cancers

Review
Efficacy, Safety, and Challenges of CAR T-Cells in the Treatment
of Solid Tumors
Qiuqiang Chen 1 , Lingeng Lu 2,3 and Wenxue Ma 4, *

1 Key Laboratory for Translational Medicine, The First Affiliated Hospital, Huzhou University School of
Medicine, Huzhou 313000, China
2 Department of Chronic Disease Epidemiology, School of Medicine, Yale School of Public Health,
New Haven, CT 06520, USA
3 Yale Cancer Center and Center for Biomedical Data Science, Yale University, 60 College Street,
New Haven, CT 06520, USA
4 Sanford Stem Cell Clinical Center, Moores Cancer Center, Department of Medicine, University of California
San Diego, La Jolla, CA 92093, USA
* Correspondence: [email protected]; Tel.: +1-858-246-1477

Simple Summary: Chimeric antigen receptor T cells (CAR T-cells) are engineered T cells that target
tumor-associated antigens. CAR T-cell therapy is a novel developed immunotherapy initially for
destroying hematological malignancies. Its great success in clinical practice of hematological malig-
nancies encourages oncologists and scientists to use CAR T-cells for the treatment of solid cancers.
However, the efficacy of CAR T-cells in solid tumors is not as good as expected in hematological ma-
lignancies. In this review, we summarized the efficacy, safety, and challenges of CAR T-cell therapy in
the clinical management of solid tumors. We also discussed the potential strategies currently applied
to improve the efficacy and safety of CAR T-cell therapy in solid tumors, and finally prospected the
future study direction for CAR T-cell therapy.

Abstract: Immunotherapy has been the fifth pillar of cancer treatment in the past decade. Chimeric
antigen receptor (CAR) T-cell therapy is a newly designed adoptive immunotherapy that is able to
Citation: Chen, Q.; Lu, L.; Ma, W.
target and further eliminate cancer cells by engaging with MHC-independent tumor-antigens. CAR
Efficacy, Safety, and Challenges of
CAR T-Cells in the Treatment of Solid
T-cell therapy has exhibited conspicuous clinical efficacy in hematological malignancies, but more
Tumors. Cancers 2022, 14, 5983. than half of patients will relapse. Of note, the efficacy of CAR T-cell therapy has been even more
https://doi.org/10.3390/ disappointing in solid tumors. These challenges mainly include (1) the failures of CAR T-cells to treat
cancers14235983 highly heterogeneous solid tumors due to the difficulty in identifying unique tumor antigen targets,
(2) the expression of target antigens in non-cancer cells, (3) the inability of CAR T-cells to effectively
Academic Editor: Gabriella D’Orazi
infiltrate solid tumors, (4) the short lifespan and lack of persistence of CAR T-cells, and (5) cytokine
Received: 3 October 2022 release syndrome and neurotoxicity. In combination with these characteristics, the ideal CAR T-cell
Accepted: 1 December 2022 therapy for solid tumors should maintain adequate T-cell response over a long term while sparing
Published: 3 December 2022 healthy tissues. This article reviewed the status, clinical application, efficacy, safety, and challenges
Publisher’s Note: MDPI stays neutral of CAR T-cell therapies, as well as the latest progress of CAR T-cell therapies for solid tumors. In
with regard to jurisdictional claims in addition, the potential strategies to improve the efficacy of CAR T-cells and prevent side effects in
published maps and institutional affil- solid tumors were also explored.
iations.
Keywords: CAR (chimeric antigen receptor); antigen; heterogeneity; efficacy; safety; T cell exhaustion;
CRS (cytokine release syndrome); ICANS (immune effector cell-associated neurotoxicity syndrome);
hematological malignancy; solid tumor
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
1. Introduction
conditions of the Creative Commons
Attribution (CC BY) license (https:// According to the American Cancer Society (www.cancer.org (accessed on 18 November
creativecommons.org/licenses/by/ 2022)), cancer remains the second most common cause of deaths in the United States, just
4.0/).

Cancers 2022, 14, 5983. https://doi.org/10.3390/cancers14235983 https://www.mdpi.com/journal/cancers

Cancers 2022, 14, 5983 2 of 30

behind heart disease. As of 2022, it is estimated that there have been 1.9 million new cancer
cases and 609,360 deaths in the United States, or about 1669 deaths every day [1].
In addition to the traditional cancer treatments, such as surgery, chemotherapy, radia-
tion therapy, and targeted therapies (e.g., Imatinib/Gleevec, Trastuzumab/Herceptin, etc.),
immunotherapy, which re-activates immune defenses against cancer cells, is a promising
new approach and has emerged as the fifth pillar of cancer treatment in the last decade.
Cell-based immunotherapy is effective in cancers. Activated T cells recognize tumor
antigens in the form of tumor antigenic peptide fragments that are presented and bound
and bind to major histocompatibility complex class (MHC) molecules on the surface of
tumor cells. However, the failure of T cells to eradicate cancer cells can result from the
expression of immune checkpoint proteins on the surface of T cells, including different
mechanisms such as T-cell exhaustion and immunosuppression [2]. Over the past decade,
a number of immune checkpoint molecules including programmed cell death protein 1
(PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), lymphocyte-activation gene 3 (LAG3),
T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), T cell immunore-
ceptor with Ig and ITIM domains (TIGIT), and B- and T-lymphocyte attenuator (BTLA, also
known as CD272) have been identified and well-studied in cancer [2,3].
One of the upmost achievements in cancer immunotherapies in the last decade has
undoubtedly been the introduction of T cell-targeted immunomodulators, the immune
checkpoint inhibitors (ICIs), such as antibodies against CTLA-4 and PD-1 or programmed
death ligand-1 (PD-L1) [4]. The first ICI antibody, Ipilimumab (Yervoy), was approved in
2011, followed by Pembrolizumab (Keytruda) and Nivolumab (Opdivo) in 2014. These ICIs
have been widely used to treat a variety of cancer types, including head and neck, lung,
kidney, bladder, lymphoma, and melanoma, etc. [2]. These ICI-based immunotherapies are
not focused on in this review.
Chimeric antigen receptor (CAR) T cell (CAR T-cell)-based immunotherapy is another
greatest achievement in cancer immunotherapies. CARs are generated by artificially fusing
a tumor-specific antibody single-chain variable fragment (scFv) to the CD3ζ chain of the T
cell receptor (TCR) via a transmembrane linker domain. The scFv specifically recognize
specific antigens expressed on the cancer cell surface, or intracellular antigens if the scFv is
expressed as intracellular antibody (intrabody) or delivered into the cells [5]. Thus, CAR
is a combination of antibody-derived extracellular proteins, typically derived from the
intracellular signaling module of an antibody and T-cell signaling proteins [6]. The fusion
constructs are then transfected into autologous or allogeneic cytolytic T lymphocytes as
CAR T-cells.
CAR T-cells recognize and target tumor antigens through the binding of CAR to
tumor-associated antigen (TAA) or tumor-specific antigen (TSA) independent of the TCR-
MHC/peptide interaction [6]. CAR T-cells have emerged as an effective novel cancer
therapy for hematological malignancies [7].
The flow of the production, application and monitoring of CAR T-cells is summarized
in Figure 1. CAR T-cell activity can be monitored with flow cytometric assay [8]. In
clinical application, lymphodepletion is needed before the infusion of CAR T-cells to
patients, so that the persistence of infused CAR T-cells can be effectively prolonged, and
the effectiveness of tumor treatment can be improved [9].
The specificity of T cells against tumor cells is mediated by CAR proteins. At present,
pan-B cell CD19 CAR T-cells have shown unprecedented response rates in treating hema-
tological malignancies including refractory (R/R) B cell malignancies [10–12]. In 2017,
autologous anti-CD19 CAR T-cells received the first regulatory approval from the US Food
and Drug Administration (FDA) for the treatment of pediatric B-cell acute lymphoblastic
leukemia (B-ALL), diffuse large B cell lymphoma (DLBCL), and, more recently, mantle cell
lymphoma (MCL) [13].
T cells isolated from a cancer patient (for making autologous CAR T-cells), or other
healthy donors (for making allogeneic CAR T-cells) are activated using artificial antigen-
presenting cells (aAPCs), transfected with the CAR-encoding viral vector, and then ex-
Cancers 2022, 14, 5983 3 of 30

panded to large numbers in a bioreactor. After expansion, the cells are washed followed by
the infusion back to the patient or concentrated and cryopreserved for future use. Activity
of circulating CAR T-cells can be monitored with flow cytometry at different time points
(e.g., 1 month, 3 months, 6 months, etc.) [14] by staining the antibodies of CD62L (circulating
innate lymphoid cell precursors) [15], CD45RO (memory T cells), CD45RA (naïve T cells),
CD4 (T helper cells), and CD8 (cytotoxic T lymphocytes). Subpopulations were defined
as CD62L+ CD45RO+ central memory T cells (TCM ), CD62L- CD45RO+ effector memory
Cancers 2022, 14, 5983 3 of 32
T cells (TEM ), CD62L- CD45RA+ cells (TEMRA ), and CD62L+ CD45RA+ naïve T cells [8].
Cytokines including intracellular IFN-γ etc. can also be measured by flow cytometry [16].

activity monitoring
Figure 1. Production, application, and activity monitoring of
of CAR
CAR T-cells.
T-cells.

A decade
T cells ago, CD19-targeting
isolated CAR T-cells
from a cancer patient showed
(for making efficacy inCAR
autologous patients with or
T-cells), chronic
other
lymphocytic
healthy donors leukemia (CLL)allogeneic
(for making [17] and ALLCAR [18]. However,
T-cells) the wide
are activated usingclinical application
artificial antigen-
of CAR T-cells
presenting cellsin(aAPCs),
both diseases has stalled.
transfected Patients
with the with CLL viral
CAR-encoding did not respond
vector, and to CD19
then ex-
CAR
panded T-cells as often
to large as expected,
numbers at least in
in a bioreactor. partexpansion,
After because of thechallenges
cells areofwashed
producing CAR
followed
T-cell
by theusing
infusionautologous
back to theT cells from
patient orthe patients with
concentrated andunderlying
cryopreserveddiseases, or long
for future use.term
Ac-
chemotherapy treatment, which can lead to lymphocytopenia
tivity of circulating CAR T-cells can be monitored with flow cytometry at different[19]. Meanwhile, severe
time
lymphocytopenia
points (e.g., 1 month, caused by chemotherapy
3 months, is associated
6 months, etc.) with reduced
[14] by staining survival of
the antibodies [20,21].
CD62L It
is important to note that lymphocytopenia is a different concept
(circulating innate lymphoid cell precursors) [15], CD45RO (memory T cells), CD45RA from lymphodepletion.
Lymphocytopenia
(naïve T cells), CD4 is (T
a disorder that lacks
helper cells), lymphocytes.
and CD8 (cytotoxicInT contrast, lymphodepletion
lymphocytes). Subpopulations is to
purposely eradicate regulatory T-cells (Tregs) and other immunosuppressive
were defined as CD62L+ CD45RO+ central memory T cells (TCM), CD62L- CD45RO+ effec- cells through
treatments
tor memoryand to make
T cells (TEM),a CD62L-
room forCD45RA+
the new CAR T-cells,
cells (T thereby increasing CAR T-cell
EMRA), and CD62L+ CD45RA+ naïve T
expansion prolonging persistence [21–23].
cells [8]. Cytokines including intracellular IFN-γ etc. can also be measured by flow cytom-
Due to the impressive results of CAR T-cells in hematological malignancies, many
etry [16].
scientists in academia
A decade and industries
ago, CD19-targeting CAR have beenshowed
T-cells attempting to extend
efficacy CARwith
in patients T- therapy
chronic
to solid tumors. In recent years, the concept of CAR T-cells has been used in the de-
lymphocytic leukemia (CLL) [17] and ALL [18]. However, the wide clinical application of
velopment of other cell-based immunotherapies. According to the updated Cancer Cell
CAR T-cells in both diseases has stalled. Patients with CLL did not respond to CD19 CAR
Immunotherapy Pipeline 2022 from Cancer Research Institute, the number of active cell
T-cells as often as expected, at least in part because of challenges of producing CAR T-cell
therapies developed in 2022 including CAR T-cells, TCR T-cells, NK and NKT cells, TIL
using autologous T cells from the patients with underlying diseases, or long term chemo-
cells, and tumor-associated antigen (TAA)/tumor-specific antigen (TSA) targeted T cells
therapy treatment, which can lead to lymphocytopenia [19]. Meanwhile, severe lympho-
from preclinical to clinical trials and marketing is 2756, compared to 2031 in 2021. Of these,
cytopenia caused by chemotherapy is associated with reduced survival [20,21]. It is im-
1432 are active CAR T-cell therapies, 280 more than 1150 in 2021 [24]. Figure 2 illustrates
portant to note that lymphocytopenia is a different concept from lymphodepletion. Lym-
the changes in the number of active CAR T-cell therapies in the past two years.
phocytopenia is a disorder that lacks lymphocytes. In contrast, lymphodepletion is to pur-
However, CAR T-cell therapy has been disappointing in the treatment of solid tumors
posely eradicate regulatory T-cells (Tregs) and other immunosuppressive cells through
and faces many challenges. Currently, a single antigen-targeted CAR T-cell therapy for
treatments and to make a room for the new CAR T-cells, thereby increasing CAR T-cell
solid tumors frequently fails, and no dual antigens-targeted CAR T-cell therapies have
expansion
been prolonging
approved persistence
for marketing yet in[21–23].
the world. To overcome tumor-defense mechanisms
Due to the impressive results
including immunosuppression (immunosuppressive of CAR T-cells in cytokines
hematological
secretedmalignancies, many
by solid tumors),
scientists in academia and industries have been attempting to extend
antigen escape, and physical barriers to infiltrate into solid tumors, more sophisticated CAR T- therapy to
solid tumors. In recent years, the concept of CAR T-cells has
engineering approaches are required to develop effective CAR T-cell therapies.been used in the development
of other cell-based immunotherapies. According to the updated Cancer Cell Immunother-
apy Pipeline 2022 from Cancer Research Institute, the number of active cell therapies de-
veloped in 2022 including CAR T-cells, TCR T-cells, NK and NKT cells, TIL cells, and
tumor-associated antigen (TAA)/tumor-specific antigen (TSA) targeted T cells from pre-
clinical to clinical trials and marketing is 2756, compared to 2031 in 2021. Of these, 1432
are active CAR T-cell therapies, 280 more than 1150 in 2021 [24]. Figure 2 illustrates the
Cancers 2022,14,
Cancers2022, 14,5983
5983 4 of4 32
of 30

