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CAR T cell therapy: A new era for cancer treatment (Review)

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DOI: 10.3892/or.2019.7335

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CAR T cell therapy: A new era for cancer treatment (Review)

RIMJHIM MOHANTY, CHITRAN ROY CHOWDHURY, SOLOMON AREGA,
PRAKRITI SEN, POOJA GANGULY and NILADRI GANGULY

Cancer Biology Laboratory, School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT) University,
Bhubaneswar, Odisha 751024, India

Received XXXXX; Accepted XXXXX

DOI: 10.3892/or_xxxxxxxx

Abstract. Cancer has recently been identified as the leading potential targets by pharmaceutical companies and research
cause of mortality worldwide. Several conventional treatments organizations, and trials have been ongoing in this direction.
and cytotoxic immunotherapies have been developed and made Although this therapeutic regimen is currently confined to
available to the market. Considering the complex behavior of treating hematologic cancer, the increasing involvement
tumors and the involvement of numerous genetic and cellular of several auxiliary techniques, such as bispecific CAR,
factors involved in tumorigenesis and metastasis, there is a need Tan‑CAR, inhibitory‑CAR, combined antigens, the clustered
to develop a promising immunotherapy that targets tumors at regularly interspaced short palindromic repeats gene‑editing
both the cellular and genetic levels. Chimeric antigen receptor tool and nanoparticle delivery, may substantially improve its
(CAR) T cell therapy has emerged as a novel therapeutic T overall anticancer effects. CAR therapy has the potential to
cell engineering practice, in which T cells derived from patient offer a rapid and safer treatment regime to treat non‑solid and
blood are engineered in vitro to express artificial receptors solid tumors. The present review presents an insight into the
targeted to a specific tumor antigen. These directly identify advantages and the advances of CAR immunotherapy and
the tumor antigen without the involvement of the major histo- presents the emerging discrepancy of CAR therapy over usual
compatibility complex. The use of this therapy in the last few forms of therapy, such as chemotherapy and radiotherapy.
years has been successful, with a reduction in remission rates
of up to 80% for hematologic cancer, particularly for acute
lymphoblastic leukemia (ALL) and non‑Hodgkin lymphomas, Contents
such as large B cell lymphoma. Recently, anti‑CD19 CAR
therapy, or UCART19, has been shown to be efficacious in 1. Introduction
treating relapsed/refractory hematologic cancer. Several other 2. CAR T cell therapy
cell surface tumor antigens, such as CD20 and CD22, found 3. A rchitectural ideology of T cell engineering and CAR
in the majority of leukemias and lymphomas are considered design
4. Mode of delivery
5. Challenges of CAR T cell therapy
6. Advanced features of CAR T cell therapy
7. Advantages of CAR therapy over other therapies
Correspondence to: Dr Niladri Ganguly, Cancer Biology 8. CAR T cell therapy trials for solid tumors
Laboratory, School of Biotechnology, Kalinga Institute of Industrial
9. Success rates of approved therapies
Technology (KIIT) University, Campus‑11, Patia, Bhubaneswar,
Odisha 751024, India
10. Future prospects
E‑mail: [email protected]; [email protected]

Abbreviations: ALL, acute lymphocytic leukemia, CLL, chronic 1. Introduction

lymphocytic leukemia; CAR, chimeric antigen receptor; TCR, T cell
receptor; mAb, monoclonal antibody; NK, natural killer; FDA, The Chimeric antigen receptors (CARs) are unique receptors that
United States Food and Drug Administration; scFv, single‑chain are designed to target a specific tumor antigen to functionally
variable fragment, CRISPR, clustered regularly interspaced short reprogram T lymphocytes. As T lymphocytes are genetically
palindromic repeats; PAM, protospacer adjacent motif; Treg, engineered to express these artificial receptors to target cancer
regulatory T cell; PD‑1, programmed death 1; PD‑L1, programmed cells, the type of therapy may be termed immunotherapy,
death‑ligand  1; CTLA‑4, cytotoxic T  lymphocyte‑associated
gene therapy or cancer therapy (1). The human defense system
protein 4; ICOS, inducible T‑cell costimulator; CR rate, complete
response rate to the treatment or disappearance of cancer
can efficiently identify self and non‑self molecules, including
bacteria, viruses and abnormal cancer cells. The identification
Key words: chimeric antigen receptor T cell therapy, NK‑CAR of tumor cells is based on their acquired antigenicity and immu-
therapy, CRISPR‑CAR, bispecific CAR, tandem CAR, inhibitory nogenicity via the expression of foreign antigens (2). However,
CAR cancer cells have the potential to subvert the immune system to
their advantage, resulting in inadequate antitumor immunity,
2 MOHANTY et al: CAR T CELL THERAPY IN CANCER (REVIEW)

