Published OnlineFirst July 16, 2018; DOI: 10.1158/2159-8290.

CD-18-0297

REVIEW

CARs versus BiTEs: A Comparison

between T Cell–Redirection Strategies
for Cancer Treatment
Clare Y. Slaney1,2, Pin Wang3, Phillip K. Darcy1,2, and Michael H. Kershaw1,2

ABSTRACT The redirection of T cells against tumors holds much promise for the treatment of
cancer. Two main approaches for T-cell redirection involve their genetic modifica-
tion with chimeric antigen receptors (CAR), or the use of recombinant proteins designated bispecific
T-cell engagers (BiTE). These approaches have demonstrated dramatic effects in patients with hemato-
logic cancers, although limited effect against solid cancers. Here, we review and compare the successes
and challenges of these two types of immunotherapies, with special focus on their mechanisms, and
discuss strategies to improve their efficacy against cancer.

Significance: CAR and BiTE cancer therapies have generated much excitement, but although the thera-
pies are potentially competitive, information directly comparing the two is difficult to obtain. Here, we
present the fundamentals of each approach and compare the range and level of functions they can elicit
from T cells, and their efficacy against cancers. Cancer Discov; 8(8); 924–34. ©2018 AACR.

INTRODUCTION ously, BiTEs mediate T-cell responses and killing of tumor

cells. Importantly, the T-cell/target cell adherence facilitated
Immunotherapy that utilizes the body’s immune system
by BiTE is independent of MHC haplotype (1).
against tumors is a promising field of cancer research. In recent
A variety of formats for bispecific antibodies have been
years, exciting technologies and platforms have been developed
developed in addition to BiTEs, including bispecific IgG, dia-
for the intricate design and production of anticancer immune
bodies, and conjugates, which have been the subject of some
reagents using antibodies in the form of single-chain variable
comprehensive recent reviews (2, 3). BiTEs have the advan-
fragments (scFv; Fig. 1). The scFv technology has been adopted
tages of relatively simple recombinant production and puri-
and used in some promising cancer immunotherapies, includ-
fication, and a molecular weight enabling tissue penetration.
ing bispecific T-cell engager (BiTE) and chimeric antigen
BiTEs have also advanced through the regulatory framework,
receptor (CAR). Both strategies use scFv to direct cytotoxic T
gaining FDA approval for certain indications, and will be the
lymphocytes (CTL) to specific surface antigens on cancer cells
focus of this review.
to facilitate a polyclonal T-cell response to tumor antigens.
The other area that has a high level of excitement for using
A BiTE is a recombinant bispecific protein that has two
scFv technologies is the CAR T-cell approach for adoptive cell
linked scFvs from two different antibodies, one targeting a
transfer. A CAR is a synthetic receptor comprising an extra-
cell-surface molecule on T cells (for example, CD3ε) and the
cellular tumor antigen recognition domain, which is fused
other targeting antigens on the surface of malignant cells.
to the CD3ζ chain for intracellular signaling and contains
The two scFvs are linked together by a short flexible linker
one or more costimulatory domains (Fig. 2). The binding of
(Fig. 1). By binding to tumor antigens and T cells simultane-
scFv to tumor cells triggers the T-cell receptor (TCR) intracel-
lular domain and leads to T-cell responses against antigen-
1
Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, expressing cells.
Victoria, Australia. 2Sir Peter MacCallum Department of Oncology, Univer- Various molecular formats of CAR have been developed,
sity of Melbourne, Parkville, Victoria, Australia. 3National Key Laboratory
differing in their extracellular, transmembrane, and cytoplas-
of Medical Immunology and Institute of Immunology, Second Military
Medical University, Shanghai, China. mic domains, which have been the subject of some previous
Corresponding Authors: Michael H. Kershaw, Peter MacCallum Cancer Cen-
excellent reviews (4). In this review, we focus on CARs in
tre, 305 Grattan Street, Melbourne, Victoria 3000, Australia. Phone: 613- which the predominant cytoplasmic signaling domains are
8559-7092; Fax: 613-8559-5039; E-mail: michael.kershaw@petermac.org; derived from CD3ζ and the costimulatory molecules CD28 or
and Clare Y. Slaney, clare.slaney@petermac.org CD137, which endow T cells with significant antitumor activ-
doi: 10.1158/2159-8290.CD-18-0297 ity, and which have received FDA approval for the treatment
©2018 American Association for Cancer Research. of some hematologic malignancies.

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 Published OnlineFirst July 16, 2018; DOI: 10.1158/2159-8290.CD-18-0297

CAR T-cell and BiTE Therapies for Cancers REVIEW

scFv
T cell
A Antigen C
Antigen
binding site binding site

Variable
Light chain
BiTE CD3
Anti-CD3 scFv
BiTE
Constant
Anti-TAA scFv
Heavy chain Linkers

TAA

IgG1 IgG2

B
VH Linker VL Linker VH Linker VL
Tumor cell
IgG1 scFv IgG2 scFv

Figure 1.  The design of a BiTE. A, Schematic representation of the derivation and structure of a BiTE generated from two antibodies, with specificity
for a T-cell activation molecule and a tumor-associated antigen (TAA). B, Schema of a BiTE gene used to produce the recombinant BiTE protein. Linkers
were constructed between VH and VL domains of the scFv and between the two scFvs. C, A BiTE creates an immunologic synapse by binding simultane-
ously to a tumor cell, via TAA, and a T cell, via CD3.

