimen after induction
chemotherapy to eradicate LSC and minimal residual disease
(MRD) in AML [
AML-related surface antigens as candidates for CAR
Due to its potent and durable anti-tumor activity, CAR
T cell therapy has been recently regarded as a promising
curative therapy against B-lineage malignancies. The
reason for these positive results is that CD19 is an ideal
target for B-cell malignancies [
]. As is well known, new
tumor-related antigens may arise following somatic
mutations in the dividing tumor cells, which can serve as
valuable therapeutic targets. These antigens are
classified as tumor-specific antigens and mutation-causing
over-expression antigens [
]. CD19 is a unique
tumor-specific antigen expressed on the tumor cells of
B-lineage malignancies but not on normal cells.
Unfortunately, truly AML-specific surface antigens have not
been identified to date. Most of the antigens currently
studied are mutation-causing over-expression antigens,
which result in fatal “on-target/off-tumor toxicity” of
CAR T cell treatments because of the expression of
these antigens on normal tissue. Therefore, one
prerequisite for developing clinically effective CAR
therapies is the confirmation of specific AML-associated
surface targets. Theoretically, these antigens should
meet the following specific requirements [
]: 1) a
confirmed AML surface antigen; 2) expressed on as few
normal tissues as possible; 3) expressed in an
adequately large percentage of AML patients; 4)
homogenously expressed on the tumor cells of a given patient;
and 5) exerts an essential function in the
pathophysiology and/or biology of AML [
In addition to the above-mentioned targets used in
CAR T cell therapy to treat AML, several other surface
molecules, which are listed in Table 1, have been
identified and may be useful for directing the future
exploration of CAR T cells in AML based on their distribution
in normal tissue and specific involvement in potential
Our group currently select optimal AML targets for
future study based on the safe and effective results of
matured antibody technology depicted in Table 2. In
addition, our group allowed that the new trend to target
the LSCs rather than tumor cells for CAR T cell therapy
may lead to better cancer treatment. Because the
socalled LSCs, which are not effectively eliminated by
current treatments, retain extensive self-renewal and
tumourigenic potential that induces tumor proliferation
and progression, it has been long proposed that AML
has a high rate of relapse [
]. As previously mentioned,
Restricted to hematopoietic
cells of myeloid lineage
Expression may identify minimal
residual disease and predict relapse
CD123 is a typical LSC target in AML, and it has been
reported that CD123-CAR T cells may be a promising
tool as a chemotherapy-free myeloablative conditioning
regimen for HSCT, which is particularly critical to avoid
]. As shown in Table 1, CD47 is
overexpressed on LSCs and can be detected in almost all AML
samples, and its expression is often associated with
worse outcomes [
]. AML LSCs escape macrophage
phagocytosis by the recognition between CD47 on the
LSCs and extracellular region of signal regulatory
protein alpha (SIRPα) on the macrophages [
]. By contrast,
CD47 is faintly expressed in most normal tissues [
These findings make CD47 an ideal marker of AML
LSCs. T-cell immunoglobulin mucin-3 (TIM-3) is
another ideal marker of AML LSCs and is highly expressed
in LSCs in most types of AML (except for M3) but is
not expressed in normal LSCs [
]. TIM-3 plays an
important role in the viability, proliferation, and
differentiation of AML LSCs [
], as well as in the exhaustion of
CD8+ T cells. Several recent studies have shown that
AML relapse after CAR T cell therapy is directly
associated with the significant up-regulation of TIM-3
receptors on T cells. TIM-3 pathways are also involved in the
exhaustion of CAR T cells and the dysfunction of AML
]. This pathway is worth further exploration as a
potential target in the clinical setting.