Figure 2.
Figure 2. Active
Active CAR
CART-cell
T-celltherapies
therapiesdeveloped
developed in in
2022 and
2022 2021.
and (a) A
2021. (a)total of 1432
A total active
of 1432 CAR CAR
active T-
cell therapies have been developed in 2022, including preclinical research (854), phase I (314),
T-cell therapies have been developed in 2022, including preclinical research (854), phase I (314), phase
II (243), phase III and market trials of 21. (b) A total of 1150 active CAR T-cell therapies were devel-
phase II (243), phase III and market trials of 21. (b) A total of 1150 active CAR T-cell therapies were
oped in 2021, including 633 preclinical research, 279 phase I, 227 phase II, 11 phase III and market
developed
trials. in 2021, including 633 preclinical research, 279 phase I, 227 phase II, 11 phase III and
market trials.
However, CAR T-cell therapy has been disappointing in the treatment of solid tu-
This article reviewed the status of CAR T-cell therapy in the treatment of solid tumors
mors and faces many challenges. Currently, a single antigen-targeted CAR T-cell therapy
(and also briefly summarized the treatment status of CAR T-cells in hematological ma-
for solid tumors frequently fails, and no dual antigens-targeted CAR T-cell therapies have
lignancies), possible causes of failure, potential solutions, and the progress of CAR T-cell
been approved for marketing yet in the world. To overcome tumor-defense mechanisms
therapy
including forimmunosuppression
solid tumors and discusses the possible significance
(immunosuppressive of CARbyT-cell
cytokines secreted solidtherapy
tumors),for
cancer treatment in the future.
antigen escape, and physical barriers to infiltrate into solid tumors, more sophisticated
engineering approaches are required to develop effective CAR T-cell therapies.
2. Status of CAR T-Cell Therapy in Hematological Malignancies
This article reviewed the status of CAR T-cell therapy in the treatment of solid tumors
(andCAR T-cell therapies
also briefly summarized have
thebeen approved
treatment statusfor the treatment
of CAR of the following
T-cells in hematological types
malig-
of cancer, including B-ALL in children and young adults up to age
nancies), possible causes of failure, potential solutions, and the progress of CAR T-cell 25, DLBCL, primary
mediastinal
therapy for solidlargetumors
B-cell and
lymphoma,
discusseslarge B-cell lymphoma
the possible significancetransformed
of CAR T-cell from follicular
therapy for
lymphoma, high grade
cancer treatment in the future. B-cell lymphoma, aggressive B-cell lymphoma not otherwise spec-
ified (NOS), follicular lymphoma, mantle cell lymphoma, and multiple myeloma. CAR
T-cell therapy
2. Status of CAR is for patients
T-Cell who in
Therapy have tried two or Malignancies
Hematological more treatments, but their cancer has
not improved or has relapsed. However, the efficacy of CAR T-cells in hematological malig-
CAR T-cell therapies have been approved for the treatment of the following types of
nancies is limited and has many challenges for its clinical applications [25]. The following
cancer, including B-ALL in children and young adults up to age 25, DLBCL, primary me-
paragraph is a quick overview on CAR T-cell therapy for hematological malignancies as
diastinal large B-cell lymphoma, large B-cell lymphoma transformed from follicular lym-
the basis of CAR T-cell therapy for solid tumor.
phoma, high grade B-cell lymphoma, aggressive B-cell lymphoma not otherwise specified
Since 2017, the FDA has approved six CAR T-cell therapies for the treatment of CD19 or B-
(NOS), follicular lymphoma, mantle cell lymphoma, and multiple myeloma. CAR T-cell
cell maturation antigen (BCMA, also referred as TNFRSF17, CD269) expressing hematological
therapy is for patients who have tried two or more treatments, but their cancer has not
malignancies including
improved or has relapsed. ALL,However,
non-Hodgkin lymphoma
the efficacy of CAR (NHL), andinmultiple
T-cells myeloma
hematological (MM).
malig-
Brexucabtagene
nancies is limited (Tecartus) is the first
and has many CAR T-cell
challenges for itsproduct
clinicalapproved
applications for [25].
patients
Thewith MCL, a
following
very aggressive disease with the worst prognosis among B-cell lymphoma
paragraph is a quick overview on CAR T-cell therapy for hematological malignancies as and a poor response
to
thechemotherapy.
basis of CAR T-cell Othertherapy
CAR T-cell products
for solid tumor.approved for the treatment of malignancies
are tisagenlecleucel (Kymriah)
Since 2017, the FDA has approved sixfor ALL, and
CAR axicabtagene ciloleucel
T-cell therapies for the(Yescarta)
treatmentfor DLBCL.
of CD19
BCMA
or B-cell maturation antigen (BCMA, also referred as TNFRSF17, CD269) expressingoffers
is one of the most specific and highly expressed antigens on myeloma cells and he- a
promising
matological malignancies including ALL, non-Hodgkin lymphoma (NHL), and multiplefor
target in R/R MM [26]. BCMA as a target of CAR-T therapy has been approved
the treatment
myeloma (MM).of MM. As of September
Brexucabtagene of 2022,
(Tecartus) is there areCAR
the first currently
T-cellfour approved
product CAR T-cell
approved for
products available
patients with MCL,for NHL,
a very two for B-ALL
aggressive diseaseand withone
the for MM,
worst with more
prognosis amongrelated products
B-cell lym-
currently
phoma and in the pipeline
a poor of clinical
response development Other
to chemotherapy. for theCAR
malignancies. All theapproved
T-cell products FDA-approved for
CAR T-cell product names, indications, manufacturers, approval dates,
the treatment of malignancies are tisagenlecleucel (Kymriah) for ALL, and axicabtagene and one-time infusion
costs are summarized
ciloleucel (Yescarta) for in DLBCL.
Table 1. BCMA is one of the most specific and highly expressed
Cancers 2022, 14, 5983 5 of 30

Table 1. FDA-Approved CAR T-cell Therapies in hematological malignancies.

Indications Cost of
Generic Name Brand Target Targeted Time FDA
(Patient Manufacturers One-Time
(Rx) Name Antigen Diseases Approved
Population) Infusion
Children and
B-cell acute
young adults
lymphoblastic
with R/R
leukemia (ALL)
B-cell ALL
30 August 2017
Tisagenlecleucel Kymriah B-cell USD 475,000
CD19 Adults with Novartis
non-Hodgkin
R/R
lymphoma
B cell NHL
(NHL)
R/R follicular Adults with
28 May 2022
lymphoma (FL) R/R FL
B-cell
Adults with
non-Hodgkin
R/R
lymphoma
Axicabtagene Yescarta CD19 B cell NHL USD 373,000
(NHL) Kite Pharma 18 October 2017
Follicular Adults with
lymphoma (FL) R/R FL
Mantle cell
Adults with
lymphoma
R/R MCL
(MCL)
Brexucabtagene Tecartus CD19 Kite Pharma 1 October 2021 USD 373,000
B-cell acute Adults with
lymphoblastic R/R
leukemia (ALL) B-ALL
B-cell
Adults with
Lisocabtagene non-Hodgkin Bristol Myers
Breyanzi CD19 R/R large B 5 February 2021 USD 432,055
maraleucel lymphoma Squibb
cell NHL
(NHL)
Axicabtagene R/R follicular Adults with 5 March 2021
Yescarta CD19 Kite Pharma USD 373,000
ciloleucel lymphoma (FL) DLBCL 1 April 2022
Ciltacabtagene
Carvykti Janssen Biotech 28 February 2022 USD 465,000
autoleucel Multiple Adults with
BCMA
Idecabtagene Myeloma (MM) R/R MM Bristol Myers
Abecma 27 March 2021 USD 419,500
vicleucel Squibb
Abbreviations: ALL: acute lymphoblastic leukemia; BCMA: B cell maturation antigen; DLBCL, diffuse large
B-cell lymphoma; FDA, U.S. Food and Drug Administration; FL, follicular lymphoma; PMBCL, primary me-
diastinal B-cell lymphoma; R/R, relapsed/refractory. MCL: Mantle cell lymphoma; MM, Multiple Myeloma;
NHL: non-Hodgkin lymphoma.