Recent advances and positive clinical results from BiTE
and CAR T-cell therapies have generated hope and excite-
ment. However, both therapies are facing similar dilemmas,
such as toxicity concerns in hematologic cancers and a lack
of response in solid cancers. In this article, we review and
compare the recent progress in the application of BiTE and
CAR T-cell therapies, summarize the current understanding
of their anticancer mechanisms, and discuss strategies to
improve their efficacy against cancers (Table 1).
Tumor cell
TAA
TCR Extracellular domain (scFv)
Hinge and TM domains CAR
Signaling domains
Figure 2.  The design of a CAR T cell. A CAR comprises an extracellular
domain, which is an scFv targeting TAA. The scFv is followed by a hinge of
varying length and flexibility (CD8 as an example) and various combina-
tions of endodomains that provide T-cell activation signals, such as CD3ζ,
and a costimulation signal, such as CD28. TM, transmembrane.
MECHANISM KEY POINTS
T-cell Types
T-cell populations are made up predominantly of two cell
subsets, expressing either CD4 or CD8. The main function
of CD8+ T cells is considered to be cytolysis of tumor cells,
whereas CD4+ T cells secrete cytokines to regulate immune
responses. In the CAR T-cell approach, CD4+ T cells and CD8+
T cells are usually mixed and genetically modified with CAR
and infused back to the patients in clinical trials. Although
there is no doubt that CD8+ CAR T cells are the major players
in killing cancer cells, CD4+ T cells armed with a CAR can also
be activated via CAR and showed cytolytic activities. Although
CD4+ CAR T cells eradicated leukemia cells more slowly, these
CD4+ CAR T cells persisted longer in vivo (5).
Clinical trials, mixing CAR-equipped CD4+ and CD8+ T cells
at a fixed ratio in treating patients, have generated remarkable
responses. A study using a defined ratio of CD4+:CD8+ CAR T
cells treating refractory B-cell acute lymphoblastic leukemia
(B-ALL) achieved bone marrow disease remission in 27 of 29
evaluable patients (6). In that study, 27 patients received a 1:1
ratio of CD4+:CD8+ CAR T cells, whereas 2 patients received
alternate ratios. Of the 2 patients receiving alternate ratios,
one relapsed and one achieved minimal residual disease. The
low patient numbers and intratrial differences in treatment
regimens did not allow a statistically meaningful comparison
between patients receiving a 1:1 and other ratios. Interest-
ingly, a similar approach using a defined mixture of CD4+
and CD8+ CAR T cells in patients with relapsed/refractory
B-cell non-Hodgkin lymphoma (NHL) achieved 64% com-
plete responses (7). This was comparable with other studies
using CAR T cells of variable composition (8, 9), although
differences in doses and treatment regimens did not allow a
direct comparison of efficacy.

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REVIEW Slaney et al.

Table 1. Comparison of CAR T cells and BiTEs

CAR T cell BiTE

Structure A synthetic gene construct encoding an scFv against A recombinant protein composed of two linked
tumor antigen linked to activation and costimulatory scFvs; one binds to CD3 on T cells and the other
motifs. to target a tumor antigen on tumor cells.
Effector cell types Engineered CD8+ and CD4+ T cells (5). Less-differentiated Endogenous CD8+ and CD4+ T cells (13). Antigen-
subsets displaying better antitumor activity experienced TEM but not TN effective (14).
in vivo (TSCM and TCM; ref. 10).
Immune synapse Atypical (15). Typical (17–19).
Serial killing Yes (16). Yes (22).
Killing mechanisms Perforin and granzyme B (16), Fas/Fas-L, or TNF/TNF-R. Perforin and granzyme B (17).
Trafficking Active. Trafficking of CAR T cells involves comprehen- Passive. Biodistribution depends on factors
sive interactions between various molecules and related to rates of diffusion through vascular
cell–cell interactions (57). endothelium, fluid flow rates, and interaction
with target.
Toxicity CRS, neurotoxicity, B-cell aplasia (31, 49). CRS, neurotoxicity, B-cell aplasia (62, 64).
Clinical applications Pretreatment lymphodepleting regime using cyclophos- No lymphodepletion regime required. Premedi-
phamide and fludarabine. cate with dexamethasone. Repeat administra-
Premedicate with acetaminophen and an H1-antihista- tion necessary, including continuous i.v. infusion
mine. One infusion. regimens.
FDA approval Yescarta was approved to treat adult patients with Blinatumomab was approved to treat relapsed/
relapsed/refractory large B-cell lymphoma in 2017. refractory B-ALL in 2014 and 2017.
Kymriah was approved to treat patients up to 25 years of
age with refractory/relapsed B-ALL in 2017.
Other characteristics Individually produced for each patient. “Off the shelf” reagents.

