Please cite this article in press as: Wagner et al.
, CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?, Molecular Therapy (2020), https://
doi.org/10.1016/j.ymthe.2020.09.015
Review
CAR T Cell Therapy for Solid Tumors:
Bright Future or Dark Reality?
Jessica Wagner,1 Elizabeth Wickman,1,2 Christopher DeRenzo,1 and Stephen Gottschalk1
1Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA; 2Graduate School of Biomedical
Sciences, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
Chimeric antigen receptor (CAR) T cell therapy has garnered
significant excitement due to its success for hematological ma-
lignancies in clinical studies leading to the US Food and Drug
Administration (FDA) approval of three CD19-targeted CAR
T cell products. In contrast, the clinical experience with CAR
T cell therapy for solid tumors and brain tumors has been
less encouraging, with only a few patients achieving complete
responses. Clinical and preclinical studies have identified mul-
tiple “roadblocks,” including (1) a limited array of targetable
antigens and heterogeneous antigen expression, (2) limited
T cell fitness and survival before reaching tumor sites, (3) an
inability of T cells to efficiently traffic to tumor sites and pene-
trate physical barriers, and (4) an immunosuppressive tumor
microenvironment. Herein, we review these challenges and
discuss strategies that investigators have taken to improve the
effector function of CAR T cells for the adoptive immuno-
therapy of solid tumors.
Adoptive cell therapies utilizing T cells expressing chimeric antigen
receptors (CARs) have been propelled to the forefront of experi-
mental cell therapies due to their clinical success for hematological
malignancies targeting a range of antigens, including CD19, CD22,
CD30, kappa, and B cell maturation antigen (BCMA).1–10 The land-
scape of CAR T cell therapies for hematological malignancies,
including successes and challenges, has been the subject of multiple
recent reviews.11–13 We therefore do not discuss these in detail,
except for highlighting the “lessons learned” as they relate to target-
ing solid tumors, including brain tumors. First, CAR T cells can
eradicate chemorefractory cancer cells regardless of their underlying
oncogenic driver mutations; second, lymphodepleting chemo-
therapy is at present a sine qua non to enable expansion and persis-
tence of infused CAR T cells; third, inclusion of at least one co-stim-
ulatory signaling domain in the CAR is critical for its success;
fourth: antigen loss variants have emerged as one mechanism of
therapeutic failure, even for antigens that are highly and homoge-
neously expressed such as CD19; and fifth, CAR T cells can induce
significant side effects, including cytokine release syndrome (CRS)
and neurotoxicity.11–15
In contrast to CAR T cell therapy for hematological malignancies,
CAR T cell therapies for solid tumors, including brain tumors, have
shown limited antitumor activity in early phase clinical testing despite
targeting a variety of target antigens and tumor types.16–26 This ther-
Molecular Therapy Vol. 28 No 11 November 2020 ª 2020 The American Society of Gene and Cell Therapy.
apeutic failure is most likely multifactorial and includes (1) CAR
design and CAR T cell generation, (2) a limited array of targetable an-
tigens and heterogeneous antigen expression (aka “antigen
dilemma”), (3) limited T cell fitness, (4) inefficient homing to and
penetration of solid tumors, and (5) the hostile tumor microenviron-
ment (TME) (Figure 1).27–30 Herein, we review CAR design, the cur-
rent clinical experience with autologous CAR T cells for solid tumors,
including brain tumors, and genetic approaches to overcome the
aforementioned roadblocks. Since allogeneic CAR T cells are at pre-
sent mainly explored for hematological malignancies in early phase
clinical studies, we refer the interested reader to recently published re-
views.31,32 We also do not review CRS and neurotoxicity since several
recent reviews have covered this topic.14,33
Design of CARs
CARs have a modular design that consists of an antigen-binding
domain, most commonly a single-chain variable fragment (scFv)
derived from a monoclonal antibody (mAb), a hinge and transmem-
brane domain, and an intracellular signaling domain.27,34–38 In addi-
tion to scFvs, natural ligands of receptors such as cytokines or pep-
tides that bind cell surface molecules are also being explored as
antigen-binding domains.22,39,40 While most CARs recognize epi-
topes of cell surface proteins in a major histocompatibility complex
(MHC)-independent manner, scFvs have also been incorporated
into CARs that recognize a peptide in the context of a human leuko-
cyte antigen (HLA) molecule.41–43 While this approach allows CAR
T cells to recognize intracellular molecules, it renders target antigen
recognition dependent on a particular HLA type, restricting its appli-
cation to a subset of patients. In addition, CAR T cells become sensi-
tive to decreased HLA expression and defects in the antigen process-
ing pathway, with both pathways used by tumor cells to actively evade
immune responses.44
First-generation CARs contained only a T cell activation domain,
and the most commonly utilized domain is derived from
CD3z.37,38 Since optimal T cell activation also relies on co-stimula-
tion, signaling domains from co-stimulatory molecules were
https://doi.org/10.1016/j.ymthe.2020.09.015.
Correspondence: Stephen Gottschalk, Department of Bone Marrow Trans-
plantation and Cellular Therapy, St. Jude Children’s Research Hospital, 262 Danny
Thomas Place, MS321, Memphis, TN 38105, USA.
E-mail: stephen.gottschalk@stjude.org
1
 Please cite this article in press as: Wagner et al., CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?, Molecular Therapy (2020), https://
doi.org/10.1016/j.ymthe.2020.09.015
www.moleculartherapy.org
Review
Figure 1. CAR T Cell Immunotherapy “Roadblocks” for Solid Tumors
Top left: antigen dilemma. Heterogeneous antigen expression results in immune
escape variants, and antigen expression in normal tissues can lead to on target/off
cancer toxicity. Top right: CAR T cell fitness. Expansion, contractions, and persis-
tence of CAR T cells is often limited after infusion. Bottom left: homing/penetration.
CAR T cells have a limited ability to traffic to and penetrate solid tumors. Bottom
right: microenvironment. The solid tumor microenvironment is hostile, limiting CAR
T cell effector function.
included into CARs. Initial studies focused on domains derived
from the canonical co-stimulatory molecules CD28 and 41BB.37,38
Since then, signaling domains from a broad range of molecules
have been explored, including OX40, CD27, and ICOS.37,45–49
CD28 and 41BB co-stimulation in the context of CAR T cells has
been extensively studied, including detailed phosphoproteomic
and single-cell RNA sequencing (RNA-seq) analyses.50–52 They acti-
vate different pathways within T cells, with CD28 signaling promot-
ing glycolytic metabolism and an effector memory phenotype, in
contrast to 41BB signaling, which induces oxidative metabolism
and a central memory phenotype.53 Depending on the number of
co-stimulatory molecules included in the CAR signaling domain,
CARs are either designated second (one domain) or third (two
domains) generation. In preclinical studies, the benefit of including
two co-stimulatory molecules in CARs is model-dependent.54,55
Results of one clinical study, comparing CD28-CAR versus
CD28.41BB-CAR T cells targeting CD19, suggest that third-genera-
tion CARs endow T cells with a greater ability to expand after infu-
sion in humans.56
2 Molecular Therapy Vol. 28 No 11 November 2020
Optimizing CAR function remains a challenge since there is an intri-
cate interplay between the functional (antigen recognition and
signaling domains) and nonfunctional components (hinge and trans-
membrane domain) of CARs.57,58 In addition, the location of the
epitope within the targeted cell surface molecule determines CAR
T cell activity.59 For example, epitopes that are proximal to the plasma
membrane induce greater CAR T cell activation than do distal epi-
topes.57,59,60 Studies have also highlighted that too much CAR
T cell activation is detrimental to T cell function. For example,
CARs with two of three mutated immunoreceptor tyrosine-based
activation motifs (ITAMs) in the CD3z chain endow CAR T cells
with improved effector function.61 In addition, excessive CD28 co-
stimulation has detrimental effects,62 and mutations in the YMXM
motif of CD28 that reduce its signaling activity improve T cell func-
tion.63 In addition to CAR signaling after activation, baseline (aka
tonic) signaling determines CAR T cell activity.64,65 Recently, investi-
gators have also focused on studying the immunological synapse that
is formed between CAR T cells and target cells, and they have corre-
lated synapse formation with CAR T cell effectiveness.66,67
Lastly, several studies have highlighted limitations of CARs with stan-
dard co-stimulatory molecules, which most likely cannot be over-
come by further refining its structure.27,37,38 Foremost, CARs only
provide signal 1 (activation) and signal 2 (co-stimulation) of canon-
ical T cell activation and not signal 3 (inflammatory cytokines). While
CAR-activated T cells initially produce cytokines, they do not secrete
sufficient amounts of cytokines after recursive exposure to antigen-
positive target cells, leading to rapid erosion of their effector func-
tion.68–70 This occurs even in the absence of an immunosuppressive
TME, highlighting that additional modification of CAR T cells or
combinatorial therapies are needed to sustain and/or enhance their
effector function. These are discussed in detail in the section
Improving CAR T Cell Fitness.
CAR T Cells for Solid Tumors and Brain Tumors—Clinical
Experience
CAR T cells have been evaluated in early phase clinical studies for
more than a decade, targeting a broad range of tumor antigens,
including B7-H3, CAIX, CEACAM5, CD133, CD171, epidermal
growth factor receptor (EGFR), EGFRvIII, FRa, GD2, GPC3,
HER2, interleukin (IL)-13Ra2, mesothelin, MUC1, prostate-specific
membrane antigen (PSMA), ROR1, and vascular endothelial growth
factor (VEGF)-R2.19,20,22–26,71–82 Table 1 lists selected published
studies, and Table 2 lists ongoing clinical studies. Early studies
focused on first-generation CARs specific for CAIX, CD171, FRa,
GD2, or IL-13Ra2.16,20,22,83 Two studies utilized polyclonal, activated
CAR T cells (CAIX, FRa);20,84 two studies focused on CAR T cell
clones (CD171, IL-13Ra2);75,83,85 and one study compared GD2-
CAR T cells with GD2-CAR/Epstein-Barr virus (EBV)-specific
T cells.81,82 First-generation CAR T cells did not expand after infusion
and had limited antitumor activity, except for the study with GD2-
CAR/EBV-specific T cells, in which 3 of 11 patients had a complete
response (CR). Despite having no antitumor activity, CAIX-CAR
T cells induced “on target/off cancer” toxicity in the form of
 Please cite this article in press as: Wagner et al., CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?, Molecular Therapy (2020), https://
doi.org/10.1016/j.ymthe.2020.09.015