and tumor survival and progression (3). Immunotherapy is tion to other small functional molecules, enhances induction of
also termed biotherapy as the immune system in the body is the cytolytic function of the engineered T cell. Together, this
naturally capable of detecting pathogens and cancerous cells. coordination of a ‘living drug’ in the immune system fights
In recent years, immunotherapy has emerged as an important against cancer (12). In addition, CAR T cells can remain stable
branch of treatment for similar types of disease; however, its for several years in the body as long‑term memory cells. This
protective mechanism may differ (4). Certain immunotherapies feature allows them to recognize and kill cancer cells encoun-
boost the immune system, whereas others directly target the tered in the circulation in the case of relapse. Another advantage
cancer cells. Each treatment type has its advantages and disad- of CAR T cells is that they specifically target only tumor cells
vantages depending on the disease type (5). Tisagenlecleucel and not auto‑antigens. Therefore, it is safe and nonlethal to host
(Kymriah) is a medication used to treat patients with acute cells (13). Once the synthetic immunoreceptor is expressed on
lymphoblastic leukemia (ALL) up to the age of 25  years. the surface of an engineered T cell, its scFv specifically binds
Similarly, axicabtagene ciloleucel (Yescarta) is approved for to target antigens expressed on a cancer cell. This binding
patients with large B‑cell lymphoma, such as non‑Hodgkin subsequently results in the transduction of an activating signal
lymphoma (NHL), and those with cancer in a refractory and into the genetically edited immune cell. The T cell then elicits
recurrent state or whose cancer is non‑responsive to other treat- its effector anticancer function (14). CAR T cell therapy is
ments (6). With increasing awareness of the immune system, considered to be the first approach to reconfigure T lympho-
a number of innovative immunotherapies are being developed cytes with an antibody‑specific scFv fragment obtained from
using methods which include inducing the immune system to monoclonal antibodies (mAbs) by replacing different parts of
function in an impertinent manner to target malignant cells. the TCRα and β chains. A recent report stated the potential of
Another approach involves the administration of immune CAR γδ T cells in curing mucosal‑derived malignant tumors
components, such as synthetic, modified immune proteins that as a novel strategy for CAR T cell therapy (15). The hybrid
are genetically engineered to target tumor antigens (7). TCR functionally expresses and recognizes the analogous
CAR T cell treatment has achieved success in treating target antigen molecules in a non‑MHC‑restricted manner.
hematopoietic malignancies; however, its effectiveness against Depending on the diverse biomarker selection and structural
solid tumors remains to be determined. In the following complexity, different generations of CAR model have been
sections, the rapid progression in implementing the adoptive developed (16).
transfer of T cells and their mechanism of tumor cell eradica-
tion are discussed. First‑generation CARs. The first‑generation CAR T model
comprises a CD3ζ chain as a key transmitter of signals
2. CAR T cell therapy from endogenous TCRs. Following a successful outcome
in pre‑clinical trials, this type of drug entered into phase I
CAR is an emerging immunotherapy for several malignancies. clinical trials for leukemia, lymphoma and various other types
This therapeutic approach is an experimental form of gene of cancer, including ovarian cancer and neuroblastoma (17).
therapy that redirects T lymphocytes to eradicate cancerous Despite the inadequate antitumor action owing to the lack
cells. The initial step in this therapy is leukapheresis or the of activation, persistent exposure to the tumor environment
isolation of a patient's peripheral blood  (8,9). Apheresis is has resulted in continued therapeutic effects in patients with
widely used to isolate blood from patients and separate it B‑cell lymphoma infused with α‑CD20‑CD3ζ CAR T cells
into its components, which are then genetically altered before and a number of patients with neuroblastoma treated with
re‑injecting them into the patient's body. Currently, apheresis scFv‑CD3ζ CAR T cells  (18). The heavy and light chains
is used by blood banks to collect platelets and other blood constitute structural parts of the B cell receptor or antibodies,
components for the treatment of several diseases, including called scFv, which are fused to the CD3 domain or T cell‑acti-
hematologic and renal disorders. Therefore, it is regarded as a vating ζ chain of the TCR to create non‑MHC‑restricted
safe practice for healthy individuals and patients (10) (Fig. 1). activating receptor molecules. These modified molecules are
capable of enhancing T cell antigen detection and cytotoxicity
3. Architectural ideology of T cell engineering and CAR by specifically targeting tumor cells (19). The original ‘true’
design CAR was designed by integrating an scFv antibody receptor
directly with the CD3ζ domain. This model was subsequently
The uniqueness of chimeric receptors exists in their ability named the ‘T body approach’ and these synthetic signaling
to fuse or split discrete vital functions, such as recognition, receptors are now known as CARs or chimeric immune recep-
co‑stimulation and activation, in different chains of a receptor tors (20).
molecule by imitating the complexity of the native T cell
receptor (TCR) structure (11). T cells do not usually require Second‑generation CARs. The success of first‑generation
costimulation for activation and to initiate proliferation, but CARs in phase 1 clinical trials paved the way for second‑gener-
in the process of establishing CAR T cells, the activation ation CAR T cell therapy. This model of CAR T cells was
and proliferation of T cells require the presence of costimu- established to elicit a more effective anti‑leukemic response
latory molecules, which also assist in CAR T cell cytokine in phase I clinical trials with complete remission rates of up
production. The strategy involves constructing an engineered to 90% in patients with recurrent B‑cell ALL (B‑ALL). Here,
chimeric receptor for T cells based on the integration of scFv the second‑generation anti‑CD19 T cells were integrated into
fragments in the hinge area that separates scFv from the cell a 4‑1BB or CD28 co‑stimulatory domain attached to the CD3
membrane. The exposure of scFv on the cell surface, in addi- domain (21). However, significant concerns remain regarding
 ONCOLOGY REPORTS 00: OR-227153 Mohanty, 0000 3

Figure 1. Adoptive CAR T cell therapy. CAR T cell therapy can be defined as a treatment in which a patient's T cells are genetically modified in the laboratory
to kill cancer cells. The mechanism of adoptive CART‑cell therapy includes the following steps: Collection of patient blood, isolation of T cells from the
peripheral blood sample by the process of leukapheresis, transduction of cells by a vector encoding the CAR gene, expansion of CAR T cells in vitro and
introduction into the patient to fight against cancer. CAR, chimeric antigen receptor.