In addition to cell subsets, T cells can exist in several dif-
ferentiation states that have different functional abilities. T
cells can be classified into naïve T cells (TN) and four main
antigen-experienced or activated subtypes: stem cell memory
(TSCM), central memory (TCM), effector memory (TEM), and
effector (TEFF) T cells. It is now evident that less differentiated
CAR T cells have better efficacy in vivo. In CD19-CAR–treated
patients with relapsed/refractory B-cell malignancies, the fre-
quency of TSCM cells within the transferred lymphocytes cor-
related with their in vivo expansion and persistence during the
first 6 weeks after infusion (10). Preclinical studies showed
that TSCM cells had greater resistance to cell death caused by
repetitive encounter with antigen and had better migration
ability to secondary lymphoid organs (10). It is proposed that
TCM are capable of self-renewal and give rise to shorter-lived
TEM and TEFF cells that are incapable of self-renewal (11).
Therefore, although TEM and TEFF can be present at high fre-
quency at the peak of the anticancer immune response, their
persistence is poor. Accordingly, strategies favoring the gen-
eration and preservation of TSCM and TCM have been applied
in the CAR T-cell therapy field (12).
Similar to CAR T-cell therapy, BiTE therapies involve poly-
clonal T-cell responses, which are independent of MHC and
TCR recognition and costimulation. It has been demon-
strated that when incubated with BiTEs, both CD8+ and
CD4+ T cells can be activated and induce target tumor cell
death, with CD8+ cells killing faster than CD4+ T cells (13).
In contrast to CAR T-cell therapy, where less differentiated
T cells showed better efficacy, in BiTE treatment, antigen-
experienced T-cell subsets mediate BiTE-induced tumor cell
death, whereas naïve T cells are not activated when engaged
by BiTE (1). In an early study, Bargou and colleagues reported
that following injection of the anti-CD19 BiTE blinatu-
momab in patients with relapsed and refractory NHL, T cells
were redistributed and activated. T cells transiently declined
in numbers within 24 to 48 hours, as the T cells adhered to
blood vessels endothelium and/or extravasated. T cells then
returned to baseline numbers with an activated phenotype,
driven by an expansion of CD4+ and CD8+ TEM cells. T-cell lev-
els eventually exceeded pretreatment levels, and this increase
was due to the expansion of TEM cells, but not naïve T cells
(14). Despite some differences in the optimal cell phenotype
for each approach, both therapies mediated tumor cell killing
independent of MHC and provide an opportunity to redirect
T-cell cytotoxicity to tumor cells.
T-cell Cytotoxicity
CAR T cells can induce target tumor cell death through the
formation of synapses, cytokine secretion, perforin/granzyme
B release and/or death receptor ligation. Interestingly, CAR-
mediated immune synapse formation has been reported to
differ from the traditional TCR-induced synapse, in that
the CAR-induced synapse has a much smaller actin ring and
diffuse distribution of LCK. In addition, the microtubule-
organizing center is distant from the immune synapse in
comparison with the classic TCR-induced synapse, which is