The challenges and corresponding strategies of CAR T cell
therapy in treating AML
CAR-redirected T cells are an emerging powerful tool
for treating patients with cancer, with an especially high
rate of long-term complete remission achieved by CAR
AML acute myeloid leukemia, CDC complement dependent cytotoxicity, ADCC antibody-dependent cell-mediated cytotoxicity, LSC leukemia stem cell
T cell treatments in relapsed/refractory CD19+ ALL
17, 19, 92
]. Over the past few years, several
groups have concertedly focused on translating CAR T
cell therapy to AML, and they have demonstrated that
CAR T cells can eradicate AML in both preclinical and
clinical trials. Thus, the efficacy of anti-AML CAR T
cells appears to be equivalent to that of anti-ALL CAR T
cells. Nevertheless, critical questions remain in this field.
Here, we will outline the challenges of CAR T cell
therapies when applied to AML, and focus on discussing the
available and potentially feasible strategies to optimize
the efficacy and safety of CAR T cell therapy (Fig. 4).
Cytokine release syndrome
When CAR T cells exert a clinical effect, persistence
and proliferation are required; however, these activities
may also cause significant toxicity. The most common
and harmful toxicity is cytokine release syndrome
(CRS), a rapid and evident inflammatory systemic
response caused by dramatic increases in many
inflammatory cytokines (e.g., soluble IL-2R, IL-6 levels,
ferritin, C-reactive protein (CRP), etc.) that occur with
the in vivo activation and exponential proliferation of
CAR T cells. [
As previously reported by Wang et al., one AML
patient treated with approximately 4 × 108 anti-CD33 CAR
T cells experienced CRS [
]. Another group submitted
an abstract that described a single patient treated with
anti-CD123 CAR T cells who showing severe CRS in the
absence of overt off-target cytotoxicity [
Many studies have indicated that IL-6 is a central
mediator of CRS-related toxicity [
]. Furthermore, several
clinical studies have proved that the combined
administration of tocilizumab, an anti-IL-6R antagonist, and
systemic corticosteroids showed successful and rapid relief
of CRS following CAR T cell infusions [
]. The clinical
treatment algorithm for CRS has been well reviewed;
please refer to reference 95 [
Strategies of further optimizing the treatment
algorithms for CRS are currently under investigation
(ClinicalTrials.gov number, NCT02906371), and gene-editing
technology could be applied to CAR T cells to avoid
CRS-related toxicities. For example, either gene
silencing or the CRISPR/Cas9 system can be used to disturb
IL-6 and other CRS-related cytokines in T cells prior to
transduction with CARs. Additionally, T cells could
simultaneously express a corresponding scFv specific to
the IL-6 receptor such as tocilizumab as well as CARs
in order to block the IL-6 receptors actively avoiding
CRS (Fig. 5h).
In all, the mechanisms by which CAR T cells cause
CRS are varied and poorly understood. How to
effectively control the CRS toxicity of CAR T cells is one of
the most important challenges for improving the field of
CAR T cell therapies overall.
Because on-target/off-tumor toxicity results from the
expression of tumor-associated antigens (TAAs) on normal
tissue, minimizing the risk of toxicity is critical in the
successful implementation of CAR T cell therapy. The first
step in this process is to select more specific
AMLassociated surface targets, as mentioned above. However,
it is highly difficult to identify surface antigens that are
uniquely expressed on malignant myeloid tumors. There
are many reports regarding insignificant myelosuppression
caused by CAR T cells in preclinical models of AML. In
addition, one AML patient enrolled in NCT01864902
experienced moderate hepatotoxicity and a transient
reduction in marrow blasts following infusion with
antiCD33 CAR T cells [
]. Another clinical trial with
antiLeY CARs in AML did not reveal any major off-target
In consideration of the serious consequences of the
“on-target/off-tumor” toxicities reported in other clinical
], we should prepare corresponding
strategies to address the “on-target/off-tumor” effects that
may arise at any time.
The expression of CARs using mRNA electroporation of
T cells ensures the gradual loss of surface CAR
expression as T cells divide, which may be a useful strategy for
determining the potential toxicity of novel constructs.
One group transiently expressed an mRNA CAR
construct targeting CD33 to avoid prolonged toxicity ,
whereas another clinical study is currently ongoing in
which T cells expressing anti-CD123 CARs via mRNA
electroporation were infused into patients with AML
(ClinicalTrials.gov number, NCT02623582) to evaluate
efficacy and safety.