its efficacy and safety and making it more robust. Anticipating prolonged proliferation, elevated cytokine production and
these concerns, second‑generation CARs aimed at integrating effective function (25). For example, a third‑generation CAR
intracellular signaling domains from different co‑stimulatory consisting of α‑CD19‑CD3ζ‑CD28‑4‑1BB reported complete
molecules, such as CD28, 4‑1BB or CD137, inducible T remission rates by infiltrating and lysing cancer tissue in patients
cell costimulator (ICOS) or CD278, OX40 or CD134 fused with chronic lymphocyte leukemia (26). In addition, certain
to the cytoplasmic tail of the CAR, thus amplifying the CAR T cells function as memory cells, thus preventing tumor
signal (22). First‑generation CARs contain a CD3ζ chain as relapse. Despite the significant curative effect, the irrepress-
a key transmitter of signals from endogenous TCRs, whereas ible activity of CARs and their increased antitumor efficacy
second‑generation CARs contain a CD3ζ chain and a single is associated with life‑threatening and unfavorable outcomes,
costimulatory molecule, which is why the receptor is known with increased secretion of pro‑inflammatory cytokines,
as a second‑generation CAR. For example, anti‑CD19 CARs multi‑organ dysfunction, pulmonary failure and death (27).
consisting of 4‑1BB or CD28 signaling domains produced
notable complete response (CR) rates in patients with relapsed Fourth‑generation CARs. All earlier CARs were based on
and refractory B‑cell malignancies  (23). CR represents a precise stratagem and helped in mediating the T cell anti-
complete response or disappearance of all signs of cancer cancer response. However, these suffered from limitations,
in response to treatment [The United States Food and Drug including a lack of antitumor activity against solid tumors
Administration (FDA) 2007]. Consequently, CD28‑based owing to large phenotypic heterogeneity and deterioration
CARs have a rapid proliferative reaction, thus enhancing T attributed to antigen‑negative cancer cells. These shortcom-
effector cell functions, whereas 4‑1BB‑based CARs lead to ings led to the development of a novel CAR stratagem (28).
improved T cell accumulation (24). The fourth‑generation CAR was introduced to establish the
tumor background via the inducible expression of transgenic
Third‑generation CARs. To extend the antitumor efficacy, immune modifiers, such as interleukin (IL)‑12, which activate
third‑generation CARs comprise two signaling domains innate immune cells and enhance T cell activation to reduce
and the CD3 ζ chain, such as CD3 ζ‑CD28‑OX40 and antigen‑negative cancer cells in the marked lesion  (29,30)
CD3ζ‑CD28‑4‑1BB, to achieve improved activation signal, (Fig. 2).
4 MOHANTY et al: CAR T CELL THERAPY IN CANCER (REVIEW)

Figure 2. Architectural evolution of CAR T cell design. The CAR is constructed from the extracellular antigen‑binding domain derived from monoclonal
antibody (scFv) and intracellular signaling domains, linked by a hinge and a transmembrane domain. First‑generation CARs contain the CD3ζ chain of the T
cell receptor complex, whereas second‑generation receptors contain one costimulatory molecule (CD28) and third‑generation CARs contain two costimulatory
molecules, respectively, such as CD28 and OX40. CARs are usually introduced into primary T cells using a vector. CD3ζ usually maintains the cytotoxic
effector function of CAR‑T cells. CD28 (blue) is important for T cell proliferation and cytokine secretion. 4‑1BB (purple) is another co‑stimulatory domain
that promotes T cell survival and in vivo T cell persistence. Each CAR generation increases in complexity relating to the addition of costimulatory molecules
or the induced promoter for cytokine IL‑12 in fourth‑generation CAR, CAR, chimeric antigen receptor; scFv, single‑chain variable fragment; IL, interleukin;
TM, transmembrane.

4. Mode of delivery sion into non‑dividing and dividing cells both in vitro and
in vivo (33).
Gene therapy involves the delivery of DNA into cells and this
can be accomplished using a number of methods summarized Non‑viral delivery methods
below. The most traditional method utilizes recombinant Transposon transfection. The mechanism of transposon trans-
viruses (also known as viral vectors), biological nanoparticles fection is different from that of viral transfection. Delivery
and non‑viral methods based on the direct delivery of naked via transposons is a non‑viral process that uses transposon
DNA. DNA and a transposase enzyme for stable gene transfer. Two
important vectors, namely piggyBac (PB) and sleeping beauty
Viral vector. Viral vectors, such as γ‑retrovirus, lentivirus (SB), are frequently used (34). Transposons shift from one gene
and adenovirus vectors are generally used in gene therapy. position to another position via a cut‑and‑paste mechanism. The
Retrovirus transduction is one of the commonly used mechanism of the transposon system using SB and PB involves
delivery methods in gene therapy. The method involves a four steps: i) the transposase enzyme helps in recognition and
reverse transcriptase that promotes the stable integration of binding to the transposon; ii) a synaptic complex is produced by
artificial genes into the host genome (31). To create a vector, the coupling and binding of the repeat elements at both ends of
γ‑retroviral coding sequences are substituted by a gene of the transposon; iii) the transposon excises the genetic element
interest. The retroviral vectors possess an innate capability to be transposed, and iv) the excised element is reintegrated
to disturb the genomic section and results in neoplastic trans- into the target location (35). The transposase for both vectors
formation. Thus, γ‑retroviral vectors have been used in gene (SB and PB) consists of the DNA‑binding domain, transposable
therapy applications (32). Lentiviral vectors are retroviruses catalytic domain and nuclear localization signal. The SB vector
derived from human immunodeficiency virus‑1, which serve is a safer substitute for viral vectors owing to its innately low
as a major tool for the delivery of transgenes into mamma- enhancer activity, non‑pathogenic source and minimum epigen-
lian cells. The advantage of using lentivirus vectors is their etic alterations at the incorporation site (36). By contrast, the
efficient transduction and their stable integration and expres- PB vector has been shown to provide a large load capacity and
 ONCOLOGY REPORTS 00: OR-227153 Mohanty, 0000 5