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CAR T-cell and BiTE Therapies for Cancers
proximal to the LCK accumulation. The adhesion molecule
LFA1 does not accumulate at the synapse, which could result
in rapid detachment from the dying tumor cells, thereby
enabling CAR T cells to act rapidly (15). In fact, CAR T-cell
serial killing (one T cell kills multiple targets) requires a sig-
nificantly shorter time than TCR-mediated serial killing of
cancer cells (16).
Similar to CAR T cells, it has been reported that, when
incubated with BiTEs, both CD8+ and CD4+ T cells could be
activated to kill target tumor cells in vitro, via perforin and
granzyme B (17), although roles for TNF and FAS do not
appear to have been investigated. However, initial studies
suggest that differences exist between the synapse formed
between T cells and target cells when mediated by BiTEs
compared with CARs. The linkage of tumor cells and T cells
by BiTE leads to the formation of cytolytic synapses that are
similar to the ones formed during regular cytotoxic T-cell
recognition (17–19). Once activated, these T cells start to
proliferate and also upregulate the expression of activation
markers, such as CD69 and CD25 (20). Although the reason
for the difference in synapses formed by CARs and BiTEs is
not clear, it potentially arises because BiTEs engage the natu-
ral CD3 complex, whereas CARs do not.
Both the size and the position of the epitope to the cell
surface determine the extent of BiTE-mediated cancer cell
killing (21). Activated T cells kill the target cancer cells via
triggering apoptosis, as evidenced by caspase 3/7 activation,
PARP cleavage, DNA fragmentation, and membrane blebbing
(17). Similar to CAR T cells, it has been shown that BiTEs can
induce serial killing by T cells, with one T-cell able to kill sev-
eral target cells (22). However, unlike CAR-redirected T-cell
function, BiTE-redirected function is sensitive to the dose of
BiTE administered. Although this may have advantages in
tuning the response to avoid an overreaction and potential
toxic levels of cytokine, an excess of BiTE may completely
coat CD3 and target antigen separately, thereby reducing
the opportunity for coengagement of T cells and target cells
(23–25).
Although direct cancer cell lysis is seen as the most potent
means to reduce cancer burden by T cells, large amounts of
cytokines can be secreted by both CAR- and BiTE-redirected
means (26). These effector cytokines have the potential to
change the tumor microenvironment and induce endog-
enous antitumor immunity. This potential was evident in a
study that demonstrated the ability of CAR T cell–secreted
cytokines to change the recruitment and activation of endog-
enous macrophages and create a favorable milieu for antican-
cer immune response (27). Interestingly, studies using both
BiTEs and CARs suggest that cytokines secreted upon target
cell ligation can contribute to lysis of antigen-negative tumor
cells in close proximity to the antigen-specific engagement,
so-called bystander lysis (28, 29), although this is not always
the case for all target antigens (30).
T-cell Numbers and Persistence
The resolution of malignant disease requires large num-
bers of responding T cells and their continued action over
prolonged time periods. In the case of CAR T cells, these
requirements are best achieved by expansion in vivo and long-
term engraftment following transfer. In some hematologic
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REVIEW
malignancies, CAR T cells have been demonstrated to expand
up to, or even exceeding, 1,000-fold (31, 32) and to reach
greater than 20% of circulating lymphocytes (33). In clini-
cal studies targeting CD19 on B-cell malignancies, absolute
counts of circulating CAR T cells can vary among patients,
but still achieve levels from several thousand per mL of blood
up to several hundred thousand per mL (8, 34, 35).
In the case of BiTEs, a large pool of antigen-experienced T
cells can be exploited to provide substantial numbers of redi-
rected T cells, with less reliance on expansion. Nevertheless,
increased numbers of circulating T cells have been demon-
strated following BiTE administration, suggesting expansion
of the pool of T cells with the potential to respond against
tumor cells. Increases of the order of 2- to 4-fold in circulat-
ing T cells have been described (14, 36, 37). However, because
the number of antigen-experienced T cells (able to respond
independently of costimulation) comprises only a propor-
tion of total T cells, the actual level of expansion of respond-
ing T cells is likely several folds higher than that reflected in
increases in total circulating T cells.
The provision of large numbers of tumor-reactive T cells in
the solid-tumor setting presents different challenges for the
BiTE and CAR T-cell approaches. Whereas extensive expan-
sion and prolonged persistence has been demonstrated for
CAR T cells in hematologic cancers, little if any expansion of
CAR T cells is usually demonstrated when treating solid can-
cers (38–40), except for isolated reports of expansion exceed-
ing 100-fold (41, 42). CAR T cells directed against blood
cancers may receive further costimulatory signals from cancer
cells, or other antigen-positive leukocytes, in a microenviron-
ment that is less immunosuppressive than that found in solid
tumors. This may explain the observed differences in T-cell
expansion in liquid cancers compared with solid cancers.
Approaches combining CAR T-cell transfer with vaccines, to
provide T-cell support away from the tumor microenviron-
ment, are being used to enhance CAR T-cell proliferation and
persistence in the solid-tumor setting (43, 44).
Because the BiTE approach is potentially less reliant on
T-cell expansion, it may be more successful against solid
tumors. However, the success of this approach is dependent
on the sustained loading of BiTEs on T cells, which may
be compromised during the process of penetration of solid
tumors.
Differences between the CAR and BiTE approaches also exist
when considering tumor recurrence. CAR T cells are able to