CD19 is an attractive target for cancer immunotherapy because it is expressed in the

majority of B-cell malignancies, including 80% of ALLs, 88% of B-cell lymphomas, and 100%
of B-cell leukemia, while its expression is very limited in normal B cells [27,28]. The “ideal”
antigens currently approved for immunotherapy of hematological malignancies are CD19,
CD20, and BCMA [27,28]. CAR T-cells eliminate tumor cells by directly targeting tumor cells
expressing CD19 or CD20 or BCMA, thereby inducing a selective toxicity of the targeted
tumor cells. A decade ago, anti-CD19 CAR T-cells first showed efficacy in patients with CLL
and ALL. In addition, CD22 is also expressed on most B-ALL and is usually retained even
after CD19 loss [29]. CD22 CAR T-cells have been shown to be effective in B-ALL patients who
are not eligible for CD19 CAR T-cell therapy [30]. Unfortunately, most of the patients with
B-ALL eventually relapse after CD22 CAR T-cell therapy [31] due to reduced or diminished
CD22 expression levels in B-cell lymphoblasts (immature B cells). CD22 CAR T-cell therapy
might still be a salvage therapy for B-ALL patients with an incomplete loss of CD22 expression
(except a standard treatment). Nevertheless, a previous report showed that the second infusion
of humanized CD22 CAR T-cells partially produced a suboptimal anti-leukemia response,
with no expansion of CAR T-cells [30]. An ongoing clinical trial of autologous CD22 CAR
T-cells for relapsed or R/R B-cell malignancies or ALL are conducted at Stanford Cancer
Cancers 2022, 14, 5983 6 of 30

Institute (Palo Alto, CA, USA). In addition, a Phase I study of CD22 CAR T-cells in patients
with R/R hairy cell leukemia and its variant is ongoing at National Institutes of Health (NIH)
Clinical Center (Bethesda, MD, USA). The participants would be followed closely for six
months, and then less frequently for at least five years. CAR T-cell therapies targeting either
CD19 or CD22 alone have potent antitumor effects, but antigen escape-mediated recurrence
frequently occurs. Dual CAR targeting might be applied to overcome the issue [32,33]. Loss of
target antigens [25,29,34,35] or poor CAR T-cell persistence [17,18] are primary factors limiting
the efficacy of CAR T-cell therapies.
Among the current FDA-approved CAR T-cell products, none of them are used for
the treatment of acute myeloid leukemia (AML). One of the challenges is no ideal antigens
existing on AML cells. However, several potential target antigens (e.g., NKG2D ligands, C-
type lectin-like molecule-1 (CLL-1), FMA-like tyrosine kinase 3 (FLT3), CD33, and CD23) are
under investigation for AML treatment with CAR T-cell therapy [36]. Given that targeted
antigens are usually shared between AML cells and myeloid progenitors, switchable CAR-T
cells constitute a key strategy in the construction, thereby increasing safety. In addition,
CD123, the transmembrane alpha chain of the IL-3 receptor, is strongly expressed on AML
cells and is thought as a promising target. Khawanky et al. developed the third generation
of anti-CD123 CAR T-cells with a humanized CSL362-based scFv and a CD28-OX40-CD3ζ
intracellular signaling domain [37], and demonstrated anti-AML activity without affecting
the healthy hematopoietic system, or causing epithelial tissue damage in a xenograft model
of MOLM-13 [37].
A phase I clinical trial (NCT04318678) of anti-CD123 CAR T-cell therapy is under
investigation for the treatment of AML/myelodysplastic syndrome (MDS), T- or B-ALL
or blastic plasmacytoid dendritic cell neoplasm (BPDCN). The primary purpose was to
find the maximum (highest) dose of CD123 CAR T-cells, at which it is safe to the patients.
The side effects of the chemotherapy as well as the CD123 CAR T-cell product were also
reported on the recipient’s body, and the primary endpoint included overall survival.
According to the latest data from Cancer Research Institute [24], the total number of
Cancers 2022, 14, 5983 active CAR T-cell therapies for leukemia conducted in 2022 are 134. Of these 134 therapies,7 of 32
78 CAR T-cell products targeted CD19, 26 targeted BCMA, 8 targeted CD20, 11 targeted
CD22 and CD123, respectively (Figure 3).

100
Number of Active CAR T-cell

CD19
Therapy in Leukemia 2022

78 BCMA
80 CD22
CD20
60 CD123

40
26
20
11 11
8
0
BC 19

C 2
C 20
3
C A
2

12
M
D

D
D
D
C

Figure 3.3. Active

Figure Active CAR
CAR T-cell
T-cell therapies
therapies in
in leukemia
leukemia in
in 2022.
2022. A total of
of 134
134 CAR
CAR T-cell
T-cell trials
trials were
were
conducted in 2022 based on different targets.
conducted in 2022 based on different targets.

Durable
Durable remission
remission after
after CAR
CAR T-cell
T-cell therapy
therapy in
in hematological
hematological malignancies
malignancies is is not
not
guaranteed.
guaranteed. More than 50% of patients with B-cell ALL ALL have
have been
been reported
reported toto relapse
relapse
within
within 1212 months
months after
after the
the treatment
treatment with
with CD19
CD19 or
or CD22
CD22 CARCAR T-cells
T-cells[38,39].
[38,39]. Subgroup
Subgroup
analysis of patients with B-cell malignancies showed that the ORR of
analysis of patients with B-cell malignancies showed that the ORR of patients patients withwith
ALL,ALL,
HL,
NHL and CLL were 79% (95% CI: 70–86%), 37% (95% CI: 21–56%),
HL, NHL and CLL were 79% (95% CI: 70%–86%), 37% (95% CI: 21%–56%), 50% (95% CI:50% (95% CI: 23–78%)
and 68% (95%
23%–78%) andCI:
68%45–84%),
(95% CI:respectively [40]. Relapse[40].
45%–84%), respectively ratesRelapse
are as high
ratesas
are75% in patients
as high as 75%
with hematological malignancies treated with CD19 CAR T-cells [41]. One
in patients with hematological malignancies treated with CD19 CAR T-cells [41]. One pos- possibility is
sibility is due to the increased contraction and transient persistence of CAR T-cells when
exposed to antigen for a prolonged period [34].
Antigen loss or escape is another common cause of resistance to CD19-targeted im-
munotherapy [29,41]. Antigen variant (mutations and/or splicing variants in CD19 gene)-
Cancers 2022, 14, 5983 7 of 30

due to the increased contraction and transient persistence of CAR T-cells when exposed to
antigen for a prolonged period [34].
Antigen loss or escape is another common cause of resistance to CD19-targeted im-
munotherapy [29,41]. Antigen variant (mutations and/or splicing variants in CD19 gene)-
caused escape accounts for 7% to 25% relapse of patients treated with CD19-targeted CAR
T-cells due to resistance of CAR T-cells [42]. CD19 CAR T-cell therapy also can lead to
a deficient or low expression of CD19, which, in turn, result in resistance to the therapy,
consequently bringing to DLBCL progression. Spiegel et al. reported that more than 50%
of DLBCL patients treated with CD19 CAR T-cells experienced progressive disease because
CD19 was absent or low in these patients [39].
According to recent studies, the global average success rates of CAR T-cell therapy is
50–80%. Since 2011, several large clinical trials have demonstrated that CD19 CAR T-cells
have CR rates of 68% to 93% in patients with R/R B-ALL. Relapse remains common over
time, occurring in about 40% to 50% of patients [43,44]. However, efficacy data are scarce on
the patients with high-risk features including BCR-ABL+ , TP53 mutation, extramedullary
disease (including CNS leukemia), or relapse after transplantation [43].
A bi-specific CAR T-cell targeting CD19 and/or CD22 (CD19-22.BB.z-CAR) was de-
veloped to prevent disease recurrence in these patients after CD19 CAR T-cell therapy,
and a Phase I clinical trial (NCT03233854) in patients with R/R B-ALL and DLBCL was
conducted. The primary endpoint was feasibility and safety of manufacturing, and the
secondary endpoint was efficacy. The results showed that B-ALL patients (n = 17) had
88% CR response rate, 29% CR for DLBCL patients (n = 21), while 50% (5 of 10) of the
patients with B-ALL and 29% (4 of 14) of the patients with DLBCL relapsed because of
CD19deficiency/low . However, these relapses were not associated with CD22deficiency/low
disease [39]. CD22 stimulation of CD19/22 CAR T-cell products also showed a reduced
cytokine production when compared with CD19 alone [39]. Targets of CAR T-cell therapies
for hematological malignancies include such as CD19, BCMA, CD22, CD20, CD123, TAA,
CD33, CD30, CD38, and CS1.
CLL patients do not respond to CAR T-cell therapy as often as expected, in part due
to the challenges of manufacturing products from patients whose T cells were unsuitable,
due to either the underlying disease or exposure to years of chemotherapy. In patients
with ALL, severe toxic effects led to delays in testing of modified dosing strategies, and
even to completely discontinue trials. The toxic effects are the result of highly potent CAR
T-cells, which leads to a significant cytokine elevation and increases blood–brain barrier
(BBB) permeability and cerebral edema in a series of high-profile cases [45].
In addition to CD19 deficiency or low expression levels, the barriers of an effective
CAR T-cell therapy also include severe life-threatening toxicity, such as cytokine release
syndrome (CRS), the most common type of toxicity [46,47], modest anti-tumor activity,
antigen escape, restricted CAR T-cell trafficking, and limited tumor infiltration [25].

3. CAR T-Cell Therapy in Solid Tumors

With the promising results of CAR T-cell therapy in the treatment of hematological
malignancies, scientists have begun to extend CAR T-cell therapy to metastatic solid tumors,
including lung, ovarian, breast, prostate, liver, kidney, stomach, pancreatic, and colon cancer.
However, response rates of CAR-T therapy in patients with solid tumors much lower than
those with hematological malignancies. The ORR of CAR T-cell therapy in patients with
solid malignancies was 20% (95% CI: 11–34%) vs. 71% (95% CI: 62–79%) in those with
hematological malignancies. Disappointing CAR T-cell therapies in the treatment of solid
tumors [48,49] indicate the potential issues in solid tumors as follows: (1) TAA antigen
identification (particularly tumor-specific antigens), expression level, and susceptibility to
CAR T-cells, (2) tumor infiltration, CAR T-cells may not be able to penetrate tumor tissue
through the vascular endothelium, and (3) survival of CAR T-cells in TME.
Cancers 2022, 14, 5983 8 of 30

In 2010, a patient with colon cancer metastasis to the lung and liver died after ERBB2-
targeting CAR T-cell therapy [50]. The potential cause of death may be that CAR T-cells
recognize low levels of ERBB2 on lung epithelial cells, thereby triggering cytokine storms.
In 2021, Tmunity Therapeutics, a clinical-stage biotherapeutics company, halted the
development of its lead CAR T-cell product after the deaths of two patients in a clinical
trial. The patients reportedly died from immune effector cell-associated neurotoxicity syn-
drome (ICANS) (Carroll J. Exclusive: Carl June’s Tmunity encounters a lethal roadblock as
2 patient deaths derail lead trial, raise red flag forcing rethink of CAR T-cells for solid tu-
mors. Endpoints News. 2 June 2021. Accessed on 3 June 2021. https://bit.ly/3wPYWm0).
The greatest challenge of CAR T-cell therapy for solid tumors is to find a tumor antigen
that is uniquely expressed on the surface of solid tumor cells to provide CAR T-cell specific
target. However, a major limitation of CAR T-cell therapy is that most proteins are tumor-
associated antigens (TAAs), which are also expressed at low levels in normal cells, making
it difficult for CAR T-cells to specifically target tumor cells without impairing healthy cells.
Additionally, CAR T-cells are limited in trafficking to and infiltrating solid tumors as an
immunosuppressive tumor microenvironment and physical tumor barrier [25]. Current
CAR T-cell products for solid tumors are single-target CAR T-cells, dual-target CAR T-cell
therapies have not yet been approved for marketing.
CAR T-cell therapy shows a strong clinical efficacy in hematological malignancies with a
complete remission (CR) of around 30–40% in treating advanced B-cell malignancies [51], but not
yet in solid tumors except for some individual cases [52]. According to 2022 AACR, Haanen and
colleagues conducted a clinical trial to evaluate the early efficacy and safety of the CAR T-cell
product targeting CLDN6. Among the 14 patients who were evaluable at six weeks after infusion,
4 patients with testicular cancer and 2 with ovarian cancer experienced a partial response (PR),
with an overall response rate of nearly 43% (https://www.aacr.org/about-the-aacr/newsroom/
news-releases/new-car-t-cell-therapy-for-solid-tumors-was-safe-and-showed-early-efficacy/#:
Cancers 2022, 14, 5983 ~:text=Among%20the%20study%20participants%20who,at%2012%20weeks%20after%20infusion 9 of 32
(accessed on 18 November 2022)).
According to the updated data from Cancer Research Institute [24], the total number of
active CAR T-cell therapies for solid tumors conducted in 2022 was 27. Of these 27 therapies,
target EGFR. These newly developed T-cell therapies in solid tumor are summarized in
4 CAR T-cell products target HER2, 6 target MSLN, 7 target GPC2/3, and 10 target EGFR.
Figurenewly
These 4. developed T-cell therapies in solid tumor are summarized in Figure 4.
Therapy in Solid Tumors 2022

15
Number of Active CAR T-cell

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solid tumors has not been supported. The following are some main CAR T-cell products developed for
the treatment of solid tumor. Except a few of them are discussed in the text based on their
attention and clinical accidents in clinical trials, others cannot be described here due to the
spatial limitation, but they are summarized in Table 2.