additional competent transposition action in in vivo studies. PB aplasia, tumor lysis syndrome and anaphylaxis (49). The prolif-
vector‑mediated CAR T therapy is considered safer than that eration of CAR T cells produces cytokines in the body which
based on the SB vector. Therefore, use of the PB vector results in kill cancer cells. The symptoms of CRS‑associated toxicity
greater CAR production, as evident from the production of CAR range from mild symptoms of fatigue, nausea, headache, fever
targeting CD19 using a PB vector (37). and chills to serious symptoms including a lowering of blood
pressure, tachycardia and capillary leakage. Another side
Electroporation. Electroporation has evolved as a useful effect is the presence of CAR T cells targeting antigens on the
technique for modifying genes of diverse cell types. The surface of B cells or T cells that not only target cancer cells but
target cells are exposed to electric fields to temporarily disrupt also normal cells (50), resulting in B cell aplasia. Therefore, a
their cell membranes. This allows the charged molecules to thorough investigation is essential to measure the properties
enter the cells. Square‑wave and pulse‑based systems are of B cell aplasia. Similarly, tumor lysis syndrome can result in
new electroporation devices (38). The Lonza Nucleofector toxicity by the collapse of dead cells generally in the begin-
II Electroporator performs effective genetic alteration of T ning of cancer treatment. It could also cause organ damage and
cells using certain electric parameters and electroporation be life‑threatening to the patient (51).
buffers (39). The electroporation of human T cells has been
reported to be associated with ~40‑60% of gene expression 6. Advanced features of CAR T cell therapy
and 80% of cell viability (40). One of the limitations of elec-
troporation is the low transfection efficiency and redundant Although cancer cells have multiple lineages and heteroge-
cell damage (41). neity, they possess common target antigens, such as CD19,
CD20, CD22 and numerous others that allow CAR T cells to
Nanoparticles. The most critical step in CAR T cell therapy recognize tumor cells irrespective of cell lineage. Therefore,
is T cell activation, for which cells need to be incubated with recent advances in this technique include more precise target
the viral vector including a CAR gene. However, stable inser- antigens expressed by tumor cells (13,52). This review places
tional mutagenesis can be dangerous as its possible effects additional emphasis on new studies of CAR T cell therapy that
on humans remain to be fully elucidated (42). Retroviral or distinguish diverse CAR T cells which can enhance tumor
lentiviral vectors offer permanent gene integration into host cell death. Certain models are already being implemented
cells, with the potential integration in close proximity with a and others are in the clinical investigation status, including
proto‑oncogene which can result in an oncogene. The various NK‑CAR and clustered regularly interspaced short palin-
disadvantages associated with these transfer methods suggest dromic repeats (CRISPR)‑CAR therapy (Fig. 3Α‑Ε).
that a safer alternative is needed (43).
The study by Smith et al elucidated the use of nanotech- Bispecific CARs. A bispecific receptor is one that contains two
nology to resolve the tumor‑targeting problem and make the distinct antigen recognition domains attached and placed with
therapy cost‑effective. They reported that polymeric nano- two distinct intracellular signaling domains that are expressed
carriers can efficiently deliver leukemia‑specific CAR genes as two different CARs on a single cell surface. At present,
targeted to specific ligands on the host T cells in situ (44). bispecific CAR CD19/CD20 has been introduced as a novel
A number of nanoparticles used for gene delivery have been synthetic molecule that can recognize and bind to more than one
investigated preclinically and reported. For example, magnetic targeted tumor antigen on the cancer cell surface. Therefore, it
nanoparticles, such as Fe3O4, integrate into the cell‑penetrating can create a synergistic cascade of effector molecules when it
peptide complexes. These oligonucleotides maintain stable encounters two tumor antigens (53). Additionally, the bispecific
cell transfection and plasmid transfection in addition to gene CAR conserves the cytolytic capacity of T cells, i.e., if one of
silencing and splice correction  (45). A recently published the objective molecules is not accessible to CAR T cells due
study by Smith et al at the Fred Hutchinson Cancer Research to a cellular hindrance such as mutation of a target antigen or
Center, Seattle, confirmed that polymeric nanoparticles loss of the target antigen commonly found in malignant cells,
carrying DNA effectively introduced CAR genes into T cell a bispecific CAR can counterbalance the tumor evasion (54).
nuclei and recognized leukemia cells distinctively (46). The Investigated therapeutics include CD3 of T cells and tumor
use of nanoparticles to replace viral vectors for gene delivery antigens, such as CD19, on malignant cells. The curative
has proved advantageous, as evident from the restriction of ability is evident from blinatumomab, an approved bispecific T
unspecific pro‑ or anti‑inflammatory effects or pro‑ or antipro- cell‑engager against relapsed/refractory B‑ALL (55). Positive
liferation effects (47). results have been reported in patients <15 years of age with
relapsed/refractory ALL, with a heightened response in 26/36
5. Challenges of CAR T cell therapy (72%) patients in 9 months. Blinatumomab was discovered
while studying a patient who was negative for minimal residual
Although CAR therapy has emerged as a promising anticancer disease (MRD) and who was found to be MRD‑positive
approach, it is not free from challenges that require optimi- following consolidated chemotherapy (56). Numerous other
zation. For example, the enhanced persistence and improved bispecific CARs have been investigated preclinically by
cytotoxic profile of CAR T cell therapy are active areas of several research organizations, including CD20/CD19 and
research requiring long‑term follow‑up in clinical trials (48). CD20/CD3 (57,58). Targeting T cells with bispecific CARs
In addition, a number of serious side effects have been known was shown to eliminate transplanted pediatric ALL in a study,
to be frequently associated with CAR T cell therapy, including whereas T cells targeted by CD20 CAR did not control the
neurological toxicity, cytokine release syndrome (CRS), B cell disease (59).
6 MOHANTY et al: CAR T CELL THERAPY IN CANCER (REVIEW)