engraft long-term and provide an ongoing source of tumor-
reactive T cells to respond against recurring tumors, even before
they become evident. This has been demonstrated in mouse
tumor models, in which mice that rejected a primary tumor also
rejected a secondary challenge with tumor cells (43). In contrast,
without continuous administration of BiTEs, recurrent tumors
have the opportunity to grow large before detection, which may
render subsequent treatment with BiTEs less effective.
CLINICAL APPLICATIONS
CAR T-cell therapies have generated fantastic responses
in B-cell malignancies and revolutionized the field of cancer
immunotherapy. CD19 CAR T-cell therapy has been tested in
treating different B-cell malignancies, including NHL (7, 45,
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REVIEW
46), chronic lymphocytic leukemia (CLL; refs. 47, 48), and ALL
(6, 31, 34, 49, 50). In these trials, the relapsed/refractory patients
who had limited treatment options were treated with CD19
CAR T cells, and the treatment generated remarkably high
rates of complete responses, over 50% in NHL (7, 8, 46), 90% in
ALL (6, 49, 50), and over 70% complete response and/or partial
response in CLL (48). In 2017, two CD19 CAR T-cell adoptive
transfer treatments were approved by the FDA. Yescarta (axi-
cabtagene ciloleucel; Kite Pharma) was approved to treat adult
patients with relapsed or refractory large B-cell lymphoma.
Kymriah (tisagenlecleucel; Novartis) was approved for the treat-
ment of patients up to 25 years of age with refractory/relapsed
B-ALL. Preconditioning that depletes endogenous lymphocytes
is important for CAR T-cell therapy; without this, significant
clinical benefit is rarely observed (51).
Given the remarkable initial response rate observed using
CAR T cells in some blood cancers, but a significant relapse
frequency at present, the question remains as to whether
this form of therapy would be best applied as a stand-alone
treatment or as a bridge to stem-cell transplantation. The use
of CAR T cells as a bridge to transplant has some attractive
features including dramatic reduction in disease burden often
to undetectable levels, perhaps enabling a subsequent less-
intensive conditioning regimen prior to transplant. In the CAR
T-cell setting, transplantation following conditioning would
have the added benefit of eliminating CAR T cells to allow
recovery of normal B cells, because B-cell aplasia can persist
long-term in a substantial proportion of patients with leuke-
mia/lymphoma achieving complete responses. This is likely to
continue to be an important option for clinicians in the treat-
ment of hematologic cancers. However, the increased cost of
an additional transplant procedure, together with transplant-
related toxicities if using allogeneic donors, indicates that with
improvements to cell production and efficacy, and controlled
gene expression or inclusion of suicide genes for CAR T cells, a
stand-alone approach would be an ultimate goal.
Although highly effective against CD19 B-cell malignan-
cies, CAR T-cell therapy did not generate the same potent
response against other lymphomas/leukemias. In addition,
the loss of CD19 expression in patients can limit the CD19-
CAR therapeutic benefit. Recently, a few additional CAR
targets have been tested. CD22 in treating patients with ALL
who are refractory to CD19 CAR T-cell therapy (52), CD20 in
indolent B-cell and mantle cell lymphomas (53), B-cell matu-
ration antigen (BCMA) in multiple myeloma (54), and Lewis
Y in acute myeloid leukemia (AML; ref. 55) have been tested
in the clinic and generated encouraging results.
In solid-cancer clinical trials, CAR T cells have not dem-
onstrated the same dramatic effect as seen in hematologic
cancers. The reasons are believed to be the solid-tumor immu-
nosuppressive microenvironment, impaired T-cell trafficking,
and the suboptimal phenotype of the infused CAR T cells (11,
56, 57). A number of tumor antigens have been targeted in
solid cancers, such as GD2 in neuroblastoma (58), HER2 in
glioblastoma and other solid cancers (40, 59), and EGFRvIII
in glioblastoma (60). Interestingly, a recent inspiring study
reported that CAR T-cell treatment targeting IL13Rα-2 has
successfully eradicated a case of glioblastoma, and the com-
plete response lasted several months (61). Although some of
the trials have observed some response, the overall outcomes
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Slaney et al.
are less satisfactory than the ones used in blood cancers.
There is great enthusiasm to improve CAR T-cell treatment
efficacy against solid cancers in preclinical research, and this
is discussed in the next section.
Similar to CAR T cells, BiTEs have been used in clinical
trials targeting a variety of antigens. Blinatumomab was
the first clinically tested and FDA-approved BiTE, receiving
accelerated approval in 2014 for the treatment of CD19+
Philadelphia chromosome–negative (Ph−) relapsed and refrac-
tory B-ALL. In July 2017, the FDA further approved its use to
include the treatment of Philadelphia chromosome–positive
(Ph+) relapsed or refractory B-ALL.
A phase II trial (ALCANTARA, NCT02000427) enrolled
45 patients with Ph+ relapsed or refractory B-ALL for
the blinatumomab treatment. In this trial, 16 patients
(36%) achieved complete remission (CR) or CR with partial
hematologic recovery, and the median duration of remis-
sion was 6.7 months (62). Thus, blinatumomab has led to
promising results for patients with Ph+ relapsed/refractory
ALL. A recent phase II trial (TOWER, NCT02013167) com-
pared blinatumomab with standard-care chemotherapy
in 405 patients with Ph− relapsed and refractory B-ALL.
Blinatumomab demonstrated a significant improvement
in overall survival (OS) and higher rates of hematologic
remission than chemotherapy. The median OS was 7.7
months with blinatumomab compared with 4 months
with chemotherapy (63).
Apart from ALL, blinatumomab has also shown promise
in treating NHL. The initial clinical studies used short infu-
sion of blinatumomab and did not show efficacy but did
show toxicities such as cytokine release syndrome (CRS) and