www.moleculartherapy.org

Review

Table 1. Selected Published CAR T Cell Clinical Studies for Solid Tumors and Brain Tumors

Antigen Diagnosis Signaling Domain Vector T Cell Product IL-2 after T Cells Comment References
Intravenous Administration, No Lymphodepleting Chemotherapy
FRa OVCA z RV ATCsa Y 14/14 NR 21

12/12 NR; on target/off cancer 19,20

CAIX RCC z RV ATCs N
toxicity—bile ducts
83
CD171 NB z Plasmid T cell clone N 1/6 PR
17
EGFRvIII HGG 4-1BB LV ATCs N 1/10 SD
81,82
GD2 NB z RV ATCs, VSTs N 3/11 CR
23
HER2 sarcoma CD28z RV ATCs N 4/17 SD
26
HER2 HGG CD28z RV VSTs N 3/17 SD
80
Mesothelin PCA 41BBz mRNA ATCs N 2/6 SD
Intravenous Administration after Lymphodepleting Chemotherapy
24
CD133 HCC, PCA, CRC 41BBz LV ATCs N 3/23 PR, 14/23 SD
CRC, PDCA, stomach, 7/14 SD; on target/off cancer 25
CEACAM5 z RV ATCs Y
esophagus toxicity—lung
86
EGFR PCA 41BBz LV ATCs N 4/16 PR, 8/16 SD, 2/16 NE
b 16
EGFRvIII HGG CD28.41BBz RV ATCs Y 17/18 NR, 1/18 NE due to TRM
GD2 NB CD28.OX40z RVc ATCs N 5/11 SD 87

88
GPC3 HCC CD28z LV ATCs N 2/13 PR, 1/13 SD, 4/13 NE
b
1/1 TRM ; on target/off cancer 18
HER2 CRC CD28.41BBz LV ATCs Y
toxicity—lung
89,90
HER2 sarcoma CD28z RV ATCs N 1/10 CR; 3/10 SD
Mesothelin MPM, PCA, OVCA 41BBz LV ATCs N 11/15d SD 91

71
PSMA prostate z RV ATCs Y 2/5 PR
4/6 mixed response; 72
ROR1 TNBC, NSCLC 41BBz LV ATCs N
1/6 SD; 1/6 not reported
VEGF-R2 metastatic CA – RV ATCs Y 1/23 PR e

Intravenous Administration after Allotransplant

f
GD2 NB z RV VSTs N 3/3 PD
Locoregional Administration
73
B7-H3 meningioma 41BBz LV ATCs N 1/1 evidence of ALV
74
IL-13Ra2 HGG z plasmid T cell clone N 1/3 tumor necrosis
a 75
IL-13Ra2 HGG z plasmid T cell clone N imaging study; outcome not reported
22
IL-13Ra2 HGG 41BBz LV ATCs N 1/1 CR
Mesothelin MPM, lung CA, breast CA CD28z RVc ATCs N 2/21g CR, 5/21 PR, 4/21 SD 76

h 92
MUC1 SVA 41BBz or CD28z LV ATCs N 1/1 tumor necrosis
ALV, antigen loss variant; ATC, activated T cell; CA, cancer; CR, complete response; CRC, colorectal cancer; HCC, hepatocellular carcinoma; HGG, high-grade glioma; LV, lentivirus;
MPM, malignant pleural mesothelioma; N, no; NB, neuroblastoma; NE, not evaluable; NR, no response; OVCA, ovarian cancer; PCA, pancreatic adenocarcinoma; PR, partial response;
RCC, renal cell carcinoma; RV, retrovirus; SD, stable disease; SVA, seminal vesicle adenocarcinoma; VST, virus-specific T cell; Y, yes.
a
Several patients received allogeneic cell products.
b
Pulmonary complications.
c
CAR vector also encoded inducible caspase-9.
d
6 of 15 patients received lymphodepleting chemotherapy.
e
ClinicalTrials.gov: NCT01218867.
f
ClinicalTrials.gov: NCT01460901.
g
18 of 21 patients received lymphodepleting chemotherapy and 13 of 21 patients received pembrolizumab.
h
41BBz CAR vector also encoded IL-12.