Table 2. Most targeted antigens in clinical trials of CAR T-cell therapy in solid tumors.
Cancers 2022, 14, 5983 9 of 30

developed for the treatment of solid tumor. Except a few of them are discussed in the text
based on their attention and clinical accidents in clinical trials, others cannot be described
here due to the spatial limitation, but they are summarized in Table 2.

Table 2. Most targeted antigens in clinical trials of CAR T-cell therapy in solid tumors.

Antigen Cancer Phase Identifier (ID)

EGFR Lung, colorectal, ovarian, pancreatic, renal cancers Phase 1/2 NCT01869166
HER2 Central nervous system tumor, pediatric glioma Phase 1 NCT03500991
EGFR806 Central nervous system tumor, pediatric glioma Phase 1 NCT03638167
Mesothelin Ovarian, cervical, pancreatic, lung Phase 1/2 NCT01583686
PSCA Lung Phase 1 NCT03198052
MUC1 Advanced solid tumors, lung Phase 1/2 NCT03179007, NCT03525782
Claudin 18.2 Advanced solid tumor Phase 1 NCT03874897
EpCAM Colon, pancreatic, prostate, gastric, liver Phase 1/2 NCT03013712
GD2 Brain Phase 1 NCT04099797
VEGFR2 Melanoma, brain Phase 1 NCT01218867
AFP Hepatocellular carcinoma liver cancer Phase 1 NCT03349255
Nectin4/FAP Nectin4-positive advanced malignant solid tumor Phase 1 NCT03932565
CEA Lung, colorectal, gastric, breast, pancreatic cancer Phase 1 NCT02349724
Lewis Y Advanced cancer Phase 1 NCT03851146
Glypican-3 Liver Phase 1 NCT02932956
EGFRvIII Glioblastoma and brain tumor Phase 1 NCT01454596
IL-13Rα2 Glioblastoma Phase 1 NCT02208362
CD171 Neuroblastoma Phase 1 NCT02311621
MUC16 (CA-125) Ovarian Phase 1 NCT 02498912
PSMA Prostate Phase 1 NCT01140373
AFP Hepatocellular carcinoma, liver Phase 1 NCT03349255
AXL Renal Phase 1 NCT03393936
CD20 Melanoma Phase 1 NCT03893019
CD80/86 Lung Phase 1 NCT03060343
c-MET Breast, hepatocellular Phase 1 NCT03060356, NCT03672305
DLL-3 Lung Phase 1 NCT03392064
DR5 Hepatoma Phase 1 NCT03638206
EphA2 Glioma Phase 1 NCT02575261
TAG72 Ovarian Phase 1 NCT05225363
gp100 Melanoma Phase 1 NCT03649529
MAGE-A1/3/4 Lung Phase 1 NCT03356808, NCT03535246
LMP1 Nasopharyngeal Phase 1 NCT02980315
Abbreviations: EGFR: epidermal growth factor receptor; HER2: human epidermal growth factor receptor 2;
PSCA: prostate stem cell antigen; MUC1: mucin 1; EpCAM: epithelial cell adhesion molecule; GD2: disialo-
ganglioside; VEGFR2: vascular endothelial growth factor receptor 2; AFP: alpha fetoprotein; FAP: fibroblast
activation protein; CEA: carcinoembryonic antigen; IL-13R: interleukin-13 receptor; CD171: L1 cell adhesion
molecule; MUC16: mucin 16; PSMA: prostate-specific membrane antigen; AXL: AXL receptor tyrosine ki-
nase; c-MET: tyrosine-protein kinase Met; DLL-3: delta like canonical notch ligand 3; DR5: death receptor 5;
EpHA2: ephrin type-A receptor 2; FR-α: Folate receptor alpha; gp100: glycoprotein 100; MAGE-A: melanoma-
associated antigen 3; LMP1: latent membrane protein 1.

3.1. CAR T-Cells Targeting Prostate TAA

Prostate-specific membrane antigen (PSMA) is a type II integral membrane glycopro-
tein highly expressed in prostate cancer and is a diagnostic and prognostic marker, which
is a tumor-associated antigen (TAA). PSMA levels in prostate cancer are 100 to 1000 times
higher than in normal tissues [53]. Meanwhile, high levels of PSMA are associated with the
aggressiveness of human malignancies [54,55]. In addition, PSMA is also highly expressed
in tumor neovascularization [56]. Increased PSMA expression is an independent predictor
of prostate cancer recurrence. Therefore, PSMA becomes an attractive new therapeutic
target with the most minimal tissue penetration for the development of anti-PSMA CAR
T-cell therapy in prostate cancer [57]. Anti-PSMA CAR T-cells have robust killing ability
against human prostate cancer cells and demonstrated strong expansion and cytotoxicity
potential in prostate cancer cells [58]. Clinical trials conducted by Junghans et al. [59]
Cancers 2022, 14, 5983 10 of 30

and Slovin et al. [60] confirmed the safety and efficacy of PSMA-targeted CAR T-cells for
prostate cancer. However, PSMA is also expressed in benign prostatic epithelial cells and
normal prostate tissue.
One CAR T-cell product targeting PSMA, P-PSMA-101, is from Poseida Therapeutics
(San Diego, CA, USA), which was designed to target prostate cancer cells expressing the
cell-surface antigen PSMA (https://poseida.com/science/pipeline (1 December 2022)).
In preclinical studies, P-PSMA-101 has been shown to eliminate tumor cells to unde-
tectable levels in 100% of animals, with only one incidence of relapse in the lower dose
(NCT04249947). Based on published literature, no other product candidate has shown
complete elimination of solid tumors in this preclinical model. A phase I clinical trial of
P-PSMA-101 CAR T-cells in patients with metastatic castration resistant prostate cancer
(mCRPC) has been conducted and a dose escalation trial of P-PSMA-101 is ongoing.
Another CAR T-cell product targeting PSMA, TmPSA01, is from Tmunity Therapeutics
(Philadelphia, PA, USA), which is a pioneer and lead CAR T-cell product, which is at the
forefront of advancing CAR T-cell applications. Unfortunately, TmPSA01 has been halted
after two patients died of neurotoxicity. This work identified potential barriers to CAR T-
cell therapy for solid tumors. Notably, the researchers identified cases of ICANS. Details
of the news were first reported by endpoints and confirmed by Tmunity. This news has
profound implications for the broader push to toward cell therapy as a treatment for solid
tumors (https://www.fiercebiotech.com/biotech/tmunity-stops-solid-tumor-car-t-trial-after-
2-patients-die (accessed on 30 October 2022)). At Tmunity, the setback has led to the end
of the CART-PSMA-TGFβRDN study and the start of work on subsequent candidates with
improved safety profile. Tmunity is aiming to file an IND in the second half of the year.
In addition to PSMA, prostate stem cell antigen (PSCA), another TAA for prostate
cancer, is a membrane glycoprotein predominantly expressed in prostate cancer, which is
expressed in 94% (105/112) of primary prostate tumors and 100% (9/9) of bone metastases [61].
In vivo studies have showed that anti-PSCA monoclonal antibodies inhibit tumor growth and
metastasis formation, making PSCA potentially useful in immunotherapy programs for the
treatment of prostate cancer [62,63]. Although the expression of PSCA is upregulated in most
of prostate cancers, its biological role in prostate cancer remains unclear.

3.2. CAR T-Cells Targeting MSLN (ATA2271)

Mesothelin (MSLN) is a cell-surface antigen associated with tumor invasion, which
is strongly expressed in many solid tumor types, including mesothelioma, lung cancer,
breast cancer, and pancreatic cancer [64]. MSLN has emerged as an important target in
CAR T-cell therapy. Phase I clinical trials have shown that MSLN-targeted CAR T-cell
therapy is safe, but its efficacy is very limited due to insufficient tumor infiltration and the
persistence of CAR T-cells [65]. Lv et al. constructed MSLN CAR T-cells using MSLN scFv,
CD3ζ, CD28, and DAP10 intracellular signaling domain (M28z10) to target MSLN. The
results of in vitro experiments showed that M28z10 T cells exhibited strong cytotoxicity and
cytokine-secreting ability to gastric cancer cells. The in vivo experimental results showed
that M28z10 T cells could induce gastric cancer regression and prolong the mouse survival
in different xenograft mouse models [66].
The expression rate of MSLN was various among pathological types of solid cancer
(serous 97%, clear cell 83%, endometrioid 77%, mucinous 71%, carcinosarcoma 65%),
pancreatic adenocarcinoma (ductal 75%, ampullary 81%), endometrial carcinoma (clear
cell 71%, serous 57%, carcinosarcoma 50%, endometrioid 45%), malignant mesothelioma
(69%) and lung adenocarcinoma (55%) [67]. The highest prevalence of positive MSLN was
found in ovarian cancer. Kachala et al. reported that MSLN overexpression is a tumor
aggressive marker and is associated with increased risk of recurrence and decreased overall
survival (OS) [68]. MSLN CAR T-cell therapy has the potential to treat a variety of solid
malignancies that are overexpressed MSLN [64]. Schoutrop et al. evaluated the efficacy
of MSLN-directed CAR T-cell therapy in an orthotopic mouse model of ovarian cancer.
Cancers 2022, 14, 5983 11 of 30

The results showed that MSLN CAR T-cell therapy significantly prolonged survival, but
sustained tumor control was not observed [69].
Malignant pleural mesothelioma (MPM) is a rare but highly aggressive malignancy
with limited treatment options [70]. It is characterized by resistance to treatment and poor
survival [71]. The median OS of patients with MPM after the first-line treatment with
cisplatin and pemetrexed was only 13 to 16 months, which was prolonged to 18.8 months
after an addition of bevacizumab, but at the cost of increased toxicity [72]. MPM treatment
guidelines from the National Comprehensive Cancer Network (NCCN) include the use
of ICIs as the second-line treatment. Although the expression levels of PD-L1 and tumor
mutation burden (TMB) are very low in patients with MPM [73], the responses still occurred
to PD-L1 blockade [70]. The scientists at Memorial Sloan Kettering Cancer Center (MSKCC)
developed and conducted the first-in-human phase I study of a regional, autologous, MSLN-
targeted CAR T-cell therapy [74]. The results showed that intrapleural administration of
0.3 to 60 M mesothelin-targeted CAR T-cells/kg was safe and well tolerated in 27 patients
(25 with MPM, one with metastatic lung cancer, another with metastatic breast cancer), and
CAR T-cells were detected in peripheral blood for >100 days in 39% of patients. The median
OS of patients receiving CAR T-cell infusion was 23.9 months (83% 1-year OS rate). Eight
patients had a stable condition for ≥6 months; two patients showed complete metabolic
responses after positron emission tomography (PET) scanning [74].
In addition to the traditional therapies described above, MSLN-targeted CAR T-cell
therapy for patients with advanced mesothelioma using next-generation PD1DNR and
1XX CAR technology is also being tested in clinical trials. ATA2271, a next-generation
autologous CAR T-cell therapy targeting MSLN, manufactured by Atara Biotherapeutics,
Inc. (San Francisco, CA, USA) is currently under clinical investigation in patients with
MPM. According to Atara Biotherapeutics, ATA2271 targets hard-to-treat solid tumors
using proprietary 1XX CAR signaling and intrinsic PD-1 checkpoint inhibition.
An ongoing Phase 1 dose-escalation trial of advanced mesothelioma has demonstrated
the early safety and durability of armored CAR T-cells in patients. The preliminary results
of the next generation autologous MSLN-targeted CAR T-cell ATA2271 were presented at
2021ESMO Immuno-Oncology Conference (9 December 2021). But on 18 February 2022,
scientists at MSKCC notified the FDA of a fatal serious adverse event (SAE) associated with
a patient treated with autologous CAR T-cells.
According to the press release issued by Atala Biotherapy on 28 February 2022 (https://
investors.atarabio.com/news-events/press-releases/detail/265/atara-biotherapeutics-provides-
update-on-ata2271-autologous (18 November 2022)): the first 6 patients enrolled in the two
lowest dose groups received either 1 × 106 cells/kg (patients 1–3) or 3 × 106 cells/kg (patient
4–6) of ATA2271 intrapleural treatment. No dose-limiting toxicities have been reported in either
cohort. The reported patient event was related to the first patient in a third, higher-dose cohort
(6 × 106 cells/kg). The temporary suspension of ATA2271 study enrollment does not affect
the ongoing work to promote IND; ATA3271 is a separate, off-the-shelf, allogeneic ATA3271.
ATA3219, Tabelecleucel (tab-cel), and ATA188 all utilize Atara’s allogeneic EBV T-cell plat-
form, the safety and tolerability have been validated by clinical studies and experience in
approximately 400 patients in various disease areas where CRS has not been observed to date.
The ongoing (from 30 September 2020 to September 2023) Phase 1 trial of MSLN-
targeted CAR T-cell therapy in patients with mesothelioma is sponsored by MSKCC
(https://clinicaltrials.gov/ct2/show/NCT04577326 (18 November 2022)). Intrapleural
administration of ATA2271 was well-tolerated at the lowest dose levels, and no CAR T-cell
related adverse events (AEs) of Grade > 2 observed and no AEs of Grade > 3 have been
observed in the study to date. All four patients had received at least four prior lines of
therapy. Importantly, ATA2271 CAR T-cells persisted in peripheral blood of patients for
more than 4 weeks and were associated with upregulated effector cytokines.
Cancers 2022, 14, 5983 12 of 30