neurotoxicity (14). Subsequently, lessons were learned from
the ALL studies for continuous infusion of blinatumomab.
A clinical trial using continuous infusion of blinatumomab
60 μg/m2/day in NHL (4-fold higher than ALL) has proven to
be feasible. In this phase I trial, single-agent blinatumomab
showed promising activity with an objective response rate of
69% (24/35 patients; ref. 64).
As with CAR T cells, treatment with BiTEs can lead to
high initial response rates but a significant relapse rate, and
the use of BiTEs in the hematologic setting offers an option
as a bridge to transplantation. As with CAR T-cell therapy,
dramatic reductions in disease and complete responses are
observed in many patients but, in contrast to CAR T cells
where T cells persist long-term, BiTEs require continuous
administration to provide protection against disease recur-
rence. Therefore, hematopoietic stem-cell transplant follow-
ing BiTE therapy may be beneficial. Longer-term follow-up of
complete responders receiving BiTEs (or CAR T cells) will be
informative to determine the feasibility of using T-cell redi-
rection as a stand-alone therapy.
Other tumor antigens have also been explored for BiTE
therapy. For example, CD3–CD33 (AMG330) BiTE has been
constructed to treat patients with relapsed/refractory AML
(NCT02715011).
For treating solid tumors, CD3-coupled BiTEs specific for
EPCAM (solitomab), CEA, and prostate-specific membrane
antigen (PSMA) have all been tested. Limited responses were
observed in treating patients with solid tumors, and toxicity
was observed. In a trial using solitomab treating different
 Published OnlineFirst July 16, 2018; DOI: 10.1158/2159-8290.CD-18-0297
CAR T-cell and BiTE Therapies for Cancers
solid tumors, transient elevation of liver enzymes and diarrhea
as dose-limiting toxicities were observed. This may be due to
the targeting of EPCAM to normal tissue in bowel epithelial
and bile ducts. The toxicity precluded further dose escalation
(65). Although some tumor response was observed, higher
doses were limited by toxicity. New targets, such as gpc3 to
treat hepatocellular carcinoma, have also been tested (66).
TOXICITIES
CAR T-cell therapy can be associated with some toxicities.
The most commonly observed toxicity is CRS, a systemic
response due to elevated levels of cytokines. Studies have dem-
onstrated that high levels of 24 serum cytokines, such as IL6
and IFNγ in the first months after CAR T-cell infusion, were
associated with severe CRS (67). CRS occurs several hours
to 14 days after CAR T-cell infusion, and symptoms include
fever, fatigue, anorexia, hypotension, tachycardia, and capil-
lary leak, which can be life-threatening. It was demonstrated
that the patients with highly elevated levels of IL6 are the ones
with maximum CAR T-cell activation and proliferation (49).
Anti-IL6 receptor antibody (tocilizumab) that blocks IL6 from
binding to its receptor can reverse the life-threatening CRS
without inhibiting CAR T-cell treatment efficacy (31).
Neurotoxicity, also termed CAR T cell–related encephalopa-
thy syndrome (CRES), has also been reported (31, 48, 49).
CRES is typically manifested by symptoms of confusion and
delirium and occasionally by seizures and cerebral edema (6,
68). The pathogenesis of neurologic toxicity remains unclear,
although it has been proposed that CRES could be due to
cytokine-mediated endothelial activation and passive diffusion
of cytokines into the brain (69) and/or trafficking of T cells
into the central nervous system (CNS; ref. 68). Although CD19
CAR T cells were identified in the cerebrospinal fluid (CSF),
levels of the transgene in the brain did not correlate with rates
of high-grade CRES events (32). CRES is generally reversible
using anti-IL6 receptor and/or corticosteroids (68, 69).
B-cell aplasia is also associated with CD19 CAR T-cell
therapy, because CD19 is expressed on normal B cells, but
injections of immunoglobulin can be used to maintain levels
of circulating antibodies (49). Infections with microorganisms
can also occur following CAR T-cell transfer, which was largely
due to depletion of endogenous leukocytes by precondition-
ing regimens (70). Bacterial infections predominated, which
included bacteremia and more localized infections to sites
including gastrointestinal tissue. Viral and fungal infections
were also experienced. These infections are largely manageable
using antibiotics, although some fatalities have resulted.
Although leukodepleting preconditioning is not required
for BiTE therapy, toxicities after blinatumomab treatment are
common, but mostly manageable. The most frequent adverse
effects were CRS and neurologic adverse effects including
headache, tremor, aphasia, and seizure (62, 64). These effects
might be associated with T-cell migration to the CNS or a
transient cytokine storm in serum. Currently, the blinatu-
momab treatment dosage and administration schedule is
based on body weight, and patients are required to be pre-
medicated with dexamethasone, to reduce cytokine produc-
tion while retaining cytolytic activity (71, 72). In addition, the
treatment of blinatumomab also depleted normal B cells and
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Research.
REVIEW
plasma precursor cells, resulting in substantial hypogamma-
globulinemia. This presents risk of infections, which can be
reduced by administration of intravenous immunoglobulin.
RESEARCH IN ENHANCING TREATMENT
EFFICACY AGAINST SOLID CANCERS
Both BiTE and CAR T-cell treatment strategies have pro-
duced remarkable effects in hematologic malignancies; how-
ever, the efficacy for both was generally poor against solid
cancers. Considerable effort is being invested in enhancing
the treatment efficacy against solid cancers.
For CAR T-cell therapy, the hurdles for treating solid can-
cers lie in the combination of the immunosuppressive effect
of the tumor microenvironment and inefficient penetration
of CAR T cells into tumors. The tumor microenvironment
is a complex assortment of cell types including malignant
cells, fibroblasts, endothelium, and immunoregulatory cells.