Molecular Therapy Vol. 28 No 11 November 2020 3

4

Review
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Molecular Therapy Vol. 28 No 11 November 2020

Table 2. Selected Actively Recruiting CAR T Cell Clinical Studies for Solid Tumors and Brain Tumors

Antigen Diagnosis Signaling Domain Additional Engineering Vector T Cell Product Other Therapy NCT No.: ClinicalTrials.gov
Intravenous Administration, No Lymphodepleting Chemotherapy Listed on ClinicalTrials.gov Site
HCC – TGF-b-CAR, IL-7-CCL19 – ATCs N NCT03198546
GPC3
HCC – N – ATCs N NCT04121273
PSMA solid tumors – N – ATCs N NCT04429451
Intravenous Administration after Lymphodepleting Chemotherapy
B7-H3 solid tumors – iC9 LV ATCs N NCT04432649
CD171 NB 41BBz or CD28.41BBz N LV ATCs N NCT02311621
Claudin 18.2 solid tumors – N – ATCs N NCT03874897
EGFR806 solid tumors 41BBz CD19-CAR LV ATCs N NCT03618381
IL-13Ra2 Melanoma 41BBz N LV ATCs IL-2 NCT04119024
NB – iC9, IL-15 RV ATCs N NCT03721068
GD2 solid tumors – iC9 – ATCs N NCT03373097
solid tumors – C7R RV ATCs N NCT03635632
DIPG, HGG – C7R RV ATCs N NCT04099797
NB – IL-15 RV iNKT N NCT03294954
NB – N – ATCs N NCT02761915
DIPG 41BBz iC9 RV ATCs N NCT04196413
solid tumors 41BBz N RV ATCs N NCT02932956
GPC3 HCC 41BBz N RV ATCs N NCT02905188
HCC – N – ATCs N NCT03884751
solid tumors – PD-1 KO – ATCs N NCT03747965
Mesothelin
solid tumors – N LV ATCs N NCT03054298
esophageal CA – PD-1 KO – ATCs IL-2 NCT03706326
MUC-1 NSCLC – PD-1 KO – ATCs IL-2 NCT03525782
breast Cancer 41BBz N LV ATCs N NCT04020575
prostate CA 41BBz N LV ATCs N NCT03873805
PSCA
solid tumors z iMC RV ATCs N NCT02744287
solid tumors – iC9 gene editing ATCs N NCT04249947
PSMA prostate CA – DNR TGFb LV ATCs N NCT03089203
prostate CA – DNR TGF-b LV ATCs N NCT04227275
Intravenous and Locoregional Administration After Lymphodepleting Chemotherapy
MUC16ecto solid tumors 41BBz IL-12 LV ATCs N NCT02498912
(Continued on next page)
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Review

cholangitis.19,20 In hindsight, limited antitumor activity was not sur-

caspase-9; iMC, inducible Myd88.CD40; ipi, ipilimumab; KO, knockout; LV, lentivirus; N, no; NB, neuroblastoma; nivo, nivolumab; OVCA, ovarian cancer; pembro, pembrolizumab; RV, retrovirus; SCCHN, squamous cell
ATC, activated T cell; CA, cancer; CCR, chimeric cytokine receptor; C7R, constitutive active IL-7 receptor a; DNR, dominant negative receptor; HCC, hepatocellular carcinoma; HGG, high-grade glioma; iC9, inducible
prising since CARs without co-stimulatory endodomains were
NCT No.: ClinicalTrials.gov

grafted onto T cells, and patients did not receive lymphodepleting

chemotherapy prior to the adoptive transfer of CAR T cells—two pre-
requisites of successful CAR T cell therapies for hematological malig-
NCT04185038
NCT04385173
NCT04077866
NCT03618381
NCT01818323
NCT03696030
NCT02442297
NCT03500991
NCT04003649
NCT02208362

NCT03585764
NCT02414269
nancies.1–10

In subsequent studies, CARs were evaluated with co-stimulatory

endodomains (CD28, 41BB, CD28/41BB, CD28/OX40) targeting
B7-H3, CEACAM5, CD133, CD171, EGFR, EGFRvIII, FRa, GD2,
GPC3, HER2, IL-13Ra2, mesothelin, MUC1, PSMA, ROR1, and
Other Therapy

VEGF-R2.72,86–89,91,92 Similar to first-generation CAR T cells, adop-

Pembro tive transfer of second- or third-generation CAR T cells or CAR vi-
nivo/ipi
TMZ
TMZ

rus-specific T cells did not result in significant T cell expansion in

N

N
N
N
N
N

two studies, and variable expansion in one study.23,26,87 Thus, inclu-

sion of a co-stimulatory domain into the CAR by itself is not sufficient
to consistently induce systemic T cell expansion. In contrast, lympho-
T Cell Product

depleting chemotherapy prior to CAR T cell infusion resulted in a sig-

nificant expansion of CAR T cells irrespective of the utilized co-stim-
ATCs
ATCs
ATCs
ATCs
ATCs
ATCs
ATCs
ATCs
ATCs
ATCs

ATCs
ATCs

ulatory domain and targeted antigen.9,93,94 Despite significant in vivo

expansion, antitumor activity remained limited (Table 1). However,
single case reports of patients with recurrent refractory solid tumors
or brain tumors, who achieved CRs after CAR T cell therapy, have
been reported (Table 1).22,90 Clinical studies have also highlighted
Vector

safety concerns. Two patients died of acute respiratory failure after

RV
RV

RV

RV
LV

LV

LV

LV
LV
LV

LV
–

having received lymphodepleting chemotherapy, a high dose (1 to

6
1010) of third-generation CAR T cells, and IL-2.16,18 CAR
T cells either recognized an antigen that is expressed (HER2) or not
expressed (EGFRvIII) on lung endothelial cells, suggesting that on
Additional Engineering

target/off cancer toxicity as well as unspecific CAR T cell activation

can result in fatal respiratory failure. Adoptive transfer of high cell
IL-4/IL-2 CCR

doses (1
109 to 5
1010) of CEACAM5-CAR T cells in conjunction

with IL-2 also resulted in pulmonary toxicities (mild pulmonary
edema), which was attributed to CEACAM5 expression on lung
iC9
N
N
N
N

N
N
N
N
N

epithelium.25

Tumor tissue after CAR T cell infusion has only been studied sys-
Locoregional Administration after Lymphodepleting Chemotherapy
Locoregional Administration, No Lymphodepleting Chemotherapy

tematically in one study in which patients with high-grade glioma

Signaling Domain

(HGG) received EGFRvIII-CAR T cells.17 Collectively, the analysis

showed that CAR T cells were able to migrate to and proliferate
at tumor sites. CAR T cells killed tumor cells as evidenced by
CD28z

CD28z

CD28z
41BBz

41BBz

41BBz
41BBz
41BBz

41BBz

the selection of target antigen-negative tumors, and, strikingly,

–
–
–

induced a reactive immunosuppressive environment as judged

by expression of IDO1 and PD-L1, and an influx of regulatory
carcinoma of the head and neck; Y, yes.