3.3. Other Tumor Antigens Used for CAR T-Cells in Solid Tumors
3.3.1. MUC1
MUC1 is a transmembrane glycoprotein that is aberrantly glycosylated and overex-
pressed in a variety of epithelial cancers. Previous studies have confirmed that MUC1 is
overexpressed in NSCLC tissues [75,76], and in about 70% of ovarian cancer [77]. Tumor-
associated MUC1 (tMUC1) is different from the MUC1 expressed in normal cells and can
be used as a biomarker and therapeutic target of cancer [78]. Zhou et al. reported that
monoclonal antibody TAB004 specifically recognizes tMUC1 in all subtypes of breast cancer,
including 95% of triple-negative breast cancer (TNBC), while retaining recognition of MUC1
in normal tissue [79]. The team transduced human T cells with MUC28z, a CAR comprised
of the scFv of TAB004 coupled to CD28 and CD3ζ. The results showed that MUC28z was
well expressed on the surface of engineered activated human T cells. MUC28z CAR T-cells
showed significant target-specific cytotoxicity against a group of human TNBC cells.

3.3.2. ICAM1
Intercellular adhesion molecule-1 (ICAM1) is a cell surface transmembrane glycoprotein
receptor. ICAM1 has been reported to be overexpressed in lung cancer, pancreatic cancer [80]
and renal cell cancer [81]. High levels of ICAM1 were correlated with metastasis and poor
prognosis in cancer patients [80]. ICAM1 expression is increased in TNBC patients and can be
up to 200-fold increase in lung metastases of TNBC patients [82]. The authors demonstrated
ICAM1 generated a phage-displayed scFv library using splenocytes from ICAM1-immunized
mice and selected a novel ICAM1-specific scFv, mG2-scFv. Using mG2-scFv as the extracellular
antigen binding domain, the team constructed ICAM1-specific CAR T-cells and demonstrated
potent and specific killing of TNBC cell lines in vitro and in vivo [83].

3.3.3. EGFR
Epidermal growth factor receptor (EGFR) is a transmembrane protein involved in cell
growth and differentiation. EGFR is overexpressed in a wide range of solid tumor types [84],
it is critical to control the growth and survival of epithelial cells, including NSCLC [85].
EGFR targeted therapies includes tyrosine kinase inhibitors (TKIs, e.g., gefitinib and er-
lotinib, afatinib, Osimertinib) [86], phosphatidylinositol 3-kinase (PI3K) inhibitors, and
antisense gene therapy. These EGFR TKIs have effectively replaced chemotherapy as the
first line treatment [87], Unfortunately, EGFR is increasingly recognized as a biomarker
of tumor resistance [84], since all patients with metastatic lung who initially benefit from
EGFR-targeted therapies eventually developed resistance [88]. EGFR-specific CAR T-cells
have been reported not only to trigger cell lysis of EGFR-positive TNBC in vitro, but
also to inhibit the growth of mouse cell lines and patient-derived xenograft (PDX) TNBC
tumors [89]. EGFR is also a target of immunotherapy [90,91]. EGFR monoclonal antibod-
ies (mAbs, e.g., cetuximab, panitumumab, nimotuzumab, and necitumumab) have been
developed for the treatment of cancer [92].
Li et al. showed that proliferation and anticancer effects of EGFR CAR T-cells in vitro
depend on time (24 to 72 h) and antigen (with and without EGFR antigen stimulation),
and the regression of EGFR-positive human lung cancer xenografts in vivo [93]. A phase
I clinical trial of EGFR CAR T-cells (NCT03182816) demonstrated that EGFR CAR T- cell
therapy was well tolerated in all nine patients in treatment of EGFR-positive advanced R/R
NSCLC patients. The results showed that EGFR CAR T-cells were detectable in peripheral
blood of eight patients, partial response (PR) was observed in one patient, stable disease
(SD) in six patients, and progressive disease (PD) in two patients. The progression-free
survival (PFS) of these 9 patients was 7.13 months (95% CI 2.71–17.10 months), and the
median OS was 15.63 months (95% CI 8.82–22.03 months) [94].

3.3.4. ROR1
Receptor tyrosine kinase-like orphan receptor 1 (ROR1), a member of ROR family,
is a protein encoded by ROR1 gene, and it is overexpressed in cancer [95]. For example,
Cancers 2022, 14, 5983 13 of 30

28.6% was found in 56 histologically confirmed lung adenocarcinoma (using a cut-off of 1),
or in 51.8% of the cases using the median value as threshold [95]. It was reported that
ROR1 repression inhibits the growth of lung adenocarcinoma regardless of EGFR status,
and leads to multiple acquired resistance mechanisms, including EGFR T790M, MET
amplification and hepatocyte growth factor (HGF) overexpression [96]. ROR1 CAR T-
cells can effectively kill lung cancer cells in a three-dimensional tumor model of NSCLC.
Wallstabe et al. reported that ROR1 CART-cell treatment not only showed strong antitumor
activity in human lung cancer cell line (A549), but also infiltrate into cancer tissue and
eradicated multiple layers of tumor cells [97]. This result provides a new strategy for the
clinical treatment of lung cancer. A clinical trial (NCT02706392) has been conducted to
evaluate autologous ROR1 CAR T-cells in patients with advanced ROR1-positive and stage
IV NSCLC; the results have not been released yet.

3.3.5. Trop2
Trophoblast cell surface antigen 2 (Trop2) is a widely expressed glycoprotein and a
member of the epithelial cell adhesion molecule (EpCAM) family in many normal tissues,
and overexpressed in a variety of human cancers, including gastric cancer [98] and breast
cancer [99]. Trop2 has potential in promoting epithelial-mesenchymal transition (EMT) in
human breast cancer [100]. Overexpression of Trop2 has prognostic significance [101]. A
study demonstrated that intra-tumoral injection of bi-specific Trop2/PD-L1 CAR T-cells can
significantly reduce the growth of gastric cancer, and the inhibitory effect is stronger than
specific Trop2 CAR T-cells [102]. These results suggest that novel Trop2/PD-L1 CAR T-cells
are involved in Trop2/PD-L1 and checkpoint blockade in gastric cancer, thereby promoting
the cytotoxicity of CAR T-cells in gastric cancer and other types of solid tumors [102].

3.3.6. TAG72
Tumor-associated glycoprotein 72 (TAG-72) is a pan-adenocarcinoma oncofetal antigen
that is highly expressed in ovarian cancers, and increased expression is associated with dis-
ease progression. The recurrence of ovarian cancer after surgery and multidrug chemother-
apy is frequent, and novel therapeutic methods are urgently needed [103]. TAG72 has been
used as a target for CAR T-cell therapy. Humanized TAG72-specific CAR T-cells have been
reported to show potential cytotoxicity and cytokine production in ovarian cancer. On the
other hand, TAG72-based CAR T cells significantly reduced the proliferative potential and
improved the survival rate of mice [104]. Shu et al. demonstrated that the co-expression
of the TAG-72 CAR and the CD47-truncated monomer CAR on T cells (dual CAR T-cell
strategy) may be effective in ovarian cancer, and applicable to other adenocarcinomas [105].

3.3.7. CA9
Carbonic anhydrase IX (CA9/ or CA IX) is an enzyme encoded by the human CA9
gene [106]. CA IX is overexpressed in many types of cancer, including clear cell renal
cell carcinoma (RCC) [107–109], cervical cancer [110], and breast and lung cancer, and
CA IX promotes tumor growth by enhancing tumor acidosis [111] as other CA family
members [112]. CAIX is a highly expressed on the surface of tumor cells in RCC [107]; thus,
CAIX is a potential therapeutic target. CAIX overexpression increased the expression of
6-Phosphofructo-2-Kinase/Fructose-2, 6-Biphosphatase 4 (PFKFB4) and EMT, and pro-
moted the migration of cervical cancer cells. CAIX can promote metastasis of cervical
cancer cells, thus its inhibitory effect can be used as a therapeutic strategy for cervical
cancer [110]. Li et al. reported that CAIX CAR T-cells combined with sunitinib induced an
effective antitumor response in an experimental model of metastatic RCC [113].

3.3.8. CD133
CD133 (Prominin 1, PROM1) is a transmembrane protein whose mRNA and glycosy-
lated forms are highly expressed in a variety of human cancer cells. CD133 is a cancer stem
cell (CSC) marker and is associated with cancer progression and patient prognosis [114,115],
Cancers 2022, 14, 5983 14 of 30