Regulatory cell types include myeloid-derived suppressor cells
(MDSC), M2 macrophages, and regulatory T cells (Treg; ref.
73). Using a preclinical model, Moon and colleagues demon-
strated that CAR T cells were able to slow down tumor growth,
but did not cause regression. The reason was the rapid loss of
functions of CAR T cells when exposed to the tumor microen-
vironment in vivo. Diminished functions included reduction
in the ability to secrete cytokines, killing ability, and phospho-
ERK expression. When CAR T cells were isolated and rested
away from the tumor, their hypofunction was reversed (74).
For targeting the suppressive tumor microenvironment,
a number of strategies have been tested. For example, using
all-trans retinoic acid (75) or GM-CSF neutralization to dis-
rupt MDSC function (76), targeting CD123 on macrophages
(77), or fibroblast activation protein on cancer-associated
fibroblasts (78) inhibiting adenosine 2A receptors (79) and
altering metabolic components such as inhibiting protein
kinase A activation (80) all demonstrated enhancement of
CAR T-cell treatment efficacy in preclinical models of solid
tumors. In addition, there has been great interest in using
checkpoint inhibitors as adjuvant therapy for CAR T-cell
treatment. Blocking a number of checkpoints, such as PD-1
(81) and LAG3 (82), has been shown to enhance CAR T-cell
treatment in solid tumors.
Other strategies that have shown promising results include
supplying CAR T cells with cytokine support to enhance
their activity within the tumor microenvironment (83). Such
approaches include inducing the local release of stimulatory
cytokines, including IL12 (84), and transducing the IL7 recep-
tor into CAR T cells to provide signal 3 for T-cell support
(85). Inhibitory cytokines present in the tumor microenviron-
ment can also be harnessed to provide support for T cells. In
this approach, T cells are genetically modified with chimeric
cytokine receptors. For example, chimeric cytokine receptors
composed of the extracellular domain of the IL4 receptor
fused to the intracellular domain of the IL7 receptor led to
activation and proliferation of T cells in the presence of the
normally inhibitory cytokine IL4 (86, 87).
To enhance CAR T-cell infiltration to the tumor bed, effort
has been made to modify chemokine receptors on CAR T cells
to enhance their migration toward tumors. For example, CAR
T cells have been modified to express CCR2b, the chemokine
August 2018 CANCER DISCOVERY | 929
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REVIEW
receptor for CCL2 that is highly expressed by the target
tumors (88). To facilitate the penetration into tumors, con-
struction of CARs targeting VEGFR2 or αvβ3 integrin over-
expressed on tumor vasculature has been reported (89, 90).
Similar to CAR T-cell therapy, the efficacy of BiTEs in treat-
ing solid tumors in preclinical models is modest. One of the
major hurdles for BiTEs is their short half-life in serum. After
injection, BiTEs are rapidly catabolized and cleared from the
circulation, leading to a short serum half-life of around 2
hours (91). To overcome the limitation, current approaches
include attaching BiTEs to heavy-chain fragments or other
novel designs to increase the serum life. A number of these
methods significantly increased the BiTE circulation time
without hampering its binding to the target cells (92).
The other major hurdle for treating solid tumors using
BiTE is the density and types of T cells already in the tumor
bed. Small-animal PET and fluorescent imaging studies have
demonstrated the penetration of BiTE into solid tumors
(93). However, the profiles of the T cells already existing in
the tumor are difficult to predict, as the intensity, location,
phenotype, and activation phenotypes of these T cells vary
dramatically among tumors (94). In addition, preexisting T
cells in the tumor may already have an exhausted phenotype
or may be Tregs. Approaches in increasing T-cell infiltration
into tumor tissue and combinational approaches to reverse
T-cell exhaustion and targeting the tumor immunosuppres-
sive microenvironment warrant further investigation to boost
the efficacy of BiTE against solid cancers.
Recent enthusiasm surrounding BiTEs extends toward
their production in vivo. One strategy is to produce geneti-
cally modified T cells for making BiTE to overcome the
limitations of BiTEs’ short half-life and passive biodistribu-
tion. Technologies were developed to generate T cells that
secrete BiTEs (ENG-T cells) targeting tumor antigens such
as EPHA2 (95), CD123 (96), and CD19 (97). These ENG-T
cells can recognize tumor cells in an antigen-dependent man-
ner in vitro and in vivo, redirecting T cells to the target cancer
cells, and have potent antitumor activity in cancer models
(95, 96). Importantly, these ENG-T cells expanded in vivo and
increased the production of BiTEs after activation, obviating
the need for continuous infusion of engager molecules (95).
Interestingly, a recent report compared the efficacy of RNA-
electroporated ENG-T cells to the RNA-electroporated CAR
T cells and concluded that the ENG-T cells showed more
efficacy in the NALM6 leukemia model (98).
Another recent promising strategy is to use an oncolytic virus
engineered to express a BiTE. Oncolytic viruses have been tested
in the clinic and have demonstrated safety and some anticancer
effects. Two recent studies engineered an oncolytic adenovi-
rus to secrete an EGFR-targeting BiTE. The BiTE-expressing
adenovirus induced a strong T-cell response against cancer cells
both in vitro and in vivo (99, 100). The use of oncolytic virus in
combination with BiTE or the oncolytic virus secreting BiTE is
another strategy for changing the tumor microenvironment.
Several oncolytic viruses are currently under development, and
whether different strains of these viruses are better than others
to be engineered to express and secrete BiTE is yet to be inves-
tigated. Post–oncolytic virus administration, large numbers of
immune cells infiltrated to the tumors, and the addition of
BiTE redirects infiltrating T cells against tumors (101).