T cells (Tregs).
CNS metastases

pleural disease
CNS tumors
CNS tumors
CNS tumors
CNS tumors

CNS tumors
CNS tumors
Diagnosis

SCCHN

Thus, CAR T cells at present rarely induce CRs in patients with solid
OVCA
HGG
HGG

tumors or brain tumors. In addition, several studies have highlighted

their potential to induce on target/off cancer toxicity. In the following
Table 2. Continued

sections we review approaches that investigators have taken to

improve their efficacy and safety, with special focus on (1) clinical
EGFR family

Mesothelin

grade CAR T cell production and T cell subsets for CAR T cell ther-
IL-13Ra2
EGFR806
Antigen

B7-H3

HER2

apy, (2) expanding the array of targetable antigens and overcoming

FRa

heterogeneous antigen expression, (3) CAR T cell fitness, (4) CAR

Molecular Therapy Vol. 28 No 11 November 2020 5

 Please cite this article in press as: Wagner et al., CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?, Molecular Therapy (2020), https://
doi.org/10.1016/j.ymthe.2020.09.015
www.moleculartherapy.org
Review
Figure 2. Strategies to Overcome CAR T Cell Immunotherapy Roadblocks for Solid Tumors
Summary of strategies that are discussed in detail in this review.
T cell trafficking and tumor penetration, and (5) counteracting the
immunosuppressive TME (Figure 2).
Improving Clinical-Grade CAR T Cell Production
Preclinical studies have highlighted that the effector function of
T cells progressively decreases as T cells differentiate from naive
(TN) to stem cell memory (TSCM), central memory (TCM), effector
memory (TEM), and terminally differentiated T cells that express
the CD45RA effector (TEMRA).95 Transcription factors govern T cell
differentiation. For example, TCF7 promotes self-renewal and mem-
ory formation, while TOX, BLIMP1, and TBET promote terminal
T cell differentiation.96–102 More recent studies have also highlighted
the critical role of epigenetic programs enforced by the de novo DNA
methyltransferase 3A (DNMT3A) in determining T cell fate.103–105 In
addition, a recent case report highlighted that disruption of TET2 in
CD19-CAR T cells, an enzyme that regulates DNA demethylation, re-
sulted in clonal T cell expansion.106
Despite these insights in T cell biology, most initial clinical studies uti-
lized polyclonal, CD3/CD28-activated T cells that are expanded in the
g-cytokine IL-2. These expansion protocols in general result in signif-
icant T cell differentiation paired with inferior effector function in
preclinical models. Investigators have explored alternative g-cyto-
kines to preserve CAR T cell function.107 For example, replacing
IL-2 with a combination of IL-7 and IL-15 for the ex vivo expansion
6 Molecular Therapy Vol. 28 No 11 November 2020
of CAR T cells increases the frequency of CD8+CD45RA+CCR7+
stem cell-like CAR T cells with superior antitumor functionality.108
Based on these findings, many clinical-grade CAR T cell production
protocols are currently using IL-7 and IL-15. In addition, IL-21 has
been shown to prevent differentiation of genetically modified
T cells paired with improved effector function in preclinical
studies.109,110 Inhibiting or activating pathways in T cells that pro-
mote or prevent T cell differentiation are alternative strategies that
have been explored. These include activating Wnt/b-catenin signaling
pathways or inhibiting mTOR or AKT signaling.111,112
Notwithstanding these advances, there is at present a lack of generally
accepted biomarkers that predict efficacy of the infused CAR T cell
product. Several studies with CD19-CAR T cells are starting to
shed light on this issue. In one study in patients with chronic lympho-
cytic leukemia (CLL), the presence of CD27+PD-1CD8+ CD19-CAR
T cells expressing high levels of the IL-6 receptor in the T cell product
correlated with antitumor activity,113 and in another study in patients
with acute lymphocytic leukemia (ALL), the presence of tumor
necrosis factor (TNF)-a+CD8+ CD19-CAR T cells, respectively.114
One recent study, using RNA-seq analysis, demonstrated that
CD19-CAR T cell clones that expand after infusion are derived
from clusters of CAR T cells in the product that express higher levels
of genes associated with T cell proliferation and cytolytic activity.115
While this study found no correlation with vector integration sites,
 Please cite this article in press as: Wagner et al., CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?, Molecular Therapy (2020), https://
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Review
another recent report demonstrated that vector integration site distri-
bution in CD19-CAR T cells is linked to clinical outcome.116 Clearly,
additional studies are needed to identify biomarkers not only for the
CAR T cell product, but also for the targeted tumor.
Besides improving culture conditions for CAR T cell generation, in-
vestigators have also focused on the genetic modification step of
T cells during CAR T cell production. Most clinical studies so far
have utilized CAR T cells that were generated with retroviral or len-
tiviral vectors with an excellent safety record.117–119 However, clin-
ical-grade production and release testing of recombinant viral vectors
is expensive, and patients require additional monitoring. Therefore,
alternative approaches have been developed, including the use of Pig-
gyBac or Sleeping Beauty transposon systems.120,121 More recently,
combining gene-editing technologies (e.g., CRISPR-Cas9, TALEN,
ZFN, meganucleases) with homology-directed DNA repair (HDR)
has enabled the integration of transgenes into specific loci.122–124
This approach allows for control of transgene expression with endog-
enous promoters. For example, CD19-CARs have been knocked in to
the T cell receptor (TCR) a constant (TRAC) locus using CRISPR-
Cas9 gene editing to drive CAR expression from the endogenous
TCR a chain promoter. This resulted in consistent CAR expression,
reduced baseline signaling, and improved antitumor activity
compared to CD19-CAR T cells generated by viral transduction.125
The clinical experience with CAR T cells generated with non-viral
methods is so far limited,78 and most ongoing studies are focused
on targeting hematological malignancies.
T Cell Subsets for CAR T Cell Therapy
Investigators have explored CAR T cell products with a defined
CD8+/CD4+ T cell subset ratio and products that were derived
from TSCM, TCM, or T helper (Th17) cells.48,126–129 These studies
have highlighted that T cell subset-derived CAR T cell populations
have improved effector function in comparison to polyclonal, CD3/
CD28-activated CAR T cells. Importantly, the co-stimulatory domain
of the CAR might have to be tailored for a specific T cell subset with
one study demonstrating superior antitumor activity when CD4+
T cells, expressing CARs with an ICOS co-stimulatory endodomain,
were combined with CD8+ T cells, expressing CARs with a 41BB
co-stimulatory endodomain.130
However, the translation of these approaches into the clinic might be
limited due to the frequency of some of these T cell subset popula-
tions; for example, TSCM cells are very low in the peripheral circula-
tion.131 In addition, sequential cycles of chemotherapy have depleted
T cell subsets, including TN cells, in heavily pretreated cancer patients,
who are the current CAR T cell study population.132
Selecting virus-specific T cells for CAR T cell generation is an alterna-
tive approach to selecting TCM and TEM cells with unknown speci-
ficity. Examples include the aforementioned clinical study with
GD2-CAR/EBV-specific T cells in which investigators demonstrated
improved persistence in comparison to GD2-CAR T cells.81 In addi-
tion, a clinical study is ongoing, which evaluates the safety and effi-
cacy of GD2-CAR/varicella zoster virus (VZV)-specific T cells (Clin-
icalTrials.gov: NCT01953900). Advantages of this approach are not
only that CAR/virus-specific T cells might have improved effector
function in comparison to their unselected CAR T cell counterparts,