Suicide gene applications
A suicide gene is a genetically encoded molecule that allows
for the selective destruction of adoptively transferred cells.
The addition of a suicide gene to cellular therapeutic
products can lead to the selective ablation of gene-modified
cells, which can mitigate or prevent collateral damage to
contiguous cells and/or tissues [
]. This approach may be
useful in abrogating the on-target and off-tumor toxicities
of CAR-directed T cells. The inducible Caspase9 (iC9)
suicide gene comprises a drug-binding domain cloned in
frame with human Caspase9. Upon the exogenous
administration of a non-therapeutic small molecule chemical
inducer of dimerization (CID), iC9 dimerizes and induces
apoptosis of the transduced cells within hours. CD44v6-,
CD33-, and CD123-directed CAR T cells all contain an iC9
suicide gene as a tool for controlling the adverse events,
which has been tested in preclinical research [
37, 68, 80
A “kill switch” is based on a tag derived from the
epidermal growth factor receptor (EGFRt) that retains the
epitope recognized by the commercially available
FDAapproved mAb cetuximab [
]. Anti-CD33- and
antiCD123-CD28ζ-EGFRt cells have been designed that can
be eliminated by cetuximab if either CRS or any
ontarget/off-tumor toxicities are observed [
When off-tumor toxicity is observed, these above
strategies could enhance the ability of either ameliorating or
abrogating these deleterious effects. Therefore, the
inclusion of up-front safeguards is in an urgent need to
prevent off-target toxicity in healthy tissues. Specific novel
strategies are described in Fig. 5a-d, but future studies
are required to expand on these ideas.
Despite the scarcity of clinical cases regarding AML
relapse after CAR T cell therapy, several preclinical studies
have been performed to explore the reasons for the
relapse. The corresponding strategies to address this issue
have also been proposed.
Reduced efficacy and LSCs
Relapse is primarily caused by the lack of effectiveness
of CAR T cells, which can be attributed to two factors:
the immunosuppressive microenvironment and LSCs.
To address the first issue, one approach is the use of
socalled “TRUCK cells”, which can induce IL-12 release
and activate innate immune cells to the targeted tumor
and thus eliminate cancer cells not recognized by CAR
T cells [
]. This strategy can enhance the efficacy of
CAR T cell therapy, thereby eliminating cancer cells and
preventing tumor relapse caused by the residual cancer
cells. To address the second issue regarding LSCs, the
best solution is to identify the optimal markers for AML
LSCs applied to CAR, which we have discussed in detail
Inhibitory receptors/pathways, such as the PD-1 and
TIM-3 pathways, induce the dysfunction and exhaustion
of CAR T cells in AML and are also the mechanism of
immune escape. Recently, several studies have indicated
that there is a significantly higher expression of PD-1
and TIM-3 on T cells in relapsed AML samples
compared with that seen in remittent or healthy donors [
]. Gene-editing technology could allow for the
permanent disruption of negative signaling pathways
]. Combined approaches using blocking antibodies
may also interrupt this interaction, thus leading to the
increased CAR T cell-induced cytotoxicity [
latest technology is the use of switch receptors that
incorporate a segment of the PD-1 receptor into the CAR
construct (Fig. 5e), thereby inducing PD-L1 expression
within the tumor microenvironment (TME) to augment
the cytokine secretion, proliferation and granzyme
expression of CAR T cells, improving tumor therapy [
A typical clinical case we observed is when an AML patient
experiences relapse following CD33-CAR T cell treatment
because leukaemic cells can selectively proliferate AML
cells with low CD33 expression to evade the identification
by CAR T cells [
]. The antigen escape-caused relapse
involves multiple mechanisms. With the exception of the
above-mentioned case, antigen loss on the tumor surface
and deleterious mutations of antigens recognized by
CART cells have been observed in ALL clinical cases [
clinical scenario is that CD19 is still present but cannot be
detected and recognized by anti-CD19 CAR-T cells as its
cell surface fragment is absent because of a deleterious
mutation or alternative splicing [
]. A new strategy to
address the antigen escape-caused relapse involves designing
CAR T cells able to be activated by multiple antigens
synchronously. Other dual-targeted CAR-T cells have been
investigated in preclinical studies. One is known as
dualsignaling CAR T cells (Fig. 5f ), which are modified by two
distinct CAR molecules with different binding domains
]. Another type is the so-called Tan-CAR T cells
(Fig. 5g), which are modified by one CAR molecule with
two different binding domains in tandem [
dual-signaling CAR and TanCAR can control antigen
escape-caused relapse because a single antigen can trigger
robust anti-tumor activity. Currently, our group is
evaluating CD33/CD123 dual-targeted CARs to prevent antigen
escape-caused relapse and may evaluate them as promising
myeloablative tools for HSCT in a follow-up study.