Figure 3. Advanced models of CAR T cells. (A) Tandem CAR: A single CAR structure targets two tumor antigens with a distinct antigen recognition domain
(scFv) linked consecutively with a single intracellular domain. (B) Bispecific CAR: Two CARs are expressed simultaneously targeting T cells, with two
distinct antigen‑recognition domains targeting two tumor antigens. (C) Physiological CAR: CAR structure comprising a ligand and receptor molecule, rather
than scFv, to recognize the paired molecule on the tumor cell. (D) Universal CAR: Extracellular domain expresses the avidin‑biotin labeled mAb, instead of
scFv, which can recognize almost all target antigens specific to mAb. (E) I‑CAR: CTLA‑4 and PD‑1 are two inhibitor receptors that can reversibly regulate
the signaling of TCR and can enhance the function of chimeric receptors. CTLA‑4 or PD‑1 are intracellular domains in I‑CARs, which trigger inhibitory
signals on T cells, such as a reduction of target cell lytic action and cytokine production. The most important factor of I‑CAR‑T is its ability to distinguish
normal and abnormal antigens on target cancer cells. CAR, chimeric antigen receptor; scFv, single‑chain variable fragment; mAb, monoclonal antibody; PD‑1,
programmed death 1; CTLA‑4, cytotoxic T lymphocyte‑associated protein 4; I‑CAR, inhibitory CAR; TM, transmembrane.

Tandem CARs (Tan‑CARs). Conventional CARs cannot meet Recently, a trivalent CAR T cell was designed by a group of
higher expectations under certain circumstances, such as the scientists by simultaneously co‑targeting multiple antigens,
downregulation or alteration of targeted antigens that can such as HER2, IL‑13 receptor α2 and ephrin A2, to overcome
occur in cancerous cells. These conditions subsequently lead interpatient changeability with a propensity to target almost
to antigenic loss or escape variations (60). To overcome this 100% of tumor cells (64).
shortcoming, scientists are attempting to develop advanced
technology, in which two particular antigen recognition sites Inhibitory CARs (I‑CARs). It has been reported that certain
are joined by a linker, placed in tandem on a single intracel- novel immunoinhibitory receptors are involved in T cell activa-
lular domain and expressed as a single CAR on a cell surface, tion and the attenuation or termination of T cell responses, such
termed a Tan‑CAR. This model enables the synchronized as programmed death‑1 (PD‑1), programmed death‑ligand 1
targeting of both antigens on a single cancer cell or tumor (PD‑L1) and cytotoxic T‑lymphocyte‑associated antigen  4
microenvironment. In this manner, it enhances the activation (CTLA‑4). These pathways are regarded as cancer immuno-
and stimulation of T cells by increasing their avidity and therapy breakthroughs (65). Recently, two immunologists won
expanding their therapeutic properties (61). Tan‑CARs have the Novel Prize in Physiology/Medicine, Dr James Allison
several significant curative implications as they are as potent and Dr Tasuku Honjo, for their profound discovery in the field
as standard disease models with single antigen‑specific CARs. of cancer immunotherapy. Dr Allison's work focused on the
Additionally, they are more efficient and less noxious in a T cell surface protein CTLA‑4; he found that the inhibition
higher disease load setting (62). This may be attributed to opti- of immune cells and antibodies against CTL‑4 eliminated
mized cytokine production and limited cell killing, as evident malignant growth and prevented new tumor formation (66).
from preclinical studies on Tan‑CAR T cells in a mouse tumor This finding was tested on 14 patients with metastatic mela-
model, which confirmed its possibility for remedial implica- noma, with relapse observed in three patients. In 2011, the
tion in human disease consisting of B‑cell antigen CD19 FDA approved an anti‑CTLA‑4 (ipilimumab) antibody as a
and human epidermal growth factor receptor 2 (HER2) (63). treatment for high‑grade melanoma (67).
 ONCOLOGY REPORTS 00: OR-227153 Mohanty, 0000 7