but these CAR/virus-specific T cells can be boosted after infusion
through their endogenous, virus-specific ab TCR either by vaccina-
tion (e.g., VZV vaccine) or reactivation of the latent virus (e.g.,
EBV). Lastly CAR-expressing gd T cells133 and invariant natural killer
(iNK)T cells have been successfully explored in preclinical studies,134
and a clinical study with iNK T cells expressing GD2-CARs and IL-15
is in progress (ClinicalTrials.gov: NCT03294954).
CAR T Cell Therapy Targets—The Antigen Dilemma
An “ideal” CAR target should be highly and homogeneously ex-
pressed throughout the tumor, across multiple patients, and have
minimal to no expression in vital normal tissues. Unfortunately, these
characteristics do not apply to most CAR targets that are currently be-
ing explored for solid tumors and brain tumors (Table 2). In general,
current targets are differentially expressed antigens, which may be
present at low levels in normal tissues. The only exceptions are EGFR-
vIII, a cancer-specific splice variant expressed in HGG, and EGFR806,
a conformational EGFR epitope that is also present in HGG.135,136
While neoantigens are a potential solution to this “antigen dilemma,”
most of these are patient specific, raising concerns about feasibility. In
addition, most neoantigens described so far are derived from intracel-
lular proteins, which are difficult to target with standard CAR
T cells.137 Investigators have therefore embarked on discovering novel
CAR targets using genomic and proteomic approaches.138–140 In
addition, there is an intense focus on engineering CARs and CAR
T cells to increase their specificity for antigens expressed on tumor
cells. These include (1) modulating the affinity of the scFv, (2) target-
ing multiple antigens, and (3) restricting CAR activity to tumor sites.
In addition, investigators have developed safety switches that can be
activated in the event of adverse events secondary to CAR T cells,
including on target/off cancer toxicity.
Modulating scFv Affinity
This approach is only applicable to antigens that are expressed at very
high levels such as gene-amplified HER2 in HER2-positive breast
cancer. For example, investigators have shown that reducing the
scFv affinity of HER2-CARs or EGFR-CARs decreases the risk of
on target/off cancer toxicity.141 In addition, in murine models, the
incidence of neurotoxicity of GD2-CAR T cells varied according to
the affinity of the expressed CAR.142 In addition to modulating
scFv affinity, recent studies have also highlighted that the choice
of hinge, transmembrane, and/or costimulatory domains of
CARs can influence antigen density requirements for CAR T cell
activation.58,143,144
Targeting Multiple Antigens
Heterogeneity is a hallmark of cancer and a major roadblock for all
targeted cancer therapies, including immunotherapy.145 Heteroge-
neous antigen expression is one consequence of tumor heterogeneity
leading to the outgrowth of tumor cell subpopulations when a single
Molecular Therapy Vol. 28 No 11 November 2020 7
 Please cite this article in press as: Wagner et al., CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?, Molecular Therapy (2020), https://
doi.org/10.1016/j.ymthe.2020.09.015
www.moleculartherapy.org
Review
antigen is targeted. Investigators have therefore focused on targeting
cancer stem cells (CSCs) or tumor-initiating cells (TICs),146–148 or
multiple antigens to prevent immune escape. In addition, multi-spe-
cific CAR T cells have the potential to reduce the risk of on target off/
cancer toxicity if they target an array of antigens present in a partic-
ular tumor and not in normal tissues. Several approaches are actively
being pursued, including expressing CARs with two scFvs. Investiga-
tors have either assembled two scFvs in a linear (aka tandem CAR)
fashion or by alternating heavy and light chains of the two scFvs
(aka loop CAR), similar to the dual-affinity retargeting (DART) bis-
pecific antibody platform.149–151 Investigators have also expressed
two or three different CARs in T cells.152 The clinical experience
with T cells expressing bispecific CARs or two CARs is currently
limited to CD19/CD22- and CD19/CD20-targeted approaches for
leukemia and/or lymphoma. An alternate approach is to design
CARs in which the antigen-binding domain is derived from a ligand
that binds multiple antigens, and the most relevant example for solid
tumors is the T1E peptide that binds to the EGFR family of recep-
tors.40 So-called “universal CARs” have also been developed to target
multiple antigens that contain an ectodomain that can be paired with
multiple antigen recognition domains. Examples include (1) avidin-
CARs/biotin-labeled scFvs, (2) CD16-CAR/mAbs, (3) anti-fluores-
cein isothiocyanate (FITC)-CARs/FITC-labeled scFvs, (4) coiled-
coil CARs (SUPRA CARs), (5) anti-PNE-CARs/PNE-scFvs, and (6)
NKG2D-CARs/ULBP2-mAbs.153–159 Lastly, bispecific T cell engagers
(BiTEs) have been expressed in T cells,160,161 and more recently
adapted to CAR T cells, opening up the opportunity to target multiple
antigens and redirecting bystander T cells to tumor cells.162
In addition to designing CAR T cells to target multiple antigens, stra-
tegies are being pursued to engineer T cells to activate bystander
T cells to recognize tumor cells (aka induce antigen/epitope
spreading). These include the transgenic expression of cytokines
(e.g., IL-18 or IL-36g) or CD40L.163–165 Likewise, one recent study
has demonstrated that engineering T cells to secrete the FLT3 ligand
promotes epitope spreading.166
Restricting CAR Activity to Tumor Sites
CAR activity can be restricted to tumor sites by several measures.
First, CAR expression can be induced once T cells have migrated to
tumor sites with small molecules, such as a doxycycline-inducible
expression system.167 Second, CAR T cells can take clues from the
TME to express a CAR. These include hypoxia-inducible expression
systems,168 as well as synthetic signaling circuits that take advantage
of Boolean logic.169–171 One of the best examples of Boolean logic-
gated CARs is the synthetic notch (synNotch) system,169–171 in which
an antigen-specific synNotch receptor induces the expression of a sec-
ond CAR upon activation. For example, EpCAM- or B7H3-specific
synNotch receptors have been used to restrict ROR1-CAR expression
to tumor sites, reducing on target/off cancer toxicity in preclinical
models.172 Other strategies include engineering T cells to express
two CARs with different specificity in which one CAR provides signal
1 and the other signal 2 to limit full CAR T cell activation to tumor
sites at which both antigens are present.173,174 With this approach
8 Molecular Therapy Vol. 28 No 11 November 2020
full CAR T cell activation has been restricted to (1) PSMA- and
PSCA-positive or (2) MUC1- and HER2-positive tumor cells in pre-
clinical models.173,174 Expressing inhibitory CARs that recognizes an
antigen expressed on normal tissues in conjunction with a tumor-spe-
cific CAR is another approach that has been evaluated using CD19 as
a tumor-specific antigen and PSMA as a model antigen expressed on
non-targeted cancer cells.175 Lastly, inactive CARs have been de-
signed (e.g., EGFR)176 that require proteolytic processing into an
active form by matrix metalloproteinases (MMPs) within the TME.
Safety Switches
Neither of the aforementioned strategies may end up being suffi-
cient to prevent on target/off cancer toxicities. Investigators have
therefore developed inducible safety switches to ablate CAR
T cells when the need arises. These safety switches can be concep-
tually divided into four approaches. The first approach relies on
activating a prodrug that is toxic to T cells. The best studied
example is the antiviral ganciclovir, which readily kills T cells that
are genetically modified with the herpes simplex virus thymidine ki-
nase (HSV-tk).177 The second approach takes advantage of dimer-
izer drugs to activate a molecule that is transgenically expressed
in CAR T cells. Several dimerizer systems have been developed;178