including pancreatic cancer [116], colorectal cancer [117] and breast cancer [118,119]. Be-
sides the application of CD133-targeted CAR T-cells in MLL leukemia [120], anti-CD133
CAR T-cells have been reported in a phase I trial including 14 patients with hepatocellular
carcinoma (HCC), 7 patients with pancreatic carcinomas, and 2 patients with colorectal
cancer [121]. The results demonstrated the feasibility, controllable toxicities, and efficacy of
anti-CD133 CAR T-cell therapy, with 3 patients achieving PR and 14 patients achieving SD
among the 23 patients enrolled.
Cancers 2022, 14, 5983 15 of 32
3.3.9. Integrin αvβ6
Integrin αvβ6 is an exciting biomarker and therapeutic target for pancreatic cancer,
and it is highly
cases [122]. expressed
It was reportedinthat
almost
CAR100% of expressing
T-cells pancreatic ductal
CXCR2adenocarcinoma
exhibited stronger(PDAC)
anti-
cases [122]. It was reported that CAR T-cells expressing CXCR2 exhibited
tumor activity against pancreatic tumor xenografts known to express αvβ6 [123]. stronger anti-
tumor activity against pancreatic tumor xenografts known to express αvβ6 [123].
4. Lesson on Safety of CAR T-Cells in Solid Tumors from Hematologic Malignancies
4. Lesson on Safety of CAR T-Cells in Solid Tumors from Hematologic Malignancies
Experience in the safety of CAR-T therapy in hematologic malignancies has accumu-
latedExperience in the safety
profound lessons, fromof CAR-T
which therapy in
physicians hematologic
may malignancies
learn to guide has practice
their clinical accumu-
lated profound lessons, from which physicians may learn to guide their clinical practice in
in patients with solid tumors, and better manage the safety issues of CAR-T therapy.
patients with solid tumors, and better manage the safety issues of CAR-T therapy.
CAR T-cell therapy may have some common mild side effects, including high fever
CAR T-cell therapy may have some common mild side effects, including high fever
and chills, dyspnea, severe nausea, vomiting, and/or diarrhea, dizziness or lightheaded-
and chills, dyspnea, severe nausea, vomiting, and/or diarrhea, dizziness or lightheaded-
ness, headaches, tachycardia, fatigue, and muscle and/or joint pain. Some serious side ef-
ness, headaches, tachycardia, fatigue, and muscle and/or joint pain. Some serious side
fects may exist, including high levels of CRS and neurotoxicity (immune effector cell-as-
effects may exist, including high levels of CRS and neurotoxicity (immune effector cell-
sociated neurotoxicity syndrome, ICANS), which make the doctors as walk on thin ice
associated neurotoxicity syndrome, ICANS), which make the doctors as walk on thin ice
when prescribing CAR T-cell therapies. In addition to above side effects, CAR T-cell ther-
when prescribing CAR T-cell therapies. In addition to above side effects, CAR T-cell therapy
apy for solid tumors also faces other safety risks, such as fatal macrophage activation syn-
for solid tumors also faces other safety risks, such as fatal macrophage activation syndrome
drome (MAS) [124] and uveitis [125], etc.
(MAS) [124] and uveitis [125], etc.
CRS, neurologic symptoms (NS) and tumor lysis syndrome (TLS) are the common
CRS, neurologic symptoms (NS) and tumor lysis syndrome (TLS) are the common
side effects caused by CAR T-cells. Of grade 3 or 4 adverse events, CRS accounts for 22%,
side effects caused by CAR T-cells. Of grade 3 or 4 adverse events, CRS accounts for 22%,
neurologic events 12%, cytopenia lasting more than 28 days 32%, infections 20%, and fe-
neurologic events 12%, cytopenia lasting more than 28 days 32%, infections 20%, and febrile
brile neutropenia 14%. Three patients died within 30 days of infusion due to disease pro-
neutropenia 14%. Three patients died within 30 days of infusion due to disease progression.
gression. No deaths were attributed to tisagenlecleucel, CRS, or cerebral edema [126].
No deaths were attributed to tisagenlecleucel, CRS, or cerebral edema [126].
Despite potentially life-threatening toxicities, the benefits of CAR T-cell therapy far
Despite potentially life-threatening toxicities, the benefits of CAR T-cell therapy far
outweigh the risks, especially as these toxicities are being mitigated with increased expe-
outweigh the risks, especially as these toxicities are being mitigated with increased experi-
rience and improved supportive therapies [42]. In clinical practice, safe use of CAR T-cells
ence and improved supportive therapies [42]. In clinical practice, safe use of CAR T-cells is
is both a skill and art. Learning to balance the efficacy and safety of CAR T cells in cancer
both a skill and art. Learning to balance the efficacy and safety of CAR T cells in cancer
treatment is critical [127]. Hopefully, one day, CAR T-cells will have both high efficacy
treatment is critical [127]. Hopefully, one day, CAR T-cells will have both high efficacy and
and high safety (Figure 5).
high safety (Figure 5).

Figure 5.
Figure 5. The
The efficacy
efficacy and
and safety
safety of
of CAR
CAR T-cell
T-cell therapy
therapy in
in the
the treatment
treatment of
of solid
solidtumors
tumorsare
arealways
always
at opposite ends of the scale. (a) the balance between safety and efficacy, (b) higher safety,lower
at opposite ends of the scale. (a) the balance between safety and efficacy, (b) higher safety, lower
efficacy: safety improves always at the expense of efficacy, (c) higher efficacy, lower safety, efficacy
efficacy: safety improves always at the expense of efficacy, (c) higher efficacy, lower safety, efficacy
improves always at the expense of safety.
improves always at the expense of safety.

4.1. Cytokine-Release Syndrome (CRS)

CRS is the most common adverse effect after CAR T-cell infusion. CRS toxicity usu-
ally occurs within the first week after CAR T-cell therapy, and typically peaks within 1–2
weeks of cell administration [128]. CRS is an acute systemic inflammatory syndrome char-
Cancers 2022, 14, 5983 15 of 30

4.1. Cytokine-Release Syndrome (CRS)

CRS is the most common adverse effect after CAR T-cell infusion. CRS toxicity usually
occurs within the first week after CAR T-cell therapy, and typically peaks within 1–2 weeks
of cell administration [128]. CRS is an acute systemic inflammatory syndrome character-
ized by high fever and chills, as well as multiple organ dysfunction (difficult breathing,
severe nausea, vomiting, and/or diarrhea, feeling dizzy or lightheaded, headaches, fast
heartbeat, feeling very tired, muscle and/or joint pain). This is caused by the release of
cytokines into the body following the activation of immune cells (especially T/CAR T-cells)
during immunotherapy [127]. CAR T-cell therapy-associated CRS remains a major hurdle
before its widespread use. CRS can be fatal if not properly identified and managed. In
addition, neurotoxicity, called CAR T-cell-related encephalopathy syndrome (CRES), is the
second most-common adverse event and can occur concurrently with or after CRS. The
management of CRS is a big concern in these indications. IL-6 is identified as a key risk
factor of CRS, which is a potential target in developing strategies to improve safety.

4.2. Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS)

ICANS is another common and unique toxicity after CAR T-cell therapy, occurring
in up to 67% of leukemia patients and 62% of lymphoma patients [129]. Particularly in
patients with ALL, this complication, however, did not prevent the product from entering
the market of hematological malignancies. It has been reported that the level of neu-
rotoxicity in patients with severe neurotoxicity is associated with the disruption of the
blood-cerebrospinal fluid (CSF) barrier, but not with the white blood cell count or CAR
T-cell number in CSF [129].
The mechanism of ICANS is relatively poor understood. Tmunity believes that if
this adverse event is going to be a problem of CAR T-cell products in solid tumors, better
understanding of the mechanism is a priority to bypass the barrier. The patients reportedly
died from immune effector cell-associated ICANS in CAR T-cell therapies. “What we are
discovering is that the cytokine profiles we see in solid tumors are completely different
from hematological cancers”, Usman “Oz” Azam, the former president and CEO of Tmu-
nity mentioned in an interview with Endpoints News (https://www.onclive.com/view/
car-t-cell-therapy-trial-in-solid-tumors-halted-following-2-patient-deaths (accessed on
18 October 2022)).

4.3. Macrophage Activation Syndrome (MAS)

Macrophage activation syndrome (MAS) is a severe life-threatening complication re-
lated to hemophagocytic lymphohistiocytosis (HLH). It is characterized by the uncontrolled
activation and proliferation of T lymphocytes and macrophages, resulting in the release
of high levels of inflammatory cytokines [130]. HLH can be divided into primary HLH
(pHLH) and secondary HLH (sHLH). The former is caused by an inherited disease, such
as severe systemic lupus erythematosus (SLE has at least a partial genetic component),
the latter is caused by other diseases including infections, malignancy, and autoimmune
diseases [131]. The clinical syndrome of HLH include fever, hepatosplenomegaly, abnormal
liver function, decreased blood cells, increased triglycerides, serum ferritin, and decreased
fibrinogen. Systemic inflammatory response rarely evolves into a fulminant hemophago-
cytic lymphohistiocytosis (HLS) and MAS, which are associated with a high mortality
rate. HLH/MAS caused by CAR T-cell therapy is an unusual manifestation of CRS with
immune-mediated multi-organ failure, poor prognosis, and a challenging diagnosis. CAR
T-cell therapy related HLH/MAS has a distinct malignancy-related PET-CT scan, show-
ing a paradoxical response of hyper-inflammation in CAR-T therapy-related HLH/MAS
patients [132]. Consistently, flow cytometry results showed the expansion of CAR T-cell
Cancers 2022, 14, 5983 16 of 30

existing in peripheral blood (PB), and the increased CAR T-cells at different follow-up time
points [132]. Anti-IL-6 therapy, steroids, anakinra (a recombinant IL-1 receptor antago-
nist) and emapalumab (an anti-IFNγ, approved by the FDA) are recommended for the
management of HLH/MAS [128,132].

5. Challenges of CAR T-Cell Therapy in Solid Tumors

CAR T-cell therapy has revolutionized the treatment of hematological malignancies,
but its use in solid tumors has been challenging. The greatest challenges in generating
CAR T-cells for the treatment of solid tumors include (1) cell recognition: solid tumors
exhibit a considerable degree of antigen heterogeneity, with only a subpopulation of the
cells expressing the target antigen. Most proteins on solid tumor cells can be targeted,
but they are also expressed even at very low levels on normal cells, making it difficult
for CAR T-cells to specifically target tumor cells without jeopardizing healthy cells. In
addition, antigen expression levels on various tumor cells may impair CAR T-cell function
because the diversity of antigens makes it difficult to identify TSA. CAR-Ts can effectively
redirect CTLs to surface antigens that are highly expressed on tumor cells. However, the
low expression of several TAAs on normal tissues hinders their safe target by CAR T-cells
due to on/off-target tumor effects [133]. (2) Cell trafficking: CAR T-cells are required as
cytotoxic CD8+ T cells to home into malignant sites after infusion, navigate the complicated
TME, form efficient interactions with cancer cells, deliver their cytotoxic activities, and
ultimately persist [134]. Yet, unlike in treating hematological malignancies, CAR T-cell
therapy is more limited in solid tumors because CAR T-cells may not be able to infiltrate
into solid tumor tissues through the vascular vessels [135]. The host and TME interactions
with CAR T-cells critically alter CAR T-cell function [25]; (3) cell surviving: TME is widely
considered to be detrimental to T cells, and CAR T-cells have limited activities in solid
tumors. The glycolytic metabolism of tumor cells makes the environment hypoxic, acidic,
and nutrient-deficient, which is easy to produce oxidative stress, thereby affecting IL-2
signaling and T cell proliferation [136]. Additionally, in an inflammatory environment,
tumor cells express ligands (e.g., Gal9 and PD-L1, etc.) that bind to the T cell inhibitory
receptors TIM-3 and PD-1, respectively, further promoting T cell exhaustion [137–139].
In addition, continuous exposure to a CD19 × CD3 bispecific molecule induces T-cell
exhaustion [140].
CAR T-cell exhaustion is a major limitation to their efficacy especially in solid tumors.
T cell exhaustion is a state of T-cell dysfunction characterized by a progressive loss of
effector function during neoplastic disease. Continuous tumor antigen stimulation, im-
munosuppressive TME, alteration of T cell-associated transcription factors, and metabolic
factors all can result in T cell exhaustion [41]. Other factors, such as CAR T manufacturing
(the structure and qualitative characteristics of CAR T structures), changes in the TME,
previous treatments, or effects of neighboring cells can also lead to CAR T-cell exhaustion
and affect response outcomes. These exhausted CAR T-cells have no proliferative capacity
and lose the ability to produce IFNγ, chemokines, and degranulation [141]. As a result,
they lose the ability to eliminate tumor cells. Furthermore, most CAR T-cells are primarily
autologous, and CAR T-cell therapy has significant deficiencies with T-cell exhaustion
potential. In summary, CAR T-cell exhaustion is believed to be due to sustained antigenic
stimulation, as well as an immunosuppression of TME, maintaining CAR T-cell effector
function, sustaining, and achieving clinical potency remains a critical challenge [41].
Moreover, other challenges also exist, which limit the therapeutic efficacy of CAR
T-cells in solid tumors. For example, the manufacture of low-quality CAR T-cells, antigen
escape (tumor cells of a significant portion of patients treated with these CAR T-cells display
either partial or complete loss of target antigen expression) [25], and severe life-threatening
toxicities. The other potential reasons for nondurable response to CAR T-cell therapy are
summarized in Figure 6.
 lytic metabolism of tumor cells makes the environment hypoxic, acidic, and nutrient-defi- 720

cient, which is easy to produce oxidative stress, thereby affecting IL-2 signaling and T cell 721

proliferation [137]. Additionally, in an inflammatory environment, tumor cells express lig- 722