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Slaney et al.
Strategies for changing the tumor microenvironment may
greatly enhance BiTE treatment efficacy in solid cancers. It
was reported that tumors upregulated PD-L1 post–BiTE
administration (102) and adding anti–PD-L1 to AMG 330
treatment enhanced killing in vitro (103). Other inhibitor
molecules to target include galectin 1, which has been docu-
mented to be linked with BiTE resistance (104). Enhancing
tumor antigen presentation has also been shown to be a
good strategy for enhancing BiTE treatment efficacy. Adding
the epigenetic modifier drugs panobinostat and azacitidine
increased CD33 expression in some cell lines and enhanced
AMG 330–induced cytotoxicity (105).
A few studies directly compared the functional efficacy of
the two strategies in their antitumor effect (106). In an early
study, Stone and colleagues compared the CAR and BiTE
treatment antitumor response in vitro using the same antican-
cer scFv against a murine fibrosarcoma epitope, 237. Although
both strategies enhanced T cell–mediated antitumor effect,
CAR T cells were more potent than the BiTE approach against
cancers expressing lower levels of antigens (107). In a recent
study, Hoseini and colleagues compared a bispecific antibody
(specific for both CD3 and GD2), with a CAR that contained
the same anti-GD2 scFv (106). Incubating GD2+ target cancer
cells with anti-GD2 CAR T cells for 24 hours led to the deple-
tion of the CARhi T cells, and, in contrast, in the presence of
BC119, the exposure of untransduced T cells to GD2+ target
cancer cells did not deplete the T cells. This GD2-specific CAR
T-cell depletion severely compromised CAR T-cell cytotoxic-
ity in vitro and impaired their in vivo antitumor efficacy. For
treating the GD2+ tumors in immunocompromised mice,
BC119 with untransduced T cells conferred rapid shrinkage
of the established tumors, whereas CAR T cell–treated tumors
showed a delayed regression for 2 weeks. The percentage of
tumor-infiltrating lymphocytes (TIL) in the BC119 treatment
was higher than that in the CAR T-cell treatment. Interest-
ingly, the ratio of CD4+ and CD8+ TILs in BC119-treated mice
was nearly equal, whereas the TILs in the CAR T cell–treated
mice were nearly all CD8+. The number and composition of
TILs could partially explain the superior antitumor effect
mediated by BiTE over CAR T-cell treatment. It is note­worthy
that the above study used immunocompromised mice with
adoptive transferred T cells, whereas in the clinic, BiTEs are
usually administered without adoptive transfer. The TIL pro-
files in immunocompetent mice are poorly studied, albeit a
few studies demonstrated that BiTE could suppress tumor
progression in these mice (108–110).
In the clinic, it is possible that patients refractory to one
therapy respond to the other. For example, in a trial run by
Maude and colleagues in 2014, 2 patients refractory to bli-
natumomab achieved CR after CD19-CAR treatment (49).
This suggests that a lack of response to one immunotherapy
against the same target may not preclude a successful therapy
outcome with another, even with the same target. In addition,
there could conceivably be some benefit from using CAR T
cells and BiTEs simultaneously against tumors. A preclinical
study targeting the alpha folate receptor using a CAR, while
also targeting EGFR using an oncolytic virus encoding a
BiTE, led to increased inhibition of tumors (111). This study
suggested that combining these approaches, and oncolytic
viruses, could address the issues of antigen heterogeneity and
 Published OnlineFirst July 16, 2018; DOI: 10.1158/2159-8290.CD-18-0297
CAR T-cell and BiTE Therapies for Cancers
immunosuppressive microenvironment of solid tumors to
enhance antitumor responses.
CONCLUSIONS
We have summarized the current clinical status of the
two immunotherapies BiTE and CAR T-cell treatment, their
modes of action, toxicities, and preclinical development. Both
platforms have demonstrated excellent efficacy in clinical
trials against hematologic malignancies and were recently
approved by the FDA for treating B-ALL. With their applica-
tions in the clinic, we will understand more about the anti-
cancer mechanisms, investigate these treatments as single
or combined methods in different situations, and eventually
expand their success to other cancers.
Despite the excitement, a few issues still remain to be
overcome. Interestingly, both platforms are facing the same
dilemma: CRS and neurologic adverse effects and moderate
anticancer effects in solid-tumor settings. Substantial effort
has been invested in finding strategies for reducing toxicities
and enhancing treatment efficacy against solid tumors, such
as targeting the immunosuppressive microenvironment and
enhancing T-cell infiltration to the tumors. Both treatments
warrant further exploration in a wide variety of tumor set-
tings.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
This work was supported by grants from Cure Cancer Australia
(1100199), the Peter MacCallum Cancer Centre Foundation, the
National Health and Medical Research Council (NHMRC) of Aus-
tralia (1103352 and 1132373), the National Breast Cancer Foun-
dation (NBCF) of Australia (IIRS-18-064), and Susan G. Komen
(16376637). P.K. Darcy and M.H. Kershaw were supported by
NHMRC Senior Research Fellowships. C.Y. Slaney was supported by
a postdoctoral fellowship from the NBCF. P. Wang was supported
by grants from the National Natural Science Foundation of China
(31470871, 81671566, and 31722019).
The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked advertisement in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Received March 29, 2018; revised May 20, 2018; accepted June 1,
2018; published first July 16, 2018.
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 Published OnlineFirst July 16, 2018; DOI: 10.1158/2159-8290.CD-18-0297

CARs versus BiTEs: A Comparison between T Cell−Redirection

Strategies for Cancer Treatment
Clare Y. Slaney, Pin Wang, Phillip K. Darcy, et al.

Cancer Discov 2018;8:924-934. Published OnlineFirst July 16, 2018.

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