of these, the inducible caspase-9 (iC9) system has been evaluated
in humans with encouraging results.179,180 Other promising strate-
gies to increase the safety profile of CAR T cells include splitting
CARs into two domains, which need to be linked with a small mole-
cule to be active,181,182 or taking advantage of the small molecule-as-
sisted shut-off (SMAsh) technology.183 The third approach relies on
expressing a molecule on the cell surface of T cells that can be tar-
geted with a clinically approved mAb (e.g., CD20 and truncated
EGFR).184,185 At present the clinical experience with these ap-
proaches is limited. Finally, the last approach relies on exploiting
intrinsic vulnerabilities of T cells with a prime example being the
tyrosine kinase inhibitor (TKI) dasatinib, which inhibits TCR and
CAR signaling through inhibition of Lck.186
Improving CAR T Cell Fitness
As mentioned in the Design of CARs section, even outside the
immunosuppressive TME, CAR T cells have a limited ability to pro-
duce cytokines, expand, and kill when recursively exposed to tumor
cells. T cell exhaustion is a widely used term to describe this T cell
response to chronic antigen exposure, and there is significant debate
in the field how to best define T cell exhaustion.187 While tradition-
ally cell surface markers, such as PD-1, LAG-3, TIM3, and TIGIT,
have been used to define T cell exhaustion, more recent studies
have highlighted that expression of transcription factors such as
TOX or epigenetic programs might be more suitable to reliably
define T cell exhaustion.100–103,105 To improve the fitness of CAR
T cells, investigators have focused on (1) providing additional sig-
nals to promote T cell activation/co-stimulation, (2) providing
signal 3 by transgenic expression of cytokines or constitutively
active cytokine receptors, (3) silencing or deleting molecules that
restrict T cell activation, and (4) transgenic expression of transcrip-
tion factors.
 Please cite this article in press as: Wagner et al., CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?, Molecular Therapy (2020), https://
doi.org/10.1016/j.ymthe.2020.09.015
www.moleculartherapy.org
Review
Provision of Additional Signals to Promote T Cell Activation/Co-
stimulation
Standard CARs only provide co-stimulation, and not directly signal 3,
a necessary signal for T cell expansion. To overcome this limitation,
investigators have incorporated the truncated cytoplasmic domain
of IL-2Rb and a STAT3-binding YXXQ motif into CD28.z-CARs tar-
geting CD19.188 Other groups have shown that T cells expressing
CD28.z-CARs that incorporate the Toll/IL-1 receptor domain of
Toll-like receptor (TLR)2 improve CAR T cell effector function.189
In both approaches, signals are provided simultaneously, which is
in contrast to physiological T cell activation in which signals are pro-
vided in a temporospatial fashion. T cells express 41BB upon activa-
tion, and studies have demonstrated that expression of the 41BB
ligand (41BBL) on the cell surface of CD28.z-CAR T cells endows
CAR T cells with superior effector function in comparison to incor-
porating the 41BB signaling domain directly into the CAR.55,190
Other ligands of the TNF superfamily have also been expressed on
the cell surface of CAR T cells, including CD40L.165 Lastly, investiga-
tors are actively exploring the activation of TLR pathways through
inducible co-stimulatory molecules containing signaling domains of
MyD88,69,191 the central signaling molecule of TLRs, IL-1b, and IL-
18.192
Transgenic Expression of Cytokines or Constitutively Active
Cytokine Receptors
Cytokines or Cytokine Receptors That Signal through the JAK/STAT
Pathway. Common g-cytokines (IL-2, IL-7, IL-15, IL-21) and IL–
12 and IL23 activate the JAK/STAT signaling pathways in T cells.
While common g-cytokines signal through JAK1/JAK2, and predom-
inantly STAT5, IL-12 and IL-23 signal through JAK2, TYK2, and
STAT3 and/or STAT4.193–195 Transgenic expression of these cyto-
kines, demonstrated in numerous preclinical studies, improves the
ability of CAR T cells to expand and/or persist, resulting in improved
antitumor activity.196–198 Investigators have not only explored secre-
tory but also membrane-bound versions of cytokines, which might be
advantageous since they have the potential to improve cytokine activ-
ity (e.g., IL-15) and also restrict most of the cytokine’s action to the
genetically modified cell.199,200 To mitigate systemic side effects of
secreted cytokines, investigators have engineered T cells in which
cytokine expression is controlled by the nuclear factor of activated
T cells (NFAT) promoter. However, while systemic side effects of
IL-12 could be mitigated in preclinical studies, one clinical study
with NFAT-IL-12-modified tumor-infiltrating lymphocytes (TILs)
indicated that the NFAT promoter is insufficient to restrict gene
expression to activated T cells.201 Another approach to reduce cyto-
kine secretion of gene-modified CAR T cells is to use a construct in
which the cytokine gene is placed 30 prime of an internal ribosomal
entry site (IRES).202 A clinical study with MUC16ecto-CAR/IRES-
IL-12 T cells is in progress (ClinicalTrials.gov: NCT02498912).
GD2-CAR T cells (ClinicalTrials.gov: NCT03721068) or GD2-CAR
iNK T cells (ClinicalTrials.gov: NCT03294954) expressing IL-15 are
also currently being evaluated in patients with neuroblastoma. In
one of these studies (ClinicalTrials.gov: NCT03721068), CAR/IL-15
T cells are also modified with the iC9 safety switch to allow for the
control of potential side effects. Alternative approaches to activate
the JAK/STAT pathways are also being pursued, including expression
of constitutively active cytokine receptors (e.g., constitutively active
IL-7 receptor a [C7R]).68
Cytokines That Signal through MyD88. IL-1b and IL-18 signal
through MyD88, and at present only transgenic expression of IL-18
has been explored by several groups of investigators. Consistently,
IL-18 improved the effector function of CAR T cells not only in xeno-
graft models but also in syngeneic murine models.163,203,204 However,
in some models, administration of CAR/IL-18 T cells was associated
with significant toxicities, which could be mitigated in one study by
expressing IL-18 under the transcriptional control of the NFAT
promoter.204
Silencing or Deletion of Molecules That Restrict T Cell Activation
T cells have developed an intricate system to limit their activation,
and investigators have conducted unbiased screens to discover key
molecules within this tightly regulated system. While the first screens
relied on short hairpin RNA (shRNA) approaches identifying the
phosphatase pp2r2d as a negative regulator of ab TCR T cell activa-
tion in tumor models,205 more recent studies have taken advantage of
CRISPR-Cas9 gene-editing technology.206 One recently published
in vitro screen identified TCEB2, SOCS1, CBLB, and RASA2 as nega-
tive regulators of ab TCR activation.206 An in vivo screen also has
highlighted REGNASE1 as a key negative regulator, and REGNASE1
knockout CD19-CAR T cells had improved antitumor activity in a
syngeneic leukemia model.207
Modulating Transcription Factors
Transcription factor networks are critical for T cell plasticity, and
several recent studies have highlighted their importance. For example,
c-Myb (1) is a transcriptional activator of TCF7, a transcription factor
critical for memory formation, and (2) represses ZEB2, a transcrip-
tion factor that promotes T cell differentiation.208 In addition, TOX
has emerged as a key transcription factor that enforces T cell exhaus-
tion, and investigators have shown that TOX- and TOX2-deficient
murine CAR T cells have improved effector function.100–102,209 Other
studies have highlighted that the nuclear receptor transcription fac-
tors NR4A1 (NUR77), NR4A2 (NURR1), and NR4A3 (NOR1)
orchestrate transcription programs that promote T cell exhaustion
and that deleting all three factors in CAR T cells is necessary for
optimal function.210 Lastly, overexpression of c-Jun (transcription