Cancers 2022, 14, 5983 ands (e.g., Gal9 and PD-L1, etc.) that bind to the T cell inhibitory receptors TIM-3 and PD- 723
17 of 30
1, respectively, further promoting T cell exhaustion [138-140]. In addition, continuous ex- 724

osure to a CD19 × CD3 bisecific molecule induces T-cell ex. 725

CAR T-cell products

Antigen expression

Severe toxicities of CAR T-cell therapy

Optimization of CAR T-cell therapy

Figure 6. Possible reasons for the failure to sustain remission after CAR T-cell therapies. (1) CAR T-cell
Figure 6. Possible reasons for the failure to sustain remission after CAR T-cell therapies. (1) CAR T- 727
products: CAR T-cell product from some patients may not be successfully manufactured due to T cell
cell products:
problems CAR T-cell
(e.g., product Tfrom
autologous cellssome patients
obtained frommay not be successfully
the patients manufactured
after chemotherapy), or thedue to 728
produced
T cell problems
CAR T-cells(e.g.,
may autologous
not expand T adequately
cells obtained from
either the in
during patients after chemotherapy),
vitro culture or after infusion or the pro-
in vivo. 729
Limited
persistence
duced CAR T-cellsofmay
CAR T-cells
not expandin other patientseither
adequately is a potential
during mechanism for disease
in vitro culture or afterrecurrence.
infusion in(2). Antigen
vivo. 730
expression: The absence or downregulation of antigens on the tumor cell surfaces, allows antigen escape
Limited persistence of CAR T-cells in other patients is a potential mechanism for disease recurrence. 731
as a mechanism of resistance to CAR T-cell therapy. (3). Severe toxicities of CAR T-cell therapy: The fatal
(2). Antigen expression:
toxicity Thetherapy
of CAR T-cell absence(e.g.,
or downregulation of antigens
CRS and/or neurotoxicity) on the atumor
prevents cell surfaces,
small percentage al- 732
of patients
lows antigen escape as a mechanism of resistance to CAR T-cell therapy. (3). Severe toxicities of CAR in
benefit from the potential therapeutic of CAR T-cell therapy. (4). Optimization of CAR T-cell therapy 733
clinical application: For the treatment of the patients with pediatric lymphoma, and solid tumors, CAR
T-cell therapy: The fatal toxicity of CAR T-cell therapy (e.g., CRS and/or neurotoxicity) prevents a 734
T- cell therapy need to be further optimized.
small percentage of patients benefit from the potential therapeutic of CAR T-cell therapy. (4). Opti- 735
6. Improve the Effectiveness and Safety of CAR T-Cells
mization of CAR T-cell therapy in clinical application: For the treatment of the patients with pediatric 736
Single-cell RNA sequencing may accelerate the understanding of CAR T-cell therapy
lymphoma, and solid tumors, CAR T- cell therapy need to be further optimized. 737
effectiveness and safety. Bai et al. used single-cell RNA sequencing and proteomics
approaches to analyze the mechanism of resistance in ALL patients treated with CD19-
targeted CAR T-cells. The authors presented 101,326 single-cell transcriptomes and surface
protein profiles from the infusion products of 12 ALL patients, the results showed significant
19
heterogeneity in antigen-specific activation states, with a deficiency of T helper 2 function
associated with CD19-positive relapse vs. durable responders (remission, >54 months) [142].
These molecular mechanisms can be potentially used to boost specific the function of
specific T cells to maintain a long-term remission.
To date, most clinical studies have focused on patients who respond to CAR T-cell
therapies only but ignoring the 50% to 60% of patients who fail CAR T-cell therapy and
relapse [143]. Future clinical trials for these patients need to be designed to develop optimal
treatment strategies for these patients.
One possibility for patients who do not respond to CAR T-cell therapy is that many
auto-CAR T patients received three or more courses of chemo treatment before cell collec-
tion, so that the collected T cells for CAR T were less suitable at baseline [144].
Unlike in hematological malignancies, another possibility for CAR T-cell therapy in
solid tumors is limited by insufficient tumor infiltration, T cell dysfunction and exhaustion.
Local delivery of CAR T cells in patients with solid tumors is a safe and feasible strategy,
which can increase the proliferation and penetration depth of CAR T-cells in tumors,
enhance the help of CD4, immune balance, and more metastasis to the metastasis site and
drainage to lymph nodes [145]. In addition, cancer cells often lose antigen expression due
to the inhibition of their antigen mRNA translations, thereby escaping antitumor immune
surveillance and attack by T cells, including CAR T-cells [146]. Moreover, the immune
evasion may also result from CAR T-cell dysfunction, unfavorable TME, or drug-resistant
cancer cells [147].
The favored antigens of CAR T-cell therapy for solid tumors usually include TAA,
HER2, MSLN, GD2, EGFR, GPC2/3, NY-ESSO-1, MUC1, PSMA, EBV, Claudin 18.2, etc.
Cadherin 17 (CDH17) is a novel oncogene, biomarker, and attractive therapeutic target
for the aggressive malignancies. Feng et al. demonstrated that anti-CDH17 CAR T-cells
Cancers 2022, 14, 5983 18 of 30

not only eradicate CDH17-expressing neuroendocrine tumors (NETs), gastric, pancreatic,

and colorectal cancers in xenograft or autochthonous mouse models, but also do not attack
normal intestinal epithelial cells, which also express CDH17 to cause toxicity [148].
Multi-target CAR T-cells is another strategy to improve the effectiveness of CAR
T-cells in solid tumors. Combined targeting of two or more tumor antigens can offset
antigen escape, thereby enhancing T-cell effector functions. This idea is derived from
dual-target CAR T-cells in hematological malignancies. Trivalent CAR T-cells that co-
target HER2, IL13Rα2, and EphA2 can overcome antigenic variation among patients with
glioblastoma [149]. Currently, no dual-target or multi-target CAR T-cell products have been
approved for marketing yet.

6.1. CD19 × CD22 CAR T-Cells

CAR T-cells targeting either CD19 or CD22 have shown remarkable activity in B-ALL.
Research results showed that some patients with B-cell tumors who received CD19 or
CD22 CAR T-cells still experienced disease progression and recurrence. The major cause
of treatment failure is antigen downregulation or loss. CD19 and CD22 are all specially
expressed on B cell malignancies. Current investigations of dual targeting antigen receptors
have demonstrated encouraging results, providing a high degree of optimism that the
efficacy and the broader application of CAR T-cell therapy will gradually increase in B-ALL
treatment [42]. A phase I trial in pediatric and young adult patients with R/R B-ALL
(n = 15) tested autologous CAR T-cells expressing both anti-CD19 and anti-CD22 and
showed a remission rate of 86% at a month after treatment with a favorable safety profile.
The 1-year OS and event-free survival (EFS) rates were 60% and 32%, respectively [150].

6.2. CD19 × BCMA CAR T-Cells

CD19 targeted CAR T-cell therapies have been successfully used in patients with B-cell
hematological malignancies, and also demonstrated consistently high antitumor efficacy in
the relapsed B-ALL, CLL and B-cell non-Hodgkin lymphoma (B-NHL) [151]. B-cell matura-
tion antigen (BCMA), also known as tumor necrosis factor receptor superfamily (TNFRSF)
member 17, is preferentially expressed in mature B lymphocytes, and its overexpression
and activation are associated with MM [152]. The potential of BCMA-targeted therapies to
improve the treatment landscape for MM has been outlined not only in preclinical models,
but also the clinical data [152]. Clinical data indicated that the combination of anti-BCMA
and anti-CD19 CAR T-cell therapies induced high response rates in patients with R/R MM.
Further clinical trials with a median follow-up time of 21.3 months have demonstrated
that the combination therapy of anti-BCMA with anti-CD19 CAR T-cells induced durable
responses in patients with R/R MM, with a median response duration of 20.3 months and
a median PFS of 18.3 months. In addition, this dual CAR T-cell treatment was associated
with a manageable long-term safety profile [153].

6.3. CD33 × CLL1 CAR T-Cells

AML is morphologically characterized by heterogeneous leukemia cells from myeloblasts
to differentiated myeloid elements [154]. Heterogeneous cells in AML can consequently offset
killing effect of single target–based CAR T-cell therapy, resulting in disease relapse. CD33 is
widely expressed in AML [155], and C-type lectin-like molecule-1 (CLL1) is highly expressed
on AML leukemia stem cells (LSC) and blasts, but not on normal hematopoietic stem cells
(HSC) [156]. Targeting both CD33 and CLL1 surface antigens may offer two distinct benefits.
Simultaneously targeting both bulk leukemia cells and LSC not only comprehensively ablates
AML disease, but also overcome the defect of single antigen loss, thereby preventing relapse. A
Phase I study has evaluated the safety and effectiveness of this CD33-CLL1 dual CAR T-cell
therapy in R/R AML patients (NCT05016063).
Cancers 2022, 14, 5983 19 of 30

6.4. HER2 × IL13Rα2 × EphA2 Trivalent CAR T-Cells

Glioblastoma (GBM) is the most common primary malignant brain tumor and is
currently incurable. CAR T-cell therapy has shown promising in the treatment of GBM.
Bielamowicz et al. [149] demonstrated that targeting three antigens of human epidermal
growth factor receptor 2 (HER2), interleukin-13 receptor subunit alpha-2 (IL13Rα2), and
ephrin-A2 (EphA2) with a single CAR T-cell product can offset antigen escape, and enhance
T-cell effector function. The results showed that combined targeting the antigens of HER2,
IL13Rα2, and EphA2 could overcome interpatient variability, with a tendency to capture
nearly 100% of tumor cells in the majority of tumors tested in their studies. The CAR T-cells
mediated robust immune responses and exhibited improved cytotoxicity and cytokine
release than optimal mono-specific and bi-specific CAR T-cells in each patient’s tumor
profile. Furthermore, even low doses of CAR T-cells can control patient-derived xenografts
(PDXs) established with GBM and improve the survival of treated mice.

7. Discussion
As one of the hottest immunotherapy technologies, CAR T-cell therapy has become
the pillar therapeutic technology of immunotherapy. The 71% overall response rate (ORR)
of CAR T-cell therapy in patients with hematological malignancies was significantly higher
than 20% in patients with solid malignancies [40], suggesting that a better understanding
of these issues and further development of CAR T-cell therapy are needed.
CAR T-cells can target antigens on tumor cell membranes only. In contrast, intracellular
antigens can be targeted only through natural or artificial T-cell receptors (TCR), which are
presented as peptides together with MHC molecules [157]. Thus, the selection of surface
antigens for CAR T-cell products in solid tumors is challenging with limited antigenic
expression in normal tissues but sufficient to induce cytotoxicity to tumors [158], given the
lack of tumor-specific antigens thus far. In addition, one of the barriers to the effectiveness of
CAR T-cell therapy in solid tumors is antigenic heterogeneity, which weakens the detection
of cancer cells by T cells and reduces the efficacy of CAR T-cell therapy.
In fact, many CAR T-cells effectively eliminate tumor cells expressing high levels
of the target antigen, but not effectively for tumor or normal cells expressing low levels
of the target antigen. The antitumor activity and “on-target, off-tumor toxicity” of CAR
T-cells are dependent on the ratio of target antigen density on tumor and normal cells [159].
Consequently, it is critical to find more stable expression and specificity of target antigens.
Scientists have been working to optimize CAR T-cell therapy. CAR T cell therapies
have greatly evolved over the past years, including numerous attempts to enhance persis-
tence, proliferation, safety, and efficacy. These efforts and creations include five generations
of engineered CAR T cells, inducible switches for CAR T cell killing or regulation, and
locoregional CAR T cell delivery, etc. However, minimizing off-target and tumor toxicity of
CAR T cells remains challenging.
Neoantigens generated by tumor-specific (somatic) mutation on the surface of solid
tumor cells, which have been shown in association with patient survival in human solid
cancer [160]. They are attractive targets for CAR T-cell therapy since their expressions are
restricted to tumor cells. Neoantigens are highly individualized, therefore, neoantigens-
based CAR T-cell therapy may need individualizing and requires that the target neoanti-
gens are membrane expressed. A couple of novel epitopes have been identified, for
example, EGFR variant III (EGFRvIII ) is a tumor-specific protein present in 25–30% of
newly diagnosed glioblastomas (GBMs), making it a potential option for CAR T-cell ther-
apy [161]. A couple of clinical trials of CAR T-cell therapy targeting EGFR mutations
(NCT02209376, NCT01454596) had been conducted [162]. Mucin 1 (MUC1) is an attractive
antigen candidate in cholangiocarcinoma (CCA). Anti-MUC1 CAR T-cells demonstrated
a significant specific killing activity against CCA cells (both KKU100 and KKU213A cell
lines) at an effector to target ratio of 5:1 [163]. Furthermore, claudin 6 (CLDN6) is a cell
surface member protein expressed on multiple solid tumor tissues and its expression
levels differ by tumor types [164] while the expression is not observed on normal adult
Cancers 2022, 14, 5983 20 of 30