In the past few years, the progress of CAR-engineered T
cells has rapidly developed and made great
achievements. Nevertheless, there still exist certain limitations
in this field that should not be ignored. One of the most
concerning issues is that there is no convincing evidence
of an AML-specific cell surface antigen that can be
safely used to maximize the usefulness of CAR T cells.
Admirably, many research groups are still confident and
have developed numerous strategies to improve the
current status of CAR T cells as a therapeutic in the
AML field, such as gene-editing technology, antibodies,
and combination therapies, most of which have been
presented in this review. If these strategies could be
successfully employed in clinical trials, the ability of
CARexpressing T cells in treating AML would be
immeasurable. In addition, we hope that this review provides
useful information regarding the overall progress of CAR T
cell therapy in the AML and injects new ideas into
future research. In conclusion, the adoptive transfer of
CAR-engineered T cells represents a valuable and
attractive therapeutic strategy that has the potential to
provide new prospects for cancer immunotherapy.
ADCC: Antibody-dependent cell-mediated cytotoxicity; ALL: Acute
lymphoblastic leukemia; allo-HSCT: Hematopoietic stem cell transplantation;
AML: Acute myeloid leukemia; ATRA: All-trans retinoic acid; CAR: Chimeric
antigen receptor; CDC: Complement dependent cytotoxicity; CIK: Cytokine
induced killer; CRS: Cytokine release syndrome; EBV-CTL: Human Epstein Barr
Virus-cytotoxic lymphocyte; EGFR: Epidermal growth factor receptor;
FLT3: Fms-like tyrosine kinase 3; FRβ: Folate receptor β; GVHD: Graft-versus-host
disease; IL12: Interleukin-12; iMC: Inducible MyD88/CD40; LAG3: Lymphocyte
activating 3; LSC: Leukemia stem cell; mAb: Monoclonal antibody;
MHC: Major histocompatibility complex; mRNA: Messenger ribonucleic acid;
NKG2D: Natural killer group 2D; PD1: Programmed death 1; scFv: Single
chain variable fragment; SIRPa: Signal regulatory protein-a; TAAs:
Tumorassociated antigens (TAAs); TCR: T cell receptor; TIM-3: T-cell immunoglobulin
mucin-3; TME: Tumor microenvironment (TME)
The authors are deeply thankful for their parents’ encouragement. We also
thank Professor Yan and Minghao Li for their generous and constant support.
Finally, Mingxue Fan would like to acknowledge Sicong Geng and “MayDay”
(Ashin, Monster, Stone, Masa, Ming); if we had never met, I would not have
had the courage to follow my dreams.
This work was supported by the National Basic Research Program of China
(2013CB932500), the National Natural Science Foundation of China
(60976004), and the “985” grants of East China Normal University (ECNU).
Availability of data and materials
The material supporting the conclusion of this review has been included
within the article.
Mingxue Fan designed the study. All authors read and approved the final
Ethics approval and consent to participate
This is not applicable for this review.
Consent for publication
This is not applicable for this review.
The authors declare that they have no competing interests.
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