factor AP-1) in CAR T cells increases their functional capacities
and reduces terminal T cell differentiation, resulting in improved
antitumor activity.211
CAR T Cell Trafficking and Tumor Penetration
In hematological malignancies, CAR T cells and their corresponding
target malignancies share hematopoietic origins, and thus have a
higher propensity to migrate to similar areas such bone marrow or
lymph nodes. In contrast, many solid tumors do not attract T cells.
These tumors are called cold tumors, and they can be further subdi-
vided into tumors that do not (cold, ignored) or have (cold, excluded)
Molecular Therapy Vol. 28 No 11 November 2020 9
 Please cite this article in press as: Wagner et al., CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?, Molecular Therapy (2020), https://
doi.org/10.1016/j.ymthe.2020.09.015
www.moleculartherapy.org
Review
Figure 3. Immunosuppressive Solid Tumor Microenvironment
Scheme of the solid tumor microenvironment that consists of (1) tumor vasculature;
(2) stromal cells, including cancer-associated fibroblasts; (3) immunosuppressive
cells, including myeloid cells, macrophages, and regulatory T cells (Tregs); (4)
suppressive metabolites; (5) inhibitory ligands, including PD-1; and (6) suppressive
cytokines. See text for additional details.
a T cell infiltrate in their periphery.212 T cell migration into tumors is
influenced not only by chemokines and adhesion molecules, but also
the tumor vasculature and the presence of other immune cells within
the tumor.212–214 Recent studies have highlighted the role of tissue-
resident memory T cells for cancer immunotherapies, which express
a unique pattern of adhesion molecules and residency markers.215
Engineering approaches have so far mainly focused on taking
advantage of chemokines that are secreted by tumor cells for which
T cells do not express the corresponding chemokine receptor (e.g.,
CXCL8 by melanoma or CCL2 by neuroblastoma).216,217 In preclin-
ical models, transgenic expression of chemokine receptors on tumor-
specific T cells, including CAR T cells, has been shown to enhance
their ability to home to tumor sites, resulting in improved antitumor
activity,216,217 and this approach is being evaluated in a clinical
study for the adoptive immunotherapy of TILs in melanoma
(ClinicalTrials.gov: NCT01740557). Alternatively, combining the
10 Molecular Therapy Vol. 28 No 11 November 2020
infusion of CAR T cells with the local delivery of an oncolytic adeno-
virus encoding RANTES and IL-15 has improved homing to and
persistence of CAR T cells at tumor sites in preclinical models.218
The tumor stroma itself can be extremely dense with various cell types
protecting the tumor. Targeting the extracellular matrix (ECM) and
degrading these physically protective proteins has also shown
promise preclinically. Heparin sulfate proteoglycans (HSPGs) are
expressed in the ECM as well as neuronal tissue during develop-
ment.219,220 Many cancer cells, especially neuroblastoma, express
high levels of HSPGs. Expression of heparinase on CAR T cells to
degrade HSPGs has demonstrated improved tumor infiltration and
antitumor activity in vivo.221 However, the addition of ECM-degrad-
ing enzymes to standard chemotherapies has provided mixed results,
with some clinical trials showing decreased survival in patients with
the combination therapy.222
Counteracting the Immunosuppressive TME
Solid tumor cells are intermixed with suppressive cell populations
such as tumor-associated macrophages (TAMs), myeloid-derived
suppressor cells (MDSCs), Tregs, and cancer-associated fibroblasts
(CAFs) (Figure 3).223–225 Tumor cells and/or cells of the TME express
a broad range of immune checkpoints, including PD-L1 and ligands
for LAG-3, TIM-3, and TIGIT. MDSCs, Tregs, and/or CAFs also
secrete immunosuppressive cytokines such as transforming growth
factor (TGF)-b and IL-10.226,227 In addition, amino acids that are
critical for T cell function such as arginine and glutamine are often
depleted within the TME, contributing to limited T cell fitness
(Figure 3).228,229 Some of the approaches that counteract the immu-
nosuppressive microenvironment rely on strategies that were
discussed in the section Improving CAR T Cell Fitness. For example,
transgenic expression of IL-15 in cytotoxic T cells counteracts Treg-
mediated suppression.230 Herein, we review additional strategies,
including (1) directly counteracting immunosuppressive factors, (2)
hijacking cytokines or growth factors to promote T cell effector func-
tion, (3) improving T cell metabolism in the TME, and (4) targeting
non-malignant cells of the tumor stroma.
Directly Counteracting Immunosuppressive Factors
Several preclinical studies have shown that combining PD-1/PD-L1
blockade with CAR T cell therapy improves antitumor activity.231
In an effort to reduce side effects from systemic checkpoint blockade,
CAR T cells were genetically modified to express a PD-1/CD28 switch
or a truncated PD-1 receptor that functions as a dominant negative
receptor (DNR).70,232 In addition, PD-1 was deleted in CAR T cells
by CRISPR-Cas9 gene editing.233–235 All three approaches resulted
in improved effector function of CAR T cells in preclinical models.
Thus far the clinical experience with these approaches is limited,
and only PD-1 knockout abTCR T cells have been evaluated clini-
cally.236 Other approaches include silencing CTLA-4 or FAS expres-
sion on the cell surface of tumor-specific or CAR T cells.237,238 More
recently, investigators demonstrated that expression of a DNR FAS
receptor also improved the effector function of adoptively transferred
tumor-specific T cells.239
 Please cite this article in press as: Wagner et al., CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?, Molecular Therapy (2020), https://
doi.org/10.1016/j.ymthe.2020.09.015
www.moleculartherapy.org
Review
Hijacking Cytokines or Growth Factors to Promote T Cell
Effector Function
The TME is rich with cytokines that either inhibit T cell function or
for which T cells do not express the corresponding receptors. The
inhibitory effects of these cytokines can be negated by expressing
DNRs or cytokine switch receptors (CSRs) that convert an inhibitory
signal into a growth-promoting signal. Examples of cytokine DNRs
include DNR-TGF-b receptors.240 DNR-TGF-b-expressing EBV-
specific T cells for EBV+ lymphoma have been evaluated in early
phase clinical studies, demonstrating safety and suggesting improved
efficacy in comparison to their unmodified counterparts.241 CSRs
have been developed to take advantage of IL-4 produced within the
TME, including IL-4/IL-2, IL-4/IL-7, and IL-4/IL-21 CSRs.242–244 In
addition, a TGF-b CSR, consisting of a TGF-b-specific scFv and a
CD28z signaling domain, has shown promise in preclinical CAR
T cell therapy models.245 Of these, the IL-4/IL-2 CSR is currently
being evaluated in one early phase clinical study in the context of
TE1-CAR T cells (ClinicalTrials.gov: NCT01818323). Lastly, col-
ony-stimulating factor-1 (CSF-1) is an example of a cytokine that is
present in the TME and for which T cells do not express the cognate
receptor. One preclinical study has shown that expression of CSF-1R
in CAR T cells improves their effector function in a CSF-1-dependent
manner.246
Improving Metabolic Fitness of CAR T Cells in the TME
The TME is hypoxic and deprived of critical nutrients that are
required for T cell proliferation. In addition, the TME (1) is enriched
in metabolic end products that are immunosuppressive, for example,
R-2-hydroxyglutarate in tumors with isocitrate dehydrogenase 1/2
mutations;247–249 (2) has increased concentrations of electrolytes,
such as potassium, that are immunosuppressive;250 and (3) harbors
reactive oxygen species (ROS) that impair T cell function.251,252
Several strategies are being actively explored to improve the metabolic
function of adoptively transferred T cells. These include ex vivo
loading of tumor-specific T cells with arginine,228 or the genetic