tissue [165]. Dr. John Haanen from Netherlands Cancer Institute presented the results
of a first-in-human open-label, multicenter clinical trial to evaluate the safety and pre-
liminary efficacy of a CAR T-cell product targeting CLDN6 during 2022 AACR annual
meeting (https://www.aacr.org/about-the-aacr/newsroom/news-releases/new-car-t-cell-
therapy-for-solid-tumors-was-safe-and-showed-early-efficacy (accessed on 18 November
2022)). According to the preliminary data of phase I/II clinical trial (NCT04503278), the
safety profile of new CAR T-cell products is acceptable, with early signs of efficacy as a
monotherapy and in combination with mRNA vaccine in patients with solid tumors. Pre-
clinical models showed that a CLDN6-encoding mRNA vaccine (CARVac) in combination
with the CAR T-cell therapy favors CAR T-cell expansion and higher persistence in the
blood. This, in turn, increase tumor cell killing [166].
Besides the efficacy and side effects (e.g., CRS and ICANS, etc.) of CAR T-cells in
the treatment of solid tumors, another issue to consider is the preparation time and cost.
It generally takes from 2 weeks to 1 month to complete the preparation of CAR T-cells
from peripheral blood lymphocytes obtained from patients. Most of CAR T-cell products
are made of autologous T cells. The promise of autologous cell therapy as a personalized
medical intervention is enormous. However, the estimated total cost of autologous CAR
T-cell therapy products produced using current manufacturing methods is astronomical
($150,000–$475,000 per treatment), making it harder to compete with “off-the-shelf” cell
therapies. So, can allogeneic CAR T-cells be used to achieve the same therapeutic goal?
Allogeneic CAR T-cells are often made from T cells donated by healthy donors or umbilical
cord blood, so that CAR T-cells can be made and cryopreserved in advance and become the
off-the-shelf products, ensuring treatment timeline and saving cost.
Donor-derived allogeneic CAR T-cells have several potential advantages over autolo-
gous approaches, such as the immediate availability of frozen batches for patient treatment,
standardization of the CAR T-cell product, timing for multiple cell modifications, redosing
or combination of CAR T-cells to different targets, and cost reduction using an industrial
process. Most importantly, the initial overall treatment response rate of most current au-
tologous CAR T-cell therapies can reach around 90%, with a 5-year sustained remission
rate of 58% (from DLBCL data). However, the ORR of the patients to allogeneic CAR
T-cell therapy was 67%, the rate of PFS at 6 months was 27%, and the OS was 55% [167].
This efficacy discrepancy suggests that there exist challenges in allogeneic CAR T-cells.
The efficacy depends on the persistence of CAR T-cells in the body of patients after infu-
sion. Also, the comparison of autologous with allogeneic CAR T-cells is mainly based on
the expansion of CAR T-cells in vivo and the cell detectability at a certain time after the
CAR T-cell infusion. The persistence of CAR T-cells is critical for later tumor recurrence.
Benjamin et al. reported that allogeneic CAR T-cells were detected in 3 of 21 patients with
R/R B-ALL treated with allogeneic CAR T-cells after 42 days, and only 1 patient after
120 days [167]. In contract, the median survival time of autologous CAR T-cells using the
first marketed model reached 168 days, and a significant number of patients had detectable
autologous CAR T-cells even at 20 months [168]. Therefore, autologous CAR T-cells are
more advantageous from the perspective of expansion level. In addition, allogeneic CAR
T-cell therapy had a 91% probability of CRS, and 14% were grade 3 and 4 adverse events.
In addition, neurotoxicity was also observed in 38% of the patients. Thus, allogeneic
CAR T-cells have higher and more severe of side effects than those of autologous CAR
T-cells [169], suggesting that transplant reactions may also exist. In addition, allogeneic
CAR T-cells cause life-threatening graft-versus-host disease (GVHD) and may be rapidly
cleared by the host immune system [170]. In conclusion, autologous CAR T-cell therapy
has advantages over allogeneic therapy in terms of efficacy, durability, side effects, and
treatment burden. Unfortunately, the leukocytes obtained from the patients at the time of
preparation are mostly after multiple treatments, which may affect the quality of patients’
own T cells. If it is defective, the efficacy of autologous CAR T-cells may be poor. An idea is
that if people could have their own T cells cryopreserved when they are young and healthy,
just in case they need them in the future, as that would be a way to save for a rainy day.
Cancers 2022, 14, 5983 21 of 30

In addition, T cell exhaustion limits the efficacy of CAR T-cell therapy [52]. T cell
dysfunction associated with T cell exhaustion is a major obstacle to its efficacy, especially
in the solid tumors treated with CAR T-cells [41]. Additional suppressive TME, and the
inefficient CAR T-cell trafficking into solid tumors also contribute to the low response rate
of solid tumor cells to CAR T-cells [171].
For these reasons, the efficacy and safety of CAR T-cells in solid tumors can be im-
proved by identifying appropriate tumor-associated (particularly specific) antigens, modify-
ing the structure of CAR to enhance the efficacy, specificity, and survival of CAR T-cells, and
optimizing the targeting of TME in solid tumor (e.g., lung cancer), exploring combination
therapies (i.e., combining with immune checkpoint inhibitors, dual CAR T-cells or trivalent
CAR T-cells), or establishing natural ligand-receptor-based CAR T-cells.
Novel technologies are under development to construct new CAR T-cell products and
improve CAR T-cell therapy efficacy with safety improvement. Atara Biotherapeutics’ 1XX
technology uses one rather than three immunoreceptor tyrosine-based activation motifs
(iTAMs) in creating CAR T-cells. This new technique may help prevent the differentiation
and exhaustion of counterproductive T-cells and enhance the antitumor activity of CAR
T-cells. CAR T-ddBCMA is an autologous anti-BCMA CAR T-cell therapy that uses a
novel synthetic binding domain, called a D-Domain, instead of a typical scFv binder [172].
Similarly, CAR T-ddBCMA developed at Arcellx Inc. is an autologous CAR T-cell therapy
that encodes a novel non-scFv synthetic binding domain-targeting BCMA with a 4-1BB
(CD137, TNFRS9) costimulatory motif and CD3ζ T-cell activation domain. This new
product is computationally designed to be highly stable and reduce immunogenicity.
According to data from a phase 1 trial (NCT04155749), durable responses and 100% ORR
was demonstrated in R/R MM patients with deep and durable responses along with poor
prognostic factors.
T-Charge is a novel CAR T-cell cell therapy platform developed by Novartis, which
makes CAR T-cells expanded primarily in the patients, eliminating the need for prolonged
cell culture in vitro. This could reduce the entire “vein to doorway” timeline by at least
half and result in more potent drugs with a better ability to self-proliferate in the body.
Novartis presented some positive first-in-human data for two products targeting CD19
and BCMA at 2021 ASH annual meeting, including a product candidate called YTB323.
However, this autologous approach has several drawbacks in terms of production time,
cost, manufacturing delay, and dependence on the functional fitness of the patient’s T cells,
often reduced by disease or previous treatment [173].
The research on CAR T-cell in treatment of solid tumors is still in its infancy, and
the beneficial results of the preliminary trials have provided a theoretical basis for their
application in the subsequent clinical treatment of solid tumors. While some of these
techniques are not currently directly used to treat solid tumors, they may be one day. With
the continuous innovation of CAR T design concepts and treatment regimen, CAR T-cell
therapy is expected to become main approaches of solid tumor treatment. It should be
noted that due to the limited space and the materials collected, this review may not be
able to exhaustedly summarize all the potential issues and aspects of each issue, and the
omissions are unpreventable and apologized.

8. Conclusions
CAR T-cell therapy is effective in hematological malignancies. However, more than
half of patients will have a relapse. Of note, CAR T-cell therapy has been even more
disappointing in solid tumors. This may be attributed to the antigenic heterogeneity in
solid tumors, the risk of on-target off-tumor toxicity, T-cell dysfunction associated with
T-cell exhaustion, suppressive TME, and inefficient transport of CAR T-cell trafficking into
solid tumors as the major obstacles to the efficacy of CAR T-cell therapy in solid tumors.
Cancers 2022, 14, 5983 22 of 30

9. Outlooks
Cancer immunotherapy comes in many forms, including targeted antibodies, cancer
vaccines, adoptive cell transfer (ACT), oncolytic viruses (using viruses to infect and destroy
cancer cells), checkpoint inhibitors, cytokines, and adjuvants. ACT includes CAR T-cell
therapy and tumor-infiltrating lymphocyte (TIL) therapy. CAR T-cell therapy does not
always work for every patient and every type of cancer, and some types of cancer are
associated with potentially severe but manageable side effects. Although scientists have not
yet fully grasped the immune system’s cancer-fighting capabilities, immunotherapy has
helped prolong and save the lives of many cancer patients. With the development of modern
science, immunotherapy has the potential to become more precise, more personalized, and
more effective and with less side effects than current cancer treatments.
The development of CAR T-cells especially for the treatment of solid tumors is
progressing. A better more powerful and longer-lived T cells could be engineered, re-
programmed, and developed with the help of a regularly clustered regularly interspaced
short palindromic repeats (CRISPR) tool to accelerate the design of improved T cell ther-
apies and improve treatment of leukemia and solid cancers [174]. The development of
CRISPR-based reprogramming of human immune cells has opened the door to the applica-
tion of reprogrammed cellular therapies to treat cancer.
Telomeres are TTAGGG repeats that are located at chromosome ends, and their length
determines cellular lifespan. Recent studies have shown that intercellular transfer of
telomeres rescues T cell senescence and promotes long-term immune memory [175]. Based
on this finding, scientists may be able to try to lengthen telomeres during T-cell activation,
so that the life span of engineered CAR T-cells can be prolonged.
We look forward to new scientific results that will be soon applied to the clinic for the
benefit of cancer.

Author Contributions: Q.C. and W.M. conceptualized the article and prepared figures; Q.C., L.L.
and W.M. wrote and edited the manuscript. W.M. finalized the figures and manuscript. All authors
have read and agreed to the published version of the manuscript.
Funding: This work was supported by the grant of Huzhou Science and Technology Bureau, Zhejiang
Province, China (2020GZ41).
Acknowledgments: This paper reflects the views of relevant and latest publications and existing
literature to which the authors have been exposed.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

ALL acute lymphoblastic leukemia

AML acute myeloid leukemia
BCMA B-cell maturation antigen
CAR chimeric antigen receptor
CI checkpoint inhibitor
CRS cytokine release syndrome
CLL chronic lymphocytic leukemia
DLBCL diffuse large B-cell lymphoma
EFS Event-free survival
FDA Food and Drug Administration
HL Hodgkin’s lymphoma
HLA human leukocyte antigen
HSC hematopoietic stem cells
ICANS immune effector cell-associated neurotoxicity syndrome
IFN-γ interferon-gamma
IL interleukin
LSC Leukemia stem cells
Cancers 2022, 14, 5983 23 of 30

MAS macrophage activation syndrome

MHC major histocompatibility complex
MM multiple myeloma
NHL B-cell non-Hodgkin lymphoma
NK natural killer
NKT natural killer T
NS neurologic symptoms
NSCLC non-small-cell lung carcinoma
OS objective response
PBMC peripheral blood mononuclear cells
PD progressive disease
PD-1 programmed cell death protein 1
PD-L1 programmed death-ligand 1
PFS progression-free survival
PSMA prostate-specific membrane antigen
R/R relapsed and refractory
TIL tumor infiltrating lymphocyte
TLS tumor lysis syndrome
TMB tumor mutational burden
TME tumor microenvironment

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