Similarly, Dr Honjo's team investigated a novel T cell Experimental trials may have been initiated but the outcomes
protein, PD‑1, which was discovered in 1992  (68). They have not yet been published.
observed that PD‑L1 presenting on healthy cells and malignant
cells bind to PD‑1. They reported another similar molecule, Universal CARs (uCARs). Although scFv is specifically directed
PD‑L2, that also binds to PD‑1. Based on these findings, they against tumor‑associated antigens, the recognition specificity
published a report that malignant cells produced PD‑L1 and potential of CAR T cells is inadequate. Therefore, uCARs
blocked PD‑L1 using a counteracting antigen to inhibit tumor were developed to overcome this limitation. To construct
growth  (69). The first clinical trials to target malignancy a universal CAR, biotin or anti‑fluorescein isothiocyanate
were launched in 2006, which indicated significant viability (FITC) scFv is used as a targeting region, which is fused to
in a number of patients in 2012. The FDA approved the a transmembrane domain with one or two endodomains (80).
main PD‑1 checkpoint inhibitors, nivolumab and pembroli- The uCAR‑expressing T cells can efficiently recognize and
zumab (70,71), for treating melanoma in 2014 (72). However, remove cancer cells through the binding of FITC‑labeled or
a single treatment may not be sufficient, which is the reason biotinylated antigen‑specific mAbs, which, in turn, activate
that a consolidated treatment targeting CTLA‑4 and PD‑1 is the T cells and stimulate their proliferation and production of
currently being investigated. Both Dr Allison and Dr Honjo cytokines (81). The mSA2 CAR T cell is a uCAR, which can
encouraged the merging of various strategies to generate more be fused to a biotinylated tumor‑specific antibody to specifi-
advanced forms of the drug for the immune system to inhibit cally target various types of tumor. As with interferon γ, it is
tumor cells more effectively (73). well equipped for interceding malignant cell lysis and cyto-
Certain tumor cells contain a high level of PD‑L1, which kine production (82). Human clinical trials involving uCAR T
assists in evading immune attack. It has already been inves- cells are ongoing. Two children with relapsed and refractory
tigated that PD‑1‑ or CTLA‑4‑based I‑CARs can efficiently B‑ALL achieved molecular and cytogenic remission following
control the cytotoxicity, secretion of cytokines, and prolif- uCAR T therapy. Clinical trials have been initiated on uCAR
eration and cytotoxicity stimulated by endogenous TCR or T cell therapy specific to CD19‑positive cells (NCT03166878
an activating chimeric receptor (74). I‑CARs are designed to and NCT03229876). However, detailed information and
control the actions of CAR T cells through inhibitory recep- conclusions are not yet available (83).
tors. This advanced feature unites the action of two chimeric
receptors; one of these generates a dominant‑negative signal Natural killer (NK)‑CARs. NK cells are a type of cytotoxic
that restricts the responses of activated CAR T cells by T cell that are essential for natural immunity. The function of
the activating receptor. I‑CARs can inhibit the activator NK cells is similar to that of cytotoxic T cells in the adaptive
CAR response to antigens expressed only by normal cells, immune response of vertebrates. Immune cells generally iden-
thus differentiating between cancer and normal cells  (75). tify MHC complexes expressed on infectious cell surfaces to
Therefore, using genetic engineering to inhibit T cell inhibi- elicit cytokine production, thus generating an immune response,
tion physiology and regulate T cell response can be harnessed culminating in the apoptosis or death of infectious cells (84).
in an antigen‑selective manner. This has been experimentally NK cells are the only immune cells that identify infected cells
confirmed preclinically in a mouse model by designing an in the absence of MHC and antibodies, thus eliciting a rapid
I‑CAR using surface antigen recognition domains CTLA‑4 immune response. These are known as ‘natural killers’ as they
and PD‑1. In mice lacking CTLA‑4 receptor, substantial T cell do not require activation to destroy cells that are devoid of ‘self’
activation and proliferation were observed, ultimately leading MHC class I molecule markers (85), making them important
to rigorous systemic autoimmune disease  (76). Similarly, for destructive cells with missing MHC I markers.
PD‑1 is another the inhibitory receptor, which was found to be Cancer cells that do not cause any inflammation are treated
particularly expressed by activated T cells causing glomerulo- as self by the immune system and do not stimulate a T cell
nephritis and arthritis in C57BL/6 mice and certain non‑obese response. NK cells produce a number of cytokines, including
diabetic mice affected by insulitis (77). tumor necrosis factor α, interferon γ and IL‑10, which act as
immune suppressors (86). The activation of NK cells leads to the
Physiological CARs. Initially, several CAR constructions gradual formation of cytolytic effectors cells, such as dendritic
contained scFv of murine origin. This is associated with the cells, macrophages and neutrophils, which consequently facili-
risk of an immune response to the modified cells, and the tate antigen‑specific T and B cell responses. NK cell‑mediated
resulting anaphylaxis of CAR T cell transfer cannot be avoided. tumor cell lysis involves several receptors, including NKp44,
These disadvantages limit the persistence of the infused cells. NKp46, NKG2D, NKp30 and DNAM. Malignant cells usually
Therefore, besides conventional CAR, a physiological CAR express NKG2D in addition to ULBP and MICA (87,88). The
has been developed, also known as a receptor‑ligand CAR, clinical effectiveness of CAR T cells has been shown for ALL;
which can recognize and bind to tumor antigens, such as however, this therapeutic approach has not been confirmed
HER3 and HER4 (78). The physiological CAR consists of an for acute myeloid leukemia (AML), suggesting the need for
antigen receptor and a CD3ζ intracellular signaling domain other therapeutic options (89). Hypothetically, automotive NK
with or without a transmembrane region, which can also be cells have an additional favorable toxic effect in comparison
engineered into immune cells to target the ligands expressed to CAR T cells, particularly for avoiding unfavorable effects
on tumor cells. This approach increases the capability of T such as CRS. Additionally, in contrast to T cells, donor NK
lymphocytes to distinguish tumor‑related targets and elimi- cells do not target non‑hematopoietic cells, indicating that
nate cancer cells (79). This physiological CAR is an emerging NK cell‑mediated antitumor activity may be activated in the
field of CAR T cell therapy with limited published reports. absence of graft‑vs.‑host disease (90) (Fig. 4).
8 MOHANTY et al: CAR T CELL THERAPY IN CANCER (REVIEW)

Figure 4. T cells and NK cells in the CAR platform. NK cells are fundamental components of the innate immune system and serve an essential role in host
immunity against cancer. Currently, CAR‑engineered NK cells are targeted to NKGD2, CD16 antigens; upon encounter with a tumor antigen on the cancer
cell, the NK‑CAR secretes perforins and granzymes, which directly mediate the cytotoxicity of the targeted tumor cell. CAR T cells secrete cytokines, such
as interferons and TNF‑a, which also mediate cell cytotoxicity and eventually cancer cell death. CAR, chimeric antigen receptor; NK, natural killer; NKGD2,
natural killer group 2 member D.