modification of CAR T cells with enzymes that are critical for arginine
re-synthesis, including argininosuccinate synthase or ornithine trans-
carbamylase.253 Investigators have also focused on manipulating
glutamine metabolism in the TME, and recent studies have high-
lighted that glutamine antagonists and transient inhibition of gluta-
minase increase T cell effector function.254,255 To protect CAR
T cells from ROS, investigators have genetically modified T cells to
secrete catalase. This not only protected T cells from ROS damage
but also protected unmodified bystander immune cells.256 Lastly,
small molecules such a metformin are being explored to reduce tumor
hypoxia.257
Targeting Non-malignant Cells of the Tumor Stroma
CAFs, MDSCs, TAMs, and tumor-associated endothelial cells are
present in the tumor stroma, and while not malignant, they promote
tumor growth/metastasis and immunosuppression and/or secrete
components to the ECM that hinder immune cell penetration.
CAFs have been targeted by several groups with fibroblast activation
protein (FAP)-CAR T cells. While FAP-CAR T cells had potent
antitumor activity in preclinical models, some safety concerns were
raised due to FAP expression on stromal cells in healthy tissues;
nevertheless, this approach has been translated into the clinic.258–261
TAMs have been targeted with CD123- or folate receptor b (FRb)-
CAR T cells in preclinical models,262,263 and CSF1R-CARs have
been developed, opening up the possibility to target CSF1R-positive
TAMs.264 MDSCs express high levels of NKG2D ligands, and in
one preclinical model, combining NKG2D-CAR NK cells with
GD2-CAR T cells resulted in improved antitumor activity for neuro-
blastoma.265 Lastly, endothelial cells of the tumor vasculature have
been targeted with CARs specific for VEGF-R2, PSMA, or the
EDB/EIIIB splice variant of fibronectin in preclinical models.266–268
Of these approaches, only VEGF-R2 CAR T cells have been success-
fully evaluated in one clinical study. While the results have not been
published, they are available (ClinicalTrials.gov: NCT01218867), and
out of 23 infused patients, only 1 patient achieved a partial response.
Combinatorial Therapies
While this review has focused on additional genetic modification to
improve the effector function of CAR T cells, numerous studies are
underway to combine CAR T cell therapy with other treatment mo-
dalities to improve outcomes by creating therapeutic synergies.269,270
These include efforts, as already mentioned in this review, to combine
CAR T cell therapy with cytokine administration, checkpoint
blockade, oncolytic viruses, radiation, and/or vaccines. Examples
for oncolytics/CAR T cell therapy combinations include the adminis-
tration of oncolytic viruses encoding BiTEs, cytokines, and/or check-
point inhibitors,271–273 or oncolytic viruses that encode CAR tar-
gets.274,275 In addition, investigators have explored the use of T cells
to directly deliver viruses into tumors.276–278 Checkpoint blockade/
CAR T cell therapy combinations are actively being pursued,279
including the aforementioned clinical studies combining mesothe-
lin-CAR T cells with pembrolizumab for the treatment of mesotheli-
oma.76 Furthermore, several studies have highlighted the benefit of
combining radiotherapy with the administration of CAR T cells,
and early clinical studies for patients with B cell lymphoma have pro-
duced encouraging results.280 Recent preclinical studies have also
demonstrated the benefit of boosting CAR T cells outside the immu-
nosuppressive TME with amphiphile CAR ligands or RNA-based
vaccines.281,282
Other approaches include combining CAR T cells with small mole-
cules that target cancer cells that do not affect CAR T cells, with
the best example being combining the BTK inhibitor ibrutinib with
CD19-CAR T cell therapies for lymphoma.283 In addition, combining
CAR T cells with chemotherapeutic agents that upregulate the expres-
sion of the targeted antigens is an attractive option to enhance the
antitumor activity of CAR T cells that is actively being pursued.284
Finally, combinational treatment of HER2-CAR T cells with the
smac-mimetic birinapant, which promotes apoptosis, significantly
enhanced antitumor activity in a TNF-dependent manner.285 Thus,
there is a myriad of combinatorial approaches, and selection of an
optimal combination will most likely depend on the targeted tumor
type and disease status. For example, brain tumors, which often
Molecular Therapy Vol. 28 No 11 November 2020 11
 Please cite this article in press as: Wagner et al., CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?, Molecular Therapy (2020), https://
doi.org/10.1016/j.ymthe.2020.09.015
www.moleculartherapy.org
Review
present as local disease or with limited sites of recurrences, might be
ideal tumors to combine local radiation or delivery of an oncolytic vi-
rus with CAR T cell therapy. In contrast, combining local delivery of
agents with CAR T cell therapy for metastatic cancer should only be
pursued if treating selected metastases induces an abscopal effect,286
leading to the regression of distant, untreated metastases. Feasibility
and costs are also a major consideration; in this regard, repurposing
FDA-approved drugs for combinatorial CAR T cell therapy should
actively be explored as for other cancer therapeutics.287,288
Conclusions
CAR T cells for solid tumors so far have limited activity in early phase
clinical studies, raising concerns of whether CAR T cells can be devel-
oped into a curative therapeutic approach in the future. While
currently ineffective, clinical and preclinical studies have identified
roadblocks, including (1) an antigen dilemma, (2) T cell fitness and
survival before reaching the tumor site, (3) inability of T cells to effi-
ciently traffic to tumor sites and penetrate physical barriers, and (4)
an immunosuppressive TME. In the last decade, investigators have
developed elegant solutions and/or countermeasures in preclinical
studies, engineering CAR T cells with significantly improved effector
function and/or combining them with other treatment modalities.
Thus, we strongly believe that the future of CAR T cells for solid tu-
mors is bright, and they will not become a mere footnote in the history
of cancer immunotherapy. A key challenge will be how to prioritize
and streamline the clinical evaluation of the developed approaches.
In addition, there is a continued need to advance our understanding
in basic CAR T cell biology and how cancer cells and resident immune
cells interact with adoptively transferred CAR T cells. Lastly, future
CAR T cell design might benefit from considering the “ten principles
for good design” that were put forward by the eminent industrial
designer Dieter Rams:289 good design (1) is innovative, (2) makes a
product useful, (3) is aesthetic, (4) makes a product understandable,
(5) is unobtrusive, (6) is honest, (7) is long-lasting, (8) is thorough
down to the last detail, (9) is environmentally friendly, and (10) in-
volves as little design as possible.
AUTHOR CONTRIBUTIONS
J.W., E.W., and S.G. wrote the manuscript. All authors (J.W., E.W.,
C.D., and S.G.) reviewed and edited the manuscript.
CONFLICTS OF INTEREST
J.W., C.D., and S.G. have patent applications in the fields of T cell and/
or gene therapy for cancer. S.G. has a research collaboration with
TESSA Therapeutics, is a DSMB member of Immatics, and is on
the Scientific Advisory Board of Tidal.
ACKNOWLEDGMENTS
The work was supported by National Institutes of Health grant
R01NS106379 (to S.G.); the Alex's Lemonade Stand Foundation
and the Cure4Cam Foundation (ALSF; Young Investigator Grant;
to J.W.); the Alliance for Cancer Gene Therapy (ACGT; to S.G.);
and by the American Lebanese Syrian Associated Charities (to C.D.
and S.G.). The content is solely the responsibility of the authors
12 Molecular Therapy Vol. 28 No 11 November 2020
and does not necessarily represent the official views of the NIH. Fig-
ures were created with BioRender.
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