CRISPR CARs. CRISPR is a gene modifying tool that uses a signaling, thereby initiating successful internalization and
guide gRNA to modify a DNA sequence. As this technology stimulation of the CAR for single and repeated exposure to a
is an integration‑free gene incorporation system, it offers a tumor antigen. It also delays effector T cell differentiation and
foolproof and competent gene knock‑in process. Following the exhaustion (97). Multiplex genome editing is another attrac-
significant progress associated with CRISPR technology, it has tive application of the CRISPR/Cas9 tool. Proficient genomic
the potential to emerge as a promising immunotherapy (91). disturbance of multiple gene loci to create a universal donor
The CRISPR system can be directly applied to mammalian cell and a potent effector T lymphocyte targeted to different
cells via transfection using a plasmid that contains both inhibitory pathways, such as PD‑1 and CTLA4, is estab-
nuclease and sgRNA. Cas9 encodes a large molecule with a lished by incorporating several gRNAs into a CAR vector.
multifunctional DNA endonuclease and is known to excise Furthermore, two‑fold knockout of the TCR gene and HLA
dsDNA from 3‑bp upstream of the protospacer adjacent class I can effectively permit the generation of an allogeneic
motif (PAM) (92). Once the nuclease binds to its gRNA, the uCAR T cell, in addition to CAR T cells that are universally
compound scans for an integral target DNA sequence (93). Fas‑resistant via three‑fold gene disruption (98).
The PAM sequence has a significant role in recognizing self
and non‑self sequences. The local PAM sequence is usually 7. Advantages of CAR therapy over other therapies
used as a ‘spy’ on the nuclease sequence 5‑NGG‑3, in which N
is any of the four deoxyribonucleic acid bases (94). The most notable advantage of CAR T cell therapy over other
Recently published reports suggest that CRISPR tech- cancer therapies is the abrupt time intervention and single
nology can deliver the CAR gene to the TRAC locus of T cells. infusion of CAR T cells. Additionally, 2‑3 weeks of proper
In this regard, CRISPR‑edited universal‑CAR T cell therapy care and observation is sufficient for the patient. CAR T cell
has been used in humans (NCT03166878 and NCT03229876). therapy is regarded as a ‘drug of the present day’ and its
This technology is rapidly developing with the potential for efficacy may persist for decades as the cells can survive in
gene correction (82). It is reported that anti‑CD19 CAR to the host body in the long term, with a constant ability to find
the TCR α constant locus (TRAC locus) not only results in and destroy cancer cells during relapse (2,3). Currently, CAR
increased T cell potency but also in the consistent expression T cell therapy is licensed for use in patients for whom trans-
of CAR in peripheral blood T cells (95). CRISPR‑modified plantation has not been curative and who relapse following
cells have been shown to perform well in generally constructed transplant. CAR T cell therapy is expected to be a substitute
CAR T cells in a mouse model of ALL (96). It has also been for different types of transplant (99). Clinical trials on blood
demonstrated that targeting the TRAC locus turns on CAR cancer have shown that, even in patients with a refractory
 ONCOLOGY REPORTS 00: OR-227153 Mohanty, 0000 9

Table I. List of chimeric antigen receptor therapy clinical trials.

Target antigen Type of cancer Clinical trial ID

CD19 BALL NCT01044069

CD19 B‑CLL NCT00466531
CD19 Leukemia NCT01416974
CD19 Lymphoma NCT00586391
CD19 B‑NHL/CLL NCT00608270
CD20 Mantel cell leukemia/B‑NHL NCT00621452
CD22 Non‑Hodgkins Lymphoma NCT02315612
CD19/CD20 B‑Non Hodgkins Lymphoma NCT00621452
CD19/CD22 B‑cell malignancy NCT03185494
CD133 Hepatocellular carcinoma NCT02541370
CD171 Neuroblastoma NCT02311621
PMSA Prostate cancer NCT001140373
CEA Breast cancer NCT00673829
CEA Colorectal cancer NCT00673827
CEA Lungs cancer NCT00673322
HER‑2 Lungs cancer NCT00889954
HER‑2 Osteosarcoma NCT00902044
HER‑2 Glioblastoma NCT01109095
CD30 Lymphoma NCT02274584
FAP Malignant pleural mesothelioma NCT01722149
NKGD2 Leukemia NCT02203825
GD2 Neuroblastoma, osteosarcoma NCT03356795
EGFRVIII Glioblastoma NCT02309373
Mesothelin Pancreatic cancer NCT02706782
CD38/CD123 B‑Cell Malignancies NCT03125577
ROR1 Chronic lymphocytic leukemia NCT02194374
MUC16 Ovarian carcinoma NCT02498912
GPC3 Lung squamous cell carcinoma NCT03198546
VEGFRII Renal cancer NCT01218867

PMSA, prostate‑specific membrane antigen; ROR1, receptor tyrosine kinase‑like orphan receptor 1; NKGD2, natural killer group 2 member D,
expressed in natural killer cells; GD2, disganglioside molecule expressed on tumors of neuroectodermal origin; EGFRVIII, epidermal growth
factor receptor variant III; Mesothelin, differentiation antigen expressed on mesothelial cells and overexpressed in numerous human tumors;
MUC16, also known as CA125, is a biomarker for ovarian cancer; GPC3, glypican 3, is a cell surface protein overexpressed in numerous solid
tumors; VEGFRII, vascular endothelial growth factor receptor II.

condition in which cancer reverted following several trans- CARs, one representing a CD28‑CD3d signaling domain
plants, CAR T cell therapy was successful in completely (CAR) and the other missing an intracellular signaling domain
eradicating the disease (100). Additionally, with CAR T cells, (dCAR). The adoptive transfer of allospecific regulatory T
patients can live life without the risk of relapse and benefit cells (Tregs) provides a better safeguard from graft rejection
from a sanatory treatment, such as stem cell transplantation. compared with that of polyclonal Tregs (104). CAR comprising
Therefore, CAR T cell therapy can be referred to as a ‘living the ICOS signaling domain liaises with the effective anti-
drug’ (101). tumor effect on epidermal growth factor receptor variant
III (EGFRvIII)‑expressing glioma  (105). The preclinical
8. CAR T cell therapy trials for solid tumors evaluation of CAR T cell therapy targeting the tumor antigen
5T4 in ovarian cancer has been associated with a successful
Several clinical trials are currently examining the use of CAR outcome  (106). An unexpected evolutionary finding was
T cell therapy against solid tumors and other diseases. Reports reported during the investigation of CAR targeting autoimmune
suggest that mesothelin‑specific CAR mRNA‑engineered diseases; therapy lacking autoimmune diseases that purposely
T cells can induce antitumor activity in solid malignan- target only the disease‑causing cells, chimeric autoantibody
cies (102,103). Furthermore, CAR technology has been used receptor (CAAR), contain pemphigus vulgaris autoantigen,
in organ transplantation using two novel HLA‑A2‑specific desmoglein (Dsg3), combined to CD137‑CD3d signaling
10 MOHANTY et al: CAR T CELL THERAPY IN CANCER (REVIEW)

domains. Dsg3 CAAR‑T cells exhibit accurate toxicity against with its safety, cost‑effectiveness and quality require thor-
anti‑Dsg3‑expressing cells (107). Certain solid tumor CAR ough investigation in order to implement this therapy for all
targets are undergoing research with varied genetic products cancer types. Another approach is to integrate CAR T cells
arising from gene mutations (EGFRvIII)  (108) or modi- with different types of immunotherapy to enhance its effec-
fied glycosylation patterns (MUC1) (109), and cancer‑testis tiveness. For example, CARs may be combined with certain
antigen‑derived peptides (MAGE), CAR specifically targets checkpoint inhibitors, which limit tumor defense mechanisms
certain overexpressed antigens in breast cancer, lung cancer against T cells. It is expected that future CAR T cell therapy
and pancreatic cancer, such as carcinoembryonic antigen (110) regimens will target several diverse molecules for a particular
GD2, prostate‑specific membrane antigen, HER2/ERBB2, type of tumor, such that CAR T cells can efficiently recognize
MUC16 (111) and mesothelin or tumor‑affiliated stoma (fibro- cancerous cells even if these undergo mutations in their target
blast activation protein and vascular endothelial growth factor molecules.
receptor) (112). A number of CAR T therapies are already available to
the market, but these are expensive, for example, $475,000
9. Success rates of approved therapies (€400,000) for Yescarta and $373,000 (€316,000) for
Kymriah. According to experts, when hospitalization
As per the 2018 records on the total clinical trials conducted expenses and the costs of other drugs required for the
in the immuno‑oncology domain, 220 trials involving CAR treatment are also considered, the cost increases to almost
T therapy were performed to identify specific targets. $1,500,000 per cancer patient. Therefore, a possible solu-
Successfully developed CAR T cell drugs that are available tion is to reduce the cost of allogeneic CAR T treatment by
to the market include CTL019 (Kymriah) (113), KTE‑C19 supplying T lymphocytes from a healthy individual that can
(Yescarta) (114) and JCAR015 (115). These have been be readily utilized when a patient requires it, rather than
developed by companies known to be antecedents of CAR genetically modifying each patient's T cells individually.
T cell therapy development, such as Novartis in associa- Considerable scientific challenges also exist with regard to
tion with the University of Pennsylvania, Kite Pharma with immunology, in addition to manufacturing, transportation
National Cancer Institute, and Juno Therapeutics with and banking solutions to enhance the extensive treatment of
Sloan Kettering, respectively, and are used to treat ALL, patients. Therefore, scientists are investigating measures to
NHL and ALL. These CAR T therapies represent a defining overcome clinical challenges in terms of regulations. CAR
moment in 2017 in the field of oncology. The first two thera- T cells are available in several scientific frameworks, which
pies specific to CD19 and approved by the FDA included may vary widely in different countries. These combined
Kymriah (tisagenlecleucel‑T) and Yescarta (axicabtagene- challenges and technology require standardization; however,
ciloleucel) by Novartis and Kite Pharma/Gilead Sciences, CAR T cells offer patients hope of advanced treatment. As
respectively (116). the first therapy is already available in the market, there is
The global ELIANA trial reported a high success rate, potential for a specific and improved alternative becoming
with a 3‑month complete remission rate of 83% with tisagenle- available in upcoming decades.
cleucel, and a 6‑month survival rate of 89% (117). Another trial,
ZUMA‑1, reported an equivalent results with an 82% overall Acknowledgements
response rate and 54% of complete remission rate following
a single infusion of the therapeutic regime in 8 months. This We acknowledge the Science and Engineering Research Board
indicates that the remission rate was more pronounced in pedi- for the generous funding.
atric B‑ALL than that in adult relapsed/refractory DLBCL;
however, reactivity was high in both diseases (118). Funding
Furthermore, multiple clinical investigations have been
implemented by Cellectis following UCART19. Cellectis is Funding from the Science and Engineering Board, Department
currently leading with two successful FDA Investigational New of Science and Technology, Government of India is duly
Drug (IND)‑approved allogeneic CAR T approaches (119): acknowledged. ECR/2016/000584.
UCART123 for patients with blastic plasmacytoid dendritic
cell neoplasm and AML  (120) and UCART22 IND for Availability of data and materials
patients with B‑ALL (121). Two other ongoing clinical trials
are UCARTCS1 for suppressing CS1‑expressing hematologic Not applicable.
malignancies (122) and UCART38 for CD38‑expressing hema-
tologic malignancies. UCART38 is specifically developed to Authors' contributions
target T cell ALL, multiple myeloma, mantle cell lymphoma
and NHL (123,124). A list of CAR therapy clinical trials are RM and NG conceptualized and co‑wrote the manuscript.
listed in Table I. CRC, SA, PS and PG contributed to the literature review,
organization and writing of various sections of the manuscript.
10. Future prospects NG is the PI and grant holder.

Although immunotherapy has achieved clinical success in Ethical approval and consent to participate
treating blood cancer, the same success rates have not been
observed for solid tumors. The various challenges associated Not applicable.
 ONCOLOGY REPORTS 00: OR-227153 Mohanty, 0000 11

Patient consent for publication 22. Till BG, Jensen MC, Wang J, Qian X, Gopal AK, Maloney DG,
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